A Textbook of Pharmaceutical Microbiology (With Experiments) 9389307341

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A Textbook of

Pharmaceutical Microbiology (With Experiments) Textbook of Pharmaceutical Mircobiology is meant for B. Pharmacy curriculum as suggested by AICTE. This book is also essential for pharmacy students because of its relevance in pharmaceutical industry, particularly in manufacturing of parenteral products, ophthalmic products, vaccines, etc., and their aseptic processing and sterilization. This book contains five sections divided into twelve chapters. Section A deals with the scope, history and classification of microorganisms. Sections B and C describe the nutrients and isolation of microorganisms, microbial genetics and sterilization techniques. Section D details the basics of immunology and microbial assays and Section E contains experiments. The book covers the following topics: •

Microorganism structure, their classification and functions and microbial genetics.

•

Sterilization processes in laboratory as well as in hospitals.

•

Staining techniques of bacteria.

•

Basics of immunology: history, different antibody classes, antigen-antibody reactions, hypersensitivity reactions, and immunological tolerance.

•

Gene transfer, replication, transcription and translation processes. Prahlad Singh Mehra is Assistant Professor, Abhilashi College of Pharmacy, Mandi, Himachal Pradesh. He obtained his B. Pharmacy degree from Shri Guru Ram Rai Institute of Technology and Science, Dehradun and M.Tech. degree in Biotechnology from West Bengal University of Technolgy, Kolkata. At present, he is involved in teaching Pharmaceutical Microbiology and Pharmaceutical Biotechnology.

He has published research papers in journals of national and international repute, viz., Journal of Ethnopharmacology (Science Direct), Current Cardiology Review (Bentham Science) and Journal of Biological Sciences (Online). He has also participated in various symposia/workshops on pharmaceutical sciences and biotechnology. His areas of interest are fermentation technology, enzymology, molecular biology and rDNA technology. 978-93-89307-34-4

` 365/9 789389 307344

A TEXTBOOK OF

A TEXTBOOK OF

Pharmaceutical Microbiology

Pharmaceutical Microbiology

(WITH EXPERIMENTS)

(WITH EXPERIMENTS)

A TEXTBOOK OF

A TEXTBOOK OF

Pharmaceutical Microbiology

Pharmaceutical Microbiology

(WITH EXPERIMENTS)

(WITH EXPERIMENTS)

A TEXTBOOK OF

A TEXTBOOK OF

Pharmaceutical Microbiology

Pharmaceutical Microbiology

(WITH EXPERIMENTS)

(WITH EXPERIMENTS)

Prahlad Singh Mehra

Prahlad Singh Mehra

Assistant Professor Abhilashi College of Pharmacy Ner Chowk, Mandi, Himachal Pradesh

Assistant Professor Abhilashi College of Pharmacy Ner Chowk, Mandi, Himachal Pradesh

A TEXTBOOK OF

A TEXTBOOK OF

Pharmaceutical Microbiology

Pharmaceutical Microbiology

(WITH EXPERIMENTS)

(WITH EXPERIMENTS)

Prahlad Singh Mehra

Prahlad Singh Mehra

Assistant Professor Abhilashi College of Pharmacy Ner Chowk, Mandi, Himachal Pradesh

Assistant Professor Abhilashi College of Pharmacy Ner Chowk, Mandi, Himachal Pradesh

©Copyright 2019 I.K. International Pvt. Ltd., New Delhi-110002. This book may not be duplicated in any way without the express written consent of the publisher, except in the form of brief excerpts or quotations for the purposes of review. The information contained herein is for the personal use of the reader and may not be incorporated in any commercial programs, other books, databases, or any kind of software without written consent of the publisher. Making copies of this book or any portion for any purpose other than your own is a violation of copyright laws. Limits of Liability/disclaimer of Warranty: The author and publisher have used their best efforts in preparing this book. The author make no representation or warranties with respect to the accuracy or completeness of the contents of this book, and specifically disclaim any implied warranties of merchantability or fitness of any particular purpose. There are no warranties which extend beyond the descriptions contained in this paragraph. No warranty may be created or extended by sales representatives or written sales materials. The accuracy and completeness of the information provided herein and the opinions stated herein are not guaranteed or warranted to produce any particulars results, and the advice and strategies contained herein may not be suitable for every individual. Neither Dreamtech Press nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Trademarks: All brand names and product names used in this book are trademarks, registered trademarks, or trade names of their respective holders. Dreamtech Press is not associated with any product or vendor mentioned in this book. ISBN: 978-93-89307-34-4 EISBN: 978-93-89447-23-1 Edition: 2019

Dedicated to My parents and All the students of B. Pharmacy

Dedicated to My parents and All the students of B. Pharmacy

Dedicated to My parents and All the students of B. Pharmacy

Dedicated to My parents and All the students of B. Pharmacy

FOREWORD

FOREWORD

The book entitled “A Textbook of Pharmaceutical Microbiology” authored by Prahlad Singh Mehra, Asst. Professor, Abhilashi College of Pharmacy, Mandi, Himachal Pradesh has been written basically for the pharmacy students. The book will be useful to update the basic concepts of microbiology, biotechnology and immunology. The role of microorganisms in the production of various pharmaceutical products is very important so this book contains different methods of identification and isolation of microorganisms with their applications. The text covers the pharmaceutical microbiology curriculum. Accordingly this book serves as a valuable source of information for teachers and students of pharmacy as well as to the allied professionals. This text is reliable and is surely going to be accepted well. To meet the textbook requirements this book summarizes important aspects from various areas of pharmaceutical microbiology. Every attempt has been made to make each chapter independent and self-contained for use as per the AICTE syllabus by the author. I wish the book to be a landmark in subject of Pharmaceutical Microbiology.

The book entitled “A Textbook of Pharmaceutical Microbiology” authored by Prahlad Singh Mehra, Asst. Professor, Abhilashi College of Pharmacy, Mandi, Himachal Pradesh has been written basically for the pharmacy students. The book will be useful to update the basic concepts of microbiology, biotechnology and immunology. The role of microorganisms in the production of various pharmaceutical products is very important so this book contains different methods of identification and isolation of microorganisms with their applications. The text covers the pharmaceutical microbiology curriculum. Accordingly this book serves as a valuable source of information for teachers and students of pharmacy as well as to the allied professionals. This text is reliable and is surely going to be accepted well. To meet the textbook requirements this book summarizes important aspects from various areas of pharmaceutical microbiology. Every attempt has been made to make each chapter independent and self-contained for use as per the AICTE syllabus by the author. I wish the book to be a landmark in subject of Pharmaceutical Microbiology.

Dr. Denesh Kumar Chatanta (Head) Ph.D., PGDCA, PGDBI. Abhilashi Institute of Life Science, Mandi, H.P. Formerly- Head Biotechnology Shoolini Institute of Life Science and Business Management Solan, H.P.

Dr. Denesh Kumar Chatanta (Head) Ph.D., PGDCA, PGDBI. Abhilashi Institute of Life Science, Mandi, H.P. Formerly- Head Biotechnology Shoolini Institute of Life Science and Business Management Solan, H.P.

FOREWORD

FOREWORD

The book entitled “A Textbook of Pharmaceutical Microbiology” authored by Prahlad Singh Mehra, Asst. Professor, Abhilashi College of Pharmacy, Mandi, Himachal Pradesh has been written basically for the pharmacy students. The book will be useful to update the basic concepts of microbiology, biotechnology and immunology. The role of microorganisms in the production of various pharmaceutical products is very important so this book contains different methods of identification and isolation of microorganisms with their applications. The text covers the pharmaceutical microbiology curriculum. Accordingly this book serves as a valuable source of information for teachers and students of pharmacy as well as to the allied professionals. This text is reliable and is surely going to be accepted well. To meet the textbook requirements this book summarizes important aspects from various areas of pharmaceutical microbiology. Every attempt has been made to make each chapter independent and self-contained for use as per the AICTE syllabus by the author. I wish the book to be a landmark in subject of Pharmaceutical Microbiology.

The book entitled “A Textbook of Pharmaceutical Microbiology” authored by Prahlad Singh Mehra, Asst. Professor, Abhilashi College of Pharmacy, Mandi, Himachal Pradesh has been written basically for the pharmacy students. The book will be useful to update the basic concepts of microbiology, biotechnology and immunology. The role of microorganisms in the production of various pharmaceutical products is very important so this book contains different methods of identification and isolation of microorganisms with their applications. The text covers the pharmaceutical microbiology curriculum. Accordingly this book serves as a valuable source of information for teachers and students of pharmacy as well as to the allied professionals. This text is reliable and is surely going to be accepted well. To meet the textbook requirements this book summarizes important aspects from various areas of pharmaceutical microbiology. Every attempt has been made to make each chapter independent and self-contained for use as per the AICTE syllabus by the author. I wish the book to be a landmark in subject of Pharmaceutical Microbiology.

Dr. Denesh Kumar Chatanta (Head) Ph.D., PGDCA, PGDBI. Abhilashi Institute of Life Science, Mandi, H.P. Formerly- Head Biotechnology Shoolini Institute of Life Science and Business Management Solan, H.P.

Dr. Denesh Kumar Chatanta (Head) Ph.D., PGDCA, PGDBI. Abhilashi Institute of Life Science, Mandi, H.P. Formerly- Head Biotechnology Shoolini Institute of Life Science and Business Management Solan, H.P.

PREFACE

PREFACE

The study of microbiology for the pharmacy students is very important and essential. This is so because its direct connection with the pharmaceutical industry particularly in the manufacture of parenteral products, ophthalmic products and vaccines etc. and their aseptic processing and sterilization. This book consists of 5 sections divided into 12 chapters. First section deals with the scope, history and classification of microorganisms. Section B and C describes the nutrients and isolation of microorganisms, microbial genetics and sterilization techniques. However, in Section D gives the details of basics of immunology and microbial assays and Section E contains all experiments. I acknowledge the help rendered by all the faculty members of Abhilashi College of Pharmacy Prof. D.N. Sharma, Asst. Prof., namely Bhupendra Singh, Deepak Awasthi, Ravi Shankar Yadav, Manjir Sharma Kataki, Ananya Rajkumari, Gitanjali, Nishant Gautam, Thakra Ram Chaudhary, Amit Kumar, Navdeep Singh and Preeti Aneja and Dr. R.K. Abhilashi chairman of Abhilashi Education Society. I am thankful to Asst. Prof. Rashmi and Nitin Jain, School of Pharmaceutical Sciences; Shoolini University, H.P; Asst. Prof. Ratendra Kumar, Department of Pharmacy, National University Jodhpur, Asst. Prof. Rajendra Awasthi, Laureate Institute of Pharmacy, H.P; Asst. Prof. Laxmi Goswami, SGRRITS Dehradun; Asst. Prof. Sumit Durgapal, Jyoti Upadhyay, Bhawna Goyal and Vikram Karki, Himalayan Institute of Pharmacy and Research, Dehradun; Asst. Prof. Kuldeep Singh Yadav, Sagar Institute of Technology and Management, U.P.; Asst. Prof. Gaurav Mittal, Dayanad Dinanath College, Institute of Pharmacy, Kanpur; Asst. Prof. Anil Waldia, Asst. Prof. Richa Gupta, SBSPGI Balawala, Dehradun; Lect. Kaushal Prasad Mishra, School of Studies in Pharmaceutical Science, Jiwaji University Gwalior. Asst. Prof. Sanjay Singh, Rohit Bist and Priyanka Mittal Siddharth Institute of Pharmacy, Dehradun; Mr. Kapil Verma, ASBASJSM, College of Pharmacy Bela, Punjab; Asst. Prof. Gaurav Verma, Shree Dev Bhoomi Institute of Education Science and Technology, Dehradun; Asst. Prof. Vikash Kumar, Allahabad Agriculture Institute, U.P. Asst. Prof. Prakash Kumar, Anand Engineering College, Agra, U.P; Lect. Anjana Sharma, Modern Education College, Annadale, Shimla and many others for their best wishes and valuable suggestions.

The study of microbiology for the pharmacy students is very important and essential. This is so because its direct connection with the pharmaceutical industry particularly in the manufacture of parenteral products, ophthalmic products and vaccines etc. and their aseptic processing and sterilization. This book consists of 5 sections divided into 12 chapters. First section deals with the scope, history and classification of microorganisms. Section B and C describes the nutrients and isolation of microorganisms, microbial genetics and sterilization techniques. However, in Section D gives the details of basics of immunology and microbial assays and Section E contains all experiments. I acknowledge the help rendered by all the faculty members of Abhilashi College of Pharmacy Prof. D.N. Sharma, Asst. Prof., namely Bhupendra Singh, Deepak Awasthi, Ravi Shankar Yadav, Manjir Sharma Kataki, Ananya Rajkumari, Gitanjali, Nishant Gautam, Thakra Ram Chaudhary, Amit Kumar, Navdeep Singh and Preeti Aneja and Dr. R.K. Abhilashi chairman of Abhilashi Education Society. I am thankful to Asst. Prof. Rashmi and Nitin Jain, School of Pharmaceutical Sciences; Shoolini University, H.P; Asst. Prof. Ratendra Kumar, Department of Pharmacy, National University Jodhpur, Asst. Prof. Rajendra Awasthi, Laureate Institute of Pharmacy, H.P; Asst. Prof. Laxmi Goswami, SGRRITS Dehradun; Asst. Prof. Sumit Durgapal, Jyoti Upadhyay, Bhawna Goyal and Vikram Karki, Himalayan Institute of Pharmacy and Research, Dehradun; Asst. Prof. Kuldeep Singh Yadav, Sagar Institute of Technology and Management, U.P.; Asst. Prof. Gaurav Mittal, Dayanad Dinanath College, Institute of Pharmacy, Kanpur; Asst. Prof. Anil Waldia, Asst. Prof. Richa Gupta, SBSPGI Balawala, Dehradun; Lect. Kaushal Prasad Mishra, School of Studies in Pharmaceutical Science, Jiwaji University Gwalior. Asst. Prof. Sanjay Singh, Rohit Bist and Priyanka Mittal Siddharth Institute of Pharmacy, Dehradun; Mr. Kapil Verma, ASBASJSM, College of Pharmacy Bela, Punjab; Asst. Prof. Gaurav Verma, Shree Dev Bhoomi Institute of Education Science and Technology, Dehradun; Asst. Prof. Vikash Kumar, Allahabad Agriculture Institute, U.P. Asst. Prof. Prakash Kumar, Anand Engineering College, Agra, U.P; Lect. Anjana Sharma, Modern Education College, Annadale, Shimla and many others for their best wishes and valuable suggestions.

PREFACE

PREFACE

The study of microbiology for the pharmacy students is very important and essential. This is so because its direct connection with the pharmaceutical industry particularly in the manufacture of parenteral products, ophthalmic products and vaccines etc. and their aseptic processing and sterilization. This book consists of 5 sections divided into 12 chapters. First section deals with the scope, history and classification of microorganisms. Section B and C describes the nutrients and isolation of microorganisms, microbial genetics and sterilization techniques. However, in Section D gives the details of basics of immunology and microbial assays and Section E contains all experiments. I acknowledge the help rendered by all the faculty members of Abhilashi College of Pharmacy Prof. D.N. Sharma, Asst. Prof., namely Bhupendra Singh, Deepak Awasthi, Ravi Shankar Yadav, Manjir Sharma Kataki, Ananya Rajkumari, Gitanjali, Nishant Gautam, Thakra Ram Chaudhary, Amit Kumar, Navdeep Singh and Preeti Aneja and Dr. R.K. Abhilashi chairman of Abhilashi Education Society. I am thankful to Asst. Prof. Rashmi and Nitin Jain, School of Pharmaceutical Sciences; Shoolini University, H.P; Asst. Prof. Ratendra Kumar, Department of Pharmacy, National University Jodhpur, Asst. Prof. Rajendra Awasthi, Laureate Institute of Pharmacy, H.P; Asst. Prof. Laxmi Goswami, SGRRITS Dehradun; Asst. Prof. Sumit Durgapal, Jyoti Upadhyay, Bhawna Goyal and Vikram Karki, Himalayan Institute of Pharmacy and Research, Dehradun; Asst. Prof. Kuldeep Singh Yadav, Sagar Institute of Technology and Management, U.P.; Asst. Prof. Gaurav Mittal, Dayanad Dinanath College, Institute of Pharmacy, Kanpur; Asst. Prof. Anil Waldia, Asst. Prof. Richa Gupta, SBSPGI Balawala, Dehradun; Lect. Kaushal Prasad Mishra, School of Studies in Pharmaceutical Science, Jiwaji University Gwalior. Asst. Prof. Sanjay Singh, Rohit Bist and Priyanka Mittal Siddharth Institute of Pharmacy, Dehradun; Mr. Kapil Verma, ASBASJSM, College of Pharmacy Bela, Punjab; Asst. Prof. Gaurav Verma, Shree Dev Bhoomi Institute of Education Science and Technology, Dehradun; Asst. Prof. Vikash Kumar, Allahabad Agriculture Institute, U.P. Asst. Prof. Prakash Kumar, Anand Engineering College, Agra, U.P; Lect. Anjana Sharma, Modern Education College, Annadale, Shimla and many others for their best wishes and valuable suggestions.

The study of microbiology for the pharmacy students is very important and essential. This is so because its direct connection with the pharmaceutical industry particularly in the manufacture of parenteral products, ophthalmic products and vaccines etc. and their aseptic processing and sterilization. This book consists of 5 sections divided into 12 chapters. First section deals with the scope, history and classification of microorganisms. Section B and C describes the nutrients and isolation of microorganisms, microbial genetics and sterilization techniques. However, in Section D gives the details of basics of immunology and microbial assays and Section E contains all experiments. I acknowledge the help rendered by all the faculty members of Abhilashi College of Pharmacy Prof. D.N. Sharma, Asst. Prof., namely Bhupendra Singh, Deepak Awasthi, Ravi Shankar Yadav, Manjir Sharma Kataki, Ananya Rajkumari, Gitanjali, Nishant Gautam, Thakra Ram Chaudhary, Amit Kumar, Navdeep Singh and Preeti Aneja and Dr. R.K. Abhilashi chairman of Abhilashi Education Society. I am thankful to Asst. Prof. Rashmi and Nitin Jain, School of Pharmaceutical Sciences; Shoolini University, H.P; Asst. Prof. Ratendra Kumar, Department of Pharmacy, National University Jodhpur, Asst. Prof. Rajendra Awasthi, Laureate Institute of Pharmacy, H.P; Asst. Prof. Laxmi Goswami, SGRRITS Dehradun; Asst. Prof. Sumit Durgapal, Jyoti Upadhyay, Bhawna Goyal and Vikram Karki, Himalayan Institute of Pharmacy and Research, Dehradun; Asst. Prof. Kuldeep Singh Yadav, Sagar Institute of Technology and Management, U.P.; Asst. Prof. Gaurav Mittal, Dayanad Dinanath College, Institute of Pharmacy, Kanpur; Asst. Prof. Anil Waldia, Asst. Prof. Richa Gupta, SBSPGI Balawala, Dehradun; Lect. Kaushal Prasad Mishra, School of Studies in Pharmaceutical Science, Jiwaji University Gwalior. Asst. Prof. Sanjay Singh, Rohit Bist and Priyanka Mittal Siddharth Institute of Pharmacy, Dehradun; Mr. Kapil Verma, ASBASJSM, College of Pharmacy Bela, Punjab; Asst. Prof. Gaurav Verma, Shree Dev Bhoomi Institute of Education Science and Technology, Dehradun; Asst. Prof. Vikash Kumar, Allahabad Agriculture Institute, U.P. Asst. Prof. Prakash Kumar, Anand Engineering College, Agra, U.P; Lect. Anjana Sharma, Modern Education College, Annadale, Shimla and many others for their best wishes and valuable suggestions.

x CONTENTS PREFACE

x CONTENTS PREFACE

I am also thankful to all my teachers: Prof. Vijay Juyal, Dr. Firoz Anwer, Asst. Prof. Divya Juyal, Prof. Preeti Kothiyal, Dr. Shon Roy Chaudhary and Dr. Jaya Bandyopadhyay. My sincere thanks to Shri Vrajesh Makhijani (Director), I.K. International Publishing House Pvt. Ltd., New Delhi and his dedicated and dynamic team for publishing this book elegantly and speedily. In truce sense I derived much inspiration and blessings from my mother, respected Smt. Devki Devi Mehra, and respected father Shri Mahendra Singh Mehra. Also I take opportunity to express my thanks to all my family members especially to my sister Mrs. Laxmi and my brother-in-law Mr. Khushal Singh and brother Mr. Tarendra Singh and Gaurav Singh. It is the privilege to offer my sincere gratitude to the co-author of this book Miss Pratibha Sharma, M. Pharmacy (Pharmacology) Soolini University, Solan, Himachal Pradesh. Who actually helped me sincerely while I was simply engrossed in preparing and also finalizing this manuscript. From the core of my heart, I am grateful to her indeed. Comments, suggestions and constructive criticisms towards improvements of future editions of this volume are very much welcomed. In fact, they underline the shortcomings which are looked into and rectified in the interest of our esteemed readers.

I am also thankful to all my teachers: Prof. Vijay Juyal, Dr. Firoz Anwer, Asst. Prof. Divya Juyal, Prof. Preeti Kothiyal, Dr. Shon Roy Chaudhary and Dr. Jaya Bandyopadhyay. My sincere thanks to Shri Vrajesh Makhijani (Director), I.K. International Publishing House Pvt. Ltd., New Delhi and his dedicated and dynamic team for publishing this book elegantly and speedily. In truce sense I derived much inspiration and blessings from my mother, respected Smt. Devki Devi Mehra, and respected father Shri Mahendra Singh Mehra. Also I take opportunity to express my thanks to all my family members especially to my sister Mrs. Laxmi and my brother-in-law Mr. Khushal Singh and brother Mr. Tarendra Singh and Gaurav Singh. It is the privilege to offer my sincere gratitude to the co-author of this book Miss Pratibha Sharma, M. Pharmacy (Pharmacology) Soolini University, Solan, Himachal Pradesh. Who actually helped me sincerely while I was simply engrossed in preparing and also finalizing this manuscript. From the core of my heart, I am grateful to her indeed. Comments, suggestions and constructive criticisms towards improvements of future editions of this volume are very much welcomed. In fact, they underline the shortcomings which are looked into and rectified in the interest of our esteemed readers.

P.S. Mehra E-Mail: [email protected]

P.S. Mehra E-Mail: [email protected]

x CONTENTS PREFACE

x CONTENTS PREFACE

I am also thankful to all my teachers: Prof. Vijay Juyal, Dr. Firoz Anwer, Asst. Prof. Divya Juyal, Prof. Preeti Kothiyal, Dr. Shon Roy Chaudhary and Dr. Jaya Bandyopadhyay. My sincere thanks to Shri Vrajesh Makhijani (Director), I.K. International Publishing House Pvt. Ltd., New Delhi and his dedicated and dynamic team for publishing this book elegantly and speedily. In truce sense I derived much inspiration and blessings from my mother, respected Smt. Devki Devi Mehra, and respected father Shri Mahendra Singh Mehra. Also I take opportunity to express my thanks to all my family members especially to my sister Mrs. Laxmi and my brother-in-law Mr. Khushal Singh and brother Mr. Tarendra Singh and Gaurav Singh. It is the privilege to offer my sincere gratitude to the co-author of this book Miss Pratibha Sharma, M. Pharmacy (Pharmacology) Soolini University, Solan, Himachal Pradesh. Who actually helped me sincerely while I was simply engrossed in preparing and also finalizing this manuscript. From the core of my heart, I am grateful to her indeed. Comments, suggestions and constructive criticisms towards improvements of future editions of this volume are very much welcomed. In fact, they underline the shortcomings which are looked into and rectified in the interest of our esteemed readers.

I am also thankful to all my teachers: Prof. Vijay Juyal, Dr. Firoz Anwer, Asst. Prof. Divya Juyal, Prof. Preeti Kothiyal, Dr. Shon Roy Chaudhary and Dr. Jaya Bandyopadhyay. My sincere thanks to Shri Vrajesh Makhijani (Director), I.K. International Publishing House Pvt. Ltd., New Delhi and his dedicated and dynamic team for publishing this book elegantly and speedily. In truce sense I derived much inspiration and blessings from my mother, respected Smt. Devki Devi Mehra, and respected father Shri Mahendra Singh Mehra. Also I take opportunity to express my thanks to all my family members especially to my sister Mrs. Laxmi and my brother-in-law Mr. Khushal Singh and brother Mr. Tarendra Singh and Gaurav Singh. It is the privilege to offer my sincere gratitude to the co-author of this book Miss Pratibha Sharma, M. Pharmacy (Pharmacology) Soolini University, Solan, Himachal Pradesh. Who actually helped me sincerely while I was simply engrossed in preparing and also finalizing this manuscript. From the core of my heart, I am grateful to her indeed. Comments, suggestions and constructive criticisms towards improvements of future editions of this volume are very much welcomed. In fact, they underline the shortcomings which are looked into and rectified in the interest of our esteemed readers.

P.S. Mehra E-Mail: [email protected]

P.S. Mehra E-Mail: [email protected]

CONTENTS Foreword

CONTENTS vii

Preface

ix

Foreword Preface

SECTION A 1. Introduction to Scope of Microbiology 1.1 Introduction 1.2 Scope of Microbiology

vii ix SECTION A

3 3 4

1. Introduction to Scope of Microbiology 1.1 Introduction 1.2 Scope of Microbiology

3 3 4

2. Structure of Bacterial Cell 2.1 History of the Cell 2.2 Description of Bacterial Cell Structure 2.3 Shape of Bacterial Cell 2.3.1 Spherical (Cocci) 2.3.2 Rod Shaped or Cylindrical 2.3.3 Spiral 2.4 Difference between Eukaryotic and Prokaryotic Cell

9 9 10 14 14 15 16 17

2. Structure of Bacterial Cell 2.1 History of the Cell 2.2 Description of Bacterial Cell Structure 2.3 Shape of Bacterial Cell 2.3.1 Spherical (Cocci) 2.3.2 Rod Shaped or Cylindrical 2.3.3 Spiral 2.4 Difference between Eukaryotic and Prokaryotic Cell

9 9 10 14 14 15 16 17

3. Classification of Microbes and Their Taxonomy 3.1 Taxonomy 3.2 Classification 3.3 Classification of Actinomycetes 3.4 Classification of Bacteria 3.5 Classification of Rickettsiae 3.5.1 Rickettsias 3.5.2 Chlamydias 3.6 Classification of Spirochaetes 3.7 Classification of Viruses

19 19 19 20 21 22 22 23 23 23

3. Classification of Microbes and Their Taxonomy 3.1 Taxonomy 3.2 Classification 3.3 Classification of Actinomycetes 3.4 Classification of Bacteria 3.5 Classification of Rickettsiae 3.5.1 Rickettsias 3.5.2 Chlamydias 3.6 Classification of Spirochaetes 3.7 Classification of Viruses

19 19 19 20 21 22 22 23 23 23

4. Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy 4.1 Simple Staining 4.2 Gram Staining 4.3 Acid Fast Staining 4.4 Endospore Staining

25 26 26 28 28

4. Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy 4.1 Simple Staining 4.2 Gram Staining 4.3 Acid Fast Staining 4.4 Endospore Staining

25 26 26 28 28

CONTENTS Foreword

CONTENTS vii

Preface

ix SECTION A

1. Introduction to Scope of Microbiology 1.1 Introduction 1.2 Scope of Microbiology

Foreword

vii

Preface

ix SECTION A

3 3 4

1. Introduction to Scope of Microbiology 1.1 Introduction 1.2 Scope of Microbiology

3 3 4

2. Structure of Bacterial Cell 2.1 History of the Cell 2.2 Description of Bacterial Cell Structure 2.3 Shape of Bacterial Cell 2.3.1 Spherical (Cocci) 2.3.2 Rod Shaped or Cylindrical 2.3.3 Spiral 2.4 Difference between Eukaryotic and Prokaryotic Cell

9 9 10 14 14 15 16 17

2. Structure of Bacterial Cell 2.1 History of the Cell 2.2 Description of Bacterial Cell Structure 2.3 Shape of Bacterial Cell 2.3.1 Spherical (Cocci) 2.3.2 Rod Shaped or Cylindrical 2.3.3 Spiral 2.4 Difference between Eukaryotic and Prokaryotic Cell

9 9 10 14 14 15 16 17

3. Classification of Microbes and Their Taxonomy 3.1 Taxonomy 3.2 Classification 3.3 Classification of Actinomycetes 3.4 Classification of Bacteria 3.5 Classification of Rickettsiae 3.5.1 Rickettsias 3.5.2 Chlamydias 3.6 Classification of Spirochaetes 3.7 Classification of Viruses

19 19 19 20 21 22 22 23 23 23

3. Classification of Microbes and Their Taxonomy 3.1 Taxonomy 3.2 Classification 3.3 Classification of Actinomycetes 3.4 Classification of Bacteria 3.5 Classification of Rickettsiae 3.5.1 Rickettsias 3.5.2 Chlamydias 3.6 Classification of Spirochaetes 3.7 Classification of Viruses

19 19 19 20 21 22 22 23 23 23

4. Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy 4.1 Simple Staining 4.2 Gram Staining 4.3 Acid Fast Staining 4.4 Endospore Staining

25 26 26 28 28

4. Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy 4.1 Simple Staining 4.2 Gram Staining 4.3 Acid Fast Staining 4.4 Endospore Staining

25 26 26 28 28

xii

xii

CONTENTS

4.5 4.6 4.7 4.8

Capsule Staining Flagella Staining Negative Staining Microscopy 4.8.1 Light or Compound Microscope 4.8.2 Electron Microscope

29 29 30 30 32 35

CONTENTS

4.5 4.6 4.7 4.8

Capsule Staining Flagella Staining Negative Staining Microscopy 4.8.1 Light or Compound Microscope 4.8.2 Electron Microscope

SECTION B

29 29 30 30 32 35

SECTION B

5. Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc. 5.1 Nutrition, Cultivation and Isolation of Bacteria 5.1.1 Autotrophic Bacteria: (Source of carbon is CO2) 5.1.2 Heterotrophic Bacteria 5.1.3 Growth Curve 5.1.4 Cultivation of Bacteria 5.1.5 Isolation of Bacteria 5.1.6 Pure Culture Isolation 5.2 Nutrition, Cultivation and Isolation of Actinomycetes 5.2.1 Nutritional Habit of Actinomycetes 5.2.2 Cultivation and Isolation of Actinomycetes 5.3 Nutrition, Cultivation and Isolation of Fungi 5.3.1 Nutritional Habit of Fungi 5.3.2 Cultivation and Isolation of Fungi 5.4 Nutrition, Cultivation and Isolation of Rickettsia 5.4.1 Nutrition 5.4.2 Cultivation 5.4.3 Isolation 5.5 Nutrition, Cultivation and Isolation of Viruses 5.5.1 Nutritional Habit of Viruses 5.5.2 Living Characters 5.5.3 Non-living Characters 5.5.4 Cultivation and Isolation of Virus (Human Virus) 5.5.5 Animal Inoculation 5.5.6 Chick Embryo 5.5.7 Cell Culture

41 41 41 43 44 45 48 51 55 55 55 56 56 56 57 57 57 57 58 58 58 58 58 59 59 61

5. Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc. 5.1 Nutrition, Cultivation and Isolation of Bacteria 5.1.1 Autotrophic Bacteria: (Source of carbon is CO2) 5.1.2 Heterotrophic Bacteria 5.1.3 Growth Curve 5.1.4 Cultivation of Bacteria 5.1.5 Isolation of Bacteria 5.1.6 Pure Culture Isolation 5.2 Nutrition, Cultivation and Isolation of Actinomycetes 5.2.1 Nutritional Habit of Actinomycetes 5.2.2 Cultivation and Isolation of Actinomycetes 5.3 Nutrition, Cultivation and Isolation of Fungi 5.3.1 Nutritional Habit of Fungi 5.3.2 Cultivation and Isolation of Fungi 5.4 Nutrition, Cultivation and Isolation of Rickettsia 5.4.1 Nutrition 5.4.2 Cultivation 5.4.3 Isolation 5.5 Nutrition, Cultivation and Isolation of Viruses 5.5.1 Nutritional Habit of Viruses 5.5.2 Living Characters 5.5.3 Non-living Characters 5.5.4 Cultivation and Isolation of Virus (Human Virus) 5.5.5 Animal Inoculation 5.5.6 Chick Embryo 5.5.7 Cell Culture

41 41 41 43 44 45 48 51 55 55 55 56 56 56 57 57 57 57 58 58 58 58 58 59 59 61

6. Microbial Genetics and Variation 6.1 Chromosome 6.2 Gene 6.3 Deoxyribonucleic Acid (DNA) 6.3.1 A Brief History of DNA 6.3.2 The Structure of DNA 6.3.3 Watson & Crick’s Observations 6.4 Structure of RNA

63 63 64 65 66 66 68 68

6. Microbial Genetics and Variation 6.1 Chromosome 6.2 Gene 6.3 Deoxyribonucleic Acid (DNA) 6.3.1 A Brief History of DNA 6.3.2 The Structure of DNA 6.3.3 Watson & Crick’s Observations 6.4 Structure of RNA

63 63 64 65 66 66 68 68

xii

xii

CONTENTS

4.5 4.6 4.7 4.8

Capsule Staining Flagella Staining Negative Staining Microscopy 4.8.1 Light or Compound Microscope 4.8.2 Electron Microscope

29 29 30 30 32 35

SECTION B

CONTENTS

4.5 4.6 4.7 4.8

Capsule Staining Flagella Staining Negative Staining Microscopy 4.8.1 Light or Compound Microscope 4.8.2 Electron Microscope

29 29 30 30 32 35

SECTION B

5. Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc. 5.1 Nutrition, Cultivation and Isolation of Bacteria 5.1.1 Autotrophic Bacteria: (Source of carbon is CO2) 5.1.2 Heterotrophic Bacteria 5.1.3 Growth Curve 5.1.4 Cultivation of Bacteria 5.1.5 Isolation of Bacteria 5.1.6 Pure Culture Isolation 5.2 Nutrition, Cultivation and Isolation of Actinomycetes 5.2.1 Nutritional Habit of Actinomycetes 5.2.2 Cultivation and Isolation of Actinomycetes 5.3 Nutrition, Cultivation and Isolation of Fungi 5.3.1 Nutritional Habit of Fungi 5.3.2 Cultivation and Isolation of Fungi 5.4 Nutrition, Cultivation and Isolation of Rickettsia 5.4.1 Nutrition 5.4.2 Cultivation 5.4.3 Isolation 5.5 Nutrition, Cultivation and Isolation of Viruses 5.5.1 Nutritional Habit of Viruses 5.5.2 Living Characters 5.5.3 Non-living Characters 5.5.4 Cultivation and Isolation of Virus (Human Virus) 5.5.5 Animal Inoculation 5.5.6 Chick Embryo 5.5.7 Cell Culture

41 41 41 43 44 45 48 51 55 55 55 56 56 56 57 57 57 57 58 58 58 58 58 59 59 61

5. Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc. 5.1 Nutrition, Cultivation and Isolation of Bacteria 5.1.1 Autotrophic Bacteria: (Source of carbon is CO2) 5.1.2 Heterotrophic Bacteria 5.1.3 Growth Curve 5.1.4 Cultivation of Bacteria 5.1.5 Isolation of Bacteria 5.1.6 Pure Culture Isolation 5.2 Nutrition, Cultivation and Isolation of Actinomycetes 5.2.1 Nutritional Habit of Actinomycetes 5.2.2 Cultivation and Isolation of Actinomycetes 5.3 Nutrition, Cultivation and Isolation of Fungi 5.3.1 Nutritional Habit of Fungi 5.3.2 Cultivation and Isolation of Fungi 5.4 Nutrition, Cultivation and Isolation of Rickettsia 5.4.1 Nutrition 5.4.2 Cultivation 5.4.3 Isolation 5.5 Nutrition, Cultivation and Isolation of Viruses 5.5.1 Nutritional Habit of Viruses 5.5.2 Living Characters 5.5.3 Non-living Characters 5.5.4 Cultivation and Isolation of Virus (Human Virus) 5.5.5 Animal Inoculation 5.5.6 Chick Embryo 5.5.7 Cell Culture

41 41 41 43 44 45 48 51 55 55 55 56 56 56 57 57 57 57 58 58 58 58 58 59 59 61

6. Microbial Genetics and Variation 6.1 Chromosome 6.2 Gene 6.3 Deoxyribonucleic Acid (DNA) 6.3.1 A Brief History of DNA 6.3.2 The Structure of DNA 6.3.3 Watson & Crick’s Observations 6.4 Structure of RNA

63 63 64 65 66 66 68 68

6. Microbial Genetics and Variation 6.1 Chromosome 6.2 Gene 6.3 Deoxyribonucleic Acid (DNA) 6.3.1 A Brief History of DNA 6.3.2 The Structure of DNA 6.3.3 Watson & Crick’s Observations 6.4 Structure of RNA

63 63 64 65 66 66 68 68

CONTENTS

xiii

6.4.1 Types of RNA 6.4.2 Biological Roles of RNA 6.4.3 Difference between DNA and RNA Genetic Code Genotype and Phenotype Replication Transcription Translation Process of Gene Transfer 6.10.1 Transformation 6.10.2 Conjugation 6.10.3 Transduction Polymerase Chain Reaction (PCR) 6.11.1 Application of PCR Mutation, Mutagen and Mutagenesis 6.12.1 Somatic vs. Gametic Mutations 6.12.2 Spontaneous vs. Induced Mutations 6.12.3 Molecular Basis of Mutation

70 73 73 74 74 75 77 78 82 82 83 84 85 87 88 89 89 89

7. Sterility Testing 7.1 Culture Media 7.2 Test Procedures 7.2.1 Method A: Membrane Filtration 7.2.2 Method B: Direct Inoculation 7.3 Sterility Test for Catgut 7.4 Limulus Amoebocyte Lysate Test (LAL test)

91 92 94 95 97 100 100

6.5 6.6 6.7 6.8 6.9 6.10

6.11 6.12

CONTENTS

6.4.1 Types of RNA 6.4.2 Biological Roles of RNA 6.4.3 Difference between DNA and RNA Genetic Code Genotype and Phenotype Replication Transcription Translation Process of Gene Transfer 6.10.1 Transformation 6.10.2 Conjugation 6.10.3 Transduction Polymerase Chain Reaction (PCR) 6.11.1 Application of PCR Mutation, Mutagen and Mutagenesis 6.12.1 Somatic vs. Gametic Mutations 6.12.2 Spontaneous vs. Induced Mutations 6.12.3 Molecular Basis of Mutation

70 73 73 74 74 75 77 78 82 82 83 84 85 87 88 89 89 89

7. Sterility Testing 7.1 Culture Media 7.2 Test Procedures 7.2.1 Method A: Membrane Filtration 7.2.2 Method B: Direct Inoculation 7.3 Sterility Test for Catgut 7.4 Limulus Amoebocyte Lysate Test (LAL test)

91 92 94 95 97 100 100

6.5 6.6 6.7 6.8 6.9 6.10

6.11 6.12

SECTION C

SECTION C

8. Disinfectants, Antiseptics, and Preservatives 8.1 Disinfectants 8.1.1 Properties of an Ideal Disinfectant 8.1.2 Factors Affecting the Disinfection Process 8.1.3 Evaluation of Disinfectants 8.1.4 Determination of Zone of Inhibition 8.1.5 Determination of Minimum Inhibitory Concentration (MIC) 8.2 Antiseptics 8.3 Preservatives

103 103 104 104 105 107

9. Sterilization 9.1 Microbial Death Kinetics 9.2 Expressions of Resistance 9.2.1 D-value 9.2.2 Z-value 9.3 Sterility Assurance

CONTENTS

103 103 104 104 105 107

107 108 108

8. Disinfectants, Antiseptics, and Preservatives 8.1 Disinfectants 8.1.1 Properties of an Ideal Disinfectant 8.1.2 Factors Affecting the Disinfection Process 8.1.3 Evaluation of Disinfectants 8.1.4 Determination of Zone of Inhibition 8.1.5 Determination of Minimum Inhibitory Concentration (MIC) 8.2 Antiseptics 8.3 Preservatives

111 112 113 113 113 113

9. Sterilization 9.1 Microbial Death Kinetics 9.2 Expressions of Resistance 9.2.1 D-value 9.2.2 Z-value 9.3 Sterility Assurance

111 112 113 113 113 113

xiii

6.4.1 Types of RNA 6.4.2 Biological Roles of RNA 6.4.3 Difference between DNA and RNA Genetic Code Genotype and Phenotype Replication Transcription Translation Process of Gene Transfer 6.10.1 Transformation 6.10.2 Conjugation 6.10.3 Transduction Polymerase Chain Reaction (PCR) 6.11.1 Application of PCR Mutation, Mutagen and Mutagenesis 6.12.1 Somatic vs. Gametic Mutations 6.12.2 Spontaneous vs. Induced Mutations 6.12.3 Molecular Basis of Mutation

70 73 73 74 74 75 77 78 82 82 83 84 85 87 88 89 89 89

7. Sterility Testing 7.1 Culture Media 7.2 Test Procedures 7.2.1 Method A: Membrane Filtration 7.2.2 Method B: Direct Inoculation 7.3 Sterility Test for Catgut 7.4 Limulus Amoebocyte Lysate Test (LAL test)

91 92 94 95 97 100 100

6.5 6.6 6.7 6.8 6.9 6.10

6.11 6.12

xiii

SECTION C

CONTENTS

107 108 108

xiii

6.4.1 Types of RNA 6.4.2 Biological Roles of RNA 6.4.3 Difference between DNA and RNA Genetic Code Genotype and Phenotype Replication Transcription Translation Process of Gene Transfer 6.10.1 Transformation 6.10.2 Conjugation 6.10.3 Transduction Polymerase Chain Reaction (PCR) 6.11.1 Application of PCR Mutation, Mutagen and Mutagenesis 6.12.1 Somatic vs. Gametic Mutations 6.12.2 Spontaneous vs. Induced Mutations 6.12.3 Molecular Basis of Mutation

70 73 73 74 74 75 77 78 82 82 83 84 85 87 88 89 89 89

7. Sterility Testing 7.1 Culture Media 7.2 Test Procedures 7.2.1 Method A: Membrane Filtration 7.2.2 Method B: Direct Inoculation 7.3 Sterility Test for Catgut 7.4 Limulus Amoebocyte Lysate Test (LAL test)

91 92 94 95 97 100 100

6.5 6.6 6.7 6.8 6.9 6.10

6.11 6.12

SECTION C

8. Disinfectants, Antiseptics, and Preservatives 8.1 Disinfectants 8.1.1 Properties of an Ideal Disinfectant 8.1.2 Factors Affecting the Disinfection Process 8.1.3 Evaluation of Disinfectants 8.1.4 Determination of Zone of Inhibition 8.1.5 Determination of Minimum Inhibitory Concentration (MIC) 8.2 Antiseptics 8.3 Preservatives

103 103 104 104 105 107

103 103 104 104 105 107

107 108 108

8. Disinfectants, Antiseptics, and Preservatives 8.1 Disinfectants 8.1.1 Properties of an Ideal Disinfectant 8.1.2 Factors Affecting the Disinfection Process 8.1.3 Evaluation of Disinfectants 8.1.4 Determination of Zone of Inhibition 8.1.5 Determination of Minimum Inhibitory Concentration (MIC) 8.2 Antiseptics 8.3 Preservatives

9. Sterilization 9.1 Microbial Death Kinetics 9.2 Expressions of Resistance 9.2.1 D-value 9.2.2 Z-value 9.3 Sterility Assurance

111 112 113 113 113 113

9. Sterilization 9.1 Microbial Death Kinetics 9.2 Expressions of Resistance 9.2.1 D-value 9.2.2 Z-value 9.3 Sterility Assurance

111 112 113 113 113 113

107 108 108

xiv CONTENTS 9.4

9.5

9.6 9.7

9.8

xiv CONTENTS

Sterilization Methods 9.4.1 Physical Agents 9.4.2 Chemical Sterilization 9.4.3 Gaseous Sterilization Factory and Hospital Hygiene: Control of Microbial Contamination During Manufacture 9.5.1 Sterile Manufacturing of Products in Factory or Industry Clean Rooms 9.6.1 Manufacture of Sterile Products Nosocomial Infection 9.7.1 Route of Transmission of Nosocomial Infection 9.7.2 Prevention of Nosocomial Infection Control of Hospital Infection

115 115 120 124

9.4

9.5 124 124 125 127 129 129 130 130

9.6 9.7

9.8

Sterilization Methods 9.4.1 Physical Agents 9.4.2 Chemical Sterilization 9.4.3 Gaseous Sterilization Factory and Hospital Hygiene: Control of Microbial Contamination During Manufacture 9.5.1 Sterile Manufacturing of Products in Factory or Industry Clean Rooms 9.6.1 Manufacture of Sterile Products Nosocomial Infection 9.7.1 Route of Transmission of Nosocomial Infection 9.7.2 Prevention of Nosocomial Infection Control of Hospital Infection

SECTION D 10. Immunology 10.1 History of Immunology 10.2 Non-specific Defence Mechanisms (Innate Immune System) 10.2.1 Skin and Mucous Membranes 10.2.2 Phagocytosis 10.2.3 Complement System 10.2.4 Classical Pathway 10.2.5 Alternative Pathway 10.2.6 Exotoxin and Endotoxin 10.2.7 Inflammation 10.3 Antigen or Immunogen 10.3.1 Characteristics of Antigen 10.4 Epitopes 10.5 Cross-reactivity 10.6 Haptens 10.7 Antibodies 10.7.1 Immunoglobulin Classes 10.8 Monoclonal Antibodies and their Application 10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 10.8.2 Applications 10.9 Active and Passive Immunity 10.9.1 Naturally Acquired Active Immunity 10.9.2 Artificially Acquired Active Immunity 10.9.3 Naturally Acquired Passive Immunity 10.9.4 Artificially Acquired Passive Immunity 10.10 Cellular and Humoral Immunity

9.5

9.6 9.7

9.8

124 124 125 127 129 129 130 130

SECTION D 137 137 140 140 140 140 141 143 143 144 145 145 146 146 147 147 148 150 150 152 152 152 153 153 153 153

xiv CONTENTS 9.4

115 115 120 124

10. Immunology 10.1 History of Immunology 10.2 Non-specific Defence Mechanisms (Innate Immune System) 10.2.1 Skin and Mucous Membranes 10.2.2 Phagocytosis 10.2.3 Complement System 10.2.4 Classical Pathway 10.2.5 Alternative Pathway 10.2.6 Exotoxin and Endotoxin 10.2.7 Inflammation 10.3 Antigen or Immunogen 10.3.1 Characteristics of Antigen 10.4 Epitopes 10.5 Cross-reactivity 10.6 Haptens 10.7 Antibodies 10.7.1 Immunoglobulin Classes 10.8 Monoclonal Antibodies and their Application 10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 10.8.2 Applications 10.9 Active and Passive Immunity 10.9.1 Naturally Acquired Active Immunity 10.9.2 Artificially Acquired Active Immunity 10.9.3 Naturally Acquired Passive Immunity 10.9.4 Artificially Acquired Passive Immunity 10.10 Cellular and Humoral Immunity

137 137 140 140 140 140 141 143 143 144 145 145 146 146 147 147 148 150 150 152 152 152 153 153 153 153

xiv CONTENTS

Sterilization Methods 9.4.1 Physical Agents 9.4.2 Chemical Sterilization 9.4.3 Gaseous Sterilization Factory and Hospital Hygiene: Control of Microbial Contamination During Manufacture 9.5.1 Sterile Manufacturing of Products in Factory or Industry Clean Rooms 9.6.1 Manufacture of Sterile Products Nosocomial Infection 9.7.1 Route of Transmission of Nosocomial Infection 9.7.2 Prevention of Nosocomial Infection Control of Hospital Infection

115 115 120 124

9.5 124 124 125 127 129 129 130 130

SECTION D 10. Immunology 10.1 History of Immunology 10.2 Non-specific Defence Mechanisms (Innate Immune System) 10.2.1 Skin and Mucous Membranes 10.2.2 Phagocytosis 10.2.3 Complement System 10.2.4 Classical Pathway 10.2.5 Alternative Pathway 10.2.6 Exotoxin and Endotoxin 10.2.7 Inflammation 10.3 Antigen or Immunogen 10.3.1 Characteristics of Antigen 10.4 Epitopes 10.5 Cross-reactivity 10.6 Haptens 10.7 Antibodies 10.7.1 Immunoglobulin Classes 10.8 Monoclonal Antibodies and their Application 10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 10.8.2 Applications 10.9 Active and Passive Immunity 10.9.1 Naturally Acquired Active Immunity 10.9.2 Artificially Acquired Active Immunity 10.9.3 Naturally Acquired Passive Immunity 10.9.4 Artificially Acquired Passive Immunity 10.10 Cellular and Humoral Immunity

9.4

9.6 9.7

9.8

Sterilization Methods 9.4.1 Physical Agents 9.4.2 Chemical Sterilization 9.4.3 Gaseous Sterilization Factory and Hospital Hygiene: Control of Microbial Contamination During Manufacture 9.5.1 Sterile Manufacturing of Products in Factory or Industry Clean Rooms 9.6.1 Manufacture of Sterile Products Nosocomial Infection 9.7.1 Route of Transmission of Nosocomial Infection 9.7.2 Prevention of Nosocomial Infection Control of Hospital Infection

115 115 120 124 124 124 125 127 129 129 130 130

SECTION D 137 137 140 140 140 140 141 143 143 144 145 145 146 146 147 147 148 150 150 152 152 152 153 153 153 153

10. Immunology 10.1 History of Immunology 10.2 Non-specific Defence Mechanisms (Innate Immune System) 10.2.1 Skin and Mucous Membranes 10.2.2 Phagocytosis 10.2.3 Complement System 10.2.4 Classical Pathway 10.2.5 Alternative Pathway 10.2.6 Exotoxin and Endotoxin 10.2.7 Inflammation 10.3 Antigen or Immunogen 10.3.1 Characteristics of Antigen 10.4 Epitopes 10.5 Cross-reactivity 10.6 Haptens 10.7 Antibodies 10.7.1 Immunoglobulin Classes 10.8 Monoclonal Antibodies and their Application 10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 10.8.2 Applications 10.9 Active and Passive Immunity 10.9.1 Naturally Acquired Active Immunity 10.9.2 Artificially Acquired Active Immunity 10.9.3 Naturally Acquired Passive Immunity 10.9.4 Artificially Acquired Passive Immunity 10.10 Cellular and Humoral Immunity

137 137 140 140 140 140 141 143 143 144 145 145 146 146 147 147 148 150 150 152 152 152 153 153 153 153

CONTENTS

10.11 10.12 10.13 10.14

Antigen-Antibody Reaction Immunological Tolerance Autoimmunity Hypersensitivity Reaction

xv 154 159 160 161

11. Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids 11.1 Antimicrobial Assay of Antibiotics 11.2 Assay of Vitamins 11.2.1 Microbial Assay of Vitamin A 11.2.2 Microbial Assay of Vitamins D 11.2.3 Microbial Assay of Calcium Pantothenate 11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) 11.3 Microbial Assay of Amino Acids

167 167 172 172 174 176 177 177

CONTENTS

10.11 10.12 10.13 10.14

Antigen-Antibody Reaction Immunological Tolerance Autoimmunity Hypersensitivity Reaction

154 159 160 161

11. Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids 11.1 Antimicrobial Assay of Antibiotics 11.2 Assay of Vitamins 11.2.1 Microbial Assay of Vitamin A 11.2.2 Microbial Assay of Vitamins D 11.2.3 Microbial Assay of Calcium Pantothenate 11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) 11.3 Microbial Assay of Amino Acids

SECTION E

xv

167 167 172 172 174 176 177 177

SECTION E

12. Experiments Microbiology Laboratory Safety Rules and Procedures Experiment No. 1 Experiment No. 2 Experiment No. 3 Experiment No. 4 Experiment No. 5 Experiment No. 6 Experiment No. 11 Experiment No. 12 Experiment No. 13 Experiment No. 14 Experiment No. 15 Experiment No. 16

181 181 183 186 195 197 198 199 200 201 206 208 210 211

12. Experiments Microbiology Laboratory Safety Rules and Procedures Experiment No. 1 Experiment No. 2 Experiment No. 3 Experiment No. 4 Experiment No. 5 Experiment No. 6 Experiment No. 11 Experiment No. 12 Experiment No. 13 Experiment No. 14 Experiment No. 15 Experiment No. 16

181 181 183 186 195 197 198 199 200 201 206 208 210 211

References

215

References

215

Index

217

Index

217

CONTENTS

10.11 10.12 10.13 10.14

Antigen-Antibody Reaction Immunological Tolerance Autoimmunity Hypersensitivity Reaction

11. Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids 11.1 Antimicrobial Assay of Antibiotics 11.2 Assay of Vitamins 11.2.1 Microbial Assay of Vitamin A 11.2.2 Microbial Assay of Vitamins D 11.2.3 Microbial Assay of Calcium Pantothenate 11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) 11.3 Microbial Assay of Amino Acids

xv 154 159 160 161 167 167 172 172 174 176 177 177

CONTENTS

10.11 10.12 10.13 10.14

Antigen-Antibody Reaction Immunological Tolerance Autoimmunity Hypersensitivity Reaction

11. Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids 11.1 Antimicrobial Assay of Antibiotics 11.2 Assay of Vitamins 11.2.1 Microbial Assay of Vitamin A 11.2.2 Microbial Assay of Vitamins D 11.2.3 Microbial Assay of Calcium Pantothenate 11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) 11.3 Microbial Assay of Amino Acids

SECTION E 12. Experiments Microbiology Laboratory Safety Rules and Procedures Experiment No. 1 Experiment No. 2 Experiment No. 3 Experiment No. 4 Experiment No. 5 Experiment No. 6 Experiment No. 11 Experiment No. 12 Experiment No. 13 Experiment No. 14 Experiment No. 15 Experiment No. 16

xv 154 159 160 161 167 167 172 172 174 176 177 177

SECTION E 181 181 183 186 195 197 198 199 200 201 206 208 210 211

12. Experiments Microbiology Laboratory Safety Rules and Procedures Experiment No. 1 Experiment No. 2 Experiment No. 3 Experiment No. 4 Experiment No. 5 Experiment No. 6 Experiment No. 11 Experiment No. 12 Experiment No. 13 Experiment No. 14 Experiment No. 15 Experiment No. 16

181 181 183 186 195 197 198 199 200 201 206 208 210 211

References

215

References

215

Index

217

Index

217

SECTION A

SECTION A

SECTION A

SECTION A

1.1

1

1

Introduction to Scope of Microbiology

Introduction to Scope of Microbiology

INTRODUCTION

1.1

INTRODUCTION

Microbiology is the study of living organisms of microscopic size or microorganisms; which includes viruses, bacteria, fungi, algae etc. In short; microbiology refers to the study of life and organisms that are too small to be seen with the naked eye. It is concerned with their form, structure, reproduction, physiology, metabolism and classification. It also includes the study of their distribution in nature, their relationship with each other and to other living organisms, their effects on human beings and other animals, and plants. Microorganisms are closely associated with the health and welfare of human beings, some microorganisms are beneficial and some are harmful or detrimental. Example: microorganisms are involved in the making of yoghurt, cheese and wine, in the production of antibiotics (e.g. penicillin, streptomycin, erythromycin, kanamycin etc.) interferons, alcohols and in the production of domestic and industrial waste etc. Microorganisms can cause disease, spoil food and deteriorate materials like iron pipes, glass lenses, and wood pilings. Microbiology is a broad term which includes virology, mycology, parasitology, bacteriology and other branches. A microbiologist is a specialist in microbiology. Bacteria and other microorganisms were first observed by Antonie van Leeuwenhoek in 1676 using a single-lens microscope of his own design. In 1876 Robert Koch, a German bacteriologist proved that anthrax is caused by a microbe bacillus anthracis. After that a large number of microorganisms have been discovered, those produce diseases in human beings and other living organisms.

Microbiology is the study of living organisms of microscopic size or microorganisms; which includes viruses, bacteria, fungi, algae etc. In short; microbiology refers to the study of life and organisms that are too small to be seen with the naked eye. It is concerned with their form, structure, reproduction, physiology, metabolism and classification. It also includes the study of their distribution in nature, their relationship with each other and to other living organisms, their effects on human beings and other animals, and plants. Microorganisms are closely associated with the health and welfare of human beings, some microorganisms are beneficial and some are harmful or detrimental. Example: microorganisms are involved in the making of yoghurt, cheese and wine, in the production of antibiotics (e.g. penicillin, streptomycin, erythromycin, kanamycin etc.) interferons, alcohols and in the production of domestic and industrial waste etc. Microorganisms can cause disease, spoil food and deteriorate materials like iron pipes, glass lenses, and wood pilings. Microbiology is a broad term which includes virology, mycology, parasitology, bacteriology and other branches. A microbiologist is a specialist in microbiology. Bacteria and other microorganisms were first observed by Antonie van Leeuwenhoek in 1676 using a single-lens microscope of his own design. In 1876 Robert Koch, a German bacteriologist proved that anthrax is caused by a microbe bacillus anthracis. After that a large number of microorganisms have been discovered, those produce diseases in human beings and other living organisms.

1

1

Introduction to Scope of Microbiology

Introduction to Scope of Microbiology

1.1

INTRODUCTION

Microbiology is the study of living organisms of microscopic size or microorganisms; which includes viruses, bacteria, fungi, algae etc. In short; microbiology refers to the study of life and organisms that are too small to be seen with the naked eye. It is concerned with their form, structure, reproduction, physiology, metabolism and classification. It also includes the study of their distribution in nature, their relationship with each other and to other living organisms, their effects on human beings and other animals, and plants. Microorganisms are closely associated with the health and welfare of human beings, some microorganisms are beneficial and some are harmful or detrimental. Example: microorganisms are involved in the making of yoghurt, cheese and wine, in the production of antibiotics (e.g. penicillin, streptomycin, erythromycin, kanamycin etc.) interferons, alcohols and in the production of domestic and industrial waste etc. Microorganisms can cause disease, spoil food and deteriorate materials like iron pipes, glass lenses, and wood pilings. Microbiology is a broad term which includes virology, mycology, parasitology, bacteriology and other branches. A microbiologist is a specialist in microbiology. Bacteria and other microorganisms were first observed by Antonie van Leeuwenhoek in 1676 using a single-lens microscope of his own design. In 1876 Robert Koch, a German bacteriologist proved that anthrax is caused by a microbe bacillus anthracis. After that a large number of microorganisms have been discovered, those produce diseases in human beings and other living organisms.

1.1

INTRODUCTION

Microbiology is the study of living organisms of microscopic size or microorganisms; which includes viruses, bacteria, fungi, algae etc. In short; microbiology refers to the study of life and organisms that are too small to be seen with the naked eye. It is concerned with their form, structure, reproduction, physiology, metabolism and classification. It also includes the study of their distribution in nature, their relationship with each other and to other living organisms, their effects on human beings and other animals, and plants. Microorganisms are closely associated with the health and welfare of human beings, some microorganisms are beneficial and some are harmful or detrimental. Example: microorganisms are involved in the making of yoghurt, cheese and wine, in the production of antibiotics (e.g. penicillin, streptomycin, erythromycin, kanamycin etc.) interferons, alcohols and in the production of domestic and industrial waste etc. Microorganisms can cause disease, spoil food and deteriorate materials like iron pipes, glass lenses, and wood pilings. Microbiology is a broad term which includes virology, mycology, parasitology, bacteriology and other branches. A microbiologist is a specialist in microbiology. Bacteria and other microorganisms were first observed by Antonie van Leeuwenhoek in 1676 using a single-lens microscope of his own design. In 1876 Robert Koch, a German bacteriologist proved that anthrax is caused by a microbe bacillus anthracis. After that a large number of microorganisms have been discovered, those produce diseases in human beings and other living organisms.

4 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Some important events in the history of microbiology: 1546 Fracastoro suggests that invisible organisms cause disease. 1676 Antony van Leeuwenhoek discovers “animalcules”. 1786 Muller produces first classification of bacteria. 1798 Jenner introduces cowpox vaccination for smallpox. 1839 Matthias Schleiden and Theodor Schwann, formalize the theory that all living things are composed of cells. 1867 Pasture devised the process of destroying bacteria known as pasteurization. 1867 Lister publishes his work on antiseptic surgery. 1876 Koch demonstrates that anthrax is caused by Bacillus anthracis. 1881 Pasture develops anthrax vaccine. 1885 Pasture develops rabies vaccine. 1902 Karl Landsteiner discovers blood groups. 1928 Griffith discovers bacterial transformation. 1929 Fleming discovers penicillin. 1944 Selman Waksman discovers streptomycin. 1954 Jonas Salk develops the first polio vaccine. 1983 Isolation and characterization of HIV (human immunodeficiency virus) by Luc- Montagnier and Robert Gallo. 1988 Polymerase chain reaction invented by Karry Mullis. 1990 First clinical trials in gene therapy testing, vaccine for hemophillus influenza, a cause of meningitis is introduced. 2000 Discovered that Vibrio cholerae has two separate chromosomes. 2002 Plasmodium falciparum, malarial parasite of humans, genome sequenced by Malcolm J. Gardner and coworkers of USA. 2002 Isolation of human gene CEM 15 that inhibits HIV-1 infection, by Ann M.S. Heely et al., of USA.

Some important events in the history of microbiology: 1546 Fracastoro suggests that invisible organisms cause disease. 1676 Antony van Leeuwenhoek discovers “animalcules”. 1786 Muller produces first classification of bacteria. 1798 Jenner introduces cowpox vaccination for smallpox. 1839 Matthias Schleiden and Theodor Schwann, formalize the theory that all living things are composed of cells. 1867 Pasture devised the process of destroying bacteria known as pasteurization. 1867 Lister publishes his work on antiseptic surgery. 1876 Koch demonstrates that anthrax is caused by Bacillus anthracis. 1881 Pasture develops anthrax vaccine. 1885 Pasture develops rabies vaccine. 1902 Karl Landsteiner discovers blood groups. 1928 Griffith discovers bacterial transformation. 1929 Fleming discovers penicillin. 1944 Selman Waksman discovers streptomycin. 1954 Jonas Salk develops the first polio vaccine. 1983 Isolation and characterization of HIV (human immunodeficiency virus) by Luc- Montagnier and Robert Gallo. 1988 Polymerase chain reaction invented by Karry Mullis. 1990 First clinical trials in gene therapy testing, vaccine for hemophillus influenza, a cause of meningitis is introduced. 2000 Discovered that Vibrio cholerae has two separate chromosomes. 2002 Plasmodium falciparum, malarial parasite of humans, genome sequenced by Malcolm J. Gardner and coworkers of USA. 2002 Isolation of human gene CEM 15 that inhibits HIV-1 infection, by Ann M.S. Heely et al., of USA.

1.2

1.2

SCOPE OF MICROBIOLOGY

SCOPE OF MICROBIOLOGY

1.

Role of Microorganisms in the Production of Antibiotics Antibiotics are capable of inhibiting the growth of microorganisms. Antibiotics are mainly of 2 types; bacteriostatics (inhibit the growth of bacteria) and bactericidals (kill the bacteria). Alexander Flaming (1928) discovers the penicillin from Penicillium notatum.

1.

4 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Some important events in the history of microbiology: 1546 Fracastoro suggests that invisible organisms cause disease. 1676 Antony van Leeuwenhoek discovers “animalcules”. 1786 Muller produces first classification of bacteria. 1798 Jenner introduces cowpox vaccination for smallpox. 1839 Matthias Schleiden and Theodor Schwann, formalize the theory that all living things are composed of cells. 1867 Pasture devised the process of destroying bacteria known as pasteurization. 1867 Lister publishes his work on antiseptic surgery. 1876 Koch demonstrates that anthrax is caused by Bacillus anthracis. 1881 Pasture develops anthrax vaccine. 1885 Pasture develops rabies vaccine. 1902 Karl Landsteiner discovers blood groups. 1928 Griffith discovers bacterial transformation. 1929 Fleming discovers penicillin. 1944 Selman Waksman discovers streptomycin. 1954 Jonas Salk develops the first polio vaccine. 1983 Isolation and characterization of HIV (human immunodeficiency virus) by Luc- Montagnier and Robert Gallo. 1988 Polymerase chain reaction invented by Karry Mullis. 1990 First clinical trials in gene therapy testing, vaccine for hemophillus influenza, a cause of meningitis is introduced. 2000 Discovered that Vibrio cholerae has two separate chromosomes. 2002 Plasmodium falciparum, malarial parasite of humans, genome sequenced by Malcolm J. Gardner and coworkers of USA. 2002 Isolation of human gene CEM 15 that inhibits HIV-1 infection, by Ann M.S. Heely et al., of USA.

Some important events in the history of microbiology: 1546 Fracastoro suggests that invisible organisms cause disease. 1676 Antony van Leeuwenhoek discovers “animalcules”. 1786 Muller produces first classification of bacteria. 1798 Jenner introduces cowpox vaccination for smallpox. 1839 Matthias Schleiden and Theodor Schwann, formalize the theory that all living things are composed of cells. 1867 Pasture devised the process of destroying bacteria known as pasteurization. 1867 Lister publishes his work on antiseptic surgery. 1876 Koch demonstrates that anthrax is caused by Bacillus anthracis. 1881 Pasture develops anthrax vaccine. 1885 Pasture develops rabies vaccine. 1902 Karl Landsteiner discovers blood groups. 1928 Griffith discovers bacterial transformation. 1929 Fleming discovers penicillin. 1944 Selman Waksman discovers streptomycin. 1954 Jonas Salk develops the first polio vaccine. 1983 Isolation and characterization of HIV (human immunodeficiency virus) by Luc- Montagnier and Robert Gallo. 1988 Polymerase chain reaction invented by Karry Mullis. 1990 First clinical trials in gene therapy testing, vaccine for hemophillus influenza, a cause of meningitis is introduced. 2000 Discovered that Vibrio cholerae has two separate chromosomes. 2002 Plasmodium falciparum, malarial parasite of humans, genome sequenced by Malcolm J. Gardner and coworkers of USA. 2002 Isolation of human gene CEM 15 that inhibits HIV-1 infection, by Ann M.S. Heely et al., of USA.

1.2

1.2

1.

SCOPE OF MICROBIOLOGY

Role of Microorganisms in the Production of Antibiotics Antibiotics are capable of inhibiting the growth of microorganisms. Antibiotics are mainly of 2 types; bacteriostatics (inhibit the growth of bacteria) and bactericidals (kill the bacteria). Alexander Flaming (1928) discovers the penicillin from Penicillium notatum.

Role of Microorganisms in the Production of Antibiotics Antibiotics are capable of inhibiting the growth of microorganisms. Antibiotics are mainly of 2 types; bacteriostatics (inhibit the growth of bacteria) and bactericidals (kill the bacteria). Alexander Flaming (1928) discovers the penicillin from Penicillium notatum.

1.

SCOPE OF MICROBIOLOGY

Role of Microorganisms in the Production of Antibiotics Antibiotics are capable of inhibiting the growth of microorganisms. Antibiotics are mainly of 2 types; bacteriostatics (inhibit the growth of bacteria) and bactericidals (kill the bacteria). Alexander Flaming (1928) discovers the penicillin from Penicillium notatum.

INTRODUCTION TO SCOPE OF MICROBIOLOGY

5

INTRODUCTION TO SCOPE OF MICROBIOLOGY

List of some bacteria which produce antibiotics are given below.

5

List of some bacteria which produce antibiotics are given below.

S.No

Name of bacterium

Name of antibiotic

S.No

Name of bacterium

Name of antibiotic

1. 2. 3. 4. 5. 6. 7. 8.

Streptomyces aureofaciens Streptomyces venezuelae Streptomyces rimosus Streptomyces erythraeus Streptomyces fradiae Streptomyces kanamyceteus Streptomyces griseus Bacillus subtilis

Aureomycin (Tetracycline) Chloramphenicol Oxytetracyline Erythromycin Neomycin Kanamycin Streptomycin Bacitracin

1. 2. 3. 4. 5. 6. 7. 8.

Streptomyces aureofaciens Streptomyces venezuelae Streptomyces rimosus Streptomyces erythraeus Streptomyces fradiae Streptomyces kanamyceteus Streptomyces griseus Bacillus subtilis

Aureomycin (Tetracycline) Chloramphenicol Oxytetracyline Erythromycin Neomycin Kanamycin Streptomycin Bacitracin

List of some fungi which produce antibiotics are given below.

List of some fungi which produce antibiotics are given below.

S.No

Name of fungi

Name of Antibiotics

S.No

Name of fungi

Name of Antibiotics

1. 2. 3. 4.

Penicillium notatum Penicillium chrysogenum Claviceps purpurea Penicillium griseofulvum

Penicllin Penicllin Ergot Griseofulvin

1. 2. 3. 4.

Penicillium notatum Penicillium chrysogenum Claviceps purpurea Penicillium griseofulvum

Penicllin Penicllin Ergot Griseofulvin

2. In Alcohol Industry (a) Yeast is used in the fermentation of carbohydrate to produce alcohol. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:

2. In Alcohol Industry (a) Yeast is used in the fermentation of carbohydrate to produce alcohol. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:

(b) Clostridium acetobutylicum takes part in the manufacture of butyl alcohol.

(b) Clostridium acetobutylicum takes part in the manufacture of butyl alcohol.

3. In Vinegar Industry The production of vinegar (acetic acid) takes place with the help of bacterial actions. In the first step, the carbohydrates undergo fermentation and converted into ethyl alcohol through the action of yeast.

3. In Vinegar Industry The production of vinegar (acetic acid) takes place with the help of bacterial actions. In the first step, the carbohydrates undergo fermentation and converted into ethyl alcohol through the action of yeast.

In the second step the aerobic bacteria for example, Acetobactor aceti and Mycoderma aceti, oxidize the alcohol into acetic acid.

In the second step the aerobic bacteria for example, Acetobactor aceti and Mycoderma aceti, oxidize the alcohol into acetic acid.

INTRODUCTION TO SCOPE OF MICROBIOLOGY

5

INTRODUCTION TO SCOPE OF MICROBIOLOGY

List of some bacteria which produce antibiotics are given below.

5

List of some bacteria which produce antibiotics are given below.

S.No

Name of bacterium

Name of antibiotic

S.No

Name of bacterium

Name of antibiotic

1. 2. 3. 4. 5. 6. 7. 8.

Streptomyces aureofaciens Streptomyces venezuelae Streptomyces rimosus Streptomyces erythraeus Streptomyces fradiae Streptomyces kanamyceteus Streptomyces griseus Bacillus subtilis

Aureomycin (Tetracycline) Chloramphenicol Oxytetracyline Erythromycin Neomycin Kanamycin Streptomycin Bacitracin

1. 2. 3. 4. 5. 6. 7. 8.

Streptomyces aureofaciens Streptomyces venezuelae Streptomyces rimosus Streptomyces erythraeus Streptomyces fradiae Streptomyces kanamyceteus Streptomyces griseus Bacillus subtilis

Aureomycin (Tetracycline) Chloramphenicol Oxytetracyline Erythromycin Neomycin Kanamycin Streptomycin Bacitracin

List of some fungi which produce antibiotics are given below.

List of some fungi which produce antibiotics are given below.

S.No

Name of fungi

Name of Antibiotics

S.No

Name of fungi

Name of Antibiotics

1. 2. 3. 4.

Penicillium notatum Penicillium chrysogenum Claviceps purpurea Penicillium griseofulvum

Penicllin Penicllin Ergot Griseofulvin

1. 2. 3. 4.

Penicillium notatum Penicillium chrysogenum Claviceps purpurea Penicillium griseofulvum

Penicllin Penicllin Ergot Griseofulvin

2. In Alcohol Industry (a) Yeast is used in the fermentation of carbohydrate to produce alcohol. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:

2. In Alcohol Industry (a) Yeast is used in the fermentation of carbohydrate to produce alcohol. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:

(b) Clostridium acetobutylicum takes part in the manufacture of butyl alcohol.

(b) Clostridium acetobutylicum takes part in the manufacture of butyl alcohol.

3. In Vinegar Industry The production of vinegar (acetic acid) takes place with the help of bacterial actions. In the first step, the carbohydrates undergo fermentation and converted into ethyl alcohol through the action of yeast.

3. In Vinegar Industry The production of vinegar (acetic acid) takes place with the help of bacterial actions. In the first step, the carbohydrates undergo fermentation and converted into ethyl alcohol through the action of yeast.

In the second step the aerobic bacteria for example, Acetobactor aceti and Mycoderma aceti, oxidize the alcohol into acetic acid.

In the second step the aerobic bacteria for example, Acetobactor aceti and Mycoderma aceti, oxidize the alcohol into acetic acid.

6 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4. Vitamin Production There are various types of vitamins obtained from the microorganisms. (a) Vitamins obtained from the bacteria are:

4. Vitamin Production There are various types of vitamins obtained from the microorganisms. (a) Vitamins obtained from the bacteria are:

S. No

Name of bacteria

Name of vitamin

S. No

Name of bacteria

Name of vitamin

1. 2.

Clostridium butylicum Pseudomonas denitrificans

Riboflavins (B2) Cobalamins (B12)

1. 2.

Clostridium butylicum Pseudomonas denitrificans

Riboflavins (B2) Cobalamins (B12)

(b) Vitamins A, C, D and E are mostly found in algae. (c) Fungi (yeast) have high content of Vita B-1, Vita B-12 and Vita-C.

(b) Vitamins A, C, D and E are mostly found in algae. (c) Fungi (yeast) have high content of Vita B-1, Vita B-12 and Vita-C.

5. Production of Acids and Enzymes List of microorganisms from which acids and enzymes are obtained.

5. Production of Acids and Enzymes List of microorganisms from which acids and enzymes are obtained.

S. No

Name of microorganism

Acid

S. No

Name of microorganism

Acid

1. 2. 3. 4.

Penicillium glaucum Aspergillus gallomyces Mucor Aspergillus niger

Gallic acid Gallic acid Citric acid Gluconic acid

1. 2. 3. 4.

Penicillium glaucum Aspergillus gallomyces Mucor Aspergillus niger

Gallic acid Gallic acid Citric acid Gluconic acid

Enzymes

Enzymes

S. No

Name of microorganism

Enzyme

S. No

Name of microorganism

Enzyme

1. 2. 3. 4.

Aspergillus oryzae Bacillus subtilis Saccharomyces cerevisiae Streptococcus pyrogens

Amylase Protease Invertase Streptokinase

1. 2. 3. 4.

Aspergillus oryzae Bacillus subtilis Saccharomyces cerevisiae Streptococcus pyrogens

Amylase Protease Invertase Streptokinase

6. Used in Baking Industry Yeast is used in the manufacture of bread. Kneaded flour is mixed with yeast and allowed to ferment. Yeast converts starch into sugars and sugar into alcohol and carbon dioxide (CO2) with the help of enzyme, zymase. CO2 is released when effervescence takes place due to which bread becomes spongy (porous) and gets swollen and is of light weight.

6. Used in Baking Industry Yeast is used in the manufacture of bread. Kneaded flour is mixed with yeast and allowed to ferment. Yeast converts starch into sugars and sugar into alcohol and carbon dioxide (CO2) with the help of enzyme, zymase. CO2 is released when effervescence takes place due to which bread becomes spongy (porous) and gets swollen and is of light weight.

7. Used in Cosmetics and Perfumes (a) Some species of lichens like Evernia and Ramatina are used for making perfumery goods and soaps. (b) A mucilaginous substance Carrageenin is excreted form sea weeds used in the manufacture of icecream, paints, shampoo etc.

7. Used in Cosmetics and Perfumes (a) Some species of lichens like Evernia and Ramatina are used for making perfumery goods and soaps. (b) A mucilaginous substance Carrageenin is excreted form sea weeds used in the manufacture of icecream, paints, shampoo etc.

8. Used in the Production of Dairy Products Almost all milk products such as curd, butter, cheese, and ghee are manufactured form bacterial activity.

8. Used in the Production of Dairy Products Almost all milk products such as curd, butter, cheese, and ghee are manufactured form bacterial activity.

6 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4. Vitamin Production There are various types of vitamins obtained from the microorganisms. (a) Vitamins obtained from the bacteria are:

4. Vitamin Production There are various types of vitamins obtained from the microorganisms. (a) Vitamins obtained from the bacteria are:

S. No

Name of bacteria

Name of vitamin

S. No

Name of bacteria

Name of vitamin

1. 2.

Clostridium butylicum Pseudomonas denitrificans

Riboflavins (B2) Cobalamins (B12)

1. 2.

Clostridium butylicum Pseudomonas denitrificans

Riboflavins (B2) Cobalamins (B12)

(b) Vitamins A, C, D and E are mostly found in algae. (c) Fungi (yeast) have high content of Vita B-1, Vita B-12 and Vita-C.

(b) Vitamins A, C, D and E are mostly found in algae. (c) Fungi (yeast) have high content of Vita B-1, Vita B-12 and Vita-C.

5. Production of Acids and Enzymes List of microorganisms from which acids and enzymes are obtained.

5. Production of Acids and Enzymes List of microorganisms from which acids and enzymes are obtained.

S. No

Name of microorganism

Acid

S. No

Name of microorganism

Acid

1. 2. 3. 4.

Penicillium glaucum Aspergillus gallomyces Mucor Aspergillus niger

Gallic acid Gallic acid Citric acid Gluconic acid

1. 2. 3. 4.

Penicillium glaucum Aspergillus gallomyces Mucor Aspergillus niger

Gallic acid Gallic acid Citric acid Gluconic acid

Enzymes

Enzymes

S. No

Name of microorganism

Enzyme

S. No

Name of microorganism

Enzyme

1. 2. 3. 4.

Aspergillus oryzae Bacillus subtilis Saccharomyces cerevisiae Streptococcus pyrogens

Amylase Protease Invertase Streptokinase

1. 2. 3. 4.

Aspergillus oryzae Bacillus subtilis Saccharomyces cerevisiae Streptococcus pyrogens

Amylase Protease Invertase Streptokinase

6. Used in Baking Industry Yeast is used in the manufacture of bread. Kneaded flour is mixed with yeast and allowed to ferment. Yeast converts starch into sugars and sugar into alcohol and carbon dioxide (CO2) with the help of enzyme, zymase. CO2 is released when effervescence takes place due to which bread becomes spongy (porous) and gets swollen and is of light weight.

6. Used in Baking Industry Yeast is used in the manufacture of bread. Kneaded flour is mixed with yeast and allowed to ferment. Yeast converts starch into sugars and sugar into alcohol and carbon dioxide (CO2) with the help of enzyme, zymase. CO2 is released when effervescence takes place due to which bread becomes spongy (porous) and gets swollen and is of light weight.

7. Used in Cosmetics and Perfumes (a) Some species of lichens like Evernia and Ramatina are used for making perfumery goods and soaps. (b) A mucilaginous substance Carrageenin is excreted form sea weeds used in the manufacture of icecream, paints, shampoo etc.

7. Used in Cosmetics and Perfumes (a) Some species of lichens like Evernia and Ramatina are used for making perfumery goods and soaps. (b) A mucilaginous substance Carrageenin is excreted form sea weeds used in the manufacture of icecream, paints, shampoo etc.

8. Used in the Production of Dairy Products Almost all milk products such as curd, butter, cheese, and ghee are manufactured form bacterial activity.

8. Used in the Production of Dairy Products Almost all milk products such as curd, butter, cheese, and ghee are manufactured form bacterial activity.

INTRODUCTION TO SCOPE OF MICROBIOLOGY

7

INTRODUCTION TO SCOPE OF MICROBIOLOGY

List of microorganisms used in dairy industry are:

7

List of microorganisms used in dairy industry are:

S.No.

Name of microorganism

Product

S.No.

Name of microorganism

Product

1. 2. 3. 4. 5.

Lactobacillus lactis Lactobacillus vulgaricus Streptococcus lactis Streptococcus lactis Streptococcus lactis and Streptococcus cremoris

Cheese Yoghurt Curd Butter Butter milk

1. 2. 3. 4. 5.

Lactobacillus lactis Lactobacillus vulgaricus Streptococcus lactis Streptococcus lactis Streptococcus lactis and Streptococcus cremoris

Cheese Yoghurt Curd Butter Butter milk

9. Used in Agriculture or Soil Fertility Nitrogen is an essential for the synthesis of proteins, nucleic acids, and other nitrogen containing compounds. The plants take nitrogen in the form of nitrates. Nitrogen fixation is done by many organisms: (a) Algae examples are: Anabina, Nostoc (b) Bacteria: Nitrosomonas and Nitrobactor

9. Used in Agriculture or Soil Fertility Nitrogen is an essential for the synthesis of proteins, nucleic acids, and other nitrogen containing compounds. The plants take nitrogen in the form of nitrates. Nitrogen fixation is done by many organisms: (a) Algae examples are: Anabina, Nostoc (b) Bacteria: Nitrosomonas and Nitrobactor

(c) Soil free bacteria: Azotobactor, Clostridium (d) Root nodules of leguminous plants: Rhizobium leguminosarum. (e) Saprophyte fungi disintegrate the decaying organic matter and thus help in release of minerals can begins absorbed by plants.

(c) Soil free bacteria: Azotobactor, Clostridium (d) Root nodules of leguminous plants: Rhizobium leguminosarum. (e) Saprophyte fungi disintegrate the decaying organic matter and thus help in release of minerals can begins absorbed by plants.

10. Used as Food Many algae Gelidium, Gracilaria etc. produce agar-agar, a jelly-like substances used in the manufacture of icecream. Saragassum and macrocystis are used as food for marine as well as domestic animals. Some seaweeds for example, Laminaria contains a lot of iodine which is an important mineral in the thyroid gland. Lichens like Reindeer moss (Cladonia rangiferina) of the arctic region is eaten by reindeers and cattles. Iceland moss (Cetraria islandica) have been used as food by man in Iceland.

10. Used as Food Many algae Gelidium, Gracilaria etc. produce agar-agar, a jelly-like substances used in the manufacture of icecream. Saragassum and macrocystis are used as food for marine as well as domestic animals. Some seaweeds for example, Laminaria contains a lot of iodine which is an important mineral in the thyroid gland. Lichens like Reindeer moss (Cladonia rangiferina) of the arctic region is eaten by reindeers and cattles. Iceland moss (Cetraria islandica) have been used as food by man in Iceland.

INTRODUCTION TO SCOPE OF MICROBIOLOGY

7

INTRODUCTION TO SCOPE OF MICROBIOLOGY

List of microorganisms used in dairy industry are:

7

List of microorganisms used in dairy industry are:

S.No.

Name of microorganism

Product

S.No.

Name of microorganism

Product

1. 2. 3. 4. 5.

Lactobacillus lactis Lactobacillus vulgaricus Streptococcus lactis Streptococcus lactis Streptococcus lactis and Streptococcus cremoris

Cheese Yoghurt Curd Butter Butter milk

1. 2. 3. 4. 5.

Lactobacillus lactis Lactobacillus vulgaricus Streptococcus lactis Streptococcus lactis Streptococcus lactis and Streptococcus cremoris

Cheese Yoghurt Curd Butter Butter milk

9. Used in Agriculture or Soil Fertility Nitrogen is an essential for the synthesis of proteins, nucleic acids, and other nitrogen containing compounds. The plants take nitrogen in the form of nitrates. Nitrogen fixation is done by many organisms: (a) Algae examples are: Anabina, Nostoc (b) Bacteria: Nitrosomonas and Nitrobactor

9. Used in Agriculture or Soil Fertility Nitrogen is an essential for the synthesis of proteins, nucleic acids, and other nitrogen containing compounds. The plants take nitrogen in the form of nitrates. Nitrogen fixation is done by many organisms: (a) Algae examples are: Anabina, Nostoc (b) Bacteria: Nitrosomonas and Nitrobactor

(c) Soil free bacteria: Azotobactor, Clostridium (d) Root nodules of leguminous plants: Rhizobium leguminosarum. (e) Saprophyte fungi disintegrate the decaying organic matter and thus help in release of minerals can begins absorbed by plants.

(c) Soil free bacteria: Azotobactor, Clostridium (d) Root nodules of leguminous plants: Rhizobium leguminosarum. (e) Saprophyte fungi disintegrate the decaying organic matter and thus help in release of minerals can begins absorbed by plants.

10. Used as Food Many algae Gelidium, Gracilaria etc. produce agar-agar, a jelly-like substances used in the manufacture of icecream. Saragassum and macrocystis are used as food for marine as well as domestic animals. Some seaweeds for example, Laminaria contains a lot of iodine which is an important mineral in the thyroid gland. Lichens like Reindeer moss (Cladonia rangiferina) of the arctic region is eaten by reindeers and cattles. Iceland moss (Cetraria islandica) have been used as food by man in Iceland.

10. Used as Food Many algae Gelidium, Gracilaria etc. produce agar-agar, a jelly-like substances used in the manufacture of icecream. Saragassum and macrocystis are used as food for marine as well as domestic animals. Some seaweeds for example, Laminaria contains a lot of iodine which is an important mineral in the thyroid gland. Lichens like Reindeer moss (Cladonia rangiferina) of the arctic region is eaten by reindeers and cattles. Iceland moss (Cetraria islandica) have been used as food by man in Iceland.

2.1

2

2

Structure of Bacterial Cell

Structure of Bacterial Cell

HISTORY OF THE CELL

2.1

HISTORY OF THE CELL

1. The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans are multicellular. (Humans have an estimated 100 trillion or 1014 cells, a typical cell size is 10—m and a typical cell mass is 1 nanogram.) The largest known cell is an unfertilized ostrich egg cell. 2. The word cell comes from the Latin cellula, meaning a small room. The descriptive term for the smallest living biological structure was coined by Robert Hooke in his book. He published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in. 3. The cell theory, first given by Matthias Jakob Schleiden and Theodor Schwann in 1839, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells. 4. Cells are mainly of two types: Prokaryotic cell (e.g. bacteria, virus) and eukaryotic cell (e.g. plant cell and animal cell). 5. All biological systems have the following characteristics: (a) The ability to reproduce. (b) The ability to ingest and metabolize them for energy and growth. (c) The ability to excrete waste products. (d) The ability to react to change in their environment.

1. The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans are multicellular. (Humans have an estimated 100 trillion or 1014 cells, a typical cell size is 10—m and a typical cell mass is 1 nanogram.) The largest known cell is an unfertilized ostrich egg cell. 2. The word cell comes from the Latin cellula, meaning a small room. The descriptive term for the smallest living biological structure was coined by Robert Hooke in his book. He published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in. 3. The cell theory, first given by Matthias Jakob Schleiden and Theodor Schwann in 1839, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells. 4. Cells are mainly of two types: Prokaryotic cell (e.g. bacteria, virus) and eukaryotic cell (e.g. plant cell and animal cell). 5. All biological systems have the following characteristics: (a) The ability to reproduce. (b) The ability to ingest and metabolize them for energy and growth. (c) The ability to excrete waste products. (d) The ability to react to change in their environment.

2

2

Structure of Bacterial Cell

Structure of Bacterial Cell

2.1

HISTORY OF THE CELL

1. The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans are multicellular. (Humans have an estimated 100 trillion or 1014 cells, a typical cell size is 10—m and a typical cell mass is 1 nanogram.) The largest known cell is an unfertilized ostrich egg cell. 2. The word cell comes from the Latin cellula, meaning a small room. The descriptive term for the smallest living biological structure was coined by Robert Hooke in his book. He published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in. 3. The cell theory, first given by Matthias Jakob Schleiden and Theodor Schwann in 1839, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells. 4. Cells are mainly of two types: Prokaryotic cell (e.g. bacteria, virus) and eukaryotic cell (e.g. plant cell and animal cell). 5. All biological systems have the following characteristics: (a) The ability to reproduce. (b) The ability to ingest and metabolize them for energy and growth. (c) The ability to excrete waste products. (d) The ability to react to change in their environment.

2.1

HISTORY OF THE CELL

1. The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans are multicellular. (Humans have an estimated 100 trillion or 1014 cells, a typical cell size is 10—m and a typical cell mass is 1 nanogram.) The largest known cell is an unfertilized ostrich egg cell. 2. The word cell comes from the Latin cellula, meaning a small room. The descriptive term for the smallest living biological structure was coined by Robert Hooke in his book. He published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in. 3. The cell theory, first given by Matthias Jakob Schleiden and Theodor Schwann in 1839, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells. 4. Cells are mainly of two types: Prokaryotic cell (e.g. bacteria, virus) and eukaryotic cell (e.g. plant cell and animal cell). 5. All biological systems have the following characteristics: (a) The ability to reproduce. (b) The ability to ingest and metabolize them for energy and growth. (c) The ability to excrete waste products. (d) The ability to react to change in their environment.

10 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 2.1. Structure of bacterial cell (prokaryotic cell).

2.2

DESCRIPTION OF BACTERIAL CELL STRUCTURE

Fig. 2.1. Structure of bacterial cell (prokaryotic cell).

2.2

DESCRIPTION OF BACTERIAL CELL STRUCTURE

Capsule: Glycocalyx meaning sugar coat is the general term used for general substances that surround cell. The bacterial glycocalyx is a viscous gelatinous polymer that is external to cell wall and composed of polysaccharide, polypeptide or both. Cell wall: The bacterial cell is surrounded by a rigid cell wall present outside the plasma membrane. It provides shape to the cell. Its main function is to prevent the cell from expanding and eventually bursting because of uptake of water, since most bacteria live in hypotonic environment. Cell wall of gram-positive bacteria: The gram-positive cell wall is characterized by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during gram staining procedures. Peptidoglycan is a polymer of NAG (N-acetyl glucosamine) and NAMA (N-acetyl muramic acid). Cell wall of gram-negative bacteria: The cell wall of gram-negative bacteria is more complex than those of gram-positive bacteria. The most interesting difference is the presence of an outer membrane that surrounds a thin underlying layer of peptidoglycan. The walls of gram-negative bacteria rich in lipids in contrast to those of gram-positive bacteria. Gramnegative bacteria mainly contain lipopolysaccharides (LPS), phospholipids and proteins.

Capsule: Glycocalyx meaning sugar coat is the general term used for general substances that surround cell. The bacterial glycocalyx is a viscous gelatinous polymer that is external to cell wall and composed of polysaccharide, polypeptide or both. Cell wall: The bacterial cell is surrounded by a rigid cell wall present outside the plasma membrane. It provides shape to the cell. Its main function is to prevent the cell from expanding and eventually bursting because of uptake of water, since most bacteria live in hypotonic environment. Cell wall of gram-positive bacteria: The gram-positive cell wall is characterized by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during gram staining procedures. Peptidoglycan is a polymer of NAG (N-acetyl glucosamine) and NAMA (N-acetyl muramic acid). Cell wall of gram-negative bacteria: The cell wall of gram-negative bacteria is more complex than those of gram-positive bacteria. The most interesting difference is the presence of an outer membrane that surrounds a thin underlying layer of peptidoglycan. The walls of gram-negative bacteria rich in lipids in contrast to those of gram-positive bacteria. Gramnegative bacteria mainly contain lipopolysaccharides (LPS), phospholipids and proteins.

10 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 2.1. Structure of bacterial cell (prokaryotic cell).

2.2

DESCRIPTION OF BACTERIAL CELL STRUCTURE

Capsule: Glycocalyx meaning sugar coat is the general term used for general substances that surround cell. The bacterial glycocalyx is a viscous gelatinous polymer that is external to cell wall and composed of polysaccharide, polypeptide or both. Cell wall: The bacterial cell is surrounded by a rigid cell wall present outside the plasma membrane. It provides shape to the cell. Its main function is to prevent the cell from expanding and eventually bursting because of uptake of water, since most bacteria live in hypotonic environment. Cell wall of gram-positive bacteria: The gram-positive cell wall is characterized by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during gram staining procedures. Peptidoglycan is a polymer of NAG (N-acetyl glucosamine) and NAMA (N-acetyl muramic acid). Cell wall of gram-negative bacteria: The cell wall of gram-negative bacteria is more complex than those of gram-positive bacteria. The most interesting difference is the presence of an outer membrane that surrounds a thin underlying layer of peptidoglycan. The walls of gram-negative bacteria rich in lipids in contrast to those of gram-positive bacteria. Gramnegative bacteria mainly contain lipopolysaccharides (LPS), phospholipids and proteins.

Fig. 2.1. Structure of bacterial cell (prokaryotic cell).

2.2

DESCRIPTION OF BACTERIAL CELL STRUCTURE

Capsule: Glycocalyx meaning sugar coat is the general term used for general substances that surround cell. The bacterial glycocalyx is a viscous gelatinous polymer that is external to cell wall and composed of polysaccharide, polypeptide or both. Cell wall: The bacterial cell is surrounded by a rigid cell wall present outside the plasma membrane. It provides shape to the cell. Its main function is to prevent the cell from expanding and eventually bursting because of uptake of water, since most bacteria live in hypotonic environment. Cell wall of gram-positive bacteria: The gram-positive cell wall is characterized by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during gram staining procedures. Peptidoglycan is a polymer of NAG (N-acetyl glucosamine) and NAMA (N-acetyl muramic acid). Cell wall of gram-negative bacteria: The cell wall of gram-negative bacteria is more complex than those of gram-positive bacteria. The most interesting difference is the presence of an outer membrane that surrounds a thin underlying layer of peptidoglycan. The walls of gram-negative bacteria rich in lipids in contrast to those of gram-positive bacteria. Gramnegative bacteria mainly contain lipopolysaccharides (LPS), phospholipids and proteins.

STRUCTURE OF BACTERIAL CELL

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STRUCTURE OF BACTERIAL CELL

11

Fig. 2.2. Schematic diagram of the peptidoglycan sheet of Staphylococcus aureus. G = N-acetyl-glucosamine; M = N-acetyl-muramic acid; L-ala = L-alanine; D-ala = D-alanine; D-glu = D-glutamic acid; L-lys = L-lysine.

Fig. 2.2. Schematic diagram of the peptidoglycan sheet of Staphylococcus aureus. G = N-acetyl-glucosamine; M = N-acetyl-muramic acid; L-ala = L-alanine; D-ala = D-alanine; D-glu = D-glutamic acid; L-lys = L-lysine.

The gram-negative cell envelope (Fig. 2.3) is even more complicated; essentially, it contains lipoprotein molecules attached covalently to the oligosaccharide backbone and in addition, on its outer side, a layer of lipopolysaccharide (LPS) and protein attached by hydrophobic interactions and divalent metal cations, Ca2+ and Mg2+. On the inner side is a layer of phospholipid (PL). The lipopolysaccharide has toxic properties and is also known as endotoxin. (Endotoxins are the structural component of bacteria, which is released after bacterial cell lysed.)

The gram-negative cell envelope (Fig. 2.3) is even more complicated; essentially, it contains lipoprotein molecules attached covalently to the oligosaccharide backbone and in addition, on its outer side, a layer of lipopolysaccharide (LPS) and protein attached by hydrophobic interactions and divalent metal cations, Ca2+ and Mg2+. On the inner side is a layer of phospholipid (PL). The lipopolysaccharide has toxic properties and is also known as endotoxin. (Endotoxins are the structural component of bacteria, which is released after bacterial cell lysed.)

Fig. 2.3. Diagram showing detailed structure of the envelope of a gram-negative bacteria.

Fig. 2.3. Diagram showing detailed structure of the envelope of a gram-negative bacteria.

STRUCTURE OF BACTERIAL CELL

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STRUCTURE OF BACTERIAL CELL

11

Fig. 2.2. Schematic diagram of the peptidoglycan sheet of Staphylococcus aureus. G = N-acetyl-glucosamine; M = N-acetyl-muramic acid; L-ala = L-alanine; D-ala = D-alanine; D-glu = D-glutamic acid; L-lys = L-lysine.

Fig. 2.2. Schematic diagram of the peptidoglycan sheet of Staphylococcus aureus. G = N-acetyl-glucosamine; M = N-acetyl-muramic acid; L-ala = L-alanine; D-ala = D-alanine; D-glu = D-glutamic acid; L-lys = L-lysine.

The gram-negative cell envelope (Fig. 2.3) is even more complicated; essentially, it contains lipoprotein molecules attached covalently to the oligosaccharide backbone and in addition, on its outer side, a layer of lipopolysaccharide (LPS) and protein attached by hydrophobic interactions and divalent metal cations, Ca2+ and Mg2+. On the inner side is a layer of phospholipid (PL). The lipopolysaccharide has toxic properties and is also known as endotoxin. (Endotoxins are the structural component of bacteria, which is released after bacterial cell lysed.)

The gram-negative cell envelope (Fig. 2.3) is even more complicated; essentially, it contains lipoprotein molecules attached covalently to the oligosaccharide backbone and in addition, on its outer side, a layer of lipopolysaccharide (LPS) and protein attached by hydrophobic interactions and divalent metal cations, Ca2+ and Mg2+. On the inner side is a layer of phospholipid (PL). The lipopolysaccharide has toxic properties and is also known as endotoxin. (Endotoxins are the structural component of bacteria, which is released after bacterial cell lysed.)

Fig. 2.3. Diagram showing detailed structure of the envelope of a gram-negative bacteria.

Fig. 2.3. Diagram showing detailed structure of the envelope of a gram-negative bacteria.

12 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

12 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Cytoplasmic membrane: The bacterial cytoplasmic membrane is composed of a phospholipid bilayer which is 5 to 10 nm in thickness and has all the general functions of a cell membrane such as acting as a permeability barrier for most molecules and serving as the location for the transport of molecules into the cell. It acts as a semipermeable membrane controlling the inflow and outflow of metabolites to and from the protoplasm. In this function, plasma membrane has selective permeability. (Note: Staining of bacteria depends upon the chemical and physical nature of cytoplasmic membrane and cytoplasm.) Mesosome: The outer membrane of cytoplasm forms much coiled invagination called mesosome. The surface of mesosome has many respiratory enzymes, which takes part in respiration. It is absent in eukaryotic cells. Nucleus: In prokaryotes nuclear membrane and nucleolus are absent. It contains a single chromosome consisting of a circular DNA filament. The genetic material DNA is present in the cytoplasm without histon proteins. Ribosomes: Bacteria contain a group of ribosomes called polyribosomes present in the cytoplasm of the cell. It is 70S type in bacterial cell having two subunits— the large unit is 50S and the smaller unit 30S. Ribosomes help in protein synthesis. Flagellum: The bacterial flagella are a long, filaments and whip-like structures that produce through the cytoplasmic membrane and are responsible for motility. The flagella are nothing but surface appendages invariably found in motile bacteria, and appear generally as filaments having diameter ranging between 12–20 Pm and length between 6-8 —m. It is only about 0.01 to 0.02 —m thick. A flagellum has three basic parts: Basal body: It is embedded in the cell envelope. Hook: The flagellar hook is a single peptide unit. It penetrates the cell wall and connects the main filaments to the hook at the surface.

Cytoplasmic membrane: The bacterial cytoplasmic membrane is composed of a phospholipid bilayer which is 5 to 10 nm in thickness and has all the general functions of a cell membrane such as acting as a permeability barrier for most molecules and serving as the location for the transport of molecules into the cell. It acts as a semipermeable membrane controlling the inflow and outflow of metabolites to and from the protoplasm. In this function, plasma membrane has selective permeability. (Note: Staining of bacteria depends upon the chemical and physical nature of cytoplasmic membrane and cytoplasm.) Mesosome: The outer membrane of cytoplasm forms much coiled invagination called mesosome. The surface of mesosome has many respiratory enzymes, which takes part in respiration. It is absent in eukaryotic cells. Nucleus: In prokaryotes nuclear membrane and nucleolus are absent. It contains a single chromosome consisting of a circular DNA filament. The genetic material DNA is present in the cytoplasm without histon proteins. Ribosomes: Bacteria contain a group of ribosomes called polyribosomes present in the cytoplasm of the cell. It is 70S type in bacterial cell having two subunits— the large unit is 50S and the smaller unit 30S. Ribosomes help in protein synthesis. Flagellum: The bacterial flagella are a long, filaments and whip-like structures that produce through the cytoplasmic membrane and are responsible for motility. The flagella are nothing but surface appendages invariably found in motile bacteria, and appear generally as filaments having diameter ranging between 12–20 Pm and length between 6-8 —m. It is only about 0.01 to 0.02 —m thick. A flagellum has three basic parts: Basal body: It is embedded in the cell envelope. Hook: The flagellar hook is a single peptide unit. It penetrates the cell wall and connects the main filaments to the hook at the surface.

Fig. 2.4. Structure of bacterial flagella.

Fig. 2.4. Structure of bacterial flagella.

12 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

12 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Cytoplasmic membrane: The bacterial cytoplasmic membrane is composed of a phospholipid bilayer which is 5 to 10 nm in thickness and has all the general functions of a cell membrane such as acting as a permeability barrier for most molecules and serving as the location for the transport of molecules into the cell. It acts as a semipermeable membrane controlling the inflow and outflow of metabolites to and from the protoplasm. In this function, plasma membrane has selective permeability. (Note: Staining of bacteria depends upon the chemical and physical nature of cytoplasmic membrane and cytoplasm.) Mesosome: The outer membrane of cytoplasm forms much coiled invagination called mesosome. The surface of mesosome has many respiratory enzymes, which takes part in respiration. It is absent in eukaryotic cells. Nucleus: In prokaryotes nuclear membrane and nucleolus are absent. It contains a single chromosome consisting of a circular DNA filament. The genetic material DNA is present in the cytoplasm without histon proteins. Ribosomes: Bacteria contain a group of ribosomes called polyribosomes present in the cytoplasm of the cell. It is 70S type in bacterial cell having two subunits— the large unit is 50S and the smaller unit 30S. Ribosomes help in protein synthesis. Flagellum: The bacterial flagella are a long, filaments and whip-like structures that produce through the cytoplasmic membrane and are responsible for motility. The flagella are nothing but surface appendages invariably found in motile bacteria, and appear generally as filaments having diameter ranging between 12–20 Pm and length between 6-8 —m. It is only about 0.01 to 0.02 —m thick. A flagellum has three basic parts: Basal body: It is embedded in the cell envelope. Hook: The flagellar hook is a single peptide unit. It penetrates the cell wall and connects the main filaments to the hook at the surface.

Cytoplasmic membrane: The bacterial cytoplasmic membrane is composed of a phospholipid bilayer which is 5 to 10 nm in thickness and has all the general functions of a cell membrane such as acting as a permeability barrier for most molecules and serving as the location for the transport of molecules into the cell. It acts as a semipermeable membrane controlling the inflow and outflow of metabolites to and from the protoplasm. In this function, plasma membrane has selective permeability. (Note: Staining of bacteria depends upon the chemical and physical nature of cytoplasmic membrane and cytoplasm.) Mesosome: The outer membrane of cytoplasm forms much coiled invagination called mesosome. The surface of mesosome has many respiratory enzymes, which takes part in respiration. It is absent in eukaryotic cells. Nucleus: In prokaryotes nuclear membrane and nucleolus are absent. It contains a single chromosome consisting of a circular DNA filament. The genetic material DNA is present in the cytoplasm without histon proteins. Ribosomes: Bacteria contain a group of ribosomes called polyribosomes present in the cytoplasm of the cell. It is 70S type in bacterial cell having two subunits— the large unit is 50S and the smaller unit 30S. Ribosomes help in protein synthesis. Flagellum: The bacterial flagella are a long, filaments and whip-like structures that produce through the cytoplasmic membrane and are responsible for motility. The flagella are nothing but surface appendages invariably found in motile bacteria, and appear generally as filaments having diameter ranging between 12–20 Pm and length between 6-8 —m. It is only about 0.01 to 0.02 —m thick. A flagellum has three basic parts: Basal body: It is embedded in the cell envelope. Hook: The flagellar hook is a single peptide unit. It penetrates the cell wall and connects the main filaments to the hook at the surface.

Fig. 2.4. Structure of bacterial flagella.

Fig. 2.4. Structure of bacterial flagella.

STRUCTURE OF BACTERIAL CELL

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STRUCTURE OF BACTERIAL CELL

13

Filaments: The filaments lie external to the cell and remain connected to the hook at the surface. Filament is composed of a protein called flagellin which has a characteristic to connect just like myosin. It does not have any ATPase activity. On the basis of the number and position of flagellum bacteria are divided into the following forms: (i) Atrichous: Bacteria without flagella. e.g., Pasteurella. (ii) Monotrichous: One flagellum is present at one end, e.g., Vibrio, Pseudomonas thiobacillus. (iii) Lophotrichous: Two or more flagella are present at one end, e.g., Spirillum serpens. (iv) Amphitrichous: Flagella are present at both the ends, e.g., Nitrosomonas. (v) Peritrichous: Flagella are found in whole body, e.g., E. coli, Clostridium tetani.

Filaments: The filaments lie external to the cell and remain connected to the hook at the surface. Filament is composed of a protein called flagellin which has a characteristic to connect just like myosin. It does not have any ATPase activity. On the basis of the number and position of flagellum bacteria are divided into the following forms: (i) Atrichous: Bacteria without flagella. e.g., Pasteurella. (ii) Monotrichous: One flagellum is present at one end, e.g., Vibrio, Pseudomonas thiobacillus. (iii) Lophotrichous: Two or more flagella are present at one end, e.g., Spirillum serpens. (iv) Amphitrichous: Flagella are present at both the ends, e.g., Nitrosomonas. (v) Peritrichous: Flagella are found in whole body, e.g., E. coli, Clostridium tetani.

Fig. 2.5. Structure of different types of flagella.

Fig. 2.5. Structure of different types of flagella.

Function of Flagella 1. Flagella are fully responsible for the bacterial motility. 2. Deflagellation by mechanical means renders the motile cells immotile. 3. The apparent movement of the bacterial cell usually takes place by the rotation of the flagella either in the clockwise or anticlockwise direction along its long axis. 4. Bacterial cell possesses the inherent capacity to alter both the direction of rotation and the speed; besides, the meticulous adjustment of frequency of ‘stops’ and ‘starts’ by the appropriate movement of the flagella. 5. Evidently, the flagellated peritrichal bacteria usually swim in a straight line over moderate distances. In actual practice, these swim-across straight line runs are interrupted frequently by abrupt alterations in the direction that ultimately leads to tumbling. Therefore, the movement of the bacteria is believed to be zig-zag.

Function of Flagella 1. Flagella are fully responsible for the bacterial motility. 2. Deflagellation by mechanical means renders the motile cells immotile. 3. The apparent movement of the bacterial cell usually takes place by the rotation of the flagella either in the clockwise or anticlockwise direction along its long axis. 4. Bacterial cell possesses the inherent capacity to alter both the direction of rotation and the speed; besides, the meticulous adjustment of frequency of ‘stops’ and ‘starts’ by the appropriate movement of the flagella. 5. Evidently, the flagellated peritrichal bacteria usually swim in a straight line over moderate distances. In actual practice, these swim-across straight line runs are interrupted frequently by abrupt alterations in the direction that ultimately leads to tumbling. Therefore, the movement of the bacteria is believed to be zig-zag.

STRUCTURE OF BACTERIAL CELL

13

STRUCTURE OF BACTERIAL CELL

13

Filaments: The filaments lie external to the cell and remain connected to the hook at the surface. Filament is composed of a protein called flagellin which has a characteristic to connect just like myosin. It does not have any ATPase activity. On the basis of the number and position of flagellum bacteria are divided into the following forms: (i) Atrichous: Bacteria without flagella. e.g., Pasteurella. (ii) Monotrichous: One flagellum is present at one end, e.g., Vibrio, Pseudomonas thiobacillus. (iii) Lophotrichous: Two or more flagella are present at one end, e.g., Spirillum serpens. (iv) Amphitrichous: Flagella are present at both the ends, e.g., Nitrosomonas. (v) Peritrichous: Flagella are found in whole body, e.g., E. coli, Clostridium tetani.

Filaments: The filaments lie external to the cell and remain connected to the hook at the surface. Filament is composed of a protein called flagellin which has a characteristic to connect just like myosin. It does not have any ATPase activity. On the basis of the number and position of flagellum bacteria are divided into the following forms: (i) Atrichous: Bacteria without flagella. e.g., Pasteurella. (ii) Monotrichous: One flagellum is present at one end, e.g., Vibrio, Pseudomonas thiobacillus. (iii) Lophotrichous: Two or more flagella are present at one end, e.g., Spirillum serpens. (iv) Amphitrichous: Flagella are present at both the ends, e.g., Nitrosomonas. (v) Peritrichous: Flagella are found in whole body, e.g., E. coli, Clostridium tetani.

Fig. 2.5. Structure of different types of flagella.

Fig. 2.5. Structure of different types of flagella.

Function of Flagella 1. Flagella are fully responsible for the bacterial motility. 2. Deflagellation by mechanical means renders the motile cells immotile. 3. The apparent movement of the bacterial cell usually takes place by the rotation of the flagella either in the clockwise or anticlockwise direction along its long axis. 4. Bacterial cell possesses the inherent capacity to alter both the direction of rotation and the speed; besides, the meticulous adjustment of frequency of ‘stops’ and ‘starts’ by the appropriate movement of the flagella. 5. Evidently, the flagellated peritrichal bacteria usually swim in a straight line over moderate distances. In actual practice, these swim-across straight line runs are interrupted frequently by abrupt alterations in the direction that ultimately leads to tumbling. Therefore, the movement of the bacteria is believed to be zig-zag.

Function of Flagella 1. Flagella are fully responsible for the bacterial motility. 2. Deflagellation by mechanical means renders the motile cells immotile. 3. The apparent movement of the bacterial cell usually takes place by the rotation of the flagella either in the clockwise or anticlockwise direction along its long axis. 4. Bacterial cell possesses the inherent capacity to alter both the direction of rotation and the speed; besides, the meticulous adjustment of frequency of ‘stops’ and ‘starts’ by the appropriate movement of the flagella. 5. Evidently, the flagellated peritrichal bacteria usually swim in a straight line over moderate distances. In actual practice, these swim-across straight line runs are interrupted frequently by abrupt alterations in the direction that ultimately leads to tumbling. Therefore, the movement of the bacteria is believed to be zig-zag.

14 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

14 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6. It has been observed that the phenomenon of smooth swimming in a fixed direction is invariably mediated by the rotation of flagella in an anticlockwise direction; whereas, the process of tumbling in a zig-zag direction is usually caused by the rotation of flagella in a clockwise direction. 7. The presence of ‘polar flagella’ in bacteria affords a distinct change in the direction that usually takes place by the reciprocal alteration in the direction of rotation.

6. It has been observed that the phenomenon of smooth swimming in a fixed direction is invariably mediated by the rotation of flagella in an anticlockwise direction; whereas, the process of tumbling in a zig-zag direction is usually caused by the rotation of flagella in a clockwise direction. 7. The presence of ‘polar flagella’ in bacteria affords a distinct change in the direction that usually takes place by the reciprocal alteration in the direction of rotation.

Pili Pili are hallow, non-helical, shorter and thinner than flagellum and are used for attachment and than for motility. On the basis of function at least 8 types of pili are present. A special type of pili is the F pili or sex pili helps in transfer of genetic material during bacterial mating.

Pili Pili are hallow, non-helical, shorter and thinner than flagellum and are used for attachment and than for motility. On the basis of function at least 8 types of pili are present. A special type of pili is the F pili or sex pili helps in transfer of genetic material during bacterial mating.

2.3 SHAPE OF BACTERIAL CELL

2.3 SHAPE OF BACTERIAL CELL

Morphologically, three distinct forms of bacteria are recognized. 1. Spherical 2. Rod shaped or cylindrical 3. Spiral

Morphologically, three distinct forms of bacteria are recognized. 1. Spherical 2. Rod shaped or cylindrical 3. Spiral

2.3.1 Spherical (Cocci) These are spherical in shape and commonly known as cocci. Majority of bacteria are cocci in their shape. They are of different types.

2.3.1 Spherical (Cocci) These are spherical in shape and commonly known as cocci. Majority of bacteria are cocci in their shape. They are of different types.

Fig. 2.6. Structure of different forms of cocci.

Fig. 2.6. Structure of different forms of cocci.

14 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

14 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6. It has been observed that the phenomenon of smooth swimming in a fixed direction is invariably mediated by the rotation of flagella in an anticlockwise direction; whereas, the process of tumbling in a zig-zag direction is usually caused by the rotation of flagella in a clockwise direction. 7. The presence of ‘polar flagella’ in bacteria affords a distinct change in the direction that usually takes place by the reciprocal alteration in the direction of rotation.

6. It has been observed that the phenomenon of smooth swimming in a fixed direction is invariably mediated by the rotation of flagella in an anticlockwise direction; whereas, the process of tumbling in a zig-zag direction is usually caused by the rotation of flagella in a clockwise direction. 7. The presence of ‘polar flagella’ in bacteria affords a distinct change in the direction that usually takes place by the reciprocal alteration in the direction of rotation.

Pili Pili are hallow, non-helical, shorter and thinner than flagellum and are used for attachment and than for motility. On the basis of function at least 8 types of pili are present. A special type of pili is the F pili or sex pili helps in transfer of genetic material during bacterial mating.

Pili Pili are hallow, non-helical, shorter and thinner than flagellum and are used for attachment and than for motility. On the basis of function at least 8 types of pili are present. A special type of pili is the F pili or sex pili helps in transfer of genetic material during bacterial mating.

2.3 SHAPE OF BACTERIAL CELL

2.3 SHAPE OF BACTERIAL CELL

Morphologically, three distinct forms of bacteria are recognized. 1. Spherical 2. Rod shaped or cylindrical 3. Spiral

Morphologically, three distinct forms of bacteria are recognized. 1. Spherical 2. Rod shaped or cylindrical 3. Spiral

2.3.1 Spherical (Cocci) These are spherical in shape and commonly known as cocci. Majority of bacteria are cocci in their shape. They are of different types.

2.3.1 Spherical (Cocci) These are spherical in shape and commonly known as cocci. Majority of bacteria are cocci in their shape. They are of different types.

Fig. 2.6. Structure of different forms of cocci.

Fig. 2.6. Structure of different forms of cocci.

STRUCTURE OF BACTERIAL CELL

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STRUCTURE OF BACTERIAL CELL

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a. Monococci or coccus: When they occur singly, e.g., Micrococcus lutteus. b. Diplococci: When bacteria are present in pairs, e.g., Diplococcus pneumonia. c. Streptococci: When bacteria are occurring in chain, e.g., Streptococcus lactis. d. Staphylococci: When bacteria are occurring in groups, e.g., Streptococcus aureus. e. Sarcina: f. Tetrad: When they occurr in a group of four.

a. Monococci or coccus: When they occur singly, e.g., Micrococcus lutteus. b. Diplococci: When bacteria are present in pairs, e.g., Diplococcus pneumonia. c. Streptococci: When bacteria are occurring in chain, e.g., Streptococcus lactis. d. Staphylococci: When bacteria are occurring in groups, e.g., Streptococcus aureus. e. Sarcina: f. Tetrad: When they occurr in a group of four.

2.3.2 Rod Shaped or Cylindrical They are also known as bacillus. Bacillus derived from Greek word Bacillum meaning stick. These are also of different types: a. Coccobacillus: This is the intermediate form between the cocci and bacilli, e.g., Haemophilus influenza, Chlamydia trachomatis and Aggregatibacter actinomycetemcomitans.

2.3.2 Rod Shaped or Cylindrical They are also known as bacillus. Bacillus derived from Greek word Bacillum meaning stick. These are also of different types: a. Coccobacillus: This is the intermediate form between the cocci and bacilli, e.g., Haemophilus influenza, Chlamydia trachomatis and Aggregatibacter actinomycetemcomitans.

Fig. 2.7. Structure of different forms of bacilli.

Fig. 2.7. Structure of different forms of bacilli.

b. Bacillus: When only one rod-shaped bacterium is present it is called bacillum, e.g., Rhodospirillum sodomense. c. Diplobacillus: When they occur in pairs, e.g., Bacillus subtilis. d. Palisades: This is another form of bacteria which is seen in the above diagram. e. Streptobacillus: e.g., Bacillus anthracis.

STRUCTURE OF BACTERIAL CELL

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b. Bacillus: When only one rod-shaped bacterium is present it is called bacillum, e.g., Rhodospirillum sodomense. c. Diplobacillus: When they occur in pairs, e.g., Bacillus subtilis. d. Palisades: This is another form of bacteria which is seen in the above diagram. e. Streptobacillus: e.g., Bacillus anthracis.

STRUCTURE OF BACTERIAL CELL

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a. Monococci or coccus: When they occur singly, e.g., Micrococcus lutteus. b. Diplococci: When bacteria are present in pairs, e.g., Diplococcus pneumonia. c. Streptococci: When bacteria are occurring in chain, e.g., Streptococcus lactis. d. Staphylococci: When bacteria are occurring in groups, e.g., Streptococcus aureus. e. Sarcina: f. Tetrad: When they occurr in a group of four.

a. Monococci or coccus: When they occur singly, e.g., Micrococcus lutteus. b. Diplococci: When bacteria are present in pairs, e.g., Diplococcus pneumonia. c. Streptococci: When bacteria are occurring in chain, e.g., Streptococcus lactis. d. Staphylococci: When bacteria are occurring in groups, e.g., Streptococcus aureus. e. Sarcina: f. Tetrad: When they occurr in a group of four.

2.3.2 Rod Shaped or Cylindrical They are also known as bacillus. Bacillus derived from Greek word Bacillum meaning stick. These are also of different types: a. Coccobacillus: This is the intermediate form between the cocci and bacilli, e.g., Haemophilus influenza, Chlamydia trachomatis and Aggregatibacter actinomycetemcomitans.

2.3.2 Rod Shaped or Cylindrical They are also known as bacillus. Bacillus derived from Greek word Bacillum meaning stick. These are also of different types: a. Coccobacillus: This is the intermediate form between the cocci and bacilli, e.g., Haemophilus influenza, Chlamydia trachomatis and Aggregatibacter actinomycetemcomitans.

Fig. 2.7. Structure of different forms of bacilli.

Fig. 2.7. Structure of different forms of bacilli.

b. Bacillus: When only one rod-shaped bacterium is present it is called bacillum, e.g., Rhodospirillum sodomense. c. Diplobacillus: When they occur in pairs, e.g., Bacillus subtilis. d. Palisades: This is another form of bacteria which is seen in the above diagram. e. Streptobacillus: e.g., Bacillus anthracis.

b. Bacillus: When only one rod-shaped bacterium is present it is called bacillum, e.g., Rhodospirillum sodomense. c. Diplobacillus: When they occur in pairs, e.g., Bacillus subtilis. d. Palisades: This is another form of bacteria which is seen in the above diagram. e. Streptobacillus: e.g., Bacillus anthracis.

16 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

16 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

2.3.3 Spiral Such type of bacteria have one or more twists, they are not straight.

2.3.3 Spiral Such type of bacteria have one or more twists, they are not straight.

Fig. 2.8. (A) Vibrio (B) Spirilla (C) Spirochete.

a. Vibrio: These are small and comma-shaped bacteria, e.g., Vibrio cholerae which cause cholera. b. Spirilla: These are coiled and twisted like a screw, e.g., Spirellum rubrum. c. Spirochetes: These are highly coild, e.g., Spirochete stenostrepta.

Structure of Eukaryotic cell

Fig. 2.9. Structure of animal cell.

Fig. 2.8. (A) Vibrio (B) Spirilla (C) Spirochete.

a. Vibrio: These are small and comma-shaped bacteria, e.g., Vibrio cholerae which cause cholera. b. Spirilla: These are coiled and twisted like a screw, e.g., Spirellum rubrum. c. Spirochetes: These are highly coild, e.g., Spirochete stenostrepta.

Structure of Eukaryotic cell

Fig. 2.9. Structure of animal cell.

16 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

16 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

2.3.3 Spiral Such type of bacteria have one or more twists, they are not straight.

2.3.3 Spiral Such type of bacteria have one or more twists, they are not straight.

Fig. 2.8. (A) Vibrio (B) Spirilla (C) Spirochete.

a. Vibrio: These are small and comma-shaped bacteria, e.g., Vibrio cholerae which cause cholera. b. Spirilla: These are coiled and twisted like a screw, e.g., Spirellum rubrum. c. Spirochetes: These are highly coild, e.g., Spirochete stenostrepta.

Structure of Eukaryotic cell

Fig. 2.9. Structure of animal cell.

Fig. 2.8. (A) Vibrio (B) Spirilla (C) Spirochete.

a. Vibrio: These are small and comma-shaped bacteria, e.g., Vibrio cholerae which cause cholera. b. Spirilla: These are coiled and twisted like a screw, e.g., Spirellum rubrum. c. Spirochetes: These are highly coild, e.g., Spirochete stenostrepta.

Structure of Eukaryotic cell

Fig. 2.9. Structure of animal cell.

STRUCTURE OF BACTERIAL CELL

17

STRUCTURE OF BACTERIAL CELL

2.4 DIFFERENCE BETWEEN EUKARYOTIC AND PROKARYOTIC CELL

2.4 DIFFERENCE BETWEEN EUKARYOTIC AND PROKARYOTIC CELL

S.No. Prokaryotic Cell

Eukaryotic cell

S.No. Prokaryotic Cell

The size range is greater than 5zm. Absent. Cell wall is absent in animal cell but present in plant cell and other cells. Sterols present but do not carry out respiration and photosynthesis.

01. 02. 03.

The size range is 0.5-1.5 zm Capsule is present. Cell wall is present.

04.

Peptidoglycan is absent. Mesosome is absent.

05. 06.

Nucleus is well developed having proper nuclear membrane and nucleolus DNA is linear and present in the nucleus.

07.

DNA is associated with the histone proteins. Ribosomes is 80S (60S+40S) All the cell organelles like mitochondria, endoplasmic reticulum, golgy complex, lysosomes, centrosomes, chloroplast, microtubules etc. are present Cell division takes place by Mitosis (fission or budding). Meiosis Flagella are microscopic size, e.g., fungi, protozoa, plants, animals.

09. 10. 11.

Cytoplasmic membrane does not contain sterols but part of respiratory and some photosynthetic machinery is present. Cell wall is made up of peptidoglycan. Mesosome is present which contains respiratory enzymes. Nuclear membrane and nucleolus is absent, i.e., nucleus is absent. DNA is circular and present in the cytoplasm. Histone protein is absent Ribosome is 70S (50S+30S) Cell organelles are absent.

01. 02. 03.

The size range is 0.5-1.5 zm Capsule is present. Cell wall is present.

04.

Cytoplasmic membrane does not contain sterols but part of respiratory and some photosynthetic machinery is present. Cell wall is made up of peptidoglycan. Mesosome is present which contains respiratory enzymes. Nuclear membrane and nucleolus is absent, i.e., nucleus is absent. DNA is circular and present in the cytoplasm. Histone protein is absent Ribosome is 70S (50S+30S) Cell organelles are absent.

05. 06. 07. 08. 09. 10. 11.

12. 13.

Cell division takes place by binary fission (simple division). Flagella are submicroscopic in size, e.g., virus, bacteria, cynobacteria

STRUCTURE OF BACTERIAL CELL

08.

12. 13.

Cell division takes place by binary fission (simple division). Flagella are submicroscopic in size, e.g., virus, bacteria, cynobacteria

17

Eukaryotic cell The size range is greater than 5zm. Absent. Cell wall is absent in animal cell but present in plant cell and other cells. Sterols present but do not carry out respiration and photosynthesis.

Peptidoglycan is absent. Mesosome is absent. Nucleus is well developed having proper nuclear membrane and nucleolus DNA is linear and present in the nucleus. DNA is associated with the histone proteins. Ribosomes is 80S (60S+40S) All the cell organelles like mitochondria, endoplasmic reticulum, golgy complex, lysosomes, centrosomes, chloroplast, microtubules etc. are present Cell division takes place by Mitosis (fission or budding). Meiosis Flagella are microscopic size, e.g., fungi, protozoa, plants, animals.

STRUCTURE OF BACTERIAL CELL

2.4 DIFFERENCE BETWEEN EUKARYOTIC AND PROKARYOTIC CELL

2.4 DIFFERENCE BETWEEN EUKARYOTIC AND PROKARYOTIC CELL

S.No. Prokaryotic Cell

Eukaryotic cell

S.No. Prokaryotic Cell

The size range is greater than 5zm. Absent. Cell wall is absent in animal cell but present in plant cell and other cells. Sterols present but do not carry out respiration and photosynthesis.

01. 02. 03.

The size range is 0.5-1.5 zm Capsule is present. Cell wall is present.

04.

Peptidoglycan is absent. Mesosome is absent.

05. 06.

Nucleus is well developed having proper nuclear membrane and nucleolus DNA is linear and present in the nucleus.

07.

DNA is associated with the histone proteins. Ribosomes is 80S (60S+40S) All the cell organelles like mitochondria, endoplasmic reticulum, golgy complex, lysosomes, centrosomes, chloroplast, microtubules etc. are present Cell division takes place by Mitosis (fission or budding). Meiosis Flagella are microscopic size, e.g., fungi, protozoa, plants, animals.

09. 10. 11.

Cytoplasmic membrane does not contain sterols but part of respiratory and some photosynthetic machinery is present. Cell wall is made up of peptidoglycan. Mesosome is present which contains respiratory enzymes. Nuclear membrane and nucleolus is absent, i.e., nucleus is absent. DNA is circular and present in the cytoplasm. Histone protein is absent Ribosome is 70S (50S+30S) Cell organelles are absent.

01. 02. 03.

The size range is 0.5-1.5 zm Capsule is present. Cell wall is present.

04.

Cytoplasmic membrane does not contain sterols but part of respiratory and some photosynthetic machinery is present. Cell wall is made up of peptidoglycan. Mesosome is present which contains respiratory enzymes. Nuclear membrane and nucleolus is absent, i.e., nucleus is absent. DNA is circular and present in the cytoplasm. Histone protein is absent Ribosome is 70S (50S+30S) Cell organelles are absent.

05. 06. 07. 08. 09. 10. 11.

12. 13.

Cell division takes place by binary fission (simple division). Flagella are submicroscopic in size, e.g., virus, bacteria, cynobacteria

08.

12. 13.

Cell division takes place by binary fission (simple division). Flagella are submicroscopic in size, e.g., virus, bacteria, cynobacteria

17

17

Eukaryotic cell The size range is greater than 5zm. Absent. Cell wall is absent in animal cell but present in plant cell and other cells. Sterols present but do not carry out respiration and photosynthesis.

Peptidoglycan is absent. Mesosome is absent. Nucleus is well developed having proper nuclear membrane and nucleolus DNA is linear and present in the nucleus. DNA is associated with the histone proteins. Ribosomes is 80S (60S+40S) All the cell organelles like mitochondria, endoplasmic reticulum, golgy complex, lysosomes, centrosomes, chloroplast, microtubules etc. are present Cell division takes place by Mitosis (fission or budding). Meiosis Flagella are microscopic size, e.g., fungi, protozoa, plants, animals.

3

3

Classification of Microbes and Their Taxonomy

Classification of Microbes and Their Taxonomy

3.1 TAXONOMY

3.1 TAXONOMY

Taxonomy is the practice and science of classification. The word finds its roots in the Greek taxis (meaning ‘order’, arrangement’) and nomos (‘law’ or ‘science’). Taxonomy uses taxonomic units, known as taxa (singular taxon). Taxonomic scheme, is a particular classification, arranged in a hierarchical structure.

Taxonomy is the practice and science of classification. The word finds its roots in the Greek taxis (meaning ‘order’, arrangement’) and nomos (‘law’ or ‘science’). Taxonomy uses taxonomic units, known as taxa (singular taxon). Taxonomic scheme, is a particular classification, arranged in a hierarchical structure.

3.2 CLASSIFICATION

3.2 CLASSIFICATION

“The arrangement of living things into categories is called classification”. Living organisms have been classified as follows. 1. Two-Kingdom Classification: This system of classification was given by Carolus Linnaeus (1707-1778). He classified living organisms into two groups; plantae and animalia. 2. Three-Kingdom Classification or Haeckel’s Kingdom Protista (1866): Haeckel suggested that a third kingdom protista and included those organisms which are neither plants nor animals. The organisms come under the protista includes: bacteria, algae, fungi and protozoa. 3. Copland’s Four-Kingdom Classification System (1956): a. Kingdom monera: He includes one another kingdom known as kingdom monera or mycota now called cynobacteria, e.g., bacteria and bleu green algae. b. Kingdom protista: True nucleate organisms. But they do not have the characters of typical plants and typical animals, e.g., red and brown algae, fungi and protozoa. c. Kingdom plantae: Organisms with cell wall and chloroplasts. d. Kingdom Animalia: Multicelluar organisms.

“The arrangement of living things into categories is called classification”. Living organisms have been classified as follows. 1. Two-Kingdom Classification: This system of classification was given by Carolus Linnaeus (1707-1778). He classified living organisms into two groups; plantae and animalia. 2. Three-Kingdom Classification or Haeckel’s Kingdom Protista (1866): Haeckel suggested that a third kingdom protista and included those organisms which are neither plants nor animals. The organisms come under the protista includes: bacteria, algae, fungi and protozoa. 3. Copland’s Four-Kingdom Classification System (1956): a. Kingdom monera: He includes one another kingdom known as kingdom monera or mycota now called cynobacteria, e.g., bacteria and bleu green algae. b. Kingdom protista: True nucleate organisms. But they do not have the characters of typical plants and typical animals, e.g., red and brown algae, fungi and protozoa. c. Kingdom plantae: Organisms with cell wall and chloroplasts. d. Kingdom Animalia: Multicelluar organisms.

3

3

Classification of Microbes and Their Taxonomy

Classification of Microbes and Their Taxonomy

3.1 TAXONOMY

3.1 TAXONOMY

Taxonomy is the practice and science of classification. The word finds its roots in the Greek taxis (meaning ‘order’, arrangement’) and nomos (‘law’ or ‘science’). Taxonomy uses taxonomic units, known as taxa (singular taxon). Taxonomic scheme, is a particular classification, arranged in a hierarchical structure.

Taxonomy is the practice and science of classification. The word finds its roots in the Greek taxis (meaning ‘order’, arrangement’) and nomos (‘law’ or ‘science’). Taxonomy uses taxonomic units, known as taxa (singular taxon). Taxonomic scheme, is a particular classification, arranged in a hierarchical structure.

3.2 CLASSIFICATION

3.2 CLASSIFICATION

“The arrangement of living things into categories is called classification”. Living organisms have been classified as follows. 1. Two-Kingdom Classification: This system of classification was given by Carolus Linnaeus (1707-1778). He classified living organisms into two groups; plantae and animalia. 2. Three-Kingdom Classification or Haeckel’s Kingdom Protista (1866): Haeckel suggested that a third kingdom protista and included those organisms which are neither plants nor animals. The organisms come under the protista includes: bacteria, algae, fungi and protozoa. 3. Copland’s Four-Kingdom Classification System (1956): a. Kingdom monera: He includes one another kingdom known as kingdom monera or mycota now called cynobacteria, e.g., bacteria and bleu green algae. b. Kingdom protista: True nucleate organisms. But they do not have the characters of typical plants and typical animals, e.g., red and brown algae, fungi and protozoa. c. Kingdom plantae: Organisms with cell wall and chloroplasts. d. Kingdom Animalia: Multicelluar organisms.

“The arrangement of living things into categories is called classification”. Living organisms have been classified as follows. 1. Two-Kingdom Classification: This system of classification was given by Carolus Linnaeus (1707-1778). He classified living organisms into two groups; plantae and animalia. 2. Three-Kingdom Classification or Haeckel’s Kingdom Protista (1866): Haeckel suggested that a third kingdom protista and included those organisms which are neither plants nor animals. The organisms come under the protista includes: bacteria, algae, fungi and protozoa. 3. Copland’s Four-Kingdom Classification System (1956): a. Kingdom monera: He includes one another kingdom known as kingdom monera or mycota now called cynobacteria, e.g., bacteria and bleu green algae. b. Kingdom protista: True nucleate organisms. But they do not have the characters of typical plants and typical animals, e.g., red and brown algae, fungi and protozoa. c. Kingdom plantae: Organisms with cell wall and chloroplasts. d. Kingdom Animalia: Multicelluar organisms.

20 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 4. Five-kingdom classification: Given by Whittakar (1969) this system of classification was based on 3 levels of cellular organization which involved to accommodate 3 principle modes of nutrition: photosynthesis, absorption and ingestion. He divided the living organisms into 5 kingdoms: monera, protista, fungi, plantae and animalia. Kingdom Monera: (Kingdom of Prokaryotes). a. Eubacteria (bacteria, cynobacteria, blue green algae) b. Archiabacteria. (ancient bacteraia.)

3.3 CLASSIFICATION OF ACTINOMYCETES

20 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 4. Five-kingdom classification: Given by Whittakar (1969) this system of classification was based on 3 levels of cellular organization which involved to accommodate 3 principle modes of nutrition: photosynthesis, absorption and ingestion. He divided the living organisms into 5 kingdoms: monera, protista, fungi, plantae and animalia. Kingdom Monera: (Kingdom of Prokaryotes). a. Eubacteria (bacteria, cynobacteria, blue green algae) b. Archiabacteria. (ancient bacteraia.)

3.3 CLASSIFICATION OF ACTINOMYCETES

1. These are mold-like bacteria which were once thought to be fungi. Now they have been shown as prokaryotes and resemble with bacteria in cell structure and chemical composition. 2. These are gram-positive rod-shaped to filamentous aerobic and generally non-motile in the vegetative phase. Due to their rod-shaped arrangement, they are called filamentous bacteria. 3. Most of genera have high percentage of G+C. 4. Actinomycetes are anaerobic to facultative aerobic, filamentous microcolony, whereas streptomyces has intact and abundant aerial mycelium with long chain spores. 5. Frankia is a nitrogen fixing actinomycetes which produce true mycelium and live symbiotically with non-leguminous plants. 6. Streptomyces is mycelia forming actinobacteria that lives in soil, produces aerial as well as substrate mycelium. The aerial hyphae differentitae to form asexual conidiospores in chains. They are blue, grey, green red, violet or yellow colour. They impart “earthy” odour to soil after rain which is due to the presence of geosmines (volatile organic compound), some important antibiotics namely, streptomycin chloramphenicol and tetracycline are produced from this genus. 7. Helicobacteria is a recently discovered actinobacteria. This is the only phototrophic gram-negative bacteria, required organic carbon sources. They have a unique type of bacteriochlorophyll called bacteriochlorophyll g, e.g., Helicobacillus and Helicobacter. Actinobacteria are a group of grampositive bacteria with high G+C ratio. They can be terrestrial or aquatic.

1. These are mold-like bacteria which were once thought to be fungi. Now they have been shown as prokaryotes and resemble with bacteria in cell structure and chemical composition. 2. These are gram-positive rod-shaped to filamentous aerobic and generally non-motile in the vegetative phase. Due to their rod-shaped arrangement, they are called filamentous bacteria. 3. Most of genera have high percentage of G+C. 4. Actinomycetes are anaerobic to facultative aerobic, filamentous microcolony, whereas streptomyces has intact and abundant aerial mycelium with long chain spores. 5. Frankia is a nitrogen fixing actinomycetes which produce true mycelium and live symbiotically with non-leguminous plants. 6. Streptomyces is mycelia forming actinobacteria that lives in soil, produces aerial as well as substrate mycelium. The aerial hyphae differentitae to form asexual conidiospores in chains. They are blue, grey, green red, violet or yellow colour. They impart “earthy” odour to soil after rain which is due to the presence of geosmines (volatile organic compound), some important antibiotics namely, streptomycin chloramphenicol and tetracycline are produced from this genus. 7. Helicobacteria is a recently discovered actinobacteria. This is the only phototrophic gram-negative bacteria, required organic carbon sources. They have a unique type of bacteriochlorophyll called bacteriochlorophyll g, e.g., Helicobacillus and Helicobacter. Actinobacteria are a group of grampositive bacteria with high G+C ratio. They can be terrestrial or aquatic.

Classification These are divided into 7 familes. The classification is based of hyphal and reproductive structures. Family 1: Streptomycetaceae: Hyphae non-fragmented, aerial mycelium with chains of spores with 5 to 50 or more conidia per chain, e.g., Streptomyces, Microdlobaspone and sporictilhya.

Classification These are divided into 7 familes. The classification is based of hyphal and reproductive structures. Family 1: Streptomycetaceae: Hyphae non-fragmented, aerial mycelium with chains of spores with 5 to 50 or more conidia per chain, e.g., Streptomyces, Microdlobaspone and sporictilhya.

20 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

20 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4. Five-kingdom classification: Given by Whittakar (1969) this system of classification was based on 3 levels of cellular organization which involved to accommodate 3 principle modes of nutrition: photosynthesis, absorption and ingestion. He divided the living organisms into 5 kingdoms: monera, protista, fungi, plantae and animalia. Kingdom Monera: (Kingdom of Prokaryotes). a. Eubacteria (bacteria, cynobacteria, blue green algae) b. Archiabacteria. (ancient bacteraia.)

3.3 CLASSIFICATION OF ACTINOMYCETES

4. Five-kingdom classification: Given by Whittakar (1969) this system of classification was based on 3 levels of cellular organization which involved to accommodate 3 principle modes of nutrition: photosynthesis, absorption and ingestion. He divided the living organisms into 5 kingdoms: monera, protista, fungi, plantae and animalia. Kingdom Monera: (Kingdom of Prokaryotes). a. Eubacteria (bacteria, cynobacteria, blue green algae) b. Archiabacteria. (ancient bacteraia.)

3.3 CLASSIFICATION OF ACTINOMYCETES

1. These are mold-like bacteria which were once thought to be fungi. Now they have been shown as prokaryotes and resemble with bacteria in cell structure and chemical composition. 2. These are gram-positive rod-shaped to filamentous aerobic and generally non-motile in the vegetative phase. Due to their rod-shaped arrangement, they are called filamentous bacteria. 3. Most of genera have high percentage of G+C. 4. Actinomycetes are anaerobic to facultative aerobic, filamentous microcolony, whereas streptomyces has intact and abundant aerial mycelium with long chain spores. 5. Frankia is a nitrogen fixing actinomycetes which produce true mycelium and live symbiotically with non-leguminous plants. 6. Streptomyces is mycelia forming actinobacteria that lives in soil, produces aerial as well as substrate mycelium. The aerial hyphae differentitae to form asexual conidiospores in chains. They are blue, grey, green red, violet or yellow colour. They impart “earthy” odour to soil after rain which is due to the presence of geosmines (volatile organic compound), some important antibiotics namely, streptomycin chloramphenicol and tetracycline are produced from this genus. 7. Helicobacteria is a recently discovered actinobacteria. This is the only phototrophic gram-negative bacteria, required organic carbon sources. They have a unique type of bacteriochlorophyll called bacteriochlorophyll g, e.g., Helicobacillus and Helicobacter. Actinobacteria are a group of grampositive bacteria with high G+C ratio. They can be terrestrial or aquatic.

1. These are mold-like bacteria which were once thought to be fungi. Now they have been shown as prokaryotes and resemble with bacteria in cell structure and chemical composition. 2. These are gram-positive rod-shaped to filamentous aerobic and generally non-motile in the vegetative phase. Due to their rod-shaped arrangement, they are called filamentous bacteria. 3. Most of genera have high percentage of G+C. 4. Actinomycetes are anaerobic to facultative aerobic, filamentous microcolony, whereas streptomyces has intact and abundant aerial mycelium with long chain spores. 5. Frankia is a nitrogen fixing actinomycetes which produce true mycelium and live symbiotically with non-leguminous plants. 6. Streptomyces is mycelia forming actinobacteria that lives in soil, produces aerial as well as substrate mycelium. The aerial hyphae differentitae to form asexual conidiospores in chains. They are blue, grey, green red, violet or yellow colour. They impart “earthy” odour to soil after rain which is due to the presence of geosmines (volatile organic compound), some important antibiotics namely, streptomycin chloramphenicol and tetracycline are produced from this genus. 7. Helicobacteria is a recently discovered actinobacteria. This is the only phototrophic gram-negative bacteria, required organic carbon sources. They have a unique type of bacteriochlorophyll called bacteriochlorophyll g, e.g., Helicobacillus and Helicobacter. Actinobacteria are a group of grampositive bacteria with high G+C ratio. They can be terrestrial or aquatic.

Classification These are divided into 7 familes. The classification is based of hyphal and reproductive structures. Family 1: Streptomycetaceae: Hyphae non-fragmented, aerial mycelium with chains of spores with 5 to 50 or more conidia per chain, e.g., Streptomyces, Microdlobaspone and sporictilhya.

Classification These are divided into 7 familes. The classification is based of hyphal and reproductive structures. Family 1: Streptomycetaceae: Hyphae non-fragmented, aerial mycelium with chains of spores with 5 to 50 or more conidia per chain, e.g., Streptomyces, Microdlobaspone and sporictilhya.

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family 2: Family 3:

Family 4:

Family 5: Family 6: Family 7:

21

Nocardiaceae: Hyphae typically fragmented e.g., Nocardia, Pseudonocardia. Microomononsporaceae: Hyphae non-fragmented conidia borne singly or in pairs or in short chains, e.g., Micromonospora, Thermonospora, Thermoactinomycetes, Actinobifida. Actinplanaceae: Sporangie bear the spores. The hyphal diameter varies from 0.2 to 2.0 ìm, e.g. Streptosporangium. Actinoplanes. Plasmobispora and dactylosporangium. Dermatophilaceae: Hyphal fragments divide to form large numbers of round, motile structures, e.g., Geodermatophilus. Frankiaceae: It is strictly associated with the root of nonleguminous plant and form root nodules, e.g., Frankia. Actinomycetaceae: No true mycelium is produced, usually strictly to facultative anaerobic, e.g., Actinomyces.

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family 2: Family 3:

Family 4:

Family 5: Family 6: Family 7:

21

Nocardiaceae: Hyphae typically fragmented e.g., Nocardia, Pseudonocardia. Microomononsporaceae: Hyphae non-fragmented conidia borne singly or in pairs or in short chains, e.g., Micromonospora, Thermonospora, Thermoactinomycetes, Actinobifida. Actinplanaceae: Sporangie bear the spores. The hyphal diameter varies from 0.2 to 2.0 ìm, e.g. Streptosporangium. Actinoplanes. Plasmobispora and dactylosporangium. Dermatophilaceae: Hyphal fragments divide to form large numbers of round, motile structures, e.g., Geodermatophilus. Frankiaceae: It is strictly associated with the root of nonleguminous plant and form root nodules, e.g., Frankia. Actinomycetaceae: No true mycelium is produced, usually strictly to facultative anaerobic, e.g., Actinomyces.

3.4 CLASSIFICATION OF BACTERIA

3.4 CLASSIFICATION OF BACTERIA

Bacteria are classified mainly into two categories. 1. Aerobic bacteria or aerobe: Aerobic bacteria are an organism that can survive and grow in an oxygenated environment. 2. Anaerobic bacteria: An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. Bacteria can be of different types according to their relationship with oxygen. They are: a. Obligate aerobes: Obligate aerobes require oxygen for aerobic cellular respiration. In a process known as cellular respiration. These organisms use oxygen to oxidize substrates in order to obtain energy. b. Obligate anaerobes: Which cannot use oxygen for growth and are even harmed by it. c. Facultative anaerobes: Which can grow without oxygen but can utilize oxygen if present. d. Microaerophiles: These are the organisms that may use oxygen but only at low concentrations. e. Aerotolerant: Organisms can survive in the presence of oxygen, but they are anaerobic because they do not use it as a terminal electron acceptor.

Bacteria are classified mainly into two categories. 1. Aerobic bacteria or aerobe: Aerobic bacteria are an organism that can survive and grow in an oxygenated environment. 2. Anaerobic bacteria: An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. Bacteria can be of different types according to their relationship with oxygen. They are: a. Obligate aerobes: Obligate aerobes require oxygen for aerobic cellular respiration. In a process known as cellular respiration. These organisms use oxygen to oxidize substrates in order to obtain energy. b. Obligate anaerobes: Which cannot use oxygen for growth and are even harmed by it. c. Facultative anaerobes: Which can grow without oxygen but can utilize oxygen if present. d. Microaerophiles: These are the organisms that may use oxygen but only at low concentrations. e. Aerotolerant: Organisms can survive in the presence of oxygen, but they are anaerobic because they do not use it as a terminal electron acceptor.

Classification 1. Aerobic gram-positive bacteria: for example, Clostridium tetani, Clostridium botulinum, Corynebacterium tuberculosis, Bacillus anthrasis, Micrococcus, Staphylococcus, Streptococcus. 2. Aerobic gram-negative bacteria (cocci and rods): for example, Pseudomonas aeruginosa, Xanthomonas, Agrobacteriaum, Methylococcus, Acetobactor.

Classification 1. Aerobic gram-positive bacteria: for example, Clostridium tetani, Clostridium botulinum, Corynebacterium tuberculosis, Bacillus anthrasis, Micrococcus, Staphylococcus, Streptococcus. 2. Aerobic gram-negative bacteria (cocci and rods): for example, Pseudomonas aeruginosa, Xanthomonas, Agrobacteriaum, Methylococcus, Acetobactor.

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family 2: Family 3:

Family 4:

Family 5: Family 6: Family 7:

21

Nocardiaceae: Hyphae typically fragmented e.g., Nocardia, Pseudonocardia. Microomononsporaceae: Hyphae non-fragmented conidia borne singly or in pairs or in short chains, e.g., Micromonospora, Thermonospora, Thermoactinomycetes, Actinobifida. Actinplanaceae: Sporangie bear the spores. The hyphal diameter varies from 0.2 to 2.0 ìm, e.g. Streptosporangium. Actinoplanes. Plasmobispora and dactylosporangium. Dermatophilaceae: Hyphal fragments divide to form large numbers of round, motile structures, e.g., Geodermatophilus. Frankiaceae: It is strictly associated with the root of nonleguminous plant and form root nodules, e.g., Frankia. Actinomycetaceae: No true mycelium is produced, usually strictly to facultative anaerobic, e.g., Actinomyces.

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family 2: Family 3:

Family 4:

Family 5: Family 6: Family 7:

21

Nocardiaceae: Hyphae typically fragmented e.g., Nocardia, Pseudonocardia. Microomononsporaceae: Hyphae non-fragmented conidia borne singly or in pairs or in short chains, e.g., Micromonospora, Thermonospora, Thermoactinomycetes, Actinobifida. Actinplanaceae: Sporangie bear the spores. The hyphal diameter varies from 0.2 to 2.0 ìm, e.g. Streptosporangium. Actinoplanes. Plasmobispora and dactylosporangium. Dermatophilaceae: Hyphal fragments divide to form large numbers of round, motile structures, e.g., Geodermatophilus. Frankiaceae: It is strictly associated with the root of nonleguminous plant and form root nodules, e.g., Frankia. Actinomycetaceae: No true mycelium is produced, usually strictly to facultative anaerobic, e.g., Actinomyces.

3.4 CLASSIFICATION OF BACTERIA

3.4 CLASSIFICATION OF BACTERIA

Bacteria are classified mainly into two categories. 1. Aerobic bacteria or aerobe: Aerobic bacteria are an organism that can survive and grow in an oxygenated environment. 2. Anaerobic bacteria: An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. Bacteria can be of different types according to their relationship with oxygen. They are: a. Obligate aerobes: Obligate aerobes require oxygen for aerobic cellular respiration. In a process known as cellular respiration. These organisms use oxygen to oxidize substrates in order to obtain energy. b. Obligate anaerobes: Which cannot use oxygen for growth and are even harmed by it. c. Facultative anaerobes: Which can grow without oxygen but can utilize oxygen if present. d. Microaerophiles: These are the organisms that may use oxygen but only at low concentrations. e. Aerotolerant: Organisms can survive in the presence of oxygen, but they are anaerobic because they do not use it as a terminal electron acceptor.

Bacteria are classified mainly into two categories. 1. Aerobic bacteria or aerobe: Aerobic bacteria are an organism that can survive and grow in an oxygenated environment. 2. Anaerobic bacteria: An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. Bacteria can be of different types according to their relationship with oxygen. They are: a. Obligate aerobes: Obligate aerobes require oxygen for aerobic cellular respiration. In a process known as cellular respiration. These organisms use oxygen to oxidize substrates in order to obtain energy. b. Obligate anaerobes: Which cannot use oxygen for growth and are even harmed by it. c. Facultative anaerobes: Which can grow without oxygen but can utilize oxygen if present. d. Microaerophiles: These are the organisms that may use oxygen but only at low concentrations. e. Aerotolerant: Organisms can survive in the presence of oxygen, but they are anaerobic because they do not use it as a terminal electron acceptor.

Classification 1. Aerobic gram-positive bacteria: for example, Clostridium tetani, Clostridium botulinum, Corynebacterium tuberculosis, Bacillus anthrasis, Micrococcus, Staphylococcus, Streptococcus. 2. Aerobic gram-negative bacteria (cocci and rods): for example, Pseudomonas aeruginosa, Xanthomonas, Agrobacteriaum, Methylococcus, Acetobactor.

Classification 1. Aerobic gram-positive bacteria: for example, Clostridium tetani, Clostridium botulinum, Corynebacterium tuberculosis, Bacillus anthrasis, Micrococcus, Staphylococcus, Streptococcus. 2. Aerobic gram-negative bacteria (cocci and rods): for example, Pseudomonas aeruginosa, Xanthomonas, Agrobacteriaum, Methylococcus, Acetobactor.

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22 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

3. Anaerobic gram-positive bacteria: for example, Peptococcus, Peptostreptococus, Rumicoccus. 4. Anaerobic gram negative bacteria: for example, Acidaminococcus, Cardiobacterium, Pasteurella, Photobacterium, Salmonella, Vibrio cholera. 5. Mycobacteria: Exhibits filamentous growth, for example, M. Tuberculosis. 6. Chemoautotrophic bacteria: for example, Nitrosomonas and Nitrobactor.

3. Anaerobic gram-positive bacteria: for example, Peptococcus, Peptostreptococus, Rumicoccus. 4. Anaerobic gram negative bacteria: for example, Acidaminococcus, Cardiobacterium, Pasteurella, Photobacterium, Salmonella, Vibrio cholera. 5. Mycobacteria: Exhibits filamentous growth, for example, M. Tuberculosis. 6. Chemoautotrophic bacteria: for example, Nitrosomonas and Nitrobactor.

Cyanobacteria: These types of bacteria grows in diverse type of habitats, fresh water, marine, terrestrial, free living and some occur in hot water springs and some grow at higher temperature 770C. e.g.: Trichodesmium erythrgeum (this bacteria cause red sea).

Cyanobacteria: These types of bacteria grows in diverse type of habitats, fresh water, marine, terrestrial, free living and some occur in hot water springs and some grow at higher temperature 770C. e.g.: Trichodesmium erythrgeum (this bacteria cause red sea).

3.5 CLASSIFICATION OF RICKETTSIAE

3.5 CLASSIFICATION OF RICKETTSIAE

This group includes two subgroups: 1. Rickettsias 2. Chlamydias

This group includes two subgroups: 1. Rickettsias 2. Chlamydias

3.5.1 Rickettsias They are obligate intracellular parasites or mutualistic with eukaryotic host (vertebrates or arthopods). Their cell wall contains muramic acid. Mainly they are rod-shaped coccoid and often pleomorphic that stain gram-negative and lack flagella. The intracellular parasitic species are associated with the reticuloendothelial and vascular endothelial cells or erythrocytes of vertebrates and often with various organs of arthropods, which may act as primary hosts. The mutualistic species are found in insects. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups. Genera of rickettsias cause various diseases in human beings. a. Rocky mountain spotted fever: caused by Rickettsia rickettsii and transmitted by ticks. b. Endemic murine typhus caused by Rickettsia typhi and transmitted by rat fleas. c. Epidemic typhus caused by Rickettsia prowazekii.

3.5.1 Rickettsias They are obligate intracellular parasites or mutualistic with eukaryotic host (vertebrates or arthopods). Their cell wall contains muramic acid. Mainly they are rod-shaped coccoid and often pleomorphic that stain gram-negative and lack flagella. The intracellular parasitic species are associated with the reticuloendothelial and vascular endothelial cells or erythrocytes of vertebrates and often with various organs of arthropods, which may act as primary hosts. The mutualistic species are found in insects. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups. Genera of rickettsias cause various diseases in human beings. a. Rocky mountain spotted fever: caused by Rickettsia rickettsii and transmitted by ticks. b. Endemic murine typhus caused by Rickettsia typhi and transmitted by rat fleas. c. Epidemic typhus caused by Rickettsia prowazekii.

Rickettsias are classified as below: Order: Rickettsiaceae Family I : Rickettsiaceae Tribe I: Rickettsieae, e.g., Rickettsia, Rochalimaea, Coxiella Tribe II: Ehrlichieae, e.g., Ehrilichina, Cowdria, Neorickttesia Tribe III: Wolbachieae, e.g., Wolbacia, Rickettsiella

Rickettsias are classified as below: Order: Rickettsiaceae Family I : Rickettsiaceae Tribe I: Rickettsieae, e.g., Rickettsia, Rochalimaea, Coxiella Tribe II: Ehrlichieae, e.g., Ehrilichina, Cowdria, Neorickttesia Tribe III: Wolbachieae, e.g., Wolbacia, Rickettsiella

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22 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

3. Anaerobic gram-positive bacteria: for example, Peptococcus, Peptostreptococus, Rumicoccus. 4. Anaerobic gram negative bacteria: for example, Acidaminococcus, Cardiobacterium, Pasteurella, Photobacterium, Salmonella, Vibrio cholera. 5. Mycobacteria: Exhibits filamentous growth, for example, M. Tuberculosis. 6. Chemoautotrophic bacteria: for example, Nitrosomonas and Nitrobactor.

3. Anaerobic gram-positive bacteria: for example, Peptococcus, Peptostreptococus, Rumicoccus. 4. Anaerobic gram negative bacteria: for example, Acidaminococcus, Cardiobacterium, Pasteurella, Photobacterium, Salmonella, Vibrio cholera. 5. Mycobacteria: Exhibits filamentous growth, for example, M. Tuberculosis. 6. Chemoautotrophic bacteria: for example, Nitrosomonas and Nitrobactor.

Cyanobacteria: These types of bacteria grows in diverse type of habitats, fresh water, marine, terrestrial, free living and some occur in hot water springs and some grow at higher temperature 770C. e.g.: Trichodesmium erythrgeum (this bacteria cause red sea).

Cyanobacteria: These types of bacteria grows in diverse type of habitats, fresh water, marine, terrestrial, free living and some occur in hot water springs and some grow at higher temperature 770C. e.g.: Trichodesmium erythrgeum (this bacteria cause red sea).

3.5 CLASSIFICATION OF RICKETTSIAE

3.5 CLASSIFICATION OF RICKETTSIAE

This group includes two subgroups: 1. Rickettsias 2. Chlamydias

This group includes two subgroups: 1. Rickettsias 2. Chlamydias

3.5.1 Rickettsias They are obligate intracellular parasites or mutualistic with eukaryotic host (vertebrates or arthopods). Their cell wall contains muramic acid. Mainly they are rod-shaped coccoid and often pleomorphic that stain gram-negative and lack flagella. The intracellular parasitic species are associated with the reticuloendothelial and vascular endothelial cells or erythrocytes of vertebrates and often with various organs of arthropods, which may act as primary hosts. The mutualistic species are found in insects. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups. Genera of rickettsias cause various diseases in human beings. a. Rocky mountain spotted fever: caused by Rickettsia rickettsii and transmitted by ticks. b. Endemic murine typhus caused by Rickettsia typhi and transmitted by rat fleas. c. Epidemic typhus caused by Rickettsia prowazekii.

3.5.1 Rickettsias They are obligate intracellular parasites or mutualistic with eukaryotic host (vertebrates or arthopods). Their cell wall contains muramic acid. Mainly they are rod-shaped coccoid and often pleomorphic that stain gram-negative and lack flagella. The intracellular parasitic species are associated with the reticuloendothelial and vascular endothelial cells or erythrocytes of vertebrates and often with various organs of arthropods, which may act as primary hosts. The mutualistic species are found in insects. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups. Genera of rickettsias cause various diseases in human beings. a. Rocky mountain spotted fever: caused by Rickettsia rickettsii and transmitted by ticks. b. Endemic murine typhus caused by Rickettsia typhi and transmitted by rat fleas. c. Epidemic typhus caused by Rickettsia prowazekii.

Rickettsias are classified as below: Order: Rickettsiaceae Family I : Rickettsiaceae Tribe I: Rickettsieae, e.g., Rickettsia, Rochalimaea, Coxiella Tribe II: Ehrlichieae, e.g., Ehrilichina, Cowdria, Neorickttesia Tribe III: Wolbachieae, e.g., Wolbacia, Rickettsiella

Rickettsias are classified as below: Order: Rickettsiaceae Family I : Rickettsiaceae Tribe I: Rickettsieae, e.g., Rickettsia, Rochalimaea, Coxiella Tribe II: Ehrlichieae, e.g., Ehrilichina, Cowdria, Neorickttesia Tribe III: Wolbachieae, e.g., Wolbacia, Rickettsiella

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family II: Family III:

23

Bartonellaceae, e.g., Bartonella, Grahamella Anaplasmataceae, e.g., Anaplasma, Aegyptianella, Haemobartonella, Eperythrozoon

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family II: Family III:

23

Bartonellaceae, e.g., Bartonella, Grahamella Anaplasmataceae, e.g., Anaplasma, Aegyptianella, Haemobartonella, Eperythrozoon

3.5.2 Chlamydias These are non-motile gram-negative, obligate parasites, coccoid bacteria multiply within membrane bounded vacuole in the cytoplasm of the cell of humans, other mammals and birds. The multiplication occurs by means of unique development cycle, which divides by fission. They are pathogenic. Specialized techniques are involved for their cultivation. They are present in two different forms during their life cycle. A large, metabolically active reticule body which is non-infectious with flexible cell wall and a smaller forms are present which is called elementary body. Chlamydias are classified as below:

3.5.2 Chlamydias These are non-motile gram-negative, obligate parasites, coccoid bacteria multiply within membrane bounded vacuole in the cytoplasm of the cell of humans, other mammals and birds. The multiplication occurs by means of unique development cycle, which divides by fission. They are pathogenic. Specialized techniques are involved for their cultivation. They are present in two different forms during their life cycle. A large, metabolically active reticule body which is non-infectious with flexible cell wall and a smaller forms are present which is called elementary body. Chlamydias are classified as below:

Order: Chlamydiales Family I: Chlamydiaceae Genus: Chlamydia.

Order: Chlamydiales Family I: Chlamydiaceae Genus: Chlamydia.

3.6 CLASSIFICATION OF SPIROCHAETES

3.6 CLASSIFICATION OF SPIROCHAETES

They are elongated, motile, flexible bacteria which are twisted spirally around the long axis. Characteristically there are varying number of fine fibrils between the cell wall and the cytoplasmic membrane of the bacterial cell. The spiral shape and serpentine motility of the cell depends on the integrity of these filaments. Spirochaetes do not possess flagella but are motile. Spirochaetes belong to the order spirochaetales which is divided into two: 1. Spirochaetaceae (saprophytes) 2. Treponemataceae (human pathogen): it consists of 3 genera. i. Borrelia, e.g., Borrelia Vencentii ii. Leptospira, e.g., Leptospira autumnatis, L. australis, L. bataviae, L. canicola, L. hebdomadis, L. pyrogen etc iii. Treponema, e.g., Treponema caratium. T. pallidum, T. pertenue, and T. vincentii.

They are elongated, motile, flexible bacteria which are twisted spirally around the long axis. Characteristically there are varying number of fine fibrils between the cell wall and the cytoplasmic membrane of the bacterial cell. The spiral shape and serpentine motility of the cell depends on the integrity of these filaments. Spirochaetes do not possess flagella but are motile. Spirochaetes belong to the order spirochaetales which is divided into two: 1. Spirochaetaceae (saprophytes) 2. Treponemataceae (human pathogen): it consists of 3 genera. i. Borrelia, e.g., Borrelia Vencentii ii. Leptospira, e.g., Leptospira autumnatis, L. australis, L. bataviae, L. canicola, L. hebdomadis, L. pyrogen etc iii. Treponema, e.g., Treponema caratium. T. pallidum, T. pertenue, and T. vincentii.

3.7 CLASSIFICATION OF VIRUSES

3.7 CLASSIFICATION OF VIRUSES

Viruses may be defined as “a genetic element enclosed in a protein coat”. “a core of nucleic acids either DNA or RNA surrounded by a protein.” Virus particles are composed of a core of genetic material, either DNA or RNA, surrounded by a coat of protein. The function of the coat is to protect the viral genes from inactivation by adverse environmental factors, such as

Viruses may be defined as “a genetic element enclosed in a protein coat”. “a core of nucleic acids either DNA or RNA surrounded by a protein.” Virus particles are composed of a core of genetic material, either DNA or RNA, surrounded by a coat of protein. The function of the coat is to protect the viral genes from inactivation by adverse environmental factors, such as

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family II: Family III:

23

Bartonellaceae, e.g., Bartonella, Grahamella Anaplasmataceae, e.g., Anaplasma, Aegyptianella, Haemobartonella, Eperythrozoon

CLASSIFICATION OF MICROBES AND THEIR TAXONOMY

Family II: Family III:

23

Bartonellaceae, e.g., Bartonella, Grahamella Anaplasmataceae, e.g., Anaplasma, Aegyptianella, Haemobartonella, Eperythrozoon

3.5.2 Chlamydias These are non-motile gram-negative, obligate parasites, coccoid bacteria multiply within membrane bounded vacuole in the cytoplasm of the cell of humans, other mammals and birds. The multiplication occurs by means of unique development cycle, which divides by fission. They are pathogenic. Specialized techniques are involved for their cultivation. They are present in two different forms during their life cycle. A large, metabolically active reticule body which is non-infectious with flexible cell wall and a smaller forms are present which is called elementary body. Chlamydias are classified as below:

3.5.2 Chlamydias These are non-motile gram-negative, obligate parasites, coccoid bacteria multiply within membrane bounded vacuole in the cytoplasm of the cell of humans, other mammals and birds. The multiplication occurs by means of unique development cycle, which divides by fission. They are pathogenic. Specialized techniques are involved for their cultivation. They are present in two different forms during their life cycle. A large, metabolically active reticule body which is non-infectious with flexible cell wall and a smaller forms are present which is called elementary body. Chlamydias are classified as below:

Order: Chlamydiales Family I: Chlamydiaceae Genus: Chlamydia.

Order: Chlamydiales Family I: Chlamydiaceae Genus: Chlamydia.

3.6 CLASSIFICATION OF SPIROCHAETES

3.6 CLASSIFICATION OF SPIROCHAETES

They are elongated, motile, flexible bacteria which are twisted spirally around the long axis. Characteristically there are varying number of fine fibrils between the cell wall and the cytoplasmic membrane of the bacterial cell. The spiral shape and serpentine motility of the cell depends on the integrity of these filaments. Spirochaetes do not possess flagella but are motile. Spirochaetes belong to the order spirochaetales which is divided into two: 1. Spirochaetaceae (saprophytes) 2. Treponemataceae (human pathogen): it consists of 3 genera. i. Borrelia, e.g., Borrelia Vencentii ii. Leptospira, e.g., Leptospira autumnatis, L. australis, L. bataviae, L. canicola, L. hebdomadis, L. pyrogen etc iii. Treponema, e.g., Treponema caratium. T. pallidum, T. pertenue, and T. vincentii.

They are elongated, motile, flexible bacteria which are twisted spirally around the long axis. Characteristically there are varying number of fine fibrils between the cell wall and the cytoplasmic membrane of the bacterial cell. The spiral shape and serpentine motility of the cell depends on the integrity of these filaments. Spirochaetes do not possess flagella but are motile. Spirochaetes belong to the order spirochaetales which is divided into two: 1. Spirochaetaceae (saprophytes) 2. Treponemataceae (human pathogen): it consists of 3 genera. i. Borrelia, e.g., Borrelia Vencentii ii. Leptospira, e.g., Leptospira autumnatis, L. australis, L. bataviae, L. canicola, L. hebdomadis, L. pyrogen etc iii. Treponema, e.g., Treponema caratium. T. pallidum, T. pertenue, and T. vincentii.

3.7 CLASSIFICATION OF VIRUSES

3.7 CLASSIFICATION OF VIRUSES

Viruses may be defined as “a genetic element enclosed in a protein coat”. “a core of nucleic acids either DNA or RNA surrounded by a protein.” Virus particles are composed of a core of genetic material, either DNA or RNA, surrounded by a coat of protein. The function of the coat is to protect the viral genes from inactivation by adverse environmental factors, such as

Viruses may be defined as “a genetic element enclosed in a protein coat”. “a core of nucleic acids either DNA or RNA surrounded by a protein.” Virus particles are composed of a core of genetic material, either DNA or RNA, surrounded by a coat of protein. The function of the coat is to protect the viral genes from inactivation by adverse environmental factors, such as

24 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

24 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

tissue nuclease enzymes which would otherwise digest a naked viral chromosome during its passage from cell to cell within a host. In a number of viruses the coat also plays an important role in the attachment of the virus to receptors on susceptible cells, and in many bacterial viruses the coat is further modified to facilitate the insertion of the viral genome through the tough structural barrier of the bacterial cell wall.

tissue nuclease enzymes which would otherwise digest a naked viral chromosome during its passage from cell to cell within a host. In a number of viruses the coat also plays an important role in the attachment of the virus to receptors on susceptible cells, and in many bacterial viruses the coat is further modified to facilitate the insertion of the viral genome through the tough structural barrier of the bacterial cell wall.

Classification 1. Viruses containing double strand DNA (ds DNA), e.g., Pox virus, Herps virus, Adenovirus, Coliphage-T2, Coliphage-T3, Coliphage T4, T6, T7. 2. Viruses containing single strand DNA (ss.DNA), e.g., Geminivirus, S13 E coliphage. 3. Viruses containing double strand RNA (ds.RNA), e.g., Reovirus, Wound tumour virus. 4. Viruses containing single strand RNA (ss.RNA), e.g., TMV (Tobacco mosaic virus), Poliomyelitis virus, Bacteriophase virus.

Classification 1. Viruses containing double strand DNA (ds DNA), e.g., Pox virus, Herps virus, Adenovirus, Coliphage-T2, Coliphage-T3, Coliphage T4, T6, T7. 2. Viruses containing single strand DNA (ss.DNA), e.g., Geminivirus, S13 E coliphage. 3. Viruses containing double strand RNA (ds.RNA), e.g., Reovirus, Wound tumour virus. 4. Viruses containing single strand RNA (ss.RNA), e.g., TMV (Tobacco mosaic virus), Poliomyelitis virus, Bacteriophase virus.

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24 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

tissue nuclease enzymes which would otherwise digest a naked viral chromosome during its passage from cell to cell within a host. In a number of viruses the coat also plays an important role in the attachment of the virus to receptors on susceptible cells, and in many bacterial viruses the coat is further modified to facilitate the insertion of the viral genome through the tough structural barrier of the bacterial cell wall.

tissue nuclease enzymes which would otherwise digest a naked viral chromosome during its passage from cell to cell within a host. In a number of viruses the coat also plays an important role in the attachment of the virus to receptors on susceptible cells, and in many bacterial viruses the coat is further modified to facilitate the insertion of the viral genome through the tough structural barrier of the bacterial cell wall.

Classification 1. Viruses containing double strand DNA (ds DNA), e.g., Pox virus, Herps virus, Adenovirus, Coliphage-T2, Coliphage-T3, Coliphage T4, T6, T7. 2. Viruses containing single strand DNA (ss.DNA), e.g., Geminivirus, S13 E coliphage. 3. Viruses containing double strand RNA (ds.RNA), e.g., Reovirus, Wound tumour virus. 4. Viruses containing single strand RNA (ss.RNA), e.g., TMV (Tobacco mosaic virus), Poliomyelitis virus, Bacteriophase virus.

Classification 1. Viruses containing double strand DNA (ds DNA), e.g., Pox virus, Herps virus, Adenovirus, Coliphage-T2, Coliphage-T3, Coliphage T4, T6, T7. 2. Viruses containing single strand DNA (ss.DNA), e.g., Geminivirus, S13 E coliphage. 3. Viruses containing double strand RNA (ds.RNA), e.g., Reovirus, Wound tumour virus. 4. Viruses containing single strand RNA (ss.RNA), e.g., TMV (Tobacco mosaic virus), Poliomyelitis virus, Bacteriophase virus.

4

4

Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy

Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy

Some microorganisms cannot be studied properly because they are transparent, practically colourless, therefore, staining procedure are used to increase their visibility and other additional information to identify them. The chemical substances which are commonly used to stain the bacteria are known as dyes. Chemically a dye is defined as an organic compound containing a benzene ring plus a chromophore and auxochrome group. Such dyes are either acidic, basic or neutral. The acidic dye (picric acid, acid fuchsin, and eosin) are anionic (negative charge on them) and stain the cytoplasmic components of the cell which are more alkaline in nature. On the other hand the basic dyes, e.g., crystal violet, methylene blue, safranin are cataionic (positive charge on them) and combine those components of cell which are acidic in nature, e.g., nucleic acid and natural dye are complex of acid and basic dyes. There are two kinds of staining procedures—simple and differential. Single stains employ single dye and in differential staining more than one staining are used.

Some microorganisms cannot be studied properly because they are transparent, practically colourless, therefore, staining procedure are used to increase their visibility and other additional information to identify them. The chemical substances which are commonly used to stain the bacteria are known as dyes. Chemically a dye is defined as an organic compound containing a benzene ring plus a chromophore and auxochrome group. Such dyes are either acidic, basic or neutral. The acidic dye (picric acid, acid fuchsin, and eosin) are anionic (negative charge on them) and stain the cytoplasmic components of the cell which are more alkaline in nature. On the other hand the basic dyes, e.g., crystal violet, methylene blue, safranin are cataionic (positive charge on them) and combine those components of cell which are acidic in nature, e.g., nucleic acid and natural dye are complex of acid and basic dyes. There are two kinds of staining procedures—simple and differential. Single stains employ single dye and in differential staining more than one staining are used.

4

4

Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy

Identification of Microbes: Stains and Type of Staining Techniques, Electron Microscopy

Some microorganisms cannot be studied properly because they are transparent, practically colourless, therefore, staining procedure are used to increase their visibility and other additional information to identify them. The chemical substances which are commonly used to stain the bacteria are known as dyes. Chemically a dye is defined as an organic compound containing a benzene ring plus a chromophore and auxochrome group. Such dyes are either acidic, basic or neutral. The acidic dye (picric acid, acid fuchsin, and eosin) are anionic (negative charge on them) and stain the cytoplasmic components of the cell which are more alkaline in nature. On the other hand the basic dyes, e.g., crystal violet, methylene blue, safranin are cataionic (positive charge on them) and combine those components of cell which are acidic in nature, e.g., nucleic acid and natural dye are complex of acid and basic dyes. There are two kinds of staining procedures—simple and differential. Single stains employ single dye and in differential staining more than one staining are used.

Some microorganisms cannot be studied properly because they are transparent, practically colourless, therefore, staining procedure are used to increase their visibility and other additional information to identify them. The chemical substances which are commonly used to stain the bacteria are known as dyes. Chemically a dye is defined as an organic compound containing a benzene ring plus a chromophore and auxochrome group. Such dyes are either acidic, basic or neutral. The acidic dye (picric acid, acid fuchsin, and eosin) are anionic (negative charge on them) and stain the cytoplasmic components of the cell which are more alkaline in nature. On the other hand the basic dyes, e.g., crystal violet, methylene blue, safranin are cataionic (positive charge on them) and combine those components of cell which are acidic in nature, e.g., nucleic acid and natural dye are complex of acid and basic dyes. There are two kinds of staining procedures—simple and differential. Single stains employ single dye and in differential staining more than one staining are used.

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26 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Stains are used intentionally in a variety of fields, including in research (biological staining), technology (metal staining), and art (wood staining, stained glass). Types of staining techniques: Staining is the method of morphological characterization of bacteria.

Stains are used intentionally in a variety of fields, including in research (biological staining), technology (metal staining), and art (wood staining, stained glass). Types of staining techniques: Staining is the method of morphological characterization of bacteria.

4.1 SIMPLE STAINING

4.1 SIMPLE STAINING

The purpose of simple staining technique is to determine the cell shape (bacilli or cocci), size and arrangement of bacterial cells. Simple staining is performed by using basic dyes, e.g., crystal violet, methylene blue, carbol fuchsin etc.

The purpose of simple staining technique is to determine the cell shape (bacilli or cocci), size and arrangement of bacterial cells. Simple staining is performed by using basic dyes, e.g., crystal violet, methylene blue, carbol fuchsin etc.

Procedure

Procedure

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free, i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 4. Air-dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air-dry the slide and examine under the microscope using immersion oil.

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free, i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 4. Air-dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air-dry the slide and examine under the microscope using immersion oil.

Result: The stained bacteria are deep violet in colour. (colour depends upon the stain used).

Result: The stained bacteria are deep violet in colour. (colour depends upon the stain used).

4.2 GRAM STAINING

4.2 GRAM STAINING

Gram staining is useful to classify the bacteria into two major groups, Grampositive and Gram-negative. In this process the fixed bacterial smear is subjected to four different reagents: crystal violet (primary stain), iodine solution (mordent), alcohol (decolourizing agent) and safranin (counter-stain). Gram-positive bacteria retain the crystal violet stain and appear deep violet or dark blue in colour while Gram-negative bacteria decolourize with alcohol and stained by safranin which appears pink or red in colour. The Gram-negative cell wall is thin and contains high lipid contents in addition to protein and mucopeptides. The higher amount of lipid is rapidly dissolved by alcohol resulting in the formation of large pores in the cell wall which cause leakage of crystal violet-iodine (CV-I) complex and resulting in the decolourization of the bacterial cell, which later takes the colour of safranin and appears red in color. In contrast the cell walls of Gram-positive bacteria are thick and composed of mainly proteins (peptidoglycan) and cross-linked mucopeptides. When treated with alcohol it causes dehydration and closure of cell wall pores, therefore not

Gram staining is useful to classify the bacteria into two major groups, Grampositive and Gram-negative. In this process the fixed bacterial smear is subjected to four different reagents: crystal violet (primary stain), iodine solution (mordent), alcohol (decolourizing agent) and safranin (counter-stain). Gram-positive bacteria retain the crystal violet stain and appear deep violet or dark blue in colour while Gram-negative bacteria decolourize with alcohol and stained by safranin which appears pink or red in colour. The Gram-negative cell wall is thin and contains high lipid contents in addition to protein and mucopeptides. The higher amount of lipid is rapidly dissolved by alcohol resulting in the formation of large pores in the cell wall which cause leakage of crystal violet-iodine (CV-I) complex and resulting in the decolourization of the bacterial cell, which later takes the colour of safranin and appears red in color. In contrast the cell walls of Gram-positive bacteria are thick and composed of mainly proteins (peptidoglycan) and cross-linked mucopeptides. When treated with alcohol it causes dehydration and closure of cell wall pores, therefore not

26 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

26 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Stains are used intentionally in a variety of fields, including in research (biological staining), technology (metal staining), and art (wood staining, stained glass). Types of staining techniques: Staining is the method of morphological characterization of bacteria.

Stains are used intentionally in a variety of fields, including in research (biological staining), technology (metal staining), and art (wood staining, stained glass). Types of staining techniques: Staining is the method of morphological characterization of bacteria.

4.1 SIMPLE STAINING

4.1 SIMPLE STAINING

The purpose of simple staining technique is to determine the cell shape (bacilli or cocci), size and arrangement of bacterial cells. Simple staining is performed by using basic dyes, e.g., crystal violet, methylene blue, carbol fuchsin etc.

The purpose of simple staining technique is to determine the cell shape (bacilli or cocci), size and arrangement of bacterial cells. Simple staining is performed by using basic dyes, e.g., crystal violet, methylene blue, carbol fuchsin etc.

Procedure

Procedure

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free, i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 4. Air-dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air-dry the slide and examine under the microscope using immersion oil.

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free, i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 4. Air-dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air-dry the slide and examine under the microscope using immersion oil.

Result: The stained bacteria are deep violet in colour. (colour depends upon the stain used).

Result: The stained bacteria are deep violet in colour. (colour depends upon the stain used).

4.2 GRAM STAINING

4.2 GRAM STAINING

Gram staining is useful to classify the bacteria into two major groups, Grampositive and Gram-negative. In this process the fixed bacterial smear is subjected to four different reagents: crystal violet (primary stain), iodine solution (mordent), alcohol (decolourizing agent) and safranin (counter-stain). Gram-positive bacteria retain the crystal violet stain and appear deep violet or dark blue in colour while Gram-negative bacteria decolourize with alcohol and stained by safranin which appears pink or red in colour. The Gram-negative cell wall is thin and contains high lipid contents in addition to protein and mucopeptides. The higher amount of lipid is rapidly dissolved by alcohol resulting in the formation of large pores in the cell wall which cause leakage of crystal violet-iodine (CV-I) complex and resulting in the decolourization of the bacterial cell, which later takes the colour of safranin and appears red in color. In contrast the cell walls of Gram-positive bacteria are thick and composed of mainly proteins (peptidoglycan) and cross-linked mucopeptides. When treated with alcohol it causes dehydration and closure of cell wall pores, therefore not

Gram staining is useful to classify the bacteria into two major groups, Grampositive and Gram-negative. In this process the fixed bacterial smear is subjected to four different reagents: crystal violet (primary stain), iodine solution (mordent), alcohol (decolourizing agent) and safranin (counter-stain). Gram-positive bacteria retain the crystal violet stain and appear deep violet or dark blue in colour while Gram-negative bacteria decolourize with alcohol and stained by safranin which appears pink or red in colour. The Gram-negative cell wall is thin and contains high lipid contents in addition to protein and mucopeptides. The higher amount of lipid is rapidly dissolved by alcohol resulting in the formation of large pores in the cell wall which cause leakage of crystal violet-iodine (CV-I) complex and resulting in the decolourization of the bacterial cell, which later takes the colour of safranin and appears red in color. In contrast the cell walls of Gram-positive bacteria are thick and composed of mainly proteins (peptidoglycan) and cross-linked mucopeptides. When treated with alcohol it causes dehydration and closure of cell wall pores, therefore not

IDENTIFICATION OF MICROBES . . . .

27

IDENTIFICATION OF MICROBES . . . .

27

allowing the loss of CV-I complex and the cell remains violet or deep blue in color. Feature of Gram-positive and Gram-negative Bacteria.

allowing the loss of CV-I complex and the cell remains violet or deep blue in color. Feature of Gram-positive and Gram-negative Bacteria.

S.No Gram-positive bacteria

Gram-negative bacteria

S.No Gram-positive bacteria

Gram-negative bacteria

01.

Cell wall is thick.

Cell wall is thin.

01.

Cell wall is thick.

Cell wall is thin.

02.

Low lipid content (1-4%) in the cell wall.

High lipid content (11-12%) in the cell wall.

02.

Low lipid content (1-4%) in the cell wall.

High lipid content (11-12%) in the cell wall.

03.

Lysosome damage the cell wall.

No effect of lysosome.

03.

Lysosome damage the cell wall.

No effect of lysosome.

04.

Cell wall is mainly made up of peptidoglycan.

Cell wall is made up of lipopolysaccharides.

04.

Cell wall is mainly made up of peptidoglycan.

Cell wall is made up of lipopolysaccharides.

05.

Susceptible to antibiotics, e.g., penicillin, streptomycin.

Resistant to antibiotics.

05.

Susceptible to antibiotics, e.g., penicillin, streptomycin.

Resistant to antibiotics.

06.

Basal body of flagellum consists of 2 rings. Basal body of flagellum consists of 2 rings.

06.

Basal body of flagellum consists of 2 rings. Basal body of flagellum consists of 2 rings.

07.

Few types of amino acids are present.

Several types of amino acids are present.

07.

Few types of amino acids are present.

Several types of amino acids are present.

08.

Teichoic acids present in the cell wall.

Absent.

08.

Teichoic acids present in the cell wall.

Absent.

09.

They mainly produce exotoxins.

They produce mainly endotoxins.

09.

They mainly produce exotoxins.

They produce mainly endotoxins.

10.

Cocci, e.g., streptococcci, diplococcus pneumoniae, streptococci. Bacilli, e.g., corynebacterium diptheriae, mycobacterium tuberculosis, mycobacterium leprae, clostridium botulinum, clostridium perfringens, Bacillus anthrasis.

Cocci, e.g., Neisseria gonorrhoeae, Neisseria meningitides. Bacilli, e.g., E. coli, Salmonella typhosa, Klebsiella pneumoniae, Shigella dysenteriae, Hemophilus ducreyi, Haemophilus influenzae, Pseudomonas aeruginosa, Pasteurella tularensis, Brucella abortus.

10.

Cocci, e.g., streptococcci, diplococcus pneumoniae, streptococci. Bacilli, e.g., corynebacterium diptheriae, mycobacterium tuberculosis, mycobacterium leprae, clostridium botulinum, clostridium perfringens, Bacillus anthrasis.

Cocci, e.g., Neisseria gonorrhoeae, Neisseria meningitides. Bacilli, e.g., E. coli, Salmonella typhosa, Klebsiella pneumoniae, Shigella dysenteriae, Hemophilus ducreyi, Haemophilus influenzae, Pseudomonas aeruginosa, Pasteurella tularensis, Brucella abortus.

Procedure of Gram Staining

Procedure of Gram Staining

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry the slide and heat fix it. 4. Cool the slide and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash the slide with distilled water for few seconds, using wash bottle. 6. Cover each slide with Gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more colour flows from the smear. Note: Gram-positive bacteria are not affected while Gram-negative bacteria are decolourized completely. 8. Wash the slide with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slide with distilled water and dry it properly. 10. Examine the slide under microscope using immersion oil.

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry the slide and heat fix it. 4. Cool the slide and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash the slide with distilled water for few seconds, using wash bottle. 6. Cover each slide with Gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more colour flows from the smear. Note: Gram-positive bacteria are not affected while Gram-negative bacteria are decolourized completely. 8. Wash the slide with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slide with distilled water and dry it properly. 10. Examine the slide under microscope using immersion oil.

Result

Result

A Gram-positive bacterium shows violet or deep blue colour while a Gramnegative bacterium shows purple or red colour.

A Gram-positive bacterium shows violet or deep blue colour while a Gramnegative bacterium shows purple or red colour.

IDENTIFICATION OF MICROBES . . . .

27

IDENTIFICATION OF MICROBES . . . .

27

allowing the loss of CV-I complex and the cell remains violet or deep blue in color. Feature of Gram-positive and Gram-negative Bacteria.

allowing the loss of CV-I complex and the cell remains violet or deep blue in color. Feature of Gram-positive and Gram-negative Bacteria.

S.No Gram-positive bacteria

Gram-negative bacteria

S.No Gram-positive bacteria

Gram-negative bacteria

01.

Cell wall is thick.

Cell wall is thin.

01.

Cell wall is thick.

Cell wall is thin.

02.

Low lipid content (1-4%) in the cell wall.

High lipid content (11-12%) in the cell wall.

02.

Low lipid content (1-4%) in the cell wall.

High lipid content (11-12%) in the cell wall.

03.

Lysosome damage the cell wall.

No effect of lysosome.

03.

Lysosome damage the cell wall.

No effect of lysosome.

04.

Cell wall is mainly made up of peptidoglycan.

Cell wall is made up of lipopolysaccharides.

04.

Cell wall is mainly made up of peptidoglycan.

Cell wall is made up of lipopolysaccharides.

05.

Susceptible to antibiotics, e.g., penicillin, streptomycin.

Resistant to antibiotics.

05.

Susceptible to antibiotics, e.g., penicillin, streptomycin.

Resistant to antibiotics.

06.

Basal body of flagellum consists of 2 rings. Basal body of flagellum consists of 2 rings.

06.

Basal body of flagellum consists of 2 rings. Basal body of flagellum consists of 2 rings.

07.

Few types of amino acids are present.

Several types of amino acids are present.

07.

Few types of amino acids are present.

Several types of amino acids are present.

08.

Teichoic acids present in the cell wall.

Absent.

08.

Teichoic acids present in the cell wall.

Absent.

09.

They mainly produce exotoxins.

They produce mainly endotoxins.

09.

They mainly produce exotoxins.

They produce mainly endotoxins.

10.

Cocci, e.g., streptococcci, diplococcus pneumoniae, streptococci. Bacilli, e.g., corynebacterium diptheriae, mycobacterium tuberculosis, mycobacterium leprae, clostridium botulinum, clostridium perfringens, Bacillus anthrasis.

Cocci, e.g., Neisseria gonorrhoeae, Neisseria meningitides. Bacilli, e.g., E. coli, Salmonella typhosa, Klebsiella pneumoniae, Shigella dysenteriae, Hemophilus ducreyi, Haemophilus influenzae, Pseudomonas aeruginosa, Pasteurella tularensis, Brucella abortus.

10.

Cocci, e.g., streptococcci, diplococcus pneumoniae, streptococci. Bacilli, e.g., corynebacterium diptheriae, mycobacterium tuberculosis, mycobacterium leprae, clostridium botulinum, clostridium perfringens, Bacillus anthrasis.

Cocci, e.g., Neisseria gonorrhoeae, Neisseria meningitides. Bacilli, e.g., E. coli, Salmonella typhosa, Klebsiella pneumoniae, Shigella dysenteriae, Hemophilus ducreyi, Haemophilus influenzae, Pseudomonas aeruginosa, Pasteurella tularensis, Brucella abortus.

Procedure of Gram Staining 1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry the slide and heat fix it. 4. Cool the slide and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash the slide with distilled water for few seconds, using wash bottle. 6. Cover each slide with Gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more colour flows from the smear. Note: Gram-positive bacteria are not affected while Gram-negative bacteria are decolourized completely. 8. Wash the slide with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slide with distilled water and dry it properly. 10. Examine the slide under microscope using immersion oil.

Procedure of Gram Staining 1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry the slide and heat fix it. 4. Cool the slide and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash the slide with distilled water for few seconds, using wash bottle. 6. Cover each slide with Gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more colour flows from the smear. Note: Gram-positive bacteria are not affected while Gram-negative bacteria are decolourized completely. 8. Wash the slide with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slide with distilled water and dry it properly. 10. Examine the slide under microscope using immersion oil.

Result

Result

A Gram-positive bacterium shows violet or deep blue colour while a Gramnegative bacterium shows purple or red colour.

A Gram-positive bacterium shows violet or deep blue colour while a Gramnegative bacterium shows purple or red colour.

28 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

28 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4.3 ACID FAST STAINING

4.3 ACID FAST STAINING

Principle

Principle

Acid fast staining was developed by Poul Ehrlich in 1882 which was later modified by Ziehl-Neelsen. So this staining is also known as Ziehl-Neelsen staining. This staining is used for those bacteria which contain high contents of a lipid called mycolic acid in the cell wall, that makes penetration by stains extremely difficult. In this staining procedure bacterial smear is treated with carbol fuchsin followed by heat fixing and treatment with acid alcohol and methylene blue. Acid fast staining is useful for the identification of members of mycobacterium especially in staining of sputum that have mycobacterium tuberculosis, which causes tuberculosis. This bacillus is the only acid fast organism commonly found in the sputum.

Acid fast staining was developed by Poul Ehrlich in 1882 which was later modified by Ziehl-Neelsen. So this staining is also known as Ziehl-Neelsen staining. This staining is used for those bacteria which contain high contents of a lipid called mycolic acid in the cell wall, that makes penetration by stains extremely difficult. In this staining procedure bacterial smear is treated with carbol fuchsin followed by heat fixing and treatment with acid alcohol and methylene blue. Acid fast staining is useful for the identification of members of mycobacterium especially in staining of sputum that have mycobacterium tuberculosis, which causes tuberculosis. This bacillus is the only acid fast organism commonly found in the sputum.

Procedure

Procedure

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry and heat-fix the smear. 4. Flood the bacterial smear with carbol fuchsin and keep for 3 to 5 minutes above the water bath for steaming. 5. Cool the slide and wash with distilled water. 6. Decolourize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 7. Add the methylene blue and keep for 1 to 2 minutes. 8. Wash the slide with distilled water. 9. Air-dry the slide and examine under the microscope using immersion oil.

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry and heat-fix the smear. 4. Flood the bacterial smear with carbol fuchsin and keep for 3 to 5 minutes above the water bath for steaming. 5. Cool the slide and wash with distilled water. 6. Decolourize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 7. Add the methylene blue and keep for 1 to 2 minutes. 8. Wash the slide with distilled water. 9. Air-dry the slide and examine under the microscope using immersion oil.

4.4 ENDOSPORE STAINING

4.4 ENDOSPORE STAINING

An endospore is dormant, tough, and non-reproductive structure produced by certain gram-positive bacteria. The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to heat, radiation, lysozyme, temperature, starvation, chemicals and other agents which are typically lethal to the organisms. Because of high content of calcium and dipicolinic acid these bacteria are heat resistant. Bacterial cell produces endospores during unfavourable environmental conditions when depletion of an essential nutrients takes place. Endospores are formed by members of seven genera, e.g., Bacillus, Clostridium, Coxiella, Desulfotomaculum, Sporolactobacillus, Sporomusa and Theromoactinomyces.

An endospore is dormant, tough, and non-reproductive structure produced by certain gram-positive bacteria. The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to heat, radiation, lysozyme, temperature, starvation, chemicals and other agents which are typically lethal to the organisms. Because of high content of calcium and dipicolinic acid these bacteria are heat resistant. Bacterial cell produces endospores during unfavourable environmental conditions when depletion of an essential nutrients takes place. Endospores are formed by members of seven genera, e.g., Bacillus, Clostridium, Coxiella, Desulfotomaculum, Sporolactobacillus, Sporomusa and Theromoactinomyces.

28 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

28 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4.3 ACID FAST STAINING

4.3 ACID FAST STAINING

Principle

Principle

Acid fast staining was developed by Poul Ehrlich in 1882 which was later modified by Ziehl-Neelsen. So this staining is also known as Ziehl-Neelsen staining. This staining is used for those bacteria which contain high contents of a lipid called mycolic acid in the cell wall, that makes penetration by stains extremely difficult. In this staining procedure bacterial smear is treated with carbol fuchsin followed by heat fixing and treatment with acid alcohol and methylene blue. Acid fast staining is useful for the identification of members of mycobacterium especially in staining of sputum that have mycobacterium tuberculosis, which causes tuberculosis. This bacillus is the only acid fast organism commonly found in the sputum.

Acid fast staining was developed by Poul Ehrlich in 1882 which was later modified by Ziehl-Neelsen. So this staining is also known as Ziehl-Neelsen staining. This staining is used for those bacteria which contain high contents of a lipid called mycolic acid in the cell wall, that makes penetration by stains extremely difficult. In this staining procedure bacterial smear is treated with carbol fuchsin followed by heat fixing and treatment with acid alcohol and methylene blue. Acid fast staining is useful for the identification of members of mycobacterium especially in staining of sputum that have mycobacterium tuberculosis, which causes tuberculosis. This bacillus is the only acid fast organism commonly found in the sputum.

Procedure

Procedure

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry and heat-fix the smear. 4. Flood the bacterial smear with carbol fuchsin and keep for 3 to 5 minutes above the water bath for steaming. 5. Cool the slide and wash with distilled water. 6. Decolourize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 7. Add the methylene blue and keep for 1 to 2 minutes. 8. Wash the slide with distilled water. 9. Air-dry the slide and examine under the microscope using immersion oil.

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Air-dry and heat-fix the smear. 4. Flood the bacterial smear with carbol fuchsin and keep for 3 to 5 minutes above the water bath for steaming. 5. Cool the slide and wash with distilled water. 6. Decolourize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 7. Add the methylene blue and keep for 1 to 2 minutes. 8. Wash the slide with distilled water. 9. Air-dry the slide and examine under the microscope using immersion oil.

4.4 ENDOSPORE STAINING

4.4 ENDOSPORE STAINING

An endospore is dormant, tough, and non-reproductive structure produced by certain gram-positive bacteria. The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to heat, radiation, lysozyme, temperature, starvation, chemicals and other agents which are typically lethal to the organisms. Because of high content of calcium and dipicolinic acid these bacteria are heat resistant. Bacterial cell produces endospores during unfavourable environmental conditions when depletion of an essential nutrients takes place. Endospores are formed by members of seven genera, e.g., Bacillus, Clostridium, Coxiella, Desulfotomaculum, Sporolactobacillus, Sporomusa and Theromoactinomyces.

An endospore is dormant, tough, and non-reproductive structure produced by certain gram-positive bacteria. The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to heat, radiation, lysozyme, temperature, starvation, chemicals and other agents which are typically lethal to the organisms. Because of high content of calcium and dipicolinic acid these bacteria are heat resistant. Bacterial cell produces endospores during unfavourable environmental conditions when depletion of an essential nutrients takes place. Endospores are formed by members of seven genera, e.g., Bacillus, Clostridium, Coxiella, Desulfotomaculum, Sporolactobacillus, Sporomusa and Theromoactinomyces.

IDENTIFICATION OF MICROBES . . . .

29

Procedure

IDENTIFICATION OF MICROBES . . . .

29

Procedure

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Flood the smears with malachite green and heat the slides to steaming for 5 minutes, adding more stain to the smear from time to time. 4. Wash the slides under slowly running tap water and counter-stain with safranin for 30 seconds. 5. Wash the smear with distilled water and air-dry the slide. 6. Examine the slide under microscope using immersion oil.

1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Flood the smears with malachite green and heat the slides to steaming for 5 minutes, adding more stain to the smear from time to time. 4. Wash the slides under slowly running tap water and counter-stain with safranin for 30 seconds. 5. Wash the smear with distilled water and air-dry the slide. 6. Examine the slide under microscope using immersion oil.

4.5 CAPSULE STAINING

4.5 CAPSULE STAINING

Some bacterial cells are surrounded by mucilaginous substances forming a viscous coat around the cell; this structure is known as capsule. It is composed of polysaccharides but may contain other materials, e.g., bacillus anthracis has a capsule of poly-D-glutamic acid. The capsule is well developed in some species of bacteria like Streptococcus pneumoniae, Clostridium perfringens and Klebsiella pneumoniae. The diagnosis of pneumonia and other diseases is determined by capsule staining.

Some bacterial cells are surrounded by mucilaginous substances forming a viscous coat around the cell; this structure is known as capsule. It is composed of polysaccharides but may contain other materials, e.g., bacillus anthracis has a capsule of poly-D-glutamic acid. The capsule is well developed in some species of bacteria like Streptococcus pneumoniae, Clostridium perfringens and Klebsiella pneumoniae. The diagnosis of pneumonia and other diseases is determined by capsule staining.

Procedure

Procedure

1. Wash the glass slide and air-dry it. 2. Put a drop of nigrosin or India ink close to one end of the glass slide. 3. Add a loopful of bacterial culture into the drop of stain and mix properly with the help of a loop. 4. Prepare a smear of suspended culture using edge of a second slide and allow the slide to air-dry. 5. Flood the smear with methylene blue for 3 minutes and wash with water. 6. Air-dry the slide and examine under the microscope using immersion oil.

1. Wash the glass slide and air-dry it. 2. Put a drop of nigrosin or India ink close to one end of the glass slide. 3. Add a loopful of bacterial culture into the drop of stain and mix properly with the help of a loop. 4. Prepare a smear of suspended culture using edge of a second slide and allow the slide to air-dry. 5. Flood the smear with methylene blue for 3 minutes and wash with water. 6. Air-dry the slide and examine under the microscope using immersion oil.

4.6 FLAGELLA STAINING

4.6 FLAGELLA STAINING

Flagella usually refers to a thread-like structure that essentially provides motility for certain microorganisms and protozoa (one, few, or many per cell), and for spermatozoa (one per cell). This method is used to stain the bacterial flagella to identify whether the bacterial cell is flagellated or non-flagellated.

Flagella usually refers to a thread-like structure that essentially provides motility for certain microorganisms and protozoa (one, few, or many per cell), and for spermatozoa (one per cell). This method is used to stain the bacterial flagella to identify whether the bacterial cell is flagellated or non-flagellated.

Procedure

Procedure

1. Smear of bacterial culture was prepared on a clean glass slide. 2. Made a heavy wax line on slide sides. 3. Add 1mL of flagella staining solution to the smear and allow staining for 15 minutes. 4. Flood the stain by adding tap water.

IDENTIFICATION OF MICROBES . . . .

1. Smear of bacterial culture was prepared on a clean glass slide. 2. Made a heavy wax line on slide sides. 3. Add 1mL of flagella staining solution to the smear and allow staining for 15 minutes. 4. Flood the stain by adding tap water.

29

Procedure 1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Flood the smears with malachite green and heat the slides to steaming for 5 minutes, adding more stain to the smear from time to time. 4. Wash the slides under slowly running tap water and counter-stain with safranin for 30 seconds. 5. Wash the smear with distilled water and air-dry the slide. 6. Examine the slide under microscope using immersion oil.

IDENTIFICATION OF MICROBES . . . .

29

Procedure 1. Wash the glass slide properly and air-dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear. 3. Flood the smears with malachite green and heat the slides to steaming for 5 minutes, adding more stain to the smear from time to time. 4. Wash the slides under slowly running tap water and counter-stain with safranin for 30 seconds. 5. Wash the smear with distilled water and air-dry the slide. 6. Examine the slide under microscope using immersion oil.

4.5 CAPSULE STAINING

4.5 CAPSULE STAINING

Some bacterial cells are surrounded by mucilaginous substances forming a viscous coat around the cell; this structure is known as capsule. It is composed of polysaccharides but may contain other materials, e.g., bacillus anthracis has a capsule of poly-D-glutamic acid. The capsule is well developed in some species of bacteria like Streptococcus pneumoniae, Clostridium perfringens and Klebsiella pneumoniae. The diagnosis of pneumonia and other diseases is determined by capsule staining.

Some bacterial cells are surrounded by mucilaginous substances forming a viscous coat around the cell; this structure is known as capsule. It is composed of polysaccharides but may contain other materials, e.g., bacillus anthracis has a capsule of poly-D-glutamic acid. The capsule is well developed in some species of bacteria like Streptococcus pneumoniae, Clostridium perfringens and Klebsiella pneumoniae. The diagnosis of pneumonia and other diseases is determined by capsule staining.

Procedure

Procedure

1. Wash the glass slide and air-dry it. 2. Put a drop of nigrosin or India ink close to one end of the glass slide. 3. Add a loopful of bacterial culture into the drop of stain and mix properly with the help of a loop. 4. Prepare a smear of suspended culture using edge of a second slide and allow the slide to air-dry. 5. Flood the smear with methylene blue for 3 minutes and wash with water. 6. Air-dry the slide and examine under the microscope using immersion oil.

1. Wash the glass slide and air-dry it. 2. Put a drop of nigrosin or India ink close to one end of the glass slide. 3. Add a loopful of bacterial culture into the drop of stain and mix properly with the help of a loop. 4. Prepare a smear of suspended culture using edge of a second slide and allow the slide to air-dry. 5. Flood the smear with methylene blue for 3 minutes and wash with water. 6. Air-dry the slide and examine under the microscope using immersion oil.

4.6 FLAGELLA STAINING

4.6 FLAGELLA STAINING

Flagella usually refers to a thread-like structure that essentially provides motility for certain microorganisms and protozoa (one, few, or many per cell), and for spermatozoa (one per cell). This method is used to stain the bacterial flagella to identify whether the bacterial cell is flagellated or non-flagellated.

Flagella usually refers to a thread-like structure that essentially provides motility for certain microorganisms and protozoa (one, few, or many per cell), and for spermatozoa (one per cell). This method is used to stain the bacterial flagella to identify whether the bacterial cell is flagellated or non-flagellated.

Procedure

Procedure

1. Smear of bacterial culture was prepared on a clean glass slide. 2. Made a heavy wax line on slide sides. 3. Add 1mL of flagella staining solution to the smear and allow staining for 15 minutes. 4. Flood the stain by adding tap water.

1. Smear of bacterial culture was prepared on a clean glass slide. 2. Made a heavy wax line on slide sides. 3. Add 1mL of flagella staining solution to the smear and allow staining for 15 minutes. 4. Flood the stain by adding tap water.

30 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 5. Add the carbol fuchsin for 1 min and rinse with water. 6. Air-dry the slide and examine under microscope using immersion oil.

30 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 5. Add the carbol fuchsin for 1 min and rinse with water. 6. Air-dry the slide and examine under microscope using immersion oil.

4.7 NEGATIVE STAINING

4.7 NEGATIVE STAINING

Negative staining provides the simplest and often quickest means of gaining information about the cell shape, cell breakage and retractile inclusions in cell such as sulphur and poly-B-hydroxy butyrate granules and about endospores. In negative staining technique a simple strain is used which stain the background not the bacterial cell, e.g., nigrosin or Indian ink, an acidic stain is used. The acidic stain, carrying a negative charge, is repelled by the bacteria which also carry a negative charge on their surface therefore bacterial cells appear transparent and unstained upon examination. A stain that stains the background and does not stain the bacterial cell is called negative staining. The advantages of negative staining are: 1. Cell appears shriveled or distorted because no heat fixing is done. 2. Capsulated bacteria that are difficult to stain can be observed by this technique.

Negative staining provides the simplest and often quickest means of gaining information about the cell shape, cell breakage and retractile inclusions in cell such as sulphur and poly-B-hydroxy butyrate granules and about endospores. In negative staining technique a simple strain is used which stain the background not the bacterial cell, e.g., nigrosin or Indian ink, an acidic stain is used. The acidic stain, carrying a negative charge, is repelled by the bacteria which also carry a negative charge on their surface therefore bacterial cells appear transparent and unstained upon examination. A stain that stains the background and does not stain the bacterial cell is called negative staining. The advantages of negative staining are: 1. Cell appears shriveled or distorted because no heat fixing is done. 2. Capsulated bacteria that are difficult to stain can be observed by this technique.

Procedure

Procedure

1. Take a glass slide, wash and dry it properly. 2. Place one drop of nigrosin at one end of the glass slide. 3. Take a loopful of the inoculum from the broth culture with the help of inoculating loop and mix it properly with nigrosin. 4. Now speared the droplet properly across the slide with the help of another slide. 5. Allow the smear to air-dry.

1. Take a glass slide, wash and dry it properly. 2. Place one drop of nigrosin at one end of the glass slide. 3. Take a loopful of the inoculum from the broth culture with the help of inoculating loop and mix it properly with nigrosin. 4. Now speared the droplet properly across the slide with the help of another slide. 5. Allow the smear to air-dry.

Result: Spherical cell occurring singly as well as in clusters appear transparent against the blue back ground.

Result: Spherical cell occurring singly as well as in clusters appear transparent against the blue back ground.

Precautions

Precautions

1. Never heat-fix the slide. 2. Properly mix the slide with culture then spread it.

1. Never heat-fix the slide. 2. Properly mix the slide with culture then spread it.

4.8 MICROSCOPY

4.8 MICROSCOPY

During the 1st century AD (year 100) Romans discovered that if you held the thick glass lenses over an object, the object would look larger. Someone also discovered that you can focus the rays of the sun with one of these special “glasses” and start a fire. These early lenses were called magnifiers or burning glasses. The word lens is derived from the latin word lentil, as they were named because they resembled the shape of a lentil bean (look up lens in a dictionary).

During the 1st century AD (year 100) Romans discovered that if you held the thick glass lenses over an object, the object would look larger. Someone also discovered that you can focus the rays of the sun with one of these special “glasses” and start a fire. These early lenses were called magnifiers or burning glasses. The word lens is derived from the latin word lentil, as they were named because they resembled the shape of a lentil bean (look up lens in a dictionary).

30 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

30 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

5. Add the carbol fuchsin for 1 min and rinse with water. 6. Air-dry the slide and examine under microscope using immersion oil.

5. Add the carbol fuchsin for 1 min and rinse with water. 6. Air-dry the slide and examine under microscope using immersion oil.

4.7 NEGATIVE STAINING

4.7 NEGATIVE STAINING

Negative staining provides the simplest and often quickest means of gaining information about the cell shape, cell breakage and retractile inclusions in cell such as sulphur and poly-B-hydroxy butyrate granules and about endospores. In negative staining technique a simple strain is used which stain the background not the bacterial cell, e.g., nigrosin or Indian ink, an acidic stain is used. The acidic stain, carrying a negative charge, is repelled by the bacteria which also carry a negative charge on their surface therefore bacterial cells appear transparent and unstained upon examination. A stain that stains the background and does not stain the bacterial cell is called negative staining. The advantages of negative staining are: 1. Cell appears shriveled or distorted because no heat fixing is done. 2. Capsulated bacteria that are difficult to stain can be observed by this technique.

Negative staining provides the simplest and often quickest means of gaining information about the cell shape, cell breakage and retractile inclusions in cell such as sulphur and poly-B-hydroxy butyrate granules and about endospores. In negative staining technique a simple strain is used which stain the background not the bacterial cell, e.g., nigrosin or Indian ink, an acidic stain is used. The acidic stain, carrying a negative charge, is repelled by the bacteria which also carry a negative charge on their surface therefore bacterial cells appear transparent and unstained upon examination. A stain that stains the background and does not stain the bacterial cell is called negative staining. The advantages of negative staining are: 1. Cell appears shriveled or distorted because no heat fixing is done. 2. Capsulated bacteria that are difficult to stain can be observed by this technique.

Procedure

Procedure

1. Take a glass slide, wash and dry it properly. 2. Place one drop of nigrosin at one end of the glass slide. 3. Take a loopful of the inoculum from the broth culture with the help of inoculating loop and mix it properly with nigrosin. 4. Now speared the droplet properly across the slide with the help of another slide. 5. Allow the smear to air-dry.

1. Take a glass slide, wash and dry it properly. 2. Place one drop of nigrosin at one end of the glass slide. 3. Take a loopful of the inoculum from the broth culture with the help of inoculating loop and mix it properly with nigrosin. 4. Now speared the droplet properly across the slide with the help of another slide. 5. Allow the smear to air-dry.

Result: Spherical cell occurring singly as well as in clusters appear transparent against the blue back ground.

Result: Spherical cell occurring singly as well as in clusters appear transparent against the blue back ground.

Precautions

Precautions

1. Never heat-fix the slide. 2. Properly mix the slide with culture then spread it.

1. Never heat-fix the slide. 2. Properly mix the slide with culture then spread it.

4.8 MICROSCOPY

4.8 MICROSCOPY

During the 1st century AD (year 100) Romans discovered that if you held the thick glass lenses over an object, the object would look larger. Someone also discovered that you can focus the rays of the sun with one of these special “glasses” and start a fire. These early lenses were called magnifiers or burning glasses. The word lens is derived from the latin word lentil, as they were named because they resembled the shape of a lentil bean (look up lens in a dictionary).

During the 1st century AD (year 100) Romans discovered that if you held the thick glass lenses over an object, the object would look larger. Someone also discovered that you can focus the rays of the sun with one of these special “glasses” and start a fire. These early lenses were called magnifiers or burning glasses. The word lens is derived from the latin word lentil, as they were named because they resembled the shape of a lentil bean (look up lens in a dictionary).

IDENTIFICATION OF MICROBES . . . .

31

IDENTIFICATION OF MICROBES . . . .

31

In 1590, two Dutch spectacle makers, Zaccharias Janssen and his father Hans started experimenting with these lenses. They put several lenses in a tube and made a very important discovery. The object near the end of the tube appeared to be greatly enlarged, much larger than any simple magnifying glass could achieve by itself. They had just invented the compound microscope (which is a microscope that uses two or more lenses). Galileo heard of their experiments and started experimenting on his own. He described the principles of lenses and light rays and improved both the microscope and telescope. Anthony Leeuwenhoek of Holland became very interested in lenses while working with magnifying glasses in a dry goods store. He used the magnifying glass to count threads in woven cloth. He became so interested that he learned how to make lenses. By grinding and polishing, he was able to make small lenses with great curvatures. These rounder lenses produced greater magnification, and his microscopes were able to magnify up to 270X. Anthony Leeuwenhoek became more involved in science and with his new improved microscope was able to see things that no man had ever seen before. He saw bacteria, yeast, blood cells and many tiny animals swimming about in a drop of water. From his great contributions, many discoveries and research papers, Anthony Leeuwenhoek (1632-1723) has since been called the “Father of Microscopy”. Robert Hooke, an Englishman (who is sometimes called the “English Father of Microscopy”), also spent much of his life working with microscopes and improved their design and capabilities. Today most of the microscopes come from Germany, Japan and China. Toy plastic microscopes should be avoided as they do not achieve the level of quality of the basic instruments with metal frames and glass lenses. Because of foreign production, quality microscopes have become affordable for all. Zaccharias Janssen, the inventor of the microscope would marvel at the quality of even the most basic microscopes found in schools today. In 1838—Schleiden and Schwann proposed the cell theory (Cell Doctine), which states that all plant and animal tissues are aggregates of individual cells. Advent of stains in the later part of the nineteenth century enabled the visualization of animal cells which are colorless and translucent. Invention of electron microscopes in the 1940s made it possible to study the cell’s structure in greater detail.

In 1590, two Dutch spectacle makers, Zaccharias Janssen and his father Hans started experimenting with these lenses. They put several lenses in a tube and made a very important discovery. The object near the end of the tube appeared to be greatly enlarged, much larger than any simple magnifying glass could achieve by itself. They had just invented the compound microscope (which is a microscope that uses two or more lenses). Galileo heard of their experiments and started experimenting on his own. He described the principles of lenses and light rays and improved both the microscope and telescope. Anthony Leeuwenhoek of Holland became very interested in lenses while working with magnifying glasses in a dry goods store. He used the magnifying glass to count threads in woven cloth. He became so interested that he learned how to make lenses. By grinding and polishing, he was able to make small lenses with great curvatures. These rounder lenses produced greater magnification, and his microscopes were able to magnify up to 270X. Anthony Leeuwenhoek became more involved in science and with his new improved microscope was able to see things that no man had ever seen before. He saw bacteria, yeast, blood cells and many tiny animals swimming about in a drop of water. From his great contributions, many discoveries and research papers, Anthony Leeuwenhoek (1632-1723) has since been called the “Father of Microscopy”. Robert Hooke, an Englishman (who is sometimes called the “English Father of Microscopy”), also spent much of his life working with microscopes and improved their design and capabilities. Today most of the microscopes come from Germany, Japan and China. Toy plastic microscopes should be avoided as they do not achieve the level of quality of the basic instruments with metal frames and glass lenses. Because of foreign production, quality microscopes have become affordable for all. Zaccharias Janssen, the inventor of the microscope would marvel at the quality of even the most basic microscopes found in schools today. In 1838—Schleiden and Schwann proposed the cell theory (Cell Doctine), which states that all plant and animal tissues are aggregates of individual cells. Advent of stains in the later part of the nineteenth century enabled the visualization of animal cells which are colorless and translucent. Invention of electron microscopes in the 1940s made it possible to study the cell’s structure in greater detail.

What is a microscope?

What is a microscope?

A microscope is an instrument which is used to see the objects or particles which are not seen by the naked eyes, e.g., bacteria — 0.5-10 Pm, fungi — 5-10 Pm, algae — 1 Pm to many feet, protozoa — 2-200 Pm.

A microscope is an instrument which is used to see the objects or particles which are not seen by the naked eyes, e.g., bacteria — 0.5-10 Pm, fungi — 5-10 Pm, algae — 1 Pm to many feet, protozoa — 2-200 Pm.

IDENTIFICATION OF MICROBES . . . .

31

IDENTIFICATION OF MICROBES . . . .

31

In 1590, two Dutch spectacle makers, Zaccharias Janssen and his father Hans started experimenting with these lenses. They put several lenses in a tube and made a very important discovery. The object near the end of the tube appeared to be greatly enlarged, much larger than any simple magnifying glass could achieve by itself. They had just invented the compound microscope (which is a microscope that uses two or more lenses). Galileo heard of their experiments and started experimenting on his own. He described the principles of lenses and light rays and improved both the microscope and telescope. Anthony Leeuwenhoek of Holland became very interested in lenses while working with magnifying glasses in a dry goods store. He used the magnifying glass to count threads in woven cloth. He became so interested that he learned how to make lenses. By grinding and polishing, he was able to make small lenses with great curvatures. These rounder lenses produced greater magnification, and his microscopes were able to magnify up to 270X. Anthony Leeuwenhoek became more involved in science and with his new improved microscope was able to see things that no man had ever seen before. He saw bacteria, yeast, blood cells and many tiny animals swimming about in a drop of water. From his great contributions, many discoveries and research papers, Anthony Leeuwenhoek (1632-1723) has since been called the “Father of Microscopy”. Robert Hooke, an Englishman (who is sometimes called the “English Father of Microscopy”), also spent much of his life working with microscopes and improved their design and capabilities. Today most of the microscopes come from Germany, Japan and China. Toy plastic microscopes should be avoided as they do not achieve the level of quality of the basic instruments with metal frames and glass lenses. Because of foreign production, quality microscopes have become affordable for all. Zaccharias Janssen, the inventor of the microscope would marvel at the quality of even the most basic microscopes found in schools today. In 1838—Schleiden and Schwann proposed the cell theory (Cell Doctine), which states that all plant and animal tissues are aggregates of individual cells. Advent of stains in the later part of the nineteenth century enabled the visualization of animal cells which are colorless and translucent. Invention of electron microscopes in the 1940s made it possible to study the cell’s structure in greater detail.

In 1590, two Dutch spectacle makers, Zaccharias Janssen and his father Hans started experimenting with these lenses. They put several lenses in a tube and made a very important discovery. The object near the end of the tube appeared to be greatly enlarged, much larger than any simple magnifying glass could achieve by itself. They had just invented the compound microscope (which is a microscope that uses two or more lenses). Galileo heard of their experiments and started experimenting on his own. He described the principles of lenses and light rays and improved both the microscope and telescope. Anthony Leeuwenhoek of Holland became very interested in lenses while working with magnifying glasses in a dry goods store. He used the magnifying glass to count threads in woven cloth. He became so interested that he learned how to make lenses. By grinding and polishing, he was able to make small lenses with great curvatures. These rounder lenses produced greater magnification, and his microscopes were able to magnify up to 270X. Anthony Leeuwenhoek became more involved in science and with his new improved microscope was able to see things that no man had ever seen before. He saw bacteria, yeast, blood cells and many tiny animals swimming about in a drop of water. From his great contributions, many discoveries and research papers, Anthony Leeuwenhoek (1632-1723) has since been called the “Father of Microscopy”. Robert Hooke, an Englishman (who is sometimes called the “English Father of Microscopy”), also spent much of his life working with microscopes and improved their design and capabilities. Today most of the microscopes come from Germany, Japan and China. Toy plastic microscopes should be avoided as they do not achieve the level of quality of the basic instruments with metal frames and glass lenses. Because of foreign production, quality microscopes have become affordable for all. Zaccharias Janssen, the inventor of the microscope would marvel at the quality of even the most basic microscopes found in schools today. In 1838—Schleiden and Schwann proposed the cell theory (Cell Doctine), which states that all plant and animal tissues are aggregates of individual cells. Advent of stains in the later part of the nineteenth century enabled the visualization of animal cells which are colorless and translucent. Invention of electron microscopes in the 1940s made it possible to study the cell’s structure in greater detail.

What is a microscope?

What is a microscope?

A microscope is an instrument which is used to see the objects or particles which are not seen by the naked eyes, e.g., bacteria — 0.5-10 Pm, fungi — 5-10 Pm, algae — 1 Pm to many feet, protozoa — 2-200 Pm.

A microscope is an instrument which is used to see the objects or particles which are not seen by the naked eyes, e.g., bacteria — 0.5-10 Pm, fungi — 5-10 Pm, algae — 1 Pm to many feet, protozoa — 2-200 Pm.

32 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

32 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Types of microscopes

Types of microscopes

(a) Light or optical or compound microscope. (b) Electron microscope.

(a) Light or optical or compound microscope. (b) Electron microscope.

4.8.1 Light or Compound Microscope Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

4.8.1 Light or Compound Microscope Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

1. Bright Field Microscopy Bright field microscope is commonly used by the students to see the shape, size and arrangement of micro-organisms like bacteria, protozoans, fungi algae, Plant and animal cells and tissues. So it is also called student microscope.

1. Bright Field Microscopy Bright field microscope is commonly used by the students to see the shape, size and arrangement of micro-organisms like bacteria, protozoans, fungi algae, Plant and animal cells and tissues. So it is also called student microscope.

Fig. 4.1. (A) Sketch of light microscope showing path of light. (B) Structure of microscope.

Fig. 4.1. (A) Sketch of light microscope showing path of light. (B) Structure of microscope.

32 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

32 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Types of microscopes

Types of microscopes

(a) Light or optical or compound microscope. (b) Electron microscope.

(a) Light or optical or compound microscope. (b) Electron microscope.

4.8.1 Light or Compound Microscope Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

4.8.1 Light or Compound Microscope Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

1. Bright Field Microscopy Bright field microscope is commonly used by the students to see the shape, size and arrangement of micro-organisms like bacteria, protozoans, fungi algae, Plant and animal cells and tissues. So it is also called student microscope.

1. Bright Field Microscopy Bright field microscope is commonly used by the students to see the shape, size and arrangement of micro-organisms like bacteria, protozoans, fungi algae, Plant and animal cells and tissues. So it is also called student microscope.

Fig. 4.1. (A) Sketch of light microscope showing path of light. (B) Structure of microscope.

Fig. 4.1. (A) Sketch of light microscope showing path of light. (B) Structure of microscope.

IDENTIFICATION OF MICROBES . . . .

33

IDENTIFICATION OF MICROBES . . . .

33

Principle

Principle

The bright-field light microscope is an instrument that magnifies images using two lens systems i.e. objective lens and ocular lens. Initial magnification occurs in the objective lens. Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs. The objective lenses are identified as the low-power (10X), high-dry (40X), and oil immersion objectives (100X). Each objective is also designated by other terms. These terms give either the linear magnification or the focal length. The magnification power of ocular lens is also 5X, 10X, 15X etc. So the total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of ocular lens.

The bright-field light microscope is an instrument that magnifies images using two lens systems i.e. objective lens and ocular lens. Initial magnification occurs in the objective lens. Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs. The objective lenses are identified as the low-power (10X), high-dry (40X), and oil immersion objectives (100X). Each objective is also designated by other terms. These terms give either the linear magnification or the focal length. The magnification power of ocular lens is also 5X, 10X, 15X etc. So the total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of ocular lens.

Resolving power

Resolving power

It is the ability to separate the two or more adjacent objects or points.

It is the ability to separate the two or more adjacent objects or points.

R.P. = Wave length of light in (nm)/2X numerical aperture of the objective lens.

R.P. = Wave length of light in (nm)/2X numerical aperture of the objective lens.

Numerical Aperture

Numerical Aperture

The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

N.A = n sinT;

where n = refractive index

N.A = n sinT;

where n = refractive index

For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.56.

For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.56.

Fig. 4.2. The relation between numerical aperture and resolution. The total angle is 116o, so T is 58o. NA = n sinT = 1.56 – sin58o = 1.56 – 0.58 = 1.33

Fig. 4.2. The relation between numerical aperture and resolution. The total angle is 116o, so T is 58o. NA = n sinT = 1.56 – sin58o = 1.56 – 0.58 = 1.33

Limit of resolution

Limit of resolution

It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture. The relationship between NA and resolution can be expressed as:

It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture. The relationship between NA and resolution can be expressed as:

IDENTIFICATION OF MICROBES . . . .

33

IDENTIFICATION OF MICROBES . . . .

33

Principle

Principle

The bright-field light microscope is an instrument that magnifies images using two lens systems i.e. objective lens and ocular lens. Initial magnification occurs in the objective lens. Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs. The objective lenses are identified as the low-power (10X), high-dry (40X), and oil immersion objectives (100X). Each objective is also designated by other terms. These terms give either the linear magnification or the focal length. The magnification power of ocular lens is also 5X, 10X, 15X etc. So the total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of ocular lens.

The bright-field light microscope is an instrument that magnifies images using two lens systems i.e. objective lens and ocular lens. Initial magnification occurs in the objective lens. Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs. The objective lenses are identified as the low-power (10X), high-dry (40X), and oil immersion objectives (100X). Each objective is also designated by other terms. These terms give either the linear magnification or the focal length. The magnification power of ocular lens is also 5X, 10X, 15X etc. So the total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of ocular lens.

Resolving power

Resolving power

It is the ability to separate the two or more adjacent objects or points.

It is the ability to separate the two or more adjacent objects or points.

R.P. = Wave length of light in (nm)/2X numerical aperture of the objective lens.

R.P. = Wave length of light in (nm)/2X numerical aperture of the objective lens.

Numerical Aperture

Numerical Aperture

The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

N.A = n sinT;

where n = refractive index

N.A = n sinT;

where n = refractive index

For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.56.

For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.56.

Fig. 4.2. The relation between numerical aperture and resolution. The total angle is 116o, so T is 58o. NA = n sinT = 1.56 – sin58o = 1.56 – 0.58 = 1.33

Fig. 4.2. The relation between numerical aperture and resolution. The total angle is 116o, so T is 58o. NA = n sinT = 1.56 – sin58o = 1.56 – 0.58 = 1.33

Limit of resolution

Limit of resolution

It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture. The relationship between NA and resolution can be expressed as:

It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture. The relationship between NA and resolution can be expressed as:

34 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY D = O/2NA Where d = resolution and O = wavelength of light

Fig. 4.3. Microorganisms seen by dark field microscope. (a) A mixture of radiolarian shells. (b) Photomicrograph of Treponema pallidum

34 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY D = O/2NA Where d = resolution and O = wavelength of light

Fig. 4.3. Microorganisms seen by dark field microscope. (a) A mixture of radiolarian shells. (b) Photomicrograph of Treponema pallidum

2. Dark Field Microscopy The effect produced by the dark field technique is that of a dark background against which objects are brilliantly illuminated. The condenser has a stop plate which blocks the light over the center of the field where specimen lies. Most of the light directed through the condenser does not enter the objectives, the field is essentially dark. However some of the light rays will be scattered if the transparent medium contains objects such as microbial cells. The scattered light will enter the objective and reach the eye, thus the object will appear bright in a dark microscope. 3. Fluorescence Microscopy Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wavelength and lesser energy contents. Such substances are called fluorescent and the phenomenon is termed fluorescence. In practice, microorganisms are stained with a fluorescent dye and then illuminated with blue light. The blue light is absorbed and green light is emitted by the dye.

2. Dark Field Microscopy The effect produced by the dark field technique is that of a dark background against which objects are brilliantly illuminated. The condenser has a stop plate which blocks the light over the center of the field where specimen lies. Most of the light directed through the condenser does not enter the objectives, the field is essentially dark. However some of the light rays will be scattered if the transparent medium contains objects such as microbial cells. The scattered light will enter the objective and reach the eye, thus the object will appear bright in a dark microscope. 3. Fluorescence Microscopy Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wavelength and lesser energy contents. Such substances are called fluorescent and the phenomenon is termed fluorescence. In practice, microorganisms are stained with a fluorescent dye and then illuminated with blue light. The blue light is absorbed and green light is emitted by the dye.

Fluorescence microscopy is commonly used in the clinical laboratory for the rapid detection and identification of bacterial antigens in tissue smears, sections, as well as the rapid identification of many disease-causing microorganisms. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence microscope.

Fluorescence microscopy is commonly used in the clinical laboratory for the rapid detection and identification of bacterial antigens in tissue smears, sections, as well as the rapid identification of many disease-causing microorganisms. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence microscope.

34 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

34 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

D = O/2NA Where d = resolution and O = wavelength of light

Fig. 4.3. Microorganisms seen by dark field microscope. (a) A mixture of radiolarian shells. (b) Photomicrograph of Treponema pallidum

D = O/2NA Where d = resolution and O = wavelength of light

Fig. 4.3. Microorganisms seen by dark field microscope. (a) A mixture of radiolarian shells. (b) Photomicrograph of Treponema pallidum

2. Dark Field Microscopy The effect produced by the dark field technique is that of a dark background against which objects are brilliantly illuminated. The condenser has a stop plate which blocks the light over the center of the field where specimen lies. Most of the light directed through the condenser does not enter the objectives, the field is essentially dark. However some of the light rays will be scattered if the transparent medium contains objects such as microbial cells. The scattered light will enter the objective and reach the eye, thus the object will appear bright in a dark microscope. 3. Fluorescence Microscopy Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wavelength and lesser energy contents. Such substances are called fluorescent and the phenomenon is termed fluorescence. In practice, microorganisms are stained with a fluorescent dye and then illuminated with blue light. The blue light is absorbed and green light is emitted by the dye.

2. Dark Field Microscopy The effect produced by the dark field technique is that of a dark background against which objects are brilliantly illuminated. The condenser has a stop plate which blocks the light over the center of the field where specimen lies. Most of the light directed through the condenser does not enter the objectives, the field is essentially dark. However some of the light rays will be scattered if the transparent medium contains objects such as microbial cells. The scattered light will enter the objective and reach the eye, thus the object will appear bright in a dark microscope. 3. Fluorescence Microscopy Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wavelength and lesser energy contents. Such substances are called fluorescent and the phenomenon is termed fluorescence. In practice, microorganisms are stained with a fluorescent dye and then illuminated with blue light. The blue light is absorbed and green light is emitted by the dye.

Fluorescence microscopy is commonly used in the clinical laboratory for the rapid detection and identification of bacterial antigens in tissue smears, sections, as well as the rapid identification of many disease-causing microorganisms. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence microscope.

Fluorescence microscopy is commonly used in the clinical laboratory for the rapid detection and identification of bacterial antigens in tissue smears, sections, as well as the rapid identification of many disease-causing microorganisms. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence microscope.

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IDENTIFICATION OF MICROBES . . . .

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Fig. 4.4: Microorganisms seen by phase contrast microscope. (a) Yeast cells and (b) Protozoan cells

Fig. 4.4: Microorganisms seen by phase contrast microscope. (a) Yeast cells and (b) Protozoan cells

4. Phase Contrast Microscopy Certain transparent, colourless living microorganisms and their internal organelles are impossible to detect by ordinary bright-field or dark-field microscopy because they do not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding environment or the rest of the microorganism. Microorganisms and their organelles are only visible when they absorb, reflect, refract, or diffract more light than their environment.

4. Phase Contrast Microscopy Certain transparent, colourless living microorganisms and their internal organelles are impossible to detect by ordinary bright-field or dark-field microscopy because they do not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding environment or the rest of the microorganism. Microorganisms and their organelles are only visible when they absorb, reflect, refract, or diffract more light than their environment.

So phase contrast microscopy is used to study living cells and tissues without staining like bacteria and their associated structures (such as endospores). The various cell organelles are different in there refractive index or thickness because of different composition. This technique is based on the fact that light passing through one material and into another material of a slightly different refractive index will undergo a change in phase. These differences in phase are translated into variations in brightness of the structures and hence are detectable by the eye.

So phase contrast microscopy is used to study living cells and tissues without staining like bacteria and their associated structures (such as endospores). The various cell organelles are different in there refractive index or thickness because of different composition. This technique is based on the fact that light passing through one material and into another material of a slightly different refractive index will undergo a change in phase. These differences in phase are translated into variations in brightness of the structures and hence are detectable by the eye.

4.8.2 Electron Microscope The electron microscope (EM) permits a direct study of biological ultra-structure. Practical resolving power of modern electron microscopes for biological samples is ~2 Pm. Electron Microscope is of two types. 1. Transmission electron microscope 2. Scanning electron microscope.

4.8.2 Electron Microscope The electron microscope (EM) permits a direct study of biological ultra-structure. Practical resolving power of modern electron microscopes for biological samples is ~2 Pm. Electron Microscope is of two types. 1. Transmission electron microscope 2. Scanning electron microscope.

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IDENTIFICATION OF MICROBES . . . .

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Fig. 4.4: Microorganisms seen by phase contrast microscope. (a) Yeast cells and (b) Protozoan cells

Fig. 4.4: Microorganisms seen by phase contrast microscope. (a) Yeast cells and (b) Protozoan cells

4. Phase Contrast Microscopy Certain transparent, colourless living microorganisms and their internal organelles are impossible to detect by ordinary bright-field or dark-field microscopy because they do not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding environment or the rest of the microorganism. Microorganisms and their organelles are only visible when they absorb, reflect, refract, or diffract more light than their environment.

4. Phase Contrast Microscopy Certain transparent, colourless living microorganisms and their internal organelles are impossible to detect by ordinary bright-field or dark-field microscopy because they do not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding environment or the rest of the microorganism. Microorganisms and their organelles are only visible when they absorb, reflect, refract, or diffract more light than their environment.

So phase contrast microscopy is used to study living cells and tissues without staining like bacteria and their associated structures (such as endospores). The various cell organelles are different in there refractive index or thickness because of different composition. This technique is based on the fact that light passing through one material and into another material of a slightly different refractive index will undergo a change in phase. These differences in phase are translated into variations in brightness of the structures and hence are detectable by the eye.

So phase contrast microscopy is used to study living cells and tissues without staining like bacteria and their associated structures (such as endospores). The various cell organelles are different in there refractive index or thickness because of different composition. This technique is based on the fact that light passing through one material and into another material of a slightly different refractive index will undergo a change in phase. These differences in phase are translated into variations in brightness of the structures and hence are detectable by the eye.

4.8.2 Electron Microscope The electron microscope (EM) permits a direct study of biological ultra-structure. Practical resolving power of modern electron microscopes for biological samples is ~2 Pm. Electron Microscope is of two types. 1. Transmission electron microscope 2. Scanning electron microscope.

4.8.2 Electron Microscope The electron microscope (EM) permits a direct study of biological ultra-structure. Practical resolving power of modern electron microscopes for biological samples is ~2 Pm. Electron Microscope is of two types. 1. Transmission electron microscope 2. Scanning electron microscope.

36 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 4.5. Electron microscope

1. Transmission Electron Microscopy (TEM) Electron microscopy differs from light microscopy in many respects. In electron microscopy electron beams and magnetic field are used to produce images whereas the light microscope uses light waves and glass lenses. The electron microscope provides tremendous useful magnification, because of the much higher resolution obtained with the extremely short wavelength of electron beams. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to – 400,000. For electron microscopy, the specimen to be examined is prepared as an extremely thin dry on small screens. (Fixing is done with glutaraldehyde and osmium tetroxide). Tissue are dehydrated and embedded in resin prior to sectioning with a microtome (an instrument to cut the specimen). Sections are placed on a small circular copper metal grid for viewing in the microscope) and is introduced into the instruments at a point between the magnetic condenser and the magnetic objectives; this may be viewed on a florescent screen through an airtight “window” or recorded on a photographic plate by a camera in the instrument. 2. Scanning Electron Microscope (SEM) SEM directly produces an image of the three dimensional structure of the surface of a specimen. SEM is usually a smaller, cheaper, and simpler device than a TEM. Whereas in a TEM electrons are passed through a specimen, in SEM electrons are scattered or emitted from the specimen’s surface; only surface features have evident. SEM gives the structure of surface only while TEM gives the internal structure.

36 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 4.5. Electron microscope

1. Transmission Electron Microscopy (TEM) Electron microscopy differs from light microscopy in many respects. In electron microscopy electron beams and magnetic field are used to produce images whereas the light microscope uses light waves and glass lenses. The electron microscope provides tremendous useful magnification, because of the much higher resolution obtained with the extremely short wavelength of electron beams. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to – 400,000. For electron microscopy, the specimen to be examined is prepared as an extremely thin dry on small screens. (Fixing is done with glutaraldehyde and osmium tetroxide). Tissue are dehydrated and embedded in resin prior to sectioning with a microtome (an instrument to cut the specimen). Sections are placed on a small circular copper metal grid for viewing in the microscope) and is introduced into the instruments at a point between the magnetic condenser and the magnetic objectives; this may be viewed on a florescent screen through an airtight “window” or recorded on a photographic plate by a camera in the instrument. 2. Scanning Electron Microscope (SEM) SEM directly produces an image of the three dimensional structure of the surface of a specimen. SEM is usually a smaller, cheaper, and simpler device than a TEM. Whereas in a TEM electrons are passed through a specimen, in SEM electrons are scattered or emitted from the specimen’s surface; only surface features have evident. SEM gives the structure of surface only while TEM gives the internal structure.

36 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 4.5. Electron microscope

1. Transmission Electron Microscopy (TEM) Electron microscopy differs from light microscopy in many respects. In electron microscopy electron beams and magnetic field are used to produce images whereas the light microscope uses light waves and glass lenses. The electron microscope provides tremendous useful magnification, because of the much higher resolution obtained with the extremely short wavelength of electron beams. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to – 400,000. For electron microscopy, the specimen to be examined is prepared as an extremely thin dry on small screens. (Fixing is done with glutaraldehyde and osmium tetroxide). Tissue are dehydrated and embedded in resin prior to sectioning with a microtome (an instrument to cut the specimen). Sections are placed on a small circular copper metal grid for viewing in the microscope) and is introduced into the instruments at a point between the magnetic condenser and the magnetic objectives; this may be viewed on a florescent screen through an airtight “window” or recorded on a photographic plate by a camera in the instrument. 2. Scanning Electron Microscope (SEM) SEM directly produces an image of the three dimensional structure of the surface of a specimen. SEM is usually a smaller, cheaper, and simpler device than a TEM. Whereas in a TEM electrons are passed through a specimen, in SEM electrons are scattered or emitted from the specimen’s surface; only surface features have evident. SEM gives the structure of surface only while TEM gives the internal structure.

36 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 4.5. Electron microscope

1. Transmission Electron Microscopy (TEM) Electron microscopy differs from light microscopy in many respects. In electron microscopy electron beams and magnetic field are used to produce images whereas the light microscope uses light waves and glass lenses. The electron microscope provides tremendous useful magnification, because of the much higher resolution obtained with the extremely short wavelength of electron beams. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to – 400,000. For electron microscopy, the specimen to be examined is prepared as an extremely thin dry on small screens. (Fixing is done with glutaraldehyde and osmium tetroxide). Tissue are dehydrated and embedded in resin prior to sectioning with a microtome (an instrument to cut the specimen). Sections are placed on a small circular copper metal grid for viewing in the microscope) and is introduced into the instruments at a point between the magnetic condenser and the magnetic objectives; this may be viewed on a florescent screen through an airtight “window” or recorded on a photographic plate by a camera in the instrument. 2. Scanning Electron Microscope (SEM) SEM directly produces an image of the three dimensional structure of the surface of a specimen. SEM is usually a smaller, cheaper, and simpler device than a TEM. Whereas in a TEM electrons are passed through a specimen, in SEM electrons are scattered or emitted from the specimen’s surface; only surface features have evident. SEM gives the structure of surface only while TEM gives the internal structure.

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37

Fig. 4.6. Structure taken by the electron microscope (internal structure of the eukaryotic cell).

Fig. 4.6. Structure taken by the electron microscope (internal structure of the eukaryotic cell).

Fig.4.7: Sketch of scanning electron microscope showing path of electron beams.

Fig.4.7: Sketch of scanning electron microscope showing path of electron beams.

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Fig. 4.6. Structure taken by the electron microscope (internal structure of the eukaryotic cell).

Fig. 4.6. Structure taken by the electron microscope (internal structure of the eukaryotic cell).

Fig.4.7: Sketch of scanning electron microscope showing path of electron beams.

Fig.4.7: Sketch of scanning electron microscope showing path of electron beams.

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38 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Sample preparation: Specimen is fixed, dried and coated with a thin layer of heavy metal. Specimen may be rapidly frozen and then transferred to a cooled specimen stage for directly examination. Specimen is scanned with a narrow beam of electrons. Scattered/emitted electrons are detected by detector which controls the intensity of the second beam. The second beam forms an image on a television screen. Image has 3-D quality.

Sample preparation: Specimen is fixed, dried and coated with a thin layer of heavy metal. Specimen may be rapidly frozen and then transferred to a cooled specimen stage for directly examination. Specimen is scanned with a narrow beam of electrons. Scattered/emitted electrons are detected by detector which controls the intensity of the second beam. The second beam forms an image on a television screen. Image has 3-D quality.

Difference between light and electron microscope:

Difference between light and electron microscope:

S.No.

Light Microscope

Electron Microscope

S.No.

Light Microscope

Electron Microscope

1

It is used to study the general structure of cells. Source of illumination is visible light

It is used to study the ultra-structure of cells. Source of illumination is narrow electron beams. High power electromagnets are used. Only dead organisms are examined. Images cannot be seen by human eyes directly. Resolving power is 10Å It can magnify objects up to 3,00,000 times.

1

It is used to study the general structure of cells. Source of illumination is visible light

It is used to study the ultra-structure of cells. Source of illumination is narrow electron beams. High power electromagnets are used. Only dead organisms are examined. Images cannot be seen by human eyes directly. Resolving power is 10Å It can magnify objects up to 3,00,000 times.

2. 3. 4.

Glass lens are used for magnification. Living and dead organisms can be observed directly by human eye.

5. 6.

Resolving power is 2500Å It can magnify objects up to 1000 times.

2. 3. 4.

Glass lens are used for magnification. Living and dead organisms can be observed directly by human eye.

5. 6.

Resolving power is 2500Å It can magnify objects up to 1000 times.

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38 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Sample preparation: Specimen is fixed, dried and coated with a thin layer of heavy metal. Specimen may be rapidly frozen and then transferred to a cooled specimen stage for directly examination. Specimen is scanned with a narrow beam of electrons. Scattered/emitted electrons are detected by detector which controls the intensity of the second beam. The second beam forms an image on a television screen. Image has 3-D quality.

Sample preparation: Specimen is fixed, dried and coated with a thin layer of heavy metal. Specimen may be rapidly frozen and then transferred to a cooled specimen stage for directly examination. Specimen is scanned with a narrow beam of electrons. Scattered/emitted electrons are detected by detector which controls the intensity of the second beam. The second beam forms an image on a television screen. Image has 3-D quality.

Difference between light and electron microscope:

Difference between light and electron microscope:

S.No.

Light Microscope

Electron Microscope

S.No.

Light Microscope

Electron Microscope

1

It is used to study the general structure of cells. Source of illumination is visible light

It is used to study the ultra-structure of cells. Source of illumination is narrow electron beams. High power electromagnets are used. Only dead organisms are examined. Images cannot be seen by human eyes directly. Resolving power is 10Å It can magnify objects up to 3,00,000 times.

1

It is used to study the general structure of cells. Source of illumination is visible light

It is used to study the ultra-structure of cells. Source of illumination is narrow electron beams. High power electromagnets are used. Only dead organisms are examined. Images cannot be seen by human eyes directly. Resolving power is 10Å It can magnify objects up to 3,00,000 times.

2. 3. 4.

Glass lens are used for magnification. Living and dead organisms can be observed directly by human eye.

5. 6.

Resolving power is 2500Å It can magnify objects up to 1000 times.

2. 3. 4.

Glass lens are used for magnification. Living and dead organisms can be observed directly by human eye.

5. 6.

Resolving power is 2500Å It can magnify objects up to 1000 times.

SECTION B

SECTION B

SECTION B

SECTION B

5

5

Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc.

Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc.

5.1

NUTRITION, CULTIVATION, AND ISOLATION OF BACTERIA

5.1

NUTRITION, CULTIVATION, AND ISOLATION OF BACTERIA

Bacteria exhibit different modes of nutrition. On this basis, broadly two types of bacteria can be recognized—autotrophic bacteria and heterotrophic bacteria.

Bacteria exhibit different modes of nutrition. On this basis, broadly two types of bacteria can be recognized—autotrophic bacteria and heterotrophic bacteria.

5.1.1 Autotrophic Bacteria: (Source of carbon is CO2)

5.1.1 Autotrophic Bacteria: (Source of carbon is CO2)

1. Photosynthetic bacteria

1. Photosynthetic bacteria

This type of bacteria use sunlight as a source of energy and the electron donors are H2O, H2S, S and other sulphur containing inorganic compounds.

This type of bacteria use sunlight as a source of energy and the electron donors are H2O, H2S, S and other sulphur containing inorganic compounds.

5

5

Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc.

Nutrition, Cultivation, Isolation of Bacteria, Actinomycetes, Fungi, Virus, etc.

5.1

NUTRITION, CULTIVATION, AND ISOLATION OF BACTERIA

5.1

NUTRITION, CULTIVATION, AND ISOLATION OF BACTERIA

Bacteria exhibit different modes of nutrition. On this basis, broadly two types of bacteria can be recognized—autotrophic bacteria and heterotrophic bacteria.

Bacteria exhibit different modes of nutrition. On this basis, broadly two types of bacteria can be recognized—autotrophic bacteria and heterotrophic bacteria.

5.1.1 Autotrophic Bacteria: (Source of carbon is CO2)

5.1.1 Autotrophic Bacteria: (Source of carbon is CO2)

1. Photosynthetic bacteria

1. Photosynthetic bacteria

This type of bacteria use sunlight as a source of energy and the electron donors are H2O, H2S, S and other sulphur containing inorganic compounds.

This type of bacteria use sunlight as a source of energy and the electron donors are H2O, H2S, S and other sulphur containing inorganic compounds.

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42 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

a. Green sulphur bacteria: Green sulphur bacteria contain bacteriochlorophyll which is chemically similar to chlorophyll, e.g., Chlorobium thiothrix.

a. Green sulphur bacteria: Green sulphur bacteria contain bacteriochlorophyll which is chemically similar to chlorophyll, e.g., Chlorobium thiothrix.

They usually live in anaerobic conditions such as sulphur and other sulphur containing inorganic compounds.

They usually live in anaerobic conditions such as sulphur and other sulphur containing inorganic compounds.

b. Purple sulphur bacteria:These bacteria contain a pigment called bacteriopurpurin and bacteriochalorophyll. In this case in place of H2S or elemental S, The electron donors are some other sulphur containing organic and inorganic compounds. But sulphur is not the by-product, e.g., Chromatium.

b. Purple sulphur bacteria:These bacteria contain a pigment called bacteriopurpurin and bacteriochalorophyll. In this case in place of H2S or elemental S, The electron donors are some other sulphur containing organic and inorganic compounds. But sulphur is not the by-product, e.g., Chromatium.

2. Chemoautotrophic or chemosynthetic bacteria

2. Chemoautotrophic or chemosynthetic bacteria

These bacteria are without photosynthetic pigments. These are oxidizable inorganic substances such as H2, H2S, Fe++, Mn++ NO2–, NH4+ or elemental sulphur as the source of energy. a. Nitrifying bacteria: These are mainly found in soil. Through their metabolic activities, they convert ammonia or ammonium ions into nitrates (NO3–). The formation of nitrates from NH3 or NH4+ is termed nitrification. This process occurs in two steps:

These bacteria are without photosynthetic pigments. These are oxidizable inorganic substances such as H2, H2S, Fe++, Mn++ NO2–, NH4+ or elemental sulphur as the source of energy. a. Nitrifying bacteria: These are mainly found in soil. Through their metabolic activities, they convert ammonia or ammonium ions into nitrates (NO3–). The formation of nitrates from NH3 or NH4+ is termed nitrification. This process occurs in two steps:

Step 1. Oxidation of ammonia into nitrite ions in the presence of nitrosomonas.

Step 1. Oxidation of ammonia into nitrite ions in the presence of nitrosomonas.

Step 2. Oxidation of nitrite ions into nitrate ions in the presence of nitrobactor.

Step 2. Oxidation of nitrite ions into nitrate ions in the presence of nitrobactor.

b. Iron bacteria: They are found in iron containing rocks, e.g., ferrobacillus leptothrix.

b. Iron bacteria: They are found in iron containing rocks, e.g., ferrobacillus leptothrix.

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42 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

a. Green sulphur bacteria: Green sulphur bacteria contain bacteriochlorophyll which is chemically similar to chlorophyll, e.g., Chlorobium thiothrix.

a. Green sulphur bacteria: Green sulphur bacteria contain bacteriochlorophyll which is chemically similar to chlorophyll, e.g., Chlorobium thiothrix.

They usually live in anaerobic conditions such as sulphur and other sulphur containing inorganic compounds.

They usually live in anaerobic conditions such as sulphur and other sulphur containing inorganic compounds.

b. Purple sulphur bacteria:These bacteria contain a pigment called bacteriopurpurin and bacteriochalorophyll. In this case in place of H2S or elemental S, The electron donors are some other sulphur containing organic and inorganic compounds. But sulphur is not the by-product, e.g., Chromatium.

b. Purple sulphur bacteria:These bacteria contain a pigment called bacteriopurpurin and bacteriochalorophyll. In this case in place of H2S or elemental S, The electron donors are some other sulphur containing organic and inorganic compounds. But sulphur is not the by-product, e.g., Chromatium.

2. Chemoautotrophic or chemosynthetic bacteria

2. Chemoautotrophic or chemosynthetic bacteria

These bacteria are without photosynthetic pigments. These are oxidizable inorganic substances such as H2, H2S, Fe++, Mn++ NO2–, NH4+ or elemental sulphur as the source of energy. a. Nitrifying bacteria: These are mainly found in soil. Through their metabolic activities, they convert ammonia or ammonium ions into nitrates (NO3–). The formation of nitrates from NH3 or NH4+ is termed nitrification. This process occurs in two steps:

These bacteria are without photosynthetic pigments. These are oxidizable inorganic substances such as H2, H2S, Fe++, Mn++ NO2–, NH4+ or elemental sulphur as the source of energy. a. Nitrifying bacteria: These are mainly found in soil. Through their metabolic activities, they convert ammonia or ammonium ions into nitrates (NO3–). The formation of nitrates from NH3 or NH4+ is termed nitrification. This process occurs in two steps:

Step 1. Oxidation of ammonia into nitrite ions in the presence of nitrosomonas.

Step 1. Oxidation of ammonia into nitrite ions in the presence of nitrosomonas.

Step 2. Oxidation of nitrite ions into nitrate ions in the presence of nitrobactor.

Step 2. Oxidation of nitrite ions into nitrate ions in the presence of nitrobactor.

b. Iron bacteria: They are found in iron containing rocks, e.g., ferrobacillus leptothrix.

b. Iron bacteria: They are found in iron containing rocks, e.g., ferrobacillus leptothrix.

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c. Sulphur bacteria: They are capable if utilizing H,S and free sulphur to produce H2SO4, e.g., Thiobacillus. d. Methanogens (hydrogen bacteria): These chemosynthetic bacteria produce methane (CH 4) and water from H 2 and CO 2, e.g., Hydrogenomonas.

c. Sulphur bacteria: They are capable if utilizing H,S and free sulphur to produce H2SO4, e.g., Thiobacillus. d. Methanogens (hydrogen bacteria): These chemosynthetic bacteria produce methane (CH 4) and water from H 2 and CO 2, e.g., Hydrogenomonas.

Note: After adding methanogens to a mixture of decomposers and animal waste. It is possible to use the resultant (biogas) to run a generator to produce electricity. e. Carbon monoxide bacteria: They utilize CO and them to CO2, e.g., Carboxydomonas.

Note: After adding methanogens to a mixture of decomposers and animal waste. It is possible to use the resultant (biogas) to run a generator to produce electricity. e. Carbon monoxide bacteria: They utilize CO and them to CO2, e.g., Carboxydomonas.

5.1.2 Heterotrophic Bacteria Bacteria that are unable to synthesize their own metabolites and depend on preformed organic compounds are called heterotrophic bacteria. They are of different types: 1. Photoheterotrophic bacteria: These bacteria possess bacteriochlorophyll and are able to use sunlight as their source of energy. They obtain carbon from organic compounds, made by other organisms and are called photoheterotrophic. Alcohol, fatty acids and a variety of other organic substances serves as electron donors for the photosynthetic reaction, e.g., Purple non-sulphur bacteria. 2. Chemo-heterotrophic bacteria: They take their food from another plants and animals. These bacteria are nutritionally similar to animals. They obtain both energy and carbon sources from organic compounds, made by other organisms. They may be classified into three categories. a. Parasitic bacteria: These bacteria can live in or upon living organisms, known as host, from which they obtain their food and usually shelter. They may be obligate parasites (grow only in living cells) and facultative parasites (grow on dead materials). The parasites which cause disease are called pathogens, e.g., • Diphtheria is caused by Corynebacterium diphtheriae. • Tuberculosis is caused by Mycobacterium tuberculosis. • Cholera is caused by Vibrio cholera. b. Saprophytic bacteria: Saprophytic bacteria obtain their food from dead and decaying organic matter of all living beings, e.g., Bacillus mycoides.

5.1.2 Heterotrophic Bacteria Bacteria that are unable to synthesize their own metabolites and depend on preformed organic compounds are called heterotrophic bacteria. They are of different types: 1. Photoheterotrophic bacteria: These bacteria possess bacteriochlorophyll and are able to use sunlight as their source of energy. They obtain carbon from organic compounds, made by other organisms and are called photoheterotrophic. Alcohol, fatty acids and a variety of other organic substances serves as electron donors for the photosynthetic reaction, e.g., Purple non-sulphur bacteria. 2. Chemo-heterotrophic bacteria: They take their food from another plants and animals. These bacteria are nutritionally similar to animals. They obtain both energy and carbon sources from organic compounds, made by other organisms. They may be classified into three categories. a. Parasitic bacteria: These bacteria can live in or upon living organisms, known as host, from which they obtain their food and usually shelter. They may be obligate parasites (grow only in living cells) and facultative parasites (grow on dead materials). The parasites which cause disease are called pathogens, e.g., • Diphtheria is caused by Corynebacterium diphtheriae. • Tuberculosis is caused by Mycobacterium tuberculosis. • Cholera is caused by Vibrio cholera. b. Saprophytic bacteria: Saprophytic bacteria obtain their food from dead and decaying organic matter of all living beings, e.g., Bacillus mycoides.

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c. Sulphur bacteria: They are capable if utilizing H,S and free sulphur to produce H2SO4, e.g., Thiobacillus. d. Methanogens (hydrogen bacteria): These chemosynthetic bacteria produce methane (CH 4) and water from H 2 and CO 2, e.g., Hydrogenomonas.

c. Sulphur bacteria: They are capable if utilizing H,S and free sulphur to produce H2SO4, e.g., Thiobacillus. d. Methanogens (hydrogen bacteria): These chemosynthetic bacteria produce methane (CH 4) and water from H 2 and CO 2, e.g., Hydrogenomonas.

Note: After adding methanogens to a mixture of decomposers and animal waste. It is possible to use the resultant (biogas) to run a generator to produce electricity. e. Carbon monoxide bacteria: They utilize CO and them to CO2, e.g., Carboxydomonas.

Note: After adding methanogens to a mixture of decomposers and animal waste. It is possible to use the resultant (biogas) to run a generator to produce electricity. e. Carbon monoxide bacteria: They utilize CO and them to CO2, e.g., Carboxydomonas.

5.1.2 Heterotrophic Bacteria Bacteria that are unable to synthesize their own metabolites and depend on preformed organic compounds are called heterotrophic bacteria. They are of different types: 1. Photoheterotrophic bacteria: These bacteria possess bacteriochlorophyll and are able to use sunlight as their source of energy. They obtain carbon from organic compounds, made by other organisms and are called photoheterotrophic. Alcohol, fatty acids and a variety of other organic substances serves as electron donors for the photosynthetic reaction, e.g., Purple non-sulphur bacteria. 2. Chemo-heterotrophic bacteria: They take their food from another plants and animals. These bacteria are nutritionally similar to animals. They obtain both energy and carbon sources from organic compounds, made by other organisms. They may be classified into three categories. a. Parasitic bacteria: These bacteria can live in or upon living organisms, known as host, from which they obtain their food and usually shelter. They may be obligate parasites (grow only in living cells) and facultative parasites (grow on dead materials). The parasites which cause disease are called pathogens, e.g., • Diphtheria is caused by Corynebacterium diphtheriae. • Tuberculosis is caused by Mycobacterium tuberculosis. • Cholera is caused by Vibrio cholera. b. Saprophytic bacteria: Saprophytic bacteria obtain their food from dead and decaying organic matter of all living beings, e.g., Bacillus mycoides.

5.1.2 Heterotrophic Bacteria Bacteria that are unable to synthesize their own metabolites and depend on preformed organic compounds are called heterotrophic bacteria. They are of different types: 1. Photoheterotrophic bacteria: These bacteria possess bacteriochlorophyll and are able to use sunlight as their source of energy. They obtain carbon from organic compounds, made by other organisms and are called photoheterotrophic. Alcohol, fatty acids and a variety of other organic substances serves as electron donors for the photosynthetic reaction, e.g., Purple non-sulphur bacteria. 2. Chemo-heterotrophic bacteria: They take their food from another plants and animals. These bacteria are nutritionally similar to animals. They obtain both energy and carbon sources from organic compounds, made by other organisms. They may be classified into three categories. a. Parasitic bacteria: These bacteria can live in or upon living organisms, known as host, from which they obtain their food and usually shelter. They may be obligate parasites (grow only in living cells) and facultative parasites (grow on dead materials). The parasites which cause disease are called pathogens, e.g., • Diphtheria is caused by Corynebacterium diphtheriae. • Tuberculosis is caused by Mycobacterium tuberculosis. • Cholera is caused by Vibrio cholera. b. Saprophytic bacteria: Saprophytic bacteria obtain their food from dead and decaying organic matter of all living beings, e.g., Bacillus mycoides.

44 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

44 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

c. Symbiotic bacteria: Some bacteria live symbiotically with other plants or animals, e.g., Rhizobium legumenosarum.

c. Symbiotic bacteria: Some bacteria live symbiotically with other plants or animals, e.g., Rhizobium legumenosarum.

5.1.3 Growth Curve When bacteria are cultured in appropriate fluid culture media, there would be an increase in the size of bacteria without any multiplication for some time called, lag phase. During this phase growth of bacteria takes place. This is followed by multiplication and increase in the number of bacteria to the extent that media look turbid to the naked eye known as log phase. After some time growth becomes stationary and later on declines. The following curve is obtained during the growth of bacteria:

5.1.3 Growth Curve When bacteria are cultured in appropriate fluid culture media, there would be an increase in the size of bacteria without any multiplication for some time called, lag phase. During this phase growth of bacteria takes place. This is followed by multiplication and increase in the number of bacteria to the extent that media look turbid to the naked eye known as log phase. After some time growth becomes stationary and later on declines. The following curve is obtained during the growth of bacteria:

Fig. 5.1. Growth curve of viable bacteria.

Fig. 5.1. Growth curve of viable bacteria.

Lag phase has short duration and during this phase: 1. Increase bacterial cell size. 2. Increase metabolic rate. 3. Adaptation to new environment and necessary enzymes and metabolites are synthesized for multiplication in log phase. 4. The length of lag phase depends upon: a. Type of bacteria. b. Better the culture medium, shorter the lag phase. c. Size of inoculum. d. Environmental factors like temperature, pH etc.

44 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Lag phase has short duration and during this phase: 1. Increase bacterial cell size. 2. Increase metabolic rate. 3. Adaptation to new environment and necessary enzymes and metabolites are synthesized for multiplication in log phase. 4. The length of lag phase depends upon: a. Type of bacteria. b. Better the culture medium, shorter the lag phase. c. Size of inoculum. d. Environmental factors like temperature, pH etc.

44 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

c. Symbiotic bacteria: Some bacteria live symbiotically with other plants or animals, e.g., Rhizobium legumenosarum.

c. Symbiotic bacteria: Some bacteria live symbiotically with other plants or animals, e.g., Rhizobium legumenosarum.

5.1.3 Growth Curve When bacteria are cultured in appropriate fluid culture media, there would be an increase in the size of bacteria without any multiplication for some time called, lag phase. During this phase growth of bacteria takes place. This is followed by multiplication and increase in the number of bacteria to the extent that media look turbid to the naked eye known as log phase. After some time growth becomes stationary and later on declines. The following curve is obtained during the growth of bacteria:

5.1.3 Growth Curve When bacteria are cultured in appropriate fluid culture media, there would be an increase in the size of bacteria without any multiplication for some time called, lag phase. During this phase growth of bacteria takes place. This is followed by multiplication and increase in the number of bacteria to the extent that media look turbid to the naked eye known as log phase. After some time growth becomes stationary and later on declines. The following curve is obtained during the growth of bacteria:

Fig. 5.1. Growth curve of viable bacteria.

Fig. 5.1. Growth curve of viable bacteria.

Lag phase has short duration and during this phase: 1. Increase bacterial cell size. 2. Increase metabolic rate. 3. Adaptation to new environment and necessary enzymes and metabolites are synthesized for multiplication in log phase. 4. The length of lag phase depends upon: a. Type of bacteria. b. Better the culture medium, shorter the lag phase. c. Size of inoculum. d. Environmental factors like temperature, pH etc.

Lag phase has short duration and during this phase: 1. Increase bacterial cell size. 2. Increase metabolic rate. 3. Adaptation to new environment and necessary enzymes and metabolites are synthesized for multiplication in log phase. 4. The length of lag phase depends upon: a. Type of bacteria. b. Better the culture medium, shorter the lag phase. c. Size of inoculum. d. Environmental factors like temperature, pH etc.

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Log Phase: During this phase cells start dividing and their number increases by geometric progression with time. Logarithms of viable count plotting time give straight line. In this period: 1. Bacteria have high rate of metabolism. 2. Bacteria develop best morphologically with typical biochemical reactions. 3. Bacteria are more sensitive to antibiotics. This phase depends upon: a. Nature of bacteria b. Temperature c. Rate of penetration of the medium depends on the concentration of material in the medium.

Log Phase: During this phase cells start dividing and their number increases by geometric progression with time. Logarithms of viable count plotting time give straight line. In this period: 1. Bacteria have high rate of metabolism. 2. Bacteria develop best morphologically with typical biochemical reactions. 3. Bacteria are more sensitive to antibiotics. This phase depends upon: a. Nature of bacteria b. Temperature c. Rate of penetration of the medium depends on the concentration of material in the medium.

Stationary Phase: After some time a stage comes when rate of multiplication and death becomes almost equal. It may be due to: 1. Depletion of nutrition. 2. Accumulation of toxic products. Sporulation may occur during this stage.

Stationary Phase: After some time a stage comes when rate of multiplication and death becomes almost equal. It may be due to: 1. Depletion of nutrition. 2. Accumulation of toxic products. Sporulation may occur during this stage.

Decline Phase: During this phase population of bacteria decreases due to death of cells. Factors responsible for this phase are: 1. Nutrition exhaustion. 2. Toxic accumulation. 3. Autolytic enzymes are produce in both decline and in this phase.

Decline Phase: During this phase population of bacteria decreases due to death of cells. Factors responsible for this phase are: 1. Nutrition exhaustion. 2. Toxic accumulation. 3. Autolytic enzymes are produce in both decline and in this phase.

5.1.4 Cultivation of Bacteria For the cultivation of bacteria following things are necessarily required: 1. Nutritional requirement i. All the organisms require a source of energy. Some take energy from chemical groups are designated as chemotrophs and other from radiant energy (light) are called phototrophs. ii. All organisms require a source of electron for their metabolism. Some organisms use reduced inorganic compounds as electron donors and are termed lithotrophs (some are chemolithotrophs and others photolithotrophs). Other organisms use organic compounds as electron donors and are called organotrophs (some are chemoorganotrophs and others are photoorganotrophs). iii. All organisms require carbon in some form for synthesizing cell components. iv. All organisms require nitrogen in some form for cell components. Some bacteria can use atmospheric nitrogen. v. All organisms require oxygen, sulphur, and phosphorus for cell components. Sulphur is needed for synthesis of amino acids and phosphorus is required for synthesis of nucleic acids, phospholipids and nucleotides etc. vi. All living organisms require metal ions such as K+, Ca++, Mg++, And Fe++ for normal growth and some other trace elements such as Zn++, Cu++, Mn++, Mo++. Ni++, B+++, and Co++ are also needed.

5.1.4 Cultivation of Bacteria For the cultivation of bacteria following things are necessarily required: 1. Nutritional requirement i. All the organisms require a source of energy. Some take energy from chemical groups are designated as chemotrophs and other from radiant energy (light) are called phototrophs. ii. All organisms require a source of electron for their metabolism. Some organisms use reduced inorganic compounds as electron donors and are termed lithotrophs (some are chemolithotrophs and others photolithotrophs). Other organisms use organic compounds as electron donors and are called organotrophs (some are chemoorganotrophs and others are photoorganotrophs). iii. All organisms require carbon in some form for synthesizing cell components. iv. All organisms require nitrogen in some form for cell components. Some bacteria can use atmospheric nitrogen. v. All organisms require oxygen, sulphur, and phosphorus for cell components. Sulphur is needed for synthesis of amino acids and phosphorus is required for synthesis of nucleic acids, phospholipids and nucleotides etc. vi. All living organisms require metal ions such as K+, Ca++, Mg++, And Fe++ for normal growth and some other trace elements such as Zn++, Cu++, Mn++, Mo++. Ni++, B+++, and Co++ are also needed.

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Log Phase: During this phase cells start dividing and their number increases by geometric progression with time. Logarithms of viable count plotting time give straight line. In this period: 1. Bacteria have high rate of metabolism. 2. Bacteria develop best morphologically with typical biochemical reactions. 3. Bacteria are more sensitive to antibiotics. This phase depends upon: a. Nature of bacteria b. Temperature c. Rate of penetration of the medium depends on the concentration of material in the medium.

Log Phase: During this phase cells start dividing and their number increases by geometric progression with time. Logarithms of viable count plotting time give straight line. In this period: 1. Bacteria have high rate of metabolism. 2. Bacteria develop best morphologically with typical biochemical reactions. 3. Bacteria are more sensitive to antibiotics. This phase depends upon: a. Nature of bacteria b. Temperature c. Rate of penetration of the medium depends on the concentration of material in the medium.

Stationary Phase: After some time a stage comes when rate of multiplication and death becomes almost equal. It may be due to: 1. Depletion of nutrition. 2. Accumulation of toxic products. Sporulation may occur during this stage.

Stationary Phase: After some time a stage comes when rate of multiplication and death becomes almost equal. It may be due to: 1. Depletion of nutrition. 2. Accumulation of toxic products. Sporulation may occur during this stage.

Decline Phase: During this phase population of bacteria decreases due to death of cells. Factors responsible for this phase are: 1. Nutrition exhaustion. 2. Toxic accumulation. 3. Autolytic enzymes are produce in both decline and in this phase.

Decline Phase: During this phase population of bacteria decreases due to death of cells. Factors responsible for this phase are: 1. Nutrition exhaustion. 2. Toxic accumulation. 3. Autolytic enzymes are produce in both decline and in this phase.

5.1.4 Cultivation of Bacteria For the cultivation of bacteria following things are necessarily required: 1. Nutritional requirement i. All the organisms require a source of energy. Some take energy from chemical groups are designated as chemotrophs and other from radiant energy (light) are called phototrophs. ii. All organisms require a source of electron for their metabolism. Some organisms use reduced inorganic compounds as electron donors and are termed lithotrophs (some are chemolithotrophs and others photolithotrophs). Other organisms use organic compounds as electron donors and are called organotrophs (some are chemoorganotrophs and others are photoorganotrophs). iii. All organisms require carbon in some form for synthesizing cell components. iv. All organisms require nitrogen in some form for cell components. Some bacteria can use atmospheric nitrogen. v. All organisms require oxygen, sulphur, and phosphorus for cell components. Sulphur is needed for synthesis of amino acids and phosphorus is required for synthesis of nucleic acids, phospholipids and nucleotides etc. vi. All living organisms require metal ions such as K+, Ca++, Mg++, And Fe++ for normal growth and some other trace elements such as Zn++, Cu++, Mn++, Mo++. Ni++, B+++, and Co++ are also needed.

5.1.4 Cultivation of Bacteria For the cultivation of bacteria following things are necessarily required: 1. Nutritional requirement i. All the organisms require a source of energy. Some take energy from chemical groups are designated as chemotrophs and other from radiant energy (light) are called phototrophs. ii. All organisms require a source of electron for their metabolism. Some organisms use reduced inorganic compounds as electron donors and are termed lithotrophs (some are chemolithotrophs and others photolithotrophs). Other organisms use organic compounds as electron donors and are called organotrophs (some are chemoorganotrophs and others are photoorganotrophs). iii. All organisms require carbon in some form for synthesizing cell components. iv. All organisms require nitrogen in some form for cell components. Some bacteria can use atmospheric nitrogen. v. All organisms require oxygen, sulphur, and phosphorus for cell components. Sulphur is needed for synthesis of amino acids and phosphorus is required for synthesis of nucleic acids, phospholipids and nucleotides etc. vi. All living organisms require metal ions such as K+, Ca++, Mg++, And Fe++ for normal growth and some other trace elements such as Zn++, Cu++, Mn++, Mo++. Ni++, B+++, and Co++ are also needed.

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46 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

vii. All living organisms contain vitamins and vitamin-like compounds. viii. All living organisms require water and in the case of bacteria all nutrients must be in aqueous solution. 2. Nutritional types of media: Either they are autotrophic or heterotrophic (described before in nutrition of bacteria). a. Cultivation of autotrophic bacteria: for the cultivation of autotrophic bacteria they require inorganic chemical compounds which are known so it is called chemically defined media or synthetic media. For example, chemical media required for Nitrosomonas europaea.

vii. All living organisms contain vitamins and vitamin-like compounds. viii. All living organisms require water and in the case of bacteria all nutrients must be in aqueous solution. 2. Nutritional types of media: Either they are autotrophic or heterotrophic (described before in nutrition of bacteria). a. Cultivation of autotrophic bacteria: for the cultivation of autotrophic bacteria they require inorganic chemical compounds which are known so it is called chemically defined media or synthetic media. For example, chemical media required for Nitrosomonas europaea.

S.No

Ingredient

Amount

S.No

Ingredient

Amount

01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11.

NH4Cl MgSO4:7H2O K2HPO4 CaCl2.2H2O Chelated iron MnCl2,4H2O Na2MoO 4.2H2O ZnSO4.7H2O CuSO4,5H2 O CoCl2,6H2O Distilled water

0.8 g 0.2 g 0.016 g 0.02 g 0.001 g 0.0002 g 0.0001 g 0.0001 g 0.00002 g 0.000002 g 1000 ml

01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11.

NH4Cl MgSO4:7H2O K2HPO4 CaCl2.2H2O Chelated iron MnCl2,4H2O Na2MoO 4.2H2O ZnSO4.7H2O CuSO4,5H2 O CoCl2,6H2O Distilled water

0.8 g 0.2 g 0.016 g 0.02 g 0.001 g 0.0002 g 0.0001 g 0.0001 g 0.00002 g 0.000002 g 1000 ml

The organisms can transform these compounds into carbohydrates, proteins, nucleic acids, lipids, vitamins, and other complex organic substances that constitute the living cell. b. Cultivation of heterotrophs: For the growth of heterotrophs specific media is used. The main nutrients which are required for heterotrophs are: inorganic salts, organic carbons, nitrogen compounds, amino acids and vitamins, e.g., E. coli, salmonella typhi, Proteus vulgaris, 3. Bacteriological media: There are different types of media that are used in laboratories for the growth of bacteria such as nutrient agar media, L.B agar media, nutrient broth etc. Composition for nutrient agar media, L.B. agar media and nutrient broth.

The organisms can transform these compounds into carbohydrates, proteins, nucleic acids, lipids, vitamins, and other complex organic substances that constitute the living cell. b. Cultivation of heterotrophs: For the growth of heterotrophs specific media is used. The main nutrients which are required for heterotrophs are: inorganic salts, organic carbons, nitrogen compounds, amino acids and vitamins, e.g., E. coli, salmonella typhi, Proteus vulgaris, 3. Bacteriological media: There are different types of media that are used in laboratories for the growth of bacteria such as nutrient agar media, L.B agar media, nutrient broth etc. Composition for nutrient agar media, L.B. agar media and nutrient broth.

Nutrient broth

Nutrient broth

Beef extract Peptone Water

0.3 gm 0.5 gm 100 ml

Nutrient agar media

L.B agar media

Beef extract Peptone Agar Water

Yeast extract Tryptone NaCl Agar Water

0.3 gm 0.5 gm 1.5 gm 100 ml

0.5 gm 1.0 gm 1.0 gm 1.5 gm 100 ml

Beef extract Peptone Water

4. Types of media: Many different types of media are needed to facilitate recognition enumeration and isolation of certain types of bacteria. On the basis of their function or application media may be classified as follows: a. Selective Media: These types of media enhance the growth of particular types of bacteria and do not enhance the other types of organisms.

46 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

0.3 gm 0.5 gm 100 ml

Nutrient agar media

L.B agar media

Beef extract Peptone Agar Water

Yeast extract Tryptone NaCl Agar Water

0.3 gm 0.5 gm 1.5 gm 100 ml

0.5 gm 1.0 gm 1.0 gm 1.5 gm 100 ml

4. Types of media: Many different types of media are needed to facilitate recognition enumeration and isolation of certain types of bacteria. On the basis of their function or application media may be classified as follows: a. Selective Media: These types of media enhance the growth of particular types of bacteria and do not enhance the other types of organisms.

46 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

vii. All living organisms contain vitamins and vitamin-like compounds. viii. All living organisms require water and in the case of bacteria all nutrients must be in aqueous solution. 2. Nutritional types of media: Either they are autotrophic or heterotrophic (described before in nutrition of bacteria). a. Cultivation of autotrophic bacteria: for the cultivation of autotrophic bacteria they require inorganic chemical compounds which are known so it is called chemically defined media or synthetic media. For example, chemical media required for Nitrosomonas europaea.

vii. All living organisms contain vitamins and vitamin-like compounds. viii. All living organisms require water and in the case of bacteria all nutrients must be in aqueous solution. 2. Nutritional types of media: Either they are autotrophic or heterotrophic (described before in nutrition of bacteria). a. Cultivation of autotrophic bacteria: for the cultivation of autotrophic bacteria they require inorganic chemical compounds which are known so it is called chemically defined media or synthetic media. For example, chemical media required for Nitrosomonas europaea.

S.No

Ingredient

Amount

S.No

Ingredient

Amount

01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11.

NH4Cl MgSO4:7H2O K2HPO4 CaCl2.2H2O Chelated iron MnCl2,4H2O Na2MoO 4.2H2O ZnSO4.7H2O CuSO4,5H2 O CoCl2,6H2O Distilled water

0.8 g 0.2 g 0.016 g 0.02 g 0.001 g 0.0002 g 0.0001 g 0.0001 g 0.00002 g 0.000002 g 1000 ml

01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11.

NH4Cl MgSO4:7H2O K2HPO4 CaCl2.2H2O Chelated iron MnCl2,4H2O Na2MoO 4.2H2O ZnSO4.7H2O CuSO4,5H2 O CoCl2,6H2O Distilled water

0.8 g 0.2 g 0.016 g 0.02 g 0.001 g 0.0002 g 0.0001 g 0.0001 g 0.00002 g 0.000002 g 1000 ml

The organisms can transform these compounds into carbohydrates, proteins, nucleic acids, lipids, vitamins, and other complex organic substances that constitute the living cell. b. Cultivation of heterotrophs: For the growth of heterotrophs specific media is used. The main nutrients which are required for heterotrophs are: inorganic salts, organic carbons, nitrogen compounds, amino acids and vitamins, e.g., E. coli, salmonella typhi, Proteus vulgaris, 3. Bacteriological media: There are different types of media that are used in laboratories for the growth of bacteria such as nutrient agar media, L.B agar media, nutrient broth etc. Composition for nutrient agar media, L.B. agar media and nutrient broth.

The organisms can transform these compounds into carbohydrates, proteins, nucleic acids, lipids, vitamins, and other complex organic substances that constitute the living cell. b. Cultivation of heterotrophs: For the growth of heterotrophs specific media is used. The main nutrients which are required for heterotrophs are: inorganic salts, organic carbons, nitrogen compounds, amino acids and vitamins, e.g., E. coli, salmonella typhi, Proteus vulgaris, 3. Bacteriological media: There are different types of media that are used in laboratories for the growth of bacteria such as nutrient agar media, L.B agar media, nutrient broth etc. Composition for nutrient agar media, L.B. agar media and nutrient broth.

Nutrient broth

Nutrient broth

Beef extract Peptone Water

0.3 gm 0.5 gm 100 ml

Nutrient agar media

L.B agar media

Beef extract Peptone Agar Water

Yeast extract Tryptone NaCl Agar Water

0.3 gm 0.5 gm 1.5 gm 100 ml

0.5 gm 1.0 gm 1.0 gm 1.5 gm 100 ml

4. Types of media: Many different types of media are needed to facilitate recognition enumeration and isolation of certain types of bacteria. On the basis of their function or application media may be classified as follows: a. Selective Media: These types of media enhance the growth of particular types of bacteria and do not enhance the other types of organisms.

Beef extract Peptone Water

0.3 gm 0.5 gm 100 ml

Nutrient agar media

L.B agar media

Beef extract Peptone Agar Water

Yeast extract Tryptone NaCl Agar Water

0.3 gm 0.5 gm 1.5 gm 100 ml

0.5 gm 1.0 gm 1.0 gm 1.5 gm 100 ml

4. Types of media: Many different types of media are needed to facilitate recognition enumeration and isolation of certain types of bacteria. On the basis of their function or application media may be classified as follows: a. Selective Media: These types of media enhance the growth of particular types of bacteria and do not enhance the other types of organisms.

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47

Example 1. A media in which cellulose is the carbon source will specially select for growth of cellulose utilizing bacteria when it is incubated with a soil sample containing many types of bacteria. Example 2. For the isolation of gonorrhea causing organisms, Neisseria gonorrhoeae are isolated by the use of media containing certain antibiotics which do not affect N. gonorrhoeae but inhibit the growth of other microorganisms. b. Differential Media: Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various kinds of bacteria. For example, if a mixture of bacteria is inoculated onto a blood containing agar media (blood agar), some of the bacteria may haemolyze the red blood cells, others do not. Thus, one can distinguish between hemolytic and non-hemolytic bacteria on the same medium. c. Basal media: These media include nutrient broth, containing beef extract, peptone and water which are the simplest liquid media and form basis of all laboratory media. d. Enriched media: Addition of substances such as blood and serum to basal media results in formation of enriched media. e. Storage media: These media help in preservation and storage of bacteria for a considerable long period. 5. Physical conditions required for growth: In addition to knowing the proper nutrients for the cultivation of bacteria, it is also necessary to know the physical environment like temperature, pH, and gaseous conditions in which the organisms will grow best. a. Temperature: Bacteria are grown at various temperatures, according to their growth behaviour. Bacteria are mainly of three types. i. Psychrophiles: Bacteria which can grow at O°C or lower are called psychrophiles, but they grow best at higher temperature. During isolation of such types of bacteria it is necessary to maintain the source samples at cold temperatures from the time they are collected and also chill all media before attempting isolation. ii. Mesophiles: Mesophiles grow best within a temperature range of approximately 25°C to 40°C. For example, all bacteria that are pathogenic for humans and warm-blooded animals are mesophiles, most growing best at about body temperature (37°C). iii. Thermophiles: Thermophiles grow best at temperature above 45°C. The growth range of many thermophiles extends into the mesophilic region, these species are designated facultative thermophiles. Other thermophiles cannot grow in the mesophilic range, these are termed true thermophiles or stenothermophiles.

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Example 1. A media in which cellulose is the carbon source will specially select for growth of cellulose utilizing bacteria when it is incubated with a soil sample containing many types of bacteria. Example 2. For the isolation of gonorrhea causing organisms, Neisseria gonorrhoeae are isolated by the use of media containing certain antibiotics which do not affect N. gonorrhoeae but inhibit the growth of other microorganisms. b. Differential Media: Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various kinds of bacteria. For example, if a mixture of bacteria is inoculated onto a blood containing agar media (blood agar), some of the bacteria may haemolyze the red blood cells, others do not. Thus, one can distinguish between hemolytic and non-hemolytic bacteria on the same medium. c. Basal media: These media include nutrient broth, containing beef extract, peptone and water which are the simplest liquid media and form basis of all laboratory media. d. Enriched media: Addition of substances such as blood and serum to basal media results in formation of enriched media. e. Storage media: These media help in preservation and storage of bacteria for a considerable long period. 5. Physical conditions required for growth: In addition to knowing the proper nutrients for the cultivation of bacteria, it is also necessary to know the physical environment like temperature, pH, and gaseous conditions in which the organisms will grow best. a. Temperature: Bacteria are grown at various temperatures, according to their growth behaviour. Bacteria are mainly of three types. i. Psychrophiles: Bacteria which can grow at O°C or lower are called psychrophiles, but they grow best at higher temperature. During isolation of such types of bacteria it is necessary to maintain the source samples at cold temperatures from the time they are collected and also chill all media before attempting isolation. ii. Mesophiles: Mesophiles grow best within a temperature range of approximately 25°C to 40°C. For example, all bacteria that are pathogenic for humans and warm-blooded animals are mesophiles, most growing best at about body temperature (37°C). iii. Thermophiles: Thermophiles grow best at temperature above 45°C. The growth range of many thermophiles extends into the mesophilic region, these species are designated facultative thermophiles. Other thermophiles cannot grow in the mesophilic range, these are termed true thermophiles or stenothermophiles.

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Example 1. A media in which cellulose is the carbon source will specially select for growth of cellulose utilizing bacteria when it is incubated with a soil sample containing many types of bacteria. Example 2. For the isolation of gonorrhea causing organisms, Neisseria gonorrhoeae are isolated by the use of media containing certain antibiotics which do not affect N. gonorrhoeae but inhibit the growth of other microorganisms. b. Differential Media: Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various kinds of bacteria. For example, if a mixture of bacteria is inoculated onto a blood containing agar media (blood agar), some of the bacteria may haemolyze the red blood cells, others do not. Thus, one can distinguish between hemolytic and non-hemolytic bacteria on the same medium. c. Basal media: These media include nutrient broth, containing beef extract, peptone and water which are the simplest liquid media and form basis of all laboratory media. d. Enriched media: Addition of substances such as blood and serum to basal media results in formation of enriched media. e. Storage media: These media help in preservation and storage of bacteria for a considerable long period. 5. Physical conditions required for growth: In addition to knowing the proper nutrients for the cultivation of bacteria, it is also necessary to know the physical environment like temperature, pH, and gaseous conditions in which the organisms will grow best. a. Temperature: Bacteria are grown at various temperatures, according to their growth behaviour. Bacteria are mainly of three types. i. Psychrophiles: Bacteria which can grow at O°C or lower are called psychrophiles, but they grow best at higher temperature. During isolation of such types of bacteria it is necessary to maintain the source samples at cold temperatures from the time they are collected and also chill all media before attempting isolation. ii. Mesophiles: Mesophiles grow best within a temperature range of approximately 25°C to 40°C. For example, all bacteria that are pathogenic for humans and warm-blooded animals are mesophiles, most growing best at about body temperature (37°C). iii. Thermophiles: Thermophiles grow best at temperature above 45°C. The growth range of many thermophiles extends into the mesophilic region, these species are designated facultative thermophiles. Other thermophiles cannot grow in the mesophilic range, these are termed true thermophiles or stenothermophiles.

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Example 1. A media in which cellulose is the carbon source will specially select for growth of cellulose utilizing bacteria when it is incubated with a soil sample containing many types of bacteria. Example 2. For the isolation of gonorrhea causing organisms, Neisseria gonorrhoeae are isolated by the use of media containing certain antibiotics which do not affect N. gonorrhoeae but inhibit the growth of other microorganisms. b. Differential Media: Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various kinds of bacteria. For example, if a mixture of bacteria is inoculated onto a blood containing agar media (blood agar), some of the bacteria may haemolyze the red blood cells, others do not. Thus, one can distinguish between hemolytic and non-hemolytic bacteria on the same medium. c. Basal media: These media include nutrient broth, containing beef extract, peptone and water which are the simplest liquid media and form basis of all laboratory media. d. Enriched media: Addition of substances such as blood and serum to basal media results in formation of enriched media. e. Storage media: These media help in preservation and storage of bacteria for a considerable long period. 5. Physical conditions required for growth: In addition to knowing the proper nutrients for the cultivation of bacteria, it is also necessary to know the physical environment like temperature, pH, and gaseous conditions in which the organisms will grow best. a. Temperature: Bacteria are grown at various temperatures, according to their growth behaviour. Bacteria are mainly of three types. i. Psychrophiles: Bacteria which can grow at O°C or lower are called psychrophiles, but they grow best at higher temperature. During isolation of such types of bacteria it is necessary to maintain the source samples at cold temperatures from the time they are collected and also chill all media before attempting isolation. ii. Mesophiles: Mesophiles grow best within a temperature range of approximately 25°C to 40°C. For example, all bacteria that are pathogenic for humans and warm-blooded animals are mesophiles, most growing best at about body temperature (37°C). iii. Thermophiles: Thermophiles grow best at temperature above 45°C. The growth range of many thermophiles extends into the mesophilic region, these species are designated facultative thermophiles. Other thermophiles cannot grow in the mesophilic range, these are termed true thermophiles or stenothermophiles.

48 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

48 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

List of some bacteria with regard to temperature at which they grow.

List of some bacteria with regard to temperature at which they grow.

Temperature of growth (°C)

Temperature of growth (°C)

Name of bacteria

Minimum

Optimum

Maximum

Name of bacteria

Minimum

Optimum

Maximum

Vibrio marinus strain MP-1 Vibrio psychroerythrus Pseudomonas fluorescens Streptococcus aureus Corynebacterium diphtheria Neisseria gonorrhoeae Streptococcus thermophilus Thermoactinomyces vulgaris Thermos aquaticus

-1 0 4 6.5 15 30 20 27-30 40

15 15 25-30 30-37 37 35-36 40-45 60 70-72

20 19 40 46 40 38.5 50 65-70 79

Vibrio marinus strain MP-1 Vibrio psychroerythrus Pseudomonas fluorescens Streptococcus aureus Corynebacterium diphtheria Neisseria gonorrhoeae Streptococcus thermophilus Thermoactinomyces vulgaris Thermos aquaticus

-1 0 4 6.5 15 30 20 27-30 40

15 15 25-30 30-37 37 35-36 40-45 60 70-72

20 19 40 46 40 38.5 50 65-70 79

b. Gaseous Requirement: The principal gases that effect bacterial growth are oxygen and carbon dioxide. Bacteria display such a wide variety of responses to free oxygen that it is convenient to divide them into four groups on the following bases: (i) Aerobic bacteria require oxygen for growth and can grow when incubated in an air atmosphere. (ii) Anaerobic bacteria do not use oxygen to obtain energy, moreover oxygen is toxic for them and they cannot grow when incubated in an air atmosphere. Some can tolerate low level of oxygen (tolerant anaerobes) but others (stringent or strict anaerobes) cannot tolerate even low levels and may die upon brief exposure to air. (iii) Facultative anaerobic bacteria do not require oxygen for growth, although they may use it for energy production if it is available. They are not inhibited by oxygen and usually grow as well as under an air atmosphere as they do in the absence of oxygen. (iv) Microaerophilic bacteria require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere. c. pH (Acidity or alkanity): For most bacteria the optimum pH for the growth lies between 6.5 and 7.5, and the limit generally lies somewhere between 5 and 9, but some bacteria can grow at lower pH. For example, Thiobacillus thiooxidans can grow 2-3.5 at acidic range. Some bacteria can grow at basic pH range.

b. Gaseous Requirement: The principal gases that effect bacterial growth are oxygen and carbon dioxide. Bacteria display such a wide variety of responses to free oxygen that it is convenient to divide them into four groups on the following bases: (i) Aerobic bacteria require oxygen for growth and can grow when incubated in an air atmosphere. (ii) Anaerobic bacteria do not use oxygen to obtain energy, moreover oxygen is toxic for them and they cannot grow when incubated in an air atmosphere. Some can tolerate low level of oxygen (tolerant anaerobes) but others (stringent or strict anaerobes) cannot tolerate even low levels and may die upon brief exposure to air. (iii) Facultative anaerobic bacteria do not require oxygen for growth, although they may use it for energy production if it is available. They are not inhibited by oxygen and usually grow as well as under an air atmosphere as they do in the absence of oxygen. (iv) Microaerophilic bacteria require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere. c. pH (Acidity or alkanity): For most bacteria the optimum pH for the growth lies between 6.5 and 7.5, and the limit generally lies somewhere between 5 and 9, but some bacteria can grow at lower pH. For example, Thiobacillus thiooxidans can grow 2-3.5 at acidic range. Some bacteria can grow at basic pH range.

5.1.5 Isolation of Bacteria The microbial population in our environment is very large and complex. For example, a single sneeze may disperse from 10,000 to 1,00,000 bacteria. One gram of faeces may contain 1011 bacteria. Our environment—air, soil, and water likewise consists of mixed population of bacteria plus other microbes. So to

5.1.5 Isolation of Bacteria The microbial population in our environment is very large and complex. For example, a single sneeze may disperse from 10,000 to 1,00,000 bacteria. One gram of faeces may contain 1011 bacteria. Our environment—air, soil, and water likewise consists of mixed population of bacteria plus other microbes. So to

48 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

48 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

List of some bacteria with regard to temperature at which they grow.

List of some bacteria with regard to temperature at which they grow.

Temperature of growth (°C)

Temperature of growth (°C)

Name of bacteria

Minimum

Optimum

Maximum

Name of bacteria

Minimum

Optimum

Maximum

Vibrio marinus strain MP-1 Vibrio psychroerythrus Pseudomonas fluorescens Streptococcus aureus Corynebacterium diphtheria Neisseria gonorrhoeae Streptococcus thermophilus Thermoactinomyces vulgaris Thermos aquaticus

-1 0 4 6.5 15 30 20 27-30 40

15 15 25-30 30-37 37 35-36 40-45 60 70-72

20 19 40 46 40 38.5 50 65-70 79

Vibrio marinus strain MP-1 Vibrio psychroerythrus Pseudomonas fluorescens Streptococcus aureus Corynebacterium diphtheria Neisseria gonorrhoeae Streptococcus thermophilus Thermoactinomyces vulgaris Thermos aquaticus

-1 0 4 6.5 15 30 20 27-30 40

15 15 25-30 30-37 37 35-36 40-45 60 70-72

20 19 40 46 40 38.5 50 65-70 79

b. Gaseous Requirement: The principal gases that effect bacterial growth are oxygen and carbon dioxide. Bacteria display such a wide variety of responses to free oxygen that it is convenient to divide them into four groups on the following bases: (i) Aerobic bacteria require oxygen for growth and can grow when incubated in an air atmosphere. (ii) Anaerobic bacteria do not use oxygen to obtain energy, moreover oxygen is toxic for them and they cannot grow when incubated in an air atmosphere. Some can tolerate low level of oxygen (tolerant anaerobes) but others (stringent or strict anaerobes) cannot tolerate even low levels and may die upon brief exposure to air. (iii) Facultative anaerobic bacteria do not require oxygen for growth, although they may use it for energy production if it is available. They are not inhibited by oxygen and usually grow as well as under an air atmosphere as they do in the absence of oxygen. (iv) Microaerophilic bacteria require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere. c. pH (Acidity or alkanity): For most bacteria the optimum pH for the growth lies between 6.5 and 7.5, and the limit generally lies somewhere between 5 and 9, but some bacteria can grow at lower pH. For example, Thiobacillus thiooxidans can grow 2-3.5 at acidic range. Some bacteria can grow at basic pH range.

b. Gaseous Requirement: The principal gases that effect bacterial growth are oxygen and carbon dioxide. Bacteria display such a wide variety of responses to free oxygen that it is convenient to divide them into four groups on the following bases: (i) Aerobic bacteria require oxygen for growth and can grow when incubated in an air atmosphere. (ii) Anaerobic bacteria do not use oxygen to obtain energy, moreover oxygen is toxic for them and they cannot grow when incubated in an air atmosphere. Some can tolerate low level of oxygen (tolerant anaerobes) but others (stringent or strict anaerobes) cannot tolerate even low levels and may die upon brief exposure to air. (iii) Facultative anaerobic bacteria do not require oxygen for growth, although they may use it for energy production if it is available. They are not inhibited by oxygen and usually grow as well as under an air atmosphere as they do in the absence of oxygen. (iv) Microaerophilic bacteria require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere. c. pH (Acidity or alkanity): For most bacteria the optimum pH for the growth lies between 6.5 and 7.5, and the limit generally lies somewhere between 5 and 9, but some bacteria can grow at lower pH. For example, Thiobacillus thiooxidans can grow 2-3.5 at acidic range. Some bacteria can grow at basic pH range.

5.1.5 Isolation of Bacteria The microbial population in our environment is very large and complex. For example, a single sneeze may disperse from 10,000 to 1,00,000 bacteria. One gram of faeces may contain 1011 bacteria. Our environment—air, soil, and water likewise consists of mixed population of bacteria plus other microbes. So to

5.1.5 Isolation of Bacteria The microbial population in our environment is very large and complex. For example, a single sneeze may disperse from 10,000 to 1,00,000 bacteria. One gram of faeces may contain 1011 bacteria. Our environment—air, soil, and water likewise consists of mixed population of bacteria plus other microbes. So to

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study the characteristics of one species, that species must be isolated form in selective methods first. Once the single species or pure culture is isolated, that can be maintained or preserved. All the selective methods are used to growth of the desired species and discouraging or even killing the other organisms present in the mixed culture. To isolate a particular type of bacteria from the mixed culture there are different types of selective methods are used like physical, chemical, biological and selection in nature.

study the characteristics of one species, that species must be isolated form in selective methods first. Once the single species or pure culture is isolated, that can be maintained or preserved. All the selective methods are used to growth of the desired species and discouraging or even killing the other organisms present in the mixed culture. To isolate a particular type of bacteria from the mixed culture there are different types of selective methods are used like physical, chemical, biological and selection in nature.

I. Physical Method of Selection 1. Incubation Temperature: To select the psychrophilic, mesophilic and thermophilic bacteria, cultures are incubated at different temperatures. For example, for psychrophiles the incubation temperature is 0°C to 5°C, and for thermophiles the incubation temperature is = 60°C. 2. pH of media: i. To select the acid tolerant bacteria, a low pH medium can be used. For example, to select for the lactobacilli present in cheddar cheese, the pH of the medium is maintained at 5.35 with an acetic acid/acetate buffer. Other microorganisms cannot grow at such low pH. ii. Similarly, to select for alkali-tolerant organisms, a high pH medium can be used. For example to select for the cholera-causing bacterium, vibrio cholera, from stool sample, we can use a medium with a pH of 8.5. Most of the intestinal bacteria are unable to grow at this pH. 3. Heat Treatment: To select the endospore-forming bacteria, a mixed culture can be heated to 80°C for 10 minutes before being used to inoculate culture media. Vegetative cells are killed at this temperature but the endospore forming bacteria survive and subsequently germinate at this temperature. 4. Cell size and motility: We can sometimes make use of a small cell diameter or of bacterial motility to achieve selection. For instance, Treponema species from the human oral cavity can be selected by taking advantage of both of these properties. i. A membrane filter having pore size of 0.15Pm is placed on the surface of an agar plate and gingival scrapings are placed on the filter. The unusually small size of treponemes allows them to penetrate the pores of the filter to reach the underlying agar. ii. Moreover, treponemes have the ability to swim through solid agar media, consequently, they migrate away from the filter and grow to form a hazy zone within the agar, from which they can be subcultured.

I. Physical Method of Selection 1. Incubation Temperature: To select the psychrophilic, mesophilic and thermophilic bacteria, cultures are incubated at different temperatures. For example, for psychrophiles the incubation temperature is 0°C to 5°C, and for thermophiles the incubation temperature is = 60°C. 2. pH of media: i. To select the acid tolerant bacteria, a low pH medium can be used. For example, to select for the lactobacilli present in cheddar cheese, the pH of the medium is maintained at 5.35 with an acetic acid/acetate buffer. Other microorganisms cannot grow at such low pH. ii. Similarly, to select for alkali-tolerant organisms, a high pH medium can be used. For example to select for the cholera-causing bacterium, vibrio cholera, from stool sample, we can use a medium with a pH of 8.5. Most of the intestinal bacteria are unable to grow at this pH. 3. Heat Treatment: To select the endospore-forming bacteria, a mixed culture can be heated to 80°C for 10 minutes before being used to inoculate culture media. Vegetative cells are killed at this temperature but the endospore forming bacteria survive and subsequently germinate at this temperature. 4. Cell size and motility: We can sometimes make use of a small cell diameter or of bacterial motility to achieve selection. For instance, Treponema species from the human oral cavity can be selected by taking advantage of both of these properties. i. A membrane filter having pore size of 0.15Pm is placed on the surface of an agar plate and gingival scrapings are placed on the filter. The unusually small size of treponemes allows them to penetrate the pores of the filter to reach the underlying agar. ii. Moreover, treponemes have the ability to swim through solid agar media, consequently, they migrate away from the filter and grow to form a hazy zone within the agar, from which they can be subcultured.

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study the characteristics of one species, that species must be isolated form in selective methods first. Once the single species or pure culture is isolated, that can be maintained or preserved. All the selective methods are used to growth of the desired species and discouraging or even killing the other organisms present in the mixed culture. To isolate a particular type of bacteria from the mixed culture there are different types of selective methods are used like physical, chemical, biological and selection in nature.

study the characteristics of one species, that species must be isolated form in selective methods first. Once the single species or pure culture is isolated, that can be maintained or preserved. All the selective methods are used to growth of the desired species and discouraging or even killing the other organisms present in the mixed culture. To isolate a particular type of bacteria from the mixed culture there are different types of selective methods are used like physical, chemical, biological and selection in nature.

I. Physical Method of Selection 1. Incubation Temperature: To select the psychrophilic, mesophilic and thermophilic bacteria, cultures are incubated at different temperatures. For example, for psychrophiles the incubation temperature is 0°C to 5°C, and for thermophiles the incubation temperature is = 60°C. 2. pH of media: i. To select the acid tolerant bacteria, a low pH medium can be used. For example, to select for the lactobacilli present in cheddar cheese, the pH of the medium is maintained at 5.35 with an acetic acid/acetate buffer. Other microorganisms cannot grow at such low pH. ii. Similarly, to select for alkali-tolerant organisms, a high pH medium can be used. For example to select for the cholera-causing bacterium, vibrio cholera, from stool sample, we can use a medium with a pH of 8.5. Most of the intestinal bacteria are unable to grow at this pH. 3. Heat Treatment: To select the endospore-forming bacteria, a mixed culture can be heated to 80°C for 10 minutes before being used to inoculate culture media. Vegetative cells are killed at this temperature but the endospore forming bacteria survive and subsequently germinate at this temperature. 4. Cell size and motility: We can sometimes make use of a small cell diameter or of bacterial motility to achieve selection. For instance, Treponema species from the human oral cavity can be selected by taking advantage of both of these properties. i. A membrane filter having pore size of 0.15Pm is placed on the surface of an agar plate and gingival scrapings are placed on the filter. The unusually small size of treponemes allows them to penetrate the pores of the filter to reach the underlying agar. ii. Moreover, treponemes have the ability to swim through solid agar media, consequently, they migrate away from the filter and grow to form a hazy zone within the agar, from which they can be subcultured.

I. Physical Method of Selection 1. Incubation Temperature: To select the psychrophilic, mesophilic and thermophilic bacteria, cultures are incubated at different temperatures. For example, for psychrophiles the incubation temperature is 0°C to 5°C, and for thermophiles the incubation temperature is = 60°C. 2. pH of media: i. To select the acid tolerant bacteria, a low pH medium can be used. For example, to select for the lactobacilli present in cheddar cheese, the pH of the medium is maintained at 5.35 with an acetic acid/acetate buffer. Other microorganisms cannot grow at such low pH. ii. Similarly, to select for alkali-tolerant organisms, a high pH medium can be used. For example to select for the cholera-causing bacterium, vibrio cholera, from stool sample, we can use a medium with a pH of 8.5. Most of the intestinal bacteria are unable to grow at this pH. 3. Heat Treatment: To select the endospore-forming bacteria, a mixed culture can be heated to 80°C for 10 minutes before being used to inoculate culture media. Vegetative cells are killed at this temperature but the endospore forming bacteria survive and subsequently germinate at this temperature. 4. Cell size and motility: We can sometimes make use of a small cell diameter or of bacterial motility to achieve selection. For instance, Treponema species from the human oral cavity can be selected by taking advantage of both of these properties. i. A membrane filter having pore size of 0.15Pm is placed on the surface of an agar plate and gingival scrapings are placed on the filter. The unusually small size of treponemes allows them to penetrate the pores of the filter to reach the underlying agar. ii. Moreover, treponemes have the ability to swim through solid agar media, consequently, they migrate away from the filter and grow to form a hazy zone within the agar, from which they can be subcultured.

50 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

50 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

II. Chemical Method of Selection 1. By the use of a particular type of substrate: We can select the particular bacteria by the use of a single substrate i.e., a single carbon or nitrogen source. For example, if we wish to isolate a soil bacteria utilizing a very complex organic compound D-conidendrin, a constituent of wood, if this bacteria is isolated in nutrient agar media, our chance of finding is very limited. So we prepare a liquid enrichment medium in which D-conidendrin is the source of carbon. Under these conditions only D-conidendrin utilizing bacteria will be able to grow well and isolated them. The other example is, if we wish to select for nitrogen-fixing bacteria, nitrogen gas can be supplied as the sole nitrogen source, since only nitrogen fixing bacteria will be able to grow well. 2. Use of Dilute Media: Certain aquatic bacteria such as Caulobactor species, are capable of growing with very low levels of carbon or nitrogen sources. Consequently, one way to select for such bacteria is to inoculate a mixed culture a very dilute medium. For example, a broth containing only 0.01 per cent peptone. The medium must have low enough levels of nutrients that other kinds of organisms will not be able to grow well in it. 3. Use of inhibitory or Toxic Chemicals: The addition of low levels of certain chemicals, such as dyes, bile salts, salts of heavy metals, or antibiotics, to culture media can be useful for the selection of certain kind of bacteria. i. Many Gram-negative bacteria can grow in the presence of low concentrations of various dyes that inhibit the growth of Gram-positive bacteria. ii. Similarly intestinal bacteria can grow in the presence of bile salt such as sodium deoxycholate, whereas non-intestinal bacteria are usually inhibited. iii. Campylobactor jejuni is a frequent cause diarrhoea in humans, yet diarrhoeic stool contains many other kinds of bacteria that interfere with the isolation of this species. By incorporating certain antibiotics such as vancomycin, polymyxin, and trimethoprim into the culture medium, most of the contaminants can be inhibited without affecting the growth of C. jejuni.

II. Chemical Method of Selection 1. By the use of a particular type of substrate: We can select the particular bacteria by the use of a single substrate i.e., a single carbon or nitrogen source. For example, if we wish to isolate a soil bacteria utilizing a very complex organic compound D-conidendrin, a constituent of wood, if this bacteria is isolated in nutrient agar media, our chance of finding is very limited. So we prepare a liquid enrichment medium in which D-conidendrin is the source of carbon. Under these conditions only D-conidendrin utilizing bacteria will be able to grow well and isolated them. The other example is, if we wish to select for nitrogen-fixing bacteria, nitrogen gas can be supplied as the sole nitrogen source, since only nitrogen fixing bacteria will be able to grow well. 2. Use of Dilute Media: Certain aquatic bacteria such as Caulobactor species, are capable of growing with very low levels of carbon or nitrogen sources. Consequently, one way to select for such bacteria is to inoculate a mixed culture a very dilute medium. For example, a broth containing only 0.01 per cent peptone. The medium must have low enough levels of nutrients that other kinds of organisms will not be able to grow well in it. 3. Use of inhibitory or Toxic Chemicals: The addition of low levels of certain chemicals, such as dyes, bile salts, salts of heavy metals, or antibiotics, to culture media can be useful for the selection of certain kind of bacteria. i. Many Gram-negative bacteria can grow in the presence of low concentrations of various dyes that inhibit the growth of Gram-positive bacteria. ii. Similarly intestinal bacteria can grow in the presence of bile salt such as sodium deoxycholate, whereas non-intestinal bacteria are usually inhibited. iii. Campylobactor jejuni is a frequent cause diarrhoea in humans, yet diarrhoeic stool contains many other kinds of bacteria that interfere with the isolation of this species. By incorporating certain antibiotics such as vancomycin, polymyxin, and trimethoprim into the culture medium, most of the contaminants can be inhibited without affecting the growth of C. jejuni.

III. Biological Methods of Selection A disease producing species occurring in a mixed culture can often be selected by taking advantage of its pathogenic properties. For example, a sputum sample containing Streptococcus pneumoniae is ordinarily contaminated by many other bacterial species. However, laboratory mice are extremely susceptible to infection by S. pneumoniae, and if the sputum sample is injected into the mouse the pathogen will multiply extensively.

III. Biological Methods of Selection A disease producing species occurring in a mixed culture can often be selected by taking advantage of its pathogenic properties. For example, a sputum sample containing Streptococcus pneumoniae is ordinarily contaminated by many other bacterial species. However, laboratory mice are extremely susceptible to infection by S. pneumoniae, and if the sputum sample is injected into the mouse the pathogen will multiply extensively.

50 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

50 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

II. Chemical Method of Selection 1. By the use of a particular type of substrate: We can select the particular bacteria by the use of a single substrate i.e., a single carbon or nitrogen source. For example, if we wish to isolate a soil bacteria utilizing a very complex organic compound D-conidendrin, a constituent of wood, if this bacteria is isolated in nutrient agar media, our chance of finding is very limited. So we prepare a liquid enrichment medium in which D-conidendrin is the source of carbon. Under these conditions only D-conidendrin utilizing bacteria will be able to grow well and isolated them. The other example is, if we wish to select for nitrogen-fixing bacteria, nitrogen gas can be supplied as the sole nitrogen source, since only nitrogen fixing bacteria will be able to grow well. 2. Use of Dilute Media: Certain aquatic bacteria such as Caulobactor species, are capable of growing with very low levels of carbon or nitrogen sources. Consequently, one way to select for such bacteria is to inoculate a mixed culture a very dilute medium. For example, a broth containing only 0.01 per cent peptone. The medium must have low enough levels of nutrients that other kinds of organisms will not be able to grow well in it. 3. Use of inhibitory or Toxic Chemicals: The addition of low levels of certain chemicals, such as dyes, bile salts, salts of heavy metals, or antibiotics, to culture media can be useful for the selection of certain kind of bacteria. i. Many Gram-negative bacteria can grow in the presence of low concentrations of various dyes that inhibit the growth of Gram-positive bacteria. ii. Similarly intestinal bacteria can grow in the presence of bile salt such as sodium deoxycholate, whereas non-intestinal bacteria are usually inhibited. iii. Campylobactor jejuni is a frequent cause diarrhoea in humans, yet diarrhoeic stool contains many other kinds of bacteria that interfere with the isolation of this species. By incorporating certain antibiotics such as vancomycin, polymyxin, and trimethoprim into the culture medium, most of the contaminants can be inhibited without affecting the growth of C. jejuni.

II. Chemical Method of Selection 1. By the use of a particular type of substrate: We can select the particular bacteria by the use of a single substrate i.e., a single carbon or nitrogen source. For example, if we wish to isolate a soil bacteria utilizing a very complex organic compound D-conidendrin, a constituent of wood, if this bacteria is isolated in nutrient agar media, our chance of finding is very limited. So we prepare a liquid enrichment medium in which D-conidendrin is the source of carbon. Under these conditions only D-conidendrin utilizing bacteria will be able to grow well and isolated them. The other example is, if we wish to select for nitrogen-fixing bacteria, nitrogen gas can be supplied as the sole nitrogen source, since only nitrogen fixing bacteria will be able to grow well. 2. Use of Dilute Media: Certain aquatic bacteria such as Caulobactor species, are capable of growing with very low levels of carbon or nitrogen sources. Consequently, one way to select for such bacteria is to inoculate a mixed culture a very dilute medium. For example, a broth containing only 0.01 per cent peptone. The medium must have low enough levels of nutrients that other kinds of organisms will not be able to grow well in it. 3. Use of inhibitory or Toxic Chemicals: The addition of low levels of certain chemicals, such as dyes, bile salts, salts of heavy metals, or antibiotics, to culture media can be useful for the selection of certain kind of bacteria. i. Many Gram-negative bacteria can grow in the presence of low concentrations of various dyes that inhibit the growth of Gram-positive bacteria. ii. Similarly intestinal bacteria can grow in the presence of bile salt such as sodium deoxycholate, whereas non-intestinal bacteria are usually inhibited. iii. Campylobactor jejuni is a frequent cause diarrhoea in humans, yet diarrhoeic stool contains many other kinds of bacteria that interfere with the isolation of this species. By incorporating certain antibiotics such as vancomycin, polymyxin, and trimethoprim into the culture medium, most of the contaminants can be inhibited without affecting the growth of C. jejuni.

III. Biological Methods of Selection A disease producing species occurring in a mixed culture can often be selected by taking advantage of its pathogenic properties. For example, a sputum sample containing Streptococcus pneumoniae is ordinarily contaminated by many other bacterial species. However, laboratory mice are extremely susceptible to infection by S. pneumoniae, and if the sputum sample is injected into the mouse the pathogen will multiply extensively.

III. Biological Methods of Selection A disease producing species occurring in a mixed culture can often be selected by taking advantage of its pathogenic properties. For example, a sputum sample containing Streptococcus pneumoniae is ordinarily contaminated by many other bacterial species. However, laboratory mice are extremely susceptible to infection by S. pneumoniae, and if the sputum sample is injected into the mouse the pathogen will multiply extensively.

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Nonpathogenic bacteria present in the sample will either be inhibited or killed by the defense mechanisms of the animal. In a sense, the animal serves as the selective medium.

Nonpathogenic bacteria present in the sample will either be inhibited or killed by the defense mechanisms of the animal. In a sense, the animal serves as the selective medium.

IV. Selection in Nature The principle of selection is not limited to the laboratory, it also commonly operates in nature. For instance, the occurrence of high salt concentration in bodies of water such as the dead sea water selects for extreme halophiles such as those of the genus halobacterium. The nodules that occur on the roots of leguminous plants contain bacteria of the genus Rhizobium, which is uniquely suited for nitrogen fixation in association with these plants.

IV. Selection in Nature The principle of selection is not limited to the laboratory, it also commonly operates in nature. For instance, the occurrence of high salt concentration in bodies of water such as the dead sea water selects for extreme halophiles such as those of the genus halobacterium. The nodules that occur on the roots of leguminous plants contain bacteria of the genus Rhizobium, which is uniquely suited for nitrogen fixation in association with these plants.

5.1.6 Pure Culture Isolation A culture that contains only one kind of microorganisms is called a pure culture. A culture which contains more than one kind of microorganisms is called a mixed culture. Pure culture is useful for studying morphology, staining techniques, colony characteristics, biochemical properties and susceptibility to antimicrobial agents of particular microorganisms (e.g., bacteria, fungi, actinomycetes etc). Isolation is the separating of a particular microorganism from the mixed environment or in culture media. Cultivation is the growth of microbial population in artificial environment or in culture media. There are different methods for obtaining a pure culture. One of the most common methods in laboratories is plating method. In which nutrient agar media used as a culture media. The most commonly used method for pure culture isolation are: 1. Streak-plate technique. 2. Pour plate technique. 3. Spread plate technique. 4. Serial dilution technique.

5.1.6 Pure Culture Isolation A culture that contains only one kind of microorganisms is called a pure culture. A culture which contains more than one kind of microorganisms is called a mixed culture. Pure culture is useful for studying morphology, staining techniques, colony characteristics, biochemical properties and susceptibility to antimicrobial agents of particular microorganisms (e.g., bacteria, fungi, actinomycetes etc). Isolation is the separating of a particular microorganism from the mixed environment or in culture media. Cultivation is the growth of microbial population in artificial environment or in culture media. There are different methods for obtaining a pure culture. One of the most common methods in laboratories is plating method. In which nutrient agar media used as a culture media. The most commonly used method for pure culture isolation are: 1. Streak-plate technique. 2. Pour plate technique. 3. Spread plate technique. 4. Serial dilution technique.

The other methods for obtaining pure culture are: 1. Use of enrichment media. 2. Use of differential and selective media. 3. Use of media containing antibiotics. 4. Selected technique for the cultivation of anaerobes.

The other methods for obtaining pure culture are: 1. Use of enrichment media. 2. Use of differential and selective media. 3. Use of media containing antibiotics. 4. Selected technique for the cultivation of anaerobes.

1. Streak-plate technique

1. Streak-plate technique

The streak-plate method offers a most practical method of obtaining discrete colonies and pure cultures. In this method a sterilized loop or transfer needle is dipped into a suitable diluted suspension of organisms which is than streaked on the surface of solidified agar plate. The aim of this exercise is to obtain colonies of microorganisms that are pure. Requirements: 24-hour to 48-hour nutrient broth cultures of microorganism, Nutrient agar plates, Inoculating loop, Bunsen burner, Wax marking pencil.

The streak-plate method offers a most practical method of obtaining discrete colonies and pure cultures. In this method a sterilized loop or transfer needle is dipped into a suitable diluted suspension of organisms which is than streaked on the surface of solidified agar plate. The aim of this exercise is to obtain colonies of microorganisms that are pure. Requirements: 24-hour to 48-hour nutrient broth cultures of microorganism, Nutrient agar plates, Inoculating loop, Bunsen burner, Wax marking pencil.

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Nonpathogenic bacteria present in the sample will either be inhibited or killed by the defense mechanisms of the animal. In a sense, the animal serves as the selective medium.

Nonpathogenic bacteria present in the sample will either be inhibited or killed by the defense mechanisms of the animal. In a sense, the animal serves as the selective medium.

IV. Selection in Nature The principle of selection is not limited to the laboratory, it also commonly operates in nature. For instance, the occurrence of high salt concentration in bodies of water such as the dead sea water selects for extreme halophiles such as those of the genus halobacterium. The nodules that occur on the roots of leguminous plants contain bacteria of the genus Rhizobium, which is uniquely suited for nitrogen fixation in association with these plants.

IV. Selection in Nature The principle of selection is not limited to the laboratory, it also commonly operates in nature. For instance, the occurrence of high salt concentration in bodies of water such as the dead sea water selects for extreme halophiles such as those of the genus halobacterium. The nodules that occur on the roots of leguminous plants contain bacteria of the genus Rhizobium, which is uniquely suited for nitrogen fixation in association with these plants.

5.1.6 Pure Culture Isolation A culture that contains only one kind of microorganisms is called a pure culture. A culture which contains more than one kind of microorganisms is called a mixed culture. Pure culture is useful for studying morphology, staining techniques, colony characteristics, biochemical properties and susceptibility to antimicrobial agents of particular microorganisms (e.g., bacteria, fungi, actinomycetes etc). Isolation is the separating of a particular microorganism from the mixed environment or in culture media. Cultivation is the growth of microbial population in artificial environment or in culture media. There are different methods for obtaining a pure culture. One of the most common methods in laboratories is plating method. In which nutrient agar media used as a culture media. The most commonly used method for pure culture isolation are: 1. Streak-plate technique. 2. Pour plate technique. 3. Spread plate technique. 4. Serial dilution technique.

5.1.6 Pure Culture Isolation A culture that contains only one kind of microorganisms is called a pure culture. A culture which contains more than one kind of microorganisms is called a mixed culture. Pure culture is useful for studying morphology, staining techniques, colony characteristics, biochemical properties and susceptibility to antimicrobial agents of particular microorganisms (e.g., bacteria, fungi, actinomycetes etc). Isolation is the separating of a particular microorganism from the mixed environment or in culture media. Cultivation is the growth of microbial population in artificial environment or in culture media. There are different methods for obtaining a pure culture. One of the most common methods in laboratories is plating method. In which nutrient agar media used as a culture media. The most commonly used method for pure culture isolation are: 1. Streak-plate technique. 2. Pour plate technique. 3. Spread plate technique. 4. Serial dilution technique.

The other methods for obtaining pure culture are: 1. Use of enrichment media. 2. Use of differential and selective media. 3. Use of media containing antibiotics. 4. Selected technique for the cultivation of anaerobes.

The other methods for obtaining pure culture are: 1. Use of enrichment media. 2. Use of differential and selective media. 3. Use of media containing antibiotics. 4. Selected technique for the cultivation of anaerobes.

1. Streak-plate technique

1. Streak-plate technique

The streak-plate method offers a most practical method of obtaining discrete colonies and pure cultures. In this method a sterilized loop or transfer needle is dipped into a suitable diluted suspension of organisms which is than streaked on the surface of solidified agar plate. The aim of this exercise is to obtain colonies of microorganisms that are pure. Requirements: 24-hour to 48-hour nutrient broth cultures of microorganism, Nutrient agar plates, Inoculating loop, Bunsen burner, Wax marking pencil.

The streak-plate method offers a most practical method of obtaining discrete colonies and pure cultures. In this method a sterilized loop or transfer needle is dipped into a suitable diluted suspension of organisms which is than streaked on the surface of solidified agar plate. The aim of this exercise is to obtain colonies of microorganisms that are pure. Requirements: 24-hour to 48-hour nutrient broth cultures of microorganism, Nutrient agar plates, Inoculating loop, Bunsen burner, Wax marking pencil.

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52 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Procedure

Procedure

1. Label all the plates on the bottom with the name of the organism to be inoculated with a wax marking pencil. 2. Hold the tube containing the broth of microorganism in the left hand. 3. Sterilize the loop holding in the right hand, remove the cotton wool plug using the little finger of the right hand and immediately flame the mouth of the tube containing broth (microorganism). 4. Introduce the loop into the broth and withdraw one loopful of culture. 5. Flame the mouth of the tube, replace the cotton wool plug and place the tube in the test tube rack. 6. Lift the Petri plate cover with the left hand and hold it at an angle of 60°. 7. Place the inoculum (the loop containing the droplet of broth) on the agar surface at the edge farthest lines across the surface of area. 8. Reflame and cool the loop and turn the Petri plate to 90°, touch the loop to a corner of the culture in area 1 and streak the inoculum across the agar in area 2 as above. 9. The rest of the agar surface is now used to complete the streaking (as shown in the figure below). 10. Replace the lid of the Petri plate, after completing the streaking and sterilize the loop by flaming. 11. Incubate all the plates in incubator (at the temperature in which microorganisms grow best) in an inverted position for 2 or 3 days (as their growing ability). 12. By taking an isolated colonies from this incubated plate streak the next plate for pure culture isolation and streak in 3 to 4 plates.

1. Label all the plates on the bottom with the name of the organism to be inoculated with a wax marking pencil. 2. Hold the tube containing the broth of microorganism in the left hand. 3. Sterilize the loop holding in the right hand, remove the cotton wool plug using the little finger of the right hand and immediately flame the mouth of the tube containing broth (microorganism). 4. Introduce the loop into the broth and withdraw one loopful of culture. 5. Flame the mouth of the tube, replace the cotton wool plug and place the tube in the test tube rack. 6. Lift the Petri plate cover with the left hand and hold it at an angle of 60°. 7. Place the inoculum (the loop containing the droplet of broth) on the agar surface at the edge farthest lines across the surface of area. 8. Reflame and cool the loop and turn the Petri plate to 90°, touch the loop to a corner of the culture in area 1 and streak the inoculum across the agar in area 2 as above. 9. The rest of the agar surface is now used to complete the streaking (as shown in the figure below). 10. Replace the lid of the Petri plate, after completing the streaking and sterilize the loop by flaming. 11. Incubate all the plates in incubator (at the temperature in which microorganisms grow best) in an inverted position for 2 or 3 days (as their growing ability). 12. By taking an isolated colonies from this incubated plate streak the next plate for pure culture isolation and streak in 3 to 4 plates.

Observation: After incubation examine each of the plates for the growth of colonies.

Observation: After incubation examine each of the plates for the growth of colonies.

Fig. 5.2. Streak plate culture showing isolated colony of bacteria.

Fig. 5.2. Streak plate culture showing isolated colony of bacteria.

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52 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Procedure

Procedure

1. Label all the plates on the bottom with the name of the organism to be inoculated with a wax marking pencil. 2. Hold the tube containing the broth of microorganism in the left hand. 3. Sterilize the loop holding in the right hand, remove the cotton wool plug using the little finger of the right hand and immediately flame the mouth of the tube containing broth (microorganism). 4. Introduce the loop into the broth and withdraw one loopful of culture. 5. Flame the mouth of the tube, replace the cotton wool plug and place the tube in the test tube rack. 6. Lift the Petri plate cover with the left hand and hold it at an angle of 60°. 7. Place the inoculum (the loop containing the droplet of broth) on the agar surface at the edge farthest lines across the surface of area. 8. Reflame and cool the loop and turn the Petri plate to 90°, touch the loop to a corner of the culture in area 1 and streak the inoculum across the agar in area 2 as above. 9. The rest of the agar surface is now used to complete the streaking (as shown in the figure below). 10. Replace the lid of the Petri plate, after completing the streaking and sterilize the loop by flaming. 11. Incubate all the plates in incubator (at the temperature in which microorganisms grow best) in an inverted position for 2 or 3 days (as their growing ability). 12. By taking an isolated colonies from this incubated plate streak the next plate for pure culture isolation and streak in 3 to 4 plates.

1. Label all the plates on the bottom with the name of the organism to be inoculated with a wax marking pencil. 2. Hold the tube containing the broth of microorganism in the left hand. 3. Sterilize the loop holding in the right hand, remove the cotton wool plug using the little finger of the right hand and immediately flame the mouth of the tube containing broth (microorganism). 4. Introduce the loop into the broth and withdraw one loopful of culture. 5. Flame the mouth of the tube, replace the cotton wool plug and place the tube in the test tube rack. 6. Lift the Petri plate cover with the left hand and hold it at an angle of 60°. 7. Place the inoculum (the loop containing the droplet of broth) on the agar surface at the edge farthest lines across the surface of area. 8. Reflame and cool the loop and turn the Petri plate to 90°, touch the loop to a corner of the culture in area 1 and streak the inoculum across the agar in area 2 as above. 9. The rest of the agar surface is now used to complete the streaking (as shown in the figure below). 10. Replace the lid of the Petri plate, after completing the streaking and sterilize the loop by flaming. 11. Incubate all the plates in incubator (at the temperature in which microorganisms grow best) in an inverted position for 2 or 3 days (as their growing ability). 12. By taking an isolated colonies from this incubated plate streak the next plate for pure culture isolation and streak in 3 to 4 plates.

Observation: After incubation examine each of the plates for the growth of colonies.

Observation: After incubation examine each of the plates for the growth of colonies.

Fig. 5.2. Streak plate culture showing isolated colony of bacteria.

Fig. 5.2. Streak plate culture showing isolated colony of bacteria.

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2. Pour-plate technique

2. Pour-plate technique

The pour plate method is based on diluting the mixed culture with liquefied nutrient agar in such a manner that the colonies formed on the plate are countable. Generally a loopful of bacteria is inoculated into tube 1 and dilute into tube 2 and then tube 3. Requirements: Mixed bacterial culture, Petri plates, Hot plate, Inoculum loop, Beaker, Distilled water, Incubator etc.

The pour plate method is based on diluting the mixed culture with liquefied nutrient agar in such a manner that the colonies formed on the plate are countable. Generally a loopful of bacteria is inoculated into tube 1 and dilute into tube 2 and then tube 3. Requirements: Mixed bacterial culture, Petri plates, Hot plate, Inoculum loop, Beaker, Distilled water, Incubator etc.

Procedure

Procedure

1. Inoculate tube 1 with a loopful of bacteria and after properly mixing, transfer a loopful of bacterial culture in tube 2. Shake the tube 2 properly and transfer the loopful of culture from tube 2 to tube 3. 2. Note that all the three tubes contain liquefied nutrient agar which are placed in hot water beaker. This beaker is further kept on hot plate. 3. Pour the nutrient agar media on the three petry plates. After solidifying the nutrient agar medium in the plates, incubate at 25°C for 24 to 48 hours in inverted position in an incubator. Result: the colonies of similar shape, size and colour will be visible.

1. Inoculate tube 1 with a loopful of bacteria and after properly mixing, transfer a loopful of bacterial culture in tube 2. Shake the tube 2 properly and transfer the loopful of culture from tube 2 to tube 3. 2. Note that all the three tubes contain liquefied nutrient agar which are placed in hot water beaker. This beaker is further kept on hot plate. 3. Pour the nutrient agar media on the three petry plates. After solidifying the nutrient agar medium in the plates, incubate at 25°C for 24 to 48 hours in inverted position in an incubator. Result: the colonies of similar shape, size and colour will be visible.

3. Spread-plate Technique

3. Spread-plate Technique

In some cases when the inoculum is transferred into Petri plates the bacterial cells or microbial cells are not separated from each other and develop mixed colonies. Hence isolation of pure culture is difficult. Therefore, spread plate technique is employed. In this technique microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod called spreader when the Petri plate is spinning on a turn table. Requirements: L-shaped glass rod, 95% ethanol, Bunsen burner, Inoculum loop, Beaker, Incubator, Nutrient broth culture of Staphylococcus aureus and Staphylococcus pneumonae.

In some cases when the inoculum is transferred into Petri plates the bacterial cells or microbial cells are not separated from each other and develop mixed colonies. Hence isolation of pure culture is difficult. Therefore, spread plate technique is employed. In this technique microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod called spreader when the Petri plate is spinning on a turn table. Requirements: L-shaped glass rod, 95% ethanol, Bunsen burner, Inoculum loop, Beaker, Incubator, Nutrient broth culture of Staphylococcus aureus and Staphylococcus pneumonae.

Procedure

Procedure

1. Take 3 petri plates and label them A, B and C respectively. 2. Aseptically inoculate the plate A with S. aureus plate B with S pneumonae and plate C with mixed of S. aureus and S. pneumonae. 3. Sterile the bent glass tube over the bunsen burner by using 95% ethanol and cool it for 30 second. 4. Very gently touch the spreader on the surface of agar plate in every direction by rotating the plate or by keeping the plate on turn table. 5. When properly spread the petri plate or stops the turn table put the lid over the petri plate and incubate all the plates at 25°C to 30°C for 24 hours in inverted position in an incubator.

NUTRITION, CULTIVATION, ISOLATION OF BACTERIA....

1. Take 3 petri plates and label them A, B and C respectively. 2. Aseptically inoculate the plate A with S. aureus plate B with S pneumonae and plate C with mixed of S. aureus and S. pneumonae. 3. Sterile the bent glass tube over the bunsen burner by using 95% ethanol and cool it for 30 second. 4. Very gently touch the spreader on the surface of agar plate in every direction by rotating the plate or by keeping the plate on turn table. 5. When properly spread the petri plate or stops the turn table put the lid over the petri plate and incubate all the plates at 25°C to 30°C for 24 hours in inverted position in an incubator.

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2. Pour-plate technique

2. Pour-plate technique

The pour plate method is based on diluting the mixed culture with liquefied nutrient agar in such a manner that the colonies formed on the plate are countable. Generally a loopful of bacteria is inoculated into tube 1 and dilute into tube 2 and then tube 3. Requirements: Mixed bacterial culture, Petri plates, Hot plate, Inoculum loop, Beaker, Distilled water, Incubator etc.

The pour plate method is based on diluting the mixed culture with liquefied nutrient agar in such a manner that the colonies formed on the plate are countable. Generally a loopful of bacteria is inoculated into tube 1 and dilute into tube 2 and then tube 3. Requirements: Mixed bacterial culture, Petri plates, Hot plate, Inoculum loop, Beaker, Distilled water, Incubator etc.

Procedure

Procedure

1. Inoculate tube 1 with a loopful of bacteria and after properly mixing, transfer a loopful of bacterial culture in tube 2. Shake the tube 2 properly and transfer the loopful of culture from tube 2 to tube 3. 2. Note that all the three tubes contain liquefied nutrient agar which are placed in hot water beaker. This beaker is further kept on hot plate. 3. Pour the nutrient agar media on the three petry plates. After solidifying the nutrient agar medium in the plates, incubate at 25°C for 24 to 48 hours in inverted position in an incubator. Result: the colonies of similar shape, size and colour will be visible.

1. Inoculate tube 1 with a loopful of bacteria and after properly mixing, transfer a loopful of bacterial culture in tube 2. Shake the tube 2 properly and transfer the loopful of culture from tube 2 to tube 3. 2. Note that all the three tubes contain liquefied nutrient agar which are placed in hot water beaker. This beaker is further kept on hot plate. 3. Pour the nutrient agar media on the three petry plates. After solidifying the nutrient agar medium in the plates, incubate at 25°C for 24 to 48 hours in inverted position in an incubator. Result: the colonies of similar shape, size and colour will be visible.

3. Spread-plate Technique

3. Spread-plate Technique

In some cases when the inoculum is transferred into Petri plates the bacterial cells or microbial cells are not separated from each other and develop mixed colonies. Hence isolation of pure culture is difficult. Therefore, spread plate technique is employed. In this technique microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod called spreader when the Petri plate is spinning on a turn table. Requirements: L-shaped glass rod, 95% ethanol, Bunsen burner, Inoculum loop, Beaker, Incubator, Nutrient broth culture of Staphylococcus aureus and Staphylococcus pneumonae.

In some cases when the inoculum is transferred into Petri plates the bacterial cells or microbial cells are not separated from each other and develop mixed colonies. Hence isolation of pure culture is difficult. Therefore, spread plate technique is employed. In this technique microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod called spreader when the Petri plate is spinning on a turn table. Requirements: L-shaped glass rod, 95% ethanol, Bunsen burner, Inoculum loop, Beaker, Incubator, Nutrient broth culture of Staphylococcus aureus and Staphylococcus pneumonae.

Procedure

Procedure

1. Take 3 petri plates and label them A, B and C respectively. 2. Aseptically inoculate the plate A with S. aureus plate B with S pneumonae and plate C with mixed of S. aureus and S. pneumonae. 3. Sterile the bent glass tube over the bunsen burner by using 95% ethanol and cool it for 30 second. 4. Very gently touch the spreader on the surface of agar plate in every direction by rotating the plate or by keeping the plate on turn table. 5. When properly spread the petri plate or stops the turn table put the lid over the petri plate and incubate all the plates at 25°C to 30°C for 24 hours in inverted position in an incubator.

1. Take 3 petri plates and label them A, B and C respectively. 2. Aseptically inoculate the plate A with S. aureus plate B with S pneumonae and plate C with mixed of S. aureus and S. pneumonae. 3. Sterile the bent glass tube over the bunsen burner by using 95% ethanol and cool it for 30 second. 4. Very gently touch the spreader on the surface of agar plate in every direction by rotating the plate or by keeping the plate on turn table. 5. When properly spread the petri plate or stops the turn table put the lid over the petri plate and incubate all the plates at 25°C to 30°C for 24 hours in inverted position in an incubator.

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54 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Result: After incubation observe the shape, size, colour of colonies in each plate. Some of the colonies grow individually do not overlap the other colony. The single colony is picked up and purified through sub-culturing.

Result: After incubation observe the shape, size, colour of colonies in each plate. Some of the colonies grow individually do not overlap the other colony. The single colony is picked up and purified through sub-culturing.

4. Serial dilution technique

4. Serial dilution technique

In this method a known amount (1gm/ml) of soil or water sample is suspended into known volume of sterile distilled water (9 ml) so that total volume becomes 10 ml. known volume (1 ml) of suspension is transferred into the next flask containing 9 ml of sterile water. By this way dilute all the tubes and finally take 1 ml of each test tube and pour into the nutrient agar plates. Incubate and observe the colonies.

In this method a known amount (1gm/ml) of soil or water sample is suspended into known volume of sterile distilled water (9 ml) so that total volume becomes 10 ml. known volume (1 ml) of suspension is transferred into the next flask containing 9 ml of sterile water. By this way dilute all the tubes and finally take 1 ml of each test tube and pour into the nutrient agar plates. Incubate and observe the colonies.

Fig. 5.3. Pour plate technique is used for isolation of pure cultures of bacteria. Step1: One loopful of original suspension is transferred to tube 1 (liquid agar media). Similar transfers are made from 1 to 2, up to 7. Step 2: Contents of each tubes are poured into separate Petri dishes. Step 3: After incubation, plates are examined for the one which contains well separated colonies. From which pure bacterial culture can be isolated by transferring a portion of a colony to a tube of sterile medium.

Fig. 5.3. Pour plate technique is used for isolation of pure cultures of bacteria. Step1: One loopful of original suspension is transferred to tube 1 (liquid agar media). Similar transfers are made from 1 to 2, up to 7. Step 2: Contents of each tubes are poured into separate Petri dishes. Step 3: After incubation, plates are examined for the one which contains well separated colonies. From which pure bacterial culture can be isolated by transferring a portion of a colony to a tube of sterile medium.

Procedure

Procedure

1. Prepare one test tube containing nutrient broth media and another 7 blank test tube containing water, autoclave all the tube and make sterile. 2. In nutrient broth tube mixed with 2 or more bacterial culture.

1. Prepare one test tube containing nutrient broth media and another 7 blank test tube containing water, autoclave all the tube and make sterile. 2. In nutrient broth tube mixed with 2 or more bacterial culture.

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54 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Result: After incubation observe the shape, size, colour of colonies in each plate. Some of the colonies grow individually do not overlap the other colony. The single colony is picked up and purified through sub-culturing.

Result: After incubation observe the shape, size, colour of colonies in each plate. Some of the colonies grow individually do not overlap the other colony. The single colony is picked up and purified through sub-culturing.

4. Serial dilution technique

4. Serial dilution technique

In this method a known amount (1gm/ml) of soil or water sample is suspended into known volume of sterile distilled water (9 ml) so that total volume becomes 10 ml. known volume (1 ml) of suspension is transferred into the next flask containing 9 ml of sterile water. By this way dilute all the tubes and finally take 1 ml of each test tube and pour into the nutrient agar plates. Incubate and observe the colonies.

In this method a known amount (1gm/ml) of soil or water sample is suspended into known volume of sterile distilled water (9 ml) so that total volume becomes 10 ml. known volume (1 ml) of suspension is transferred into the next flask containing 9 ml of sterile water. By this way dilute all the tubes and finally take 1 ml of each test tube and pour into the nutrient agar plates. Incubate and observe the colonies.

Fig. 5.3. Pour plate technique is used for isolation of pure cultures of bacteria. Step1: One loopful of original suspension is transferred to tube 1 (liquid agar media). Similar transfers are made from 1 to 2, up to 7. Step 2: Contents of each tubes are poured into separate Petri dishes. Step 3: After incubation, plates are examined for the one which contains well separated colonies. From which pure bacterial culture can be isolated by transferring a portion of a colony to a tube of sterile medium.

Fig. 5.3. Pour plate technique is used for isolation of pure cultures of bacteria. Step1: One loopful of original suspension is transferred to tube 1 (liquid agar media). Similar transfers are made from 1 to 2, up to 7. Step 2: Contents of each tubes are poured into separate Petri dishes. Step 3: After incubation, plates are examined for the one which contains well separated colonies. From which pure bacterial culture can be isolated by transferring a portion of a colony to a tube of sterile medium.

Procedure

Procedure

1. Prepare one test tube containing nutrient broth media and another 7 blank test tube containing water, autoclave all the tube and make sterile. 2. In nutrient broth tube mixed with 2 or more bacterial culture.

1. Prepare one test tube containing nutrient broth media and another 7 blank test tube containing water, autoclave all the tube and make sterile. 2. In nutrient broth tube mixed with 2 or more bacterial culture.

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3. After mixing transfer 1 ml of the bacterial suspension from tube number 1 to tube number 2 containing sterile water. 4. Take 1 ml of bacterial suspension form tube number 2 and transfer to tube number 3. By this way make serial dilution up to the 7 tubes. 5. Transfer 1 ml of the bacterial suspension each from tube numbers 1 to 7 into the Petri plate’s numbers 1 to 7 containing nutrient agar media with the help of sterile pipettes. 6. Leave the plates for 5 to 10 minutes and remove the bacterial suspension from the plate with the help of micro pipette which is not socked by agar plates. 7. Incubate all the agar plates for 24 to 48 hours at 37°C in inverted position in an incubator.

3. After mixing transfer 1 ml of the bacterial suspension from tube number 1 to tube number 2 containing sterile water. 4. Take 1 ml of bacterial suspension form tube number 2 and transfer to tube number 3. By this way make serial dilution up to the 7 tubes. 5. Transfer 1 ml of the bacterial suspension each from tube numbers 1 to 7 into the Petri plate’s numbers 1 to 7 containing nutrient agar media with the help of sterile pipettes. 6. Leave the plates for 5 to 10 minutes and remove the bacterial suspension from the plate with the help of micro pipette which is not socked by agar plates. 7. Incubate all the agar plates for 24 to 48 hours at 37°C in inverted position in an incubator.

Observations: Examine the plates for appearance of individual colonies growing throughout the agar medium.

Observations: Examine the plates for appearance of individual colonies growing throughout the agar medium.

5.2

5.2

NUTRITION, CULTIVATION AND ISOLATION OF ACTINOMYCETES

NUTRITION, CULTIVATION AND ISOLATION OF ACTINOMYCETES

5.2.1 Nutritional Habit of Actinomycetes They are chemoorganotrophic. Chemoorganotrophs are organisms which use organic compounds as their energy source. These organic chemicals include glucose and acetate. Most bacteria, and Protists as well as all animals and fungi are chemoorganotrophs. Indeed, most heterotrophs are chemoorganotrophs. However, chemolithotrophs instead use inorganic compounds as a source of energy.

5.2.1 Nutritional Habit of Actinomycetes They are chemoorganotrophic. Chemoorganotrophs are organisms which use organic compounds as their energy source. These organic chemicals include glucose and acetate. Most bacteria, and Protists as well as all animals and fungi are chemoorganotrophs. Indeed, most heterotrophs are chemoorganotrophs. However, chemolithotrophs instead use inorganic compounds as a source of energy.

5.2.2 Cultivation and Isolation of Actinomycetes The samples of soil, water and various vegetable materials used in experiments were collected locally and different locations around the world. Actinomycetes are well cultivated in nutrient agar media. Composition of L.B. agar media is: Tryptone-1%, Yeast extract-0.5%, NaCl-0.5%, Agar-1.5%, and Dist.water100mL. A nutrient agar media was overlaid with a 0.22 to 0.45ìm pore cellulose ester membrane filter, and the surface of the filter was inoculated with bacterial culture. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycetes bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycetes colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth. Offers a general method for their selective isolation.

5.2.2 Cultivation and Isolation of Actinomycetes The samples of soil, water and various vegetable materials used in experiments were collected locally and different locations around the world. Actinomycetes are well cultivated in nutrient agar media. Composition of L.B. agar media is: Tryptone-1%, Yeast extract-0.5%, NaCl-0.5%, Agar-1.5%, and Dist.water100mL. A nutrient agar media was overlaid with a 0.22 to 0.45ìm pore cellulose ester membrane filter, and the surface of the filter was inoculated with bacterial culture. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycetes bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycetes colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth. Offers a general method for their selective isolation.

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3. After mixing transfer 1 ml of the bacterial suspension from tube number 1 to tube number 2 containing sterile water. 4. Take 1 ml of bacterial suspension form tube number 2 and transfer to tube number 3. By this way make serial dilution up to the 7 tubes. 5. Transfer 1 ml of the bacterial suspension each from tube numbers 1 to 7 into the Petri plate’s numbers 1 to 7 containing nutrient agar media with the help of sterile pipettes. 6. Leave the plates for 5 to 10 minutes and remove the bacterial suspension from the plate with the help of micro pipette which is not socked by agar plates. 7. Incubate all the agar plates for 24 to 48 hours at 37°C in inverted position in an incubator.

3. After mixing transfer 1 ml of the bacterial suspension from tube number 1 to tube number 2 containing sterile water. 4. Take 1 ml of bacterial suspension form tube number 2 and transfer to tube number 3. By this way make serial dilution up to the 7 tubes. 5. Transfer 1 ml of the bacterial suspension each from tube numbers 1 to 7 into the Petri plate’s numbers 1 to 7 containing nutrient agar media with the help of sterile pipettes. 6. Leave the plates for 5 to 10 minutes and remove the bacterial suspension from the plate with the help of micro pipette which is not socked by agar plates. 7. Incubate all the agar plates for 24 to 48 hours at 37°C in inverted position in an incubator.

Observations: Examine the plates for appearance of individual colonies growing throughout the agar medium.

Observations: Examine the plates for appearance of individual colonies growing throughout the agar medium.

5.2

5.2

NUTRITION, CULTIVATION AND ISOLATION OF ACTINOMYCETES

NUTRITION, CULTIVATION AND ISOLATION OF ACTINOMYCETES

5.2.1 Nutritional Habit of Actinomycetes They are chemoorganotrophic. Chemoorganotrophs are organisms which use organic compounds as their energy source. These organic chemicals include glucose and acetate. Most bacteria, and Protists as well as all animals and fungi are chemoorganotrophs. Indeed, most heterotrophs are chemoorganotrophs. However, chemolithotrophs instead use inorganic compounds as a source of energy.

5.2.1 Nutritional Habit of Actinomycetes They are chemoorganotrophic. Chemoorganotrophs are organisms which use organic compounds as their energy source. These organic chemicals include glucose and acetate. Most bacteria, and Protists as well as all animals and fungi are chemoorganotrophs. Indeed, most heterotrophs are chemoorganotrophs. However, chemolithotrophs instead use inorganic compounds as a source of energy.

5.2.2 Cultivation and Isolation of Actinomycetes The samples of soil, water and various vegetable materials used in experiments were collected locally and different locations around the world. Actinomycetes are well cultivated in nutrient agar media. Composition of L.B. agar media is: Tryptone-1%, Yeast extract-0.5%, NaCl-0.5%, Agar-1.5%, and Dist.water100mL. A nutrient agar media was overlaid with a 0.22 to 0.45ìm pore cellulose ester membrane filter, and the surface of the filter was inoculated with bacterial culture. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycetes bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycetes colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth. Offers a general method for their selective isolation.

5.2.2 Cultivation and Isolation of Actinomycetes The samples of soil, water and various vegetable materials used in experiments were collected locally and different locations around the world. Actinomycetes are well cultivated in nutrient agar media. Composition of L.B. agar media is: Tryptone-1%, Yeast extract-0.5%, NaCl-0.5%, Agar-1.5%, and Dist.water100mL. A nutrient agar media was overlaid with a 0.22 to 0.45ìm pore cellulose ester membrane filter, and the surface of the filter was inoculated with bacterial culture. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycetes bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycetes colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth. Offers a general method for their selective isolation.

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56 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

The isolation of actinomycetes from the mixed microflora present in the nature is complicated by their characteristic slow growth relative to that of the other soil bacteria. This has resulted in the development of selective isolation procedures based primarily on one or the both of the following approaches: 1. Nutritional selection: In which media are formulated with nutrients which are preferentially utilized by actinomycetes. 2. Selective inhibition: In which compounds such as antibiotics are incorporated into media to selectively inhibit non-actinomycetes bacteria.

The isolation of actinomycetes from the mixed microflora present in the nature is complicated by their characteristic slow growth relative to that of the other soil bacteria. This has resulted in the development of selective isolation procedures based primarily on one or the both of the following approaches: 1. Nutritional selection: In which media are formulated with nutrients which are preferentially utilized by actinomycetes. 2. Selective inhibition: In which compounds such as antibiotics are incorporated into media to selectively inhibit non-actinomycetes bacteria.

Methods

Methods

Membrane filter: the membrane filters used were obtained from millipore Crop. All filters were 90 mm in diameter, were composed of mixed esters of cellulose, and were steam sterilized before use.

Membrane filter: the membrane filters used were obtained from millipore Crop. All filters were 90 mm in diameter, were composed of mixed esters of cellulose, and were steam sterilized before use.

5.3 NUTRITION, CULTIVATION AND ISOLATION OF FUNGI

5.3 NUTRITION, CULTIVATION AND ISOLATION OF FUNGI

5.3.1 Nutritional Habit of Fungi Fungi are heterotrophic in nature. Due to lack of chlorophyll pigments they are unable to synthesize their food molecule and obtained from other organisms, therefore they are either parasites or saprophytes. Obligate parasites: These fungi take their food from host plants and hence, death of host plants, causes the death of parasitic fungi. Such fungi are called obligate parasites. Facultative saprophytes: Some fungi which are usually parasitic but after the death of host plant, they are able to absorb their food from the decaying body of the plant are called facultative saprophytes. Obligate parasite: Those fungi which always take their food from decaying organic matter like decaying leather, foods, bread, fruits etc. are called obligate saprophytes, e.g., yeast, rhizopus, penicillium etc. Facultative parasite: Those fungi which are usually saprophytes but can live parasitically under some conditions are called facultative parasites.

5.3.1 Nutritional Habit of Fungi Fungi are heterotrophic in nature. Due to lack of chlorophyll pigments they are unable to synthesize their food molecule and obtained from other organisms, therefore they are either parasites or saprophytes. Obligate parasites: These fungi take their food from host plants and hence, death of host plants, causes the death of parasitic fungi. Such fungi are called obligate parasites. Facultative saprophytes: Some fungi which are usually parasitic but after the death of host plant, they are able to absorb their food from the decaying body of the plant are called facultative saprophytes. Obligate parasite: Those fungi which always take their food from decaying organic matter like decaying leather, foods, bread, fruits etc. are called obligate saprophytes, e.g., yeast, rhizopus, penicillium etc. Facultative parasite: Those fungi which are usually saprophytes but can live parasitically under some conditions are called facultative parasites.

5.3.2 Cultivation and Isolation of Fungi Sabouarud’s Dextrose Agar (SDA) is the most suitable medium as fungal growth is favoured by a high sugar concentration and is relatively tolerant to acidity (pH 5.4). The agar is prepared as slopes in test tubes stoppered with cottonwool as most of the fungi are aerobic. Chloramphenicol is incorporated in the culture medium to prevent contamination by bacteria. Similarly, addition of cycloheximide can suppress the contaminating fungi. The fungal growth can be identified by its colour and morphology on visual examination and pigmentation on the reverse.

5.3.2 Cultivation and Isolation of Fungi Sabouarud’s Dextrose Agar (SDA) is the most suitable medium as fungal growth is favoured by a high sugar concentration and is relatively tolerant to acidity (pH 5.4). The agar is prepared as slopes in test tubes stoppered with cottonwool as most of the fungi are aerobic. Chloramphenicol is incorporated in the culture medium to prevent contamination by bacteria. Similarly, addition of cycloheximide can suppress the contaminating fungi. The fungal growth can be identified by its colour and morphology on visual examination and pigmentation on the reverse.

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56 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

The isolation of actinomycetes from the mixed microflora present in the nature is complicated by their characteristic slow growth relative to that of the other soil bacteria. This has resulted in the development of selective isolation procedures based primarily on one or the both of the following approaches: 1. Nutritional selection: In which media are formulated with nutrients which are preferentially utilized by actinomycetes. 2. Selective inhibition: In which compounds such as antibiotics are incorporated into media to selectively inhibit non-actinomycetes bacteria.

The isolation of actinomycetes from the mixed microflora present in the nature is complicated by their characteristic slow growth relative to that of the other soil bacteria. This has resulted in the development of selective isolation procedures based primarily on one or the both of the following approaches: 1. Nutritional selection: In which media are formulated with nutrients which are preferentially utilized by actinomycetes. 2. Selective inhibition: In which compounds such as antibiotics are incorporated into media to selectively inhibit non-actinomycetes bacteria.

Methods

Methods

Membrane filter: the membrane filters used were obtained from millipore Crop. All filters were 90 mm in diameter, were composed of mixed esters of cellulose, and were steam sterilized before use.

Membrane filter: the membrane filters used were obtained from millipore Crop. All filters were 90 mm in diameter, were composed of mixed esters of cellulose, and were steam sterilized before use.

5.3 NUTRITION, CULTIVATION AND ISOLATION OF FUNGI

5.3 NUTRITION, CULTIVATION AND ISOLATION OF FUNGI

5.3.1 Nutritional Habit of Fungi Fungi are heterotrophic in nature. Due to lack of chlorophyll pigments they are unable to synthesize their food molecule and obtained from other organisms, therefore they are either parasites or saprophytes. Obligate parasites: These fungi take their food from host plants and hence, death of host plants, causes the death of parasitic fungi. Such fungi are called obligate parasites. Facultative saprophytes: Some fungi which are usually parasitic but after the death of host plant, they are able to absorb their food from the decaying body of the plant are called facultative saprophytes. Obligate parasite: Those fungi which always take their food from decaying organic matter like decaying leather, foods, bread, fruits etc. are called obligate saprophytes, e.g., yeast, rhizopus, penicillium etc. Facultative parasite: Those fungi which are usually saprophytes but can live parasitically under some conditions are called facultative parasites.

5.3.1 Nutritional Habit of Fungi Fungi are heterotrophic in nature. Due to lack of chlorophyll pigments they are unable to synthesize their food molecule and obtained from other organisms, therefore they are either parasites or saprophytes. Obligate parasites: These fungi take their food from host plants and hence, death of host plants, causes the death of parasitic fungi. Such fungi are called obligate parasites. Facultative saprophytes: Some fungi which are usually parasitic but after the death of host plant, they are able to absorb their food from the decaying body of the plant are called facultative saprophytes. Obligate parasite: Those fungi which always take their food from decaying organic matter like decaying leather, foods, bread, fruits etc. are called obligate saprophytes, e.g., yeast, rhizopus, penicillium etc. Facultative parasite: Those fungi which are usually saprophytes but can live parasitically under some conditions are called facultative parasites.

5.3.2 Cultivation and Isolation of Fungi Sabouarud’s Dextrose Agar (SDA) is the most suitable medium as fungal growth is favoured by a high sugar concentration and is relatively tolerant to acidity (pH 5.4). The agar is prepared as slopes in test tubes stoppered with cottonwool as most of the fungi are aerobic. Chloramphenicol is incorporated in the culture medium to prevent contamination by bacteria. Similarly, addition of cycloheximide can suppress the contaminating fungi. The fungal growth can be identified by its colour and morphology on visual examination and pigmentation on the reverse.

5.3.2 Cultivation and Isolation of Fungi Sabouarud’s Dextrose Agar (SDA) is the most suitable medium as fungal growth is favoured by a high sugar concentration and is relatively tolerant to acidity (pH 5.4). The agar is prepared as slopes in test tubes stoppered with cottonwool as most of the fungi are aerobic. Chloramphenicol is incorporated in the culture medium to prevent contamination by bacteria. Similarly, addition of cycloheximide can suppress the contaminating fungi. The fungal growth can be identified by its colour and morphology on visual examination and pigmentation on the reverse.

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Microscope examination is done to evaluate the morphology of hyphae, spores and other structures. Teased mounts are made in lactophenol blue and examined under a microscope. The morphology of various spores is characteristic of different fungi. Slide cultures useful for studying the exact morphology of the fungus. By the addition of some antibiotic and by preventing the contamination, pure culture of the fungus is isolated.

Microscope examination is done to evaluate the morphology of hyphae, spores and other structures. Teased mounts are made in lactophenol blue and examined under a microscope. The morphology of various spores is characteristic of different fungi. Slide cultures useful for studying the exact morphology of the fungus. By the addition of some antibiotic and by preventing the contamination, pure culture of the fungus is isolated.

5.4

5.4

NUTRITION, CULTIVATION AND ISOLATION OF RICKETTSIA

NUTRITION, CULTIVATION AND ISOLATION OF RICKETTSIA

5.4.1 Nutrition Rickettsias are obligate parasites means that they obtain their food from other organisms. They are intracellular parasites and found mainly in eukaryotic hosts like vertebrates and arthropods which are the primary host. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups.

5.4.1 Nutrition Rickettsias are obligate parasites means that they obtain their food from other organisms. They are intracellular parasites and found mainly in eukaryotic hosts like vertebrates and arthropods which are the primary host. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups.

5.4.2 Cultivation It is unable to grow in cell free media except R. quintana which grows on blood agar. It is cultivated in yolk sac of developing chick embryo. Pure preparation of reckettsiae may be obtained by different centrifugation of yolk sac suspension. It may grow on chorioallantoic membrane. In cell culture, the generation time is 8 to 10 hours at 34°C (the incubation temperature is between 26°C to 30°C). Laboratory animals like mice and guinea pig are helpful for isolation of rickettsiae from patients. It may be propagated in arthropods. Rickettsial growth is enhanced in the presence of sulfonamide. Obviously these rickettsial diseases are made more severe by sulfonamides.

5.4.2 Cultivation It is unable to grow in cell free media except R. quintana which grows on blood agar. It is cultivated in yolk sac of developing chick embryo. Pure preparation of reckettsiae may be obtained by different centrifugation of yolk sac suspension. It may grow on chorioallantoic membrane. In cell culture, the generation time is 8 to 10 hours at 34°C (the incubation temperature is between 26°C to 30°C). Laboratory animals like mice and guinea pig are helpful for isolation of rickettsiae from patients. It may be propagated in arthropods. Rickettsial growth is enhanced in the presence of sulfonamide. Obviously these rickettsial diseases are made more severe by sulfonamides.

5.4.3 Isolation Prepare a nutrient agar media having pH range 7.0 to 7.4 and autoclaved properly. Take a loopful culture from macerated ovarian or embryonic material obtained under sterile condition, is taken and stick on the surface of Petri plate having standard nutrient agar media, incubate the plate at 26°C for 48 hours. After that examine the Petri plates, which shows several minute yellowish grey, semi-transparent, shining, oval or round, mucoid colonies (sometimes with several other colonies when contamination occurs). Take a single colony from the Petri plate having the above characteristics and stick on other nutrient agar plate that incubates at 26°C for 48 hours when colonies arise, store the plate at low temperature (4°C to -20°C).

5.4.3 Isolation Prepare a nutrient agar media having pH range 7.0 to 7.4 and autoclaved properly. Take a loopful culture from macerated ovarian or embryonic material obtained under sterile condition, is taken and stick on the surface of Petri plate having standard nutrient agar media, incubate the plate at 26°C for 48 hours. After that examine the Petri plates, which shows several minute yellowish grey, semi-transparent, shining, oval or round, mucoid colonies (sometimes with several other colonies when contamination occurs). Take a single colony from the Petri plate having the above characteristics and stick on other nutrient agar plate that incubates at 26°C for 48 hours when colonies arise, store the plate at low temperature (4°C to -20°C).

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Microscope examination is done to evaluate the morphology of hyphae, spores and other structures. Teased mounts are made in lactophenol blue and examined under a microscope. The morphology of various spores is characteristic of different fungi. Slide cultures useful for studying the exact morphology of the fungus. By the addition of some antibiotic and by preventing the contamination, pure culture of the fungus is isolated.

Microscope examination is done to evaluate the morphology of hyphae, spores and other structures. Teased mounts are made in lactophenol blue and examined under a microscope. The morphology of various spores is characteristic of different fungi. Slide cultures useful for studying the exact morphology of the fungus. By the addition of some antibiotic and by preventing the contamination, pure culture of the fungus is isolated.

5.4

5.4

NUTRITION, CULTIVATION AND ISOLATION OF RICKETTSIA

NUTRITION, CULTIVATION AND ISOLATION OF RICKETTSIA

5.4.1 Nutrition Rickettsias are obligate parasites means that they obtain their food from other organisms. They are intracellular parasites and found mainly in eukaryotic hosts like vertebrates and arthropods which are the primary host. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups.

5.4.1 Nutrition Rickettsias are obligate parasites means that they obtain their food from other organisms. They are intracellular parasites and found mainly in eukaryotic hosts like vertebrates and arthropods which are the primary host. Rickettsias are transmitted to humans primarily by arthropods such as ticks and lice and are responsible for a number of diseases known as the spotted fever groups.

5.4.2 Cultivation It is unable to grow in cell free media except R. quintana which grows on blood agar. It is cultivated in yolk sac of developing chick embryo. Pure preparation of reckettsiae may be obtained by different centrifugation of yolk sac suspension. It may grow on chorioallantoic membrane. In cell culture, the generation time is 8 to 10 hours at 34°C (the incubation temperature is between 26°C to 30°C). Laboratory animals like mice and guinea pig are helpful for isolation of rickettsiae from patients. It may be propagated in arthropods. Rickettsial growth is enhanced in the presence of sulfonamide. Obviously these rickettsial diseases are made more severe by sulfonamides.

5.4.2 Cultivation It is unable to grow in cell free media except R. quintana which grows on blood agar. It is cultivated in yolk sac of developing chick embryo. Pure preparation of reckettsiae may be obtained by different centrifugation of yolk sac suspension. It may grow on chorioallantoic membrane. In cell culture, the generation time is 8 to 10 hours at 34°C (the incubation temperature is between 26°C to 30°C). Laboratory animals like mice and guinea pig are helpful for isolation of rickettsiae from patients. It may be propagated in arthropods. Rickettsial growth is enhanced in the presence of sulfonamide. Obviously these rickettsial diseases are made more severe by sulfonamides.

5.4.3 Isolation Prepare a nutrient agar media having pH range 7.0 to 7.4 and autoclaved properly. Take a loopful culture from macerated ovarian or embryonic material obtained under sterile condition, is taken and stick on the surface of Petri plate having standard nutrient agar media, incubate the plate at 26°C for 48 hours. After that examine the Petri plates, which shows several minute yellowish grey, semi-transparent, shining, oval or round, mucoid colonies (sometimes with several other colonies when contamination occurs). Take a single colony from the Petri plate having the above characteristics and stick on other nutrient agar plate that incubates at 26°C for 48 hours when colonies arise, store the plate at low temperature (4°C to -20°C).

5.4.3 Isolation Prepare a nutrient agar media having pH range 7.0 to 7.4 and autoclaved properly. Take a loopful culture from macerated ovarian or embryonic material obtained under sterile condition, is taken and stick on the surface of Petri plate having standard nutrient agar media, incubate the plate at 26°C for 48 hours. After that examine the Petri plates, which shows several minute yellowish grey, semi-transparent, shining, oval or round, mucoid colonies (sometimes with several other colonies when contamination occurs). Take a single colony from the Petri plate having the above characteristics and stick on other nutrient agar plate that incubates at 26°C for 48 hours when colonies arise, store the plate at low temperature (4°C to -20°C).

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58 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

5.5

5.5

NUTRITION, CULTIVATION, AND ISOLATION OF VIRUSES

NUTRITION, CULTIVATION, AND ISOLATION OF VIRUSES

5.5.1 Nutritional Habit of Viruses 1. Bawden in 1935 defined viruses as “obligate parasite” and connecting link between living and non-living things. Viruses are ultramicroscopic infectious particles ranging from 20 to 350 mm in diameter.

5.5.1 Nutritional Habit of Viruses 1. Bawden in 1935 defined viruses as “obligate parasite” and connecting link between living and non-living things. Viruses are ultramicroscopic infectious particles ranging from 20 to 350 mm in diameter.

5.5.2 Living Characters 2. They are nucleoproteins and contain either DNA or RNA surrounded by a protein coat. 3. They replicate, although inside the living cells. 4. Capable of synthesizing protein for thin layer coat. 5. They cause diseases like bacteria and fungi.

5.5.2 Living Characters 2. They are nucleoproteins and contain either DNA or RNA surrounded by a protein coat. 3. They replicate, although inside the living cells. 4. Capable of synthesizing protein for thin layer coat. 5. They cause diseases like bacteria and fungi.

5.5.3 Non-living Characters 1. They do not have protoplasm. 2. They do not have enzyme system. 3. They do not have to respire. 4. They can be catalyzed.

5.5.3 Non-living Characters 1. They do not have protoplasm. 2. They do not have enzyme system. 3. They do not have to respire. 4. They can be catalyzed.

They are perfect obligate intracellular parasites which means that they depend upon specific host cells like animal cell, plant cell and bacterial cell etc. for their reproduction and development. The virus particles attached to the host cell by adsorption with the help of tail fibres and dissolve the cell wall of host cell. Now the DNA of virus enter into the host cell and attack the host cell machinery and synthesizing series of enzymes, which are essential for the reproduction of virus.

They are perfect obligate intracellular parasites which means that they depend upon specific host cells like animal cell, plant cell and bacterial cell etc. for their reproduction and development. The virus particles attached to the host cell by adsorption with the help of tail fibres and dissolve the cell wall of host cell. Now the DNA of virus enter into the host cell and attack the host cell machinery and synthesizing series of enzymes, which are essential for the reproduction of virus.

5.5.4 Cultivation and Isolation of Virus (Human Virus) The cultivation of viruses from material taken from lesions is an important step in the diagnosis of many viral diseases. Studies of the basic biology and multiplication processes of human viruses also require that they are grown in the laboratory under experimental conditions. Since they are obligate intracellular parasites and cannot grow on inanimate culture medium. Three methods are used for their cultivation: 1. Animals inoculation 2. Chick embryo 3. Cell culture

5.5.4 Cultivation and Isolation of Virus (Human Virus) The cultivation of viruses from material taken from lesions is an important step in the diagnosis of many viral diseases. Studies of the basic biology and multiplication processes of human viruses also require that they are grown in the laboratory under experimental conditions. Since they are obligate intracellular parasites and cannot grow on inanimate culture medium. Three methods are used for their cultivation: 1. Animals inoculation 2. Chick embryo 3. Cell culture

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5.5

5.5

NUTRITION, CULTIVATION, AND ISOLATION OF VIRUSES

NUTRITION, CULTIVATION, AND ISOLATION OF VIRUSES

5.5.1 Nutritional Habit of Viruses 1. Bawden in 1935 defined viruses as “obligate parasite” and connecting link between living and non-living things. Viruses are ultramicroscopic infectious particles ranging from 20 to 350 mm in diameter.

5.5.1 Nutritional Habit of Viruses 1. Bawden in 1935 defined viruses as “obligate parasite” and connecting link between living and non-living things. Viruses are ultramicroscopic infectious particles ranging from 20 to 350 mm in diameter.

5.5.2 Living Characters 2. They are nucleoproteins and contain either DNA or RNA surrounded by a protein coat. 3. They replicate, although inside the living cells. 4. Capable of synthesizing protein for thin layer coat. 5. They cause diseases like bacteria and fungi.

5.5.2 Living Characters 2. They are nucleoproteins and contain either DNA or RNA surrounded by a protein coat. 3. They replicate, although inside the living cells. 4. Capable of synthesizing protein for thin layer coat. 5. They cause diseases like bacteria and fungi.

5.5.3 Non-living Characters 1. They do not have protoplasm. 2. They do not have enzyme system. 3. They do not have to respire. 4. They can be catalyzed.

5.5.3 Non-living Characters 1. They do not have protoplasm. 2. They do not have enzyme system. 3. They do not have to respire. 4. They can be catalyzed.

They are perfect obligate intracellular parasites which means that they depend upon specific host cells like animal cell, plant cell and bacterial cell etc. for their reproduction and development. The virus particles attached to the host cell by adsorption with the help of tail fibres and dissolve the cell wall of host cell. Now the DNA of virus enter into the host cell and attack the host cell machinery and synthesizing series of enzymes, which are essential for the reproduction of virus.

They are perfect obligate intracellular parasites which means that they depend upon specific host cells like animal cell, plant cell and bacterial cell etc. for their reproduction and development. The virus particles attached to the host cell by adsorption with the help of tail fibres and dissolve the cell wall of host cell. Now the DNA of virus enter into the host cell and attack the host cell machinery and synthesizing series of enzymes, which are essential for the reproduction of virus.

5.5.4 Cultivation and Isolation of Virus (Human Virus) The cultivation of viruses from material taken from lesions is an important step in the diagnosis of many viral diseases. Studies of the basic biology and multiplication processes of human viruses also require that they are grown in the laboratory under experimental conditions. Since they are obligate intracellular parasites and cannot grow on inanimate culture medium. Three methods are used for their cultivation: 1. Animals inoculation 2. Chick embryo 3. Cell culture

5.5.4 Cultivation and Isolation of Virus (Human Virus) The cultivation of viruses from material taken from lesions is an important step in the diagnosis of many viral diseases. Studies of the basic biology and multiplication processes of human viruses also require that they are grown in the laboratory under experimental conditions. Since they are obligate intracellular parasites and cannot grow on inanimate culture medium. Three methods are used for their cultivation: 1. Animals inoculation 2. Chick embryo 3. Cell culture

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Fig. 5.4. The morphology of a variety of virus particles.

Fig. 5.4. The morphology of a variety of virus particles.

5.5.5 Animal Inoculation It is one of the oldest methods for the cultivation of viruses. The poliomyelitis virus after intraspinal or intracerebral inoculation in monkeys causes typical paralytic diseases and so isolation of viruses. Smallpox virus may be inoculated in the scarified skin or cornea of rabbit. Brain tissues of rabied dog when inoculated intracerebrally in mice or rabbit develop encephalitis. Growth of virus in animals may be known by the disease, visible classical lesions or death. Sometimes immunity in experimental animal may interfere with the growth of viruses in that animal. It is not out of place to mention the other utility of animal inoculation, i.e., to study pathogenesis, immune response, and epidemiology.

5.5.5 Animal Inoculation It is one of the oldest methods for the cultivation of viruses. The poliomyelitis virus after intraspinal or intracerebral inoculation in monkeys causes typical paralytic diseases and so isolation of viruses. Smallpox virus may be inoculated in the scarified skin or cornea of rabbit. Brain tissues of rabied dog when inoculated intracerebrally in mice or rabbit develop encephalitis. Growth of virus in animals may be known by the disease, visible classical lesions or death. Sometimes immunity in experimental animal may interfere with the growth of viruses in that animal. It is not out of place to mention the other utility of animal inoculation, i.e., to study pathogenesis, immune response, and epidemiology.

5.5.6 Chick Embryo Fertile chicken eggs, 10-12 days old, have been used as a convenient cell system in which to grow a number of human pathogenic viruses. Fig. 5.6. shows that viruses generally have preferences for particular tissues within the embryo. Influenza viruses, for example, can be grown in the cells of the membrane bounding the amniotic cavity, while smallpox virus will grow in the

5.5.6 Chick Embryo Fertile chicken eggs, 10-12 days old, have been used as a convenient cell system in which to grow a number of human pathogenic viruses. Fig. 5.6. shows that viruses generally have preferences for particular tissues within the embryo. Influenza viruses, for example, can be grown in the cells of the membrane bounding the amniotic cavity, while smallpox virus will grow in the

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Fig. 5.4. The morphology of a variety of virus particles.

Fig. 5.4. The morphology of a variety of virus particles.

5.5.5 Animal Inoculation It is one of the oldest methods for the cultivation of viruses. The poliomyelitis virus after intraspinal or intracerebral inoculation in monkeys causes typical paralytic diseases and so isolation of viruses. Smallpox virus may be inoculated in the scarified skin or cornea of rabbit. Brain tissues of rabied dog when inoculated intracerebrally in mice or rabbit develop encephalitis. Growth of virus in animals may be known by the disease, visible classical lesions or death. Sometimes immunity in experimental animal may interfere with the growth of viruses in that animal. It is not out of place to mention the other utility of animal inoculation, i.e., to study pathogenesis, immune response, and epidemiology.

5.5.5 Animal Inoculation It is one of the oldest methods for the cultivation of viruses. The poliomyelitis virus after intraspinal or intracerebral inoculation in monkeys causes typical paralytic diseases and so isolation of viruses. Smallpox virus may be inoculated in the scarified skin or cornea of rabbit. Brain tissues of rabied dog when inoculated intracerebrally in mice or rabbit develop encephalitis. Growth of virus in animals may be known by the disease, visible classical lesions or death. Sometimes immunity in experimental animal may interfere with the growth of viruses in that animal. It is not out of place to mention the other utility of animal inoculation, i.e., to study pathogenesis, immune response, and epidemiology.

5.5.6 Chick Embryo Fertile chicken eggs, 10-12 days old, have been used as a convenient cell system in which to grow a number of human pathogenic viruses. Fig. 5.6. shows that viruses generally have preferences for particular tissues within the embryo. Influenza viruses, for example, can be grown in the cells of the membrane bounding the amniotic cavity, while smallpox virus will grow in the

5.5.6 Chick Embryo Fertile chicken eggs, 10-12 days old, have been used as a convenient cell system in which to grow a number of human pathogenic viruses. Fig. 5.6. shows that viruses generally have preferences for particular tissues within the embryo. Influenza viruses, for example, can be grown in the cells of the membrane bounding the amniotic cavity, while smallpox virus will grow in the

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60 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 5.5. The cytopathic effect of a virus on a tissue culture cell monolayer.

Fig. 5.5. The cytopathic effect of a virus on a tissue culture cell monolayer.

Fig. 5.6. A chick embryo showing the inoculation routes for virus cultivation.

Fig. 5.6. A chick embryo showing the inoculation routes for virus cultivation.

chorioallantoic membrane. The growth of smallpox virus in the embryo is recognized by the formation of characteristic pock marks on the membrane. Influenza virus replication is detected by exploiting the ability of these particles to cause erythrocytes to clump together. Fluid from the amniotic cavity of the infected embryo is titrated for its haemagglutinating activity.

chorioallantoic membrane. The growth of smallpox virus in the embryo is recognized by the formation of characteristic pock marks on the membrane. Influenza virus replication is detected by exploiting the ability of these particles to cause erythrocytes to clump together. Fluid from the amniotic cavity of the infected embryo is titrated for its haemagglutinating activity.

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60 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 5.5. The cytopathic effect of a virus on a tissue culture cell monolayer.

Fig. 5.5. The cytopathic effect of a virus on a tissue culture cell monolayer.

Fig. 5.6. A chick embryo showing the inoculation routes for virus cultivation.

Fig. 5.6. A chick embryo showing the inoculation routes for virus cultivation.

chorioallantoic membrane. The growth of smallpox virus in the embryo is recognized by the formation of characteristic pock marks on the membrane. Influenza virus replication is detected by exploiting the ability of these particles to cause erythrocytes to clump together. Fluid from the amniotic cavity of the infected embryo is titrated for its haemagglutinating activity.

chorioallantoic membrane. The growth of smallpox virus in the embryo is recognized by the formation of characteristic pock marks on the membrane. Influenza virus replication is detected by exploiting the ability of these particles to cause erythrocytes to clump together. Fluid from the amniotic cavity of the infected embryo is titrated for its haemagglutinating activity.

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5.5.7 Cell Culture Cells from human or other primate sources are obtained from an intact tissue, e.g., human embryo, kidney or liver cells. The cells are dispersed by digestion with trypsin and the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium. The cells will metabolize and grow and after a few days of incubation at 37°C will form a continuous film or monolayer one cell thick. These cells are then capable of supporting viral replication. Cell cultures may be divided into three types according to their history. 1. Primary cell cultures, which are prepared directly from tissues. 2. Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 3. There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses.

5.5.7 Cell Culture Cells from human or other primate sources are obtained from an intact tissue, e.g., human embryo, kidney or liver cells. The cells are dispersed by digestion with trypsin and the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium. The cells will metabolize and grow and after a few days of incubation at 37°C will form a continuous film or monolayer one cell thick. These cells are then capable of supporting viral replication. Cell cultures may be divided into three types according to their history. 1. Primary cell cultures, which are prepared directly from tissues. 2. Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 3. There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses.

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 5.5). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and various known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture.

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 5.5). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and various known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture.

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5.5.7 Cell Culture Cells from human or other primate sources are obtained from an intact tissue, e.g., human embryo, kidney or liver cells. The cells are dispersed by digestion with trypsin and the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium. The cells will metabolize and grow and after a few days of incubation at 37°C will form a continuous film or monolayer one cell thick. These cells are then capable of supporting viral replication. Cell cultures may be divided into three types according to their history. 1. Primary cell cultures, which are prepared directly from tissues. 2. Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 3. There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses.

5.5.7 Cell Culture Cells from human or other primate sources are obtained from an intact tissue, e.g., human embryo, kidney or liver cells. The cells are dispersed by digestion with trypsin and the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium. The cells will metabolize and grow and after a few days of incubation at 37°C will form a continuous film or monolayer one cell thick. These cells are then capable of supporting viral replication. Cell cultures may be divided into three types according to their history. 1. Primary cell cultures, which are prepared directly from tissues. 2. Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 3. There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses.

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 5.5). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and various known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture.

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 5.5). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and various known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture.

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6

Microbial Genetics and Variation

Microbial Genetics and Variation

Microbial genetics is mainly related with the area within biotechnology and genetic engineering. It studies genetics of very small (micro) organisms.. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes. It also involves the study of genetic processes taking place in these microorganisms i.e., recombianant DNA technology etc. Prokaryotic cell contains a single circular chromosome (e.g. bacteria, cynobacteria etc) while eukaryotic cell contains many chromosomes Human contains 23 pairs of chromosome. What is chromosome, gene and DNA?

Microbial genetics is mainly related with the area within biotechnology and genetic engineering. It studies genetics of very small (micro) organisms.. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes. It also involves the study of genetic processes taking place in these microorganisms i.e., recombianant DNA technology etc. Prokaryotic cell contains a single circular chromosome (e.g. bacteria, cynobacteria etc) while eukaryotic cell contains many chromosomes Human contains 23 pairs of chromosome. What is chromosome, gene and DNA?

6.1 CHROMOSOME

6.1 CHROMOSOME

A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNAbound proteins, which serve to package the DNA and control its functions. Typically eukaryotic cells have large linear chromosomes and prokaryotic cells have smaller circular chromosomes. Furthermore, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. Prokaryotes do not possess nuclei. Instead, their DNA is organized in a structure called the circular or nucleoid and they do not contain histone proteins. Eukaryotic cells (with nuclei such as those found in plants, yeast, and animals) possess multiple large linear chromosomes contained in the cell’s nucleus. In eukaryotes, nuclear chromosomes are packaged by proteins called histone proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. This stylistic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome. The chromosome is X-shaped because it is

A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNAbound proteins, which serve to package the DNA and control its functions. Typically eukaryotic cells have large linear chromosomes and prokaryotic cells have smaller circular chromosomes. Furthermore, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. Prokaryotes do not possess nuclei. Instead, their DNA is organized in a structure called the circular or nucleoid and they do not contain histone proteins. Eukaryotic cells (with nuclei such as those found in plants, yeast, and animals) possess multiple large linear chromosomes contained in the cell’s nucleus. In eukaryotes, nuclear chromosomes are packaged by proteins called histone proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. This stylistic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome. The chromosome is X-shaped because it is

6

6

Microbial Genetics and Variation

Microbial Genetics and Variation

Microbial genetics is mainly related with the area within biotechnology and genetic engineering. It studies genetics of very small (micro) organisms.. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes. It also involves the study of genetic processes taking place in these microorganisms i.e., recombianant DNA technology etc. Prokaryotic cell contains a single circular chromosome (e.g. bacteria, cynobacteria etc) while eukaryotic cell contains many chromosomes Human contains 23 pairs of chromosome. What is chromosome, gene and DNA?

Microbial genetics is mainly related with the area within biotechnology and genetic engineering. It studies genetics of very small (micro) organisms.. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes. It also involves the study of genetic processes taking place in these microorganisms i.e., recombianant DNA technology etc. Prokaryotic cell contains a single circular chromosome (e.g. bacteria, cynobacteria etc) while eukaryotic cell contains many chromosomes Human contains 23 pairs of chromosome. What is chromosome, gene and DNA?

6.1 CHROMOSOME

6.1 CHROMOSOME

A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNAbound proteins, which serve to package the DNA and control its functions. Typically eukaryotic cells have large linear chromosomes and prokaryotic cells have smaller circular chromosomes. Furthermore, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. Prokaryotes do not possess nuclei. Instead, their DNA is organized in a structure called the circular or nucleoid and they do not contain histone proteins. Eukaryotic cells (with nuclei such as those found in plants, yeast, and animals) possess multiple large linear chromosomes contained in the cell’s nucleus. In eukaryotes, nuclear chromosomes are packaged by proteins called histone proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. This stylistic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome. The chromosome is X-shaped because it is

A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNAbound proteins, which serve to package the DNA and control its functions. Typically eukaryotic cells have large linear chromosomes and prokaryotic cells have smaller circular chromosomes. Furthermore, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. Prokaryotes do not possess nuclei. Instead, their DNA is organized in a structure called the circular or nucleoid and they do not contain histone proteins. Eukaryotic cells (with nuclei such as those found in plants, yeast, and animals) possess multiple large linear chromosomes contained in the cell’s nucleus. In eukaryotes, nuclear chromosomes are packaged by proteins called histone proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. This stylistic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome. The chromosome is X-shaped because it is

64 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

64 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.1. Showing the structure of chromosome, gene, and DNA strand contains exons and introns.

Fig. 6.1. Showing the structure of chromosome, gene, and DNA strand contains exons and introns.

dividing. Introns are regions often found in eukaryote genes that are removed in the splicing process (after the DNA is transcribed into RNA). Only the exons encode the protein. This diagram labels a region of only 50 or so bases as a gene. In reality, most genes are hundreds of times larger. A chromosome is made up of a group of genes. In case of prokaryotes only exons are present (no introns). But both are present in eukaryotic cells.

dividing. Introns are regions often found in eukaryote genes that are removed in the splicing process (after the DNA is transcribed into RNA). Only the exons encode the protein. This diagram labels a region of only 50 or so bases as a gene. In reality, most genes are hundreds of times larger. A chromosome is made up of a group of genes. In case of prokaryotes only exons are present (no introns). But both are present in eukaryotic cells.

6.2 GENE

6.2 GENE

A gene is the basic unit of heredity in a living organisms. All living things depend on genes. Genes hold the information to build and maintain an organism’s cells and pass genetic traits to offspring. A modern working definition of a gene is “a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions, and or other functional sequence regions”. A gene is the basic instruction, a sequence of nucleic acid (DNA or, in the case of certain viruses RNA). Exons are the coding regions in the DNA whereas interons are the non-coding regions which are not translated into proteins. During the protein synthesis (or in mature RNA) these interons are spliced out and the proteins are synthesized from the coding regions or from exons.

A gene is the basic unit of heredity in a living organisms. All living things depend on genes. Genes hold the information to build and maintain an organism’s cells and pass genetic traits to offspring. A modern working definition of a gene is “a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions, and or other functional sequence regions”. A gene is the basic instruction, a sequence of nucleic acid (DNA or, in the case of certain viruses RNA). Exons are the coding regions in the DNA whereas interons are the non-coding regions which are not translated into proteins. During the protein synthesis (or in mature RNA) these interons are spliced out and the proteins are synthesized from the coding regions or from exons.

64 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

64 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.1. Showing the structure of chromosome, gene, and DNA strand contains exons and introns.

Fig. 6.1. Showing the structure of chromosome, gene, and DNA strand contains exons and introns.

dividing. Introns are regions often found in eukaryote genes that are removed in the splicing process (after the DNA is transcribed into RNA). Only the exons encode the protein. This diagram labels a region of only 50 or so bases as a gene. In reality, most genes are hundreds of times larger. A chromosome is made up of a group of genes. In case of prokaryotes only exons are present (no introns). But both are present in eukaryotic cells.

dividing. Introns are regions often found in eukaryote genes that are removed in the splicing process (after the DNA is transcribed into RNA). Only the exons encode the protein. This diagram labels a region of only 50 or so bases as a gene. In reality, most genes are hundreds of times larger. A chromosome is made up of a group of genes. In case of prokaryotes only exons are present (no introns). But both are present in eukaryotic cells.

6.2 GENE

6.2 GENE

A gene is the basic unit of heredity in a living organisms. All living things depend on genes. Genes hold the information to build and maintain an organism’s cells and pass genetic traits to offspring. A modern working definition of a gene is “a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions, and or other functional sequence regions”. A gene is the basic instruction, a sequence of nucleic acid (DNA or, in the case of certain viruses RNA). Exons are the coding regions in the DNA whereas interons are the non-coding regions which are not translated into proteins. During the protein synthesis (or in mature RNA) these interons are spliced out and the proteins are synthesized from the coding regions or from exons.

A gene is the basic unit of heredity in a living organisms. All living things depend on genes. Genes hold the information to build and maintain an organism’s cells and pass genetic traits to offspring. A modern working definition of a gene is “a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions, and or other functional sequence regions”. A gene is the basic instruction, a sequence of nucleic acid (DNA or, in the case of certain viruses RNA). Exons are the coding regions in the DNA whereas interons are the non-coding regions which are not translated into proteins. During the protein synthesis (or in mature RNA) these interons are spliced out and the proteins are synthesized from the coding regions or from exons.

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6.3 DEOXYRIBONUCLEIC ACID (DNA)

MICROBIAL GENETICS AND VARIATION

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6.3 DEOXYRIBONUCLEIC ACID (DNA)

• DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. • The main role of DNA molecule is the long term storage of information. • The DNA segments that carry this genetic information are called genes.

• DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. • The main role of DNA molecule is the long term storage of information. • The DNA segments that carry this genetic information are called genes.

DNA is composed of polynucleotide chains that are held together by weak, non-covalent bonds between pairs of bases. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. This is called the complementary sequence.

DNA is composed of polynucleotide chains that are held together by weak, non-covalent bonds between pairs of bases. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. This is called the complementary sequence.

Fig. 6.2. (A): Schematic modal of the double helix, one turn of the helix (34 Å or 3.4 Pm), contains nearly 10.5 base pairs. Each base pair is displaced or twisted from the previous one by about 36°. Thus, the X-ray crystal structure of DNA it takes a stalk of about 10 base pairs to go completely around the helix 360°. (B): detailed structure of polynucleotide polymer (the structure shows pairing between purines and pyramidines).

Fig. 6.2. (A): Schematic modal of the double helix, one turn of the helix (34 Å or 3.4 Pm), contains nearly 10.5 base pairs. Each base pair is displaced or twisted from the previous one by about 36°. Thus, the X-ray crystal structure of DNA it takes a stalk of about 10 base pairs to go completely around the helix 360°. (B): detailed structure of polynucleotide polymer (the structure shows pairing between purines and pyramidines).

Sugar and base alone are called a nucleoside (Sugar + base = nucleoside). Adding a phosphate to a nucleoside created a nucleotide (Sugar + base + phosphate = Nucleotide). This is by making a glycosidic bond between the base and sugar and by making a phosphoester bond between the sugar and phosphoric acid. As shown in diagram on the next page: DNA composed of phosphate, deoxyribose sugar and four different nitrogenous bases. The bases in DNA fall into two classes, purines and

Sugar and base alone are called a nucleoside (Sugar + base = nucleoside). Adding a phosphate to a nucleoside created a nucleotide (Sugar + base + phosphate = Nucleotide). This is by making a glycosidic bond between the base and sugar and by making a phosphoester bond between the sugar and phosphoric acid. As shown in diagram on the next page: DNA composed of phosphate, deoxyribose sugar and four different nitrogenous bases. The bases in DNA fall into two classes, purines and

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6.3 DEOXYRIBONUCLEIC ACID (DNA)

MICROBIAL GENETICS AND VARIATION

65

6.3 DEOXYRIBONUCLEIC ACID (DNA)

• DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. • The main role of DNA molecule is the long term storage of information. • The DNA segments that carry this genetic information are called genes.

• DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. • The main role of DNA molecule is the long term storage of information. • The DNA segments that carry this genetic information are called genes.

DNA is composed of polynucleotide chains that are held together by weak, non-covalent bonds between pairs of bases. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. This is called the complementary sequence.

DNA is composed of polynucleotide chains that are held together by weak, non-covalent bonds between pairs of bases. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. This is called the complementary sequence.

Fig. 6.2. (A): Schematic modal of the double helix, one turn of the helix (34 Å or 3.4 Pm), contains nearly 10.5 base pairs. Each base pair is displaced or twisted from the previous one by about 36°. Thus, the X-ray crystal structure of DNA it takes a stalk of about 10 base pairs to go completely around the helix 360°. (B): detailed structure of polynucleotide polymer (the structure shows pairing between purines and pyramidines).

Fig. 6.2. (A): Schematic modal of the double helix, one turn of the helix (34 Å or 3.4 Pm), contains nearly 10.5 base pairs. Each base pair is displaced or twisted from the previous one by about 36°. Thus, the X-ray crystal structure of DNA it takes a stalk of about 10 base pairs to go completely around the helix 360°. (B): detailed structure of polynucleotide polymer (the structure shows pairing between purines and pyramidines).

Sugar and base alone are called a nucleoside (Sugar + base = nucleoside). Adding a phosphate to a nucleoside created a nucleotide (Sugar + base + phosphate = Nucleotide). This is by making a glycosidic bond between the base and sugar and by making a phosphoester bond between the sugar and phosphoric acid. As shown in diagram on the next page: DNA composed of phosphate, deoxyribose sugar and four different nitrogenous bases. The bases in DNA fall into two classes, purines and

Sugar and base alone are called a nucleoside (Sugar + base = nucleoside). Adding a phosphate to a nucleoside created a nucleotide (Sugar + base + phosphate = Nucleotide). This is by making a glycosidic bond between the base and sugar and by making a phosphoester bond between the sugar and phosphoric acid. As shown in diagram on the next page: DNA composed of phosphate, deoxyribose sugar and four different nitrogenous bases. The bases in DNA fall into two classes, purines and

66 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

66 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.3. Formation of the nucleotide by removal if water.

Fig. 6.3. Formation of the nucleotide by removal if water.

pyramidines. The purines are adenine and guanine and the pyramidines are cytosine and thymine (but in case of RNA thymine base is replaced by uracil). The purines are derived from the double ringed structure while pyramidines are single ringed structure. The bases are attached to the deoxyribose by glycosidic linkages at N1 of the pyramidines or at N9 of the purines. Adenine bind with Thymine (A:T) with the help of two hydrogen bonds, and guanine bind with cytosine (G:C) through three hydrogen bonds in a DNA double helix.

pyramidines. The purines are adenine and guanine and the pyramidines are cytosine and thymine (but in case of RNA thymine base is replaced by uracil). The purines are derived from the double ringed structure while pyramidines are single ringed structure. The bases are attached to the deoxyribose by glycosidic linkages at N1 of the pyramidines or at N9 of the purines. Adenine bind with Thymine (A:T) with the help of two hydrogen bonds, and guanine bind with cytosine (G:C) through three hydrogen bonds in a DNA double helix.

6.3.1 A Brief History of DNA Edward Chargaff showed that DNA from several species contains equal amounts of A and T, and equal amounts of G and C (1950s). Chargaff’s rules: %A=%T %G=%C 1 purine: 1 pyrimidine.

6.3.1 A Brief History of DNA Edward Chargaff showed that DNA from several species contains equal amounts of A and T, and equal amounts of G and C (1950s). Chargaff’s rules: %A=%T %G=%C 1 purine: 1 pyrimidine.

6.3.2 The Structure of DNA In 1953 Watson and Crick discovered the double helical model or structure of DNA and they got the Noble Prize for this. Before the discovery of Watson and Crick they knew the following evidence about the DNA. 1. Bases, sugars and phosphate groups all linked into a polynucleotide chain. 2. Erwin Chargaff’s rules: A = T and G = C. 3. Rosalind Franklin and Maurice Wilkins had shown using X-ray diffraction that the molecule was a helical structure.

6.3.2 The Structure of DNA In 1953 Watson and Crick discovered the double helical model or structure of DNA and they got the Noble Prize for this. Before the discovery of Watson and Crick they knew the following evidence about the DNA. 1. Bases, sugars and phosphate groups all linked into a polynucleotide chain. 2. Erwin Chargaff’s rules: A = T and G = C. 3. Rosalind Franklin and Maurice Wilkins had shown using X-ray diffraction that the molecule was a helical structure.

66 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

66 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.3. Formation of the nucleotide by removal if water.

Fig. 6.3. Formation of the nucleotide by removal if water.

pyramidines. The purines are adenine and guanine and the pyramidines are cytosine and thymine (but in case of RNA thymine base is replaced by uracil). The purines are derived from the double ringed structure while pyramidines are single ringed structure. The bases are attached to the deoxyribose by glycosidic linkages at N1 of the pyramidines or at N9 of the purines. Adenine bind with Thymine (A:T) with the help of two hydrogen bonds, and guanine bind with cytosine (G:C) through three hydrogen bonds in a DNA double helix.

pyramidines. The purines are adenine and guanine and the pyramidines are cytosine and thymine (but in case of RNA thymine base is replaced by uracil). The purines are derived from the double ringed structure while pyramidines are single ringed structure. The bases are attached to the deoxyribose by glycosidic linkages at N1 of the pyramidines or at N9 of the purines. Adenine bind with Thymine (A:T) with the help of two hydrogen bonds, and guanine bind with cytosine (G:C) through three hydrogen bonds in a DNA double helix.

6.3.1 A Brief History of DNA Edward Chargaff showed that DNA from several species contains equal amounts of A and T, and equal amounts of G and C (1950s). Chargaff’s rules: %A=%T %G=%C 1 purine: 1 pyrimidine.

6.3.1 A Brief History of DNA Edward Chargaff showed that DNA from several species contains equal amounts of A and T, and equal amounts of G and C (1950s). Chargaff’s rules: %A=%T %G=%C 1 purine: 1 pyrimidine.

6.3.2 The Structure of DNA In 1953 Watson and Crick discovered the double helical model or structure of DNA and they got the Noble Prize for this. Before the discovery of Watson and Crick they knew the following evidence about the DNA. 1. Bases, sugars and phosphate groups all linked into a polynucleotide chain. 2. Erwin Chargaff’s rules: A = T and G = C. 3. Rosalind Franklin and Maurice Wilkins had shown using X-ray diffraction that the molecule was a helical structure.

6.3.2 The Structure of DNA In 1953 Watson and Crick discovered the double helical model or structure of DNA and they got the Noble Prize for this. Before the discovery of Watson and Crick they knew the following evidence about the DNA. 1. Bases, sugars and phosphate groups all linked into a polynucleotide chain. 2. Erwin Chargaff’s rules: A = T and G = C. 3. Rosalind Franklin and Maurice Wilkins had shown using X-ray diffraction that the molecule was a helical structure.

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Fig. 6.4. Purines and pyramidines. The dotted lines indicate the sites of attachment of the bases to the sugar.

Fig. 6.4. Purines and pyramidines. The dotted lines indicate the sites of attachment of the bases to the sugar.

Fig. 6.5. Three-dimensional structure of DNA given by Watson and Crick.

Fig. 6.5. Three-dimensional structure of DNA given by Watson and Crick.

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MICROBIAL GENETICS AND VARIATION

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Fig. 6.4. Purines and pyramidines. The dotted lines indicate the sites of attachment of the bases to the sugar.

Fig. 6.4. Purines and pyramidines. The dotted lines indicate the sites of attachment of the bases to the sugar.

Fig. 6.5. Three-dimensional structure of DNA given by Watson and Crick.

Fig. 6.5. Three-dimensional structure of DNA given by Watson and Crick.

68 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

68 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Watson and Crick used this information and build three-dimensional models for the structure of DNA.

Watson and Crick used this information and build three-dimensional models for the structure of DNA.

6.3.3 Watson & Crick’s Observations 1. DNA consists of two chains in a right handed double helix. 2. The diameter is 2 Pm. 3. The 2 chains are antiparallel (opposite polarity). 4. The sugar-phosphate backbone is on the outside and the bases are on the inside. 5. The bases are held together across the two strands by weak hydrogen bonds. 6. The base pairs are 0.34 mm apart. A complete 360 degree turn requires 10 base pairs and is 3.4 Pm long. 7. Because of the way the bases bond with each other, the 2 sugar-phosphate backbones are not equally spaced and 2 distinct grooves of unequal size (a major and a minor groove) are present. (These different size grooves are important in DNA protein interactions.

6.3.3 Watson & Crick’s Observations 1. DNA consists of two chains in a right handed double helix. 2. The diameter is 2 Pm. 3. The 2 chains are antiparallel (opposite polarity). 4. The sugar-phosphate backbone is on the outside and the bases are on the inside. 5. The bases are held together across the two strands by weak hydrogen bonds. 6. The base pairs are 0.34 mm apart. A complete 360 degree turn requires 10 base pairs and is 3.4 Pm long. 7. Because of the way the bases bond with each other, the 2 sugar-phosphate backbones are not equally spaced and 2 distinct grooves of unequal size (a major and a minor groove) are present. (These different size grooves are important in DNA protein interactions.

6.4 STRUCTURE OF RNA

6.4 STRUCTURE OF RNA

RNA contains ribose and uracil, and is usually single-stranded composed of polyribonucleotide chains.

RNA contains ribose and uracil, and is usually single-stranded composed of polyribonucleotide chains.

Fig. 6.6. Structure of RNA

Fig. 6.6. Structure of RNA

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Watson and Crick used this information and build three-dimensional models for the structure of DNA.

Watson and Crick used this information and build three-dimensional models for the structure of DNA.

6.3.3 Watson & Crick’s Observations 1. DNA consists of two chains in a right handed double helix. 2. The diameter is 2 Pm. 3. The 2 chains are antiparallel (opposite polarity). 4. The sugar-phosphate backbone is on the outside and the bases are on the inside. 5. The bases are held together across the two strands by weak hydrogen bonds. 6. The base pairs are 0.34 mm apart. A complete 360 degree turn requires 10 base pairs and is 3.4 Pm long. 7. Because of the way the bases bond with each other, the 2 sugar-phosphate backbones are not equally spaced and 2 distinct grooves of unequal size (a major and a minor groove) are present. (These different size grooves are important in DNA protein interactions.

6.3.3 Watson & Crick’s Observations 1. DNA consists of two chains in a right handed double helix. 2. The diameter is 2 Pm. 3. The 2 chains are antiparallel (opposite polarity). 4. The sugar-phosphate backbone is on the outside and the bases are on the inside. 5. The bases are held together across the two strands by weak hydrogen bonds. 6. The base pairs are 0.34 mm apart. A complete 360 degree turn requires 10 base pairs and is 3.4 Pm long. 7. Because of the way the bases bond with each other, the 2 sugar-phosphate backbones are not equally spaced and 2 distinct grooves of unequal size (a major and a minor groove) are present. (These different size grooves are important in DNA protein interactions.

6.4 STRUCTURE OF RNA

6.4 STRUCTURE OF RNA

RNA contains ribose and uracil, and is usually single-stranded composed of polyribonucleotide chains.

RNA contains ribose and uracil, and is usually single-stranded composed of polyribonucleotide chains.

Fig. 6.6. Structure of RNA

Fig. 6.6. Structure of RNA

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Fig. 6.7. Double helical characteristics of RNA: In an RNA molecule having regions of complementary sequences, the intervening (noncomplementary) stretches of RNA may become “looped out” to form one of the structures illustrated in the figure. (a) hairpin (b) bulge (c) loop.

Fig. 6.7. Double helical characteristics of RNA: In an RNA molecule having regions of complementary sequences, the intervening (noncomplementary) stretches of RNA may become “looped out” to form one of the structures illustrated in the figure. (a) hairpin (b) bulge (c) loop.

Fig. 6.8. The pseudoknot structure is formed by base paring between noncontiguous complementary sequence.

Fig. 6.8. The pseudoknot structure is formed by base paring between noncontiguous complementary sequence.

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Fig. 6.7. Double helical characteristics of RNA: In an RNA molecule having regions of complementary sequences, the intervening (noncomplementary) stretches of RNA may become “looped out” to form one of the structures illustrated in the figure. (a) hairpin (b) bulge (c) loop.

Fig. 6.7. Double helical characteristics of RNA: In an RNA molecule having regions of complementary sequences, the intervening (noncomplementary) stretches of RNA may become “looped out” to form one of the structures illustrated in the figure. (a) hairpin (b) bulge (c) loop.

Fig. 6.8. The pseudoknot structure is formed by base paring between noncontiguous complementary sequence.

Fig. 6.8. The pseudoknot structure is formed by base paring between noncontiguous complementary sequence.

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6.4.1 Types of RNA In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each differs from the others by size, function, and general stability. Messenger RNA (mRNA): It forms 5% of total RNA. 1. The final form of mRNA: the main part has useful codon for protein synthesis. At the 5' end is an initiation or starting codon AUG which codes for methionine amino acid in the eukaryotes and Nformylmethionine in prokaryotes. 2. At the 3' end is the termination codon or stopping codon it can be UAA, UAG and UGA. 3. At the 5' end there is cap for attachment with the ribosome. 4. At 3' end there is a poly A tail (AAAA…..n) part which is cut off before mRNA leaves the nucleus.

6.4.1 Types of RNA In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each differs from the others by size, function, and general stability. Messenger RNA (mRNA): It forms 5% of total RNA. 1. The final form of mRNA: the main part has useful codon for protein synthesis. At the 5' end is an initiation or starting codon AUG which codes for methionine amino acid in the eukaryotes and Nformylmethionine in prokaryotes. 2. At the 3' end is the termination codon or stopping codon it can be UAA, UAG and UGA. 3. At the 5' end there is cap for attachment with the ribosome. 4. At 3' end there is a poly A tail (AAAA…..n) part which is cut off before mRNA leaves the nucleus.

Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5' terminal of mRNA is “capped” by a 7-methylguanosine triphosphate that is linked to an adjacent 2'-O-methyl ribonucleoside at its 5’hydroxyl through the three phosphates. The mRNA molecules frequently contain internal 6-methyladenylates and other 2'-O-ribose methylated nucleotides. The cap is involved in the recognition of mRNA by the translating machinery, and it probably helps stabilize the mRNA by preventing the attack of 5’exonucleases. The protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5' or capped terminal. The other end of most mRNA molecules, the 3'-hydroxyl terminal, has an attached polymer of adenylate (AAAAA……n) residues 20–250 nucleotides in length. The specific function of the poly (A) “tail” at the 3’-hydroxyl terminal of mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific mRNA by preventing the attack of 3'-exonucleases. Some mRNAs, including those for some histones, do not contain poly (A). The poly(A) tail, because it will form a base pair with oligodeoxythymidine polymers attached to a solid substrate like cellulose, can be used to separatemRNA from other species of RNA, including mRNA milecules that lack this tail. In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must be formed by processing from a precursor molecule before entering the cytoplasm. Thus, in mammalian nuclei, the immediate products of gene transcription constitute a fourth class of RNA molecules. These nuclear RNA molecules are very heterogeneous in size and are quite large. The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, whereas the molecular weight of mRNA

Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5' terminal of mRNA is “capped” by a 7-methylguanosine triphosphate that is linked to an adjacent 2'-O-methyl ribonucleoside at its 5’hydroxyl through the three phosphates. The mRNA molecules frequently contain internal 6-methyladenylates and other 2'-O-ribose methylated nucleotides. The cap is involved in the recognition of mRNA by the translating machinery, and it probably helps stabilize the mRNA by preventing the attack of 5’exonucleases. The protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5' or capped terminal. The other end of most mRNA molecules, the 3'-hydroxyl terminal, has an attached polymer of adenylate (AAAAA……n) residues 20–250 nucleotides in length. The specific function of the poly (A) “tail” at the 3’-hydroxyl terminal of mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific mRNA by preventing the attack of 3'-exonucleases. Some mRNAs, including those for some histones, do not contain poly (A). The poly(A) tail, because it will form a base pair with oligodeoxythymidine polymers attached to a solid substrate like cellulose, can be used to separatemRNA from other species of RNA, including mRNA milecules that lack this tail. In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must be formed by processing from a precursor molecule before entering the cytoplasm. Thus, in mammalian nuclei, the immediate products of gene transcription constitute a fourth class of RNA molecules. These nuclear RNA molecules are very heterogeneous in size and are quite large. The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, whereas the molecular weight of mRNA

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70 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6.4.1 Types of RNA In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each differs from the others by size, function, and general stability. Messenger RNA (mRNA): It forms 5% of total RNA. 1. The final form of mRNA: the main part has useful codon for protein synthesis. At the 5' end is an initiation or starting codon AUG which codes for methionine amino acid in the eukaryotes and Nformylmethionine in prokaryotes. 2. At the 3' end is the termination codon or stopping codon it can be UAA, UAG and UGA. 3. At the 5' end there is cap for attachment with the ribosome. 4. At 3' end there is a poly A tail (AAAA…..n) part which is cut off before mRNA leaves the nucleus.

6.4.1 Types of RNA In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each differs from the others by size, function, and general stability. Messenger RNA (mRNA): It forms 5% of total RNA. 1. The final form of mRNA: the main part has useful codon for protein synthesis. At the 5' end is an initiation or starting codon AUG which codes for methionine amino acid in the eukaryotes and Nformylmethionine in prokaryotes. 2. At the 3' end is the termination codon or stopping codon it can be UAA, UAG and UGA. 3. At the 5' end there is cap for attachment with the ribosome. 4. At 3' end there is a poly A tail (AAAA…..n) part which is cut off before mRNA leaves the nucleus.

Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5' terminal of mRNA is “capped” by a 7-methylguanosine triphosphate that is linked to an adjacent 2'-O-methyl ribonucleoside at its 5’hydroxyl through the three phosphates. The mRNA molecules frequently contain internal 6-methyladenylates and other 2'-O-ribose methylated nucleotides. The cap is involved in the recognition of mRNA by the translating machinery, and it probably helps stabilize the mRNA by preventing the attack of 5’exonucleases. The protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5' or capped terminal. The other end of most mRNA molecules, the 3'-hydroxyl terminal, has an attached polymer of adenylate (AAAAA……n) residues 20–250 nucleotides in length. The specific function of the poly (A) “tail” at the 3’-hydroxyl terminal of mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific mRNA by preventing the attack of 3'-exonucleases. Some mRNAs, including those for some histones, do not contain poly (A). The poly(A) tail, because it will form a base pair with oligodeoxythymidine polymers attached to a solid substrate like cellulose, can be used to separatemRNA from other species of RNA, including mRNA milecules that lack this tail. In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must be formed by processing from a precursor molecule before entering the cytoplasm. Thus, in mammalian nuclei, the immediate products of gene transcription constitute a fourth class of RNA molecules. These nuclear RNA molecules are very heterogeneous in size and are quite large. The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, whereas the molecular weight of mRNA

Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5' terminal of mRNA is “capped” by a 7-methylguanosine triphosphate that is linked to an adjacent 2'-O-methyl ribonucleoside at its 5’hydroxyl through the three phosphates. The mRNA molecules frequently contain internal 6-methyladenylates and other 2'-O-ribose methylated nucleotides. The cap is involved in the recognition of mRNA by the translating machinery, and it probably helps stabilize the mRNA by preventing the attack of 5’exonucleases. The protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5' or capped terminal. The other end of most mRNA molecules, the 3'-hydroxyl terminal, has an attached polymer of adenylate (AAAAA……n) residues 20–250 nucleotides in length. The specific function of the poly (A) “tail” at the 3’-hydroxyl terminal of mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific mRNA by preventing the attack of 3'-exonucleases. Some mRNAs, including those for some histones, do not contain poly (A). The poly(A) tail, because it will form a base pair with oligodeoxythymidine polymers attached to a solid substrate like cellulose, can be used to separatemRNA from other species of RNA, including mRNA milecules that lack this tail. In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must be formed by processing from a precursor molecule before entering the cytoplasm. Thus, in mammalian nuclei, the immediate products of gene transcription constitute a fourth class of RNA molecules. These nuclear RNA molecules are very heterogeneous in size and are quite large. The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, whereas the molecular weight of mRNA

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Fig. 6.9. The expression of genetic information in DNA into the form of an mRNA transcript. This is subsequently translated by ribosomes into a specific protein molecule.

Fig. 6.9. The expression of genetic information in DNA into the form of an mRNA transcript. This is subsequently translated by ribosomes into a specific protein molecule.

molecules is generally less than 2 × 106. Heterogeneous nuclear RNA molecules are processed to generate the mRNA molecules which then enter the cytoplasm to serves as templates for protein synthesis. Transfer RNA (tRNA): It forms 15% of total RNA. tRNA molecules vary in length from 74 to 95 nucleotides. They are also generated by nuclear processing of precursor molecules. The tRNA molecule serves as an adapter for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA molecules as a class have many features in common. The primary structure, i.e., the nucleotide sequence of all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears like a cloverleaf. All tRNA molecules contain four main arms. The acceptor arm helps in the attachment of amino acid. The amino acid is attached to the 3'-OH group of the A moiety of the acceptor arm. The D, T, C, and extra arms help define a specific tRNA. Although tRNAs are quite stable in prokaryotes, they are somewhat less stable in eukaryotes. The opposite is true for mRNAs, which are quite unstable in prokaryotes but generally stable in eukaryotic organisms.

molecules is generally less than 2 × 106. Heterogeneous nuclear RNA molecules are processed to generate the mRNA molecules which then enter the cytoplasm to serves as templates for protein synthesis. Transfer RNA (tRNA): It forms 15% of total RNA. tRNA molecules vary in length from 74 to 95 nucleotides. They are also generated by nuclear processing of precursor molecules. The tRNA molecule serves as an adapter for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA molecules as a class have many features in common. The primary structure, i.e., the nucleotide sequence of all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears like a cloverleaf. All tRNA molecules contain four main arms. The acceptor arm helps in the attachment of amino acid. The amino acid is attached to the 3'-OH group of the A moiety of the acceptor arm. The D, T, C, and extra arms help define a specific tRNA. Although tRNAs are quite stable in prokaryotes, they are somewhat less stable in eukaryotes. The opposite is true for mRNAs, which are quite unstable in prokaryotes but generally stable in eukaryotic organisms.

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Fig. 6.9. The expression of genetic information in DNA into the form of an mRNA transcript. This is subsequently translated by ribosomes into a specific protein molecule.

Fig. 6.9. The expression of genetic information in DNA into the form of an mRNA transcript. This is subsequently translated by ribosomes into a specific protein molecule.

molecules is generally less than 2 × 106. Heterogeneous nuclear RNA molecules are processed to generate the mRNA molecules which then enter the cytoplasm to serves as templates for protein synthesis. Transfer RNA (tRNA): It forms 15% of total RNA. tRNA molecules vary in length from 74 to 95 nucleotides. They are also generated by nuclear processing of precursor molecules. The tRNA molecule serves as an adapter for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA molecules as a class have many features in common. The primary structure, i.e., the nucleotide sequence of all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears like a cloverleaf. All tRNA molecules contain four main arms. The acceptor arm helps in the attachment of amino acid. The amino acid is attached to the 3'-OH group of the A moiety of the acceptor arm. The D, T, C, and extra arms help define a specific tRNA. Although tRNAs are quite stable in prokaryotes, they are somewhat less stable in eukaryotes. The opposite is true for mRNAs, which are quite unstable in prokaryotes but generally stable in eukaryotic organisms.

molecules is generally less than 2 × 106. Heterogeneous nuclear RNA molecules are processed to generate the mRNA molecules which then enter the cytoplasm to serves as templates for protein synthesis. Transfer RNA (tRNA): It forms 15% of total RNA. tRNA molecules vary in length from 74 to 95 nucleotides. They are also generated by nuclear processing of precursor molecules. The tRNA molecule serves as an adapter for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA molecules as a class have many features in common. The primary structure, i.e., the nucleotide sequence of all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears like a cloverleaf. All tRNA molecules contain four main arms. The acceptor arm helps in the attachment of amino acid. The amino acid is attached to the 3'-OH group of the A moiety of the acceptor arm. The D, T, C, and extra arms help define a specific tRNA. Although tRNAs are quite stable in prokaryotes, they are somewhat less stable in eukaryotes. The opposite is true for mRNAs, which are quite unstable in prokaryotes but generally stable in eukaryotic organisms.

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Fig. 6.10. Typical aminoacyl tRNA in which the amino acid is attached to the 3' CCA terminal. The anticodon, TØC, and dihydrouracil (D) arms are indicated, as are the positions of the intramolecular hydrogen bonding between these base pairs.

Fig. 6.10. Typical aminoacyl tRNA in which the amino acid is attached to the 3' CCA terminal. The anticodon, TØC, and dihydrouracil (D) arms are indicated, as are the positions of the intramolecular hydrogen bonding between these base pairs.

Ribosomal RNA (rRNA): It forms the 80% of the total RNA. A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into specific protein molecules information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. rRNA produced from the nucleolus in the nucleus then passed to the cytoplasm through the nuclear pore. The ribosome has 2 subunits called Svedberg units. In prokaryotes the subdivided are 30S and 50S where together they are called 70S and in case of eukaryotes these subunits are 40S and 60S but together they are called 80S.

Ribosomal RNA (rRNA): It forms the 80% of the total RNA. A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into specific protein molecules information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. rRNA produced from the nucleolus in the nucleus then passed to the cytoplasm through the nuclear pore. The ribosome has 2 subunits called Svedberg units. In prokaryotes the subdivided are 30S and 50S where together they are called 70S and in case of eukaryotes these subunits are 40S and 60S but together they are called 80S.

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Fig. 6.10. Typical aminoacyl tRNA in which the amino acid is attached to the 3' CCA terminal. The anticodon, TØC, and dihydrouracil (D) arms are indicated, as are the positions of the intramolecular hydrogen bonding between these base pairs.

Fig. 6.10. Typical aminoacyl tRNA in which the amino acid is attached to the 3' CCA terminal. The anticodon, TØC, and dihydrouracil (D) arms are indicated, as are the positions of the intramolecular hydrogen bonding between these base pairs.

Ribosomal RNA (rRNA): It forms the 80% of the total RNA. A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into specific protein molecules information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. rRNA produced from the nucleolus in the nucleus then passed to the cytoplasm through the nuclear pore. The ribosome has 2 subunits called Svedberg units. In prokaryotes the subdivided are 30S and 50S where together they are called 70S and in case of eukaryotes these subunits are 40S and 60S but together they are called 80S.

Ribosomal RNA (rRNA): It forms the 80% of the total RNA. A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into specific protein molecules information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. rRNA produced from the nucleolus in the nucleus then passed to the cytoplasm through the nuclear pore. The ribosome has 2 subunits called Svedberg units. In prokaryotes the subdivided are 30S and 50S where together they are called 70S and in case of eukaryotes these subunits are 40S and 60S but together they are called 80S.

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The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to ribosomes and its translation. Recent studies suggest that an rRNA component performs—the peptidyl transferase activity and thus is an enzyme—a ribozymes.

The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to ribosomes and its translation. Recent studies suggest that an rRNA component performs—the peptidyl transferase activity and thus is an enzyme—a ribozymes.

6.4.2 Biological Roles of RNA 1. RNA is the genetic material of some viruses. 2. RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery. 3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4. RNA serves as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of certain mRNAs. 5. Some RNAs are enzymes that catalyze essential reactions in the cell (RNase P ribozyme, large rRNA, self-splicing introns, etc).

6.4.2 Biological Roles of RNA 1. RNA is the genetic material of some viruses. 2. RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery. 3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4. RNA serves as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of certain mRNAs. 5. Some RNAs are enzymes that catalyze essential reactions in the cell (RNase P ribozyme, large rRNA, self-splicing introns, etc).

6.4.3 Difference between DNA and RNA

6.4.3 Difference between DNA and RNA

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The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to ribosomes and its translation. Recent studies suggest that an rRNA component performs—the peptidyl transferase activity and thus is an enzyme—a ribozymes.

The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to ribosomes and its translation. Recent studies suggest that an rRNA component performs—the peptidyl transferase activity and thus is an enzyme—a ribozymes.

6.4.2 Biological Roles of RNA 1. RNA is the genetic material of some viruses. 2. RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery. 3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4. RNA serves as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of certain mRNAs. 5. Some RNAs are enzymes that catalyze essential reactions in the cell (RNase P ribozyme, large rRNA, self-splicing introns, etc).

6.4.2 Biological Roles of RNA 1. RNA is the genetic material of some viruses. 2. RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery. 3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4. RNA serves as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of certain mRNAs. 5. Some RNAs are enzymes that catalyze essential reactions in the cell (RNase P ribozyme, large rRNA, self-splicing introns, etc).

6.4.3 Difference between DNA and RNA

6.4.3 Difference between DNA and RNA

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6.5 GENETIC CODE

6.5 GENETIC CODE

The genetic information passes into the form of triplet code which is called the genetic code. The genomic of an organism is inscribed in DNA, or in the case of some viruses, RNA. The portion of the genome that codes for a protein or RNA is referred to as a gene. Those genes that code for proteins are composed of triplet code or tri-nucleotide units called codons, each coding for a single amino acid. Each nucleotide subunit consists of a phosphate, deoxyribose sugar and one of the 4 nitrogenous nucleotide bases (A, G, C and U). There are 4³ = 64 different codon combinations possible with a triplet codon of three nucleotides; all 64 codons are assigned for either amino acids or stop signals during translation. If, for example, an RNA sequence, UUUAAACCC is considered and the reading frame starts with the first U, there are three codons, namely, UUU, AAA and CCC, each of which specifies one amino acid. Translation starts with a initiation codon or start codon and stop with stopping codon. The most common start codon is AUG which is read as methionine or, in bacteria it is formylmethionine. The stopping codons have been given names as, UAG, UGA, and UAA. The table shows the 64 codons and the amino acid for each. The direction of the mRNA is 5' to 3'. The codon AUG both codes for methionine and serves as an initiation site: the first AUG in an mRNS’s coding region is where translation into protein begins.

The genetic information passes into the form of triplet code which is called the genetic code. The genomic of an organism is inscribed in DNA, or in the case of some viruses, RNA. The portion of the genome that codes for a protein or RNA is referred to as a gene. Those genes that code for proteins are composed of triplet code or tri-nucleotide units called codons, each coding for a single amino acid. Each nucleotide subunit consists of a phosphate, deoxyribose sugar and one of the 4 nitrogenous nucleotide bases (A, G, C and U). There are 4³ = 64 different codon combinations possible with a triplet codon of three nucleotides; all 64 codons are assigned for either amino acids or stop signals during translation. If, for example, an RNA sequence, UUUAAACCC is considered and the reading frame starts with the first U, there are three codons, namely, UUU, AAA and CCC, each of which specifies one amino acid. Translation starts with a initiation codon or start codon and stop with stopping codon. The most common start codon is AUG which is read as methionine or, in bacteria it is formylmethionine. The stopping codons have been given names as, UAG, UGA, and UAA. The table shows the 64 codons and the amino acid for each. The direction of the mRNA is 5' to 3'. The codon AUG both codes for methionine and serves as an initiation site: the first AUG in an mRNS’s coding region is where translation into protein begins.

6.6 GENOTYPE AND PHENOTYPE

6.6 GENOTYPE AND PHENOTYPE

A genotype refers to the actual set of genes that an organism carries inside. When these genes are expressed under observable conditions, they are called phenotypes and the expressions are called phenotypic expressions. The fact is, phenotypes are dependent on the genes they inherit. However, their expression is also influenced by environmental factors. The influence of the environment modifies the role that the genes play to a certain extent. The expression of the genes, modified by environmental factors, produces a phenotype. Genotype and phenotype represent very real differences between genetic composition and expressed form. A genotype basically determines the type of traits that a phenotype can have. For instance, the genotypic traits of an organism will determine his susceptibility to a certain disease. However, the phenotypical aspect of the organism displays observable aspects of this disease. The symptoms related to the particular aspect of the disease, the presence or even the absence of such a disease are phenotypic expression.

A genotype refers to the actual set of genes that an organism carries inside. When these genes are expressed under observable conditions, they are called phenotypes and the expressions are called phenotypic expressions. The fact is, phenotypes are dependent on the genes they inherit. However, their expression is also influenced by environmental factors. The influence of the environment modifies the role that the genes play to a certain extent. The expression of the genes, modified by environmental factors, produces a phenotype. Genotype and phenotype represent very real differences between genetic composition and expressed form. A genotype basically determines the type of traits that a phenotype can have. For instance, the genotypic traits of an organism will determine his susceptibility to a certain disease. However, the phenotypical aspect of the organism displays observable aspects of this disease. The symptoms related to the particular aspect of the disease, the presence or even the absence of such a disease are phenotypic expression.

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6.5 GENETIC CODE

6.5 GENETIC CODE

The genetic information passes into the form of triplet code which is called the genetic code. The genomic of an organism is inscribed in DNA, or in the case of some viruses, RNA. The portion of the genome that codes for a protein or RNA is referred to as a gene. Those genes that code for proteins are composed of triplet code or tri-nucleotide units called codons, each coding for a single amino acid. Each nucleotide subunit consists of a phosphate, deoxyribose sugar and one of the 4 nitrogenous nucleotide bases (A, G, C and U). There are 4³ = 64 different codon combinations possible with a triplet codon of three nucleotides; all 64 codons are assigned for either amino acids or stop signals during translation. If, for example, an RNA sequence, UUUAAACCC is considered and the reading frame starts with the first U, there are three codons, namely, UUU, AAA and CCC, each of which specifies one amino acid. Translation starts with a initiation codon or start codon and stop with stopping codon. The most common start codon is AUG which is read as methionine or, in bacteria it is formylmethionine. The stopping codons have been given names as, UAG, UGA, and UAA. The table shows the 64 codons and the amino acid for each. The direction of the mRNA is 5' to 3'. The codon AUG both codes for methionine and serves as an initiation site: the first AUG in an mRNS’s coding region is where translation into protein begins.

The genetic information passes into the form of triplet code which is called the genetic code. The genomic of an organism is inscribed in DNA, or in the case of some viruses, RNA. The portion of the genome that codes for a protein or RNA is referred to as a gene. Those genes that code for proteins are composed of triplet code or tri-nucleotide units called codons, each coding for a single amino acid. Each nucleotide subunit consists of a phosphate, deoxyribose sugar and one of the 4 nitrogenous nucleotide bases (A, G, C and U). There are 4³ = 64 different codon combinations possible with a triplet codon of three nucleotides; all 64 codons are assigned for either amino acids or stop signals during translation. If, for example, an RNA sequence, UUUAAACCC is considered and the reading frame starts with the first U, there are three codons, namely, UUU, AAA and CCC, each of which specifies one amino acid. Translation starts with a initiation codon or start codon and stop with stopping codon. The most common start codon is AUG which is read as methionine or, in bacteria it is formylmethionine. The stopping codons have been given names as, UAG, UGA, and UAA. The table shows the 64 codons and the amino acid for each. The direction of the mRNA is 5' to 3'. The codon AUG both codes for methionine and serves as an initiation site: the first AUG in an mRNS’s coding region is where translation into protein begins.

6.6 GENOTYPE AND PHENOTYPE

6.6 GENOTYPE AND PHENOTYPE

A genotype refers to the actual set of genes that an organism carries inside. When these genes are expressed under observable conditions, they are called phenotypes and the expressions are called phenotypic expressions. The fact is, phenotypes are dependent on the genes they inherit. However, their expression is also influenced by environmental factors. The influence of the environment modifies the role that the genes play to a certain extent. The expression of the genes, modified by environmental factors, produces a phenotype. Genotype and phenotype represent very real differences between genetic composition and expressed form. A genotype basically determines the type of traits that a phenotype can have. For instance, the genotypic traits of an organism will determine his susceptibility to a certain disease. However, the phenotypical aspect of the organism displays observable aspects of this disease. The symptoms related to the particular aspect of the disease, the presence or even the absence of such a disease are phenotypic expression.

A genotype refers to the actual set of genes that an organism carries inside. When these genes are expressed under observable conditions, they are called phenotypes and the expressions are called phenotypic expressions. The fact is, phenotypes are dependent on the genes they inherit. However, their expression is also influenced by environmental factors. The influence of the environment modifies the role that the genes play to a certain extent. The expression of the genes, modified by environmental factors, produces a phenotype. Genotype and phenotype represent very real differences between genetic composition and expressed form. A genotype basically determines the type of traits that a phenotype can have. For instance, the genotypic traits of an organism will determine his susceptibility to a certain disease. However, the phenotypical aspect of the organism displays observable aspects of this disease. The symptoms related to the particular aspect of the disease, the presence or even the absence of such a disease are phenotypic expression.

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A phenotypic trait may be visible to eye, such as the colour of a flower or the texture of hair, or it may require special tests for its identification, as in the determiniation of the respiratory quotient or the serological test for blood type. Each phenotypic trait of an organism is caused and determined by at least two Mendelian factors which are now called genes. The genes may be defined as hereditary units which reside in a long DNA molecule (the main constituent molecule of chromosome) and contains coded instructions or information for the production of proteins, the organic substances which ultimately cause and determine a phenotypic trait in a given environment.

A phenotypic trait may be visible to eye, such as the colour of a flower or the texture of hair, or it may require special tests for its identification, as in the determiniation of the respiratory quotient or the serological test for blood type. Each phenotypic trait of an organism is caused and determined by at least two Mendelian factors which are now called genes. The genes may be defined as hereditary units which reside in a long DNA molecule (the main constituent molecule of chromosome) and contains coded instructions or information for the production of proteins, the organic substances which ultimately cause and determine a phenotypic trait in a given environment.

Summary

Summary

1. Genotype decides the genetics and inherited traits of an organism, but phenotypes refer to the actual display of these traits. 2. Genotypes are decided by inherited genes, while phenotypes are determined by the effect of environmental factors. 3. The genotype largely determines the ultimate phenotype of an organism. 4. The more complex a biological process, the more is the effect of environmental factors on it and therefore the chances of a predominant phenotype.

1. Genotype decides the genetics and inherited traits of an organism, but phenotypes refer to the actual display of these traits. 2. Genotypes are decided by inherited genes, while phenotypes are determined by the effect of environmental factors. 3. The genotype largely determines the ultimate phenotype of an organism. 4. The more complex a biological process, the more is the effect of environmental factors on it and therefore the chances of a predominant phenotype.

6.7 REPLICATION

6.7 REPLICATION

It is a process in which parental DNA strand forms two daughter strands or the copy of parental strand. Each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. DNA polymerase is the enzyme that synthesizes the new DNA strands by adding nucleotides matched to the template strand, a number of other enzymes are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA polymerase always synthesizes a new strand 5'  3' direction. During replication one strand acts as continuous or leading strand (3'  5') in which DNA strand is synthesized continuously and another one is discontinuous or lagging strand (5'  3') in which okazaki fragments are formed which is removed by the enzyme exonuclease. DNA polymerase also has a proof reading property i.e., it repairs the miss matched nucleotides. DNA polymerase joins 1000 base pairs per second and after joining 107 base pairs the possibilities of one miss-matching which is corrected by the DNA polymerase. This correction of DNA polymerase is known as proof reading property.

It is a process in which parental DNA strand forms two daughter strands or the copy of parental strand. Each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. DNA polymerase is the enzyme that synthesizes the new DNA strands by adding nucleotides matched to the template strand, a number of other enzymes are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA polymerase always synthesizes a new strand 5'  3' direction. During replication one strand acts as continuous or leading strand (3'  5') in which DNA strand is synthesized continuously and another one is discontinuous or lagging strand (5'  3') in which okazaki fragments are formed which is removed by the enzyme exonuclease. DNA polymerase also has a proof reading property i.e., it repairs the miss matched nucleotides. DNA polymerase joins 1000 base pairs per second and after joining 107 base pairs the possibilities of one miss-matching which is corrected by the DNA polymerase. This correction of DNA polymerase is known as proof reading property.

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A phenotypic trait may be visible to eye, such as the colour of a flower or the texture of hair, or it may require special tests for its identification, as in the determiniation of the respiratory quotient or the serological test for blood type. Each phenotypic trait of an organism is caused and determined by at least two Mendelian factors which are now called genes. The genes may be defined as hereditary units which reside in a long DNA molecule (the main constituent molecule of chromosome) and contains coded instructions or information for the production of proteins, the organic substances which ultimately cause and determine a phenotypic trait in a given environment.

A phenotypic trait may be visible to eye, such as the colour of a flower or the texture of hair, or it may require special tests for its identification, as in the determiniation of the respiratory quotient or the serological test for blood type. Each phenotypic trait of an organism is caused and determined by at least two Mendelian factors which are now called genes. The genes may be defined as hereditary units which reside in a long DNA molecule (the main constituent molecule of chromosome) and contains coded instructions or information for the production of proteins, the organic substances which ultimately cause and determine a phenotypic trait in a given environment.

Summary

Summary

1. Genotype decides the genetics and inherited traits of an organism, but phenotypes refer to the actual display of these traits. 2. Genotypes are decided by inherited genes, while phenotypes are determined by the effect of environmental factors. 3. The genotype largely determines the ultimate phenotype of an organism. 4. The more complex a biological process, the more is the effect of environmental factors on it and therefore the chances of a predominant phenotype.

1. Genotype decides the genetics and inherited traits of an organism, but phenotypes refer to the actual display of these traits. 2. Genotypes are decided by inherited genes, while phenotypes are determined by the effect of environmental factors. 3. The genotype largely determines the ultimate phenotype of an organism. 4. The more complex a biological process, the more is the effect of environmental factors on it and therefore the chances of a predominant phenotype.

6.7 REPLICATION

6.7 REPLICATION

It is a process in which parental DNA strand forms two daughter strands or the copy of parental strand. Each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. DNA polymerase is the enzyme that synthesizes the new DNA strands by adding nucleotides matched to the template strand, a number of other enzymes are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA polymerase always synthesizes a new strand 5'  3' direction. During replication one strand acts as continuous or leading strand (3'  5') in which DNA strand is synthesized continuously and another one is discontinuous or lagging strand (5'  3') in which okazaki fragments are formed which is removed by the enzyme exonuclease. DNA polymerase also has a proof reading property i.e., it repairs the miss matched nucleotides. DNA polymerase joins 1000 base pairs per second and after joining 107 base pairs the possibilities of one miss-matching which is corrected by the DNA polymerase. This correction of DNA polymerase is known as proof reading property.

It is a process in which parental DNA strand forms two daughter strands or the copy of parental strand. Each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. DNA polymerase is the enzyme that synthesizes the new DNA strands by adding nucleotides matched to the template strand, a number of other enzymes are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA polymerase always synthesizes a new strand 5'  3' direction. During replication one strand acts as continuous or leading strand (3'  5') in which DNA strand is synthesized continuously and another one is discontinuous or lagging strand (5'  3') in which okazaki fragments are formed which is removed by the enzyme exonuclease. DNA polymerase also has a proof reading property i.e., it repairs the miss matched nucleotides. DNA polymerase joins 1000 base pairs per second and after joining 107 base pairs the possibilities of one miss-matching which is corrected by the DNA polymerase. This correction of DNA polymerase is known as proof reading property.

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Fig. 6.11. Showing the different enzymes used in DNA replication.

Fig. 6.11. Showing the different enzymes used in DNA replication.

Fig. 6.12. Process of DNA replication.

Fig. 6.12. Process of DNA replication.

Process of DNA replication 1. 2. 3. 4.

Segments of single-stranded DNA are called template strands. Segments of single-stranded DNA are called template strands. Gyrase (a type of topoisomerase) relaxes the supercoiled DNA. Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate). (Hydrolysis of ATP causes a shape change in DNA helicase.)

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Process of DNA replication 1. 2. 3. 4.

Segments of single-stranded DNA are called template strands. Segments of single-stranded DNA are called template strands. Gyrase (a type of topoisomerase) relaxes the supercoiled DNA. Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate). (Hydrolysis of ATP causes a shape change in DNA helicase.)

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Fig. 6.11. Showing the different enzymes used in DNA replication.

Fig. 6.11. Showing the different enzymes used in DNA replication.

Fig. 6.12. Process of DNA replication.

Fig. 6.12. Process of DNA replication.

Process of DNA replication 1. 2. 3. 4.

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Segments of single-stranded DNA are called template strands. Segments of single-stranded DNA are called template strands. Gyrase (a type of topoisomerase) relaxes the supercoiled DNA. Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate). (Hydrolysis of ATP causes a shape change in DNA helicase.)

Process of DNA replication 1. 2. 3. 4.

Segments of single-stranded DNA are called template strands. Segments of single-stranded DNA are called template strands. Gyrase (a type of topoisomerase) relaxes the supercoiled DNA. Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate). (Hydrolysis of ATP causes a shape change in DNA helicase.)

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5. Single strand binding (SSB) proteins prevent single strands from rewinding. 6. Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase adds nucleotides. 7. DNA polymerase adds nucleotides 5' to 3' on both strands beginning at the RNA primer. 8. DNA polymerase has a proof reading property bases added and replaces incorrect nucleotides. 9. Leading strand synthesis continues in a 5' to 3' direction. 10. Discontinuous synthesis produces 5' to 3' DNA segments called Okazaki fragments. 11. Exonuclease enzymes remove RNA primers. 12. Ligase forms bonds between sugar-phosphate backbone.

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5. Single strand binding (SSB) proteins prevent single strands from rewinding. 6. Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase adds nucleotides. 7. DNA polymerase adds nucleotides 5' to 3' on both strands beginning at the RNA primer. 8. DNA polymerase has a proof reading property bases added and replaces incorrect nucleotides. 9. Leading strand synthesis continues in a 5' to 3' direction. 10. Discontinuous synthesis produces 5' to 3' DNA segments called Okazaki fragments. 11. Exonuclease enzymes remove RNA primers. 12. Ligase forms bonds between sugar-phosphate backbone.

6.8 TRANSCRIPTION

6.8 TRANSCRIPTION

It is the process in which RNA synthesis takes place from DNA template strand. Similar to DNA polymerase (adds deoxyribonucleotides in replication), RNA polymerase adds new ribonucleotides to the synthesizing RNA strand. During transcription, DNA sequence is read by RNA plymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. RNA polymerase also has a proof reading property. Although it is very accurate, but less accurate than replication (one mistake occurs in 10,000 nucleotides added, compared to one in 10,000,000 for replication). This difference reflects the lack of extensive proof reading mechanisms for transcription. Transcription by RNA polymerase proceeds in a series of steps which are grouped into three phases: initiation, elongation and termination. Note: RNA polymerase does not need a primer like replication. 1. Initiation: In this step RNA polymerase bind to the promoter. Now the promoter-polymerase complex undergoes structural changes required to initiation to proceed and DNA starts unwind and producing a bubble. Transcription always occurs in a 5' to 3' direction. That is the new ribonucleotides is added to the 3' end of the growing chain. In transcription only one stand act as a template strand on which the RNA strand is built. 2. Elongation: Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase. This transition requires further conformational changes in polymerase that lead it to grip the template more firmly and adds more ribonucleotides to the strand. RNA polymerase also performs proofreading functions. 3. Termination: Once the polymerase has transcribed the length of gene, it must stop and release the RNA product. This step is called termination.

It is the process in which RNA synthesis takes place from DNA template strand. Similar to DNA polymerase (adds deoxyribonucleotides in replication), RNA polymerase adds new ribonucleotides to the synthesizing RNA strand. During transcription, DNA sequence is read by RNA plymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. RNA polymerase also has a proof reading property. Although it is very accurate, but less accurate than replication (one mistake occurs in 10,000 nucleotides added, compared to one in 10,000,000 for replication). This difference reflects the lack of extensive proof reading mechanisms for transcription. Transcription by RNA polymerase proceeds in a series of steps which are grouped into three phases: initiation, elongation and termination. Note: RNA polymerase does not need a primer like replication. 1. Initiation: In this step RNA polymerase bind to the promoter. Now the promoter-polymerase complex undergoes structural changes required to initiation to proceed and DNA starts unwind and producing a bubble. Transcription always occurs in a 5' to 3' direction. That is the new ribonucleotides is added to the 3' end of the growing chain. In transcription only one stand act as a template strand on which the RNA strand is built. 2. Elongation: Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase. This transition requires further conformational changes in polymerase that lead it to grip the template more firmly and adds more ribonucleotides to the strand. RNA polymerase also performs proofreading functions. 3. Termination: Once the polymerase has transcribed the length of gene, it must stop and release the RNA product. This step is called termination.

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5. Single strand binding (SSB) proteins prevent single strands from rewinding. 6. Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase adds nucleotides. 7. DNA polymerase adds nucleotides 5' to 3' on both strands beginning at the RNA primer. 8. DNA polymerase has a proof reading property bases added and replaces incorrect nucleotides. 9. Leading strand synthesis continues in a 5' to 3' direction. 10. Discontinuous synthesis produces 5' to 3' DNA segments called Okazaki fragments. 11. Exonuclease enzymes remove RNA primers. 12. Ligase forms bonds between sugar-phosphate backbone.

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5. Single strand binding (SSB) proteins prevent single strands from rewinding. 6. Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase adds nucleotides. 7. DNA polymerase adds nucleotides 5' to 3' on both strands beginning at the RNA primer. 8. DNA polymerase has a proof reading property bases added and replaces incorrect nucleotides. 9. Leading strand synthesis continues in a 5' to 3' direction. 10. Discontinuous synthesis produces 5' to 3' DNA segments called Okazaki fragments. 11. Exonuclease enzymes remove RNA primers. 12. Ligase forms bonds between sugar-phosphate backbone.

6.8 TRANSCRIPTION

6.8 TRANSCRIPTION

It is the process in which RNA synthesis takes place from DNA template strand. Similar to DNA polymerase (adds deoxyribonucleotides in replication), RNA polymerase adds new ribonucleotides to the synthesizing RNA strand. During transcription, DNA sequence is read by RNA plymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. RNA polymerase also has a proof reading property. Although it is very accurate, but less accurate than replication (one mistake occurs in 10,000 nucleotides added, compared to one in 10,000,000 for replication). This difference reflects the lack of extensive proof reading mechanisms for transcription. Transcription by RNA polymerase proceeds in a series of steps which are grouped into three phases: initiation, elongation and termination. Note: RNA polymerase does not need a primer like replication. 1. Initiation: In this step RNA polymerase bind to the promoter. Now the promoter-polymerase complex undergoes structural changes required to initiation to proceed and DNA starts unwind and producing a bubble. Transcription always occurs in a 5' to 3' direction. That is the new ribonucleotides is added to the 3' end of the growing chain. In transcription only one stand act as a template strand on which the RNA strand is built. 2. Elongation: Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase. This transition requires further conformational changes in polymerase that lead it to grip the template more firmly and adds more ribonucleotides to the strand. RNA polymerase also performs proofreading functions. 3. Termination: Once the polymerase has transcribed the length of gene, it must stop and release the RNA product. This step is called termination.

It is the process in which RNA synthesis takes place from DNA template strand. Similar to DNA polymerase (adds deoxyribonucleotides in replication), RNA polymerase adds new ribonucleotides to the synthesizing RNA strand. During transcription, DNA sequence is read by RNA plymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. RNA polymerase also has a proof reading property. Although it is very accurate, but less accurate than replication (one mistake occurs in 10,000 nucleotides added, compared to one in 10,000,000 for replication). This difference reflects the lack of extensive proof reading mechanisms for transcription. Transcription by RNA polymerase proceeds in a series of steps which are grouped into three phases: initiation, elongation and termination. Note: RNA polymerase does not need a primer like replication. 1. Initiation: In this step RNA polymerase bind to the promoter. Now the promoter-polymerase complex undergoes structural changes required to initiation to proceed and DNA starts unwind and producing a bubble. Transcription always occurs in a 5' to 3' direction. That is the new ribonucleotides is added to the 3' end of the growing chain. In transcription only one stand act as a template strand on which the RNA strand is built. 2. Elongation: Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase. This transition requires further conformational changes in polymerase that lead it to grip the template more firmly and adds more ribonucleotides to the strand. RNA polymerase also performs proofreading functions. 3. Termination: Once the polymerase has transcribed the length of gene, it must stop and release the RNA product. This step is called termination.

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Fig. 6.13. The phases of the transcription cycles: initiation, elongation and termination.

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Fig. 6.13. The phases of the transcription cycles: initiation, elongation and termination.

6.9 TRANSLATION

6.9 TRANSLATION

It is the process of formation of protein from the mature RNA. The protein synthesis on mRNA strand takes place from 5 to 3 directions. Proteins are synthesized by the addition of amino acids and formation of peptide bond between them. There are three types of RNA which help in the protein synthesis (mRNA, rRNA and tRNA are described earlier of this chapter in structure of RNA).

It is the process of formation of protein from the mature RNA. The protein synthesis on mRNA strand takes place from 5 to 3 directions. Proteins are synthesized by the addition of amino acids and formation of peptide bond between them. There are three types of RNA which help in the protein synthesis (mRNA, rRNA and tRNA are described earlier of this chapter in structure of RNA).

Steps of protein synthesis

Steps of protein synthesis

Activation of amino acid: The first step of protein synthesis is amino acid activation. It is a process in which amino acid are attached to tRNA molecules.

Activation of amino acid: The first step of protein synthesis is amino acid activation. It is a process in which amino acid are attached to tRNA molecules.

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Fig. 6.13. The phases of the transcription cycles: initiation, elongation and termination.

Fig. 6.13. The phases of the transcription cycles: initiation, elongation and termination.

6.9 TRANSLATION

6.9 TRANSLATION

It is the process of formation of protein from the mature RNA. The protein synthesis on mRNA strand takes place from 5 to 3 directions. Proteins are synthesized by the addition of amino acids and formation of peptide bond between them. There are three types of RNA which help in the protein synthesis (mRNA, rRNA and tRNA are described earlier of this chapter in structure of RNA).

It is the process of formation of protein from the mature RNA. The protein synthesis on mRNA strand takes place from 5 to 3 directions. Proteins are synthesized by the addition of amino acids and formation of peptide bond between them. There are three types of RNA which help in the protein synthesis (mRNA, rRNA and tRNA are described earlier of this chapter in structure of RNA).

Steps of protein synthesis

Steps of protein synthesis

Activation of amino acid: The first step of protein synthesis is amino acid activation. It is a process in which amino acid are attached to tRNA molecules.

Activation of amino acid: The first step of protein synthesis is amino acid activation. It is a process in which amino acid are attached to tRNA molecules.

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Amino acids are activated for protein synthesis through a reaction catalyzed by the enzyme aminoacyl-tRNA-synthetase.

Amino acids are activated for protein synthesis through a reaction catalyzed by the enzyme aminoacyl-tRNA-synthetase.

There are 20 different types of amino acids so 20 different types of aminoacyl-tRNA-synthetase occur which is specific for a particular amino acid. Initiation of protein synthesis: Protein synthesis properly is divided into three stages: initiation, elongation and termination.

There are 20 different types of amino acids so 20 different types of aminoacyl-tRNA-synthetase occur which is specific for a particular amino acid. Initiation of protein synthesis: Protein synthesis properly is divided into three stages: initiation, elongation and termination.

Initiation stage of protein synthesis

Initiation stage of protein synthesis

1. In the first step dissociation of the ribosome (70S) into its subunits 30S and 50S subunits. 2. Binding of initiation factors IF-1, IF-2 and IF-3, GTP and activated amino acids N-formylmethionyl-tRNAfmet (fmet-tRNA) to the 30S subunit of ribosome. 3. In next step mRNA attaches to the 30S subunit and form 30S initiation complex. 4. IF-3 prevents 30S subunit binding to the 50S subunit and promotes the proper mRNA binding to the 30S subunit.IF-2 helps in the binding of GTP and fmet-tRNA and directs the attachment of fmet-tRNA to the P site of the 30S subunit. IF-1 helps in the release of IF-2 and GDP from the completed 70S ribosome. It may also help in binding of 30S unit to the 50S subunit.

1. In the first step dissociation of the ribosome (70S) into its subunits 30S and 50S subunits. 2. Binding of initiation factors IF-1, IF-2 and IF-3, GTP and activated amino acids N-formylmethionyl-tRNAfmet (fmet-tRNA) to the 30S subunit of ribosome. 3. In next step mRNA attaches to the 30S subunit and form 30S initiation complex. 4. IF-3 prevents 30S subunit binding to the 50S subunit and promotes the proper mRNA binding to the 30S subunit.IF-2 helps in the binding of GTP and fmet-tRNA and directs the attachment of fmet-tRNA to the P site of the 30S subunit. IF-1 helps in the release of IF-2 and GDP from the completed 70S ribosome. It may also help in binding of 30S unit to the 50S subunit.

Elongation of the polypeptide chain: The ribosomes have 3 sites for binding of tRNA: i. A site or acceptor site or aminoacyl site: During the protein synthesis every aminoacyl-tRNA comes into the surface of ribosome after then enter into the A site. But the first aminoacyl-tRNA (N-formylmethionyltRNA) directly enters into the P site. After than all aminoacyl-tRNA enter into the A site so it is also called acceptor site. ii. P site or peptidyl site or donor site. iii. E site or exit site.

Elongation of the polypeptide chain: The ribosomes have 3 sites for binding of tRNA: i. A site or acceptor site or aminoacyl site: During the protein synthesis every aminoacyl-tRNA comes into the surface of ribosome after then enter into the A site. But the first aminoacyl-tRNA (N-formylmethionyltRNA) directly enters into the P site. After than all aminoacyl-tRNA enter into the A site so it is also called acceptor site. ii. P site or peptidyl site or donor site. iii. E site or exit site.

The first aminoacyl-tRNA (N-fmet-tRNA) directly enters into the P site. At the beginning of the elongation cycle, the P site is filled with N-formylmethionyl– tRNA and A site and Exit sites are empty. The first step of elongation is the binding of aminoacyl-tRNA corresponding to the codon in mRNA is inserted into the A site. GTP and elongation factor Ef-Tu which donate the aminoacyl-tRNA to the ribosome are required for aminoacyl-tRNA insertion. When GTP and Ef-Tu transfer the aminoacyl-tRNA to the A site hydrolysis of GTP takes place and

The first aminoacyl-tRNA (N-fmet-tRNA) directly enters into the P site. At the beginning of the elongation cycle, the P site is filled with N-formylmethionyl– tRNA and A site and Exit sites are empty. The first step of elongation is the binding of aminoacyl-tRNA corresponding to the codon in mRNA is inserted into the A site. GTP and elongation factor Ef-Tu which donate the aminoacyl-tRNA to the ribosome are required for aminoacyl-tRNA insertion. When GTP and Ef-Tu transfer the aminoacyl-tRNA to the A site hydrolysis of GTP takes place and

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Amino acids are activated for protein synthesis through a reaction catalyzed by the enzyme aminoacyl-tRNA-synthetase.

Amino acids are activated for protein synthesis through a reaction catalyzed by the enzyme aminoacyl-tRNA-synthetase.

There are 20 different types of amino acids so 20 different types of aminoacyl-tRNA-synthetase occur which is specific for a particular amino acid. Initiation of protein synthesis: Protein synthesis properly is divided into three stages: initiation, elongation and termination.

There are 20 different types of amino acids so 20 different types of aminoacyl-tRNA-synthetase occur which is specific for a particular amino acid. Initiation of protein synthesis: Protein synthesis properly is divided into three stages: initiation, elongation and termination.

Initiation stage of protein synthesis

Initiation stage of protein synthesis

1. In the first step dissociation of the ribosome (70S) into its subunits 30S and 50S subunits. 2. Binding of initiation factors IF-1, IF-2 and IF-3, GTP and activated amino acids N-formylmethionyl-tRNAfmet (fmet-tRNA) to the 30S subunit of ribosome. 3. In next step mRNA attaches to the 30S subunit and form 30S initiation complex. 4. IF-3 prevents 30S subunit binding to the 50S subunit and promotes the proper mRNA binding to the 30S subunit.IF-2 helps in the binding of GTP and fmet-tRNA and directs the attachment of fmet-tRNA to the P site of the 30S subunit. IF-1 helps in the release of IF-2 and GDP from the completed 70S ribosome. It may also help in binding of 30S unit to the 50S subunit.

1. In the first step dissociation of the ribosome (70S) into its subunits 30S and 50S subunits. 2. Binding of initiation factors IF-1, IF-2 and IF-3, GTP and activated amino acids N-formylmethionyl-tRNAfmet (fmet-tRNA) to the 30S subunit of ribosome. 3. In next step mRNA attaches to the 30S subunit and form 30S initiation complex. 4. IF-3 prevents 30S subunit binding to the 50S subunit and promotes the proper mRNA binding to the 30S subunit.IF-2 helps in the binding of GTP and fmet-tRNA and directs the attachment of fmet-tRNA to the P site of the 30S subunit. IF-1 helps in the release of IF-2 and GDP from the completed 70S ribosome. It may also help in binding of 30S unit to the 50S subunit.

Elongation of the polypeptide chain: The ribosomes have 3 sites for binding of tRNA: i. A site or acceptor site or aminoacyl site: During the protein synthesis every aminoacyl-tRNA comes into the surface of ribosome after then enter into the A site. But the first aminoacyl-tRNA (N-formylmethionyltRNA) directly enters into the P site. After than all aminoacyl-tRNA enter into the A site so it is also called acceptor site. ii. P site or peptidyl site or donor site. iii. E site or exit site.

Elongation of the polypeptide chain: The ribosomes have 3 sites for binding of tRNA: i. A site or acceptor site or aminoacyl site: During the protein synthesis every aminoacyl-tRNA comes into the surface of ribosome after then enter into the A site. But the first aminoacyl-tRNA (N-formylmethionyltRNA) directly enters into the P site. After than all aminoacyl-tRNA enter into the A site so it is also called acceptor site. ii. P site or peptidyl site or donor site. iii. E site or exit site.

The first aminoacyl-tRNA (N-fmet-tRNA) directly enters into the P site. At the beginning of the elongation cycle, the P site is filled with N-formylmethionyl– tRNA and A site and Exit sites are empty. The first step of elongation is the binding of aminoacyl-tRNA corresponding to the codon in mRNA is inserted into the A site. GTP and elongation factor Ef-Tu which donate the aminoacyl-tRNA to the ribosome are required for aminoacyl-tRNA insertion. When GTP and Ef-Tu transfer the aminoacyl-tRNA to the A site hydrolysis of GTP takes place and

The first aminoacyl-tRNA (N-fmet-tRNA) directly enters into the P site. At the beginning of the elongation cycle, the P site is filled with N-formylmethionyl– tRNA and A site and Exit sites are empty. The first step of elongation is the binding of aminoacyl-tRNA corresponding to the codon in mRNA is inserted into the A site. GTP and elongation factor Ef-Tu which donate the aminoacyl-tRNA to the ribosome are required for aminoacyl-tRNA insertion. When GTP and Ef-Tu transfer the aminoacyl-tRNA to the A site hydrolysis of GTP takes place and

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Fig. 6.14.1. Diagrammatic representation of the initiation of protein synthesis on the mRNA template strand with various initiation factors.

Fig. 6.14.1. Diagrammatic representation of the initiation of protein synthesis on the mRNA template strand with various initiation factors.

the Ef-Tu–GDP complex leaves the ribosome. Ef-Tu.GDP complex converted into Ef-Tu.GTP with the aid of second elongation factor Ef-Ts and the other aminoacyl-tRNA bind to the Ef-Tu.GTP. Aminoacyl-tRNA binding to the A site initiates the second phase of the elongation cycle by the transpeptidation reaction which is catalyzed by the peptidyl transferase located in the 50S subunit. The amino acid of the P site transferred to the A site and the peptide chain grows by one amino acid and tRNA present in the P site transfer to the exit

the Ef-Tu–GDP complex leaves the ribosome. Ef-Tu.GDP complex converted into Ef-Tu.GTP with the aid of second elongation factor Ef-Ts and the other aminoacyl-tRNA bind to the Ef-Tu.GTP. Aminoacyl-tRNA binding to the A site initiates the second phase of the elongation cycle by the transpeptidation reaction which is catalyzed by the peptidyl transferase located in the 50S subunit. The amino acid of the P site transferred to the A site and the peptide chain grows by one amino acid and tRNA present in the P site transfer to the exit

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Fig. 6.14.1. Diagrammatic representation of the initiation of protein synthesis on the mRNA template strand with various initiation factors.

Fig. 6.14.1. Diagrammatic representation of the initiation of protein synthesis on the mRNA template strand with various initiation factors.

the Ef-Tu–GDP complex leaves the ribosome. Ef-Tu.GDP complex converted into Ef-Tu.GTP with the aid of second elongation factor Ef-Ts and the other aminoacyl-tRNA bind to the Ef-Tu.GTP. Aminoacyl-tRNA binding to the A site initiates the second phase of the elongation cycle by the transpeptidation reaction which is catalyzed by the peptidyl transferase located in the 50S subunit. The amino acid of the P site transferred to the A site and the peptide chain grows by one amino acid and tRNA present in the P site transfer to the exit

the Ef-Tu–GDP complex leaves the ribosome. Ef-Tu.GDP complex converted into Ef-Tu.GTP with the aid of second elongation factor Ef-Ts and the other aminoacyl-tRNA bind to the Ef-Tu.GTP. Aminoacyl-tRNA binding to the A site initiates the second phase of the elongation cycle by the transpeptidation reaction which is catalyzed by the peptidyl transferase located in the 50S subunit. The amino acid of the P site transferred to the A site and the peptide chain grows by one amino acid and tRNA present in the P site transfer to the exit

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Fig. 6.14.2. Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labelled n-1, n, n+1 etc, represent the amino acid residues of the newly formed protein molecule.

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Fig. 6.14.2. Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labelled n-1, n, n+1 etc, represent the amino acid residues of the newly formed protein molecule.

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Fig. 6.14.2. Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labelled n-1, n, n+1 etc, represent the amino acid residues of the newly formed protein molecule.

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Fig. 6.14.2. Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labelled n-1, n, n+1 etc, represent the amino acid residues of the newly formed protein molecule.

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site finally. In the next step the ribosome unit sift one more codon forward and the peptidyl-tRNA sift to the P site and the new aminoacyl-tRNA come into the A site. This step is repeated continuously. The final phase in the elongation cycle is translocation. Three things happen simultaneously: 1. The peptidyl-tRNA moves about 20 Å from the A site to the P site. 2. The ribosome moves one codon along mRNA so that a new codon is positioned in the A site. 3. The empty tRNA leaves the P site and sifts to E site and finally leaves the ribosomes.

site finally. In the next step the ribosome unit sift one more codon forward and the peptidyl-tRNA sift to the P site and the new aminoacyl-tRNA come into the A site. This step is repeated continuously. The final phase in the elongation cycle is translocation. Three things happen simultaneously: 1. The peptidyl-tRNA moves about 20 Å from the A site to the P site. 2. The ribosome moves one codon along mRNA so that a new codon is positioned in the A site. 3. The empty tRNA leaves the P site and sifts to E site and finally leaves the ribosomes.

Termination of protein synthesis

Termination of protein synthesis

Protein synthesis stops when the ribosome reaches one of three special nonsense codons- UAA, UAG, and UGA. Three releasing factors (RF-1, RF-2 and RF-3) aid the ribosome in recognizing these codons. After the ribosome has stopped, peptidyl transferase hydrolyzes the peptide free from its tRNA and the empty tRNA is released. When the protein synthesis is completed, protein is released and all the units like mRNA, tRNA, rRNA and initiation factors are separated out.

Protein synthesis stops when the ribosome reaches one of three special nonsense codons- UAA, UAG, and UGA. Three releasing factors (RF-1, RF-2 and RF-3) aid the ribosome in recognizing these codons. After the ribosome has stopped, peptidyl transferase hydrolyzes the peptide free from its tRNA and the empty tRNA is released. When the protein synthesis is completed, protein is released and all the units like mRNA, tRNA, rRNA and initiation factors are separated out.

6.10 PROCESS OF GENE TRANSFER

6.10 PROCESS OF GENE TRANSFER

6.10.1 Transformation In 1944, Avery, Macleod and McCarty demonstrated that DNA purified from an encapsulated strain of pneumococcus was capable of imparting the ability to form capsule onto a noncapsulated strain and the resulting strain was genetically stable. This property of gene transfer by soluble DNA was called transformation. Transformation requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. Double stranded DNA is more deficient in transformation. The size of the DNA is related to the transforming ability. DNA with less than 0.3 million Dalton molecular weight usually fails to transform. The competency of the host cell requires the expression of a number of cellular properties which are specific for this task. The initial binding of the DNA on to the competent cell is reversible but as the process continues it becomes irreversible. Shortly after the irreversible binding there is a brief eclipse period during which the transforming ability of the donor DNA cannot be recovered. While traversing the bacterial membrane one of the strands of the DNA gets hydrolyzed resulting into the entry of single stranded DNA into the cytoplasm. This single stranded DNA synapses with homologous region of the host genome.

6.10.1 Transformation In 1944, Avery, Macleod and McCarty demonstrated that DNA purified from an encapsulated strain of pneumococcus was capable of imparting the ability to form capsule onto a noncapsulated strain and the resulting strain was genetically stable. This property of gene transfer by soluble DNA was called transformation. Transformation requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. Double stranded DNA is more deficient in transformation. The size of the DNA is related to the transforming ability. DNA with less than 0.3 million Dalton molecular weight usually fails to transform. The competency of the host cell requires the expression of a number of cellular properties which are specific for this task. The initial binding of the DNA on to the competent cell is reversible but as the process continues it becomes irreversible. Shortly after the irreversible binding there is a brief eclipse period during which the transforming ability of the donor DNA cannot be recovered. While traversing the bacterial membrane one of the strands of the DNA gets hydrolyzed resulting into the entry of single stranded DNA into the cytoplasm. This single stranded DNA synapses with homologous region of the host genome.

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site finally. In the next step the ribosome unit sift one more codon forward and the peptidyl-tRNA sift to the P site and the new aminoacyl-tRNA come into the A site. This step is repeated continuously. The final phase in the elongation cycle is translocation. Three things happen simultaneously: 1. The peptidyl-tRNA moves about 20 Å from the A site to the P site. 2. The ribosome moves one codon along mRNA so that a new codon is positioned in the A site. 3. The empty tRNA leaves the P site and sifts to E site and finally leaves the ribosomes.

site finally. In the next step the ribosome unit sift one more codon forward and the peptidyl-tRNA sift to the P site and the new aminoacyl-tRNA come into the A site. This step is repeated continuously. The final phase in the elongation cycle is translocation. Three things happen simultaneously: 1. The peptidyl-tRNA moves about 20 Å from the A site to the P site. 2. The ribosome moves one codon along mRNA so that a new codon is positioned in the A site. 3. The empty tRNA leaves the P site and sifts to E site and finally leaves the ribosomes.

Termination of protein synthesis

Termination of protein synthesis

Protein synthesis stops when the ribosome reaches one of three special nonsense codons- UAA, UAG, and UGA. Three releasing factors (RF-1, RF-2 and RF-3) aid the ribosome in recognizing these codons. After the ribosome has stopped, peptidyl transferase hydrolyzes the peptide free from its tRNA and the empty tRNA is released. When the protein synthesis is completed, protein is released and all the units like mRNA, tRNA, rRNA and initiation factors are separated out.

Protein synthesis stops when the ribosome reaches one of three special nonsense codons- UAA, UAG, and UGA. Three releasing factors (RF-1, RF-2 and RF-3) aid the ribosome in recognizing these codons. After the ribosome has stopped, peptidyl transferase hydrolyzes the peptide free from its tRNA and the empty tRNA is released. When the protein synthesis is completed, protein is released and all the units like mRNA, tRNA, rRNA and initiation factors are separated out.

6.10 PROCESS OF GENE TRANSFER

6.10 PROCESS OF GENE TRANSFER

6.10.1 Transformation In 1944, Avery, Macleod and McCarty demonstrated that DNA purified from an encapsulated strain of pneumococcus was capable of imparting the ability to form capsule onto a noncapsulated strain and the resulting strain was genetically stable. This property of gene transfer by soluble DNA was called transformation. Transformation requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. Double stranded DNA is more deficient in transformation. The size of the DNA is related to the transforming ability. DNA with less than 0.3 million Dalton molecular weight usually fails to transform. The competency of the host cell requires the expression of a number of cellular properties which are specific for this task. The initial binding of the DNA on to the competent cell is reversible but as the process continues it becomes irreversible. Shortly after the irreversible binding there is a brief eclipse period during which the transforming ability of the donor DNA cannot be recovered. While traversing the bacterial membrane one of the strands of the DNA gets hydrolyzed resulting into the entry of single stranded DNA into the cytoplasm. This single stranded DNA synapses with homologous region of the host genome.

6.10.1 Transformation In 1944, Avery, Macleod and McCarty demonstrated that DNA purified from an encapsulated strain of pneumococcus was capable of imparting the ability to form capsule onto a noncapsulated strain and the resulting strain was genetically stable. This property of gene transfer by soluble DNA was called transformation. Transformation requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. Double stranded DNA is more deficient in transformation. The size of the DNA is related to the transforming ability. DNA with less than 0.3 million Dalton molecular weight usually fails to transform. The competency of the host cell requires the expression of a number of cellular properties which are specific for this task. The initial binding of the DNA on to the competent cell is reversible but as the process continues it becomes irreversible. Shortly after the irreversible binding there is a brief eclipse period during which the transforming ability of the donor DNA cannot be recovered. While traversing the bacterial membrane one of the strands of the DNA gets hydrolyzed resulting into the entry of single stranded DNA into the cytoplasm. This single stranded DNA synapses with homologous region of the host genome.

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Fig. 6.14.3. Diagrammatic representation of the termination process of protein synthesis. Releasing factor RF-1 bind to the stop codon, releasing factor RF-3, with bind GDP, bind to RF-1. Hydrolysis of the peptidyl –tRNA complex is shown by the entry of H2O. N and C indicate the amino acid and carboxyl terminal of amino acids, respectively.

Fig. 6.14.3. Diagrammatic representation of the termination process of protein synthesis. Releasing factor RF-1 bind to the stop codon, releasing factor RF-3, with bind GDP, bind to RF-1. Hydrolysis of the peptidyl –tRNA complex is shown by the entry of H2O. N and C indicate the amino acid and carboxyl terminal of amino acids, respectively.

6.10.2 Conjugation Conjugation was discovered by Lederberg and Tatum in 1946. Conjugation is defined as the transfer of DNA directly from one bacterial cell to another bacterial cell by the mechanism that requires cell to cell contact. Lederberg and Tatum discovered that some bacterial cells contains F factor or fertility factor also known as conjugative plasmid and call them F+ cell and other bacterial cells do not contain F factor these are called F- cells. F+ cells are also known as male cells and F- cells are known as female cells.

6.10.2 Conjugation Conjugation was discovered by Lederberg and Tatum in 1946. Conjugation is defined as the transfer of DNA directly from one bacterial cell to another bacterial cell by the mechanism that requires cell to cell contact. Lederberg and Tatum discovered that some bacterial cells contains F factor or fertility factor also known as conjugative plasmid and call them F+ cell and other bacterial cells do not contain F factor these are called F- cells. F+ cells are also known as male cells and F- cells are known as female cells.

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Fig. 6.14.3. Diagrammatic representation of the termination process of protein synthesis. Releasing factor RF-1 bind to the stop codon, releasing factor RF-3, with bind GDP, bind to RF-1. Hydrolysis of the peptidyl –tRNA complex is shown by the entry of H2O. N and C indicate the amino acid and carboxyl terminal of amino acids, respectively.

Fig. 6.14.3. Diagrammatic representation of the termination process of protein synthesis. Releasing factor RF-1 bind to the stop codon, releasing factor RF-3, with bind GDP, bind to RF-1. Hydrolysis of the peptidyl –tRNA complex is shown by the entry of H2O. N and C indicate the amino acid and carboxyl terminal of amino acids, respectively.

6.10.2 Conjugation Conjugation was discovered by Lederberg and Tatum in 1946. Conjugation is defined as the transfer of DNA directly from one bacterial cell to another bacterial cell by the mechanism that requires cell to cell contact. Lederberg and Tatum discovered that some bacterial cells contains F factor or fertility factor also known as conjugative plasmid and call them F+ cell and other bacterial cells do not contain F factor these are called F- cells. F+ cells are also known as male cells and F- cells are known as female cells.

6.10.2 Conjugation Conjugation was discovered by Lederberg and Tatum in 1946. Conjugation is defined as the transfer of DNA directly from one bacterial cell to another bacterial cell by the mechanism that requires cell to cell contact. Lederberg and Tatum discovered that some bacterial cells contains F factor or fertility factor also known as conjugative plasmid and call them F+ cell and other bacterial cells do not contain F factor these are called F- cells. F+ cells are also known as male cells and F- cells are known as female cells.

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Fig. 6.15. Transformation in bacterial cell.

Fig. 6.15. Transformation in bacterial cell.

When conjugation takes place between F+ cell or donor cell and F- cell or the recipient cell the F factor being copied and transferred from donor to recipient cell and F- cell becomes F+ cell (i.e., male cell converts into female cell). For example, E. coli.

When conjugation takes place between F+ cell or donor cell and F- cell or the recipient cell the F factor being copied and transferred from donor to recipient cell and F- cell becomes F+ cell (i.e., male cell converts into female cell). For example, E. coli.

6.10.3 Transduction Transduction was discovered by Zinder and Lederberg in 1952. Gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by bacteriophage viral infection is known as transduction. This introduces only a small fragment of DNA. In the transduction process bacteriophage virus attached to the surface of bacteria transfers its genetic material to the bacterial cytoplasm. Phase virus

6.10.3 Transduction Transduction was discovered by Zinder and Lederberg in 1952. Gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by bacteriophage viral infection is known as transduction. This introduces only a small fragment of DNA. In the transduction process bacteriophage virus attached to the surface of bacteria transfers its genetic material to the bacterial cytoplasm. Phase virus

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Fig. 6.15. Transformation in bacterial cell.

Fig. 6.15. Transformation in bacterial cell.

When conjugation takes place between F+ cell or donor cell and F- cell or the recipient cell the F factor being copied and transferred from donor to recipient cell and F- cell becomes F+ cell (i.e., male cell converts into female cell). For example, E. coli.

When conjugation takes place between F+ cell or donor cell and F- cell or the recipient cell the F factor being copied and transferred from donor to recipient cell and F- cell becomes F+ cell (i.e., male cell converts into female cell). For example, E. coli.

6.10.3 Transduction Transduction was discovered by Zinder and Lederberg in 1952. Gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by bacteriophage viral infection is known as transduction. This introduces only a small fragment of DNA. In the transduction process bacteriophage virus attached to the surface of bacteria transfers its genetic material to the bacterial cytoplasm. Phase virus

6.10.3 Transduction Transduction was discovered by Zinder and Lederberg in 1952. Gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by bacteriophage viral infection is known as transduction. This introduces only a small fragment of DNA. In the transduction process bacteriophage virus attached to the surface of bacteria transfers its genetic material to the bacterial cytoplasm. Phase virus

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Fig. 6.16. Conjugation in bacteria.

Fig. 6.16. Conjugation in bacteria.

releases some proteiolytic enzymes which degrade the DNA of the host cell and replicates itself and forms a protein coat around each small fragment. Now the new viral particle contains bacterial as well as viral DNA fragment. This new viral particle having DNA of donor bacteria infects the new bacterial cell and transfers the genetic material to the bacterial cell. By such method transfer of genetic material of donor to recipient cell takes place.

releases some proteiolytic enzymes which degrade the DNA of the host cell and replicates itself and forms a protein coat around each small fragment. Now the new viral particle contains bacterial as well as viral DNA fragment. This new viral particle having DNA of donor bacteria infects the new bacterial cell and transfers the genetic material to the bacterial cell. By such method transfer of genetic material of donor to recipient cell takes place.

6.11 POLYMERASE CHAIN REACTION (PCR)

6.11 POLYMERASE CHAIN REACTION (PCR)

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or few copies of DNA and generating thousands to millions of copies of a particular DNA sequence. PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set-up requires several components and reagents. These include: • DNA template that contains the DNA region (target) to be amplified. • Two primers (forward and reverse) that are complementary to the parental strand of target DNA are used.

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or few copies of DNA and generating thousands to millions of copies of a particular DNA sequence. PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set-up requires several components and reagents. These include: • DNA template that contains the DNA region (target) to be amplified. • Two primers (forward and reverse) that are complementary to the parental strand of target DNA are used.

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Fig. 6.16. Conjugation in bacteria.

Fig. 6.16. Conjugation in bacteria.

releases some proteiolytic enzymes which degrade the DNA of the host cell and replicates itself and forms a protein coat around each small fragment. Now the new viral particle contains bacterial as well as viral DNA fragment. This new viral particle having DNA of donor bacteria infects the new bacterial cell and transfers the genetic material to the bacterial cell. By such method transfer of genetic material of donor to recipient cell takes place.

releases some proteiolytic enzymes which degrade the DNA of the host cell and replicates itself and forms a protein coat around each small fragment. Now the new viral particle contains bacterial as well as viral DNA fragment. This new viral particle having DNA of donor bacteria infects the new bacterial cell and transfers the genetic material to the bacterial cell. By such method transfer of genetic material of donor to recipient cell takes place.

6.11 POLYMERASE CHAIN REACTION (PCR)

6.11 POLYMERASE CHAIN REACTION (PCR)

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or few copies of DNA and generating thousands to millions of copies of a particular DNA sequence. PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set-up requires several components and reagents. These include: • DNA template that contains the DNA region (target) to be amplified. • Two primers (forward and reverse) that are complementary to the parental strand of target DNA are used.

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or few copies of DNA and generating thousands to millions of copies of a particular DNA sequence. PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set-up requires several components and reagents. These include: • DNA template that contains the DNA region (target) to be amplified. • Two primers (forward and reverse) that are complementary to the parental strand of target DNA are used.

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Fig. 6.17. Transduction process in bacteria.

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Fig. 6.17. Transduction process in bacteria.

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Fig. 6.17. Transduction process in bacteria.

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Fig. 6.17. Transduction process in bacteria.

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• Taq polymerase or DNA polymerase is used which is thermostable (does not denaturate upto 95°C). • Deoxynucleoside triphosphates (dNTPs); the building blocks from which the DNA polymerases synthesizes a new DNA strand. • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. • Monovalent cation potassium ions.

• Taq polymerase or DNA polymerase is used which is thermostable (does not denaturate upto 95°C). • Deoxynucleoside triphosphates (dNTPs); the building blocks from which the DNA polymerases synthesizes a new DNA strand. • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. • Monovalent cation potassium ions.

There are 3 steps mainly in PCR: 1. Denaturation (94°C) 2. Annealing (50-60°C) 3. Extension (72°C) 1. Denaturation step: This step is the first regular cycling event and consists of heating the reaction mixure to 94–96°C for 45 sec to 1 minute. It causes DNA melting of the DNA double strand by disrupting the hydrogen bonds between complementary bases and yielding single-stranded DNA molecules. 2. Annealing step: The reaction mixture temperature is lowered to 50–65°C for 30-45 seconds allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 3. Extension/ elongation step at 72°C: In this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'hydroxyl group at the end of the nascent (extending) DNA strand. This step mainly takes 1-2 minutes. At each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragments.

There are 3 steps mainly in PCR: 1. Denaturation (94°C) 2. Annealing (50-60°C) 3. Extension (72°C) 1. Denaturation step: This step is the first regular cycling event and consists of heating the reaction mixure to 94–96°C for 45 sec to 1 minute. It causes DNA melting of the DNA double strand by disrupting the hydrogen bonds between complementary bases and yielding single-stranded DNA molecules. 2. Annealing step: The reaction mixture temperature is lowered to 50–65°C for 30-45 seconds allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 3. Extension/ elongation step at 72°C: In this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'hydroxyl group at the end of the nascent (extending) DNA strand. This step mainly takes 1-2 minutes. At each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragments.

6.11.1 Application of PCR

6.11.1 Application of PCR

Amplification and quantification of DNA

Amplification and quantification of DNA

1. PCR amplifies the regions of DNA that it targets, PCR can be used to analyse extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. 2. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been

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1. PCR amplifies the regions of DNA that it targets, PCR can be used to analyse extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. 2. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been

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• Taq polymerase or DNA polymerase is used which is thermostable (does not denaturate upto 95°C). • Deoxynucleoside triphosphates (dNTPs); the building blocks from which the DNA polymerases synthesizes a new DNA strand. • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. • Monovalent cation potassium ions.

• Taq polymerase or DNA polymerase is used which is thermostable (does not denaturate upto 95°C). • Deoxynucleoside triphosphates (dNTPs); the building blocks from which the DNA polymerases synthesizes a new DNA strand. • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. • Monovalent cation potassium ions.

There are 3 steps mainly in PCR: 1. Denaturation (94°C) 2. Annealing (50-60°C) 3. Extension (72°C) 1. Denaturation step: This step is the first regular cycling event and consists of heating the reaction mixure to 94–96°C for 45 sec to 1 minute. It causes DNA melting of the DNA double strand by disrupting the hydrogen bonds between complementary bases and yielding single-stranded DNA molecules. 2. Annealing step: The reaction mixture temperature is lowered to 50–65°C for 30-45 seconds allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 3. Extension/ elongation step at 72°C: In this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'hydroxyl group at the end of the nascent (extending) DNA strand. This step mainly takes 1-2 minutes. At each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragments.

There are 3 steps mainly in PCR: 1. Denaturation (94°C) 2. Annealing (50-60°C) 3. Extension (72°C) 1. Denaturation step: This step is the first regular cycling event and consists of heating the reaction mixure to 94–96°C for 45 sec to 1 minute. It causes DNA melting of the DNA double strand by disrupting the hydrogen bonds between complementary bases and yielding single-stranded DNA molecules. 2. Annealing step: The reaction mixture temperature is lowered to 50–65°C for 30-45 seconds allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 3. Extension/ elongation step at 72°C: In this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'hydroxyl group at the end of the nascent (extending) DNA strand. This step mainly takes 1-2 minutes. At each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragments.

6.11.1 Application of PCR

6.11.1 Application of PCR

Amplification and quantification of DNA

Amplification and quantification of DNA

1. PCR amplifies the regions of DNA that it targets, PCR can be used to analyse extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. 2. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been

1. PCR amplifies the regions of DNA that it targets, PCR can be used to analyse extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. 2. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been

88 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.18. The different steps in PCR.

successfully used on animals, such as a 40,000 year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar. 3. PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. 4. PCR also permits identification of non-cultivable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses from tissue culture assay and animal models. 5. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

88 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.18. The different steps in PCR.

successfully used on animals, such as a 40,000 year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar. 3. PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. 4. PCR also permits identification of non-cultivable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses from tissue culture assay and animal models. 5. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

6.12 MUTATION, MUTAGEN AND MUTAGENESIS

6.12 MUTATION, MUTAGEN AND MUTAGENESIS

Mutation is a process in which sudden heritable changes in the genetic materials are called mutation which is caused by mutagens.

Mutation is a process in which sudden heritable changes in the genetic materials are called mutation which is caused by mutagens.

88 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

88 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 6.18. The different steps in PCR.

successfully used on animals, such as a 40,000 year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar. 3. PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. 4. PCR also permits identification of non-cultivable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses from tissue culture assay and animal models. 5. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

Fig. 6.18. The different steps in PCR.

successfully used on animals, such as a 40,000 year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar. 3. PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. 4. PCR also permits identification of non-cultivable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses from tissue culture assay and animal models. 5. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

6.12 MUTATION, MUTAGEN AND MUTAGENESIS

6.12 MUTATION, MUTAGEN AND MUTAGENESIS

Mutation is a process in which sudden heritable changes in the genetic materials are called mutation which is caused by mutagens.

Mutation is a process in which sudden heritable changes in the genetic materials are called mutation which is caused by mutagens.

MICROBIAL GENETICS AND VARIATION

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MICROBIAL GENETICS AND VARIATION

89

A mutagen is a physical agent or a chemical agent that causes mutation. For example, nitrous acid reacts with some DNA bases changing their identity and hydrogen bonding properties. Mutagenesis is a process of producing a mutation. If it occurs in nature without the addition of a known mutagen it is called spontaneous mutagenesis and the resulting mutations are called spontaneous mutations and if a mutagen is used the process is induced mutagenesis and the resulting mutations are induced mutagenesis. An organism exhibiting a novel phenotype as a result of the presence of a mutation is called a mutant.

A mutagen is a physical agent or a chemical agent that causes mutation. For example, nitrous acid reacts with some DNA bases changing their identity and hydrogen bonding properties. Mutagenesis is a process of producing a mutation. If it occurs in nature without the addition of a known mutagen it is called spontaneous mutagenesis and the resulting mutations are called spontaneous mutations and if a mutagen is used the process is induced mutagenesis and the resulting mutations are induced mutagenesis. An organism exhibiting a novel phenotype as a result of the presence of a mutation is called a mutant.

6.12.1 Somatic vs. Gametic Mutations The consequences of a mutation depend upon where they occur in an individual. Some mutations occur in regular body cells (somatic cells i.e. all cells other than reproductive cells); these are somatic mutations. Some mutations occur in germ line cells. These cells produce the gametes; therefore, they are gametic mutations.

6.12.1 Somatic vs. Gametic Mutations The consequences of a mutation depend upon where they occur in an individual. Some mutations occur in regular body cells (somatic cells i.e. all cells other than reproductive cells); these are somatic mutations. Some mutations occur in germ line cells. These cells produce the gametes; therefore, they are gametic mutations.

6.12.2 Spontaneous vs. Induced Mutations Some mutations arise as natural errors in DNA replication (or as a result of unknown chemical reactions); these are known as spontaneous mutations. Mutations can also be caused by physical and chemical agents; these are induced mutations.

6.12.2 Spontaneous vs. Induced Mutations Some mutations arise as natural errors in DNA replication (or as a result of unknown chemical reactions); these are known as spontaneous mutations. Mutations can also be caused by physical and chemical agents; these are induced mutations.

6.12.3 Molecular Basis of Mutation There are two basic types of mutations: 1. Base substitutions: This is the replacement of one base by another. For example, if a DNA molecule usually contains guanine at a certain position, but adenine takes the place of the guanine, then a base substitution occurs. There are two types of base substitutions: a. Transitions: Mutations resulting from tautomeric shifts in the bases of DNA involving the replacement of a purine in one strand of the DNA with the other purine and replacement of a pyrimidine in the complementary strand of the DNA with other pyrimidine or the replacement of a pyrimidine in one strand of the DNA with the other pyrimidine and replacement of a purine in the complementary strand of the DNA with other purine. b. Transversions: Base pair substitutions involving the substitution of a purine with a pyrimidine in one strand of the DNA or vice versa. 2. Frameshift mutation: A point mutation which changes the reading frame of a gene. There are two types of frameshift mutations: a. Insertions, as the name implies, these involve the insertion of one or more extra nucleotides into a DNA chain. b. Deletions, these result from the loss of one or more nucleotides from a DNA chain.

6.12.3 Molecular Basis of Mutation There are two basic types of mutations: 1. Base substitutions: This is the replacement of one base by another. For example, if a DNA molecule usually contains guanine at a certain position, but adenine takes the place of the guanine, then a base substitution occurs. There are two types of base substitutions: a. Transitions: Mutations resulting from tautomeric shifts in the bases of DNA involving the replacement of a purine in one strand of the DNA with the other purine and replacement of a pyrimidine in the complementary strand of the DNA with other pyrimidine or the replacement of a pyrimidine in one strand of the DNA with the other pyrimidine and replacement of a purine in the complementary strand of the DNA with other purine. b. Transversions: Base pair substitutions involving the substitution of a purine with a pyrimidine in one strand of the DNA or vice versa. 2. Frameshift mutation: A point mutation which changes the reading frame of a gene. There are two types of frameshift mutations: a. Insertions, as the name implies, these involve the insertion of one or more extra nucleotides into a DNA chain. b. Deletions, these result from the loss of one or more nucleotides from a DNA chain.

MICROBIAL GENETICS AND VARIATION

89

MICROBIAL GENETICS AND VARIATION

89

A mutagen is a physical agent or a chemical agent that causes mutation. For example, nitrous acid reacts with some DNA bases changing their identity and hydrogen bonding properties. Mutagenesis is a process of producing a mutation. If it occurs in nature without the addition of a known mutagen it is called spontaneous mutagenesis and the resulting mutations are called spontaneous mutations and if a mutagen is used the process is induced mutagenesis and the resulting mutations are induced mutagenesis. An organism exhibiting a novel phenotype as a result of the presence of a mutation is called a mutant.

A mutagen is a physical agent or a chemical agent that causes mutation. For example, nitrous acid reacts with some DNA bases changing their identity and hydrogen bonding properties. Mutagenesis is a process of producing a mutation. If it occurs in nature without the addition of a known mutagen it is called spontaneous mutagenesis and the resulting mutations are called spontaneous mutations and if a mutagen is used the process is induced mutagenesis and the resulting mutations are induced mutagenesis. An organism exhibiting a novel phenotype as a result of the presence of a mutation is called a mutant.

6.12.1 Somatic vs. Gametic Mutations The consequences of a mutation depend upon where they occur in an individual. Some mutations occur in regular body cells (somatic cells i.e. all cells other than reproductive cells); these are somatic mutations. Some mutations occur in germ line cells. These cells produce the gametes; therefore, they are gametic mutations.

6.12.1 Somatic vs. Gametic Mutations The consequences of a mutation depend upon where they occur in an individual. Some mutations occur in regular body cells (somatic cells i.e. all cells other than reproductive cells); these are somatic mutations. Some mutations occur in germ line cells. These cells produce the gametes; therefore, they are gametic mutations.

6.12.2 Spontaneous vs. Induced Mutations Some mutations arise as natural errors in DNA replication (or as a result of unknown chemical reactions); these are known as spontaneous mutations. Mutations can also be caused by physical and chemical agents; these are induced mutations.

6.12.2 Spontaneous vs. Induced Mutations Some mutations arise as natural errors in DNA replication (or as a result of unknown chemical reactions); these are known as spontaneous mutations. Mutations can also be caused by physical and chemical agents; these are induced mutations.

6.12.3 Molecular Basis of Mutation There are two basic types of mutations: 1. Base substitutions: This is the replacement of one base by another. For example, if a DNA molecule usually contains guanine at a certain position, but adenine takes the place of the guanine, then a base substitution occurs. There are two types of base substitutions: a. Transitions: Mutations resulting from tautomeric shifts in the bases of DNA involving the replacement of a purine in one strand of the DNA with the other purine and replacement of a pyrimidine in the complementary strand of the DNA with other pyrimidine or the replacement of a pyrimidine in one strand of the DNA with the other pyrimidine and replacement of a purine in the complementary strand of the DNA with other purine. b. Transversions: Base pair substitutions involving the substitution of a purine with a pyrimidine in one strand of the DNA or vice versa. 2. Frameshift mutation: A point mutation which changes the reading frame of a gene. There are two types of frameshift mutations: a. Insertions, as the name implies, these involve the insertion of one or more extra nucleotides into a DNA chain. b. Deletions, these result from the loss of one or more nucleotides from a DNA chain.

6.12.3 Molecular Basis of Mutation There are two basic types of mutations: 1. Base substitutions: This is the replacement of one base by another. For example, if a DNA molecule usually contains guanine at a certain position, but adenine takes the place of the guanine, then a base substitution occurs. There are two types of base substitutions: a. Transitions: Mutations resulting from tautomeric shifts in the bases of DNA involving the replacement of a purine in one strand of the DNA with the other purine and replacement of a pyrimidine in the complementary strand of the DNA with other pyrimidine or the replacement of a pyrimidine in one strand of the DNA with the other pyrimidine and replacement of a purine in the complementary strand of the DNA with other purine. b. Transversions: Base pair substitutions involving the substitution of a purine with a pyrimidine in one strand of the DNA or vice versa. 2. Frameshift mutation: A point mutation which changes the reading frame of a gene. There are two types of frameshift mutations: a. Insertions, as the name implies, these involve the insertion of one or more extra nucleotides into a DNA chain. b. Deletions, these result from the loss of one or more nucleotides from a DNA chain.

90 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

90 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

90 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

90 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

7

7

Sterility Testing

Sterility Testing

1. Sterility testing is done for detection of presence of microorganisms in the medicaments or products. 2. Aseptically prepared parental products cannot be issued in the market without doing sterility testing because the risk of contamination is high. 3. In the United Kingdom sterility tests controlled the British pharmacopoeia and the therapeutic substances regulation. 4. All the parental products of the BP must comply with the official sterility test. This does not mean that samples must be tested from every batch manufactured. 5. Occasional checks are adequate in some circumstances such as when the preparation is sterilized by a properly controlled heating process. 6. The test is based upon the principle that if microorganisms are placed in a nutritive medium and kept at a favourable temperature, the organisms will grow and their presence can be indicated by turbidity in the originally clear medium. 7. For the sterility testing the sample is taken randomly from a batch. Batch may be defined as a homogeneous collection of sealed containers prepared in such a manner that the risk of contamination is nil with certainty. 8. Every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of result which depends upon • Environmental condition of manufacture. • The volume of preparation per container and other special considerations particular to the preparation being examined. • The number of units tested.

1. Sterility testing is done for detection of presence of microorganisms in the medicaments or products. 2. Aseptically prepared parental products cannot be issued in the market without doing sterility testing because the risk of contamination is high. 3. In the United Kingdom sterility tests controlled the British pharmacopoeia and the therapeutic substances regulation. 4. All the parental products of the BP must comply with the official sterility test. This does not mean that samples must be tested from every batch manufactured. 5. Occasional checks are adequate in some circumstances such as when the preparation is sterilized by a properly controlled heating process. 6. The test is based upon the principle that if microorganisms are placed in a nutritive medium and kept at a favourable temperature, the organisms will grow and their presence can be indicated by turbidity in the originally clear medium. 7. For the sterility testing the sample is taken randomly from a batch. Batch may be defined as a homogeneous collection of sealed containers prepared in such a manner that the risk of contamination is nil with certainty. 8. Every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of result which depends upon • Environmental condition of manufacture. • The volume of preparation per container and other special considerations particular to the preparation being examined. • The number of units tested.

The table given below gives guidance on the minimum number of items recommended to be tested in relation to the number of items in the batch on the assumption that the preparation has been manufactured under conditions designed to be exclude contamination.

The table given below gives guidance on the minimum number of items recommended to be tested in relation to the number of items in the batch on the assumption that the preparation has been manufactured under conditions designed to be exclude contamination.

7

7

Sterility Testing

Sterility Testing

1. Sterility testing is done for detection of presence of microorganisms in the medicaments or products. 2. Aseptically prepared parental products cannot be issued in the market without doing sterility testing because the risk of contamination is high. 3. In the United Kingdom sterility tests controlled the British pharmacopoeia and the therapeutic substances regulation. 4. All the parental products of the BP must comply with the official sterility test. This does not mean that samples must be tested from every batch manufactured. 5. Occasional checks are adequate in some circumstances such as when the preparation is sterilized by a properly controlled heating process. 6. The test is based upon the principle that if microorganisms are placed in a nutritive medium and kept at a favourable temperature, the organisms will grow and their presence can be indicated by turbidity in the originally clear medium. 7. For the sterility testing the sample is taken randomly from a batch. Batch may be defined as a homogeneous collection of sealed containers prepared in such a manner that the risk of contamination is nil with certainty. 8. Every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of result which depends upon • Environmental condition of manufacture. • The volume of preparation per container and other special considerations particular to the preparation being examined. • The number of units tested.

1. Sterility testing is done for detection of presence of microorganisms in the medicaments or products. 2. Aseptically prepared parental products cannot be issued in the market without doing sterility testing because the risk of contamination is high. 3. In the United Kingdom sterility tests controlled the British pharmacopoeia and the therapeutic substances regulation. 4. All the parental products of the BP must comply with the official sterility test. This does not mean that samples must be tested from every batch manufactured. 5. Occasional checks are adequate in some circumstances such as when the preparation is sterilized by a properly controlled heating process. 6. The test is based upon the principle that if microorganisms are placed in a nutritive medium and kept at a favourable temperature, the organisms will grow and their presence can be indicated by turbidity in the originally clear medium. 7. For the sterility testing the sample is taken randomly from a batch. Batch may be defined as a homogeneous collection of sealed containers prepared in such a manner that the risk of contamination is nil with certainty. 8. Every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of result which depends upon • Environmental condition of manufacture. • The volume of preparation per container and other special considerations particular to the preparation being examined. • The number of units tested.

The table given below gives guidance on the minimum number of items recommended to be tested in relation to the number of items in the batch on the assumption that the preparation has been manufactured under conditions designed to be exclude contamination.

The table given below gives guidance on the minimum number of items recommended to be tested in relation to the number of items in the batch on the assumption that the preparation has been manufactured under conditions designed to be exclude contamination.

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92 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Table 1

Table 1

S.No.

Number of items in the batch

A.

Injectable preparation

1. 2.

Not more than 100 containers. More that 100 but not more than 500 containers. More than 500 containers.

3. B.

Ophthalmic and other non-injectable preparations

1. 2.

Not more than 200 containers. More than 200 containers.

C.

Surgical dressings

2.

Not more than 100 packages. More than 100 but not more than 500 packages. More than 500 packages.

D.

Bulk solids

1. 2.

Less than 4 containers. 4 containers but not more than 50 containers. More than 50 containers.

1.

3.

Minimum number of items

10% or 4 containers whichever is greater. 10 containers. 2% or 20 containers whichever is less.

5% or 2 containers whichever is greater. 10 containers.

S.No.

Number of items in the batch

A.

Injectable preparation

1. 2.

Not more than 100 containers. More that 100 but not more than 500 containers. More than 500 containers.

3. B.

Ophthalmic and other non-injectable preparations

1. 2.

Not more than 200 containers. More than 200 containers.

C.

Surgical dressings

10% or 4 packages whichever is greater.

2.

Not more than 100 packages. More than 100 but not more than 500 packages. More than 500 packages.

D.

Bulk solids

1. 2.

Less than 4 containers. 4 containers but not more than 50 containers. More than 50 containers.

1. 10 packages. 2% or 20 packages whichever is less.

Each container. 20% or 4 containers whichever is greater. 2% or 10 containers whichever is greater.

3.

Minimum number of items

10% or 4 containers whichever is greater. 10 containers. 2% or 20 containers whichever is less.

5% or 2 containers whichever is greater. 10 containers.

10% or 4 packages whichever is greater. 10 packages. 2% or 20 packages whichever is less.

Each container. 20% or 4 containers whichever is greater. 2% or 10 containers whichever is greater.

7.1 CULTURE MEDIA

7.1 CULTURE MEDIA

For the sterility testing different types of media are used. Which are given below: a. Fluid thioglycollate media - For use with clear fluid product L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Granular agar (moisture less than 15% w/w) 0.75 g Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Resazurin (0.10%fresh sol.) 1.0 mL Distilled water 1000 mL

For the sterility testing different types of media are used. Which are given below: a. Fluid thioglycollate media - For use with clear fluid product L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Granular agar (moisture less than 15% w/w) 0.75 g Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Resazurin (0.10%fresh sol.) 1.0 mL Distilled water 1000 mL

Procedure

Procedure

1. Mix the ingredients other than the thioglycollate and the resazurin, in the order given above in a mortar, with through grinding. 2. Stir in some heated distilled water, transfer to a suitable container, add the remainder of the water and complete the solution by heating in a boiling water-bath.

1. Mix the ingredients other than the thioglycollate and the resazurin, in the order given above in a mortar, with through grinding. 2. Stir in some heated distilled water, transfer to a suitable container, add the remainder of the water and complete the solution by heating in a boiling water-bath.

92 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

92 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Table 1

Table 1

S.No.

Number of items in the batch

A.

Injectable preparation

1. 2.

Not more than 100 containers. More that 100 but not more than 500 containers. More than 500 containers.

3. B.

Ophthalmic and other non-injectable preparations

1. 2.

Not more than 200 containers. More than 200 containers.

C.

2. D.

Bulk solids

1. 2.

Less than 4 containers. 4 containers but not more than 50 containers. More than 50 containers.

3.

10% or 4 containers whichever is greater. 10 containers. 2% or 20 containers whichever is less.

5% or 2 containers whichever is greater. 10 containers.

Surgical dressings Not more than 100 packages. More than 100 but not more than 500 packages. More than 500 packages.

1.

Minimum number of items

S.No.

Number of items in the batch

A.

Injectable preparation

1. 2.

Not more than 100 containers. More that 100 but not more than 500 containers. More than 500 containers.

3. B.

Ophthalmic and other non-injectable preparations

1. 2.

Not more than 200 containers. More than 200 containers.

C.

Surgical dressings

10% or 4 packages whichever is greater.

2.

Not more than 100 packages. More than 100 but not more than 500 packages. More than 500 packages.

D.

Bulk solids

1. 2.

Less than 4 containers. 4 containers but not more than 50 containers. More than 50 containers.

1. 10 packages. 2% or 20 packages whichever is less.

Each container. 20% or 4 containers whichever is greater. 2% or 10 containers whichever is greater.

3.

Minimum number of items

10% or 4 containers whichever is greater. 10 containers. 2% or 20 containers whichever is less.

5% or 2 containers whichever is greater. 10 containers.

10% or 4 packages whichever is greater. 10 packages. 2% or 20 packages whichever is less.

Each container. 20% or 4 containers whichever is greater. 2% or 10 containers whichever is greater.

7.1 CULTURE MEDIA

7.1 CULTURE MEDIA

For the sterility testing different types of media are used. Which are given below: a. Fluid thioglycollate media - For use with clear fluid product L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Granular agar (moisture less than 15% w/w) 0.75 g Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Resazurin (0.10%fresh sol.) 1.0 mL Distilled water 1000 mL

For the sterility testing different types of media are used. Which are given below: a. Fluid thioglycollate media - For use with clear fluid product L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Granular agar (moisture less than 15% w/w) 0.75 g Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Resazurin (0.10%fresh sol.) 1.0 mL Distilled water 1000 mL

Procedure

Procedure

1. Mix the ingredients other than the thioglycollate and the resazurin, in the order given above in a mortar, with through grinding. 2. Stir in some heated distilled water, transfer to a suitable container, add the remainder of the water and complete the solution by heating in a boiling water-bath.

1. Mix the ingredients other than the thioglycollate and the resazurin, in the order given above in a mortar, with through grinding. 2. Stir in some heated distilled water, transfer to a suitable container, add the remainder of the water and complete the solution by heating in a boiling water-bath.

STERILITY TESTING

93

STERILITY TESTING

93

3. Add the sodium thioglycollate, then 1M sodium hydroxide, if necessary, so that (after sterilisation) the medium will have a pH of 7.1 + 0.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution. 4. Distribute into suitable vessel which provides a ratio of surface to depth of medium such that not more than the upper half of the medium undergone a colour change, indicative of oxygen uptake at the end of the incubating period. 5. Sterilise in an autoclave at 121ºC for 20 minutes. Cool promptly to 25ºC and store at 20º to 30ºC, avoiding excess of light. If more than the upper one-third has acquired a pink colour, the medium may be restored once by reheating in a water-bath until the pink colour disappears and cooling rapidly. When ready for use, not more than the upper one-tenth of the medium should have a pink colour. Medium more than 3 weeks old should not be used.

3. Add the sodium thioglycollate, then 1M sodium hydroxide, if necessary, so that (after sterilisation) the medium will have a pH of 7.1 + 0.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution. 4. Distribute into suitable vessel which provides a ratio of surface to depth of medium such that not more than the upper half of the medium undergone a colour change, indicative of oxygen uptake at the end of the incubating period. 5. Sterilise in an autoclave at 121ºC for 20 minutes. Cool promptly to 25ºC and store at 20º to 30ºC, avoiding excess of light. If more than the upper one-third has acquired a pink colour, the medium may be restored once by reheating in a water-bath until the pink colour disappears and cooling rapidly. When ready for use, not more than the upper one-tenth of the medium should have a pink colour. Medium more than 3 weeks old should not be used.

Fluid thioglycollate medium is used by incubating it at 30º to 35ºC under aerobic conditions. b. Alternative thioglycollate medium: For use with turbid and viscid products and for devices having tubes with small lumina. L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Distilled water 1000 mL

Fluid thioglycollate medium is used by incubating it at 30º to 35ºC under aerobic conditions. b. Alternative thioglycollate medium: For use with turbid and viscid products and for devices having tubes with small lumina. L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Distilled water 1000 mL

Procedure

Procedure

1. Heat the ingredients in a suitable container until the solution is affected. 2. Mix and adjust the reaction with 1M sodium hydroxide, if necessary, so that the medium will have a pH of 7.1 + 0.2 after sterilisation. 3. Filter if necessary, place in suitable vessels and sterilise in an autoclave at 121ºC for 20 minutes.

1. Heat the ingredients in a suitable container until the solution is affected. 2. Mix and adjust the reaction with 1M sodium hydroxide, if necessary, so that the medium will have a pH of 7.1 + 0.2 after sterilisation. 3. Filter if necessary, place in suitable vessels and sterilise in an autoclave at 121ºC for 20 minutes.

Note: The medium is freshly prepared or heated in a steam-bath and allowed to cool just prior to use. Do not reheat. Use alternative thioglycollate medium in a manner that will assure anaerobic conditions for the duration of the incubation at 30º to 35ºC. c. Soya bean- casein digest medium Pancreatic digest of casein 17.0 g Papaic digest of soya bean meal 3.0 g Sodium chloride 5.0 g

Note: The medium is freshly prepared or heated in a steam-bath and allowed to cool just prior to use. Do not reheat. Use alternative thioglycollate medium in a manner that will assure anaerobic conditions for the duration of the incubation at 30º to 35ºC. c. Soya bean- casein digest medium Pancreatic digest of casein 17.0 g Papaic digest of soya bean meal 3.0 g Sodium chloride 5.0 g

STERILITY TESTING

93

STERILITY TESTING

93

3. Add the sodium thioglycollate, then 1M sodium hydroxide, if necessary, so that (after sterilisation) the medium will have a pH of 7.1 + 0.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution. 4. Distribute into suitable vessel which provides a ratio of surface to depth of medium such that not more than the upper half of the medium undergone a colour change, indicative of oxygen uptake at the end of the incubating period. 5. Sterilise in an autoclave at 121ºC for 20 minutes. Cool promptly to 25ºC and store at 20º to 30ºC, avoiding excess of light. If more than the upper one-third has acquired a pink colour, the medium may be restored once by reheating in a water-bath until the pink colour disappears and cooling rapidly. When ready for use, not more than the upper one-tenth of the medium should have a pink colour. Medium more than 3 weeks old should not be used.

3. Add the sodium thioglycollate, then 1M sodium hydroxide, if necessary, so that (after sterilisation) the medium will have a pH of 7.1 + 0.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution. 4. Distribute into suitable vessel which provides a ratio of surface to depth of medium such that not more than the upper half of the medium undergone a colour change, indicative of oxygen uptake at the end of the incubating period. 5. Sterilise in an autoclave at 121ºC for 20 minutes. Cool promptly to 25ºC and store at 20º to 30ºC, avoiding excess of light. If more than the upper one-third has acquired a pink colour, the medium may be restored once by reheating in a water-bath until the pink colour disappears and cooling rapidly. When ready for use, not more than the upper one-tenth of the medium should have a pink colour. Medium more than 3 weeks old should not be used.

Fluid thioglycollate medium is used by incubating it at 30º to 35ºC under aerobic conditions. b. Alternative thioglycollate medium: For use with turbid and viscid products and for devices having tubes with small lumina. L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Distilled water 1000 mL

Fluid thioglycollate medium is used by incubating it at 30º to 35ºC under aerobic conditions. b. Alternative thioglycollate medium: For use with turbid and viscid products and for devices having tubes with small lumina. L-Cystin 0.5 g Sodium chloride 2.5 g 5.5 g Dextrose (C6H12O6H2O) Yeast extra (water soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 5.0 g Thioglycollic acid 0.3 mL Distilled water 1000 mL

Procedure

Procedure

1. Heat the ingredients in a suitable container until the solution is affected. 2. Mix and adjust the reaction with 1M sodium hydroxide, if necessary, so that the medium will have a pH of 7.1 + 0.2 after sterilisation. 3. Filter if necessary, place in suitable vessels and sterilise in an autoclave at 121ºC for 20 minutes.

1. Heat the ingredients in a suitable container until the solution is affected. 2. Mix and adjust the reaction with 1M sodium hydroxide, if necessary, so that the medium will have a pH of 7.1 + 0.2 after sterilisation. 3. Filter if necessary, place in suitable vessels and sterilise in an autoclave at 121ºC for 20 minutes.

Note: The medium is freshly prepared or heated in a steam-bath and allowed to cool just prior to use. Do not reheat. Use alternative thioglycollate medium in a manner that will assure anaerobic conditions for the duration of the incubation at 30º to 35ºC. c. Soya bean- casein digest medium Pancreatic digest of casein 17.0 g Papaic digest of soya bean meal 3.0 g Sodium chloride 5.0 g

Note: The medium is freshly prepared or heated in a steam-bath and allowed to cool just prior to use. Do not reheat. Use alternative thioglycollate medium in a manner that will assure anaerobic conditions for the duration of the incubation at 30º to 35ºC. c. Soya bean- casein digest medium Pancreatic digest of casein 17.0 g Papaic digest of soya bean meal 3.0 g Sodium chloride 5.0 g

94 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Dibasic potassium phosphate (K2HPO4) Dextrose (C6H12O6H2O) Distilled water

94 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2.5 g 2.5 g 1000 mL

Dibasic potassium phosphate (K2HPO4) Dextrose (C6H12O6H2O) Distilled water

Procedure

2.5 g 2.5 g 1000 mL

Procedure

1. Dissolve the solids in distilled water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1M sodium hydroxide to give a final pH of 7.1 + 0.2 after sterilisation. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121ºC for 20 minutes. The media used should comply with the following tests carried out before the test on the preparation being examined. Sterility test for the media: Incubate portions of the fluid thioglycollate medium/alternative thioglycollate medium at 30º to 35ºC and soya bean casein digest medium at 20º to 25ºC for not less than 7 days. There should be no growth of microorganisms. Growth promotion test: This test is done for checking the nutritive quality of the medium. Inoculate the microorganisms listed in table given below and incubate according to the condition specified. Table 2

1. Dissolve the solids in distilled water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1M sodium hydroxide to give a final pH of 7.1 + 0.2 after sterilisation. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121ºC for 20 minutes. The media used should comply with the following tests carried out before the test on the preparation being examined. Sterility test for the media: Incubate portions of the fluid thioglycollate medium/alternative thioglycollate medium at 30º to 35ºC and soya bean casein digest medium at 20º to 25ºC for not less than 7 days. There should be no growth of microorganisms. Growth promotion test: This test is done for checking the nutritive quality of the medium. Inoculate the microorganisms listed in table given below and incubate according to the condition specified. Table 2

Medium

Test microorganisms

Incubation

Fluid thioglycollate

Bacillus subtilis Candida albicans Bacteroides vulgatus

30 to 35°C 30 to 35°C 30 to 35°C

Aerobic Aerobic Aerobic

Alternative thioglycollate

Bacteroids valgatus

30 to 35°C

Anaerobic

Soyabean-casein digest

Bacillus subtilis Candida albicans

20 to 25°C 20 to 25°C

Aerobic Aerobic

Medium

Test microorganisms

Incubation

Fluid thioglycollate

Bacillus subtilis Candida albicans Bacteroides vulgatus

30 to 35°C 30 to 35°C 30 to 35°C

Aerobic Aerobic Aerobic

Alternative thioglycollate

Bacteroids valgatus

30 to 35°C

Anaerobic

Soyabean-casein digest

Bacillus subtilis Candida albicans

20 to 25°C 20 to 25°C

Aerobic Aerobic

The test media are satisfactory if clear evidence of growth appears in all the inoculated media containers within 7 days. The tests may be conducted simultaneously with the use of the test media for sterility test purposes. The sterility test is considered invalid if the test medium shows inadequate growth response. If freshly prepared media are not used within 2 days, store them in dark, preferably at 2º to 25ºC.

The test media are satisfactory if clear evidence of growth appears in all the inoculated media containers within 7 days. The tests may be conducted simultaneously with the use of the test media for sterility test purposes. The sterility test is considered invalid if the test medium shows inadequate growth response. If freshly prepared media are not used within 2 days, store them in dark, preferably at 2º to 25ºC.

7.2 TEST PROCEDURES

7.2 TEST PROCEDURES

There are two methods: Method A or ‘Membrane filtration’ and Method B or ‘Direct Inoculation’. For liquid products where the volume in a container is 100 ml or more, only Method A should be employed.

There are two methods: Method A or ‘Membrane filtration’ and Method B or ‘Direct Inoculation’. For liquid products where the volume in a container is 100 ml or more, only Method A should be employed.

94 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

94 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Dibasic potassium phosphate (K2HPO4) Dextrose (C6H12O6H2O) Distilled water

2.5 g 2.5 g 1000 mL

Dibasic potassium phosphate (K2HPO4) Dextrose (C6H12O6H2O) Distilled water

Procedure

2.5 g 2.5 g 1000 mL

Procedure

1. Dissolve the solids in distilled water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1M sodium hydroxide to give a final pH of 7.1 + 0.2 after sterilisation. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121ºC for 20 minutes. The media used should comply with the following tests carried out before the test on the preparation being examined. Sterility test for the media: Incubate portions of the fluid thioglycollate medium/alternative thioglycollate medium at 30º to 35ºC and soya bean casein digest medium at 20º to 25ºC for not less than 7 days. There should be no growth of microorganisms. Growth promotion test: This test is done for checking the nutritive quality of the medium. Inoculate the microorganisms listed in table given below and incubate according to the condition specified. Table 2

1. Dissolve the solids in distilled water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1M sodium hydroxide to give a final pH of 7.1 + 0.2 after sterilisation. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121ºC for 20 minutes. The media used should comply with the following tests carried out before the test on the preparation being examined. Sterility test for the media: Incubate portions of the fluid thioglycollate medium/alternative thioglycollate medium at 30º to 35ºC and soya bean casein digest medium at 20º to 25ºC for not less than 7 days. There should be no growth of microorganisms. Growth promotion test: This test is done for checking the nutritive quality of the medium. Inoculate the microorganisms listed in table given below and incubate according to the condition specified. Table 2

Medium

Test microorganisms

Incubation

Fluid thioglycollate

Bacillus subtilis Candida albicans Bacteroides vulgatus

30 to 35°C 30 to 35°C 30 to 35°C

Aerobic Aerobic Aerobic

Alternative thioglycollate

Bacteroids valgatus

30 to 35°C

Anaerobic

Soyabean-casein digest

Bacillus subtilis Candida albicans

20 to 25°C 20 to 25°C

Aerobic Aerobic

Medium

Test microorganisms

Incubation

Fluid thioglycollate

Bacillus subtilis Candida albicans Bacteroides vulgatus

30 to 35°C 30 to 35°C 30 to 35°C

Aerobic Aerobic Aerobic

Alternative thioglycollate

Bacteroids valgatus

30 to 35°C

Anaerobic

Soyabean-casein digest

Bacillus subtilis Candida albicans

20 to 25°C 20 to 25°C

Aerobic Aerobic

The test media are satisfactory if clear evidence of growth appears in all the inoculated media containers within 7 days. The tests may be conducted simultaneously with the use of the test media for sterility test purposes. The sterility test is considered invalid if the test medium shows inadequate growth response. If freshly prepared media are not used within 2 days, store them in dark, preferably at 2º to 25ºC.

The test media are satisfactory if clear evidence of growth appears in all the inoculated media containers within 7 days. The tests may be conducted simultaneously with the use of the test media for sterility test purposes. The sterility test is considered invalid if the test medium shows inadequate growth response. If freshly prepared media are not used within 2 days, store them in dark, preferably at 2º to 25ºC.

7.2 TEST PROCEDURES

7.2 TEST PROCEDURES

There are two methods: Method A or ‘Membrane filtration’ and Method B or ‘Direct Inoculation’. For liquid products where the volume in a container is 100 ml or more, only Method A should be employed.

There are two methods: Method A or ‘Membrane filtration’ and Method B or ‘Direct Inoculation’. For liquid products where the volume in a container is 100 ml or more, only Method A should be employed.

STERILITY TESTING

95

STERILITY TESTING

95

7.2.1 Method A: Membrane Filtration A membrane, generally suitable for sterility test, has a nominal pore size not greater than 0.45 —m and diameter of approximately 47 mm, the effectiveness of which in retaining micro-organisms has been established.

7.2.1 Method A: Membrane Filtration A membrane, generally suitable for sterility test, has a nominal pore size not greater than 0.45 —m and diameter of approximately 47 mm, the effectiveness of which in retaining micro-organisms has been established.

Diluting fluids

Diluting fluids

Fluid A : Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks in 100 mL quantities and sterilise at 121°C for 20 minutes. Note – Where fluid A is to be used in performing the tests for sterility on a specimen of the penicillin or cephalosporin class of antibiotics, aseptically add a quantity of sterile penicillinase to the fluid A to be used to rinse the membrane(s) sufficient to inactivate any residual antibiotic activity on the membrane(s) after the solution of the specimen has been filtered. Fluid B : If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1 ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilise at 121°C for 20 minutes. Note – A sterile fluid should not have antibacterial or antifungal properties if it is to be considered suitable for dissolving, diluting or rinsing a preparation being examined for sterility.

Fluid A : Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks in 100 mL quantities and sterilise at 121°C for 20 minutes. Note – Where fluid A is to be used in performing the tests for sterility on a specimen of the penicillin or cephalosporin class of antibiotics, aseptically add a quantity of sterile penicillinase to the fluid A to be used to rinse the membrane(s) sufficient to inactivate any residual antibiotic activity on the membrane(s) after the solution of the specimen has been filtered. Fluid B : If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1 ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilise at 121°C for 20 minutes. Note – A sterile fluid should not have antibacterial or antifungal properties if it is to be considered suitable for dissolving, diluting or rinsing a preparation being examined for sterility.

Quantities of sample to be used

Quantities of sample to be used

1. For injectable preparations : Whenever possible use the whole contents of the container, but in any case not less than the quantities prescribed in table given below, diluting where necessary to about 100 ml with a suitable sterile diluent such fluid A. Table 3 Quantity in each container of injectable preparation

Table 3 Minimum quantity to be used for each culture medium

For liquids : Less than 1 mL 1 mL or more but less than 4 mL 4 mL or more but less than 20 mL 20 mL or more but less than 100 mL 100 mL or more

Quantity in each container of injectable preparation

Minimum quantity to be used for each culture medium

For liquids : Total contents of a container Half the contents of a container 2 mL 10% of the contents of a container unless otherwise specified in the monograph. Not less than half the contents of a container unless otherwise specified in the monograph.

For solids Less than 50 mg 50 mg or more but less than 200 mg 200 mg or more

1. For injectable preparations : Whenever possible use the whole contents of the container, but in any case not less than the quantities prescribed in table given below, diluting where necessary to about 100 ml with a suitable sterile diluent such fluid A.

Less than 1 mL 1 mL or more but less than 4 mL 4 mL or more but less than 20 mL 20 mL or more but less than 100 mL 100 mL or more

Total contents of a container Half the contents of a container 2 mL 10% of the contents of a container unless otherwise specified in the monograph. Not less than half the contents of a container unless otherwise specified in the monograph.

For solids Total contents of a container Half the contents of a container 100 mg

STERILITY TESTING

Less than 50 mg 50 mg or more but less than 200 mg 200 mg or more

Total contents of a container Half the contents of a container 100 mg

95

STERILITY TESTING

95

7.2.1 Method A: Membrane Filtration A membrane, generally suitable for sterility test, has a nominal pore size not greater than 0.45 —m and diameter of approximately 47 mm, the effectiveness of which in retaining micro-organisms has been established.

7.2.1 Method A: Membrane Filtration A membrane, generally suitable for sterility test, has a nominal pore size not greater than 0.45 —m and diameter of approximately 47 mm, the effectiveness of which in retaining micro-organisms has been established.

Diluting fluids

Diluting fluids

Fluid A : Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks in 100 mL quantities and sterilise at 121°C for 20 minutes. Note – Where fluid A is to be used in performing the tests for sterility on a specimen of the penicillin or cephalosporin class of antibiotics, aseptically add a quantity of sterile penicillinase to the fluid A to be used to rinse the membrane(s) sufficient to inactivate any residual antibiotic activity on the membrane(s) after the solution of the specimen has been filtered. Fluid B : If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1 ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilise at 121°C for 20 minutes. Note – A sterile fluid should not have antibacterial or antifungal properties if it is to be considered suitable for dissolving, diluting or rinsing a preparation being examined for sterility.

Fluid A : Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks in 100 mL quantities and sterilise at 121°C for 20 minutes. Note – Where fluid A is to be used in performing the tests for sterility on a specimen of the penicillin or cephalosporin class of antibiotics, aseptically add a quantity of sterile penicillinase to the fluid A to be used to rinse the membrane(s) sufficient to inactivate any residual antibiotic activity on the membrane(s) after the solution of the specimen has been filtered. Fluid B : If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1 ml of polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilise at 121°C for 20 minutes. Note – A sterile fluid should not have antibacterial or antifungal properties if it is to be considered suitable for dissolving, diluting or rinsing a preparation being examined for sterility.

Quantities of sample to be used

Quantities of sample to be used

1. For injectable preparations : Whenever possible use the whole contents of the container, but in any case not less than the quantities prescribed in table given below, diluting where necessary to about 100 ml with a suitable sterile diluent such fluid A. Table 3 Quantity in each container of injectable preparation

Table 3 Minimum quantity to be used for each culture medium

For liquids : Less than 1 mL 1 mL or more but less than 4 mL 4 mL or more but less than 20 mL 20 mL or more but less than 100 mL 100 mL or more

Quantity in each container of injectable preparation

Minimum quantity to be used for each culture medium

For liquids : Total contents of a container Half the contents of a container 2 mL 10% of the contents of a container unless otherwise specified in the monograph. Not less than half the contents of a container unless otherwise specified in the monograph.

For solids Less than 50 mg 50 mg or more but less than 200 mg 200 mg or more

1. For injectable preparations : Whenever possible use the whole contents of the container, but in any case not less than the quantities prescribed in table given below, diluting where necessary to about 100 ml with a suitable sterile diluent such fluid A.

Less than 1 mL 1 mL or more but less than 4 mL 4 mL or more but less than 20 mL 20 mL or more but less than 100 mL 100 mL or more

Total contents of a container Half the contents of a container 2 mL 10% of the contents of a container unless otherwise specified in the monograph. Not less than half the contents of a container unless otherwise specified in the monograph.

For solids Total contents of a container Half the contents of a container 100 mg

Less than 50 mg 50 mg or more but less than 200 mg 200 mg or more

Total contents of a container Half the contents of a container 100 mg

96 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. For ophthalmic and other non-injectable preparations: Take an amount within the range prescribed in column (A) of Table given below, if necessary, using the contents of more than one container, and mix thoroughly. For each medium use the amount specified in column (B) of Table given below, taken from the mixed sample. Table 4

96 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. For ophthalmic and other non-injectable preparations: Take an amount within the range prescribed in column (A) of Table given below, if necessary, using the contents of more than one container, and mix thoroughly. For each medium use the amount specified in column (B) of Table given below, taken from the mixed sample. Table 4

Type of preparation culture medium

Quantity to be mixed (A)

Quantity to be used for each (B)

Type of preparation culture medium

Quantity to be mixed (A)

Quantity to be used for each (B)

Ophthalmic solutions; other non-injectable liquid preparations

10 to 100 mL

5 to 10 mL

Ophthalmic solutions; other non-injectable liquid preparations

10 to 100 mL

5 to 10 mL

Other preparations; preparations soluble in water or appropriate solvents; insoluble preparations to be suspended or emulsified (ointments and creams) 1 to 10 g Absorbent cotton

0.5 to 1 g

Other preparations; preparations soluble in water or appropriate solvents; insoluble preparations to be suspended or emulsified (ointments and creams) 1 to 10 g

0.5 to 1 g

Not less than 1 g*

Absorbent cotton

Not less than 1 g*

Method of test

Method of test

1. For aqueous solutions: Prepare each membrane by aseptically transferring a small quantity of fluid A onto the membrane and filtering it. For each medium to be used, transfer aseptically into two separate membrane filter funnels or to separate sterile pooling vessels prior to transfer not less than the quantity of preparation being examined prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with the aid of vacuum. If the solution being examined has anti-microbial properties, wash the membrane(s) by filtering through it (them) not less than three successive quantities, each of approximately 100 mL. of sterile fluid A. The quantities of fluid used should be sufficient to allow growth of a small inoculum of organisms (approximately 50) sensitive to the antimicrobial substance in the presence of the residual inhibitory material on the membrane. After filtration, aseptically remove the membrane from the holder, cut the membrane in half, if only one is used, immerse the membrane, or one-half of the membrane, in 100 mL of soya bean-casein digest medium and incubate at 20° to 25°C for not less than 7 days. Similarly, immerse the other membrane or other half of the membrane, in 100 mL of fluid thioglycollate medium and incubate at 30° to 35°C for not less than 7 days. 2. For liquids immiscible with aqueous vehicles and suspensions: Carry out the test described under above (For aqueous solutions) but add a sufficient quantity of fluid A to the pooled sample to achieve rapid

96 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. For ophthalmic and other non-injectable preparations: Take an amount within the range prescribed in column (A) of Table given below, if necessary, using the contents of more than one container, and mix thoroughly. For each medium use the amount specified in column (B) of Table given below, taken from the mixed sample. Table 4

1. For aqueous solutions: Prepare each membrane by aseptically transferring a small quantity of fluid A onto the membrane and filtering it. For each medium to be used, transfer aseptically into two separate membrane filter funnels or to separate sterile pooling vessels prior to transfer not less than the quantity of preparation being examined prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with the aid of vacuum. If the solution being examined has anti-microbial properties, wash the membrane(s) by filtering through it (them) not less than three successive quantities, each of approximately 100 mL. of sterile fluid A. The quantities of fluid used should be sufficient to allow growth of a small inoculum of organisms (approximately 50) sensitive to the antimicrobial substance in the presence of the residual inhibitory material on the membrane. After filtration, aseptically remove the membrane from the holder, cut the membrane in half, if only one is used, immerse the membrane, or one-half of the membrane, in 100 mL of soya bean-casein digest medium and incubate at 20° to 25°C for not less than 7 days. Similarly, immerse the other membrane or other half of the membrane, in 100 mL of fluid thioglycollate medium and incubate at 30° to 35°C for not less than 7 days. 2. For liquids immiscible with aqueous vehicles and suspensions: Carry out the test described under above (For aqueous solutions) but add a sufficient quantity of fluid A to the pooled sample to achieve rapid

96 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. For ophthalmic and other non-injectable preparations: Take an amount within the range prescribed in column (A) of Table given below, if necessary, using the contents of more than one container, and mix thoroughly. For each medium use the amount specified in column (B) of Table given below, taken from the mixed sample. Table 4

Type of preparation culture medium

Quantity to be mixed (A)

Quantity to be used for each (B)

Type of preparation culture medium

Quantity to be mixed (A)

Quantity to be used for each (B)

Ophthalmic solutions; other non-injectable liquid preparations

10 to 100 mL

5 to 10 mL

Ophthalmic solutions; other non-injectable liquid preparations

10 to 100 mL

5 to 10 mL

Other preparations; preparations soluble in water or appropriate solvents; insoluble preparations to be suspended or emulsified (ointments and creams) 1 to 10 g Absorbent cotton

0.5 to 1 g

Other preparations; preparations soluble in water or appropriate solvents; insoluble preparations to be suspended or emulsified (ointments and creams) 1 to 10 g

0.5 to 1 g

Not less than 1 g*

Absorbent cotton

Not less than 1 g*

Method of test 1. For aqueous solutions: Prepare each membrane by aseptically transferring a small quantity of fluid A onto the membrane and filtering it. For each medium to be used, transfer aseptically into two separate membrane filter funnels or to separate sterile pooling vessels prior to transfer not less than the quantity of preparation being examined prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with the aid of vacuum. If the solution being examined has anti-microbial properties, wash the membrane(s) by filtering through it (them) not less than three successive quantities, each of approximately 100 mL. of sterile fluid A. The quantities of fluid used should be sufficient to allow growth of a small inoculum of organisms (approximately 50) sensitive to the antimicrobial substance in the presence of the residual inhibitory material on the membrane. After filtration, aseptically remove the membrane from the holder, cut the membrane in half, if only one is used, immerse the membrane, or one-half of the membrane, in 100 mL of soya bean-casein digest medium and incubate at 20° to 25°C for not less than 7 days. Similarly, immerse the other membrane or other half of the membrane, in 100 mL of fluid thioglycollate medium and incubate at 30° to 35°C for not less than 7 days. 2. For liquids immiscible with aqueous vehicles and suspensions: Carry out the test described under above (For aqueous solutions) but add a sufficient quantity of fluid A to the pooled sample to achieve rapid

Method of test 1. For aqueous solutions: Prepare each membrane by aseptically transferring a small quantity of fluid A onto the membrane and filtering it. For each medium to be used, transfer aseptically into two separate membrane filter funnels or to separate sterile pooling vessels prior to transfer not less than the quantity of preparation being examined prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with the aid of vacuum. If the solution being examined has anti-microbial properties, wash the membrane(s) by filtering through it (them) not less than three successive quantities, each of approximately 100 mL. of sterile fluid A. The quantities of fluid used should be sufficient to allow growth of a small inoculum of organisms (approximately 50) sensitive to the antimicrobial substance in the presence of the residual inhibitory material on the membrane. After filtration, aseptically remove the membrane from the holder, cut the membrane in half, if only one is used, immerse the membrane, or one-half of the membrane, in 100 mL of soya bean-casein digest medium and incubate at 20° to 25°C for not less than 7 days. Similarly, immerse the other membrane or other half of the membrane, in 100 mL of fluid thioglycollate medium and incubate at 30° to 35°C for not less than 7 days. 2. For liquids immiscible with aqueous vehicles and suspensions: Carry out the test described under above (For aqueous solutions) but add a sufficient quantity of fluid A to the pooled sample to achieve rapid

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filtration. Sterile enzyme preparations such as penicillinase or cellulase may be added to fluid A to aid in dissolving insoluble substances. If the substance under test contains lecithin, use fluid B for diluting. For oils and oily solutions: Filter oils or oily solutions of sufficient low viscosity without dilution through a dry membrane. Dilute viscous oils as necessary with a suitable sterile diluent such as isopropyl myristate that has been shown not to have antimicrobial properties under the conditions of the test. Allow the oil to penetrate the membrane and filter by applying pressure or suction gradually. Wash the membrane by filtering through it at least three successive quantities, each of approximately 100 mL, of sterile fluid B or any other suitable sterile diluent. Complete the tests described above (For aqueous solutions beginning at the words “After filtration”). For ointments and creams: Dilute ointments in a fatty base and emulsions of the water-in-oil type to give a fluid concentration of 1% w/ v, by heating if necessary, to not more than 40°C with a suitable sterile diluent such as isopropyl myristate previously rendered sterile by filtration through a 0.22—m membrane filter that has been shown not to have antimicrobial properties under the conditions of test. Filter as rapidly as possible and complete the test as described above (For oils and oily solutions beginning at the words “Wash the membrane by ….”) In exceptional cases, it may necessary to heat the substance to not more than 45°C and to use warm solutions for washing the membrane. Note – For ointments and oils that are insoluble in isopropyl myristate, use Method B. For soluble solids: For each medium, dissolve not less than the quantity of the substance being examined, as prescribed in Tables 3 and 4, in a suitable sterile solvent such as fluid A and carry out the test described above (For aqueous solutions) using a membrane appropriate to the chosen solvents. For sterile devices: Aseptically pass a sufficient volume of fluid B through each of not less than twenty devices so that not less than 100 mL is recovered from each device. Collect the fluids in sterile containers and filter the entire volume collected through membrane filter funnel(s) as described above (For aqueous solutions).

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filtration. Sterile enzyme preparations such as penicillinase or cellulase may be added to fluid A to aid in dissolving insoluble substances. If the substance under test contains lecithin, use fluid B for diluting. For oils and oily solutions: Filter oils or oily solutions of sufficient low viscosity without dilution through a dry membrane. Dilute viscous oils as necessary with a suitable sterile diluent such as isopropyl myristate that has been shown not to have antimicrobial properties under the conditions of the test. Allow the oil to penetrate the membrane and filter by applying pressure or suction gradually. Wash the membrane by filtering through it at least three successive quantities, each of approximately 100 mL, of sterile fluid B or any other suitable sterile diluent. Complete the tests described above (For aqueous solutions beginning at the words “After filtration”). For ointments and creams: Dilute ointments in a fatty base and emulsions of the water-in-oil type to give a fluid concentration of 1% w/ v, by heating if necessary, to not more than 40°C with a suitable sterile diluent such as isopropyl myristate previously rendered sterile by filtration through a 0.22—m membrane filter that has been shown not to have antimicrobial properties under the conditions of test. Filter as rapidly as possible and complete the test as described above (For oils and oily solutions beginning at the words “Wash the membrane by ….”) In exceptional cases, it may necessary to heat the substance to not more than 45°C and to use warm solutions for washing the membrane. Note – For ointments and oils that are insoluble in isopropyl myristate, use Method B. For soluble solids: For each medium, dissolve not less than the quantity of the substance being examined, as prescribed in Tables 3 and 4, in a suitable sterile solvent such as fluid A and carry out the test described above (For aqueous solutions) using a membrane appropriate to the chosen solvents. For sterile devices: Aseptically pass a sufficient volume of fluid B through each of not less than twenty devices so that not less than 100 mL is recovered from each device. Collect the fluids in sterile containers and filter the entire volume collected through membrane filter funnel(s) as described above (For aqueous solutions).

7.2.2 Method B: Direct Inoculation

7.2.2 Method B: Direct Inoculation

Method of test

Method of test

1. For aqueous solutions and suspensions: The tests for microbial contamination are carried out on the same sample of the preparations being examined using the above stated media. When the quantity in a single container is insufficient to carry out the tests, the combined contents

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filtration. Sterile enzyme preparations such as penicillinase or cellulase may be added to fluid A to aid in dissolving insoluble substances. If the substance under test contains lecithin, use fluid B for diluting. For oils and oily solutions: Filter oils or oily solutions of sufficient low viscosity without dilution through a dry membrane. Dilute viscous oils as necessary with a suitable sterile diluent such as isopropyl myristate that has been shown not to have antimicrobial properties under the conditions of the test. Allow the oil to penetrate the membrane and filter by applying pressure or suction gradually. Wash the membrane by filtering through it at least three successive quantities, each of approximately 100 mL, of sterile fluid B or any other suitable sterile diluent. Complete the tests described above (For aqueous solutions beginning at the words “After filtration”). For ointments and creams: Dilute ointments in a fatty base and emulsions of the water-in-oil type to give a fluid concentration of 1% w/ v, by heating if necessary, to not more than 40°C with a suitable sterile diluent such as isopropyl myristate previously rendered sterile by filtration through a 0.22—m membrane filter that has been shown not to have antimicrobial properties under the conditions of test. Filter as rapidly as possible and complete the test as described above (For oils and oily solutions beginning at the words “Wash the membrane by ….”) In exceptional cases, it may necessary to heat the substance to not more than 45°C and to use warm solutions for washing the membrane. Note – For ointments and oils that are insoluble in isopropyl myristate, use Method B. For soluble solids: For each medium, dissolve not less than the quantity of the substance being examined, as prescribed in Tables 3 and 4, in a suitable sterile solvent such as fluid A and carry out the test described above (For aqueous solutions) using a membrane appropriate to the chosen solvents. For sterile devices: Aseptically pass a sufficient volume of fluid B through each of not less than twenty devices so that not less than 100 mL is recovered from each device. Collect the fluids in sterile containers and filter the entire volume collected through membrane filter funnel(s) as described above (For aqueous solutions).

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filtration. Sterile enzyme preparations such as penicillinase or cellulase may be added to fluid A to aid in dissolving insoluble substances. If the substance under test contains lecithin, use fluid B for diluting. For oils and oily solutions: Filter oils or oily solutions of sufficient low viscosity without dilution through a dry membrane. Dilute viscous oils as necessary with a suitable sterile diluent such as isopropyl myristate that has been shown not to have antimicrobial properties under the conditions of the test. Allow the oil to penetrate the membrane and filter by applying pressure or suction gradually. Wash the membrane by filtering through it at least three successive quantities, each of approximately 100 mL, of sterile fluid B or any other suitable sterile diluent. Complete the tests described above (For aqueous solutions beginning at the words “After filtration”). For ointments and creams: Dilute ointments in a fatty base and emulsions of the water-in-oil type to give a fluid concentration of 1% w/ v, by heating if necessary, to not more than 40°C with a suitable sterile diluent such as isopropyl myristate previously rendered sterile by filtration through a 0.22—m membrane filter that has been shown not to have antimicrobial properties under the conditions of test. Filter as rapidly as possible and complete the test as described above (For oils and oily solutions beginning at the words “Wash the membrane by ….”) In exceptional cases, it may necessary to heat the substance to not more than 45°C and to use warm solutions for washing the membrane. Note – For ointments and oils that are insoluble in isopropyl myristate, use Method B. For soluble solids: For each medium, dissolve not less than the quantity of the substance being examined, as prescribed in Tables 3 and 4, in a suitable sterile solvent such as fluid A and carry out the test described above (For aqueous solutions) using a membrane appropriate to the chosen solvents. For sterile devices: Aseptically pass a sufficient volume of fluid B through each of not less than twenty devices so that not less than 100 mL is recovered from each device. Collect the fluids in sterile containers and filter the entire volume collected through membrane filter funnel(s) as described above (For aqueous solutions).

7.2.2 Method B: Direct Inoculation

7.2.2 Method B: Direct Inoculation

Method of test

Method of test

1. For aqueous solutions and suspensions: The tests for microbial contamination are carried out on the same sample of the preparations being examined using the above stated media. When the quantity in a single container is insufficient to carry out the tests, the combined contents

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1. For aqueous solutions and suspensions: The tests for microbial contamination are carried out on the same sample of the preparations being examined using the above stated media. When the quantity in a single container is insufficient to carry out the tests, the combined contents

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of the two or more containers are used to inoculate the above stated media. Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a needle. Aseptically transfer the specified volume of the material from each container to a vessel of the culture medium. Mix the liquid with the medium but not aerate excessively. Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph, at 30° to 35°C in the case of fluid thioglycollate medium and at 20° to 25°C in the case of soy bean-casein digest medium. When the material being examined renders the medium turbid so that the presence or absence of microbial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days. For oils and oily solutions: Use media to which have been added 0.1% w/v of (4-tert-octylphenoxy) polyethoxyethanol, 1% w/v of polysorbate 80 or other suitable emulsifying agent, in an appropriate concentration, shown not to have any antimicrobial properties under the conditions of test. Carry out the test as described above (For aqueous solutions and suspensions). Cultures containing oily preparations should be shaken gently each day. However when the fluid thioglycollate medium is used for the detection of anaerobic micro-organisms, shaking or mixing should be kept to a minimum to maintain anaerobic conditions. For ointments: Prepare by diluting tenfold in a sterile diluent such as fluid B or any other aqueous vehicle capable of dispersing the test material homogeneously throughout the fluid mixture. (Before use, test the dispersing agent to ascertain that in the concentration used it has no significant antimicrobial effects during the time intervals for all transfers.) Mix 10 mL of the fluid mixture so obtained with 80 mL of the medium and proceed as directed above (For aqueous solutions and suspensions). For solids: Transfer the quantity of the preparation to be examined to the quantity of the medium specified in Table 5 and mix, the conditions of incubation being same as For aqueous solutions and suspensions. Proceed as directed above (For aqueous solutions and suspensions beginning at the words “When the material being examined….”). For sterile devices: For articles of such size and shape as permit complete immersion in not more than 1000 mL of culture medium test the intact article, using the appropriate media, and incubating as directed above (For aqueous solutions and suspensions).

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of the two or more containers are used to inoculate the above stated media. Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a needle. Aseptically transfer the specified volume of the material from each container to a vessel of the culture medium. Mix the liquid with the medium but not aerate excessively. Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph, at 30° to 35°C in the case of fluid thioglycollate medium and at 20° to 25°C in the case of soy bean-casein digest medium. When the material being examined renders the medium turbid so that the presence or absence of microbial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days. For oils and oily solutions: Use media to which have been added 0.1% w/v of (4-tert-octylphenoxy) polyethoxyethanol, 1% w/v of polysorbate 80 or other suitable emulsifying agent, in an appropriate concentration, shown not to have any antimicrobial properties under the conditions of test. Carry out the test as described above (For aqueous solutions and suspensions). Cultures containing oily preparations should be shaken gently each day. However when the fluid thioglycollate medium is used for the detection of anaerobic micro-organisms, shaking or mixing should be kept to a minimum to maintain anaerobic conditions. For ointments: Prepare by diluting tenfold in a sterile diluent such as fluid B or any other aqueous vehicle capable of dispersing the test material homogeneously throughout the fluid mixture. (Before use, test the dispersing agent to ascertain that in the concentration used it has no significant antimicrobial effects during the time intervals for all transfers.) Mix 10 mL of the fluid mixture so obtained with 80 mL of the medium and proceed as directed above (For aqueous solutions and suspensions). For solids: Transfer the quantity of the preparation to be examined to the quantity of the medium specified in Table 5 and mix, the conditions of incubation being same as For aqueous solutions and suspensions. Proceed as directed above (For aqueous solutions and suspensions beginning at the words “When the material being examined….”). For sterile devices: For articles of such size and shape as permit complete immersion in not more than 1000 mL of culture medium test the intact article, using the appropriate media, and incubating as directed above (For aqueous solutions and suspensions).

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of the two or more containers are used to inoculate the above stated media. Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a needle. Aseptically transfer the specified volume of the material from each container to a vessel of the culture medium. Mix the liquid with the medium but not aerate excessively. Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph, at 30° to 35°C in the case of fluid thioglycollate medium and at 20° to 25°C in the case of soy bean-casein digest medium. When the material being examined renders the medium turbid so that the presence or absence of microbial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days. For oils and oily solutions: Use media to which have been added 0.1% w/v of (4-tert-octylphenoxy) polyethoxyethanol, 1% w/v of polysorbate 80 or other suitable emulsifying agent, in an appropriate concentration, shown not to have any antimicrobial properties under the conditions of test. Carry out the test as described above (For aqueous solutions and suspensions). Cultures containing oily preparations should be shaken gently each day. However when the fluid thioglycollate medium is used for the detection of anaerobic micro-organisms, shaking or mixing should be kept to a minimum to maintain anaerobic conditions. For ointments: Prepare by diluting tenfold in a sterile diluent such as fluid B or any other aqueous vehicle capable of dispersing the test material homogeneously throughout the fluid mixture. (Before use, test the dispersing agent to ascertain that in the concentration used it has no significant antimicrobial effects during the time intervals for all transfers.) Mix 10 mL of the fluid mixture so obtained with 80 mL of the medium and proceed as directed above (For aqueous solutions and suspensions). For solids: Transfer the quantity of the preparation to be examined to the quantity of the medium specified in Table 5 and mix, the conditions of incubation being same as For aqueous solutions and suspensions. Proceed as directed above (For aqueous solutions and suspensions beginning at the words “When the material being examined….”). For sterile devices: For articles of such size and shape as permit complete immersion in not more than 1000 mL of culture medium test the intact article, using the appropriate media, and incubating as directed above (For aqueous solutions and suspensions).

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of the two or more containers are used to inoculate the above stated media. Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a needle. Aseptically transfer the specified volume of the material from each container to a vessel of the culture medium. Mix the liquid with the medium but not aerate excessively. Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph, at 30° to 35°C in the case of fluid thioglycollate medium and at 20° to 25°C in the case of soy bean-casein digest medium. When the material being examined renders the medium turbid so that the presence or absence of microbial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days. For oils and oily solutions: Use media to which have been added 0.1% w/v of (4-tert-octylphenoxy) polyethoxyethanol, 1% w/v of polysorbate 80 or other suitable emulsifying agent, in an appropriate concentration, shown not to have any antimicrobial properties under the conditions of test. Carry out the test as described above (For aqueous solutions and suspensions). Cultures containing oily preparations should be shaken gently each day. However when the fluid thioglycollate medium is used for the detection of anaerobic micro-organisms, shaking or mixing should be kept to a minimum to maintain anaerobic conditions. For ointments: Prepare by diluting tenfold in a sterile diluent such as fluid B or any other aqueous vehicle capable of dispersing the test material homogeneously throughout the fluid mixture. (Before use, test the dispersing agent to ascertain that in the concentration used it has no significant antimicrobial effects during the time intervals for all transfers.) Mix 10 mL of the fluid mixture so obtained with 80 mL of the medium and proceed as directed above (For aqueous solutions and suspensions). For solids: Transfer the quantity of the preparation to be examined to the quantity of the medium specified in Table 5 and mix, the conditions of incubation being same as For aqueous solutions and suspensions. Proceed as directed above (For aqueous solutions and suspensions beginning at the words “When the material being examined….”). For sterile devices: For articles of such size and shape as permit complete immersion in not more than 1000 mL of culture medium test the intact article, using the appropriate media, and incubating as directed above (For aqueous solutions and suspensions).

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For transfusion or infusion assemblies or where the size of an item makes immersion impracticable and only the liquid pathway must be sterile, flush the lumen of each of twenty units with a sufficient quantity of fluid thioglycollate medium and the lumen of each of twenty units with a sufficient quantity of soyabean-casein digest medium to yield a recovery of not less than 15 mL of each medium, and incubate with not less than 100 mL of each of the two media as directed above (For aqueous solutions and suspensions). For devices in which the lumen is so small that fluid thioglycollate medium will not pass through, substitute alternative thioglycollate medium for the fluid thioglycollate medium and incubate that inoculated medium anaerobically. Where the presence of the specimen being tested, in the medium interferes with the test teriostatic or fungistatic action, rinse the article thoroughly with the minimum amount of fluid A. Recover the rinsed fluid and test as described above (For sterile devices—Method A). Precautions: The tests for sterility should be carried out under conditions designed to avoid accidental contamination of the product during the test using, for example, a laminar airflow cabinet. The precautions taken to avoid contamination must be such that they do not affect any microorganisms that should be revealed in the test. The working conditions in which the test is performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests. Observation and Interpretation of Results At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence, that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second test using twice the number of samples. If no evidence of microbial growth is found, the preparation being examined passes the test for sterility. If evidence of growth of any microorganisms is found, the preparation being examined fails the tests for sterility.

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For transfusion or infusion assemblies or where the size of an item makes immersion impracticable and only the liquid pathway must be sterile, flush the lumen of each of twenty units with a sufficient quantity of fluid thioglycollate medium and the lumen of each of twenty units with a sufficient quantity of soyabean-casein digest medium to yield a recovery of not less than 15 mL of each medium, and incubate with not less than 100 mL of each of the two media as directed above (For aqueous solutions and suspensions). For devices in which the lumen is so small that fluid thioglycollate medium will not pass through, substitute alternative thioglycollate medium for the fluid thioglycollate medium and incubate that inoculated medium anaerobically. Where the presence of the specimen being tested, in the medium interferes with the test teriostatic or fungistatic action, rinse the article thoroughly with the minimum amount of fluid A. Recover the rinsed fluid and test as described above (For sterile devices—Method A). Precautions: The tests for sterility should be carried out under conditions designed to avoid accidental contamination of the product during the test using, for example, a laminar airflow cabinet. The precautions taken to avoid contamination must be such that they do not affect any microorganisms that should be revealed in the test. The working conditions in which the test is performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests. Observation and Interpretation of Results At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence, that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second test using twice the number of samples. If no evidence of microbial growth is found, the preparation being examined passes the test for sterility. If evidence of growth of any microorganisms is found, the preparation being examined fails the tests for sterility.

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For transfusion or infusion assemblies or where the size of an item makes immersion impracticable and only the liquid pathway must be sterile, flush the lumen of each of twenty units with a sufficient quantity of fluid thioglycollate medium and the lumen of each of twenty units with a sufficient quantity of soyabean-casein digest medium to yield a recovery of not less than 15 mL of each medium, and incubate with not less than 100 mL of each of the two media as directed above (For aqueous solutions and suspensions). For devices in which the lumen is so small that fluid thioglycollate medium will not pass through, substitute alternative thioglycollate medium for the fluid thioglycollate medium and incubate that inoculated medium anaerobically. Where the presence of the specimen being tested, in the medium interferes with the test teriostatic or fungistatic action, rinse the article thoroughly with the minimum amount of fluid A. Recover the rinsed fluid and test as described above (For sterile devices—Method A). Precautions: The tests for sterility should be carried out under conditions designed to avoid accidental contamination of the product during the test using, for example, a laminar airflow cabinet. The precautions taken to avoid contamination must be such that they do not affect any microorganisms that should be revealed in the test. The working conditions in which the test is performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests. Observation and Interpretation of Results At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence, that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second test using twice the number of samples. If no evidence of microbial growth is found, the preparation being examined passes the test for sterility. If evidence of growth of any microorganisms is found, the preparation being examined fails the tests for sterility.

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For transfusion or infusion assemblies or where the size of an item makes immersion impracticable and only the liquid pathway must be sterile, flush the lumen of each of twenty units with a sufficient quantity of fluid thioglycollate medium and the lumen of each of twenty units with a sufficient quantity of soyabean-casein digest medium to yield a recovery of not less than 15 mL of each medium, and incubate with not less than 100 mL of each of the two media as directed above (For aqueous solutions and suspensions). For devices in which the lumen is so small that fluid thioglycollate medium will not pass through, substitute alternative thioglycollate medium for the fluid thioglycollate medium and incubate that inoculated medium anaerobically. Where the presence of the specimen being tested, in the medium interferes with the test teriostatic or fungistatic action, rinse the article thoroughly with the minimum amount of fluid A. Recover the rinsed fluid and test as described above (For sterile devices—Method A). Precautions: The tests for sterility should be carried out under conditions designed to avoid accidental contamination of the product during the test using, for example, a laminar airflow cabinet. The precautions taken to avoid contamination must be such that they do not affect any microorganisms that should be revealed in the test. The working conditions in which the test is performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests. Observation and Interpretation of Results At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence, that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second test using twice the number of samples. If no evidence of microbial growth is found, the preparation being examined passes the test for sterility. If evidence of growth of any microorganisms is found, the preparation being examined fails the tests for sterility.

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100 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

7.3 STERILITY TEST FOR CATGUT

7.3 STERILITY TEST FOR CATGUT

Heat and chemical methods for the sterilization of catgut are described by Carter et al. Sterilization by ionizing radiation has already become established as a satisfactory alternative and its advantage are: 1. Smaller loss of tensile strength. 2. The container is safer to handle. The glass tube used for heat and chemically sterilized gut may cut the gloves and fingers of the theatre staff and produce spicules that can damage the thread and even find their way into the operational field. These risks are absent with the sachets of plastic aluminium foil laminate used for radiation sterilization. 3. The gut is easier to use because it is only loosely coiled in the sachets.

Heat and chemical methods for the sterilization of catgut are described by Carter et al. Sterilization by ionizing radiation has already become established as a satisfactory alternative and its advantage are: 1. Smaller loss of tensile strength. 2. The container is safer to handle. The glass tube used for heat and chemically sterilized gut may cut the gloves and fingers of the theatre staff and produce spicules that can damage the thread and even find their way into the operational field. These risks are absent with the sachets of plastic aluminium foil laminate used for radiation sterilization. 3. The gut is easier to use because it is only loosely coiled in the sachets.

7.4 LIMULUS AMOEBOCYTE LYSATE TEST (LAL TEST)

7.4 LIMULUS AMOEBOCYTE LYSATE TEST (LAL TEST)

Pharmaceutical products and medical devices could only be categorized as pyrogenic or non-pyrogenic by in vivo rabbit test. Such type of test progressively has been replaced by an in vitro test specific for bacterial endotoxins, this is the LAL test. The LAL test depends upon the reaction between endotoxins and a clottable protein contained within the amoebocyte cells of the blood of the horseshoe crab (limulus polyphemus). The specificity of the reaction has been attributed to a complex cascade of enzyme mediated reactions. The standard LAL test is based on the formation of a semisolid gel between LAL and bacterial endotoxins and in conducted on the end point principle. LAL test is supplied with an identified sensitivity e.g. 0.03 endotoxin units (EU) per mL. This means that when mixed with equal volume of the material under test a gel or clot will form if the material contains 0.03 EU/mL or greater. When it is necessary to quantify endotoxins concentration in a material it is usual to test a series of doubling dilutions against the reagent in temperature controlled conditions. The greatest dilution that gives a positive, is the end point and the concentration of endotoxins in the material can be calculated by multiplying the dilution factor at the end point by the sensitivity of LAL reagent.

Pharmaceutical products and medical devices could only be categorized as pyrogenic or non-pyrogenic by in vivo rabbit test. Such type of test progressively has been replaced by an in vitro test specific for bacterial endotoxins, this is the LAL test. The LAL test depends upon the reaction between endotoxins and a clottable protein contained within the amoebocyte cells of the blood of the horseshoe crab (limulus polyphemus). The specificity of the reaction has been attributed to a complex cascade of enzyme mediated reactions. The standard LAL test is based on the formation of a semisolid gel between LAL and bacterial endotoxins and in conducted on the end point principle. LAL test is supplied with an identified sensitivity e.g. 0.03 endotoxin units (EU) per mL. This means that when mixed with equal volume of the material under test a gel or clot will form if the material contains 0.03 EU/mL or greater. When it is necessary to quantify endotoxins concentration in a material it is usual to test a series of doubling dilutions against the reagent in temperature controlled conditions. The greatest dilution that gives a positive, is the end point and the concentration of endotoxins in the material can be calculated by multiplying the dilution factor at the end point by the sensitivity of LAL reagent.

100 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

100 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

7.3 STERILITY TEST FOR CATGUT

7.3 STERILITY TEST FOR CATGUT

Heat and chemical methods for the sterilization of catgut are described by Carter et al. Sterilization by ionizing radiation has already become established as a satisfactory alternative and its advantage are: 1. Smaller loss of tensile strength. 2. The container is safer to handle. The glass tube used for heat and chemically sterilized gut may cut the gloves and fingers of the theatre staff and produce spicules that can damage the thread and even find their way into the operational field. These risks are absent with the sachets of plastic aluminium foil laminate used for radiation sterilization. 3. The gut is easier to use because it is only loosely coiled in the sachets.

Heat and chemical methods for the sterilization of catgut are described by Carter et al. Sterilization by ionizing radiation has already become established as a satisfactory alternative and its advantage are: 1. Smaller loss of tensile strength. 2. The container is safer to handle. The glass tube used for heat and chemically sterilized gut may cut the gloves and fingers of the theatre staff and produce spicules that can damage the thread and even find their way into the operational field. These risks are absent with the sachets of plastic aluminium foil laminate used for radiation sterilization. 3. The gut is easier to use because it is only loosely coiled in the sachets.

7.4 LIMULUS AMOEBOCYTE LYSATE TEST (LAL TEST)

7.4 LIMULUS AMOEBOCYTE LYSATE TEST (LAL TEST)

Pharmaceutical products and medical devices could only be categorized as pyrogenic or non-pyrogenic by in vivo rabbit test. Such type of test progressively has been replaced by an in vitro test specific for bacterial endotoxins, this is the LAL test. The LAL test depends upon the reaction between endotoxins and a clottable protein contained within the amoebocyte cells of the blood of the horseshoe crab (limulus polyphemus). The specificity of the reaction has been attributed to a complex cascade of enzyme mediated reactions. The standard LAL test is based on the formation of a semisolid gel between LAL and bacterial endotoxins and in conducted on the end point principle. LAL test is supplied with an identified sensitivity e.g. 0.03 endotoxin units (EU) per mL. This means that when mixed with equal volume of the material under test a gel or clot will form if the material contains 0.03 EU/mL or greater. When it is necessary to quantify endotoxins concentration in a material it is usual to test a series of doubling dilutions against the reagent in temperature controlled conditions. The greatest dilution that gives a positive, is the end point and the concentration of endotoxins in the material can be calculated by multiplying the dilution factor at the end point by the sensitivity of LAL reagent.

Pharmaceutical products and medical devices could only be categorized as pyrogenic or non-pyrogenic by in vivo rabbit test. Such type of test progressively has been replaced by an in vitro test specific for bacterial endotoxins, this is the LAL test. The LAL test depends upon the reaction between endotoxins and a clottable protein contained within the amoebocyte cells of the blood of the horseshoe crab (limulus polyphemus). The specificity of the reaction has been attributed to a complex cascade of enzyme mediated reactions. The standard LAL test is based on the formation of a semisolid gel between LAL and bacterial endotoxins and in conducted on the end point principle. LAL test is supplied with an identified sensitivity e.g. 0.03 endotoxin units (EU) per mL. This means that when mixed with equal volume of the material under test a gel or clot will form if the material contains 0.03 EU/mL or greater. When it is necessary to quantify endotoxins concentration in a material it is usual to test a series of doubling dilutions against the reagent in temperature controlled conditions. The greatest dilution that gives a positive, is the end point and the concentration of endotoxins in the material can be calculated by multiplying the dilution factor at the end point by the sensitivity of LAL reagent.

SECTION C

SECTION C

SECTION C

SECTION C

8

8

Disinfectants, Antiseptics, and Preservatives

Disinfectants, Antiseptics, and Preservatives

INTRODUCTION

INTRODUCTION

Disinfectants, antiseptics and preservatives are chemicals which have the ability to destroy or inhibit the growth of microorganisms and which are used for this purpose.

Disinfectants, antiseptics and preservatives are chemicals which have the ability to destroy or inhibit the growth of microorganisms and which are used for this purpose.

8.1 DISINFECTANTS

8.1 DISINFECTANTS

1. Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms, the process of which is known as disinfection. 2. Disinfectants should generally be distinguished from antibiotics as antibiotics destroy microorganisms within the body, and from antiseptics, which destroy microorganisms on living tissue. 3. The British Standards Institution further defines disinfection as not necessarily killing all microorganisms, but reducing them to a level acceptable for a defined purpose, for example, a level which is harmful neither to health nor to the quality of perishable goods. 4. The term high level disinfection indicates destruction of all microorganisms but not necessarily bacterial spores; intermediate level disinfection indicates destruction of all vegetative bacteria including Mycobacterium tuberculosis but may exclude some viruses and fungi and have little or no sporicidal activity; low level disinfection can destroy most vegetative bacteria, fungi and viruses, but this will not include spores and some of the more resistant microorganisms. 5. Some high level disinfectants have good sporicidal activity and have been ascribed the name ‘liquid chemical sterilant’ or ‘chemosterilant’ to indicate that they can effect a complete kill of all microorganisms, as in sterilization.

1. Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms, the process of which is known as disinfection. 2. Disinfectants should generally be distinguished from antibiotics as antibiotics destroy microorganisms within the body, and from antiseptics, which destroy microorganisms on living tissue. 3. The British Standards Institution further defines disinfection as not necessarily killing all microorganisms, but reducing them to a level acceptable for a defined purpose, for example, a level which is harmful neither to health nor to the quality of perishable goods. 4. The term high level disinfection indicates destruction of all microorganisms but not necessarily bacterial spores; intermediate level disinfection indicates destruction of all vegetative bacteria including Mycobacterium tuberculosis but may exclude some viruses and fungi and have little or no sporicidal activity; low level disinfection can destroy most vegetative bacteria, fungi and viruses, but this will not include spores and some of the more resistant microorganisms. 5. Some high level disinfectants have good sporicidal activity and have been ascribed the name ‘liquid chemical sterilant’ or ‘chemosterilant’ to indicate that they can effect a complete kill of all microorganisms, as in sterilization.

8

8

Disinfectants, Antiseptics, and Preservatives

Disinfectants, Antiseptics, and Preservatives

INTRODUCTION

INTRODUCTION

Disinfectants, antiseptics and preservatives are chemicals which have the ability to destroy or inhibit the growth of microorganisms and which are used for this purpose.

Disinfectants, antiseptics and preservatives are chemicals which have the ability to destroy or inhibit the growth of microorganisms and which are used for this purpose.

8.1 DISINFECTANTS

8.1 DISINFECTANTS

1. Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms, the process of which is known as disinfection. 2. Disinfectants should generally be distinguished from antibiotics as antibiotics destroy microorganisms within the body, and from antiseptics, which destroy microorganisms on living tissue. 3. The British Standards Institution further defines disinfection as not necessarily killing all microorganisms, but reducing them to a level acceptable for a defined purpose, for example, a level which is harmful neither to health nor to the quality of perishable goods. 4. The term high level disinfection indicates destruction of all microorganisms but not necessarily bacterial spores; intermediate level disinfection indicates destruction of all vegetative bacteria including Mycobacterium tuberculosis but may exclude some viruses and fungi and have little or no sporicidal activity; low level disinfection can destroy most vegetative bacteria, fungi and viruses, but this will not include spores and some of the more resistant microorganisms. 5. Some high level disinfectants have good sporicidal activity and have been ascribed the name ‘liquid chemical sterilant’ or ‘chemosterilant’ to indicate that they can effect a complete kill of all microorganisms, as in sterilization.

1. Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms, the process of which is known as disinfection. 2. Disinfectants should generally be distinguished from antibiotics as antibiotics destroy microorganisms within the body, and from antiseptics, which destroy microorganisms on living tissue. 3. The British Standards Institution further defines disinfection as not necessarily killing all microorganisms, but reducing them to a level acceptable for a defined purpose, for example, a level which is harmful neither to health nor to the quality of perishable goods. 4. The term high level disinfection indicates destruction of all microorganisms but not necessarily bacterial spores; intermediate level disinfection indicates destruction of all vegetative bacteria including Mycobacterium tuberculosis but may exclude some viruses and fungi and have little or no sporicidal activity; low level disinfection can destroy most vegetative bacteria, fungi and viruses, but this will not include spores and some of the more resistant microorganisms. 5. Some high level disinfectants have good sporicidal activity and have been ascribed the name ‘liquid chemical sterilant’ or ‘chemosterilant’ to indicate that they can effect a complete kill of all microorganisms, as in sterilization.

104 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

104 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

They are frequently used in research laboratories hospitals, dental clinics, surgery, kitchens and bathrooms to kill infectious organisms.

They are frequently used in research laboratories hospitals, dental clinics, surgery, kitchens and bathrooms to kill infectious organisms.

8.1.1 Properties of an Ideal Disinfectant The properties of an ideal disinfectant can be used to compare those of any other compounds which is in use or is about to be introduced. 1. Broad spectrum: Should always have the widest possible microbial spectrum. 2. Fast acting: Should have a rapidly lethal action on all vegetative forms and spores of bacteria and fungi, protozoa and viruses. 3. Nontoxic. 4. Easy to use. 5. Not affected by physical factors: a. Active in the presence of organic matter such as blood, sputum and faeces. b. Should be compatible with soaps, detergents and other chemicals encountered in use. 6. Should not corrode instruments and other metallic surfaces. 7. Should not cause the disintegration of cloth, rubber, plastics or other materials. 8. Economical: Should not be prohibitively costly.

8.1.1 Properties of an Ideal Disinfectant The properties of an ideal disinfectant can be used to compare those of any other compounds which is in use or is about to be introduced. 1. Broad spectrum: Should always have the widest possible microbial spectrum. 2. Fast acting: Should have a rapidly lethal action on all vegetative forms and spores of bacteria and fungi, protozoa and viruses. 3. Nontoxic. 4. Easy to use. 5. Not affected by physical factors: a. Active in the presence of organic matter such as blood, sputum and faeces. b. Should be compatible with soaps, detergents and other chemicals encountered in use. 6. Should not corrode instruments and other metallic surfaces. 7. Should not cause the disintegration of cloth, rubber, plastics or other materials. 8. Economical: Should not be prohibitively costly.

8.1.2 Factors Affecting the Disinfection Process There are many factors which affect the action of disinfectants: 1. Effect of temperature 2. Effect of pH 3. Effect of dilution 4. Effect of surface activity 5. Effect of interfering agent

8.1.2 Factors Affecting the Disinfection Process There are many factors which affect the action of disinfectants: 1. Effect of temperature 2. Effect of pH 3. Effect of dilution 4. Effect of surface activity 5. Effect of interfering agent

Effect of temperature: If the temperature of the disinfectants is increased, the effectiveness of the disinfectants also increases. Bacteriologist Robert Koch had noted that anthrex spores were more rapidly killed by the same concentration of phenol if the temperature was elevated. As the temperature is increased arithmetical progression, the rate of disinfectant increases geometrically by the first order chemical reaction. Effect of pH: Changes in pH can affect: 1. The rate of growth of innoculum: The optimal bacterial growth takes place in the range of 6-8; on either side of this the rate of growth declines. 2. The ability of the disinfectant to combine with sites on the cell surface: Change in the pH range, affects the adsorption at the cell surface. 3. The potency of the antibacterial agent itself: The degree of ionization of the drug depends on the pH. The condition of pH which favour the

Effect of temperature: If the temperature of the disinfectants is increased, the effectiveness of the disinfectants also increases. Bacteriologist Robert Koch had noted that anthrex spores were more rapidly killed by the same concentration of phenol if the temperature was elevated. As the temperature is increased arithmetical progression, the rate of disinfectant increases geometrically by the first order chemical reaction. Effect of pH: Changes in pH can affect: 1. The rate of growth of innoculum: The optimal bacterial growth takes place in the range of 6-8; on either side of this the rate of growth declines. 2. The ability of the disinfectant to combine with sites on the cell surface: Change in the pH range, affects the adsorption at the cell surface. 3. The potency of the antibacterial agent itself: The degree of ionization of the drug depends on the pH. The condition of pH which favour the

104 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

104 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

They are frequently used in research laboratories hospitals, dental clinics, surgery, kitchens and bathrooms to kill infectious organisms.

They are frequently used in research laboratories hospitals, dental clinics, surgery, kitchens and bathrooms to kill infectious organisms.

8.1.1 Properties of an Ideal Disinfectant The properties of an ideal disinfectant can be used to compare those of any other compounds which is in use or is about to be introduced. 1. Broad spectrum: Should always have the widest possible microbial spectrum. 2. Fast acting: Should have a rapidly lethal action on all vegetative forms and spores of bacteria and fungi, protozoa and viruses. 3. Nontoxic. 4. Easy to use. 5. Not affected by physical factors: a. Active in the presence of organic matter such as blood, sputum and faeces. b. Should be compatible with soaps, detergents and other chemicals encountered in use. 6. Should not corrode instruments and other metallic surfaces. 7. Should not cause the disintegration of cloth, rubber, plastics or other materials. 8. Economical: Should not be prohibitively costly.

8.1.1 Properties of an Ideal Disinfectant The properties of an ideal disinfectant can be used to compare those of any other compounds which is in use or is about to be introduced. 1. Broad spectrum: Should always have the widest possible microbial spectrum. 2. Fast acting: Should have a rapidly lethal action on all vegetative forms and spores of bacteria and fungi, protozoa and viruses. 3. Nontoxic. 4. Easy to use. 5. Not affected by physical factors: a. Active in the presence of organic matter such as blood, sputum and faeces. b. Should be compatible with soaps, detergents and other chemicals encountered in use. 6. Should not corrode instruments and other metallic surfaces. 7. Should not cause the disintegration of cloth, rubber, plastics or other materials. 8. Economical: Should not be prohibitively costly.

8.1.2 Factors Affecting the Disinfection Process There are many factors which affect the action of disinfectants: 1. Effect of temperature 2. Effect of pH 3. Effect of dilution 4. Effect of surface activity 5. Effect of interfering agent

8.1.2 Factors Affecting the Disinfection Process There are many factors which affect the action of disinfectants: 1. Effect of temperature 2. Effect of pH 3. Effect of dilution 4. Effect of surface activity 5. Effect of interfering agent

Effect of temperature: If the temperature of the disinfectants is increased, the effectiveness of the disinfectants also increases. Bacteriologist Robert Koch had noted that anthrex spores were more rapidly killed by the same concentration of phenol if the temperature was elevated. As the temperature is increased arithmetical progression, the rate of disinfectant increases geometrically by the first order chemical reaction. Effect of pH: Changes in pH can affect: 1. The rate of growth of innoculum: The optimal bacterial growth takes place in the range of 6-8; on either side of this the rate of growth declines. 2. The ability of the disinfectant to combine with sites on the cell surface: Change in the pH range, affects the adsorption at the cell surface. 3. The potency of the antibacterial agent itself: The degree of ionization of the drug depends on the pH. The condition of pH which favour the

Effect of temperature: If the temperature of the disinfectants is increased, the effectiveness of the disinfectants also increases. Bacteriologist Robert Koch had noted that anthrex spores were more rapidly killed by the same concentration of phenol if the temperature was elevated. As the temperature is increased arithmetical progression, the rate of disinfectant increases geometrically by the first order chemical reaction. Effect of pH: Changes in pH can affect: 1. The rate of growth of innoculum: The optimal bacterial growth takes place in the range of 6-8; on either side of this the rate of growth declines. 2. The ability of the disinfectant to combine with sites on the cell surface: Change in the pH range, affects the adsorption at the cell surface. 3. The potency of the antibacterial agent itself: The degree of ionization of the drug depends on the pH. The condition of pH which favour the

DISINFECTANTS, ANTISEPTICS, AND PRESERVATIVES

105

DISINFECTANTS, ANTISEPTICS, AND PRESERVATIVES

105

formation of the ions of drugs will also reduce their activity, e.g., at 7.3, 9-amino acridine, which exists at this pH entirely as the cation, will inhibit the growth of Streptococcus pyrogens.

formation of the ions of drugs will also reduce their activity, e.g., at 7.3, 9-amino acridine, which exists at this pH entirely as the cation, will inhibit the growth of Streptococcus pyrogens.

The antibacterial activity of cationic detergent such as cetrimide and acridines increases with increase the pH. Effect of dilution: At higher concentration the drug acts as a bactericidal while on dilution the drug acts as a bacteriostatic. Effect of surface activity: The addition of low concentration of surface active compounds may potentiate the biological effect of an antibacterial agent, e.g., phenols are more active in the presence of soaps. Effect of interfering agent: Interfering agents like organic acids may directly or indirectly affect the action of disinfectants. These interfering agents reduce the activity of disinfectants by adsorbing or inactivating them thus, reducing the amount available for combining with the cell it is desired to kill.

The antibacterial activity of cationic detergent such as cetrimide and acridines increases with increase the pH. Effect of dilution: At higher concentration the drug acts as a bactericidal while on dilution the drug acts as a bacteriostatic. Effect of surface activity: The addition of low concentration of surface active compounds may potentiate the biological effect of an antibacterial agent, e.g., phenols are more active in the presence of soaps. Effect of interfering agent: Interfering agents like organic acids may directly or indirectly affect the action of disinfectants. These interfering agents reduce the activity of disinfectants by adsorbing or inactivating them thus, reducing the amount available for combining with the cell it is desired to kill.

8.1.3 Evaluation of Disinfectants There are various methods being employed to evaluate the potency of the disinfectants. The evaluation of disinfectants may be classified into:

8.1.3 Evaluation of Disinfectants There are various methods being employed to evaluate the potency of the disinfectants. The evaluation of disinfectants may be classified into:

Phenol coefficient test

Phenol coefficient test

Phenol coefficient is a measure of the bacterial activity of a chemical compound in relation to phenol. When listed numerically, the figure expressing the disinfecting power of a substance by relating it to the disinfecting power of phenol may be a function of the standardized test performed.

Phenol coefficient is a measure of the bacterial activity of a chemical compound in relation to phenol. When listed numerically, the figure expressing the disinfecting power of a substance by relating it to the disinfecting power of phenol may be a function of the standardized test performed.

The phenol coefficient may be determined in the presence of a standard amount of added organic matter or in the absence of organic matter and the formula is given by:

The phenol coefficient may be determined in the presence of a standard amount of added organic matter or in the absence of organic matter and the formula is given by:

N = 1/CT

N = 1/CT

Where, C is the conc. of agent applied; T is the time for which agent is applied and N is the number of survivors

Where, C is the conc. of agent applied; T is the time for which agent is applied and N is the number of survivors

There are two methods or tests in practice for this test: a) Rideal Walker test. b) Chick Martin test. These tests were introduced during the time when typhoid fever was endemic.

There are two methods or tests in practice for this test: a) Rideal Walker test. b) Chick Martin test. These tests were introduced during the time when typhoid fever was endemic.

Rideal Walker test

Rideal Walker test

1. This test uses a strain of Salmonella typhi that is prepared for the test in a specified way, using a standard broth.

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105

1. This test uses a strain of Salmonella typhi that is prepared for the test in a specified way, using a standard broth.

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105

formation of the ions of drugs will also reduce their activity, e.g., at 7.3, 9-amino acridine, which exists at this pH entirely as the cation, will inhibit the growth of Streptococcus pyrogens.

formation of the ions of drugs will also reduce their activity, e.g., at 7.3, 9-amino acridine, which exists at this pH entirely as the cation, will inhibit the growth of Streptococcus pyrogens.

The antibacterial activity of cationic detergent such as cetrimide and acridines increases with increase the pH. Effect of dilution: At higher concentration the drug acts as a bactericidal while on dilution the drug acts as a bacteriostatic. Effect of surface activity: The addition of low concentration of surface active compounds may potentiate the biological effect of an antibacterial agent, e.g., phenols are more active in the presence of soaps. Effect of interfering agent: Interfering agents like organic acids may directly or indirectly affect the action of disinfectants. These interfering agents reduce the activity of disinfectants by adsorbing or inactivating them thus, reducing the amount available for combining with the cell it is desired to kill.

The antibacterial activity of cationic detergent such as cetrimide and acridines increases with increase the pH. Effect of dilution: At higher concentration the drug acts as a bactericidal while on dilution the drug acts as a bacteriostatic. Effect of surface activity: The addition of low concentration of surface active compounds may potentiate the biological effect of an antibacterial agent, e.g., phenols are more active in the presence of soaps. Effect of interfering agent: Interfering agents like organic acids may directly or indirectly affect the action of disinfectants. These interfering agents reduce the activity of disinfectants by adsorbing or inactivating them thus, reducing the amount available for combining with the cell it is desired to kill.

8.1.3 Evaluation of Disinfectants There are various methods being employed to evaluate the potency of the disinfectants. The evaluation of disinfectants may be classified into:

8.1.3 Evaluation of Disinfectants There are various methods being employed to evaluate the potency of the disinfectants. The evaluation of disinfectants may be classified into:

Phenol coefficient test

Phenol coefficient test

Phenol coefficient is a measure of the bacterial activity of a chemical compound in relation to phenol. When listed numerically, the figure expressing the disinfecting power of a substance by relating it to the disinfecting power of phenol may be a function of the standardized test performed.

Phenol coefficient is a measure of the bacterial activity of a chemical compound in relation to phenol. When listed numerically, the figure expressing the disinfecting power of a substance by relating it to the disinfecting power of phenol may be a function of the standardized test performed.

The phenol coefficient may be determined in the presence of a standard amount of added organic matter or in the absence of organic matter and the formula is given by:

The phenol coefficient may be determined in the presence of a standard amount of added organic matter or in the absence of organic matter and the formula is given by:

N = 1/CT

N = 1/CT

Where, C is the conc. of agent applied; T is the time for which agent is applied and N is the number of survivors

Where, C is the conc. of agent applied; T is the time for which agent is applied and N is the number of survivors

There are two methods or tests in practice for this test: a) Rideal Walker test. b) Chick Martin test. These tests were introduced during the time when typhoid fever was endemic.

There are two methods or tests in practice for this test: a) Rideal Walker test. b) Chick Martin test. These tests were introduced during the time when typhoid fever was endemic.

Rideal Walker test

Rideal Walker test

1. This test uses a strain of Salmonella typhi that is prepared for the test in a specified way, using a standard broth.

1. This test uses a strain of Salmonella typhi that is prepared for the test in a specified way, using a standard broth.

106 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

106 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

2. Serial dilution of phenol and the disinfectants under the test are inoculated with 0.2 cm2 of the culture and standards loopfuls are removed into 5 cm2 volumes of broth 2.5, 5, 7.5 and 10 minutes later and incubated at 37°C for 48 hours. 3. The phenol coefficient if obtained by dividing the lowest concentration of disinfectants showing growth after 5 minutes but not after 7.5 minutes by the lowest concentration of phenol giving the same result. For example:

2. Serial dilution of phenol and the disinfectants under the test are inoculated with 0.2 cm2 of the culture and standards loopfuls are removed into 5 cm2 volumes of broth 2.5, 5, 7.5 and 10 minutes later and incubated at 37°C for 48 hours. 3. The phenol coefficient if obtained by dividing the lowest concentration of disinfectants showing growth after 5 minutes but not after 7.5 minutes by the lowest concentration of phenol giving the same result. For example:

The phenol coefficient is: 400/80 = 5.

The phenol coefficient is: 400/80 = 5.

Chick Martin test

Chick Martin test

This type of test is done in controlled amount of organic matter in the form of standardized suspensions of yeast cells. In this test all the conditions like Rideal Walker test but the temperature is 20°C and the exposure time is 30 minutes. The Chick Martin coefficient is the mean of the highest concentration of phenol showing growth and lowest concentration preventing growth, divided by the same mean for the disinfectants under test. For example:

This type of test is done in controlled amount of organic matter in the form of standardized suspensions of yeast cells. In this test all the conditions like Rideal Walker test but the temperature is 20°C and the exposure time is 30 minutes. The Chick Martin coefficient is the mean of the highest concentration of phenol showing growth and lowest concentration preventing growth, divided by the same mean for the disinfectants under test. For example:

Phenol

Phenol

% 2.0 1.80 1.62 1.45

Tube 1 + +

Test disinfectants 2 +

% 0.47 0.41 0.37 0.33

Tubes 1 + +

2 + +

% 2.0 1.80 1.62 1.45

The phenol coefficient is

Tube 1 + +

Test disinfectants 2 +

% 0.47 0.41 0.37 0.33

Tubes 1 + +

2 + +

The phenol coefficient is

Kelsey-Sykes test

Kelsey-Sykes test

a. This test measures the capacity of disinfectants to retain its capacity when repeatedly used microbiologically. b. The standard organisms Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are added to the disinfectant in three successive lots at 0, 10, and 20 minutes.

106 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

a. This test measures the capacity of disinfectants to retain its capacity when repeatedly used microbiologically. b. The standard organisms Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are added to the disinfectant in three successive lots at 0, 10, and 20 minutes.

106 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

2. Serial dilution of phenol and the disinfectants under the test are inoculated with 0.2 cm2 of the culture and standards loopfuls are removed into 5 cm2 volumes of broth 2.5, 5, 7.5 and 10 minutes later and incubated at 37°C for 48 hours. 3. The phenol coefficient if obtained by dividing the lowest concentration of disinfectants showing growth after 5 minutes but not after 7.5 minutes by the lowest concentration of phenol giving the same result. For example:

2. Serial dilution of phenol and the disinfectants under the test are inoculated with 0.2 cm2 of the culture and standards loopfuls are removed into 5 cm2 volumes of broth 2.5, 5, 7.5 and 10 minutes later and incubated at 37°C for 48 hours. 3. The phenol coefficient if obtained by dividing the lowest concentration of disinfectants showing growth after 5 minutes but not after 7.5 minutes by the lowest concentration of phenol giving the same result. For example:

The phenol coefficient is: 400/80 = 5.

The phenol coefficient is: 400/80 = 5.

Chick Martin test

Chick Martin test

This type of test is done in controlled amount of organic matter in the form of standardized suspensions of yeast cells. In this test all the conditions like Rideal Walker test but the temperature is 20°C and the exposure time is 30 minutes. The Chick Martin coefficient is the mean of the highest concentration of phenol showing growth and lowest concentration preventing growth, divided by the same mean for the disinfectants under test. For example:

This type of test is done in controlled amount of organic matter in the form of standardized suspensions of yeast cells. In this test all the conditions like Rideal Walker test but the temperature is 20°C and the exposure time is 30 minutes. The Chick Martin coefficient is the mean of the highest concentration of phenol showing growth and lowest concentration preventing growth, divided by the same mean for the disinfectants under test. For example:

Phenol

Phenol

% 2.0 1.80 1.62 1.45

Tube 1 + +

Test disinfectants 2 +

% 0.47 0.41 0.37 0.33

Tubes 1 + +

2 + +

The phenol coefficient is

Kelsey-Sykes test a. This test measures the capacity of disinfectants to retain its capacity when repeatedly used microbiologically. b. The standard organisms Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are added to the disinfectant in three successive lots at 0, 10, and 20 minutes.

% 2.0 1.80 1.62 1.45

Tube 1 + +

Test disinfectants 2 +

% 0.47 0.41 0.37 0.33

Tubes 1 + +

2 + +

The phenol coefficient is

Kelsey-Sykes test a. This test measures the capacity of disinfectants to retain its capacity when repeatedly used microbiologically. b. The standard organisms Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are added to the disinfectant in three successive lots at 0, 10, and 20 minutes.

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c. Each increment is in contact with disinfectants for eight minutes and the sample is transferred at 8, 18 and 28 minutes to a recovery medium. The test is carried out under both clean and dirty condition.

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c. Each increment is in contact with disinfectants for eight minutes and the sample is transferred at 8, 18 and 28 minutes to a recovery medium. The test is carried out under both clean and dirty condition.

Filter paper test

Filter paper test

In this method small filter paper discs soaked with a chemical agent are used. The discs are placed on the surface of an agar plate that has been inoculated with a test organism. A new plate is used for each organism. The chemical agent which inhibits the growth of microorganisms shows the clear zone or zone of inhibition area around the disc where the bacteria have been killed.

In this method small filter paper discs soaked with a chemical agent are used. The discs are placed on the surface of an agar plate that has been inoculated with a test organism. A new plate is used for each organism. The chemical agent which inhibits the growth of microorganisms shows the clear zone or zone of inhibition area around the disc where the bacteria have been killed.

Capacity use-dilution test

Capacity use-dilution test

It uses standard preparation of certain test bacteria. a. These bacteria are added in the test tube containing different dilutions of chemical agent. b. The tubes are incubated and are observed for the presence or absence of growth. c. Chemical agents that prevent growth at the greatest dilutions are considered most effective.

It uses standard preparation of certain test bacteria. a. These bacteria are added in the test tube containing different dilutions of chemical agent. b. The tubes are incubated and are observed for the presence or absence of growth. c. Chemical agents that prevent growth at the greatest dilutions are considered most effective.

8.1.4 Determination of Zone of Inhibition

8.1.4 Determination of Zone of Inhibition

Fig. 8.1. Clear zone showing the no growth of bacteria.

Fig. 8.1. Clear zone showing the no growth of bacteria.

8.1.5 Determination of Minimum Inhibitory Concentration (MIC) Sometimes MIC called bacteriostatic values. The MIC of an antibacterial agent or a disinfectant, for a particular organism, is the lowest concentration that just prevents growth of that organism. There are two methods for describing the MIC. a. Liquid dilution methods: The different concentrations of the inhibitor (antibiotic or disinfectant) are prepared in broth and accurate volumes of

8.1.5 Determination of Minimum Inhibitory Concentration (MIC) Sometimes MIC called bacteriostatic values. The MIC of an antibacterial agent or a disinfectant, for a particular organism, is the lowest concentration that just prevents growth of that organism. There are two methods for describing the MIC. a. Liquid dilution methods: The different concentrations of the inhibitor (antibiotic or disinfectant) are prepared in broth and accurate volumes of

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c. Each increment is in contact with disinfectants for eight minutes and the sample is transferred at 8, 18 and 28 minutes to a recovery medium. The test is carried out under both clean and dirty condition.

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c. Each increment is in contact with disinfectants for eight minutes and the sample is transferred at 8, 18 and 28 minutes to a recovery medium. The test is carried out under both clean and dirty condition.

Filter paper test

Filter paper test

In this method small filter paper discs soaked with a chemical agent are used. The discs are placed on the surface of an agar plate that has been inoculated with a test organism. A new plate is used for each organism. The chemical agent which inhibits the growth of microorganisms shows the clear zone or zone of inhibition area around the disc where the bacteria have been killed.

In this method small filter paper discs soaked with a chemical agent are used. The discs are placed on the surface of an agar plate that has been inoculated with a test organism. A new plate is used for each organism. The chemical agent which inhibits the growth of microorganisms shows the clear zone or zone of inhibition area around the disc where the bacteria have been killed.

Capacity use-dilution test

Capacity use-dilution test

It uses standard preparation of certain test bacteria. a. These bacteria are added in the test tube containing different dilutions of chemical agent. b. The tubes are incubated and are observed for the presence or absence of growth. c. Chemical agents that prevent growth at the greatest dilutions are considered most effective.

It uses standard preparation of certain test bacteria. a. These bacteria are added in the test tube containing different dilutions of chemical agent. b. The tubes are incubated and are observed for the presence or absence of growth. c. Chemical agents that prevent growth at the greatest dilutions are considered most effective.

8.1.4 Determination of Zone of Inhibition

8.1.4 Determination of Zone of Inhibition

Fig. 8.1. Clear zone showing the no growth of bacteria.

Fig. 8.1. Clear zone showing the no growth of bacteria.

8.1.5 Determination of Minimum Inhibitory Concentration (MIC) Sometimes MIC called bacteriostatic values. The MIC of an antibacterial agent or a disinfectant, for a particular organism, is the lowest concentration that just prevents growth of that organism. There are two methods for describing the MIC. a. Liquid dilution methods: The different concentrations of the inhibitor (antibiotic or disinfectant) are prepared in broth and accurate volumes of

8.1.5 Determination of Minimum Inhibitory Concentration (MIC) Sometimes MIC called bacteriostatic values. The MIC of an antibacterial agent or a disinfectant, for a particular organism, is the lowest concentration that just prevents growth of that organism. There are two methods for describing the MIC. a. Liquid dilution methods: The different concentrations of the inhibitor (antibiotic or disinfectant) are prepared in broth and accurate volumes of

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Fig. 8.2. Shows the growth of microorganism in various concentration of inhibitor. Blank contains no growth because no microorganisms. At 2 and 3 milli molar no growth of microorganisms takes place because higher concentration of inhibitors. So 2 milli molar is lowest concentration of inhibitor at which no bacterial growth takes place.

a suspension containing microorganism is added to each test tube and incubated for 1-2 days in shaker incubator at 37°C, and examined for growth. The MIC lies between the lowest concentration inhibiting growth and the highest concentration allowing growth. b. Solid dilution method: Prepare the nutrient agar media as described by Cook (1954). Antibiotics or disinfectants are mixed in the nutrient agar media, after autoclaving when the temperature is near about 40°C, poured into Petri dishes.

8.2 ANTISEPTICS 1. Antisepsis is defined as destruction or inhibition of microorganisms on living tissues having the effect of limiting or preventing the harmful results of infection. 2. The chemicals used are applied to skin and mucous membranes, therefore as well as having adequate antimicrobial activity, they must not be toxic or irritating for skin. 3. Antiseptics are mostly used to reduce the microbial population on the skin prior to surgery or on the hands to help prevent spread of infection by this route. 4. Antiseptics are often lower concentrations of the agents used for disinfection.

8.3 PRESERVATIVES 1. These are included in pharmaceutical preparations to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring infection when the preparation is administered.

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Fig. 8.2. Shows the growth of microorganism in various concentration of inhibitor. Blank contains no growth because no microorganisms. At 2 and 3 milli molar no growth of microorganisms takes place because higher concentration of inhibitors. So 2 milli molar is lowest concentration of inhibitor at which no bacterial growth takes place.

a suspension containing microorganism is added to each test tube and incubated for 1-2 days in shaker incubator at 37°C, and examined for growth. The MIC lies between the lowest concentration inhibiting growth and the highest concentration allowing growth. b. Solid dilution method: Prepare the nutrient agar media as described by Cook (1954). Antibiotics or disinfectants are mixed in the nutrient agar media, after autoclaving when the temperature is near about 40°C, poured into Petri dishes.

8.2 ANTISEPTICS 1. Antisepsis is defined as destruction or inhibition of microorganisms on living tissues having the effect of limiting or preventing the harmful results of infection. 2. The chemicals used are applied to skin and mucous membranes, therefore as well as having adequate antimicrobial activity, they must not be toxic or irritating for skin. 3. Antiseptics are mostly used to reduce the microbial population on the skin prior to surgery or on the hands to help prevent spread of infection by this route. 4. Antiseptics are often lower concentrations of the agents used for disinfection.

8.3 PRESERVATIVES 1. These are included in pharmaceutical preparations to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring infection when the preparation is administered.

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Fig. 8.2. Shows the growth of microorganism in various concentration of inhibitor. Blank contains no growth because no microorganisms. At 2 and 3 milli molar no growth of microorganisms takes place because higher concentration of inhibitors. So 2 milli molar is lowest concentration of inhibitor at which no bacterial growth takes place.

a suspension containing microorganism is added to each test tube and incubated for 1-2 days in shaker incubator at 37°C, and examined for growth. The MIC lies between the lowest concentration inhibiting growth and the highest concentration allowing growth. b. Solid dilution method: Prepare the nutrient agar media as described by Cook (1954). Antibiotics or disinfectants are mixed in the nutrient agar media, after autoclaving when the temperature is near about 40°C, poured into Petri dishes.

8.2 ANTISEPTICS 1. Antisepsis is defined as destruction or inhibition of microorganisms on living tissues having the effect of limiting or preventing the harmful results of infection. 2. The chemicals used are applied to skin and mucous membranes, therefore as well as having adequate antimicrobial activity, they must not be toxic or irritating for skin. 3. Antiseptics are mostly used to reduce the microbial population on the skin prior to surgery or on the hands to help prevent spread of infection by this route. 4. Antiseptics are often lower concentrations of the agents used for disinfection.

8.3 PRESERVATIVES 1. These are included in pharmaceutical preparations to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring infection when the preparation is administered.

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Fig. 8.2. Shows the growth of microorganism in various concentration of inhibitor. Blank contains no growth because no microorganisms. At 2 and 3 milli molar no growth of microorganisms takes place because higher concentration of inhibitors. So 2 milli molar is lowest concentration of inhibitor at which no bacterial growth takes place.

a suspension containing microorganism is added to each test tube and incubated for 1-2 days in shaker incubator at 37°C, and examined for growth. The MIC lies between the lowest concentration inhibiting growth and the highest concentration allowing growth. b. Solid dilution method: Prepare the nutrient agar media as described by Cook (1954). Antibiotics or disinfectants are mixed in the nutrient agar media, after autoclaving when the temperature is near about 40°C, poured into Petri dishes.

8.2 ANTISEPTICS 1. Antisepsis is defined as destruction or inhibition of microorganisms on living tissues having the effect of limiting or preventing the harmful results of infection. 2. The chemicals used are applied to skin and mucous membranes, therefore as well as having adequate antimicrobial activity, they must not be toxic or irritating for skin. 3. Antiseptics are mostly used to reduce the microbial population on the skin prior to surgery or on the hands to help prevent spread of infection by this route. 4. Antiseptics are often lower concentrations of the agents used for disinfection.

8.3 PRESERVATIVES 1. These are included in pharmaceutical preparations to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring infection when the preparation is administered.

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2. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably during manufacture and use of non-sterile products such as oral and topical medications. In sterile products such as eye drops and multidose injections, preservatives should kill any microbial contaminants introduced inadvertently during use. 3. It is essential that a preservative is not toxic in relation to the intended route of administration of the preserved preparation. Preservatives therefore tend to be employed at low concentrations and consequently levels of antimicrobial action also tend to be of a lower order than for disinfectants or antiseptics. 4. There are around 250 chemical entities that have been identified as active components of microbiocidal products in the European Union. The aim of this chapter is to introduce the range of chemicals in common use and to indicate their activities and applications.

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2. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably during manufacture and use of non-sterile products such as oral and topical medications. In sterile products such as eye drops and multidose injections, preservatives should kill any microbial contaminants introduced inadvertently during use. 3. It is essential that a preservative is not toxic in relation to the intended route of administration of the preserved preparation. Preservatives therefore tend to be employed at low concentrations and consequently levels of antimicrobial action also tend to be of a lower order than for disinfectants or antiseptics. 4. There are around 250 chemical entities that have been identified as active components of microbiocidal products in the European Union. The aim of this chapter is to introduce the range of chemicals in common use and to indicate their activities and applications.

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2. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably during manufacture and use of non-sterile products such as oral and topical medications. In sterile products such as eye drops and multidose injections, preservatives should kill any microbial contaminants introduced inadvertently during use. 3. It is essential that a preservative is not toxic in relation to the intended route of administration of the preserved preparation. Preservatives therefore tend to be employed at low concentrations and consequently levels of antimicrobial action also tend to be of a lower order than for disinfectants or antiseptics. 4. There are around 250 chemical entities that have been identified as active components of microbiocidal products in the European Union. The aim of this chapter is to introduce the range of chemicals in common use and to indicate their activities and applications.

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2. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably during manufacture and use of non-sterile products such as oral and topical medications. In sterile products such as eye drops and multidose injections, preservatives should kill any microbial contaminants introduced inadvertently during use. 3. It is essential that a preservative is not toxic in relation to the intended route of administration of the preserved preparation. Preservatives therefore tend to be employed at low concentrations and consequently levels of antimicrobial action also tend to be of a lower order than for disinfectants or antiseptics. 4. There are around 250 chemical entities that have been identified as active components of microbiocidal products in the European Union. The aim of this chapter is to introduce the range of chemicals in common use and to indicate their activities and applications.

9

9

Sterilization

Sterilization

Sterilization is to produce complete sterile state, i.e., complete destruction or elimination of vial microorganisms (such as fungi, bacteria, viruses, spore forms, etc.) by the use of physical and chemical procedures. In recent years the pharmaceutical industry has intensified its efforts to quantitate the rate and extent of microbial destruction or elimination. The food and drug administration (FDA) has stated in its current good manufacturing practice regulations that sterilization procedures must be validated pertaining to (1) the design of the equipment and the process used to produce batch sterilization. (2) The conformation with reproducible data of a given probability level of residual microbial contamination upon completion of the sterilization process. Sterilization is an essential stage in the processing of any product destined for parenteral administration, or for contact with broken skin, mucosal surfaces or internal organs, where the threat of infection exists. In addition, sterilization of microbiological materials, soiled dressings and other contaminated items is necessary to minimize the health hazard associated with these articles. Sterilization processes involve the application of a biocidal agent or physical microbial removal process to a product or preparation with the object of killing or removing all microorganisms. These processes may involve elevated temperature, reactive gas, irradiation or filtration through a microorganism-proof filter. The success of the process depends upon a suitable choice of treatment conditions, e.g. temperature and duration of exposure. It must be remembered, however, that with all articles to be sterilized there is a potential risk of product damage, which for a pharmaceutical preparation may result in reduced therapeutic efficacy or patient acceptability. Thus, there is a need to achieve a balance between the maximum acceptable risk of failing to achieve sterility and the maximum level of product damage which is acceptable. This is best determined from knowledge of the properties of the sterilizing agent, the properties of the product to be sterilized and the nature of the likely contaminants. A suitable sterilization process may then be selected to ensure maximum microbial kill/ removal with minimum product deterioration.

Sterilization is to produce complete sterile state, i.e., complete destruction or elimination of vial microorganisms (such as fungi, bacteria, viruses, spore forms, etc.) by the use of physical and chemical procedures. In recent years the pharmaceutical industry has intensified its efforts to quantitate the rate and extent of microbial destruction or elimination. The food and drug administration (FDA) has stated in its current good manufacturing practice regulations that sterilization procedures must be validated pertaining to (1) the design of the equipment and the process used to produce batch sterilization. (2) The conformation with reproducible data of a given probability level of residual microbial contamination upon completion of the sterilization process. Sterilization is an essential stage in the processing of any product destined for parenteral administration, or for contact with broken skin, mucosal surfaces or internal organs, where the threat of infection exists. In addition, sterilization of microbiological materials, soiled dressings and other contaminated items is necessary to minimize the health hazard associated with these articles. Sterilization processes involve the application of a biocidal agent or physical microbial removal process to a product or preparation with the object of killing or removing all microorganisms. These processes may involve elevated temperature, reactive gas, irradiation or filtration through a microorganism-proof filter. The success of the process depends upon a suitable choice of treatment conditions, e.g. temperature and duration of exposure. It must be remembered, however, that with all articles to be sterilized there is a potential risk of product damage, which for a pharmaceutical preparation may result in reduced therapeutic efficacy or patient acceptability. Thus, there is a need to achieve a balance between the maximum acceptable risk of failing to achieve sterility and the maximum level of product damage which is acceptable. This is best determined from knowledge of the properties of the sterilizing agent, the properties of the product to be sterilized and the nature of the likely contaminants. A suitable sterilization process may then be selected to ensure maximum microbial kill/ removal with minimum product deterioration.

9

9

Sterilization

Sterilization

Sterilization is to produce complete sterile state, i.e., complete destruction or elimination of vial microorganisms (such as fungi, bacteria, viruses, spore forms, etc.) by the use of physical and chemical procedures. In recent years the pharmaceutical industry has intensified its efforts to quantitate the rate and extent of microbial destruction or elimination. The food and drug administration (FDA) has stated in its current good manufacturing practice regulations that sterilization procedures must be validated pertaining to (1) the design of the equipment and the process used to produce batch sterilization. (2) The conformation with reproducible data of a given probability level of residual microbial contamination upon completion of the sterilization process. Sterilization is an essential stage in the processing of any product destined for parenteral administration, or for contact with broken skin, mucosal surfaces or internal organs, where the threat of infection exists. In addition, sterilization of microbiological materials, soiled dressings and other contaminated items is necessary to minimize the health hazard associated with these articles. Sterilization processes involve the application of a biocidal agent or physical microbial removal process to a product or preparation with the object of killing or removing all microorganisms. These processes may involve elevated temperature, reactive gas, irradiation or filtration through a microorganism-proof filter. The success of the process depends upon a suitable choice of treatment conditions, e.g. temperature and duration of exposure. It must be remembered, however, that with all articles to be sterilized there is a potential risk of product damage, which for a pharmaceutical preparation may result in reduced therapeutic efficacy or patient acceptability. Thus, there is a need to achieve a balance between the maximum acceptable risk of failing to achieve sterility and the maximum level of product damage which is acceptable. This is best determined from knowledge of the properties of the sterilizing agent, the properties of the product to be sterilized and the nature of the likely contaminants. A suitable sterilization process may then be selected to ensure maximum microbial kill/ removal with minimum product deterioration.

Sterilization is to produce complete sterile state, i.e., complete destruction or elimination of vial microorganisms (such as fungi, bacteria, viruses, spore forms, etc.) by the use of physical and chemical procedures. In recent years the pharmaceutical industry has intensified its efforts to quantitate the rate and extent of microbial destruction or elimination. The food and drug administration (FDA) has stated in its current good manufacturing practice regulations that sterilization procedures must be validated pertaining to (1) the design of the equipment and the process used to produce batch sterilization. (2) The conformation with reproducible data of a given probability level of residual microbial contamination upon completion of the sterilization process. Sterilization is an essential stage in the processing of any product destined for parenteral administration, or for contact with broken skin, mucosal surfaces or internal organs, where the threat of infection exists. In addition, sterilization of microbiological materials, soiled dressings and other contaminated items is necessary to minimize the health hazard associated with these articles. Sterilization processes involve the application of a biocidal agent or physical microbial removal process to a product or preparation with the object of killing or removing all microorganisms. These processes may involve elevated temperature, reactive gas, irradiation or filtration through a microorganism-proof filter. The success of the process depends upon a suitable choice of treatment conditions, e.g. temperature and duration of exposure. It must be remembered, however, that with all articles to be sterilized there is a potential risk of product damage, which for a pharmaceutical preparation may result in reduced therapeutic efficacy or patient acceptability. Thus, there is a need to achieve a balance between the maximum acceptable risk of failing to achieve sterility and the maximum level of product damage which is acceptable. This is best determined from knowledge of the properties of the sterilizing agent, the properties of the product to be sterilized and the nature of the likely contaminants. A suitable sterilization process may then be selected to ensure maximum microbial kill/ removal with minimum product deterioration.

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9.1 MICROBIAL DEATH KINETICS

9.1 MICROBIAL DEATH KINETICS

When exposed to a killing process, populations of microorganisms generally lose their viability in an exponential fashion, independent of the initial number of organisms. This can be represented graphically with a ‘survivor curve’ drawn from a plot of the logarithm of the fraction of survivors against the exposure time or dose (Fig. 9.1). Of the typical curves obtained, all have a linear portion which may be continuous (plot A), or may be modified by an initial shoulder (B) or by a reduced rate of kill at low survivor levels (C). Furthermore, a short activation phase, representing an initial increase in viable count, may be seen during the heat treatment of certain bacterial spores. Survivor curves have been employed principally in the examination of heat sterilization methods, but can equally be applied to any biocidal process.

When exposed to a killing process, populations of microorganisms generally lose their viability in an exponential fashion, independent of the initial number of organisms. This can be represented graphically with a ‘survivor curve’ drawn from a plot of the logarithm of the fraction of survivors against the exposure time or dose (Fig. 9.1). Of the typical curves obtained, all have a linear portion which may be continuous (plot A), or may be modified by an initial shoulder (B) or by a reduced rate of kill at low survivor levels (C). Furthermore, a short activation phase, representing an initial increase in viable count, may be seen during the heat treatment of certain bacterial spores. Survivor curves have been employed principally in the examination of heat sterilization methods, but can equally be applied to any biocidal process.

Fig. 9.1. Typical survivor curves for bacterial spores exposed to moist heat or gammaradiation.

Fig. 9.1. Typical survivor curves for bacterial spores exposed to moist heat or gammaradiation.

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9.1 MICROBIAL DEATH KINETICS

9.1 MICROBIAL DEATH KINETICS

When exposed to a killing process, populations of microorganisms generally lose their viability in an exponential fashion, independent of the initial number of organisms. This can be represented graphically with a ‘survivor curve’ drawn from a plot of the logarithm of the fraction of survivors against the exposure time or dose (Fig. 9.1). Of the typical curves obtained, all have a linear portion which may be continuous (plot A), or may be modified by an initial shoulder (B) or by a reduced rate of kill at low survivor levels (C). Furthermore, a short activation phase, representing an initial increase in viable count, may be seen during the heat treatment of certain bacterial spores. Survivor curves have been employed principally in the examination of heat sterilization methods, but can equally be applied to any biocidal process.

When exposed to a killing process, populations of microorganisms generally lose their viability in an exponential fashion, independent of the initial number of organisms. This can be represented graphically with a ‘survivor curve’ drawn from a plot of the logarithm of the fraction of survivors against the exposure time or dose (Fig. 9.1). Of the typical curves obtained, all have a linear portion which may be continuous (plot A), or may be modified by an initial shoulder (B) or by a reduced rate of kill at low survivor levels (C). Furthermore, a short activation phase, representing an initial increase in viable count, may be seen during the heat treatment of certain bacterial spores. Survivor curves have been employed principally in the examination of heat sterilization methods, but can equally be applied to any biocidal process.

Fig. 9.1. Typical survivor curves for bacterial spores exposed to moist heat or gammaradiation.

Fig. 9.1. Typical survivor curves for bacterial spores exposed to moist heat or gammaradiation.

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9.2 EXPRESSIONS OF RESISTANCE

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9.2 EXPRESSIONS OF RESISTANCE

Fig. 9.2. Calculation of: (A) D-value; (B) Z-value.

Fig. 9.2. Calculation of: (A) D-value; (B) Z-value.

9.2.1 D-value The D value is the time required to destruct or eliminate more than 90% of the microorganisms (i.e., a 1 log cycle reduction in survivors; Fig. 9.2A). The calculation of the D-value assumes a linear type A survivor curve (Fig. 9.1), and must be corrected to allow for any deviation from linearity with type B or C curves.

9.2.1 D-value The D value is the time required to destruct or eliminate more than 90% of the microorganisms (i.e., a 1 log cycle reduction in survivors; Fig. 9.2A). The calculation of the D-value assumes a linear type A survivor curve (Fig. 9.1), and must be corrected to allow for any deviation from linearity with type B or C curves.

9.2.2 Z-value The number of degrees (°C or F) required for a 1 log reduction in D value (Fig. 9.2B) is called the z-value. For bacterial spores used as biological indicators for moist heat (B. stearothermophilus) and dry heat (B. subtilis) sterilization processes, mean z-values are given as 10°C and 22°C, respectively. The zvalue is not truly independent of temperature but may be considered essentially constant over the temperature ranges used in heat sterilization processes.

9.2.2 Z-value The number of degrees (°C or F) required for a 1 log reduction in D value (Fig. 9.2B) is called the z-value. For bacterial spores used as biological indicators for moist heat (B. stearothermophilus) and dry heat (B. subtilis) sterilization processes, mean z-values are given as 10°C and 22°C, respectively. The zvalue is not truly independent of temperature but may be considered essentially constant over the temperature ranges used in heat sterilization processes.

9.3 STERILITY ASSURANCE

9.3 STERILITY ASSURANCE

The term ‘sterile’, in a microbiological context, means no surviving organisms whatsoever. Thus, there are no degrees of sterility; an item is either sterile or it is not, and so there are no levels of contamination which may be considered negligible or insignificant and therefore acceptable. From the survivor curves presented, it can be seen that the elimination of viable microorganisms from a product is a time-dependent process, and will be influenced by the rate and duration of biocidal action and the initial microbial contamination level. It is also evident from Fig. 9.2A that true sterility, represented by zero survivors, can only be achieved after an infinite exposure

The term ‘sterile’, in a microbiological context, means no surviving organisms whatsoever. Thus, there are no degrees of sterility; an item is either sterile or it is not, and so there are no levels of contamination which may be considered negligible or insignificant and therefore acceptable. From the survivor curves presented, it can be seen that the elimination of viable microorganisms from a product is a time-dependent process, and will be influenced by the rate and duration of biocidal action and the initial microbial contamination level. It is also evident from Fig. 9.2A that true sterility, represented by zero survivors, can only be achieved after an infinite exposure

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9.2 EXPRESSIONS OF RESISTANCE

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9.2 EXPRESSIONS OF RESISTANCE

Fig. 9.2. Calculation of: (A) D-value; (B) Z-value.

Fig. 9.2. Calculation of: (A) D-value; (B) Z-value.

9.2.1 D-value The D value is the time required to destruct or eliminate more than 90% of the microorganisms (i.e., a 1 log cycle reduction in survivors; Fig. 9.2A). The calculation of the D-value assumes a linear type A survivor curve (Fig. 9.1), and must be corrected to allow for any deviation from linearity with type B or C curves.

9.2.1 D-value The D value is the time required to destruct or eliminate more than 90% of the microorganisms (i.e., a 1 log cycle reduction in survivors; Fig. 9.2A). The calculation of the D-value assumes a linear type A survivor curve (Fig. 9.1), and must be corrected to allow for any deviation from linearity with type B or C curves.

9.2.2 Z-value The number of degrees (°C or F) required for a 1 log reduction in D value (Fig. 9.2B) is called the z-value. For bacterial spores used as biological indicators for moist heat (B. stearothermophilus) and dry heat (B. subtilis) sterilization processes, mean z-values are given as 10°C and 22°C, respectively. The zvalue is not truly independent of temperature but may be considered essentially constant over the temperature ranges used in heat sterilization processes.

9.2.2 Z-value The number of degrees (°C or F) required for a 1 log reduction in D value (Fig. 9.2B) is called the z-value. For bacterial spores used as biological indicators for moist heat (B. stearothermophilus) and dry heat (B. subtilis) sterilization processes, mean z-values are given as 10°C and 22°C, respectively. The zvalue is not truly independent of temperature but may be considered essentially constant over the temperature ranges used in heat sterilization processes.

9.3 STERILITY ASSURANCE

9.3 STERILITY ASSURANCE

The term ‘sterile’, in a microbiological context, means no surviving organisms whatsoever. Thus, there are no degrees of sterility; an item is either sterile or it is not, and so there are no levels of contamination which may be considered negligible or insignificant and therefore acceptable. From the survivor curves presented, it can be seen that the elimination of viable microorganisms from a product is a time-dependent process, and will be influenced by the rate and duration of biocidal action and the initial microbial contamination level. It is also evident from Fig. 9.2A that true sterility, represented by zero survivors, can only be achieved after an infinite exposure

The term ‘sterile’, in a microbiological context, means no surviving organisms whatsoever. Thus, there are no degrees of sterility; an item is either sterile or it is not, and so there are no levels of contamination which may be considered negligible or insignificant and therefore acceptable. From the survivor curves presented, it can be seen that the elimination of viable microorganisms from a product is a time-dependent process, and will be influenced by the rate and duration of biocidal action and the initial microbial contamination level. It is also evident from Fig. 9.2A that true sterility, represented by zero survivors, can only be achieved after an infinite exposure

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period or radiation dose. Clearly, then, it is illogical to claim, or expect, that a sterilization procedure will guarantee sterility. Thus, the likelihood of a product being produced free of microorganisms is best expressed in terms of the probability of an organism surviving the treatment process, a possibility not entertained in the absolute term ‘sterile’. From this approach has arisen the concept of sterility assurance or a microbial safety index which gives a numerical value to the probability of a single surviving organism remaining to contaminate a processed product. For pharmaceutical products, the most frequently applied standard is that the probability, post-sterilization, of a non-sterile unit is 1 in 1 million units processed (i.e. = sl0-6). The sterilization protocol necessary to achieve this with any given organism of known D-value can be established from the inactivation factor (IF) which may be defined as: IF = 10*°

period or radiation dose. Clearly, then, it is illogical to claim, or expect, that a sterilization procedure will guarantee sterility. Thus, the likelihood of a product being produced free of microorganisms is best expressed in terms of the probability of an organism surviving the treatment process, a possibility not entertained in the absolute term ‘sterile’. From this approach has arisen the concept of sterility assurance or a microbial safety index which gives a numerical value to the probability of a single surviving organism remaining to contaminate a processed product. For pharmaceutical products, the most frequently applied standard is that the probability, post-sterilization, of a non-sterile unit is 1 in 1 million units processed (i.e. = sl0-6). The sterilization protocol necessary to achieve this with any given organism of known D-value can be established from the inactivation factor (IF) which may be defined as: IF = 10*°

Fig. 9.3. Sterility assurance. At Y, there is (literally) 10"1 bacterium in one bottle, i.e. in 10 loads of single containers, there would be one chance in 10 that one load would be positive. Likewise, at Z, there is (literally) 10-6 bacterium in one bottle, i.e. in 1 million (106) loads of single containers, there is one chance in 1 million that one load would be positive.

Fig. 9.3. Sterility assurance. At Y, there is (literally) 10"1 bacterium in one bottle, i.e. in 10 loads of single containers, there would be one chance in 10 that one load would be positive. Likewise, at Z, there is (literally) 10-6 bacterium in one bottle, i.e. in 1 million (106) loads of single containers, there is one chance in 1 million that one load would be positive.

Where t is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing radiation) and D is the D-value appropriate to the process employed. Thus, for an initial burden of 102 spores an inactivation factor of 108 will be needed to give the required sterility assurance of 10-6 (Fig. 9.3). The sterilization process will therefore need to produce sufficient lethality to achieve an 8 log cycle reduction in viable organisms; this will require exposure of the product to eight times the D-value of the reference organism (8D). In

Where t is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing radiation) and D is the D-value appropriate to the process employed. Thus, for an initial burden of 102 spores an inactivation factor of 108 will be needed to give the required sterility assurance of 10-6 (Fig. 9.3). The sterilization process will therefore need to produce sufficient lethality to achieve an 8 log cycle reduction in viable organisms; this will require exposure of the product to eight times the D-value of the reference organism (8D). In

114 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

114 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

period or radiation dose. Clearly, then, it is illogical to claim, or expect, that a sterilization procedure will guarantee sterility. Thus, the likelihood of a product being produced free of microorganisms is best expressed in terms of the probability of an organism surviving the treatment process, a possibility not entertained in the absolute term ‘sterile’. From this approach has arisen the concept of sterility assurance or a microbial safety index which gives a numerical value to the probability of a single surviving organism remaining to contaminate a processed product. For pharmaceutical products, the most frequently applied standard is that the probability, post-sterilization, of a non-sterile unit is 1 in 1 million units processed (i.e. = sl0-6). The sterilization protocol necessary to achieve this with any given organism of known D-value can be established from the inactivation factor (IF) which may be defined as: IF = 10*°

period or radiation dose. Clearly, then, it is illogical to claim, or expect, that a sterilization procedure will guarantee sterility. Thus, the likelihood of a product being produced free of microorganisms is best expressed in terms of the probability of an organism surviving the treatment process, a possibility not entertained in the absolute term ‘sterile’. From this approach has arisen the concept of sterility assurance or a microbial safety index which gives a numerical value to the probability of a single surviving organism remaining to contaminate a processed product. For pharmaceutical products, the most frequently applied standard is that the probability, post-sterilization, of a non-sterile unit is 1 in 1 million units processed (i.e. = sl0-6). The sterilization protocol necessary to achieve this with any given organism of known D-value can be established from the inactivation factor (IF) which may be defined as: IF = 10*°

Fig. 9.3. Sterility assurance. At Y, there is (literally) 10"1 bacterium in one bottle, i.e. in 10 loads of single containers, there would be one chance in 10 that one load would be positive. Likewise, at Z, there is (literally) 10-6 bacterium in one bottle, i.e. in 1 million (106) loads of single containers, there is one chance in 1 million that one load would be positive.

Fig. 9.3. Sterility assurance. At Y, there is (literally) 10"1 bacterium in one bottle, i.e. in 10 loads of single containers, there would be one chance in 10 that one load would be positive. Likewise, at Z, there is (literally) 10-6 bacterium in one bottle, i.e. in 1 million (106) loads of single containers, there is one chance in 1 million that one load would be positive.

Where t is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing radiation) and D is the D-value appropriate to the process employed. Thus, for an initial burden of 102 spores an inactivation factor of 108 will be needed to give the required sterility assurance of 10-6 (Fig. 9.3). The sterilization process will therefore need to produce sufficient lethality to achieve an 8 log cycle reduction in viable organisms; this will require exposure of the product to eight times the D-value of the reference organism (8D). In

Where t is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing radiation) and D is the D-value appropriate to the process employed. Thus, for an initial burden of 102 spores an inactivation factor of 108 will be needed to give the required sterility assurance of 10-6 (Fig. 9.3). The sterilization process will therefore need to produce sufficient lethality to achieve an 8 log cycle reduction in viable organisms; this will require exposure of the product to eight times the D-value of the reference organism (8D). In

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practice, it is generally assumed that the contaminant will have the same resistance as the test spores unless full microbiological data are available to indicate otherwise.

practice, it is generally assumed that the contaminant will have the same resistance as the test spores unless full microbiological data are available to indicate otherwise.

9.4 STERILIZATION METHODS

9.4 STERILIZATION METHODS

The mechanisms by which microorganisms are killed by heat is thought to be the coagulation of the protein of the living cell.

The mechanisms by which microorganisms are killed by heat is thought to be the coagulation of the protein of the living cell.

9.4.1 Physical Agents

9.4.1 Physical Agents

I. Sterilization by heat

I. Sterilization by heat

a. Dry heat (e.g., Hot air oven):

a. Dry heat (e.g., Hot air oven):

Temperature

Time

Temperature

Time

140°C

3 hrs

140°C

3 hrs

180°C

2 hrs

180°C

2 hrs

260°C

45 minutes

260°C

45 minutes

Normally, it can be expected that at this time and temperature all the spores and vegetative forms of microorganisms are killed. But adverse effect on many substances also takes place like cellulose materials—paper and cloth. At this temperature many chemicals are decomposed, rubber is rapidly oxidized and thermoplastic materials melt.

STERILIZATION

Normally, it can be expected that at this time and temperature all the spores and vegetative forms of microorganisms are killed. But adverse effect on many substances also takes place like cellulose materials—paper and cloth. At this temperature many chemicals are decomposed, rubber is rapidly oxidized and thermoplastic materials melt.

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practice, it is generally assumed that the contaminant will have the same resistance as the test spores unless full microbiological data are available to indicate otherwise.

practice, it is generally assumed that the contaminant will have the same resistance as the test spores unless full microbiological data are available to indicate otherwise.

9.4 STERILIZATION METHODS

9.4 STERILIZATION METHODS

The mechanisms by which microorganisms are killed by heat is thought to be the coagulation of the protein of the living cell.

The mechanisms by which microorganisms are killed by heat is thought to be the coagulation of the protein of the living cell.

9.4.1 Physical Agents

9.4.1 Physical Agents

I. Sterilization by heat

I. Sterilization by heat

a. Dry heat (e.g., Hot air oven):

a. Dry heat (e.g., Hot air oven):

Temperature

Time

Temperature

Time

140°C

3 hrs

140°C

3 hrs

180°C

2 hrs

180°C

2 hrs

260°C

45 minutes

260°C

45 minutes

Normally, it can be expected that at this time and temperature all the spores and vegetative forms of microorganisms are killed. But adverse effect on many substances also takes place like cellulose materials—paper and cloth. At this temperature many chemicals are decomposed, rubber is rapidly oxidized and thermoplastic materials melt.

Normally, it can be expected that at this time and temperature all the spores and vegetative forms of microorganisms are killed. But adverse effect on many substances also takes place like cellulose materials—paper and cloth. At this temperature many chemicals are decomposed, rubber is rapidly oxidized and thermoplastic materials melt.

116 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

116 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Therefore this method of sterilization is reserved largely for glassware, metalware, and anhydrous oils.

Therefore this method of sterilization is reserved largely for glassware, metalware, and anhydrous oils.

Applications

Applications

1. Glassware: Most of the glassware is regularly sterilized by dry heat. It includes flasks, beakers, tubes, containers, pipettes, Petri dishes and all glass syringes. 2. Other equipment such as mortar and pestles, evaporating basins, and metalware like scissors, scalpels, ointments tubes, beakers and dishes of stainless steel. 3. Oils and similar anhydrous materials. e.g., Ethyl oleate and other esters. Ingredient of ointment bases. e.g., liquid, soft and hard paraffin wool fat, wool alcohols, bee wax and medical lubricants e.g., glycerol. 4. Powders: Starch 100°C for 1hr, sulphonamides and lactose.

1. Glassware: Most of the glassware is regularly sterilized by dry heat. It includes flasks, beakers, tubes, containers, pipettes, Petri dishes and all glass syringes. 2. Other equipment such as mortar and pestles, evaporating basins, and metalware like scissors, scalpels, ointments tubes, beakers and dishes of stainless steel. 3. Oils and similar anhydrous materials. e.g., Ethyl oleate and other esters. Ingredient of ointment bases. e.g., liquid, soft and hard paraffin wool fat, wool alcohols, bee wax and medical lubricants e.g., glycerol. 4. Powders: Starch 100°C for 1hr, sulphonamides and lactose.

b. Moist heat sterilization Moist heat is believed to destroy microorganisms by causing protein coagulation or denaturation. It can kill microorganisms at lower temperature in shorter times than dry heat. Moist heat has been recognized as an efficient biocidal agent from the early days of bacteriology, when it was principally developed for the sterilization of culture media. It now finds widespread application in the processing of many thermostable products and devices. In the pharmaceutical and medical sphere it is used in the sterilization of dressings, sheets, surgical and diagnostic equipment, containers and closures, and aqueous injections, ophthalmic preparations and irrigation fluids. Sterilization by moist heat usually involves the use of steam at temperatures in the range 121-134°C, and while alternative strategies are available for processing products unstable at these high temperatures, they rarely offer the same degree of sterility assurance and should be avoided if at all possible. The elevated temperatures generally associated with moist heat sterilization methods can only be achieved by generating steam under pressure. By far the most commonly employed standard temperature/time cycles for bottled fluids and porous loads (e.g. surgical dressings) are 121°C for 15 minutes and 134°C. For the moist heat sterilization autoclave is mainly used. Autoclave is an instrument in which steam is generated at very high temperature and pressure. At this higher temperature (121°C or more) and pressure (15 lb/inch2) for 15 to 20 minutes the protein of the microorganisms denature and cell wall damage also takes place which cause lyses or death of microorganisms. As with dry heat sterilization, it is not possible to kill the spores of certain saprophytes but with the help of moist heat sterilization all the pathogens are destroyed.

b. Moist heat sterilization Moist heat is believed to destroy microorganisms by causing protein coagulation or denaturation. It can kill microorganisms at lower temperature in shorter times than dry heat. Moist heat has been recognized as an efficient biocidal agent from the early days of bacteriology, when it was principally developed for the sterilization of culture media. It now finds widespread application in the processing of many thermostable products and devices. In the pharmaceutical and medical sphere it is used in the sterilization of dressings, sheets, surgical and diagnostic equipment, containers and closures, and aqueous injections, ophthalmic preparations and irrigation fluids. Sterilization by moist heat usually involves the use of steam at temperatures in the range 121-134°C, and while alternative strategies are available for processing products unstable at these high temperatures, they rarely offer the same degree of sterility assurance and should be avoided if at all possible. The elevated temperatures generally associated with moist heat sterilization methods can only be achieved by generating steam under pressure. By far the most commonly employed standard temperature/time cycles for bottled fluids and porous loads (e.g. surgical dressings) are 121°C for 15 minutes and 134°C. For the moist heat sterilization autoclave is mainly used. Autoclave is an instrument in which steam is generated at very high temperature and pressure. At this higher temperature (121°C or more) and pressure (15 lb/inch2) for 15 to 20 minutes the protein of the microorganisms denature and cell wall damage also takes place which cause lyses or death of microorganisms. As with dry heat sterilization, it is not possible to kill the spores of certain saprophytes but with the help of moist heat sterilization all the pathogens are destroyed.

116 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

116 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Therefore this method of sterilization is reserved largely for glassware, metalware, and anhydrous oils.

Therefore this method of sterilization is reserved largely for glassware, metalware, and anhydrous oils.

Applications

Applications

1. Glassware: Most of the glassware is regularly sterilized by dry heat. It includes flasks, beakers, tubes, containers, pipettes, Petri dishes and all glass syringes. 2. Other equipment such as mortar and pestles, evaporating basins, and metalware like scissors, scalpels, ointments tubes, beakers and dishes of stainless steel. 3. Oils and similar anhydrous materials. e.g., Ethyl oleate and other esters. Ingredient of ointment bases. e.g., liquid, soft and hard paraffin wool fat, wool alcohols, bee wax and medical lubricants e.g., glycerol. 4. Powders: Starch 100°C for 1hr, sulphonamides and lactose.

1. Glassware: Most of the glassware is regularly sterilized by dry heat. It includes flasks, beakers, tubes, containers, pipettes, Petri dishes and all glass syringes. 2. Other equipment such as mortar and pestles, evaporating basins, and metalware like scissors, scalpels, ointments tubes, beakers and dishes of stainless steel. 3. Oils and similar anhydrous materials. e.g., Ethyl oleate and other esters. Ingredient of ointment bases. e.g., liquid, soft and hard paraffin wool fat, wool alcohols, bee wax and medical lubricants e.g., glycerol. 4. Powders: Starch 100°C for 1hr, sulphonamides and lactose.

b. Moist heat sterilization Moist heat is believed to destroy microorganisms by causing protein coagulation or denaturation. It can kill microorganisms at lower temperature in shorter times than dry heat. Moist heat has been recognized as an efficient biocidal agent from the early days of bacteriology, when it was principally developed for the sterilization of culture media. It now finds widespread application in the processing of many thermostable products and devices. In the pharmaceutical and medical sphere it is used in the sterilization of dressings, sheets, surgical and diagnostic equipment, containers and closures, and aqueous injections, ophthalmic preparations and irrigation fluids. Sterilization by moist heat usually involves the use of steam at temperatures in the range 121-134°C, and while alternative strategies are available for processing products unstable at these high temperatures, they rarely offer the same degree of sterility assurance and should be avoided if at all possible. The elevated temperatures generally associated with moist heat sterilization methods can only be achieved by generating steam under pressure. By far the most commonly employed standard temperature/time cycles for bottled fluids and porous loads (e.g. surgical dressings) are 121°C for 15 minutes and 134°C. For the moist heat sterilization autoclave is mainly used. Autoclave is an instrument in which steam is generated at very high temperature and pressure. At this higher temperature (121°C or more) and pressure (15 lb/inch2) for 15 to 20 minutes the protein of the microorganisms denature and cell wall damage also takes place which cause lyses or death of microorganisms. As with dry heat sterilization, it is not possible to kill the spores of certain saprophytes but with the help of moist heat sterilization all the pathogens are destroyed.

b. Moist heat sterilization Moist heat is believed to destroy microorganisms by causing protein coagulation or denaturation. It can kill microorganisms at lower temperature in shorter times than dry heat. Moist heat has been recognized as an efficient biocidal agent from the early days of bacteriology, when it was principally developed for the sterilization of culture media. It now finds widespread application in the processing of many thermostable products and devices. In the pharmaceutical and medical sphere it is used in the sterilization of dressings, sheets, surgical and diagnostic equipment, containers and closures, and aqueous injections, ophthalmic preparations and irrigation fluids. Sterilization by moist heat usually involves the use of steam at temperatures in the range 121-134°C, and while alternative strategies are available for processing products unstable at these high temperatures, they rarely offer the same degree of sterility assurance and should be avoided if at all possible. The elevated temperatures generally associated with moist heat sterilization methods can only be achieved by generating steam under pressure. By far the most commonly employed standard temperature/time cycles for bottled fluids and porous loads (e.g. surgical dressings) are 121°C for 15 minutes and 134°C. For the moist heat sterilization autoclave is mainly used. Autoclave is an instrument in which steam is generated at very high temperature and pressure. At this higher temperature (121°C or more) and pressure (15 lb/inch2) for 15 to 20 minutes the protein of the microorganisms denature and cell wall damage also takes place which cause lyses or death of microorganisms. As with dry heat sterilization, it is not possible to kill the spores of certain saprophytes but with the help of moist heat sterilization all the pathogens are destroyed.

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Autoclaving condition

STERILIZATION

Autoclaving condition

Temperature

Holding period

Temperature

Holding period

1210C

15 minutes

1210C

15 minutes

0

117

0

126 C

10 minutes

126 C

10 minutes

1340C

3 minutes

1340C

3 minutes

The standard working condition of autoclave is: Temperature 121°C at pressure 15 lb/inch2 for 15 minutes.

The standard working condition of autoclave is: Temperature 121°C at pressure 15 lb/inch2 for 15 minutes.

Fig. 9.4. Diagrammatic sketch of an autoclave.

Fig. 9.4. Diagrammatic sketch of an autoclave.

Applications

Applications

1. Some of the glassware and closures having rubber parts are sterilized in autoclaves. 2. Nearly all the injection solutions and suspensions like adrenaline, atropine sulphate, chloroquine sulphate, chlorpromazine, propanalol, lignocaine hydrochloride etc are sterilized by autoclave. 3. Variety of materials used in surgery like bandages, operating gowns, caps, mask, towels, trolley clothes, hand gloves etc are sterilized in autoclaves.

STERILIZATION

1. Some of the glassware and closures having rubber parts are sterilized in autoclaves. 2. Nearly all the injection solutions and suspensions like adrenaline, atropine sulphate, chloroquine sulphate, chlorpromazine, propanalol, lignocaine hydrochloride etc are sterilized by autoclave. 3. Variety of materials used in surgery like bandages, operating gowns, caps, mask, towels, trolley clothes, hand gloves etc are sterilized in autoclaves.

117

Autoclaving condition

STERILIZATION

Autoclaving condition

Temperature

Holding period

Temperature

Holding period

1210C

15 minutes

1210C

15 minutes

1260C

10 minutes

1260C

10 minutes

0

134 C

117

3 minutes

0

134 C

3 minutes

The standard working condition of autoclave is: Temperature 121°C at pressure 15 lb/inch2 for 15 minutes.

The standard working condition of autoclave is: Temperature 121°C at pressure 15 lb/inch2 for 15 minutes.

Fig. 9.4. Diagrammatic sketch of an autoclave.

Fig. 9.4. Diagrammatic sketch of an autoclave.

Applications 1. Some of the glassware and closures having rubber parts are sterilized in autoclaves. 2. Nearly all the injection solutions and suspensions like adrenaline, atropine sulphate, chloroquine sulphate, chlorpromazine, propanalol, lignocaine hydrochloride etc are sterilized by autoclave. 3. Variety of materials used in surgery like bandages, operating gowns, caps, mask, towels, trolley clothes, hand gloves etc are sterilized in autoclaves.

Applications 1. Some of the glassware and closures having rubber parts are sterilized in autoclaves. 2. Nearly all the injection solutions and suspensions like adrenaline, atropine sulphate, chloroquine sulphate, chlorpromazine, propanalol, lignocaine hydrochloride etc are sterilized by autoclave. 3. Variety of materials used in surgery like bandages, operating gowns, caps, mask, towels, trolley clothes, hand gloves etc are sterilized in autoclaves.

118 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY c. Other methods of moist heat sterilization 1. Sterilization of vaccine: Sterilization is carried out in a water bath thermostatically controlled temperature. The container must be immersed entirely because organisms on surfaces, above the water level may not be killed, e.g., 15 minutes at 55°C for plague, 1hr. at 56°C for cholera and typhoid. 2. Pasteurization: Pasteurization processes are used to make milk safe and improve its keeping qualities. The original method was developed by Louis Pasture to prevent the souring of wine. This is done by two methods: a. Holder method: The milk is heated as 62.8°C held there for 30 minutes and quickly cooled. This is carried out in jacketed stainless steel tanks containing agitators to ensure the correct exposure throughout the milk. b. High temperature short time method or flesh: the milk is rapidly raised to 71.1°C, held at this temperature for at least 15 seconds and quickly cold. 3. Tyndallization: This technique was developed by the bacteriologist Tyndall. This method of sterilization is used for the spore forming microorganisms. • Pack and seal the solution in its final container and maintain the whole at 80°C for 1 hr on each of 3 successive days. • In theory the first heating destroys the vegetative cells but not the bacterial spores. During the interval between the first and second heating the spores germinate and forming vegetative cells that are killed by the second heating. • The third heating is a precautionary measure to destroy cells from any spores that do not germinate until the second interval.

118 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY c. Other methods of moist heat sterilization 1. Sterilization of vaccine: Sterilization is carried out in a water bath thermostatically controlled temperature. The container must be immersed entirely because organisms on surfaces, above the water level may not be killed, e.g., 15 minutes at 55°C for plague, 1hr. at 56°C for cholera and typhoid. 2. Pasteurization: Pasteurization processes are used to make milk safe and improve its keeping qualities. The original method was developed by Louis Pasture to prevent the souring of wine. This is done by two methods: a. Holder method: The milk is heated as 62.8°C held there for 30 minutes and quickly cooled. This is carried out in jacketed stainless steel tanks containing agitators to ensure the correct exposure throughout the milk. b. High temperature short time method or flesh: the milk is rapidly raised to 71.1°C, held at this temperature for at least 15 seconds and quickly cold. 3. Tyndallization: This technique was developed by the bacteriologist Tyndall. This method of sterilization is used for the spore forming microorganisms. • Pack and seal the solution in its final container and maintain the whole at 80°C for 1 hr on each of 3 successive days. • In theory the first heating destroys the vegetative cells but not the bacterial spores. During the interval between the first and second heating the spores germinate and forming vegetative cells that are killed by the second heating. • The third heating is a precautionary measure to destroy cells from any spores that do not germinate until the second interval.

II. Sterilization by ionizing radiation

II. Sterilization by ionizing radiation

Ionizing radiation includes X-rays, Gamma rays and beta rays and these can cause breakdown of genetic material (DNA) of the microbes.

Ionizing radiation includes X-rays, Gamma rays and beta rays and these can cause breakdown of genetic material (DNA) of the microbes.

Fig. 9.5. Various types of electromagnetic waves can be arranged in a continuous spectrum according to their wavelength. Quantum energy increases as wavelength decreases. That is why the penetrating power of gamma rays and X-rays are more than IR or radio waves.

Fig. 9.5. Various types of electromagnetic waves can be arranged in a continuous spectrum according to their wavelength. Quantum energy increases as wavelength decreases. That is why the penetrating power of gamma rays and X-rays are more than IR or radio waves.

X-rays and gamma radiations penetrate well because they are uncharged, massless, and have very high energetic quenta. Ionizing radiation can be obtained from radioactive isotopes.

X-rays and gamma radiations penetrate well because they are uncharged, massless, and have very high energetic quenta. Ionizing radiation can be obtained from radioactive isotopes.

118 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

118 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

c. Other methods of moist heat sterilization 1. Sterilization of vaccine: Sterilization is carried out in a water bath thermostatically controlled temperature. The container must be immersed entirely because organisms on surfaces, above the water level may not be killed, e.g., 15 minutes at 55°C for plague, 1hr. at 56°C for cholera and typhoid. 2. Pasteurization: Pasteurization processes are used to make milk safe and improve its keeping qualities. The original method was developed by Louis Pasture to prevent the souring of wine. This is done by two methods: a. Holder method: The milk is heated as 62.8°C held there for 30 minutes and quickly cooled. This is carried out in jacketed stainless steel tanks containing agitators to ensure the correct exposure throughout the milk. b. High temperature short time method or flesh: the milk is rapidly raised to 71.1°C, held at this temperature for at least 15 seconds and quickly cold. 3. Tyndallization: This technique was developed by the bacteriologist Tyndall. This method of sterilization is used for the spore forming microorganisms. • Pack and seal the solution in its final container and maintain the whole at 80°C for 1 hr on each of 3 successive days. • In theory the first heating destroys the vegetative cells but not the bacterial spores. During the interval between the first and second heating the spores germinate and forming vegetative cells that are killed by the second heating. • The third heating is a precautionary measure to destroy cells from any spores that do not germinate until the second interval.

c. Other methods of moist heat sterilization 1. Sterilization of vaccine: Sterilization is carried out in a water bath thermostatically controlled temperature. The container must be immersed entirely because organisms on surfaces, above the water level may not be killed, e.g., 15 minutes at 55°C for plague, 1hr. at 56°C for cholera and typhoid. 2. Pasteurization: Pasteurization processes are used to make milk safe and improve its keeping qualities. The original method was developed by Louis Pasture to prevent the souring of wine. This is done by two methods: a. Holder method: The milk is heated as 62.8°C held there for 30 minutes and quickly cooled. This is carried out in jacketed stainless steel tanks containing agitators to ensure the correct exposure throughout the milk. b. High temperature short time method or flesh: the milk is rapidly raised to 71.1°C, held at this temperature for at least 15 seconds and quickly cold. 3. Tyndallization: This technique was developed by the bacteriologist Tyndall. This method of sterilization is used for the spore forming microorganisms. • Pack and seal the solution in its final container and maintain the whole at 80°C for 1 hr on each of 3 successive days. • In theory the first heating destroys the vegetative cells but not the bacterial spores. During the interval between the first and second heating the spores germinate and forming vegetative cells that are killed by the second heating. • The third heating is a precautionary measure to destroy cells from any spores that do not germinate until the second interval.

II. Sterilization by ionizing radiation

II. Sterilization by ionizing radiation

Ionizing radiation includes X-rays, Gamma rays and beta rays and these can cause breakdown of genetic material (DNA) of the microbes.

Ionizing radiation includes X-rays, Gamma rays and beta rays and these can cause breakdown of genetic material (DNA) of the microbes.

Fig. 9.5. Various types of electromagnetic waves can be arranged in a continuous spectrum according to their wavelength. Quantum energy increases as wavelength decreases. That is why the penetrating power of gamma rays and X-rays are more than IR or radio waves.

Fig. 9.5. Various types of electromagnetic waves can be arranged in a continuous spectrum according to their wavelength. Quantum energy increases as wavelength decreases. That is why the penetrating power of gamma rays and X-rays are more than IR or radio waves.

X-rays and gamma radiations penetrate well because they are uncharged, massless, and have very high energetic quenta. Ionizing radiation can be obtained from radioactive isotopes.

X-rays and gamma radiations penetrate well because they are uncharged, massless, and have very high energetic quenta. Ionizing radiation can be obtained from radioactive isotopes.

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Gamma rays for sterilization are usually derived from a cobalt-60 (60Co) source (caesium-137 may also be used), with a half-life of 5.25 years, which on disintegration emits radiation at two energy levels of 1.33 and 1.17 MeV. Mode of action: Ionizing radiation act by two ways: I. Direct method (target theory): Every cell has a particular target region which is highly sensitive to radiation, e.g., in bacteriophase virus nucleic acid is coated with a thin layer of protein which is damaged by the radiation. II. Indirect method (chemical theory): Absorption of radiation by the water within or surrounding living cells produces free radicals. These free radicals are powerful oxidizing agents capable of damaging essential molecules of the cell and therefore causing death of the microbial cells.

Also if dissolved oxygen is present:

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119

Gamma rays for sterilization are usually derived from a cobalt-60 (60Co) source (caesium-137 may also be used), with a half-life of 5.25 years, which on disintegration emits radiation at two energy levels of 1.33 and 1.17 MeV. Mode of action: Ionizing radiation act by two ways: I. Direct method (target theory): Every cell has a particular target region which is highly sensitive to radiation, e.g., in bacteriophase virus nucleic acid is coated with a thin layer of protein which is damaged by the radiation. II. Indirect method (chemical theory): Absorption of radiation by the water within or surrounding living cells produces free radicals. These free radicals are powerful oxidizing agents capable of damaging essential molecules of the cell and therefore causing death of the microbial cells.

Also if dissolved oxygen is present:

The oxidizing radicals OH, HO2 and H2O2 are more destructive than the reducing radicals H.

The oxidizing radicals OH, HO2 and H2O2 are more destructive than the reducing radicals H.

Applications

Applications

1. 2. 3. 4.

Large amount of material can be treated at once. The methods are reliable and can be accurate controlled. Frozen material can be treated . Some bacterial and viral vaccines can be sterilized without loss of antigenicity.

1. 2. 3. 4.

Large amount of material can be treated at once. The methods are reliable and can be accurate controlled. Frozen material can be treated . Some bacterial and viral vaccines can be sterilized without loss of antigenicity.

Sterilization by UV radiation

Sterilization by UV radiation

The range of UV radiation is between 1 Pm to 400 Pm. But the wavelength between 240 to 300 Pm is more harmful for the microbes, because at this

The range of UV radiation is between 1 Pm to 400 Pm. But the wavelength between 240 to 300 Pm is more harmful for the microbes, because at this

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Gamma rays for sterilization are usually derived from a cobalt-60 (60Co) source (caesium-137 may also be used), with a half-life of 5.25 years, which on disintegration emits radiation at two energy levels of 1.33 and 1.17 MeV. Mode of action: Ionizing radiation act by two ways: I. Direct method (target theory): Every cell has a particular target region which is highly sensitive to radiation, e.g., in bacteriophase virus nucleic acid is coated with a thin layer of protein which is damaged by the radiation. II. Indirect method (chemical theory): Absorption of radiation by the water within or surrounding living cells produces free radicals. These free radicals are powerful oxidizing agents capable of damaging essential molecules of the cell and therefore causing death of the microbial cells.

Also if dissolved oxygen is present:

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Gamma rays for sterilization are usually derived from a cobalt-60 (60Co) source (caesium-137 may also be used), with a half-life of 5.25 years, which on disintegration emits radiation at two energy levels of 1.33 and 1.17 MeV. Mode of action: Ionizing radiation act by two ways: I. Direct method (target theory): Every cell has a particular target region which is highly sensitive to radiation, e.g., in bacteriophase virus nucleic acid is coated with a thin layer of protein which is damaged by the radiation. II. Indirect method (chemical theory): Absorption of radiation by the water within or surrounding living cells produces free radicals. These free radicals are powerful oxidizing agents capable of damaging essential molecules of the cell and therefore causing death of the microbial cells.

Also if dissolved oxygen is present:

The oxidizing radicals OH, HO2 and H2O2 are more destructive than the reducing radicals H.

The oxidizing radicals OH, HO2 and H2O2 are more destructive than the reducing radicals H.

Applications

Applications

1. 2. 3. 4.

Large amount of material can be treated at once. The methods are reliable and can be accurate controlled. Frozen material can be treated . Some bacterial and viral vaccines can be sterilized without loss of antigenicity.

1. 2. 3. 4.

Large amount of material can be treated at once. The methods are reliable and can be accurate controlled. Frozen material can be treated . Some bacterial and viral vaccines can be sterilized without loss of antigenicity.

Sterilization by UV radiation

Sterilization by UV radiation

The range of UV radiation is between 1 Pm to 400 Pm. But the wavelength between 240 to 300 Pm is more harmful for the microbes, because at this

The range of UV radiation is between 1 Pm to 400 Pm. But the wavelength between 240 to 300 Pm is more harmful for the microbes, because at this

120 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

120 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

wavelength genetic material and protein are destroyed and death of microbes takes place. Exposures of UV rays cause the formation of purine and pyramidine dimers between adjacent base pairs in the same DNA stand.

wavelength genetic material and protein are destroyed and death of microbes takes place. Exposures of UV rays cause the formation of purine and pyramidine dimers between adjacent base pairs in the same DNA stand.

Applications

Applications

1. Irradiation of incoming air and internal air of the sterile filling areas of antibiotic plants. 2. To prevent cross-infection in hospitals and schools etc. 3. Sterilization of thermolabile substances. 4. Improvement of bacteriological quality of water used to manufacture nonsterile pharmaceuticals.

1. Irradiation of incoming air and internal air of the sterile filling areas of antibiotic plants. 2. To prevent cross-infection in hospitals and schools etc. 3. Sterilization of thermolabile substances. 4. Improvement of bacteriological quality of water used to manufacture nonsterile pharmaceuticals.

Sterilization by filtration

Sterilization by filtration

The principal applications of sterilizing-grade filters are the treatment of heatsensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. These filter are made up of: 1. Sintered ceramics: Made from finally ground porcelain or from diatomaceous earth. 2. Fibrous pads: Containing asbestos and wood cellulose. 3. Sintered glass: Made from borosilicate glass.

The principal applications of sterilizing-grade filters are the treatment of heatsensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. These filter are made up of: 1. Sintered ceramics: Made from finally ground porcelain or from diatomaceous earth. 2. Fibrous pads: Containing asbestos and wood cellulose. 3. Sintered glass: Made from borosilicate glass.

Membrane filters: These are widely used nowadays and consist of cellulose ester. Filter sterilization is used for both liquid and gases. In order to compare with other methods of sterilization, the microorganism removal efficiency of filters employed in the processing of liquids must be high. For this reason, membrane filters of 0.2-0.22 —m pore diameter are chiefly used. The principal application for filtration sterilization of gases is in the provision of sterile air to aseptic manufacturing suites, hospital isolation units and some operating theatres. Filters employed generally consist of pleated sheets of glass microfibres separated and supported by corrugated sheets of Kraft paper or aluminium; these are employed in wall or ceiling panels, overhead canopies, or laminar airflow cabinets. These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of particles greater than 0.20 —m in diameter and thus are acting as depth filters. In practice their microorganism removal efficiency is rather better than other methods.

Membrane filters: These are widely used nowadays and consist of cellulose ester. Filter sterilization is used for both liquid and gases. In order to compare with other methods of sterilization, the microorganism removal efficiency of filters employed in the processing of liquids must be high. For this reason, membrane filters of 0.2-0.22 —m pore diameter are chiefly used. The principal application for filtration sterilization of gases is in the provision of sterile air to aseptic manufacturing suites, hospital isolation units and some operating theatres. Filters employed generally consist of pleated sheets of glass microfibres separated and supported by corrugated sheets of Kraft paper or aluminium; these are employed in wall or ceiling panels, overhead canopies, or laminar airflow cabinets. These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of particles greater than 0.20 —m in diameter and thus are acting as depth filters. In practice their microorganism removal efficiency is rather better than other methods.

9.4.2 Chemical Sterilization This includes: 1. Agent acting on the cell membrane

9.4.2 Chemical Sterilization This includes: 1. Agent acting on the cell membrane

120 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

120 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

wavelength genetic material and protein are destroyed and death of microbes takes place. Exposures of UV rays cause the formation of purine and pyramidine dimers between adjacent base pairs in the same DNA stand.

wavelength genetic material and protein are destroyed and death of microbes takes place. Exposures of UV rays cause the formation of purine and pyramidine dimers between adjacent base pairs in the same DNA stand.

Applications

Applications

1. Irradiation of incoming air and internal air of the sterile filling areas of antibiotic plants. 2. To prevent cross-infection in hospitals and schools etc. 3. Sterilization of thermolabile substances. 4. Improvement of bacteriological quality of water used to manufacture nonsterile pharmaceuticals.

1. Irradiation of incoming air and internal air of the sterile filling areas of antibiotic plants. 2. To prevent cross-infection in hospitals and schools etc. 3. Sterilization of thermolabile substances. 4. Improvement of bacteriological quality of water used to manufacture nonsterile pharmaceuticals.

Sterilization by filtration

Sterilization by filtration

The principal applications of sterilizing-grade filters are the treatment of heatsensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. These filter are made up of: 1. Sintered ceramics: Made from finally ground porcelain or from diatomaceous earth. 2. Fibrous pads: Containing asbestos and wood cellulose. 3. Sintered glass: Made from borosilicate glass.

The principal applications of sterilizing-grade filters are the treatment of heatsensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. These filter are made up of: 1. Sintered ceramics: Made from finally ground porcelain or from diatomaceous earth. 2. Fibrous pads: Containing asbestos and wood cellulose. 3. Sintered glass: Made from borosilicate glass.

Membrane filters: These are widely used nowadays and consist of cellulose ester. Filter sterilization is used for both liquid and gases. In order to compare with other methods of sterilization, the microorganism removal efficiency of filters employed in the processing of liquids must be high. For this reason, membrane filters of 0.2-0.22 —m pore diameter are chiefly used. The principal application for filtration sterilization of gases is in the provision of sterile air to aseptic manufacturing suites, hospital isolation units and some operating theatres. Filters employed generally consist of pleated sheets of glass microfibres separated and supported by corrugated sheets of Kraft paper or aluminium; these are employed in wall or ceiling panels, overhead canopies, or laminar airflow cabinets. These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of particles greater than 0.20 —m in diameter and thus are acting as depth filters. In practice their microorganism removal efficiency is rather better than other methods.

Membrane filters: These are widely used nowadays and consist of cellulose ester. Filter sterilization is used for both liquid and gases. In order to compare with other methods of sterilization, the microorganism removal efficiency of filters employed in the processing of liquids must be high. For this reason, membrane filters of 0.2-0.22 —m pore diameter are chiefly used. The principal application for filtration sterilization of gases is in the provision of sterile air to aseptic manufacturing suites, hospital isolation units and some operating theatres. Filters employed generally consist of pleated sheets of glass microfibres separated and supported by corrugated sheets of Kraft paper or aluminium; these are employed in wall or ceiling panels, overhead canopies, or laminar airflow cabinets. These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of particles greater than 0.20 —m in diameter and thus are acting as depth filters. In practice their microorganism removal efficiency is rather better than other methods.

9.4.2 Chemical Sterilization This includes: 1. Agent acting on the cell membrane

9.4.2 Chemical Sterilization This includes: 1. Agent acting on the cell membrane

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a. Surface acting agent. Surface-active agents or surfactants are classified as anionic, cationic, non-ionic or ampholytic according to the ionization of the hydrophilic group in the molecule. The anionic agents include soaps which act better at acid pH and are effective against Gram-positive agents. Gram-negative bacteria are relatively resistant because of the presence of lipopolysaccharide in their cell wall. The cationic agents used for their antimicrobial activity all fall within the group known as the quaternary ammonium compounds which are variously described as QACs. The QACs are most effective against microorganisms at neutral or slightly alkaline pH and become virtually inactive below pH 3.5. Not surprisingly, anionic agents greatly reduce the activity of these cationic agents. QACs exhibit greatest activity against Gram-positive bacteria. Ampholytic or amphoteric agents can ionize to give anionic, cationic and zwiterionic (positively and negatively charged ions in the same molecule) activity. Consequently, they display both the detergent properties of the anionic surface-active agents and the antimicrobial activity of the cationic agents. They are used quite extensively in Europe for pre-surgical hand scrubbing, medical instrument disinfection and floor disinfection in hospitals. b. Phenols. Phenols are good antimicrobial agents and are rapidly bactericidal but generally are not sporicidal activity. Their activity is markedly diminished by dilution and is also reduced by organic matter. They are more active at acid pH. The main disadvantages of phenols are their caustic effect on skin and tissues and their systemic toxicity. The more highly substituted phenols are less toxic and can be used as preservatives and antiseptics; however, they are also less active than the simple phenolics, especially against Gram-negative organisms. c. Organic solvents. Alcohol is the most important example of this group. Alcohol disorganizes the lipid structure of the cell membrane by penetrating into the hydrocarbon region. It also denatures the proteins of the cell. Ethanol is used to sterilize the skin prior to cutaneous injections. It is active against gram negative, gram positive and acid fast organisms and acts best at concentration of 70%. Isopropyl alcohol is more active than ethanol and is less volatile and can be used to sterilize thermometers. Chloroform and toluene are used to keep the solutions sterile and to disrupt permeability barriers. Chloroform (CHCl3) has a narrow spectrum of activity. It has been used extensively as a preservative in pharmaceuticals.

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a. Surface acting agent. Surface-active agents or surfactants are classified as anionic, cationic, non-ionic or ampholytic according to the ionization of the hydrophilic group in the molecule. The anionic agents include soaps which act better at acid pH and are effective against Gram-positive agents. Gram-negative bacteria are relatively resistant because of the presence of lipopolysaccharide in their cell wall. The cationic agents used for their antimicrobial activity all fall within the group known as the quaternary ammonium compounds which are variously described as QACs. The QACs are most effective against microorganisms at neutral or slightly alkaline pH and become virtually inactive below pH 3.5. Not surprisingly, anionic agents greatly reduce the activity of these cationic agents. QACs exhibit greatest activity against Gram-positive bacteria. Ampholytic or amphoteric agents can ionize to give anionic, cationic and zwiterionic (positively and negatively charged ions in the same molecule) activity. Consequently, they display both the detergent properties of the anionic surface-active agents and the antimicrobial activity of the cationic agents. They are used quite extensively in Europe for pre-surgical hand scrubbing, medical instrument disinfection and floor disinfection in hospitals. b. Phenols. Phenols are good antimicrobial agents and are rapidly bactericidal but generally are not sporicidal activity. Their activity is markedly diminished by dilution and is also reduced by organic matter. They are more active at acid pH. The main disadvantages of phenols are their caustic effect on skin and tissues and their systemic toxicity. The more highly substituted phenols are less toxic and can be used as preservatives and antiseptics; however, they are also less active than the simple phenolics, especially against Gram-negative organisms. c. Organic solvents. Alcohol is the most important example of this group. Alcohol disorganizes the lipid structure of the cell membrane by penetrating into the hydrocarbon region. It also denatures the proteins of the cell. Ethanol is used to sterilize the skin prior to cutaneous injections. It is active against gram negative, gram positive and acid fast organisms and acts best at concentration of 70%. Isopropyl alcohol is more active than ethanol and is less volatile and can be used to sterilize thermometers. Chloroform and toluene are used to keep the solutions sterile and to disrupt permeability barriers. Chloroform (CHCl3) has a narrow spectrum of activity. It has been used extensively as a preservative in pharmaceuticals.

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a. Surface acting agent. Surface-active agents or surfactants are classified as anionic, cationic, non-ionic or ampholytic according to the ionization of the hydrophilic group in the molecule. The anionic agents include soaps which act better at acid pH and are effective against Gram-positive agents. Gram-negative bacteria are relatively resistant because of the presence of lipopolysaccharide in their cell wall. The cationic agents used for their antimicrobial activity all fall within the group known as the quaternary ammonium compounds which are variously described as QACs. The QACs are most effective against microorganisms at neutral or slightly alkaline pH and become virtually inactive below pH 3.5. Not surprisingly, anionic agents greatly reduce the activity of these cationic agents. QACs exhibit greatest activity against Gram-positive bacteria. Ampholytic or amphoteric agents can ionize to give anionic, cationic and zwiterionic (positively and negatively charged ions in the same molecule) activity. Consequently, they display both the detergent properties of the anionic surface-active agents and the antimicrobial activity of the cationic agents. They are used quite extensively in Europe for pre-surgical hand scrubbing, medical instrument disinfection and floor disinfection in hospitals. b. Phenols. Phenols are good antimicrobial agents and are rapidly bactericidal but generally are not sporicidal activity. Their activity is markedly diminished by dilution and is also reduced by organic matter. They are more active at acid pH. The main disadvantages of phenols are their caustic effect on skin and tissues and their systemic toxicity. The more highly substituted phenols are less toxic and can be used as preservatives and antiseptics; however, they are also less active than the simple phenolics, especially against Gram-negative organisms. c. Organic solvents. Alcohol is the most important example of this group. Alcohol disorganizes the lipid structure of the cell membrane by penetrating into the hydrocarbon region. It also denatures the proteins of the cell. Ethanol is used to sterilize the skin prior to cutaneous injections. It is active against gram negative, gram positive and acid fast organisms and acts best at concentration of 70%. Isopropyl alcohol is more active than ethanol and is less volatile and can be used to sterilize thermometers. Chloroform and toluene are used to keep the solutions sterile and to disrupt permeability barriers. Chloroform (CHCl3) has a narrow spectrum of activity. It has been used extensively as a preservative in pharmaceuticals.

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a. Surface acting agent. Surface-active agents or surfactants are classified as anionic, cationic, non-ionic or ampholytic according to the ionization of the hydrophilic group in the molecule. The anionic agents include soaps which act better at acid pH and are effective against Gram-positive agents. Gram-negative bacteria are relatively resistant because of the presence of lipopolysaccharide in their cell wall. The cationic agents used for their antimicrobial activity all fall within the group known as the quaternary ammonium compounds which are variously described as QACs. The QACs are most effective against microorganisms at neutral or slightly alkaline pH and become virtually inactive below pH 3.5. Not surprisingly, anionic agents greatly reduce the activity of these cationic agents. QACs exhibit greatest activity against Gram-positive bacteria. Ampholytic or amphoteric agents can ionize to give anionic, cationic and zwiterionic (positively and negatively charged ions in the same molecule) activity. Consequently, they display both the detergent properties of the anionic surface-active agents and the antimicrobial activity of the cationic agents. They are used quite extensively in Europe for pre-surgical hand scrubbing, medical instrument disinfection and floor disinfection in hospitals. b. Phenols. Phenols are good antimicrobial agents and are rapidly bactericidal but generally are not sporicidal activity. Their activity is markedly diminished by dilution and is also reduced by organic matter. They are more active at acid pH. The main disadvantages of phenols are their caustic effect on skin and tissues and their systemic toxicity. The more highly substituted phenols are less toxic and can be used as preservatives and antiseptics; however, they are also less active than the simple phenolics, especially against Gram-negative organisms. c. Organic solvents. Alcohol is the most important example of this group. Alcohol disorganizes the lipid structure of the cell membrane by penetrating into the hydrocarbon region. It also denatures the proteins of the cell. Ethanol is used to sterilize the skin prior to cutaneous injections. It is active against gram negative, gram positive and acid fast organisms and acts best at concentration of 70%. Isopropyl alcohol is more active than ethanol and is less volatile and can be used to sterilize thermometers. Chloroform and toluene are used to keep the solutions sterile and to disrupt permeability barriers. Chloroform (CHCl3) has a narrow spectrum of activity. It has been used extensively as a preservative in pharmaceuticals.

122 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. Agents that denature proteins Agents that can alter the structure of protein make the protein nonfunctional. a. Acids and alkalies: These agents release their free H+ and OH- ions and than through undissociated molecules, or by altering the pH of the medium they denature the proteins of the organisms, e.g., benzoic acid, propionic acid etc. 3. Agent acting on functional group of proteins a. Heavy metals: Mercury and silver have long been known to have antibacterial properties and preparations of these metals were among the earliest used antiseptics, but have been replaced by less toxic compounds. Other metals such as zinc, copper, aluminium and tin have weak antibacterial properties but are used in medicine for other functions, e.g., aluminium acetate and zinc sulphate are employed as astringents. b. Oxidizing agents: This group includes hydrogen peroxide, halogens and potassium permanganate, etc. The germicidal properties of hydrogen peroxide (H2O2 ) have been known for more than a century. It is used as an antiseptic for open wounds and ulcers where it provides additional cleansing due to its oxidation of organic debris. Its activity against the protozoa, Acanthamoeba, which can cause keratitis in contact lens wearers, has made it popular for disinfection of soft contact lenses. Concentrations of 3-6% are effective for general disinfection purposes. At high concentrations (up to 30%) and increased temperature hydrogen peroxide is sporicidal. Use has been made of this in vapour- phase hydrogen peroxide decontamination of laboratory equipment and enclosed spaces. Halogens (chlorine and iodine) have been used extensively since their introduction as disinfecting agents in the early 19th century. Preparations containing these halogens such as Dakin’s solution and tincture of iodine were early inclusions in many pharmacopoeiae and national formularies. More recent formulations of these elements have improved activity, stability and ease of use. A large number of antimicrobially active chlorine compounds are commercially available, one of the most important being liquid chlorine. Iodine has a wide spectrum of antimicrobial activity. Gramnegative and Gram-positive organisms, bacterial spores (on extended exposure), mycobacteria, fungi and viruses are all susceptible. The active agent is the elemental iodine molecule, I2. As elemental iodine is only slightly soluble in water, iodide ions are required for aqueous solutions such as aqueous iodine solution, BP 1988 (Lugol’s solution) containing 5% iodine in 10% potassium iodide solution. Iodine (2.5%) may also be dissolved in ethanol (90%) and potassium iodide (2.5%) solution to give weak iodine solution, BP 1988 (Iodine Tincture).

122 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. Agents that denature proteins Agents that can alter the structure of protein make the protein nonfunctional. a. Acids and alkalies: These agents release their free H+ and OH- ions and than through undissociated molecules, or by altering the pH of the medium they denature the proteins of the organisms, e.g., benzoic acid, propionic acid etc. 3. Agent acting on functional group of proteins a. Heavy metals: Mercury and silver have long been known to have antibacterial properties and preparations of these metals were among the earliest used antiseptics, but have been replaced by less toxic compounds. Other metals such as zinc, copper, aluminium and tin have weak antibacterial properties but are used in medicine for other functions, e.g., aluminium acetate and zinc sulphate are employed as astringents. b. Oxidizing agents: This group includes hydrogen peroxide, halogens and potassium permanganate, etc. The germicidal properties of hydrogen peroxide (H2O2 ) have been known for more than a century. It is used as an antiseptic for open wounds and ulcers where it provides additional cleansing due to its oxidation of organic debris. Its activity against the protozoa, Acanthamoeba, which can cause keratitis in contact lens wearers, has made it popular for disinfection of soft contact lenses. Concentrations of 3-6% are effective for general disinfection purposes. At high concentrations (up to 30%) and increased temperature hydrogen peroxide is sporicidal. Use has been made of this in vapour- phase hydrogen peroxide decontamination of laboratory equipment and enclosed spaces. Halogens (chlorine and iodine) have been used extensively since their introduction as disinfecting agents in the early 19th century. Preparations containing these halogens such as Dakin’s solution and tincture of iodine were early inclusions in many pharmacopoeiae and national formularies. More recent formulations of these elements have improved activity, stability and ease of use. A large number of antimicrobially active chlorine compounds are commercially available, one of the most important being liquid chlorine. Iodine has a wide spectrum of antimicrobial activity. Gramnegative and Gram-positive organisms, bacterial spores (on extended exposure), mycobacteria, fungi and viruses are all susceptible. The active agent is the elemental iodine molecule, I2. As elemental iodine is only slightly soluble in water, iodide ions are required for aqueous solutions such as aqueous iodine solution, BP 1988 (Lugol’s solution) containing 5% iodine in 10% potassium iodide solution. Iodine (2.5%) may also be dissolved in ethanol (90%) and potassium iodide (2.5%) solution to give weak iodine solution, BP 1988 (Iodine Tincture).

122 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. Agents that denature proteins Agents that can alter the structure of protein make the protein nonfunctional. a. Acids and alkalies: These agents release their free H+ and OH- ions and than through undissociated molecules, or by altering the pH of the medium they denature the proteins of the organisms, e.g., benzoic acid, propionic acid etc. 3. Agent acting on functional group of proteins a. Heavy metals: Mercury and silver have long been known to have antibacterial properties and preparations of these metals were among the earliest used antiseptics, but have been replaced by less toxic compounds. Other metals such as zinc, copper, aluminium and tin have weak antibacterial properties but are used in medicine for other functions, e.g., aluminium acetate and zinc sulphate are employed as astringents. b. Oxidizing agents: This group includes hydrogen peroxide, halogens and potassium permanganate, etc. The germicidal properties of hydrogen peroxide (H2O2 ) have been known for more than a century. It is used as an antiseptic for open wounds and ulcers where it provides additional cleansing due to its oxidation of organic debris. Its activity against the protozoa, Acanthamoeba, which can cause keratitis in contact lens wearers, has made it popular for disinfection of soft contact lenses. Concentrations of 3-6% are effective for general disinfection purposes. At high concentrations (up to 30%) and increased temperature hydrogen peroxide is sporicidal. Use has been made of this in vapour- phase hydrogen peroxide decontamination of laboratory equipment and enclosed spaces. Halogens (chlorine and iodine) have been used extensively since their introduction as disinfecting agents in the early 19th century. Preparations containing these halogens such as Dakin’s solution and tincture of iodine were early inclusions in many pharmacopoeiae and national formularies. More recent formulations of these elements have improved activity, stability and ease of use. A large number of antimicrobially active chlorine compounds are commercially available, one of the most important being liquid chlorine. Iodine has a wide spectrum of antimicrobial activity. Gramnegative and Gram-positive organisms, bacterial spores (on extended exposure), mycobacteria, fungi and viruses are all susceptible. The active agent is the elemental iodine molecule, I2. As elemental iodine is only slightly soluble in water, iodide ions are required for aqueous solutions such as aqueous iodine solution, BP 1988 (Lugol’s solution) containing 5% iodine in 10% potassium iodide solution. Iodine (2.5%) may also be dissolved in ethanol (90%) and potassium iodide (2.5%) solution to give weak iodine solution, BP 1988 (Iodine Tincture).

122 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 2. Agents that denature proteins Agents that can alter the structure of protein make the protein nonfunctional. a. Acids and alkalies: These agents release their free H+ and OH- ions and than through undissociated molecules, or by altering the pH of the medium they denature the proteins of the organisms, e.g., benzoic acid, propionic acid etc. 3. Agent acting on functional group of proteins a. Heavy metals: Mercury and silver have long been known to have antibacterial properties and preparations of these metals were among the earliest used antiseptics, but have been replaced by less toxic compounds. Other metals such as zinc, copper, aluminium and tin have weak antibacterial properties but are used in medicine for other functions, e.g., aluminium acetate and zinc sulphate are employed as astringents. b. Oxidizing agents: This group includes hydrogen peroxide, halogens and potassium permanganate, etc. The germicidal properties of hydrogen peroxide (H2O2 ) have been known for more than a century. It is used as an antiseptic for open wounds and ulcers where it provides additional cleansing due to its oxidation of organic debris. Its activity against the protozoa, Acanthamoeba, which can cause keratitis in contact lens wearers, has made it popular for disinfection of soft contact lenses. Concentrations of 3-6% are effective for general disinfection purposes. At high concentrations (up to 30%) and increased temperature hydrogen peroxide is sporicidal. Use has been made of this in vapour- phase hydrogen peroxide decontamination of laboratory equipment and enclosed spaces. Halogens (chlorine and iodine) have been used extensively since their introduction as disinfecting agents in the early 19th century. Preparations containing these halogens such as Dakin’s solution and tincture of iodine were early inclusions in many pharmacopoeiae and national formularies. More recent formulations of these elements have improved activity, stability and ease of use. A large number of antimicrobially active chlorine compounds are commercially available, one of the most important being liquid chlorine. Iodine has a wide spectrum of antimicrobial activity. Gramnegative and Gram-positive organisms, bacterial spores (on extended exposure), mycobacteria, fungi and viruses are all susceptible. The active agent is the elemental iodine molecule, I2. As elemental iodine is only slightly soluble in water, iodide ions are required for aqueous solutions such as aqueous iodine solution, BP 1988 (Lugol’s solution) containing 5% iodine in 10% potassium iodide solution. Iodine (2.5%) may also be dissolved in ethanol (90%) and potassium iodide (2.5%) solution to give weak iodine solution, BP 1988 (Iodine Tincture).

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c. Dyes. Crystal violet (Gentian violet), brilliant green and malachite green are triphenyl-methane dyes widely used to stain bacteria for microscopic examination. They also have bacteriostatic and fungistatic activity and have been applied topically for the treatment of infections. Staining of skin and clothes is a disadvantage of these agents. Due to concern about possible carcinogenicity, they are now rarely used. The acridine dyes, including proflavine, acriflavine and aminacrine, have also been employed for skin disinfection and treatment of infected wounds or burns. They are slow-acting and mainly bacteriostatic in effect, with no useful fungicidal or sporicidal activity. d. Alkylating agents. This group includes formaldehyde, glutaraldehyde, and ethylene oxide. All these cause lethal effect upon proteins. A number of aldehydes possess antimicrobial properties, including sporicidal activity; however, only two, formaldehyde and glutaraldehyde, are used for disinfection. Formaldehyde (HCHO) can be used in either the liquid or gaseous state for disinfection purposes. In the vapour phase it has been used for decontamination of safety cabinets and rooms; however, recent trends have been to combine formaldehyde vapour with low temperature steam (LTSF) for the sterilization of heat-sensitive items. Formaldehyde vapour is highly toxic and potentially carcinogenic if inhaled, thus its use must be carefully controlled. Glutaraldehyde (CHO(CH2)3CHO) has a broad spectrum of antimicrobial activity and rapid rate of kill, most vegetative bacteria being killed within a minute of exposure, although bacterial spores may require 3 hours or more. It has the further advantage of not being affected significantly by organic matter. The glutaraldehyde molecule possesses two aldehyde groupings which are highly reactive and their presence is an important component of biocidal activity. At a pH of 8, biocidal activity is the greatest but stability is poor due to polymerization. In practice, glutaraldehyde is generally supplied as an acidic 2% aqueous solution, which is stable on prolonged storage. Glutaraldehyde is employed mainly for the cold, liquid chemical sterilization of medical and surgical materials that cannot be sterilized by other methods. Endoscopes, including for example, arthroscopes, laparoscopes, cystoscopes and bronchoscopes may be decontaminated by glutaraldehyde treatment. Ethylene oxide is used to sterilize fragile, heat sensitive equipment, powders as well as components of spacecraft.

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c. Dyes. Crystal violet (Gentian violet), brilliant green and malachite green are triphenyl-methane dyes widely used to stain bacteria for microscopic examination. They also have bacteriostatic and fungistatic activity and have been applied topically for the treatment of infections. Staining of skin and clothes is a disadvantage of these agents. Due to concern about possible carcinogenicity, they are now rarely used. The acridine dyes, including proflavine, acriflavine and aminacrine, have also been employed for skin disinfection and treatment of infected wounds or burns. They are slow-acting and mainly bacteriostatic in effect, with no useful fungicidal or sporicidal activity. d. Alkylating agents. This group includes formaldehyde, glutaraldehyde, and ethylene oxide. All these cause lethal effect upon proteins. A number of aldehydes possess antimicrobial properties, including sporicidal activity; however, only two, formaldehyde and glutaraldehyde, are used for disinfection. Formaldehyde (HCHO) can be used in either the liquid or gaseous state for disinfection purposes. In the vapour phase it has been used for decontamination of safety cabinets and rooms; however, recent trends have been to combine formaldehyde vapour with low temperature steam (LTSF) for the sterilization of heat-sensitive items. Formaldehyde vapour is highly toxic and potentially carcinogenic if inhaled, thus its use must be carefully controlled. Glutaraldehyde (CHO(CH2)3CHO) has a broad spectrum of antimicrobial activity and rapid rate of kill, most vegetative bacteria being killed within a minute of exposure, although bacterial spores may require 3 hours or more. It has the further advantage of not being affected significantly by organic matter. The glutaraldehyde molecule possesses two aldehyde groupings which are highly reactive and their presence is an important component of biocidal activity. At a pH of 8, biocidal activity is the greatest but stability is poor due to polymerization. In practice, glutaraldehyde is generally supplied as an acidic 2% aqueous solution, which is stable on prolonged storage. Glutaraldehyde is employed mainly for the cold, liquid chemical sterilization of medical and surgical materials that cannot be sterilized by other methods. Endoscopes, including for example, arthroscopes, laparoscopes, cystoscopes and bronchoscopes may be decontaminated by glutaraldehyde treatment. Ethylene oxide is used to sterilize fragile, heat sensitive equipment, powders as well as components of spacecraft.

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c. Dyes. Crystal violet (Gentian violet), brilliant green and malachite green are triphenyl-methane dyes widely used to stain bacteria for microscopic examination. They also have bacteriostatic and fungistatic activity and have been applied topically for the treatment of infections. Staining of skin and clothes is a disadvantage of these agents. Due to concern about possible carcinogenicity, they are now rarely used. The acridine dyes, including proflavine, acriflavine and aminacrine, have also been employed for skin disinfection and treatment of infected wounds or burns. They are slow-acting and mainly bacteriostatic in effect, with no useful fungicidal or sporicidal activity. d. Alkylating agents. This group includes formaldehyde, glutaraldehyde, and ethylene oxide. All these cause lethal effect upon proteins. A number of aldehydes possess antimicrobial properties, including sporicidal activity; however, only two, formaldehyde and glutaraldehyde, are used for disinfection. Formaldehyde (HCHO) can be used in either the liquid or gaseous state for disinfection purposes. In the vapour phase it has been used for decontamination of safety cabinets and rooms; however, recent trends have been to combine formaldehyde vapour with low temperature steam (LTSF) for the sterilization of heat-sensitive items. Formaldehyde vapour is highly toxic and potentially carcinogenic if inhaled, thus its use must be carefully controlled. Glutaraldehyde (CHO(CH2)3CHO) has a broad spectrum of antimicrobial activity and rapid rate of kill, most vegetative bacteria being killed within a minute of exposure, although bacterial spores may require 3 hours or more. It has the further advantage of not being affected significantly by organic matter. The glutaraldehyde molecule possesses two aldehyde groupings which are highly reactive and their presence is an important component of biocidal activity. At a pH of 8, biocidal activity is the greatest but stability is poor due to polymerization. In practice, glutaraldehyde is generally supplied as an acidic 2% aqueous solution, which is stable on prolonged storage. Glutaraldehyde is employed mainly for the cold, liquid chemical sterilization of medical and surgical materials that cannot be sterilized by other methods. Endoscopes, including for example, arthroscopes, laparoscopes, cystoscopes and bronchoscopes may be decontaminated by glutaraldehyde treatment. Ethylene oxide is used to sterilize fragile, heat sensitive equipment, powders as well as components of spacecraft.

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c. Dyes. Crystal violet (Gentian violet), brilliant green and malachite green are triphenyl-methane dyes widely used to stain bacteria for microscopic examination. They also have bacteriostatic and fungistatic activity and have been applied topically for the treatment of infections. Staining of skin and clothes is a disadvantage of these agents. Due to concern about possible carcinogenicity, they are now rarely used. The acridine dyes, including proflavine, acriflavine and aminacrine, have also been employed for skin disinfection and treatment of infected wounds or burns. They are slow-acting and mainly bacteriostatic in effect, with no useful fungicidal or sporicidal activity. d. Alkylating agents. This group includes formaldehyde, glutaraldehyde, and ethylene oxide. All these cause lethal effect upon proteins. A number of aldehydes possess antimicrobial properties, including sporicidal activity; however, only two, formaldehyde and glutaraldehyde, are used for disinfection. Formaldehyde (HCHO) can be used in either the liquid or gaseous state for disinfection purposes. In the vapour phase it has been used for decontamination of safety cabinets and rooms; however, recent trends have been to combine formaldehyde vapour with low temperature steam (LTSF) for the sterilization of heat-sensitive items. Formaldehyde vapour is highly toxic and potentially carcinogenic if inhaled, thus its use must be carefully controlled. Glutaraldehyde (CHO(CH2)3CHO) has a broad spectrum of antimicrobial activity and rapid rate of kill, most vegetative bacteria being killed within a minute of exposure, although bacterial spores may require 3 hours or more. It has the further advantage of not being affected significantly by organic matter. The glutaraldehyde molecule possesses two aldehyde groupings which are highly reactive and their presence is an important component of biocidal activity. At a pH of 8, biocidal activity is the greatest but stability is poor due to polymerization. In practice, glutaraldehyde is generally supplied as an acidic 2% aqueous solution, which is stable on prolonged storage. Glutaraldehyde is employed mainly for the cold, liquid chemical sterilization of medical and surgical materials that cannot be sterilized by other methods. Endoscopes, including for example, arthroscopes, laparoscopes, cystoscopes and bronchoscopes may be decontaminated by glutaraldehyde treatment. Ethylene oxide is used to sterilize fragile, heat sensitive equipment, powders as well as components of spacecraft.

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124 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

9.4.3 Gaseous Sterilization Many substances like thermolabile solid medicaments and thermolabile equipment (article of plastics, electrical diagnostic equipment, delicate rubber items, and blankets) are not sterilized by other methods like heat and chemical sterilization. So such types of substances are sterilized by gaseous sterilization. Formaldehyde was once widely used, but at present ethylene oxide is the only compound used in pharmaceutical and medical fields. At room temperature it is a colourless gas with a characterstic ethereal order. Concentrations greater than 3% in air are highly inflammable. So the mixture containing 1 part of ethylene oxide with 9 parts of CO2 is used. It is mainly used in chamber from which at least 95% of the air has been removed so the ethylene oxide used in the absence of air. Ethylene oxide is a powerful alkylating agent and its antimicrobial activity is probably due to alkylation of the sulphydryl, imino, carboxyl and hydroxyl group of proteins and other important cell constituents.

9.4.3 Gaseous Sterilization Many substances like thermolabile solid medicaments and thermolabile equipment (article of plastics, electrical diagnostic equipment, delicate rubber items, and blankets) are not sterilized by other methods like heat and chemical sterilization. So such types of substances are sterilized by gaseous sterilization. Formaldehyde was once widely used, but at present ethylene oxide is the only compound used in pharmaceutical and medical fields. At room temperature it is a colourless gas with a characterstic ethereal order. Concentrations greater than 3% in air are highly inflammable. So the mixture containing 1 part of ethylene oxide with 9 parts of CO2 is used. It is mainly used in chamber from which at least 95% of the air has been removed so the ethylene oxide used in the absence of air. Ethylene oxide is a powerful alkylating agent and its antimicrobial activity is probably due to alkylation of the sulphydryl, imino, carboxyl and hydroxyl group of proteins and other important cell constituents.

Applications

Applications

1. Powders: Thermolabile as well as thermostable powders like talc and mize starch are sterilized by gaseous sterilization. 2. Sterilization of eye drops in plastic uni dose containers. 3. Equipment: Plastic equipment like syringes, needles, catheters, intravenous sets, blood oxygenators bottles and vials. 4. Fragile rubber articles survive more, by the treatment of ethylene oxide. 5. This method has been used for the sterilization of mills before grinding of sterile powders. 6. This method is widely used in the sterilization of blankets in the hospital wards and theaters. 7. Used in sterility testing.

9.5

FACTORY AND HOSPITAL HYGIENE: CONTROL OF MICROBIAL CONTAMINATION DURING MANUFACTURE

1. Powders: Thermolabile as well as thermostable powders like talc and mize starch are sterilized by gaseous sterilization. 2. Sterilization of eye drops in plastic uni dose containers. 3. Equipment: Plastic equipment like syringes, needles, catheters, intravenous sets, blood oxygenators bottles and vials. 4. Fragile rubber articles survive more, by the treatment of ethylene oxide. 5. This method has been used for the sterilization of mills before grinding of sterile powders. 6. This method is widely used in the sterilization of blankets in the hospital wards and theaters. 7. Used in sterility testing.

9.5

FACTORY AND HOSPITAL HYGIENE: CONTROL OF MICROBIAL CONTAMINATION DURING MANUFACTURE

9.5.1 Sterile Manufacturing of Products in Factory or Industry All the parental products and other drugs should be manufactured in sterile conditions. These areas should be free from microbial contamination. For the sterile manufacturing following equipment are necessary to meet the requirement of Drugs and Cosmetic Act. 1. Storage cabinets 2. Storage equipment for ampoules and vials 3. Ampoule washing machine 4. Ampoule drying machine

9.5.1 Sterile Manufacturing of Products in Factory or Industry All the parental products and other drugs should be manufactured in sterile conditions. These areas should be free from microbial contamination. For the sterile manufacturing following equipment are necessary to meet the requirement of Drugs and Cosmetic Act. 1. Storage cabinets 2. Storage equipment for ampoules and vials 3. Ampoule washing machine 4. Ampoule drying machine

124 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

124 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

9.4.3 Gaseous Sterilization Many substances like thermolabile solid medicaments and thermolabile equipment (article of plastics, electrical diagnostic equipment, delicate rubber items, and blankets) are not sterilized by other methods like heat and chemical sterilization. So such types of substances are sterilized by gaseous sterilization. Formaldehyde was once widely used, but at present ethylene oxide is the only compound used in pharmaceutical and medical fields. At room temperature it is a colourless gas with a characterstic ethereal order. Concentrations greater than 3% in air are highly inflammable. So the mixture containing 1 part of ethylene oxide with 9 parts of CO2 is used. It is mainly used in chamber from which at least 95% of the air has been removed so the ethylene oxide used in the absence of air. Ethylene oxide is a powerful alkylating agent and its antimicrobial activity is probably due to alkylation of the sulphydryl, imino, carboxyl and hydroxyl group of proteins and other important cell constituents.

9.4.3 Gaseous Sterilization Many substances like thermolabile solid medicaments and thermolabile equipment (article of plastics, electrical diagnostic equipment, delicate rubber items, and blankets) are not sterilized by other methods like heat and chemical sterilization. So such types of substances are sterilized by gaseous sterilization. Formaldehyde was once widely used, but at present ethylene oxide is the only compound used in pharmaceutical and medical fields. At room temperature it is a colourless gas with a characterstic ethereal order. Concentrations greater than 3% in air are highly inflammable. So the mixture containing 1 part of ethylene oxide with 9 parts of CO2 is used. It is mainly used in chamber from which at least 95% of the air has been removed so the ethylene oxide used in the absence of air. Ethylene oxide is a powerful alkylating agent and its antimicrobial activity is probably due to alkylation of the sulphydryl, imino, carboxyl and hydroxyl group of proteins and other important cell constituents.

Applications

Applications

1. Powders: Thermolabile as well as thermostable powders like talc and mize starch are sterilized by gaseous sterilization. 2. Sterilization of eye drops in plastic uni dose containers. 3. Equipment: Plastic equipment like syringes, needles, catheters, intravenous sets, blood oxygenators bottles and vials. 4. Fragile rubber articles survive more, by the treatment of ethylene oxide. 5. This method has been used for the sterilization of mills before grinding of sterile powders. 6. This method is widely used in the sterilization of blankets in the hospital wards and theaters. 7. Used in sterility testing.

9.5

FACTORY AND HOSPITAL HYGIENE: CONTROL OF MICROBIAL CONTAMINATION DURING MANUFACTURE

9.5.1 Sterile Manufacturing of Products in Factory or Industry All the parental products and other drugs should be manufactured in sterile conditions. These areas should be free from microbial contamination. For the sterile manufacturing following equipment are necessary to meet the requirement of Drugs and Cosmetic Act. 1. Storage cabinets 2. Storage equipment for ampoules and vials 3. Ampoule washing machine 4. Ampoule drying machine

1. Powders: Thermolabile as well as thermostable powders like talc and mize starch are sterilized by gaseous sterilization. 2. Sterilization of eye drops in plastic uni dose containers. 3. Equipment: Plastic equipment like syringes, needles, catheters, intravenous sets, blood oxygenators bottles and vials. 4. Fragile rubber articles survive more, by the treatment of ethylene oxide. 5. This method has been used for the sterilization of mills before grinding of sterile powders. 6. This method is widely used in the sterilization of blankets in the hospital wards and theaters. 7. Used in sterility testing.

9.5

FACTORY AND HOSPITAL HYGIENE: CONTROL OF MICROBIAL CONTAMINATION DURING MANUFACTURE

9.5.1 Sterile Manufacturing of Products in Factory or Industry All the parental products and other drugs should be manufactured in sterile conditions. These areas should be free from microbial contamination. For the sterile manufacturing following equipment are necessary to meet the requirement of Drugs and Cosmetic Act. 1. Storage cabinets 2. Storage equipment for ampoules and vials 3. Ampoule washing machine 4. Ampoule drying machine

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5. 6. 7. 8. 9. 10. 11.

125

Filling and sealing unit Sintered glass funnel Filter press Hot air oven Equipment for evaluation and quality control Autoclave Labelling and packing units.

STERILIZATION

5. 6. 7. 8. 9. 10. 11.

125

Filling and sealing unit Sintered glass funnel Filter press Hot air oven Equipment for evaluation and quality control Autoclave Labelling and packing units.

To control the microbial contamination during manufacture all the area should be clean and be dust free. There should be highly cleanliness in the aseptic filling rooms. For construction, it requires neat material and design. The ceiling walls and floors should be constructed of materials which are easy to clean and non-porous so as to prevent accumulation of dust and moisture. The floor of these areas should be made up of materials like ceramic cement applied as a thick coat on the existing floor to give continuous sealed surface. Persons those are working in these areas should be wear gloves, shoes, hats and face mask. Once they enter the aseptic area, they are not permitted to move out of the area till the manufacturing cycle is completed. Unauthorised person should not be permitted into aseptic area. Generally, cleaning is done at the end of working day or during night. It includes all the surface area like ceiling, walls, floors, counters, equipment, etc. it should be kept in mind that cleaning should never be done just prior to the beginning of the production process. For cleaning liquid disinfectant should be sprayed or wiped on all surface. Whole rooms are sterilized by the UV radiation. UV rays are antibacterial in action.

To control the microbial contamination during manufacture all the area should be clean and be dust free. There should be highly cleanliness in the aseptic filling rooms. For construction, it requires neat material and design. The ceiling walls and floors should be constructed of materials which are easy to clean and non-porous so as to prevent accumulation of dust and moisture. The floor of these areas should be made up of materials like ceramic cement applied as a thick coat on the existing floor to give continuous sealed surface. Persons those are working in these areas should be wear gloves, shoes, hats and face mask. Once they enter the aseptic area, they are not permitted to move out of the area till the manufacturing cycle is completed. Unauthorised person should not be permitted into aseptic area. Generally, cleaning is done at the end of working day or during night. It includes all the surface area like ceiling, walls, floors, counters, equipment, etc. it should be kept in mind that cleaning should never be done just prior to the beginning of the production process. For cleaning liquid disinfectant should be sprayed or wiped on all surface. Whole rooms are sterilized by the UV radiation. UV rays are antibacterial in action.

9.6 CLEAN ROOMS

9.6 CLEAN ROOMS

The air of the aseptic area can be one of the greatest sources of contamination. It is necessary to clean the air before it is allowed to enter into the aseptic area. Clean rooms are generally classified into two categories. 1. Conventional clean room systems. 2. Laminar air flow clean room system.

The air of the aseptic area can be one of the greatest sources of contamination. It is necessary to clean the air before it is allowed to enter into the aseptic area. Clean rooms are generally classified into two categories. 1. Conventional clean room systems. 2. Laminar air flow clean room system.

1. Conventional clean room system

1. Conventional clean room system

The term conventional clean room is applied where a lesser degree of cleanliness such as class 10,000 clean room which is defined as such an environment containing not more than 10,000 particles per cubic feet of 0.5 micrometre and larger size. Conventional clean room helps in handling the air, i.e., air-conditioning whereby the dust particles, humidity and temperature is controlled. This dehumidified, cooled and filtered air is made to enter the room at a velocity of 800 ft/min. Conventionally two air-conditioners one at the ceiling and other at

The term conventional clean room is applied where a lesser degree of cleanliness such as class 10,000 clean room which is defined as such an environment containing not more than 10,000 particles per cubic feet of 0.5 micrometre and larger size. Conventional clean room helps in handling the air, i.e., air-conditioning whereby the dust particles, humidity and temperature is controlled. This dehumidified, cooled and filtered air is made to enter the room at a velocity of 800 ft/min. Conventionally two air-conditioners one at the ceiling and other at

STERILIZATION

5. 6. 7. 8. 9. 10. 11.

125

Filling and sealing unit Sintered glass funnel Filter press Hot air oven Equipment for evaluation and quality control Autoclave Labelling and packing units.

STERILIZATION

5. 6. 7. 8. 9. 10. 11.

125

Filling and sealing unit Sintered glass funnel Filter press Hot air oven Equipment for evaluation and quality control Autoclave Labelling and packing units.

To control the microbial contamination during manufacture all the area should be clean and be dust free. There should be highly cleanliness in the aseptic filling rooms. For construction, it requires neat material and design. The ceiling walls and floors should be constructed of materials which are easy to clean and non-porous so as to prevent accumulation of dust and moisture. The floor of these areas should be made up of materials like ceramic cement applied as a thick coat on the existing floor to give continuous sealed surface. Persons those are working in these areas should be wear gloves, shoes, hats and face mask. Once they enter the aseptic area, they are not permitted to move out of the area till the manufacturing cycle is completed. Unauthorised person should not be permitted into aseptic area. Generally, cleaning is done at the end of working day or during night. It includes all the surface area like ceiling, walls, floors, counters, equipment, etc. it should be kept in mind that cleaning should never be done just prior to the beginning of the production process. For cleaning liquid disinfectant should be sprayed or wiped on all surface. Whole rooms are sterilized by the UV radiation. UV rays are antibacterial in action.

To control the microbial contamination during manufacture all the area should be clean and be dust free. There should be highly cleanliness in the aseptic filling rooms. For construction, it requires neat material and design. The ceiling walls and floors should be constructed of materials which are easy to clean and non-porous so as to prevent accumulation of dust and moisture. The floor of these areas should be made up of materials like ceramic cement applied as a thick coat on the existing floor to give continuous sealed surface. Persons those are working in these areas should be wear gloves, shoes, hats and face mask. Once they enter the aseptic area, they are not permitted to move out of the area till the manufacturing cycle is completed. Unauthorised person should not be permitted into aseptic area. Generally, cleaning is done at the end of working day or during night. It includes all the surface area like ceiling, walls, floors, counters, equipment, etc. it should be kept in mind that cleaning should never be done just prior to the beginning of the production process. For cleaning liquid disinfectant should be sprayed or wiped on all surface. Whole rooms are sterilized by the UV radiation. UV rays are antibacterial in action.

9.6 CLEAN ROOMS

9.6 CLEAN ROOMS

The air of the aseptic area can be one of the greatest sources of contamination. It is necessary to clean the air before it is allowed to enter into the aseptic area. Clean rooms are generally classified into two categories. 1. Conventional clean room systems. 2. Laminar air flow clean room system.

The air of the aseptic area can be one of the greatest sources of contamination. It is necessary to clean the air before it is allowed to enter into the aseptic area. Clean rooms are generally classified into two categories. 1. Conventional clean room systems. 2. Laminar air flow clean room system.

1. Conventional clean room system

1. Conventional clean room system

The term conventional clean room is applied where a lesser degree of cleanliness such as class 10,000 clean room which is defined as such an environment containing not more than 10,000 particles per cubic feet of 0.5 micrometre and larger size. Conventional clean room helps in handling the air, i.e., air-conditioning whereby the dust particles, humidity and temperature is controlled. This dehumidified, cooled and filtered air is made to enter the room at a velocity of 800 ft/min. Conventionally two air-conditioners one at the ceiling and other at

The term conventional clean room is applied where a lesser degree of cleanliness such as class 10,000 clean room which is defined as such an environment containing not more than 10,000 particles per cubic feet of 0.5 micrometre and larger size. Conventional clean room helps in handling the air, i.e., air-conditioning whereby the dust particles, humidity and temperature is controlled. This dehumidified, cooled and filtered air is made to enter the room at a velocity of 800 ft/min. Conventionally two air-conditioners one at the ceiling and other at

126 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

126 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

the bench level are fixed in a room. The working bench is placed in the central area which is comparatively clean as compared to the other parts of the room. It is very difficult to maintain a conventional clean room and its maintenance is disadvantageous for reasons such as: 1. Lots of space is wasted. 2. Temperature, humidity and dust are controlled all at one time which becomes difficult. 3. Formation of the dust pockets occurs which contaminates the area. Conventional clean room system is not suitable for the aseptic processing units but instead is used for maintaining general cleanliness in the establishment.

the bench level are fixed in a room. The working bench is placed in the central area which is comparatively clean as compared to the other parts of the room. It is very difficult to maintain a conventional clean room and its maintenance is disadvantageous for reasons such as: 1. Lots of space is wasted. 2. Temperature, humidity and dust are controlled all at one time which becomes difficult. 3. Formation of the dust pockets occurs which contaminates the area. Conventional clean room system is not suitable for the aseptic processing units but instead is used for maintaining general cleanliness in the establishment.

2. Laminar air flow clean room system

2. Laminar air flow clean room system

Laminar air flow provides complete removal of microorganisms from the working area in the laminar air hood by air moves with uniform velocity along the parallel lines, originating through high efficiency particulate air (HEPA) filters. Therefore, the entire area of the working bench receives clean air and removes all the contaminants. The effective air velocity is 100 ± 20 ft./min. The cabinet is usually made of stainless steel with no gaps or joints where spores might

Laminar air flow provides complete removal of microorganisms from the working area in the laminar air hood by air moves with uniform velocity along the parallel lines, originating through high efficiency particulate air (HEPA) filters. Therefore, the entire area of the working bench receives clean air and removes all the contaminants. The effective air velocity is 100 ± 20 ft./min. The cabinet is usually made of stainless steel with no gaps or joints where spores might

Fig. 9.6. Laminar air flow.

Fig. 9.6. Laminar air flow.

126 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

126 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

the bench level are fixed in a room. The working bench is placed in the central area which is comparatively clean as compared to the other parts of the room. It is very difficult to maintain a conventional clean room and its maintenance is disadvantageous for reasons such as: 1. Lots of space is wasted. 2. Temperature, humidity and dust are controlled all at one time which becomes difficult. 3. Formation of the dust pockets occurs which contaminates the area. Conventional clean room system is not suitable for the aseptic processing units but instead is used for maintaining general cleanliness in the establishment.

the bench level are fixed in a room. The working bench is placed in the central area which is comparatively clean as compared to the other parts of the room. It is very difficult to maintain a conventional clean room and its maintenance is disadvantageous for reasons such as: 1. Lots of space is wasted. 2. Temperature, humidity and dust are controlled all at one time which becomes difficult. 3. Formation of the dust pockets occurs which contaminates the area. Conventional clean room system is not suitable for the aseptic processing units but instead is used for maintaining general cleanliness in the establishment.

2. Laminar air flow clean room system

2. Laminar air flow clean room system

Laminar air flow provides complete removal of microorganisms from the working area in the laminar air hood by air moves with uniform velocity along the parallel lines, originating through high efficiency particulate air (HEPA) filters. Therefore, the entire area of the working bench receives clean air and removes all the contaminants. The effective air velocity is 100 ± 20 ft./min. The cabinet is usually made of stainless steel with no gaps or joints where spores might

Laminar air flow provides complete removal of microorganisms from the working area in the laminar air hood by air moves with uniform velocity along the parallel lines, originating through high efficiency particulate air (HEPA) filters. Therefore, the entire area of the working bench receives clean air and removes all the contaminants. The effective air velocity is 100 ± 20 ft./min. The cabinet is usually made of stainless steel with no gaps or joints where spores might

Fig. 9.6. Laminar air flow.

Fig. 9.6. Laminar air flow.

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127

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127

collect. Laminar flow cabinets may have a UV lamp to sterilize the shell and contents when not in use. (It is important to switch off this light off during use, as it will quickly give any exposed skin sunburn and may cause cataracts). Such hoods exist in both horizontal and vertical configurations, and there are different types of cabinets with a variety of airflow patterns and acceptable uses. NSF49 is the commonly accepted regulatory standard for these cabinets. In a typical horizontal laminar air flow air is taken from the lower front through a prefilter (Fig. 9.6) usually composed of a spun glass. This air is come out from the HEPA filter in the working bench at the velocity of 230 ± 90 cm/min. The filter removes 99.97% of the particles of size 0.20 —m to 20 —m or above, thus removing inert solid material as well as airborne microorganisms. Laminar air flow prevents the formation of eddies (Fig. 9.7) of air and then maintains the integrity of the internal environment. The HEPA filter should be replaced when the velocity of the air falls below 22 m/min. but the prefilters are changed at monthly intervals.

collect. Laminar flow cabinets may have a UV lamp to sterilize the shell and contents when not in use. (It is important to switch off this light off during use, as it will quickly give any exposed skin sunburn and may cause cataracts). Such hoods exist in both horizontal and vertical configurations, and there are different types of cabinets with a variety of airflow patterns and acceptable uses. NSF49 is the commonly accepted regulatory standard for these cabinets. In a typical horizontal laminar air flow air is taken from the lower front through a prefilter (Fig. 9.6) usually composed of a spun glass. This air is come out from the HEPA filter in the working bench at the velocity of 230 ± 90 cm/min. The filter removes 99.97% of the particles of size 0.20 —m to 20 —m or above, thus removing inert solid material as well as airborne microorganisms. Laminar air flow prevents the formation of eddies (Fig. 9.7) of air and then maintains the integrity of the internal environment. The HEPA filter should be replaced when the velocity of the air falls below 22 m/min. but the prefilters are changed at monthly intervals.

Fig. 9.7. The flow of air inside the cabinet.

Fig. 9.7. The flow of air inside the cabinet.

9.6.1 Manufacture of Sterile Products The area that contains more than 100 particles per cubic feet of 0.5—m and large size can be achieved by laminar flow of HEPA filters whereas clear rooms such as class 10,000 can be defined as “the area that contains not more than 10,000 particles per cubic feet”. The sterile area can be divided into the following categories: 1. Clean area 2. Compounding area or preparation area 3. Aseptic area. 4. Quarantine area 5. Finishing and packaging area.

9.6.1 Manufacture of Sterile Products The area that contains more than 100 particles per cubic feet of 0.5—m and large size can be achieved by laminar flow of HEPA filters whereas clear rooms such as class 10,000 can be defined as “the area that contains not more than 10,000 particles per cubic feet”. The sterile area can be divided into the following categories: 1. Clean area 2. Compounding area or preparation area 3. Aseptic area. 4. Quarantine area 5. Finishing and packaging area.

1. Clean area: This area has walls and ceilings with film coating material. There should be no holes, corners or projections. The air should be free

1. Clean area: This area has walls and ceilings with film coating material. There should be no holes, corners or projections. The air should be free

STERILIZATION

127

STERILIZATION

127

collect. Laminar flow cabinets may have a UV lamp to sterilize the shell and contents when not in use. (It is important to switch off this light off during use, as it will quickly give any exposed skin sunburn and may cause cataracts). Such hoods exist in both horizontal and vertical configurations, and there are different types of cabinets with a variety of airflow patterns and acceptable uses. NSF49 is the commonly accepted regulatory standard for these cabinets. In a typical horizontal laminar air flow air is taken from the lower front through a prefilter (Fig. 9.6) usually composed of a spun glass. This air is come out from the HEPA filter in the working bench at the velocity of 230 ± 90 cm/min. The filter removes 99.97% of the particles of size 0.20 —m to 20 —m or above, thus removing inert solid material as well as airborne microorganisms. Laminar air flow prevents the formation of eddies (Fig. 9.7) of air and then maintains the integrity of the internal environment. The HEPA filter should be replaced when the velocity of the air falls below 22 m/min. but the prefilters are changed at monthly intervals.

collect. Laminar flow cabinets may have a UV lamp to sterilize the shell and contents when not in use. (It is important to switch off this light off during use, as it will quickly give any exposed skin sunburn and may cause cataracts). Such hoods exist in both horizontal and vertical configurations, and there are different types of cabinets with a variety of airflow patterns and acceptable uses. NSF49 is the commonly accepted regulatory standard for these cabinets. In a typical horizontal laminar air flow air is taken from the lower front through a prefilter (Fig. 9.6) usually composed of a spun glass. This air is come out from the HEPA filter in the working bench at the velocity of 230 ± 90 cm/min. The filter removes 99.97% of the particles of size 0.20 —m to 20 —m or above, thus removing inert solid material as well as airborne microorganisms. Laminar air flow prevents the formation of eddies (Fig. 9.7) of air and then maintains the integrity of the internal environment. The HEPA filter should be replaced when the velocity of the air falls below 22 m/min. but the prefilters are changed at monthly intervals.

Fig. 9.7. The flow of air inside the cabinet.

Fig. 9.7. The flow of air inside the cabinet.

9.6.1 Manufacture of Sterile Products The area that contains more than 100 particles per cubic feet of 0.5—m and large size can be achieved by laminar flow of HEPA filters whereas clear rooms such as class 10,000 can be defined as “the area that contains not more than 10,000 particles per cubic feet”. The sterile area can be divided into the following categories: 1. Clean area 2. Compounding area or preparation area 3. Aseptic area. 4. Quarantine area 5. Finishing and packaging area.

9.6.1 Manufacture of Sterile Products The area that contains more than 100 particles per cubic feet of 0.5—m and large size can be achieved by laminar flow of HEPA filters whereas clear rooms such as class 10,000 can be defined as “the area that contains not more than 10,000 particles per cubic feet”. The sterile area can be divided into the following categories: 1. Clean area 2. Compounding area or preparation area 3. Aseptic area. 4. Quarantine area 5. Finishing and packaging area.

1. Clean area: This area has walls and ceilings with film coating material. There should be no holes, corners or projections. The air should be free

1. Clean area: This area has walls and ceilings with film coating material. There should be no holes, corners or projections. The air should be free

128 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

2.

3.

4.

5.

from dirt and microbes. The room should undergo a minimum of 10-15 air changes per hour. All incoming air should be passed through filters with an efficiency of atleast 95%. Preparation of compounding area: In this area formula is compounded and for this it is not essential that the area should be aseptic but controlling measures are provided to control the dust generated from weighing and compounding procedures. Cabinets and counters are generally made up of stainless steel. The ceiling, walls should be made of such materials which are impervious to water. Aseptic area: a. The ceiling walls and floor must be sealed properly so that they can be washed and disinfected when required. b. All the counters are made up of stainless steel and they are constructed in such a way that dirt particles shall not accumulate. c. As much as possible storage tanks, mixing tanks containing the compounded products, should remain outside the aseptic area and then the products are fed into aseptic area through pipeline. Quarantine area: The purpose of quarantine area is that batches can be stored physically segregated from either “in process” batches or approved batches in a lock to which access is restricted to a responsible person. Facility should be available for segregation between batches and the storage system is designed in such a way that easy access to each batch of the product can be made by an approved person in case of resampling. Labelling and packing area: In this batch numbering and over-printing of labels should take place. Adequate space is required for installation of over-printing devices and packaging machines. Only one type of product label should be processed at any one time.

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2.

3.

4.

5.

(Note: For details see sterilization methods and their applications.)

128 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

2.

3.

4.

5.

from dirt and microbes. The room should undergo a minimum of 10-15 air changes per hour. All incoming air should be passed through filters with an efficiency of atleast 95%. Preparation of compounding area: In this area formula is compounded and for this it is not essential that the area should be aseptic but controlling measures are provided to control the dust generated from weighing and compounding procedures. Cabinets and counters are generally made up of stainless steel. The ceiling, walls should be made of such materials which are impervious to water. Aseptic area: a. The ceiling walls and floor must be sealed properly so that they can be washed and disinfected when required. b. All the counters are made up of stainless steel and they are constructed in such a way that dirt particles shall not accumulate. c. As much as possible storage tanks, mixing tanks containing the compounded products, should remain outside the aseptic area and then the products are fed into aseptic area through pipeline. Quarantine area: The purpose of quarantine area is that batches can be stored physically segregated from either “in process” batches or approved batches in a lock to which access is restricted to a responsible person. Facility should be available for segregation between batches and the storage system is designed in such a way that easy access to each batch of the product can be made by an approved person in case of resampling. Labelling and packing area: In this batch numbering and over-printing of labels should take place. Adequate space is required for installation of over-printing devices and packaging machines. Only one type of product label should be processed at any one time.

(Note: For details see sterilization methods and their applications.)

from dirt and microbes. The room should undergo a minimum of 10-15 air changes per hour. All incoming air should be passed through filters with an efficiency of atleast 95%. Preparation of compounding area: In this area formula is compounded and for this it is not essential that the area should be aseptic but controlling measures are provided to control the dust generated from weighing and compounding procedures. Cabinets and counters are generally made up of stainless steel. The ceiling, walls should be made of such materials which are impervious to water. Aseptic area: a. The ceiling walls and floor must be sealed properly so that they can be washed and disinfected when required. b. All the counters are made up of stainless steel and they are constructed in such a way that dirt particles shall not accumulate. c. As much as possible storage tanks, mixing tanks containing the compounded products, should remain outside the aseptic area and then the products are fed into aseptic area through pipeline. Quarantine area: The purpose of quarantine area is that batches can be stored physically segregated from either “in process” batches or approved batches in a lock to which access is restricted to a responsible person. Facility should be available for segregation between batches and the storage system is designed in such a way that easy access to each batch of the product can be made by an approved person in case of resampling. Labelling and packing area: In this batch numbering and over-printing of labels should take place. Adequate space is required for installation of over-printing devices and packaging machines. Only one type of product label should be processed at any one time.

(Note: For details see sterilization methods and their applications.)

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2.

3.

4.

5.

from dirt and microbes. The room should undergo a minimum of 10-15 air changes per hour. All incoming air should be passed through filters with an efficiency of atleast 95%. Preparation of compounding area: In this area formula is compounded and for this it is not essential that the area should be aseptic but controlling measures are provided to control the dust generated from weighing and compounding procedures. Cabinets and counters are generally made up of stainless steel. The ceiling, walls should be made of such materials which are impervious to water. Aseptic area: a. The ceiling walls and floor must be sealed properly so that they can be washed and disinfected when required. b. All the counters are made up of stainless steel and they are constructed in such a way that dirt particles shall not accumulate. c. As much as possible storage tanks, mixing tanks containing the compounded products, should remain outside the aseptic area and then the products are fed into aseptic area through pipeline. Quarantine area: The purpose of quarantine area is that batches can be stored physically segregated from either “in process” batches or approved batches in a lock to which access is restricted to a responsible person. Facility should be available for segregation between batches and the storage system is designed in such a way that easy access to each batch of the product can be made by an approved person in case of resampling. Labelling and packing area: In this batch numbering and over-printing of labels should take place. Adequate space is required for installation of over-printing devices and packaging machines. Only one type of product label should be processed at any one time.

(Note: For details see sterilization methods and their applications.)

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9.7 NOSOCOMIAL INFECTION

9.7 NOSOCOMIAL INFECTION

Nosocomial infections also known as hospital-acquired infections, hospitalassociated infections, and hospital infections are infections that are not present in the patient at the time of admission to hospital but develop during the course of the stay in hospital. There are two forms: • Endogenous infection, self-infection, or auto-infection. The causative agent of the infection is present in the patient at the time of admission to hospital but there are no signs of infection. The infection develops during the stay in the hospital as a result of the patient’s altered resistance. • Cross-contamination followed by cross-infection. During the stay in hospital the patient comes into contact with new infective agents, becomes contaminated, and subsequently develops an infection.

Nosocomial infections also known as hospital-acquired infections, hospitalassociated infections, and hospital infections are infections that are not present in the patient at the time of admission to hospital but develop during the course of the stay in hospital. There are two forms: • Endogenous infection, self-infection, or auto-infection. The causative agent of the infection is present in the patient at the time of admission to hospital but there are no signs of infection. The infection develops during the stay in the hospital as a result of the patient’s altered resistance. • Cross-contamination followed by cross-infection. During the stay in hospital the patient comes into contact with new infective agents, becomes contaminated, and subsequently develops an infection.

While there is no clinically significant difference between the endogenous self-infection and the exogenous cross-infection, the distinction is important from the standpoint of epidemiology and prevention.

While there is no clinically significant difference between the endogenous self-infection and the exogenous cross-infection, the distinction is important from the standpoint of epidemiology and prevention.

9.7.1 Route of Transmission of Nosocomial Infection Microorganisms can be transmitted from their source to a new host through direct or indirect contact, in the air, or by vectors. 1. Droplet transmission occurs when droplets are generated from the infected person mainly during coughing, sneezing, and talking, and during the performance of certain procedures such as bronchoscopy. Transmission occurs when droplets containing germs from the infected person are propelled a short distance through the air and deposited on the host’s body. 2. Airborne transmission occurs only with microorganisms that are dispersed into the air and that are characterized by a low minimal infective dose. Only a few bacteria and viruses are present in expired air, and these are dispersed in large numbers only as a result of sneezing or coughing. 3. Vector-borne transmission is typical of countries in which insects, arthropods, and other parasites are widespread. These become contaminated by contact with excreta or secretions from an infected patient and transmit the infective organisms mechanically to other patients. 4. Direct contact between patients does not usually occur in healthcare facilities, but an infected healthcare worker can touch a patient and directly transmit a large number of microorganisms to the new host. 5. The most frequent route of transmission, however, is indirect contact. The infected patient touches and contaminates an object, an instrument, or a surface. Subsequent contact between that item and another patient is likely to contaminate the second individual who may then develop an infection.

9.7.1 Route of Transmission of Nosocomial Infection Microorganisms can be transmitted from their source to a new host through direct or indirect contact, in the air, or by vectors. 1. Droplet transmission occurs when droplets are generated from the infected person mainly during coughing, sneezing, and talking, and during the performance of certain procedures such as bronchoscopy. Transmission occurs when droplets containing germs from the infected person are propelled a short distance through the air and deposited on the host’s body. 2. Airborne transmission occurs only with microorganisms that are dispersed into the air and that are characterized by a low minimal infective dose. Only a few bacteria and viruses are present in expired air, and these are dispersed in large numbers only as a result of sneezing or coughing. 3. Vector-borne transmission is typical of countries in which insects, arthropods, and other parasites are widespread. These become contaminated by contact with excreta or secretions from an infected patient and transmit the infective organisms mechanically to other patients. 4. Direct contact between patients does not usually occur in healthcare facilities, but an infected healthcare worker can touch a patient and directly transmit a large number of microorganisms to the new host. 5. The most frequent route of transmission, however, is indirect contact. The infected patient touches and contaminates an object, an instrument, or a surface. Subsequent contact between that item and another patient is likely to contaminate the second individual who may then develop an infection.

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9.7 NOSOCOMIAL INFECTION

9.7 NOSOCOMIAL INFECTION

Nosocomial infections also known as hospital-acquired infections, hospitalassociated infections, and hospital infections are infections that are not present in the patient at the time of admission to hospital but develop during the course of the stay in hospital. There are two forms: • Endogenous infection, self-infection, or auto-infection. The causative agent of the infection is present in the patient at the time of admission to hospital but there are no signs of infection. The infection develops during the stay in the hospital as a result of the patient’s altered resistance. • Cross-contamination followed by cross-infection. During the stay in hospital the patient comes into contact with new infective agents, becomes contaminated, and subsequently develops an infection.

Nosocomial infections also known as hospital-acquired infections, hospitalassociated infections, and hospital infections are infections that are not present in the patient at the time of admission to hospital but develop during the course of the stay in hospital. There are two forms: • Endogenous infection, self-infection, or auto-infection. The causative agent of the infection is present in the patient at the time of admission to hospital but there are no signs of infection. The infection develops during the stay in the hospital as a result of the patient’s altered resistance. • Cross-contamination followed by cross-infection. During the stay in hospital the patient comes into contact with new infective agents, becomes contaminated, and subsequently develops an infection.

While there is no clinically significant difference between the endogenous self-infection and the exogenous cross-infection, the distinction is important from the standpoint of epidemiology and prevention.

While there is no clinically significant difference between the endogenous self-infection and the exogenous cross-infection, the distinction is important from the standpoint of epidemiology and prevention.

9.7.1 Route of Transmission of Nosocomial Infection Microorganisms can be transmitted from their source to a new host through direct or indirect contact, in the air, or by vectors. 1. Droplet transmission occurs when droplets are generated from the infected person mainly during coughing, sneezing, and talking, and during the performance of certain procedures such as bronchoscopy. Transmission occurs when droplets containing germs from the infected person are propelled a short distance through the air and deposited on the host’s body. 2. Airborne transmission occurs only with microorganisms that are dispersed into the air and that are characterized by a low minimal infective dose. Only a few bacteria and viruses are present in expired air, and these are dispersed in large numbers only as a result of sneezing or coughing. 3. Vector-borne transmission is typical of countries in which insects, arthropods, and other parasites are widespread. These become contaminated by contact with excreta or secretions from an infected patient and transmit the infective organisms mechanically to other patients. 4. Direct contact between patients does not usually occur in healthcare facilities, but an infected healthcare worker can touch a patient and directly transmit a large number of microorganisms to the new host. 5. The most frequent route of transmission, however, is indirect contact. The infected patient touches and contaminates an object, an instrument, or a surface. Subsequent contact between that item and another patient is likely to contaminate the second individual who may then develop an infection.

9.7.1 Route of Transmission of Nosocomial Infection Microorganisms can be transmitted from their source to a new host through direct or indirect contact, in the air, or by vectors. 1. Droplet transmission occurs when droplets are generated from the infected person mainly during coughing, sneezing, and talking, and during the performance of certain procedures such as bronchoscopy. Transmission occurs when droplets containing germs from the infected person are propelled a short distance through the air and deposited on the host’s body. 2. Airborne transmission occurs only with microorganisms that are dispersed into the air and that are characterized by a low minimal infective dose. Only a few bacteria and viruses are present in expired air, and these are dispersed in large numbers only as a result of sneezing or coughing. 3. Vector-borne transmission is typical of countries in which insects, arthropods, and other parasites are widespread. These become contaminated by contact with excreta or secretions from an infected patient and transmit the infective organisms mechanically to other patients. 4. Direct contact between patients does not usually occur in healthcare facilities, but an infected healthcare worker can touch a patient and directly transmit a large number of microorganisms to the new host. 5. The most frequent route of transmission, however, is indirect contact. The infected patient touches and contaminates an object, an instrument, or a surface. Subsequent contact between that item and another patient is likely to contaminate the second individual who may then develop an infection.

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130 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6. During general care and/or medical treatment, the hands of healthcare workers often come into close contact with patients. The hands of the clinical personnel are thus the most frequent vehicles for nosocomial infections. Transmission by this route is much more common than vectorborne or airborne transmission or other forms of direct or indirect contact.

6. During general care and/or medical treatment, the hands of healthcare workers often come into close contact with patients. The hands of the clinical personnel are thus the most frequent vehicles for nosocomial infections. Transmission by this route is much more common than vectorborne or airborne transmission or other forms of direct or indirect contact.

9.7.2 Prevention of Nosocomial Infection 1. Hand washing: Wash hands after touching blood, secretions, excretions and contaminated items, whether or not gloves are worn. Wash hands immediately after the gloves are removed, between patient contacts. Use a plain soap for routine hand washing. Use an antimicrobial agent for specific circumstances. 2. Gloves: Wear gloves while touching blood, body fluids, secretions, excretions, and contaminated items. Put on clean gloves just before touching mucous membranes and non-intact skin. 3. Mask, eye protection, face shield: Wear a mask and eye protection or a face shield during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, and excretions. 4. Gown: Wear a gown during procedures and patient-care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. 5. Patient-care equipment: Ensure that reusable equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately. 6. Environmental control: Ensure that the hospital has adequate procedures for the routine care, cleaning, and disinfection of environmental surfaces. 7. Linen: Handle used linen, soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures, and that avoids transfer of microorganisms to other patients and environments. 8. Occupational health and blood-borne pathogens: Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices. Use ventilation devices as an alternative to mouth-to-mouth resuscitation methods. 9. Place of care of the patient: Place a patient who contaminates the environment or who does not assist in maintaining appropriate hygiene in an isolated (or separate) room.

9.7.2 Prevention of Nosocomial Infection 1. Hand washing: Wash hands after touching blood, secretions, excretions and contaminated items, whether or not gloves are worn. Wash hands immediately after the gloves are removed, between patient contacts. Use a plain soap for routine hand washing. Use an antimicrobial agent for specific circumstances. 2. Gloves: Wear gloves while touching blood, body fluids, secretions, excretions, and contaminated items. Put on clean gloves just before touching mucous membranes and non-intact skin. 3. Mask, eye protection, face shield: Wear a mask and eye protection or a face shield during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, and excretions. 4. Gown: Wear a gown during procedures and patient-care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. 5. Patient-care equipment: Ensure that reusable equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately. 6. Environmental control: Ensure that the hospital has adequate procedures for the routine care, cleaning, and disinfection of environmental surfaces. 7. Linen: Handle used linen, soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures, and that avoids transfer of microorganisms to other patients and environments. 8. Occupational health and blood-borne pathogens: Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices. Use ventilation devices as an alternative to mouth-to-mouth resuscitation methods. 9. Place of care of the patient: Place a patient who contaminates the environment or who does not assist in maintaining appropriate hygiene in an isolated (or separate) room.

9.8 CONTROL OF HOSPITAL INFECTION

9.8 CONTROL OF HOSPITAL INFECTION

Infection control is the discipline concerned with preventing nosocomial or healthcare-associated infection. Infection control and hospital epidemiology are

Infection control is the discipline concerned with preventing nosocomial or healthcare-associated infection. Infection control and hospital epidemiology are

130 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

130 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

6. During general care and/or medical treatment, the hands of healthcare workers often come into close contact with patients. The hands of the clinical personnel are thus the most frequent vehicles for nosocomial infections. Transmission by this route is much more common than vectorborne or airborne transmission or other forms of direct or indirect contact.

6. During general care and/or medical treatment, the hands of healthcare workers often come into close contact with patients. The hands of the clinical personnel are thus the most frequent vehicles for nosocomial infections. Transmission by this route is much more common than vectorborne or airborne transmission or other forms of direct or indirect contact.

9.7.2 Prevention of Nosocomial Infection 1. Hand washing: Wash hands after touching blood, secretions, excretions and contaminated items, whether or not gloves are worn. Wash hands immediately after the gloves are removed, between patient contacts. Use a plain soap for routine hand washing. Use an antimicrobial agent for specific circumstances. 2. Gloves: Wear gloves while touching blood, body fluids, secretions, excretions, and contaminated items. Put on clean gloves just before touching mucous membranes and non-intact skin. 3. Mask, eye protection, face shield: Wear a mask and eye protection or a face shield during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, and excretions. 4. Gown: Wear a gown during procedures and patient-care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. 5. Patient-care equipment: Ensure that reusable equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately. 6. Environmental control: Ensure that the hospital has adequate procedures for the routine care, cleaning, and disinfection of environmental surfaces. 7. Linen: Handle used linen, soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures, and that avoids transfer of microorganisms to other patients and environments. 8. Occupational health and blood-borne pathogens: Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices. Use ventilation devices as an alternative to mouth-to-mouth resuscitation methods. 9. Place of care of the patient: Place a patient who contaminates the environment or who does not assist in maintaining appropriate hygiene in an isolated (or separate) room.

9.7.2 Prevention of Nosocomial Infection 1. Hand washing: Wash hands after touching blood, secretions, excretions and contaminated items, whether or not gloves are worn. Wash hands immediately after the gloves are removed, between patient contacts. Use a plain soap for routine hand washing. Use an antimicrobial agent for specific circumstances. 2. Gloves: Wear gloves while touching blood, body fluids, secretions, excretions, and contaminated items. Put on clean gloves just before touching mucous membranes and non-intact skin. 3. Mask, eye protection, face shield: Wear a mask and eye protection or a face shield during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, and excretions. 4. Gown: Wear a gown during procedures and patient-care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. 5. Patient-care equipment: Ensure that reusable equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately. 6. Environmental control: Ensure that the hospital has adequate procedures for the routine care, cleaning, and disinfection of environmental surfaces. 7. Linen: Handle used linen, soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures, and that avoids transfer of microorganisms to other patients and environments. 8. Occupational health and blood-borne pathogens: Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices. Use ventilation devices as an alternative to mouth-to-mouth resuscitation methods. 9. Place of care of the patient: Place a patient who contaminates the environment or who does not assist in maintaining appropriate hygiene in an isolated (or separate) room.

9.8 CONTROL OF HOSPITAL INFECTION

9.8 CONTROL OF HOSPITAL INFECTION

Infection control is the discipline concerned with preventing nosocomial or healthcare-associated infection. Infection control and hospital epidemiology are

Infection control is the discipline concerned with preventing nosocomial or healthcare-associated infection. Infection control and hospital epidemiology are

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akin to public health practice, practised within the confines of a particular healthcare delivery system rather than directed at society as a whole. Infection control addresses factors related to the spread of infections within the healthcare setting (whether patient-to-patient, from patients to staff and from staff to patients, or among-staff), including prevention (via hand hygiene/hand washing, cleaning/disinfection/ sterilization, vaccination, surveillance), monitoring/investigation of demonstrated or suspected spread of infection within a particular healthcare setting (surveillance and outbreak investigation), and management (interruption of outbreaks). It is on this basis that the common title being adopted within healthcare is infection prevention and Control. 1. Principles: Two basic principles govern the main measures that should be taken in order to prevent the spread of nosocomial infections in healthcare facilities: • Separate the infection source from the rest of the hospital. • Cut off any route of transmission.

akin to public health practice, practised within the confines of a particular healthcare delivery system rather than directed at society as a whole. Infection control addresses factors related to the spread of infections within the healthcare setting (whether patient-to-patient, from patients to staff and from staff to patients, or among-staff), including prevention (via hand hygiene/hand washing, cleaning/disinfection/ sterilization, vaccination, surveillance), monitoring/investigation of demonstrated or suspected spread of infection within a particular healthcare setting (surveillance and outbreak investigation), and management (interruption of outbreaks). It is on this basis that the common title being adopted within healthcare is infection prevention and Control. 1. Principles: Two basic principles govern the main measures that should be taken in order to prevent the spread of nosocomial infections in healthcare facilities: • Separate the infection source from the rest of the hospital. • Cut off any route of transmission.

The separation of the source has to be interpreted in a broad sense. It includes not only the isolation of infected patients but also all “aseptic techniques”—the measures that are intended to act as a barrier between infected or potentially contaminated tissue and the environment, including other patients and personnel. In recent years, increasing attention has been paid to the protection of the personnel, in particular against the transmission of blood-borne infections, e.g. AIDS and viral hepatitis B and C. Preventive measures are known as “universal” or “standard” precautions. It is impossible to avoid contact with infected tissue or potentially contaminated body fluids, excreta, and secretions. Even when they are not touched with the bare hands, they may come in contact with instruments, containers, linen, etc. All objects that come in contact with patients should be considered potentially contaminated. If an object is disposable, it should be discarded as waste. If it is reusable, transmission of infective agents must be prevented by cleaning, disinfection, or sterilization. Despite the continuing concern of hospital managers and all attempts at improvement, many health-care establishments are unable to achieve adequate levels of prevention, particularly in developing countries. An international survey of the prevalence of hospital-acquired infections was conducted in 14 countries in different regions of the world between 1983 and 1985. The results of this survey, which covered 47 hospitals of size ranging from 227 to 1502 beds (mean 614) showed a wide range of nosocomial infections, with prevalence varying from 3% to 21% (mean 8.4%) in individual hospitals. This work emphasizes the importance of the public health problem.

The separation of the source has to be interpreted in a broad sense. It includes not only the isolation of infected patients but also all “aseptic techniques”—the measures that are intended to act as a barrier between infected or potentially contaminated tissue and the environment, including other patients and personnel. In recent years, increasing attention has been paid to the protection of the personnel, in particular against the transmission of blood-borne infections, e.g. AIDS and viral hepatitis B and C. Preventive measures are known as “universal” or “standard” precautions. It is impossible to avoid contact with infected tissue or potentially contaminated body fluids, excreta, and secretions. Even when they are not touched with the bare hands, they may come in contact with instruments, containers, linen, etc. All objects that come in contact with patients should be considered potentially contaminated. If an object is disposable, it should be discarded as waste. If it is reusable, transmission of infective agents must be prevented by cleaning, disinfection, or sterilization. Despite the continuing concern of hospital managers and all attempts at improvement, many health-care establishments are unable to achieve adequate levels of prevention, particularly in developing countries. An international survey of the prevalence of hospital-acquired infections was conducted in 14 countries in different regions of the world between 1983 and 1985. The results of this survey, which covered 47 hospitals of size ranging from 227 to 1502 beds (mean 614) showed a wide range of nosocomial infections, with prevalence varying from 3% to 21% (mean 8.4%) in individual hospitals. This work emphasizes the importance of the public health problem.

2. Isolation of infected patients and standard precautions: The first essential measure in preventing the spread of nosocomial infections is

2. Isolation of infected patients and standard precautions: The first essential measure in preventing the spread of nosocomial infections is

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akin to public health practice, practised within the confines of a particular healthcare delivery system rather than directed at society as a whole. Infection control addresses factors related to the spread of infections within the healthcare setting (whether patient-to-patient, from patients to staff and from staff to patients, or among-staff), including prevention (via hand hygiene/hand washing, cleaning/disinfection/ sterilization, vaccination, surveillance), monitoring/investigation of demonstrated or suspected spread of infection within a particular healthcare setting (surveillance and outbreak investigation), and management (interruption of outbreaks). It is on this basis that the common title being adopted within healthcare is infection prevention and Control. 1. Principles: Two basic principles govern the main measures that should be taken in order to prevent the spread of nosocomial infections in healthcare facilities: • Separate the infection source from the rest of the hospital. • Cut off any route of transmission.

akin to public health practice, practised within the confines of a particular healthcare delivery system rather than directed at society as a whole. Infection control addresses factors related to the spread of infections within the healthcare setting (whether patient-to-patient, from patients to staff and from staff to patients, or among-staff), including prevention (via hand hygiene/hand washing, cleaning/disinfection/ sterilization, vaccination, surveillance), monitoring/investigation of demonstrated or suspected spread of infection within a particular healthcare setting (surveillance and outbreak investigation), and management (interruption of outbreaks). It is on this basis that the common title being adopted within healthcare is infection prevention and Control. 1. Principles: Two basic principles govern the main measures that should be taken in order to prevent the spread of nosocomial infections in healthcare facilities: • Separate the infection source from the rest of the hospital. • Cut off any route of transmission.

The separation of the source has to be interpreted in a broad sense. It includes not only the isolation of infected patients but also all “aseptic techniques”—the measures that are intended to act as a barrier between infected or potentially contaminated tissue and the environment, including other patients and personnel. In recent years, increasing attention has been paid to the protection of the personnel, in particular against the transmission of blood-borne infections, e.g. AIDS and viral hepatitis B and C. Preventive measures are known as “universal” or “standard” precautions. It is impossible to avoid contact with infected tissue or potentially contaminated body fluids, excreta, and secretions. Even when they are not touched with the bare hands, they may come in contact with instruments, containers, linen, etc. All objects that come in contact with patients should be considered potentially contaminated. If an object is disposable, it should be discarded as waste. If it is reusable, transmission of infective agents must be prevented by cleaning, disinfection, or sterilization. Despite the continuing concern of hospital managers and all attempts at improvement, many health-care establishments are unable to achieve adequate levels of prevention, particularly in developing countries. An international survey of the prevalence of hospital-acquired infections was conducted in 14 countries in different regions of the world between 1983 and 1985. The results of this survey, which covered 47 hospitals of size ranging from 227 to 1502 beds (mean 614) showed a wide range of nosocomial infections, with prevalence varying from 3% to 21% (mean 8.4%) in individual hospitals. This work emphasizes the importance of the public health problem.

The separation of the source has to be interpreted in a broad sense. It includes not only the isolation of infected patients but also all “aseptic techniques”—the measures that are intended to act as a barrier between infected or potentially contaminated tissue and the environment, including other patients and personnel. In recent years, increasing attention has been paid to the protection of the personnel, in particular against the transmission of blood-borne infections, e.g. AIDS and viral hepatitis B and C. Preventive measures are known as “universal” or “standard” precautions. It is impossible to avoid contact with infected tissue or potentially contaminated body fluids, excreta, and secretions. Even when they are not touched with the bare hands, they may come in contact with instruments, containers, linen, etc. All objects that come in contact with patients should be considered potentially contaminated. If an object is disposable, it should be discarded as waste. If it is reusable, transmission of infective agents must be prevented by cleaning, disinfection, or sterilization. Despite the continuing concern of hospital managers and all attempts at improvement, many health-care establishments are unable to achieve adequate levels of prevention, particularly in developing countries. An international survey of the prevalence of hospital-acquired infections was conducted in 14 countries in different regions of the world between 1983 and 1985. The results of this survey, which covered 47 hospitals of size ranging from 227 to 1502 beds (mean 614) showed a wide range of nosocomial infections, with prevalence varying from 3% to 21% (mean 8.4%) in individual hospitals. This work emphasizes the importance of the public health problem.

2. Isolation of infected patients and standard precautions: The first essential measure in preventing the spread of nosocomial infections is

2. Isolation of infected patients and standard precautions: The first essential measure in preventing the spread of nosocomial infections is

132 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY isolation of infected patients. The term isolation covers a broad domain of measures. The strictest form of isolation is applied in case of very infectious diseases (e.g., haemorrhagic fever, diphtheria); less stringent precautions can be taken in case of diseases such as tuberculosis, other respiratory infections, and infectious diarrhoea. Isolation of any degree is expensive, labour-intensive, and usually inconvenient or uncomfortable for both patients and healthcare personnel; its implementation should therefore be adapted to the severity of the disease and to the causative agent. Disease-specific precautions should include details of all the measures (private room, wearing of masks or gowns, etc.) to be taken in the case of a specific disease caused by a defined organism. The so-called standard precautions, essentially protect healthcare workers from blood-borne infections caused by human immunodeficiency virus and hepatitis B and C viruses. 3. Cleaning: One of the most basic measures for the maintenance of hygiene, and one that is particularly important in the hospital environment, is cleaning. The principal aim of cleaning is to remove visible dirt. It is essentially a mechanical process: the dirt is dissolved by water, diluted until it is no longer visible, and rinsed off. Soaps and detergents act as solubility-promoting agents. The microbiological effect of cleaning is also essentially mechanical: bacteria and other microorganisms are suspended in the cleaning fluid and removed from the surface. The efficacy of the cleaning process depends completely on this mechanical action, since neither soap nor detergents possess any antimicrobial activity. Thorough cleaning will remove more than 90% of microorganisms. However, careless and superficial cleaning is much less effective; it is even possible that it has a negative effect, by dispersing the microorganisms over a greater surface and increasing the chance that they may contaminate other objects. Cleaning has therefore to be carried out in a standardized manner or, better, by automated means that will guarantee an adequate level of cleanliness. Diluting and removing the dirt also removes the breeding-ground or culture medium for bacteria and fungi. Most non-sporulating bacteria and viruses survive only when they are protected by dirt or a film of organic matter; otherwise they dry out and die. Non-sporulating bacteria are unlikely to survive on clean surfaces. The effectiveness of disinfection and sterilization is increased by prior or simultaneous cleaning. 4. Sterilization: Self-evidently, an object should be sterile, i.e. free of microorganisms, after sterilization. However, sterilization is never absolute; by definition, it effects a reduction in the number of microorganisms by a factor of more than 106 (i.e., more than 99.9999% are killed). Standard reference works, such as pharmacopoeias, often state that no more than one out of 10,000,00 sterilized items may still bear microorganisms. It is

132 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY isolation of infected patients. The term isolation covers a broad domain of measures. The strictest form of isolation is applied in case of very infectious diseases (e.g., haemorrhagic fever, diphtheria); less stringent precautions can be taken in case of diseases such as tuberculosis, other respiratory infections, and infectious diarrhoea. Isolation of any degree is expensive, labour-intensive, and usually inconvenient or uncomfortable for both patients and healthcare personnel; its implementation should therefore be adapted to the severity of the disease and to the causative agent. Disease-specific precautions should include details of all the measures (private room, wearing of masks or gowns, etc.) to be taken in the case of a specific disease caused by a defined organism. The so-called standard precautions, essentially protect healthcare workers from blood-borne infections caused by human immunodeficiency virus and hepatitis B and C viruses. 3. Cleaning: One of the most basic measures for the maintenance of hygiene, and one that is particularly important in the hospital environment, is cleaning. The principal aim of cleaning is to remove visible dirt. It is essentially a mechanical process: the dirt is dissolved by water, diluted until it is no longer visible, and rinsed off. Soaps and detergents act as solubility-promoting agents. The microbiological effect of cleaning is also essentially mechanical: bacteria and other microorganisms are suspended in the cleaning fluid and removed from the surface. The efficacy of the cleaning process depends completely on this mechanical action, since neither soap nor detergents possess any antimicrobial activity. Thorough cleaning will remove more than 90% of microorganisms. However, careless and superficial cleaning is much less effective; it is even possible that it has a negative effect, by dispersing the microorganisms over a greater surface and increasing the chance that they may contaminate other objects. Cleaning has therefore to be carried out in a standardized manner or, better, by automated means that will guarantee an adequate level of cleanliness. Diluting and removing the dirt also removes the breeding-ground or culture medium for bacteria and fungi. Most non-sporulating bacteria and viruses survive only when they are protected by dirt or a film of organic matter; otherwise they dry out and die. Non-sporulating bacteria are unlikely to survive on clean surfaces. The effectiveness of disinfection and sterilization is increased by prior or simultaneous cleaning. 4. Sterilization: Self-evidently, an object should be sterile, i.e. free of microorganisms, after sterilization. However, sterilization is never absolute; by definition, it effects a reduction in the number of microorganisms by a factor of more than 106 (i.e., more than 99.9999% are killed). Standard reference works, such as pharmacopoeias, often state that no more than one out of 10,000,00 sterilized items may still bear microorganisms. It is

132 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY isolation of infected patients. The term isolation covers a broad domain of measures. The strictest form of isolation is applied in case of very infectious diseases (e.g., haemorrhagic fever, diphtheria); less stringent precautions can be taken in case of diseases such as tuberculosis, other respiratory infections, and infectious diarrhoea. Isolation of any degree is expensive, labour-intensive, and usually inconvenient or uncomfortable for both patients and healthcare personnel; its implementation should therefore be adapted to the severity of the disease and to the causative agent. Disease-specific precautions should include details of all the measures (private room, wearing of masks or gowns, etc.) to be taken in the case of a specific disease caused by a defined organism. The so-called standard precautions, essentially protect healthcare workers from blood-borne infections caused by human immunodeficiency virus and hepatitis B and C viruses. 3. Cleaning: One of the most basic measures for the maintenance of hygiene, and one that is particularly important in the hospital environment, is cleaning. The principal aim of cleaning is to remove visible dirt. It is essentially a mechanical process: the dirt is dissolved by water, diluted until it is no longer visible, and rinsed off. Soaps and detergents act as solubility-promoting agents. The microbiological effect of cleaning is also essentially mechanical: bacteria and other microorganisms are suspended in the cleaning fluid and removed from the surface. The efficacy of the cleaning process depends completely on this mechanical action, since neither soap nor detergents possess any antimicrobial activity. Thorough cleaning will remove more than 90% of microorganisms. However, careless and superficial cleaning is much less effective; it is even possible that it has a negative effect, by dispersing the microorganisms over a greater surface and increasing the chance that they may contaminate other objects. Cleaning has therefore to be carried out in a standardized manner or, better, by automated means that will guarantee an adequate level of cleanliness. Diluting and removing the dirt also removes the breeding-ground or culture medium for bacteria and fungi. Most non-sporulating bacteria and viruses survive only when they are protected by dirt or a film of organic matter; otherwise they dry out and die. Non-sporulating bacteria are unlikely to survive on clean surfaces. The effectiveness of disinfection and sterilization is increased by prior or simultaneous cleaning. 4. Sterilization: Self-evidently, an object should be sterile, i.e. free of microorganisms, after sterilization. However, sterilization is never absolute; by definition, it effects a reduction in the number of microorganisms by a factor of more than 106 (i.e., more than 99.9999% are killed). Standard reference works, such as pharmacopoeias, often state that no more than one out of 10,000,00 sterilized items may still bear microorganisms. It is

132 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY isolation of infected patients. The term isolation covers a broad domain of measures. The strictest form of isolation is applied in case of very infectious diseases (e.g., haemorrhagic fever, diphtheria); less stringent precautions can be taken in case of diseases such as tuberculosis, other respiratory infections, and infectious diarrhoea. Isolation of any degree is expensive, labour-intensive, and usually inconvenient or uncomfortable for both patients and healthcare personnel; its implementation should therefore be adapted to the severity of the disease and to the causative agent. Disease-specific precautions should include details of all the measures (private room, wearing of masks or gowns, etc.) to be taken in the case of a specific disease caused by a defined organism. The so-called standard precautions, essentially protect healthcare workers from blood-borne infections caused by human immunodeficiency virus and hepatitis B and C viruses. 3. Cleaning: One of the most basic measures for the maintenance of hygiene, and one that is particularly important in the hospital environment, is cleaning. The principal aim of cleaning is to remove visible dirt. It is essentially a mechanical process: the dirt is dissolved by water, diluted until it is no longer visible, and rinsed off. Soaps and detergents act as solubility-promoting agents. The microbiological effect of cleaning is also essentially mechanical: bacteria and other microorganisms are suspended in the cleaning fluid and removed from the surface. The efficacy of the cleaning process depends completely on this mechanical action, since neither soap nor detergents possess any antimicrobial activity. Thorough cleaning will remove more than 90% of microorganisms. However, careless and superficial cleaning is much less effective; it is even possible that it has a negative effect, by dispersing the microorganisms over a greater surface and increasing the chance that they may contaminate other objects. Cleaning has therefore to be carried out in a standardized manner or, better, by automated means that will guarantee an adequate level of cleanliness. Diluting and removing the dirt also removes the breeding-ground or culture medium for bacteria and fungi. Most non-sporulating bacteria and viruses survive only when they are protected by dirt or a film of organic matter; otherwise they dry out and die. Non-sporulating bacteria are unlikely to survive on clean surfaces. The effectiveness of disinfection and sterilization is increased by prior or simultaneous cleaning. 4. Sterilization: Self-evidently, an object should be sterile, i.e. free of microorganisms, after sterilization. However, sterilization is never absolute; by definition, it effects a reduction in the number of microorganisms by a factor of more than 106 (i.e., more than 99.9999% are killed). Standard reference works, such as pharmacopoeias, often state that no more than one out of 10,000,00 sterilized items may still bear microorganisms. It is

STERILIZATION

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therefore important to minimize the level of contamination of the material to be sterilized. This is done by sterilizing only objects that are clean (free of visible dirt) and applying the principles of good manufacturing practice. Sterilization can be achieved by both physical and chemical means. Physical methods are based on the action of heat (autoclaving, dry thermal or wet thermal sterilization), on irradiation (g-irradiation), or on mechanical separation by filtration. Chemical means include gas sterilization with ethylene oxide or other gases, and immersion in a disinfectant solution with sterilizing properties (e.g., glutaraldehyde). 5. Disinfection: The term disinfection is difficult to define, as the activity of a disinfectant process can vary widely. The guidelines of the Center for Disease Control (Garner & Favero, 1986) allow the following distinction to be followed: • High-level disinfection can be expected to destroy all microorganisms, with the exception of large numbers of bacterial spores. • Intermediate disinfection inactivates mycobacterium tuberculosis, vegetative bacteria, most viruses, and most fungi; does not necessarily kill bacterial spores. • Low-level disinfection can kill most bacteria, some viruses, and some fungi; cannot be relied on to kill resistant microorganisms such as tubercle bacilli or bacterial spores.

therefore important to minimize the level of contamination of the material to be sterilized. This is done by sterilizing only objects that are clean (free of visible dirt) and applying the principles of good manufacturing practice. Sterilization can be achieved by both physical and chemical means. Physical methods are based on the action of heat (autoclaving, dry thermal or wet thermal sterilization), on irradiation (g-irradiation), or on mechanical separation by filtration. Chemical means include gas sterilization with ethylene oxide or other gases, and immersion in a disinfectant solution with sterilizing properties (e.g., glutaraldehyde). 5. Disinfection: The term disinfection is difficult to define, as the activity of a disinfectant process can vary widely. The guidelines of the Center for Disease Control (Garner & Favero, 1986) allow the following distinction to be followed: • High-level disinfection can be expected to destroy all microorganisms, with the exception of large numbers of bacterial spores. • Intermediate disinfection inactivates mycobacterium tuberculosis, vegetative bacteria, most viruses, and most fungi; does not necessarily kill bacterial spores. • Low-level disinfection can kill most bacteria, some viruses, and some fungi; cannot be relied on to kill resistant microorganisms such as tubercle bacilli or bacterial spores.

There is no ideal disinfectant and the best compromise should be chosen according to the situation. A disinfectant solution is considered appropriate when the compromise between the antimicrobial activity and the toxicity of the product is satisfactory for the given application. Another consideration may well be the cost. The more active disinfectants are automatically the more toxic ones; potentially toxic products can be applied to inanimate objects or surfaces, whereas for disinfection of human tissues only the less toxic disinfectants can be considered. For antisepsis, different disinfectants are used for application to the intact skin (e.g., alcoholic solutions) and to mucous membranes or wounds (only aqueous solutions of non-toxic substances). Cost is a less important consideration for an antiseptic than for a disinfectant. The principal requirements for a good antiseptic are absence of toxicity and rapid and adequate activity on both the natural flora and, especially, pathogenic bacteria and other microorganisms after a very short exposure time. Essential requirements for a disinfectant are somewhat different: there must be adequate activity against bacteria, fungi, and viruses that may be present in large numbers and protected by dirt or organic matter. In addition, since disinfectants are applied in large quantities, they should be of low ecotoxicity. In general, use of the chosen disinfectant, at the appropriate concentration and for the appropriate time, should kill pathogenic microorganisms, rendering

There is no ideal disinfectant and the best compromise should be chosen according to the situation. A disinfectant solution is considered appropriate when the compromise between the antimicrobial activity and the toxicity of the product is satisfactory for the given application. Another consideration may well be the cost. The more active disinfectants are automatically the more toxic ones; potentially toxic products can be applied to inanimate objects or surfaces, whereas for disinfection of human tissues only the less toxic disinfectants can be considered. For antisepsis, different disinfectants are used for application to the intact skin (e.g., alcoholic solutions) and to mucous membranes or wounds (only aqueous solutions of non-toxic substances). Cost is a less important consideration for an antiseptic than for a disinfectant. The principal requirements for a good antiseptic are absence of toxicity and rapid and adequate activity on both the natural flora and, especially, pathogenic bacteria and other microorganisms after a very short exposure time. Essential requirements for a disinfectant are somewhat different: there must be adequate activity against bacteria, fungi, and viruses that may be present in large numbers and protected by dirt or organic matter. In addition, since disinfectants are applied in large quantities, they should be of low ecotoxicity. In general, use of the chosen disinfectant, at the appropriate concentration and for the appropriate time, should kill pathogenic microorganisms, rendering

STERILIZATION

133

STERILIZATION

133

therefore important to minimize the level of contamination of the material to be sterilized. This is done by sterilizing only objects that are clean (free of visible dirt) and applying the principles of good manufacturing practice. Sterilization can be achieved by both physical and chemical means. Physical methods are based on the action of heat (autoclaving, dry thermal or wet thermal sterilization), on irradiation (g-irradiation), or on mechanical separation by filtration. Chemical means include gas sterilization with ethylene oxide or other gases, and immersion in a disinfectant solution with sterilizing properties (e.g., glutaraldehyde). 5. Disinfection: The term disinfection is difficult to define, as the activity of a disinfectant process can vary widely. The guidelines of the Center for Disease Control (Garner & Favero, 1986) allow the following distinction to be followed: • High-level disinfection can be expected to destroy all microorganisms, with the exception of large numbers of bacterial spores. • Intermediate disinfection inactivates mycobacterium tuberculosis, vegetative bacteria, most viruses, and most fungi; does not necessarily kill bacterial spores. • Low-level disinfection can kill most bacteria, some viruses, and some fungi; cannot be relied on to kill resistant microorganisms such as tubercle bacilli or bacterial spores.

therefore important to minimize the level of contamination of the material to be sterilized. This is done by sterilizing only objects that are clean (free of visible dirt) and applying the principles of good manufacturing practice. Sterilization can be achieved by both physical and chemical means. Physical methods are based on the action of heat (autoclaving, dry thermal or wet thermal sterilization), on irradiation (g-irradiation), or on mechanical separation by filtration. Chemical means include gas sterilization with ethylene oxide or other gases, and immersion in a disinfectant solution with sterilizing properties (e.g., glutaraldehyde). 5. Disinfection: The term disinfection is difficult to define, as the activity of a disinfectant process can vary widely. The guidelines of the Center for Disease Control (Garner & Favero, 1986) allow the following distinction to be followed: • High-level disinfection can be expected to destroy all microorganisms, with the exception of large numbers of bacterial spores. • Intermediate disinfection inactivates mycobacterium tuberculosis, vegetative bacteria, most viruses, and most fungi; does not necessarily kill bacterial spores. • Low-level disinfection can kill most bacteria, some viruses, and some fungi; cannot be relied on to kill resistant microorganisms such as tubercle bacilli or bacterial spores.

There is no ideal disinfectant and the best compromise should be chosen according to the situation. A disinfectant solution is considered appropriate when the compromise between the antimicrobial activity and the toxicity of the product is satisfactory for the given application. Another consideration may well be the cost. The more active disinfectants are automatically the more toxic ones; potentially toxic products can be applied to inanimate objects or surfaces, whereas for disinfection of human tissues only the less toxic disinfectants can be considered. For antisepsis, different disinfectants are used for application to the intact skin (e.g., alcoholic solutions) and to mucous membranes or wounds (only aqueous solutions of non-toxic substances). Cost is a less important consideration for an antiseptic than for a disinfectant. The principal requirements for a good antiseptic are absence of toxicity and rapid and adequate activity on both the natural flora and, especially, pathogenic bacteria and other microorganisms after a very short exposure time. Essential requirements for a disinfectant are somewhat different: there must be adequate activity against bacteria, fungi, and viruses that may be present in large numbers and protected by dirt or organic matter. In addition, since disinfectants are applied in large quantities, they should be of low ecotoxicity. In general, use of the chosen disinfectant, at the appropriate concentration and for the appropriate time, should kill pathogenic microorganisms, rendering

There is no ideal disinfectant and the best compromise should be chosen according to the situation. A disinfectant solution is considered appropriate when the compromise between the antimicrobial activity and the toxicity of the product is satisfactory for the given application. Another consideration may well be the cost. The more active disinfectants are automatically the more toxic ones; potentially toxic products can be applied to inanimate objects or surfaces, whereas for disinfection of human tissues only the less toxic disinfectants can be considered. For antisepsis, different disinfectants are used for application to the intact skin (e.g., alcoholic solutions) and to mucous membranes or wounds (only aqueous solutions of non-toxic substances). Cost is a less important consideration for an antiseptic than for a disinfectant. The principal requirements for a good antiseptic are absence of toxicity and rapid and adequate activity on both the natural flora and, especially, pathogenic bacteria and other microorganisms after a very short exposure time. Essential requirements for a disinfectant are somewhat different: there must be adequate activity against bacteria, fungi, and viruses that may be present in large numbers and protected by dirt or organic matter. In addition, since disinfectants are applied in large quantities, they should be of low ecotoxicity. In general, use of the chosen disinfectant, at the appropriate concentration and for the appropriate time, should kill pathogenic microorganisms, rendering

134 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

134 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

an object safe for use in a patient, or human tissue free of pathogens to exclude cross-contamination.

an object safe for use in a patient, or human tissue free of pathogens to exclude cross-contamination.

6. Hand hygiene: As the hands of healthcare workers are the most frequent vehicle of nosocomial infections, hand hygiene—including both hand washing and hand disinfection—is the primary preventive measure. Thorough hand washing with adequate quantities of water and soap removes more than 90% of the transient, i.e. superficial, flora including all or most contaminants. An antimicrobial soap will further reduce the transient flora, but only if used for several minutes. Hand washing with (non-medicated) soap is essential when hands are dirty and should be routine after physical contact with a patient. Killing all transient floras with all contaminants within a short time (a few seconds) necessitates hygienic hand disinfection: only alcohol or alcoholic preparations act sufficiently fast. Hands should be disinfected with alcohol when an infected tissue or body fluid is touched without gloves.

6. Hand hygiene: As the hands of healthcare workers are the most frequent vehicle of nosocomial infections, hand hygiene—including both hand washing and hand disinfection—is the primary preventive measure. Thorough hand washing with adequate quantities of water and soap removes more than 90% of the transient, i.e. superficial, flora including all or most contaminants. An antimicrobial soap will further reduce the transient flora, but only if used for several minutes. Hand washing with (non-medicated) soap is essential when hands are dirty and should be routine after physical contact with a patient. Killing all transient floras with all contaminants within a short time (a few seconds) necessitates hygienic hand disinfection: only alcohol or alcoholic preparations act sufficiently fast. Hands should be disinfected with alcohol when an infected tissue or body fluid is touched without gloves.

134 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

134 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

an object safe for use in a patient, or human tissue free of pathogens to exclude cross-contamination.

an object safe for use in a patient, or human tissue free of pathogens to exclude cross-contamination.

6. Hand hygiene: As the hands of healthcare workers are the most frequent vehicle of nosocomial infections, hand hygiene—including both hand washing and hand disinfection—is the primary preventive measure. Thorough hand washing with adequate quantities of water and soap removes more than 90% of the transient, i.e. superficial, flora including all or most contaminants. An antimicrobial soap will further reduce the transient flora, but only if used for several minutes. Hand washing with (non-medicated) soap is essential when hands are dirty and should be routine after physical contact with a patient. Killing all transient floras with all contaminants within a short time (a few seconds) necessitates hygienic hand disinfection: only alcohol or alcoholic preparations act sufficiently fast. Hands should be disinfected with alcohol when an infected tissue or body fluid is touched without gloves.

6. Hand hygiene: As the hands of healthcare workers are the most frequent vehicle of nosocomial infections, hand hygiene—including both hand washing and hand disinfection—is the primary preventive measure. Thorough hand washing with adequate quantities of water and soap removes more than 90% of the transient, i.e. superficial, flora including all or most contaminants. An antimicrobial soap will further reduce the transient flora, but only if used for several minutes. Hand washing with (non-medicated) soap is essential when hands are dirty and should be routine after physical contact with a patient. Killing all transient floras with all contaminants within a short time (a few seconds) necessitates hygienic hand disinfection: only alcohol or alcoholic preparations act sufficiently fast. Hands should be disinfected with alcohol when an infected tissue or body fluid is touched without gloves.

SECTION D

SECTION D

SECTION D

SECTION D

10

10

Immunology

Immunology

10.1 HISTORY OF IMMUNOLOGY

10.1 HISTORY OF IMMUNOLOGY

The first recorded attempts to induce immunity deliberately were performed by the Chinese and Turks in the fifteenth century. Various reports suggest that the dried crusts derived from smallpox pustules were either inhaled into the nostrils or inserted into small cuts in the skin (a technique called variolation). In 1718, Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople, observed the positive effects of variolation on the native population and had the technique performed on her own children. The method was significantly improved by the English physician Edward Jenner, in 1798. Intrigued by the fact that milkmaids who had contracted the mild disease cowpox were subsequently immune to smallpox, which is a disfiguring and often fatal disease, Jenner reasoned that introducing fluid from a cowpox pustule into people (i.e. inoculating them) might protect them from smallpox. To test this idea, he inoculated an eight-year-old boy with fluid from a cowpox pustule and later intentionally infected the child with smallpox. As predicted, the child did not develop smallpox. Jenner’s technique of inoculating with cowpox to protect against smallpox spread quickly throughout Europe. However, for many reasons, including lack of obvious disease targets and knowledge of their causes, it was nearly a hundred years before this technique was applied to other diseases. As so often happens in science, serendipity in combination with astute observation led to the next major advance in immunology, the induction of immunity to cholera. Louis Pasteur had succeeded in growing the bacterium thought to cause fowl cholera in culture and then had shown that chickens injected with the cultured bacterium developed cholera. After returning from a summer vacation, he injected some chickens with an old culture. The chickens became ill, but, to Pasteur’s surprise, they recovered. Pasteur then grew a fresh culture of the bacterium with the intention of injecting it into some fresh chickens. But, as the story goes, his supply of chickens was limited, and therefore he used the previously injected

The first recorded attempts to induce immunity deliberately were performed by the Chinese and Turks in the fifteenth century. Various reports suggest that the dried crusts derived from smallpox pustules were either inhaled into the nostrils or inserted into small cuts in the skin (a technique called variolation). In 1718, Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople, observed the positive effects of variolation on the native population and had the technique performed on her own children. The method was significantly improved by the English physician Edward Jenner, in 1798. Intrigued by the fact that milkmaids who had contracted the mild disease cowpox were subsequently immune to smallpox, which is a disfiguring and often fatal disease, Jenner reasoned that introducing fluid from a cowpox pustule into people (i.e. inoculating them) might protect them from smallpox. To test this idea, he inoculated an eight-year-old boy with fluid from a cowpox pustule and later intentionally infected the child with smallpox. As predicted, the child did not develop smallpox. Jenner’s technique of inoculating with cowpox to protect against smallpox spread quickly throughout Europe. However, for many reasons, including lack of obvious disease targets and knowledge of their causes, it was nearly a hundred years before this technique was applied to other diseases. As so often happens in science, serendipity in combination with astute observation led to the next major advance in immunology, the induction of immunity to cholera. Louis Pasteur had succeeded in growing the bacterium thought to cause fowl cholera in culture and then had shown that chickens injected with the cultured bacterium developed cholera. After returning from a summer vacation, he injected some chickens with an old culture. The chickens became ill, but, to Pasteur’s surprise, they recovered. Pasteur then grew a fresh culture of the bacterium with the intention of injecting it into some fresh chickens. But, as the story goes, his supply of chickens was limited, and therefore he used the previously injected

10

10

Immunology

Immunology

10.1 HISTORY OF IMMUNOLOGY

10.1 HISTORY OF IMMUNOLOGY

The first recorded attempts to induce immunity deliberately were performed by the Chinese and Turks in the fifteenth century. Various reports suggest that the dried crusts derived from smallpox pustules were either inhaled into the nostrils or inserted into small cuts in the skin (a technique called variolation). In 1718, Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople, observed the positive effects of variolation on the native population and had the technique performed on her own children. The method was significantly improved by the English physician Edward Jenner, in 1798. Intrigued by the fact that milkmaids who had contracted the mild disease cowpox were subsequently immune to smallpox, which is a disfiguring and often fatal disease, Jenner reasoned that introducing fluid from a cowpox pustule into people (i.e. inoculating them) might protect them from smallpox. To test this idea, he inoculated an eight-year-old boy with fluid from a cowpox pustule and later intentionally infected the child with smallpox. As predicted, the child did not develop smallpox. Jenner’s technique of inoculating with cowpox to protect against smallpox spread quickly throughout Europe. However, for many reasons, including lack of obvious disease targets and knowledge of their causes, it was nearly a hundred years before this technique was applied to other diseases. As so often happens in science, serendipity in combination with astute observation led to the next major advance in immunology, the induction of immunity to cholera. Louis Pasteur had succeeded in growing the bacterium thought to cause fowl cholera in culture and then had shown that chickens injected with the cultured bacterium developed cholera. After returning from a summer vacation, he injected some chickens with an old culture. The chickens became ill, but, to Pasteur’s surprise, they recovered. Pasteur then grew a fresh culture of the bacterium with the intention of injecting it into some fresh chickens. But, as the story goes, his supply of chickens was limited, and therefore he used the previously injected

The first recorded attempts to induce immunity deliberately were performed by the Chinese and Turks in the fifteenth century. Various reports suggest that the dried crusts derived from smallpox pustules were either inhaled into the nostrils or inserted into small cuts in the skin (a technique called variolation). In 1718, Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople, observed the positive effects of variolation on the native population and had the technique performed on her own children. The method was significantly improved by the English physician Edward Jenner, in 1798. Intrigued by the fact that milkmaids who had contracted the mild disease cowpox were subsequently immune to smallpox, which is a disfiguring and often fatal disease, Jenner reasoned that introducing fluid from a cowpox pustule into people (i.e. inoculating them) might protect them from smallpox. To test this idea, he inoculated an eight-year-old boy with fluid from a cowpox pustule and later intentionally infected the child with smallpox. As predicted, the child did not develop smallpox. Jenner’s technique of inoculating with cowpox to protect against smallpox spread quickly throughout Europe. However, for many reasons, including lack of obvious disease targets and knowledge of their causes, it was nearly a hundred years before this technique was applied to other diseases. As so often happens in science, serendipity in combination with astute observation led to the next major advance in immunology, the induction of immunity to cholera. Louis Pasteur had succeeded in growing the bacterium thought to cause fowl cholera in culture and then had shown that chickens injected with the cultured bacterium developed cholera. After returning from a summer vacation, he injected some chickens with an old culture. The chickens became ill, but, to Pasteur’s surprise, they recovered. Pasteur then grew a fresh culture of the bacterium with the intention of injecting it into some fresh chickens. But, as the story goes, his supply of chickens was limited, and therefore he used the previously injected

138 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

138 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

chickens. Again to his surprise, the chickens were completely protected from the disease. Pasteur hypothesized and proved that aging had weakened the virulence of the pathogen and that such an attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine (from the Latin vacca, meaning “cow”), in honour of Jenner’s work with cowpox inoculation. Later on Pasture discovered the vaccine for anthrax and rabies. In 1801, Jenner prophesied the eradication of smallpox by the practice of vaccination. In 1967 the disease infected 10 million people. The World Health Organization (WHO) initiated a programme of confinement and vaccination with the object of eradicating the disease. In Somalia in 1977 the last case of naturally acquired smallpox occurred, and in 1979 the WHO announced the total eradication of smallpox, thus fulfilling Jenner’s prophecy. The science of immunology not only encompasses the body’s immune responses to bacteria and viruses but is extensively involved in: tumour recognition and subsequent rejection; the rejection of transplanted organs and tissues; the elimination of parasites from the body, allergies, and autoimmunity (the condition when the body mounts a reaction against its own tissues).

chickens. Again to his surprise, the chickens were completely protected from the disease. Pasteur hypothesized and proved that aging had weakened the virulence of the pathogen and that such an attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine (from the Latin vacca, meaning “cow”), in honour of Jenner’s work with cowpox inoculation. Later on Pasture discovered the vaccine for anthrax and rabies. In 1801, Jenner prophesied the eradication of smallpox by the practice of vaccination. In 1967 the disease infected 10 million people. The World Health Organization (WHO) initiated a programme of confinement and vaccination with the object of eradicating the disease. In Somalia in 1977 the last case of naturally acquired smallpox occurred, and in 1979 the WHO announced the total eradication of smallpox, thus fulfilling Jenner’s prophecy. The science of immunology not only encompasses the body’s immune responses to bacteria and viruses but is extensively involved in: tumour recognition and subsequent rejection; the rejection of transplanted organs and tissues; the elimination of parasites from the body, allergies, and autoimmunity (the condition when the body mounts a reaction against its own tissues).

How did Edward Jenner Discover Vaccination? Jenner worked in a rural community and most of his patients were farmers or worked on farms with cattle. In the 18th century smallpox was a very common disease and was a major cause of death. The main treatment was by a method which had brought success to a Dutch physiologist Jan Ingenhaus and was brought to England in 1721 from Turkey by Lady Mary Wortly Montague. This method involved inoculating healthy people with substances from the pustules of those who had a mild case of the disease, but this often had fatal results. In 1788 an epidemic of smallpox hit Gloucestershire and during this outbreak Jenner observed that those of his patients who worked with cattle and had come in contact with the much milder disease called cowpox never came down with smallpox. Jenner needed a way of showing that his theory actually worked. Jenner was given the opportunity on the 14 May 1796, when a young milkmaid called Sarah Nelmes came to see him with sores on her hands like blisters. Jenner identified that she had caught cowpox from the cows she handled each day. Jenner now had the opportunity to obtain the material try out his theories. He carefully extracted some liquid from her sores and then took some liquid from the sores of a patient with mild smallpox. Jenner believed that if he could inject someone with cowpox, the germs from the cowpox would make the body able to defend itself against the dangerous smallpox germs which he would inject later. Jenner approached a local farmer called Phipps and asked him if he could inoculate his son James against smallpox. He explained to the farmer that if his

How did Edward Jenner Discover Vaccination? Jenner worked in a rural community and most of his patients were farmers or worked on farms with cattle. In the 18th century smallpox was a very common disease and was a major cause of death. The main treatment was by a method which had brought success to a Dutch physiologist Jan Ingenhaus and was brought to England in 1721 from Turkey by Lady Mary Wortly Montague. This method involved inoculating healthy people with substances from the pustules of those who had a mild case of the disease, but this often had fatal results. In 1788 an epidemic of smallpox hit Gloucestershire and during this outbreak Jenner observed that those of his patients who worked with cattle and had come in contact with the much milder disease called cowpox never came down with smallpox. Jenner needed a way of showing that his theory actually worked. Jenner was given the opportunity on the 14 May 1796, when a young milkmaid called Sarah Nelmes came to see him with sores on her hands like blisters. Jenner identified that she had caught cowpox from the cows she handled each day. Jenner now had the opportunity to obtain the material try out his theories. He carefully extracted some liquid from her sores and then took some liquid from the sores of a patient with mild smallpox. Jenner believed that if he could inject someone with cowpox, the germs from the cowpox would make the body able to defend itself against the dangerous smallpox germs which he would inject later. Jenner approached a local farmer called Phipps and asked him if he could inoculate his son James against smallpox. He explained to the farmer that if his

138 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

138 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

chickens. Again to his surprise, the chickens were completely protected from the disease. Pasteur hypothesized and proved that aging had weakened the virulence of the pathogen and that such an attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine (from the Latin vacca, meaning “cow”), in honour of Jenner’s work with cowpox inoculation. Later on Pasture discovered the vaccine for anthrax and rabies. In 1801, Jenner prophesied the eradication of smallpox by the practice of vaccination. In 1967 the disease infected 10 million people. The World Health Organization (WHO) initiated a programme of confinement and vaccination with the object of eradicating the disease. In Somalia in 1977 the last case of naturally acquired smallpox occurred, and in 1979 the WHO announced the total eradication of smallpox, thus fulfilling Jenner’s prophecy. The science of immunology not only encompasses the body’s immune responses to bacteria and viruses but is extensively involved in: tumour recognition and subsequent rejection; the rejection of transplanted organs and tissues; the elimination of parasites from the body, allergies, and autoimmunity (the condition when the body mounts a reaction against its own tissues).

chickens. Again to his surprise, the chickens were completely protected from the disease. Pasteur hypothesized and proved that aging had weakened the virulence of the pathogen and that such an attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine (from the Latin vacca, meaning “cow”), in honour of Jenner’s work with cowpox inoculation. Later on Pasture discovered the vaccine for anthrax and rabies. In 1801, Jenner prophesied the eradication of smallpox by the practice of vaccination. In 1967 the disease infected 10 million people. The World Health Organization (WHO) initiated a programme of confinement and vaccination with the object of eradicating the disease. In Somalia in 1977 the last case of naturally acquired smallpox occurred, and in 1979 the WHO announced the total eradication of smallpox, thus fulfilling Jenner’s prophecy. The science of immunology not only encompasses the body’s immune responses to bacteria and viruses but is extensively involved in: tumour recognition and subsequent rejection; the rejection of transplanted organs and tissues; the elimination of parasites from the body, allergies, and autoimmunity (the condition when the body mounts a reaction against its own tissues).

How did Edward Jenner Discover Vaccination? Jenner worked in a rural community and most of his patients were farmers or worked on farms with cattle. In the 18th century smallpox was a very common disease and was a major cause of death. The main treatment was by a method which had brought success to a Dutch physiologist Jan Ingenhaus and was brought to England in 1721 from Turkey by Lady Mary Wortly Montague. This method involved inoculating healthy people with substances from the pustules of those who had a mild case of the disease, but this often had fatal results. In 1788 an epidemic of smallpox hit Gloucestershire and during this outbreak Jenner observed that those of his patients who worked with cattle and had come in contact with the much milder disease called cowpox never came down with smallpox. Jenner needed a way of showing that his theory actually worked. Jenner was given the opportunity on the 14 May 1796, when a young milkmaid called Sarah Nelmes came to see him with sores on her hands like blisters. Jenner identified that she had caught cowpox from the cows she handled each day. Jenner now had the opportunity to obtain the material try out his theories. He carefully extracted some liquid from her sores and then took some liquid from the sores of a patient with mild smallpox. Jenner believed that if he could inject someone with cowpox, the germs from the cowpox would make the body able to defend itself against the dangerous smallpox germs which he would inject later. Jenner approached a local farmer called Phipps and asked him if he could inoculate his son James against smallpox. He explained to the farmer that if his

How did Edward Jenner Discover Vaccination? Jenner worked in a rural community and most of his patients were farmers or worked on farms with cattle. In the 18th century smallpox was a very common disease and was a major cause of death. The main treatment was by a method which had brought success to a Dutch physiologist Jan Ingenhaus and was brought to England in 1721 from Turkey by Lady Mary Wortly Montague. This method involved inoculating healthy people with substances from the pustules of those who had a mild case of the disease, but this often had fatal results. In 1788 an epidemic of smallpox hit Gloucestershire and during this outbreak Jenner observed that those of his patients who worked with cattle and had come in contact with the much milder disease called cowpox never came down with smallpox. Jenner needed a way of showing that his theory actually worked. Jenner was given the opportunity on the 14 May 1796, when a young milkmaid called Sarah Nelmes came to see him with sores on her hands like blisters. Jenner identified that she had caught cowpox from the cows she handled each day. Jenner now had the opportunity to obtain the material try out his theories. He carefully extracted some liquid from her sores and then took some liquid from the sores of a patient with mild smallpox. Jenner believed that if he could inject someone with cowpox, the germs from the cowpox would make the body able to defend itself against the dangerous smallpox germs which he would inject later. Jenner approached a local farmer called Phipps and asked him if he could inoculate his son James against smallpox. He explained to the farmer that if his

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theory was correct, James would never contract smallpox. Surprisingly, the farmer agreed. Jenner made two small cuts on James’s left arm. He then poured the liquid from Sarah’s cowpox sores into the open wounds which he bandaged. James went down with cowpox but was not very ill. Six weeks later when James had recovered, Jenner vaccinated him again, this time with the smallpox virus.This was an extremely dangerous experiment. If James lived Jenner would have found a way of preventing smallpox. If James developed smallpox and died he would be a murderer. To Jenner’s relief James did not catch smallpox. His experiment had worked. In 1798 after carrying out further successful tests, he published his findings: An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Known by the Name of Cow Pox. However Jenner persevered and eventually, doctors found that vaccination did work and by 1800 most were using it. Jenner was awarded £30 000 by Parliament to enable him to continue carrying out his tests. Deaths from smallpox plummeted and vaccination spread through Europe and North America. Jenner died in Berkeley on January 26, 1823 aged 74.

theory was correct, James would never contract smallpox. Surprisingly, the farmer agreed. Jenner made two small cuts on James’s left arm. He then poured the liquid from Sarah’s cowpox sores into the open wounds which he bandaged. James went down with cowpox but was not very ill. Six weeks later when James had recovered, Jenner vaccinated him again, this time with the smallpox virus.This was an extremely dangerous experiment. If James lived Jenner would have found a way of preventing smallpox. If James developed smallpox and died he would be a murderer. To Jenner’s relief James did not catch smallpox. His experiment had worked. In 1798 after carrying out further successful tests, he published his findings: An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Known by the Name of Cow Pox. However Jenner persevered and eventually, doctors found that vaccination did work and by 1800 most were using it. Jenner was awarded £30 000 by Parliament to enable him to continue carrying out his tests. Deaths from smallpox plummeted and vaccination spread through Europe and North America. Jenner died in Berkeley on January 26, 1823 aged 74.

Definition

Definition

1. The immune system is a set of mechanisms that protect an organisms from infection by recognizing, killing and eliminating foreign pathogens or particles. 2. The immune system is able to discriminate between foreign molecules (Ag) and body own cells and molecules (This is called the self- and non-self-discrimination). 3. The system is also able to recognize host cells that are altered and that may lead to cancer.

Defence mechanism of body

1. The immune system is a set of mechanisms that protect an organisms from infection by recognizing, killing and eliminating foreign pathogens or particles. 2. The immune system is able to discriminate between foreign molecules (Ag) and body own cells and molecules (This is called the self- and non-self-discrimination). 3. The system is also able to recognize host cells that are altered and that may lead to cancer.

Defence mechanism of body

1. Non-specific defence mechanisms or natural or innate immunity. 2. Specific defence mechanisms or adaptive immunity.

1. Non-specific defence mechanisms or natural or innate immunity. 2. Specific defence mechanisms or adaptive immunity.

S. No. Innate immune system

Adaptive immune syste

S. No. Innate immune system

Adaptive immune syste

1. 2.

Pathogen and antigen specific response Lag time between exposure and maximal response It takes time in days. Cell-mediated and humoral components Exposure leads to immunological memory Found only in jawed vertebrates Lymphocyts, Ag-specific receptors, antibodies

1. 2.

Pathogen and antigen specific response Lag time between exposure and maximal response It takes time in days. Cell-mediated and humoral components Exposure leads to immunological memory Found only in jawed vertebrates Lymphocyts, Ag-specific receptors, antibodies

3. 4. 5. 6. Eg.

Response is non-specific Exposure leads to immediate maximal response Immediate response within hours. Cell-mediated and humoral components No immunological memory Found in nearly all forms of life Skin and mucosal membrane, tears, lysozymes etc.

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3. 4. 5. 6. Eg.

Response is non-specific Exposure leads to immediate maximal response Immediate response within hours. Cell-mediated and humoral components No immunological memory Found in nearly all forms of life Skin and mucosal membrane, tears, lysozymes etc.

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theory was correct, James would never contract smallpox. Surprisingly, the farmer agreed. Jenner made two small cuts on James’s left arm. He then poured the liquid from Sarah’s cowpox sores into the open wounds which he bandaged. James went down with cowpox but was not very ill. Six weeks later when James had recovered, Jenner vaccinated him again, this time with the smallpox virus.This was an extremely dangerous experiment. If James lived Jenner would have found a way of preventing smallpox. If James developed smallpox and died he would be a murderer. To Jenner’s relief James did not catch smallpox. His experiment had worked. In 1798 after carrying out further successful tests, he published his findings: An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Known by the Name of Cow Pox. However Jenner persevered and eventually, doctors found that vaccination did work and by 1800 most were using it. Jenner was awarded £30 000 by Parliament to enable him to continue carrying out his tests. Deaths from smallpox plummeted and vaccination spread through Europe and North America. Jenner died in Berkeley on January 26, 1823 aged 74.

theory was correct, James would never contract smallpox. Surprisingly, the farmer agreed. Jenner made two small cuts on James’s left arm. He then poured the liquid from Sarah’s cowpox sores into the open wounds which he bandaged. James went down with cowpox but was not very ill. Six weeks later when James had recovered, Jenner vaccinated him again, this time with the smallpox virus.This was an extremely dangerous experiment. If James lived Jenner would have found a way of preventing smallpox. If James developed smallpox and died he would be a murderer. To Jenner’s relief James did not catch smallpox. His experiment had worked. In 1798 after carrying out further successful tests, he published his findings: An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Known by the Name of Cow Pox. However Jenner persevered and eventually, doctors found that vaccination did work and by 1800 most were using it. Jenner was awarded £30 000 by Parliament to enable him to continue carrying out his tests. Deaths from smallpox plummeted and vaccination spread through Europe and North America. Jenner died in Berkeley on January 26, 1823 aged 74.

Definition

Definition

1. The immune system is a set of mechanisms that protect an organisms from infection by recognizing, killing and eliminating foreign pathogens or particles. 2. The immune system is able to discriminate between foreign molecules (Ag) and body own cells and molecules (This is called the self- and non-self-discrimination). 3. The system is also able to recognize host cells that are altered and that may lead to cancer.

Defence mechanism of body

1. The immune system is a set of mechanisms that protect an organisms from infection by recognizing, killing and eliminating foreign pathogens or particles. 2. The immune system is able to discriminate between foreign molecules (Ag) and body own cells and molecules (This is called the self- and non-self-discrimination). 3. The system is also able to recognize host cells that are altered and that may lead to cancer.

Defence mechanism of body

1. Non-specific defence mechanisms or natural or innate immunity. 2. Specific defence mechanisms or adaptive immunity.

1. Non-specific defence mechanisms or natural or innate immunity. 2. Specific defence mechanisms or adaptive immunity.

S. No. Innate immune system

Adaptive immune syste

S. No. Innate immune system

Adaptive immune syste

1. 2.

Pathogen and antigen specific response Lag time between exposure and maximal response It takes time in days. Cell-mediated and humoral components Exposure leads to immunological memory Found only in jawed vertebrates Lymphocyts, Ag-specific receptors, antibodies

1. 2.

Pathogen and antigen specific response Lag time between exposure and maximal response It takes time in days. Cell-mediated and humoral components Exposure leads to immunological memory Found only in jawed vertebrates Lymphocyts, Ag-specific receptors, antibodies

3. 4. 5. 6. Eg.

Response is non-specific Exposure leads to immediate maximal response Immediate response within hours. Cell-mediated and humoral components No immunological memory Found in nearly all forms of life Skin and mucosal membrane, tears, lysozymes etc.

3. 4. 5. 6. Eg.

Response is non-specific Exposure leads to immediate maximal response Immediate response within hours. Cell-mediated and humoral components No immunological memory Found in nearly all forms of life Skin and mucosal membrane, tears, lysozymes etc.

140 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

140 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10.2 NON-SPECIFIC DEFENCE MECHANISMS (INNATE IMMUNE SYSTEM)

10.2 NON-SPECIFIC DEFENCE MECHANISMS (INNATE IMMUNE SYSTEM)

1. 2. 3. 4. 5.

Skin and mucous membranes Phagocytosis Complement activation Exotoxin and endotoxin Inflamation

1. 2. 3. 4. 5.

Skin and mucous membranes Phagocytosis Complement activation Exotoxin and endotoxin Inflamation

10.2.1 Skin and Mucous Membranes The skins are virtually impermeable to microorganisms and enter into the skin only when damage occurs. Furthermore, many microorganisms fail to survive on the skin surface for any length of time due to the inhibitory effects of fatty acids and lactic acid in sweat and sebaceous secretions. Mucus, secreted by the membranes lining the inner surfaces of the body (like respiratory tract, gastrointestinal tract etc) acts as a protective barrier by trapping microorganisms and other foreign particles and these are subsequently removed by ciliary action linked, in the case of the respiratory tract, with coughing and sneezing. Many body secretions contain substances that exert a bactericidal action, for example the enzyme lysozyme which is found in tears, nasal secretions and saliva; hydrochloric acid in the stomach which results in a low pH; and basic polypeptides such as spermine which are found in semen. The body possesses a normal bacterial flora which, by competing for essential nutrients or by the production of inhibitory substances such as monolactams or colicins, suppresses the growth of many potential pathogens.

10.2.1 Skin and Mucous Membranes The skins are virtually impermeable to microorganisms and enter into the skin only when damage occurs. Furthermore, many microorganisms fail to survive on the skin surface for any length of time due to the inhibitory effects of fatty acids and lactic acid in sweat and sebaceous secretions. Mucus, secreted by the membranes lining the inner surfaces of the body (like respiratory tract, gastrointestinal tract etc) acts as a protective barrier by trapping microorganisms and other foreign particles and these are subsequently removed by ciliary action linked, in the case of the respiratory tract, with coughing and sneezing. Many body secretions contain substances that exert a bactericidal action, for example the enzyme lysozyme which is found in tears, nasal secretions and saliva; hydrochloric acid in the stomach which results in a low pH; and basic polypeptides such as spermine which are found in semen. The body possesses a normal bacterial flora which, by competing for essential nutrients or by the production of inhibitory substances such as monolactams or colicins, suppresses the growth of many potential pathogens.

10.2.2 Phagocytosis 1. Phagocytosis is the process of engulfing the particles such as bacteria, parasites, dead host cells, foreign debris etc by the phagocytes like monocytes, macrophages, neutrophils, dendritic cells and mast cells. These cells are called phagocytic cells. 2. These phagocytic cells have specific receptors from which microbes are bound. 3. The phagocytic cell stretches around the microbes and engulf it. 4. The microbes or pathogen trapped in a compartment called a phagosome within one minute the phagosome merges with lysosome to form a phagolysosome which is finally digested or broken down by digestive enzymes present in lysosome.

10.2.2 Phagocytosis 1. Phagocytosis is the process of engulfing the particles such as bacteria, parasites, dead host cells, foreign debris etc by the phagocytes like monocytes, macrophages, neutrophils, dendritic cells and mast cells. These cells are called phagocytic cells. 2. These phagocytic cells have specific receptors from which microbes are bound. 3. The phagocytic cell stretches around the microbes and engulf it. 4. The microbes or pathogen trapped in a compartment called a phagosome within one minute the phagosome merges with lysosome to form a phagolysosome which is finally digested or broken down by digestive enzymes present in lysosome.

10.2.3 Complement System It is a group of steps which comprises a large number of components or proteins which activate each other in a subsequential manner to produce a specified action.

10.2.3 Complement System It is a group of steps which comprises a large number of components or proteins which activate each other in a subsequential manner to produce a specified action.

140 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

140 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10.2 NON-SPECIFIC DEFENCE MECHANISMS (INNATE IMMUNE SYSTEM)

10.2 NON-SPECIFIC DEFENCE MECHANISMS (INNATE IMMUNE SYSTEM)

1. 2. 3. 4. 5.

Skin and mucous membranes Phagocytosis Complement activation Exotoxin and endotoxin Inflamation

1. 2. 3. 4. 5.

Skin and mucous membranes Phagocytosis Complement activation Exotoxin and endotoxin Inflamation

10.2.1 Skin and Mucous Membranes The skins are virtually impermeable to microorganisms and enter into the skin only when damage occurs. Furthermore, many microorganisms fail to survive on the skin surface for any length of time due to the inhibitory effects of fatty acids and lactic acid in sweat and sebaceous secretions. Mucus, secreted by the membranes lining the inner surfaces of the body (like respiratory tract, gastrointestinal tract etc) acts as a protective barrier by trapping microorganisms and other foreign particles and these are subsequently removed by ciliary action linked, in the case of the respiratory tract, with coughing and sneezing. Many body secretions contain substances that exert a bactericidal action, for example the enzyme lysozyme which is found in tears, nasal secretions and saliva; hydrochloric acid in the stomach which results in a low pH; and basic polypeptides such as spermine which are found in semen. The body possesses a normal bacterial flora which, by competing for essential nutrients or by the production of inhibitory substances such as monolactams or colicins, suppresses the growth of many potential pathogens.

10.2.1 Skin and Mucous Membranes The skins are virtually impermeable to microorganisms and enter into the skin only when damage occurs. Furthermore, many microorganisms fail to survive on the skin surface for any length of time due to the inhibitory effects of fatty acids and lactic acid in sweat and sebaceous secretions. Mucus, secreted by the membranes lining the inner surfaces of the body (like respiratory tract, gastrointestinal tract etc) acts as a protective barrier by trapping microorganisms and other foreign particles and these are subsequently removed by ciliary action linked, in the case of the respiratory tract, with coughing and sneezing. Many body secretions contain substances that exert a bactericidal action, for example the enzyme lysozyme which is found in tears, nasal secretions and saliva; hydrochloric acid in the stomach which results in a low pH; and basic polypeptides such as spermine which are found in semen. The body possesses a normal bacterial flora which, by competing for essential nutrients or by the production of inhibitory substances such as monolactams or colicins, suppresses the growth of many potential pathogens.

10.2.2 Phagocytosis 1. Phagocytosis is the process of engulfing the particles such as bacteria, parasites, dead host cells, foreign debris etc by the phagocytes like monocytes, macrophages, neutrophils, dendritic cells and mast cells. These cells are called phagocytic cells. 2. These phagocytic cells have specific receptors from which microbes are bound. 3. The phagocytic cell stretches around the microbes and engulf it. 4. The microbes or pathogen trapped in a compartment called a phagosome within one minute the phagosome merges with lysosome to form a phagolysosome which is finally digested or broken down by digestive enzymes present in lysosome.

10.2.2 Phagocytosis 1. Phagocytosis is the process of engulfing the particles such as bacteria, parasites, dead host cells, foreign debris etc by the phagocytes like monocytes, macrophages, neutrophils, dendritic cells and mast cells. These cells are called phagocytic cells. 2. These phagocytic cells have specific receptors from which microbes are bound. 3. The phagocytic cell stretches around the microbes and engulf it. 4. The microbes or pathogen trapped in a compartment called a phagosome within one minute the phagosome merges with lysosome to form a phagolysosome which is finally digested or broken down by digestive enzymes present in lysosome.

10.2.3 Complement System It is a group of steps which comprises a large number of components or proteins which activate each other in a subsequential manner to produce a specified action.

10.2.3 Complement System It is a group of steps which comprises a large number of components or proteins which activate each other in a subsequential manner to produce a specified action.

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Fig. 10.1. Shows the phagocytosis by a host cell.

Fig. 10.1. Shows the phagocytosis by a host cell.

It is the most complex system made up of about 25 proteins which are made up of 9 different types of group of proteins designated as C1 to C9. These proteins can be activated by two different ways which are called classical pathway and alternative pathway of complement activation. The activated complement cascade provides 3 useful results: phagocytosis, lysis and inflammation. The nine components of complement can be grouped into three: 1. Recognition Unit: C1 (C1q+C1r+C1s) 2. Activation Complex: C4, C2,C3 3. Membrane Attack Complex: C5, C6, C7, C8, and C9.

It is the most complex system made up of about 25 proteins which are made up of 9 different types of group of proteins designated as C1 to C9. These proteins can be activated by two different ways which are called classical pathway and alternative pathway of complement activation. The activated complement cascade provides 3 useful results: phagocytosis, lysis and inflammation. The nine components of complement can be grouped into three: 1. Recognition Unit: C1 (C1q+C1r+C1s) 2. Activation Complex: C4, C2,C3 3. Membrane Attack Complex: C5, C6, C7, C8, and C9.

10.2.4 Classical Pathway The first component of complement is C1. This is a complex of three molecules designated Clq, Clr and Cls. The classical pathway is only initiated by an immune complex (antibody-antigen complex) when Clq binds to this Ag-Ab complex.

10.2.4 Classical Pathway The first component of complement is C1. This is a complex of three molecules designated Clq, Clr and Cls. The classical pathway is only initiated by an immune complex (antibody-antigen complex) when Clq binds to this Ag-Ab complex.

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Fig. 10.1. Shows the phagocytosis by a host cell.

Fig. 10.1. Shows the phagocytosis by a host cell.

It is the most complex system made up of about 25 proteins which are made up of 9 different types of group of proteins designated as C1 to C9. These proteins can be activated by two different ways which are called classical pathway and alternative pathway of complement activation. The activated complement cascade provides 3 useful results: phagocytosis, lysis and inflammation. The nine components of complement can be grouped into three: 1. Recognition Unit: C1 (C1q+C1r+C1s) 2. Activation Complex: C4, C2,C3 3. Membrane Attack Complex: C5, C6, C7, C8, and C9.

It is the most complex system made up of about 25 proteins which are made up of 9 different types of group of proteins designated as C1 to C9. These proteins can be activated by two different ways which are called classical pathway and alternative pathway of complement activation. The activated complement cascade provides 3 useful results: phagocytosis, lysis and inflammation. The nine components of complement can be grouped into three: 1. Recognition Unit: C1 (C1q+C1r+C1s) 2. Activation Complex: C4, C2,C3 3. Membrane Attack Complex: C5, C6, C7, C8, and C9.

10.2.4 Classical Pathway The first component of complement is C1. This is a complex of three molecules designated Clq, Clr and Cls. The classical pathway is only initiated by an immune complex (antibody-antigen complex) when Clq binds to this Ag-Ab complex.

10.2.4 Classical Pathway The first component of complement is C1. This is a complex of three molecules designated Clq, Clr and Cls. The classical pathway is only initiated by an immune complex (antibody-antigen complex) when Clq binds to this Ag-Ab complex.

142 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

142 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.2. Complement system activation by different proteins.

Fig. 10.2. Complement system activation by different proteins.

The binding of Clq activates the Clr and Cls molecules associated with it to yield activated CI which now activates the C4 and split into C4a and C4b components. C4a molecule has now been recognized as an anaphylatoxin. Anaphylatoxin binds to mast cells and basophils and causes them to discharge their cytoplasmic granules. When freed from these cells, the content of these granules contract smooth muscles, causing oedema, and shortness of breath. C4b attaches to erythrocyte surfaces, bacterial cell membrane, and other antigens. It does not normally attach to C1. Now C4b component binds with the C2 and activates it which dissociates into C2a and C2b. C2b function with C4b and from C4b2a which is a C3 convertase as it can now cleave C3 into C3a and C3b. C3b is bound to the C4b2a complex to yield C4b2a3b which is also called C5 convertase. Now the phagocytic cells having the receptor for C3b on their surface, microbial cells like bacteria cell with C3b attached to it get marked for phagocytosis.

The binding of Clq activates the Clr and Cls molecules associated with it to yield activated CI which now activates the C4 and split into C4a and C4b components. C4a molecule has now been recognized as an anaphylatoxin. Anaphylatoxin binds to mast cells and basophils and causes them to discharge their cytoplasmic granules. When freed from these cells, the content of these granules contract smooth muscles, causing oedema, and shortness of breath. C4b attaches to erythrocyte surfaces, bacterial cell membrane, and other antigens. It does not normally attach to C1. Now C4b component binds with the C2 and activates it which dissociates into C2a and C2b. C2b function with C4b and from C4b2a which is a C3 convertase as it can now cleave C3 into C3a and C3b. C3b is bound to the C4b2a complex to yield C4b2a3b which is also called C5 convertase. Now the phagocytic cells having the receptor for C3b on their surface, microbial cells like bacteria cell with C3b attached to it get marked for phagocytosis.

142 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

142 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.2. Complement system activation by different proteins.

Fig. 10.2. Complement system activation by different proteins.

The binding of Clq activates the Clr and Cls molecules associated with it to yield activated CI which now activates the C4 and split into C4a and C4b components. C4a molecule has now been recognized as an anaphylatoxin. Anaphylatoxin binds to mast cells and basophils and causes them to discharge their cytoplasmic granules. When freed from these cells, the content of these granules contract smooth muscles, causing oedema, and shortness of breath. C4b attaches to erythrocyte surfaces, bacterial cell membrane, and other antigens. It does not normally attach to C1. Now C4b component binds with the C2 and activates it which dissociates into C2a and C2b. C2b function with C4b and from C4b2a which is a C3 convertase as it can now cleave C3 into C3a and C3b. C3b is bound to the C4b2a complex to yield C4b2a3b which is also called C5 convertase. Now the phagocytic cells having the receptor for C3b on their surface, microbial cells like bacteria cell with C3b attached to it get marked for phagocytosis.

The binding of Clq activates the Clr and Cls molecules associated with it to yield activated CI which now activates the C4 and split into C4a and C4b components. C4a molecule has now been recognized as an anaphylatoxin. Anaphylatoxin binds to mast cells and basophils and causes them to discharge their cytoplasmic granules. When freed from these cells, the content of these granules contract smooth muscles, causing oedema, and shortness of breath. C4b attaches to erythrocyte surfaces, bacterial cell membrane, and other antigens. It does not normally attach to C1. Now C4b component binds with the C2 and activates it which dissociates into C2a and C2b. C2b function with C4b and from C4b2a which is a C3 convertase as it can now cleave C3 into C3a and C3b. C3b is bound to the C4b2a complex to yield C4b2a3b which is also called C5 convertase. Now the phagocytic cells having the receptor for C3b on their surface, microbial cells like bacteria cell with C3b attached to it get marked for phagocytosis.

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C4b2a3b which is also known as C5 convertase, activates C5 and dissociates into C5a and C5b. C3a and C5a are released into the fluid surroundings where they serve as potent anaphylatoxins in that they cause vasoactive substances such as histamines to be released from mast cells and basophils and cause inflamation. C5b attaches to membrane of target alongwith C6, C7, C8 and C9 bring about lysis of cells.

C4b2a3b which is also known as C5 convertase, activates C5 and dissociates into C5a and C5b. C3a and C5a are released into the fluid surroundings where they serve as potent anaphylatoxins in that they cause vasoactive substances such as histamines to be released from mast cells and basophils and cause inflamation. C5b attaches to membrane of target alongwith C6, C7, C8 and C9 bring about lysis of cells.

10.2.5 Alternative Pathway The cleavage of C3 and the activation of the remainder of the complement cascade can be triggered, in the absence of complement-fixing antibody, by agents such as bacterial polysaccharide. However, C3b bound to the surface of many microbes is able to bind to a serum protein designated factor B, (this proactivator B generates Ba and Bb) which is now, in turn, cleaved by another serum protease, factor D. The resulting complex, C3b.Bb, is stabilized by another protein called properdein (P). The resultant stable complex, C3b.Bb, is a C3 convertase, analogous to C4b.2a. It cleaves C3 to form a multimolecular complex, C3b.Bb.3b, which is a C5 convertase and can generate C5b which is the focal point for the assembly of the membrane attack complex (MAC). Proteins B, D and P also amplify the effects of the classical pathway in that some of the 3b generated by this pathway interacts with these proteins to form additional C3 convertase that supplements that provided by C4b.2a. Likewise, enhanced cleavage of C5 occurs due to the dual activity of C4.2a.3b and C3b.Bb.C3b complexes.

10.2.5 Alternative Pathway The cleavage of C3 and the activation of the remainder of the complement cascade can be triggered, in the absence of complement-fixing antibody, by agents such as bacterial polysaccharide. However, C3b bound to the surface of many microbes is able to bind to a serum protein designated factor B, (this proactivator B generates Ba and Bb) which is now, in turn, cleaved by another serum protease, factor D. The resulting complex, C3b.Bb, is stabilized by another protein called properdein (P). The resultant stable complex, C3b.Bb, is a C3 convertase, analogous to C4b.2a. It cleaves C3 to form a multimolecular complex, C3b.Bb.3b, which is a C5 convertase and can generate C5b which is the focal point for the assembly of the membrane attack complex (MAC). Proteins B, D and P also amplify the effects of the classical pathway in that some of the 3b generated by this pathway interacts with these proteins to form additional C3 convertase that supplements that provided by C4b.2a. Likewise, enhanced cleavage of C5 occurs due to the dual activity of C4.2a.3b and C3b.Bb.C3b complexes.

10.2.6 Exotoxin and Endotoxin Exotoxin: Exotoxins are the proteins which are excreted by the microorganisms including bacteria, algae, fungi and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Endotoxin: It is the component of Gram-negative bacterial cell wall envelope which is composed of lipopolysaccharides (LPS), having the ability to cause disease in the host cell.

10.2.6 Exotoxin and Endotoxin Exotoxin: Exotoxins are the proteins which are excreted by the microorganisms including bacteria, algae, fungi and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Endotoxin: It is the component of Gram-negative bacterial cell wall envelope which is composed of lipopolysaccharides (LPS), having the ability to cause disease in the host cell.

S.No.

Exotoxin

Endotoxin

S.No.

Exotoxin

Endotoxin

1. 2.

These are the protein polypeptides. Excreted by the living microorganism.

1. 2.

These are the protein polypeptides. Excreted by the living microorganism.

3.

Exotoxins are heat labile, means its toxicity is destroyed rapidly on exposure to temperature 60°C They stimulate the immune system of the host to produce specific antitoxin which can neutralize toxin. They do not produce fever in the host. Exotoxins are specific for particular tissue, e.g., tetanus toxin for nervous tissue. Produced mainly by Gram-positive bacteria.

These are the lipopolysaccharides (LPS). It is the constituent of cell wall, released when disruption of cell takes place. Endotoxins are heat resistant.

3.

No antitoxin is formed. Only antibodies against LPS.

4.

Usually produce fever in the host. Non-specific in action.

5. 6.

Produced mainly by Gram-negative bacteria.

e.g.

Exotoxins are heat labile, means its toxicity is destroyed rapidly on exposure to temperature 60°C They stimulate the immune system of the host to produce specific antitoxin which can neutralize toxin. They do not produce fever in the host. Exotoxins are specific for particular tissue, e.g., tetanus toxin for nervous tissue. Produced mainly by Gram-positive bacteria.

These are the lipopolysaccharides (LPS). It is the constituent of cell wall, released when disruption of cell takes place. Endotoxins are heat resistant.

4.

5. 6. e.g.

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No antitoxin is formed. Only antibodies against LPS. Usually produce fever in the host. Non-specific in action. Produced mainly by Gram-negative bacteria.

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C4b2a3b which is also known as C5 convertase, activates C5 and dissociates into C5a and C5b. C3a and C5a are released into the fluid surroundings where they serve as potent anaphylatoxins in that they cause vasoactive substances such as histamines to be released from mast cells and basophils and cause inflamation. C5b attaches to membrane of target alongwith C6, C7, C8 and C9 bring about lysis of cells.

C4b2a3b which is also known as C5 convertase, activates C5 and dissociates into C5a and C5b. C3a and C5a are released into the fluid surroundings where they serve as potent anaphylatoxins in that they cause vasoactive substances such as histamines to be released from mast cells and basophils and cause inflamation. C5b attaches to membrane of target alongwith C6, C7, C8 and C9 bring about lysis of cells.

10.2.5 Alternative Pathway The cleavage of C3 and the activation of the remainder of the complement cascade can be triggered, in the absence of complement-fixing antibody, by agents such as bacterial polysaccharide. However, C3b bound to the surface of many microbes is able to bind to a serum protein designated factor B, (this proactivator B generates Ba and Bb) which is now, in turn, cleaved by another serum protease, factor D. The resulting complex, C3b.Bb, is stabilized by another protein called properdein (P). The resultant stable complex, C3b.Bb, is a C3 convertase, analogous to C4b.2a. It cleaves C3 to form a multimolecular complex, C3b.Bb.3b, which is a C5 convertase and can generate C5b which is the focal point for the assembly of the membrane attack complex (MAC). Proteins B, D and P also amplify the effects of the classical pathway in that some of the 3b generated by this pathway interacts with these proteins to form additional C3 convertase that supplements that provided by C4b.2a. Likewise, enhanced cleavage of C5 occurs due to the dual activity of C4.2a.3b and C3b.Bb.C3b complexes.

10.2.5 Alternative Pathway The cleavage of C3 and the activation of the remainder of the complement cascade can be triggered, in the absence of complement-fixing antibody, by agents such as bacterial polysaccharide. However, C3b bound to the surface of many microbes is able to bind to a serum protein designated factor B, (this proactivator B generates Ba and Bb) which is now, in turn, cleaved by another serum protease, factor D. The resulting complex, C3b.Bb, is stabilized by another protein called properdein (P). The resultant stable complex, C3b.Bb, is a C3 convertase, analogous to C4b.2a. It cleaves C3 to form a multimolecular complex, C3b.Bb.3b, which is a C5 convertase and can generate C5b which is the focal point for the assembly of the membrane attack complex (MAC). Proteins B, D and P also amplify the effects of the classical pathway in that some of the 3b generated by this pathway interacts with these proteins to form additional C3 convertase that supplements that provided by C4b.2a. Likewise, enhanced cleavage of C5 occurs due to the dual activity of C4.2a.3b and C3b.Bb.C3b complexes.

10.2.6 Exotoxin and Endotoxin Exotoxin: Exotoxins are the proteins which are excreted by the microorganisms including bacteria, algae, fungi and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Endotoxin: It is the component of Gram-negative bacterial cell wall envelope which is composed of lipopolysaccharides (LPS), having the ability to cause disease in the host cell.

10.2.6 Exotoxin and Endotoxin Exotoxin: Exotoxins are the proteins which are excreted by the microorganisms including bacteria, algae, fungi and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Endotoxin: It is the component of Gram-negative bacterial cell wall envelope which is composed of lipopolysaccharides (LPS), having the ability to cause disease in the host cell.

S.No.

Exotoxin

Endotoxin

S.No.

Exotoxin

Endotoxin

1. 2.

These are the protein polypeptides. Excreted by the living microorganism.

1. 2.

These are the protein polypeptides. Excreted by the living microorganism.

3.

Exotoxins are heat labile, means its toxicity is destroyed rapidly on exposure to temperature 60°C They stimulate the immune system of the host to produce specific antitoxin which can neutralize toxin. They do not produce fever in the host. Exotoxins are specific for particular tissue, e.g., tetanus toxin for nervous tissue. Produced mainly by Gram-positive bacteria.

These are the lipopolysaccharides (LPS). It is the constituent of cell wall, released when disruption of cell takes place. Endotoxins are heat resistant.

3.

No antitoxin is formed. Only antibodies against LPS.

4.

Usually produce fever in the host. Non-specific in action.

5. 6.

Produced mainly by Gram-negative bacteria.

e.g.

Exotoxins are heat labile, means its toxicity is destroyed rapidly on exposure to temperature 60°C They stimulate the immune system of the host to produce specific antitoxin which can neutralize toxin. They do not produce fever in the host. Exotoxins are specific for particular tissue, e.g., tetanus toxin for nervous tissue. Produced mainly by Gram-positive bacteria.

These are the lipopolysaccharides (LPS). It is the constituent of cell wall, released when disruption of cell takes place. Endotoxins are heat resistant.

4.

5. 6. e.g.

No antitoxin is formed. Only antibodies against LPS. Usually produce fever in the host. Non-specific in action. Produced mainly by Gram-negative bacteria.

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144 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10.2.7 Inflammation When the tissue injury occurs, due to the microbial infection inflammation takes place. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation is caused by infection. Inflammatory responses are mainly of two types: either it is localized inflammatory response or systemic inflammatory response. 1. Localized inflammatory response: When the tissue injury occurs due to the microbial infection degranulation of mast cell and macrophages takes place and release vasodilators like histamines, prostaglandins, chemokines, interleukins and TNF-D etc. These vasodilator agents cause the vasodilation of blood vessels and increase the blood volume in that area. Mast cell also responds to stimuli for complement activation. Because of vasodilation increase the vascular permeability and leading to leakage of fluid from the blood vessel and accumulate into the tissue resulting to the swelling or inflammation and redness in that area.

10.2.7 Inflammation When the tissue injury occurs, due to the microbial infection inflammation takes place. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation is caused by infection. Inflammatory responses are mainly of two types: either it is localized inflammatory response or systemic inflammatory response. 1. Localized inflammatory response: When the tissue injury occurs due to the microbial infection degranulation of mast cell and macrophages takes place and release vasodilators like histamines, prostaglandins, chemokines, interleukins and TNF-D etc. These vasodilator agents cause the vasodilation of blood vessels and increase the blood volume in that area. Mast cell also responds to stimuli for complement activation. Because of vasodilation increase the vascular permeability and leading to leakage of fluid from the blood vessel and accumulate into the tissue resulting to the swelling or inflammation and redness in that area.

Fig. 10.3. Overview of the cells and mediators involved in a local acute inflammatory response. Generally neutrophiles are the first leucocytes to migrate into the tissue followed by monocytes and lymphocytes.

Fig. 10.3. Overview of the cells and mediators involved in a local acute inflammatory response. Generally neutrophiles are the first leucocytes to migrate into the tissue followed by monocytes and lymphocytes.

2. Systemic inflammatory response: The local inflammatory response is accompanied by a systemic response known as the systemic acute phase response. This response is mediated by:

2. Systemic inflammatory response: The local inflammatory response is accompanied by a systemic response known as the systemic acute phase response. This response is mediated by:

144 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

144 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10.2.7 Inflammation When the tissue injury occurs, due to the microbial infection inflammation takes place. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation is caused by infection. Inflammatory responses are mainly of two types: either it is localized inflammatory response or systemic inflammatory response. 1. Localized inflammatory response: When the tissue injury occurs due to the microbial infection degranulation of mast cell and macrophages takes place and release vasodilators like histamines, prostaglandins, chemokines, interleukins and TNF-D etc. These vasodilator agents cause the vasodilation of blood vessels and increase the blood volume in that area. Mast cell also responds to stimuli for complement activation. Because of vasodilation increase the vascular permeability and leading to leakage of fluid from the blood vessel and accumulate into the tissue resulting to the swelling or inflammation and redness in that area.

10.2.7 Inflammation When the tissue injury occurs, due to the microbial infection inflammation takes place. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation is caused by infection. Inflammatory responses are mainly of two types: either it is localized inflammatory response or systemic inflammatory response. 1. Localized inflammatory response: When the tissue injury occurs due to the microbial infection degranulation of mast cell and macrophages takes place and release vasodilators like histamines, prostaglandins, chemokines, interleukins and TNF-D etc. These vasodilator agents cause the vasodilation of blood vessels and increase the blood volume in that area. Mast cell also responds to stimuli for complement activation. Because of vasodilation increase the vascular permeability and leading to leakage of fluid from the blood vessel and accumulate into the tissue resulting to the swelling or inflammation and redness in that area.

Fig. 10.3. Overview of the cells and mediators involved in a local acute inflammatory response. Generally neutrophiles are the first leucocytes to migrate into the tissue followed by monocytes and lymphocytes.

Fig. 10.3. Overview of the cells and mediators involved in a local acute inflammatory response. Generally neutrophiles are the first leucocytes to migrate into the tissue followed by monocytes and lymphocytes.

2. Systemic inflammatory response: The local inflammatory response is accompanied by a systemic response known as the systemic acute phase response. This response is mediated by:

2. Systemic inflammatory response: The local inflammatory response is accompanied by a systemic response known as the systemic acute phase response. This response is mediated by:

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Induction of fever. Increase the systemic hormone such as ACTH and hydrocortisone. Increase the production of WBC. Production of large number of acute phase proteins in the liver.

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Induction of fever. Increase the systemic hormone such as ACTH and hydrocortisone. Increase the production of WBC. Production of large number of acute phase proteins in the liver.

The increase in body temperature inhibits the growth of a number of pathogens and appears to enhance the immune response to the pathogen.

The increase in body temperature inhibits the growth of a number of pathogens and appears to enhance the immune response to the pathogen.

10.3 ANTIGEN OR IMMUNOGEN

10.3 ANTIGEN OR IMMUNOGEN

Antigens are the foreign particles which stimulate the immune cells to secrete antibody. When an antigen is introduced into the host, host cell induces the formation of specific antibody and T-lymphocytes that are reactive against the antigen, e.g., bacteria, virus, pollen grains, dust particles etc.

Antigens are the foreign particles which stimulate the immune cells to secrete antibody. When an antigen is introduced into the host, host cell induces the formation of specific antibody and T-lymphocytes that are reactive against the antigen, e.g., bacteria, virus, pollen grains, dust particles etc.

Immunogenicity and antigenicity

Immunogenicity and antigenicity

Immunogenicity is the ability to induce a humoral and cell mediated immune response.

Immunogenicity is the ability to induce a humoral and cell mediated immune response.

Where Tc is the Cytotoxic T cell and Th is the Helper T cell. “Antigenicity is the ability to combine specifically with the final products of the immune response (i.e. secreted antibodies and/or surface receptors on T-cells). Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true.”

Where Tc is the Cytotoxic T cell and Th is the Helper T cell. “Antigenicity is the ability to combine specifically with the final products of the immune response (i.e. secreted antibodies and/or surface receptors on T-cells). Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true.”

10.3.1 Characteristics of Antigen 1. Foreignness: It should be different from the host cell. 2. Molecular size: Generally the particles having size 1,00,000 Dalton are good antigens and size less than 5000 Dalton are poor antigens. 3. Chemical composition and complexity: Antigens composed of different amino acids or sugars have high antigenic property than single amino acids. 4. Biodegradability: If a substance is insoluble in the body fluid and cannot be converted into soluble forms by tissue enzymes it may not act as an antigen so the antigen should be digested by the phagocytes.

10.3.1 Characteristics of Antigen 1. Foreignness: It should be different from the host cell. 2. Molecular size: Generally the particles having size 1,00,000 Dalton are good antigens and size less than 5000 Dalton are poor antigens. 3. Chemical composition and complexity: Antigens composed of different amino acids or sugars have high antigenic property than single amino acids. 4. Biodegradability: If a substance is insoluble in the body fluid and cannot be converted into soluble forms by tissue enzymes it may not act as an antigen so the antigen should be digested by the phagocytes.

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Induction of fever. Increase the systemic hormone such as ACTH and hydrocortisone. Increase the production of WBC. Production of large number of acute phase proteins in the liver.

IMMUNOLOGY

• • • •

145

Induction of fever. Increase the systemic hormone such as ACTH and hydrocortisone. Increase the production of WBC. Production of large number of acute phase proteins in the liver.

The increase in body temperature inhibits the growth of a number of pathogens and appears to enhance the immune response to the pathogen.

The increase in body temperature inhibits the growth of a number of pathogens and appears to enhance the immune response to the pathogen.

10.3 ANTIGEN OR IMMUNOGEN

10.3 ANTIGEN OR IMMUNOGEN

Antigens are the foreign particles which stimulate the immune cells to secrete antibody. When an antigen is introduced into the host, host cell induces the formation of specific antibody and T-lymphocytes that are reactive against the antigen, e.g., bacteria, virus, pollen grains, dust particles etc.

Antigens are the foreign particles which stimulate the immune cells to secrete antibody. When an antigen is introduced into the host, host cell induces the formation of specific antibody and T-lymphocytes that are reactive against the antigen, e.g., bacteria, virus, pollen grains, dust particles etc.

Immunogenicity and antigenicity

Immunogenicity and antigenicity

Immunogenicity is the ability to induce a humoral and cell mediated immune response.

Immunogenicity is the ability to induce a humoral and cell mediated immune response.

Where Tc is the Cytotoxic T cell and Th is the Helper T cell. “Antigenicity is the ability to combine specifically with the final products of the immune response (i.e. secreted antibodies and/or surface receptors on T-cells). Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true.”

Where Tc is the Cytotoxic T cell and Th is the Helper T cell. “Antigenicity is the ability to combine specifically with the final products of the immune response (i.e. secreted antibodies and/or surface receptors on T-cells). Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true.”

10.3.1 Characteristics of Antigen 1. Foreignness: It should be different from the host cell. 2. Molecular size: Generally the particles having size 1,00,000 Dalton are good antigens and size less than 5000 Dalton are poor antigens. 3. Chemical composition and complexity: Antigens composed of different amino acids or sugars have high antigenic property than single amino acids. 4. Biodegradability: If a substance is insoluble in the body fluid and cannot be converted into soluble forms by tissue enzymes it may not act as an antigen so the antigen should be digested by the phagocytes.

10.3.1 Characteristics of Antigen 1. Foreignness: It should be different from the host cell. 2. Molecular size: Generally the particles having size 1,00,000 Dalton are good antigens and size less than 5000 Dalton are poor antigens. 3. Chemical composition and complexity: Antigens composed of different amino acids or sugars have high antigenic property than single amino acids. 4. Biodegradability: If a substance is insoluble in the body fluid and cannot be converted into soluble forms by tissue enzymes it may not act as an antigen so the antigen should be digested by the phagocytes.

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146 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10.4 Epitopes 1. Epitopes are the antigen determinant sites present on the surface of antigen. So an epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune cells, specifically by antibodies, B cells or T cells. 2. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes.

10.4 Epitopes 1. Epitopes are the antigen determinant sites present on the surface of antigen. So an epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune cells, specifically by antibodies, B cells or T cells. 2. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes.

Fig. 10.4. Structure of an antigen having different epitopes from where antibodies are binds.

Fig. 10.4. Structure of an antigen having different epitopes from where antibodies are binds.

3. The number of epitopes per molecule of antigen is referred to as valence of antigen. 4. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes comprise discontinuous sections of the antigen’s amino acid sequence. They interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. Most epitopes are conformational. 5. In contrast linear epitopes interact with the paratope based on their primary structure. The amino acids that make up a linear epitope are a continuous sequence of amino acids from the antigen.

3. The number of epitopes per molecule of antigen is referred to as valence of antigen. 4. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes comprise discontinuous sections of the antigen’s amino acid sequence. They interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. Most epitopes are conformational. 5. In contrast linear epitopes interact with the paratope based on their primary structure. The amino acids that make up a linear epitope are a continuous sequence of amino acids from the antigen.

10.5 CROSS-REACTIVITY

10.5 CROSS-REACTIVITY

Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies. If an antibody binds to an antigen’s epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.

Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies. If an antibody binds to an antigen’s epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.

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146 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

10.4 Epitopes 1. Epitopes are the antigen determinant sites present on the surface of antigen. So an epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune cells, specifically by antibodies, B cells or T cells. 2. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes.

10.4 Epitopes 1. Epitopes are the antigen determinant sites present on the surface of antigen. So an epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune cells, specifically by antibodies, B cells or T cells. 2. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes.

Fig. 10.4. Structure of an antigen having different epitopes from where antibodies are binds.

Fig. 10.4. Structure of an antigen having different epitopes from where antibodies are binds.

3. The number of epitopes per molecule of antigen is referred to as valence of antigen. 4. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes comprise discontinuous sections of the antigen’s amino acid sequence. They interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. Most epitopes are conformational. 5. In contrast linear epitopes interact with the paratope based on their primary structure. The amino acids that make up a linear epitope are a continuous sequence of amino acids from the antigen.

3. The number of epitopes per molecule of antigen is referred to as valence of antigen. 4. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes comprise discontinuous sections of the antigen’s amino acid sequence. They interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. Most epitopes are conformational. 5. In contrast linear epitopes interact with the paratope based on their primary structure. The amino acids that make up a linear epitope are a continuous sequence of amino acids from the antigen.

10.5 CROSS-REACTIVITY

10.5 CROSS-REACTIVITY

Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies. If an antibody binds to an antigen’s epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.

Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies. If an antibody binds to an antigen’s epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.

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Fig.10.5. Binding of haptens with carrier molecules and formation of complete antigen.

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Fig.10.5. Binding of haptens with carrier molecules and formation of complete antigen.

10.6 HAPTENS

10.6 HAPTENS

A hapten is a small molecule that can elicit an immune response only when attached to large carrier molecules such as a protein. The carrier molecules may be one that also does not elicit an immune response by itself. These carrier molecules are the incomplete antigens when bound with the haptens they form the complete antigens. Adjuvents: Adjuvents are the substances when mixed with an antigen and injected into the host enhance the immunogenicity of the antigen.

A hapten is a small molecule that can elicit an immune response only when attached to large carrier molecules such as a protein. The carrier molecules may be one that also does not elicit an immune response by itself. These carrier molecules are the incomplete antigens when bound with the haptens they form the complete antigens. Adjuvents: Adjuvents are the substances when mixed with an antigen and injected into the host enhance the immunogenicity of the antigen.

10.7 ANTIBODIES

10.7 ANTIBODIES

1. Antibodies are the antigen binding proteins present on the surface of Bcells and secreted by the plasma cells. These antibodies after being secreted from the plasma cells circulate in the blood where they search and kill the microbes. 2. When the blood or plasma is allowed to clot, the fluid phase that remains is called serum. It has been known that antibodies reside on the serum. 3. Each antibody molecule consists of four peptide chains: two identical heavy chains having molecular weight 50,000 to 70,000 Dalton and made up of 446 amino acids. Two identical light chains having molecular weight 20,000 to 25,000 Dalton and made up of 214 amino acids. 4. Each light chain binds with a heavy chain by a disulphide bond and by non-covalent hydrophobic bonds. 5. The first 110 amino acids of the amino terminal of the light chain and heavy chain are highly variable from which it binds with the antigens. 6. When the antibodies digested with papain it forms 2 Fab and 1 Fc region. The Fab fragment combines with an antigen and the Fc region does not bind with antigen.

IMMUNOLOGY

1. Antibodies are the antigen binding proteins present on the surface of Bcells and secreted by the plasma cells. These antibodies after being secreted from the plasma cells circulate in the blood where they search and kill the microbes. 2. When the blood or plasma is allowed to clot, the fluid phase that remains is called serum. It has been known that antibodies reside on the serum. 3. Each antibody molecule consists of four peptide chains: two identical heavy chains having molecular weight 50,000 to 70,000 Dalton and made up of 446 amino acids. Two identical light chains having molecular weight 20,000 to 25,000 Dalton and made up of 214 amino acids. 4. Each light chain binds with a heavy chain by a disulphide bond and by non-covalent hydrophobic bonds. 5. The first 110 amino acids of the amino terminal of the light chain and heavy chain are highly variable from which it binds with the antigens. 6. When the antibodies digested with papain it forms 2 Fab and 1 Fc region. The Fab fragment combines with an antigen and the Fc region does not bind with antigen.

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Fig.10.5. Binding of haptens with carrier molecules and formation of complete antigen.

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Fig.10.5. Binding of haptens with carrier molecules and formation of complete antigen.

10.6 HAPTENS

10.6 HAPTENS

A hapten is a small molecule that can elicit an immune response only when attached to large carrier molecules such as a protein. The carrier molecules may be one that also does not elicit an immune response by itself. These carrier molecules are the incomplete antigens when bound with the haptens they form the complete antigens. Adjuvents: Adjuvents are the substances when mixed with an antigen and injected into the host enhance the immunogenicity of the antigen.

A hapten is a small molecule that can elicit an immune response only when attached to large carrier molecules such as a protein. The carrier molecules may be one that also does not elicit an immune response by itself. These carrier molecules are the incomplete antigens when bound with the haptens they form the complete antigens. Adjuvents: Adjuvents are the substances when mixed with an antigen and injected into the host enhance the immunogenicity of the antigen.

10.7 ANTIBODIES

10.7 ANTIBODIES

1. Antibodies are the antigen binding proteins present on the surface of Bcells and secreted by the plasma cells. These antibodies after being secreted from the plasma cells circulate in the blood where they search and kill the microbes. 2. When the blood or plasma is allowed to clot, the fluid phase that remains is called serum. It has been known that antibodies reside on the serum. 3. Each antibody molecule consists of four peptide chains: two identical heavy chains having molecular weight 50,000 to 70,000 Dalton and made up of 446 amino acids. Two identical light chains having molecular weight 20,000 to 25,000 Dalton and made up of 214 amino acids. 4. Each light chain binds with a heavy chain by a disulphide bond and by non-covalent hydrophobic bonds. 5. The first 110 amino acids of the amino terminal of the light chain and heavy chain are highly variable from which it binds with the antigens. 6. When the antibodies digested with papain it forms 2 Fab and 1 Fc region. The Fab fragment combines with an antigen and the Fc region does not bind with antigen.

1. Antibodies are the antigen binding proteins present on the surface of Bcells and secreted by the plasma cells. These antibodies after being secreted from the plasma cells circulate in the blood where they search and kill the microbes. 2. When the blood or plasma is allowed to clot, the fluid phase that remains is called serum. It has been known that antibodies reside on the serum. 3. Each antibody molecule consists of four peptide chains: two identical heavy chains having molecular weight 50,000 to 70,000 Dalton and made up of 446 amino acids. Two identical light chains having molecular weight 20,000 to 25,000 Dalton and made up of 214 amino acids. 4. Each light chain binds with a heavy chain by a disulphide bond and by non-covalent hydrophobic bonds. 5. The first 110 amino acids of the amino terminal of the light chain and heavy chain are highly variable from which it binds with the antigens. 6. When the antibodies digested with papain it forms 2 Fab and 1 Fc region. The Fab fragment combines with an antigen and the Fc region does not bind with antigen.

148 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

148 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.6. Structure of antibody.

Fig. 10.6. Structure of antibody.

10.7.1 Immunoglobulin classes Based on the structure of, their heavy chain constant region, immunoglobulins are classed into five categories in human beings:

10.7.1 Immunoglobulin classes Based on the structure of, their heavy chain constant region, immunoglobulins are classed into five categories in human beings:

S.No.

Class

Heavy chain

Subclasses

light chain

S.No.

Class

Heavy chain

Subclasses

light chain

1. 2. 3. 4. 5.

IgG IgM IgA IgE IgD

J z D H G

J1, J2, J3, J4 None D1, D2 None None

O O O O O

1. 2. 3. 4. 5.

IgG IgM IgA IgE IgD

J z D H G

J1, J2, J3, J4 None D 1, D2 None None

O O O O O

or or or or or

N N N N N

1. Immunoglobulin G (IgG)

or or or or or

N N N N N

1. Immunoglobulin G (IgG)

1. IgG is the most abundant class of serum. It constitutes about 80% of total serum immunoglobulin and their serum concentration in adult human beings is between 10 to 15mgmH. The IgG molecule consists of two ã heavy chain and two ê or two ë light chains. There are four human IgG subclasses IgG1, IgG2, IgG3 & IgG4. 2. IgG1, IgG3 & IgG4 has the ability to cross the placenta and therefore provides a major line of defence against infection for the newborn. 3. IgG-3 in the most effective complement activator. 4. IgG1& IgG3 bind with Fc receptors on phagocytic cells and thus mediate opsonization. 5. It diffuses readily into the extravascular spaces where it can act in the neutralization of bacterial toxins and can bind to microorganisms enhancing the process of phagocytosis (opsonization).

148 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

1. IgG is the most abundant class of serum. It constitutes about 80% of total serum immunoglobulin and their serum concentration in adult human beings is between 10 to 15mgmH. The IgG molecule consists of two ã heavy chain and two ê or two ë light chains. There are four human IgG subclasses IgG1, IgG2, IgG3 & IgG4. 2. IgG1, IgG3 & IgG4 has the ability to cross the placenta and therefore provides a major line of defence against infection for the newborn. 3. IgG-3 in the most effective complement activator. 4. IgG1& IgG3 bind with Fc receptors on phagocytic cells and thus mediate opsonization. 5. It diffuses readily into the extravascular spaces where it can act in the neutralization of bacterial toxins and can bind to microorganisms enhancing the process of phagocytosis (opsonization).

148 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.6. Structure of antibody.

Fig. 10.6. Structure of antibody.

10.7.1 Immunoglobulin classes Based on the structure of, their heavy chain constant region, immunoglobulins are classed into five categories in human beings:

10.7.1 Immunoglobulin classes Based on the structure of, their heavy chain constant region, immunoglobulins are classed into five categories in human beings:

S.No.

Class

Heavy chain

Subclasses

light chain

S.No.

Class

Heavy chain

Subclasses

light chain

1. 2. 3. 4. 5.

IgG IgM IgA IgE IgD

J z D H G

J1, J2, J3, J4 None D1, D2 None None

O O O O O

1. 2. 3. 4. 5.

IgG IgM IgA IgE IgD

J z D H G

J1, J2, J3, J4 None D 1, D2 None None

O O O O O

or or or or or

N N N N N

1. Immunoglobulin G (IgG) 1. IgG is the most abundant class of serum. It constitutes about 80% of total serum immunoglobulin and their serum concentration in adult human beings is between 10 to 15mgmH. The IgG molecule consists of two ã heavy chain and two ê or two ë light chains. There are four human IgG subclasses IgG1, IgG2, IgG3 & IgG4. 2. IgG1, IgG3 & IgG4 has the ability to cross the placenta and therefore provides a major line of defence against infection for the newborn. 3. IgG-3 in the most effective complement activator. 4. IgG1& IgG3 bind with Fc receptors on phagocytic cells and thus mediate opsonization. 5. It diffuses readily into the extravascular spaces where it can act in the neutralization of bacterial toxins and can bind to microorganisms enhancing the process of phagocytosis (opsonization).

or or or or or

N N N N N

1. Immunoglobulin G (IgG) 1. IgG is the most abundant class of serum. It constitutes about 80% of total serum immunoglobulin and their serum concentration in adult human beings is between 10 to 15mgmH. The IgG molecule consists of two ã heavy chain and two ê or two ë light chains. There are four human IgG subclasses IgG1, IgG2, IgG3 & IgG4. 2. IgG1, IgG3 & IgG4 has the ability to cross the placenta and therefore provides a major line of defence against infection for the newborn. 3. IgG-3 in the most effective complement activator. 4. IgG1& IgG3 bind with Fc receptors on phagocytic cells and thus mediate opsonization. 5. It diffuses readily into the extravascular spaces where it can act in the neutralization of bacterial toxins and can bind to microorganisms enhancing the process of phagocytosis (opsonization).

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2. Immunoglobulin M (IgM)

2. Immunoglobulin M (IgM)

It is 5 to 10% of the total serum immunoglobulin and there serum concentrations lie between 0.5 and 2.5mgml_1. IgM is secreted by plasma cells as a pentamer in which 5 monomeric units are held together by disulfide hands.

It is 5 to 10% of the total serum immunoglobulin and there serum concentrations lie between 0.5 and 2.5mgml_1. IgM is secreted by plasma cells as a pentamer in which 5 monomeric units are held together by disulfide hands.

Fig. 10.7. Pentameric structure of IgM antibody.

Fig. 10.7. Pentameric structure of IgM antibody.

Because of its pentameric structure with 10 antigen binding sites, its valency is higher than other isotypes. IgM is the first immunoglobulin class produced in a primary response to an Ag, and it is also the first immunoglobulin to be synthesized by the neonate.

Because of its pentameric structure with 10 antigen binding sites, its valency is higher than other isotypes. IgM is the first immunoglobulin class produced in a primary response to an Ag, and it is also the first immunoglobulin to be synthesized by the neonate.

3. Immunoglobulin A (IgA)

3. Immunoglobulin A (IgA)

It constitutes only 10% to 15% of the total immunoglobulin in serum. It is the predominant immunoglobulin class in external recreations such as saliva, tears, breast milk and mucus of the bronchial, genitourinary and digestive tracts etc. Its purpose appears to be to protect the external surfaces of the body from microbial attack. The function of IgA appears to be to prevent the adherence of microorganisms to the surface of mucosal cells thus preventing them entering the body tissues. It is protected from proteolysis by combination with another protein—the secretory component.

It constitutes only 10% to 15% of the total immunoglobulin in serum. It is the predominant immunoglobulin class in external recreations such as saliva, tears, breast milk and mucus of the bronchial, genitourinary and digestive tracts etc. Its purpose appears to be to protect the external surfaces of the body from microbial attack. The function of IgA appears to be to prevent the adherence of microorganisms to the surface of mucosal cells thus preventing them entering the body tissues. It is protected from proteolysis by combination with another protein—the secretory component.

IMMUNOLOGY

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2. Immunoglobulin M (IgM)

2. Immunoglobulin M (IgM)

It is 5 to 10% of the total serum immunoglobulin and there serum concentrations lie between 0.5 and 2.5mgml_1. IgM is secreted by plasma cells as a pentamer in which 5 monomeric units are held together by disulfide hands.

It is 5 to 10% of the total serum immunoglobulin and there serum concentrations lie between 0.5 and 2.5mgml_1. IgM is secreted by plasma cells as a pentamer in which 5 monomeric units are held together by disulfide hands.

Fig. 10.7. Pentameric structure of IgM antibody.

Fig. 10.7. Pentameric structure of IgM antibody.

Because of its pentameric structure with 10 antigen binding sites, its valency is higher than other isotypes. IgM is the first immunoglobulin class produced in a primary response to an Ag, and it is also the first immunoglobulin to be synthesized by the neonate.

Because of its pentameric structure with 10 antigen binding sites, its valency is higher than other isotypes. IgM is the first immunoglobulin class produced in a primary response to an Ag, and it is also the first immunoglobulin to be synthesized by the neonate.

3. Immunoglobulin A (IgA)

3. Immunoglobulin A (IgA)

It constitutes only 10% to 15% of the total immunoglobulin in serum. It is the predominant immunoglobulin class in external recreations such as saliva, tears, breast milk and mucus of the bronchial, genitourinary and digestive tracts etc. Its purpose appears to be to protect the external surfaces of the body from microbial attack. The function of IgA appears to be to prevent the adherence of microorganisms to the surface of mucosal cells thus preventing them entering the body tissues. It is protected from proteolysis by combination with another protein—the secretory component.

It constitutes only 10% to 15% of the total immunoglobulin in serum. It is the predominant immunoglobulin class in external recreations such as saliva, tears, breast milk and mucus of the bronchial, genitourinary and digestive tracts etc. Its purpose appears to be to protect the external surfaces of the body from microbial attack. The function of IgA appears to be to prevent the adherence of microorganisms to the surface of mucosal cells thus preventing them entering the body tissues. It is protected from proteolysis by combination with another protein—the secretory component.

150 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

150 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4. Immunoglobulin E (IgE)

4. Immunoglobulin E (IgE)

Very few plasma cells in the body synthesize this immunoglobulin and the concentration of IgE in serum in also very low. It binds with very high affinity to mast cells and basophils via a site in the Fc region of the molecule. Crosslinking of the cell-bound IgE antibodies by antigen triggers the degranulation of these cells with the release of histamine, leukotrienes and other vasoactive compounds. This class may play a role in immunity to helminthic parasites but in the western world it is more commonly associated with immediate hypersensitivity reactions such as hay fever, extrinsic asthma and anaphylactic shock.

Very few plasma cells in the body synthesize this immunoglobulin and the concentration of IgE in serum in also very low. It binds with very high affinity to mast cells and basophils via a site in the Fc region of the molecule. Crosslinking of the cell-bound IgE antibodies by antigen triggers the degranulation of these cells with the release of histamine, leukotrienes and other vasoactive compounds. This class may play a role in immunity to helminthic parasites but in the western world it is more commonly associated with immediate hypersensitivity reactions such as hay fever, extrinsic asthma and anaphylactic shock.

Fig. 10.8. Binding of allergens to the IgE antibody present on the surface of mast cell and cause degranulation of mast cell and release histamines and prostaglandins as shown in the figure.

Fig. 10.8. Binding of allergens to the IgE antibody present on the surface of mast cell and cause degranulation of mast cell and release histamines and prostaglandins as shown in the figure.

5. Immunoglobulin D (IgD)

5. Immunoglobulin D (IgD)

IgD in uniquely susceptible to proteolytic degradation and has a very short half life. Immature B cells express surface IgM without IgD but as these cells mature IgD is also expressed. After activation of the B cells, surface IgD can no longer be detected and it would appear that IgD may be involved with the differentiation of B cells where it seems to be interacting with antigens.

IgD in uniquely susceptible to proteolytic degradation and has a very short half life. Immature B cells express surface IgM without IgD but as these cells mature IgD is also expressed. After activation of the B cells, surface IgD can no longer be detected and it would appear that IgD may be involved with the differentiation of B cells where it seems to be interacting with antigens.

10.8 MONOCLONAL ANTIBODIES AND THEIR APPLICATION

10.8 MONOCLONAL ANTIBODIES AND THEIR APPLICATION

10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 1. Most antigens have multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from a B cell that recognizes a particular epitope. These B-cells produce different types of antibodies for each specific epitope called polyclonal antibody.

10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 1. Most antigens have multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from a B cell that recognizes a particular epitope. These B-cells produce different types of antibodies for each specific epitope called polyclonal antibody.

150 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

150 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

4. Immunoglobulin E (IgE)

4. Immunoglobulin E (IgE)

Very few plasma cells in the body synthesize this immunoglobulin and the concentration of IgE in serum in also very low. It binds with very high affinity to mast cells and basophils via a site in the Fc region of the molecule. Crosslinking of the cell-bound IgE antibodies by antigen triggers the degranulation of these cells with the release of histamine, leukotrienes and other vasoactive compounds. This class may play a role in immunity to helminthic parasites but in the western world it is more commonly associated with immediate hypersensitivity reactions such as hay fever, extrinsic asthma and anaphylactic shock.

Very few plasma cells in the body synthesize this immunoglobulin and the concentration of IgE in serum in also very low. It binds with very high affinity to mast cells and basophils via a site in the Fc region of the molecule. Crosslinking of the cell-bound IgE antibodies by antigen triggers the degranulation of these cells with the release of histamine, leukotrienes and other vasoactive compounds. This class may play a role in immunity to helminthic parasites but in the western world it is more commonly associated with immediate hypersensitivity reactions such as hay fever, extrinsic asthma and anaphylactic shock.

Fig. 10.8. Binding of allergens to the IgE antibody present on the surface of mast cell and cause degranulation of mast cell and release histamines and prostaglandins as shown in the figure.

Fig. 10.8. Binding of allergens to the IgE antibody present on the surface of mast cell and cause degranulation of mast cell and release histamines and prostaglandins as shown in the figure.

5. Immunoglobulin D (IgD)

5. Immunoglobulin D (IgD)

IgD in uniquely susceptible to proteolytic degradation and has a very short half life. Immature B cells express surface IgM without IgD but as these cells mature IgD is also expressed. After activation of the B cells, surface IgD can no longer be detected and it would appear that IgD may be involved with the differentiation of B cells where it seems to be interacting with antigens.

IgD in uniquely susceptible to proteolytic degradation and has a very short half life. Immature B cells express surface IgM without IgD but as these cells mature IgD is also expressed. After activation of the B cells, surface IgD can no longer be detected and it would appear that IgD may be involved with the differentiation of B cells where it seems to be interacting with antigens.

10.8 MONOCLONAL ANTIBODIES AND THEIR APPLICATION

10.8 MONOCLONAL ANTIBODIES AND THEIR APPLICATION

10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 1. Most antigens have multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from a B cell that recognizes a particular epitope. These B-cells produce different types of antibodies for each specific epitope called polyclonal antibody.

10.8.1 Preparation of Monoclonal Antibodies (Hybridoma Technolgoy) 1. Most antigens have multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from a B cell that recognizes a particular epitope. These B-cells produce different types of antibodies for each specific epitope called polyclonal antibody.

IMMUNOLOGY

2.

3.

4.

5.

151

Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies, derived from a single clone and thus specific for a single epitope, are preferable. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing monoclonal antibody, which quickly became one of immunology’s key technologies. By fusing a normal activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell), they were able to generate a hybrid cell, called a hybridoma that possessed the immortal growth properties of the myeloma cell and secreted the antibody produced by the B cell. The next step is to select for the hybridomas. The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRT is hypoxanthine- guanine phosphoribosyl transferase, an enzyme involved in the synthesis of nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells. The myeloma cells that fuse with another myeloma cell

Fig. 10.9. The polyclonal antiserum produced in response to a complex antigen contains a mixture of monoclonal antibodies, each specific for one of the four epitopes shown on the antigen. In contrast a monoclonal antibody which is derived from a single plasma cell is specific for one epitope on a complex antigen. The outline of the basic method for obtaining monoclonal antibody is illustrated here.

IMMUNOLOGY

2.

3.

4.

5.

IMMUNOLOGY

2.

3.

4.

5.

Fig. 10.9. The polyclonal antiserum produced in response to a complex antigen contains a mixture of monoclonal antibodies, each specific for one of the four epitopes shown on the antigen. In contrast a monoclonal antibody which is derived from a single plasma cell is specific for one epitope on a complex antigen. The outline of the basic method for obtaining monoclonal antibody is illustrated here.

Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies, derived from a single clone and thus specific for a single epitope, are preferable. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing monoclonal antibody, which quickly became one of immunology’s key technologies. By fusing a normal activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell), they were able to generate a hybrid cell, called a hybridoma that possessed the immortal growth properties of the myeloma cell and secreted the antibody produced by the B cell. The next step is to select for the hybridomas. The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRT is hypoxanthine- guanine phosphoribosyl transferase, an enzyme involved in the synthesis of nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells. The myeloma cells that fuse with another myeloma cell

Fig. 10.9. The polyclonal antiserum produced in response to a complex antigen contains a mixture of monoclonal antibodies, each specific for one of the four epitopes shown on the antigen. In contrast a monoclonal antibody which is derived from a single plasma cell is specific for one epitope on a complex antigen. The outline of the basic method for obtaining monoclonal antibody is illustrated here.

151

Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies, derived from a single clone and thus specific for a single epitope, are preferable. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing monoclonal antibody, which quickly became one of immunology’s key technologies. By fusing a normal activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell), they were able to generate a hybrid cell, called a hybridoma that possessed the immortal growth properties of the myeloma cell and secreted the antibody produced by the B cell. The next step is to select for the hybridomas. The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRT is hypoxanthine- guanine phosphoribosyl transferase, an enzyme involved in the synthesis of nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells. The myeloma cells that fuse with another myeloma cell

151

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2.

3.

4.

5.

151

Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies, derived from a single clone and thus specific for a single epitope, are preferable. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing monoclonal antibody, which quickly became one of immunology’s key technologies. By fusing a normal activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell), they were able to generate a hybrid cell, called a hybridoma that possessed the immortal growth properties of the myeloma cell and secreted the antibody produced by the B cell. The next step is to select for the hybridomas. The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRT is hypoxanthine- guanine phosphoribosyl transferase, an enzyme involved in the synthesis of nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells. The myeloma cells that fuse with another myeloma cell

Fig. 10.9. The polyclonal antiserum produced in response to a complex antigen contains a mixture of monoclonal antibodies, each specific for one of the four epitopes shown on the antigen. In contrast a monoclonal antibody which is derived from a single plasma cell is specific for one epitope on a complex antigen. The outline of the basic method for obtaining monoclonal antibody is illustrated here.

152 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

152 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

or do not fuse at all die in the HAT medium since they are HGPRT–. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous. 6. The resulting clones of hybridoma cells, which secrete large quantities of monoclonal antibody, can be cultured indefinitely.

or do not fuse at all die in the HAT medium since they are HGPRT–. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous. 6. The resulting clones of hybridoma cells, which secrete large quantities of monoclonal antibody, can be cultured indefinitely.

10.8.2 Applications 1. Identification of odd substances produced by the body in special circumstances, e.g. hormones produced in pregnancy, or abnormal metabolites, which enable screening for genetic abnormalities, drugs, etc and other health problems. 2. Identification and quantification of circulating microorganisms, possibly in small numbers or too small to see under the microscope, e.g. infecting viruses. 3. To target and identify, neutralise or remove unwanted substances from the body, especially the blood plasma. Examples might include drugs taken in overdose. 4. If a particular type of cancer with cells widespread in the body can be shown to have different antigens than ordinary cells then it may be possible to treat cancer by targeting only the unwanted cells in the body. 5. Reducing the possibility of rejection in transplants by more accurate tissue typing between recipient and possible donors. 6. Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic use of antibodies is the diagnosis of AIDS by the ELISA test. 7. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells. 8. Monoclonal antibodies can be used to treat viral diseases, traditionally considered untreatable. 9. Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria gonorrhoeae can be typed using monoclonal antibodies.

10.8.2 Applications 1. Identification of odd substances produced by the body in special circumstances, e.g. hormones produced in pregnancy, or abnormal metabolites, which enable screening for genetic abnormalities, drugs, etc and other health problems. 2. Identification and quantification of circulating microorganisms, possibly in small numbers or too small to see under the microscope, e.g. infecting viruses. 3. To target and identify, neutralise or remove unwanted substances from the body, especially the blood plasma. Examples might include drugs taken in overdose. 4. If a particular type of cancer with cells widespread in the body can be shown to have different antigens than ordinary cells then it may be possible to treat cancer by targeting only the unwanted cells in the body. 5. Reducing the possibility of rejection in transplants by more accurate tissue typing between recipient and possible donors. 6. Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic use of antibodies is the diagnosis of AIDS by the ELISA test. 7. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells. 8. Monoclonal antibodies can be used to treat viral diseases, traditionally considered untreatable. 9. Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria gonorrhoeae can be typed using monoclonal antibodies.

10.9 ACTIVE AND PASSIVE IMMUNITY

10.9 ACTIVE AND PASSIVE IMMUNITY

10.9.1 Naturally Acquired Active Immunity Active immunity develops after antigens enter the body and the individuals immune system responds with antibodies. The exposure to antigens may be unintentional or intentional. When former, the immunity that develops is called naturally acquired active immunity.

10.9.1 Naturally Acquired Active Immunity Active immunity develops after antigens enter the body and the individuals immune system responds with antibodies. The exposure to antigens may be unintentional or intentional. When former, the immunity that develops is called naturally acquired active immunity.

152 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

152 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

or do not fuse at all die in the HAT medium since they are HGPRT–. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous. 6. The resulting clones of hybridoma cells, which secrete large quantities of monoclonal antibody, can be cultured indefinitely.

or do not fuse at all die in the HAT medium since they are HGPRT–. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous. 6. The resulting clones of hybridoma cells, which secrete large quantities of monoclonal antibody, can be cultured indefinitely.

10.8.2 Applications 1. Identification of odd substances produced by the body in special circumstances, e.g. hormones produced in pregnancy, or abnormal metabolites, which enable screening for genetic abnormalities, drugs, etc and other health problems. 2. Identification and quantification of circulating microorganisms, possibly in small numbers or too small to see under the microscope, e.g. infecting viruses. 3. To target and identify, neutralise or remove unwanted substances from the body, especially the blood plasma. Examples might include drugs taken in overdose. 4. If a particular type of cancer with cells widespread in the body can be shown to have different antigens than ordinary cells then it may be possible to treat cancer by targeting only the unwanted cells in the body. 5. Reducing the possibility of rejection in transplants by more accurate tissue typing between recipient and possible donors. 6. Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic use of antibodies is the diagnosis of AIDS by the ELISA test. 7. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells. 8. Monoclonal antibodies can be used to treat viral diseases, traditionally considered untreatable. 9. Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria gonorrhoeae can be typed using monoclonal antibodies.

10.8.2 Applications 1. Identification of odd substances produced by the body in special circumstances, e.g. hormones produced in pregnancy, or abnormal metabolites, which enable screening for genetic abnormalities, drugs, etc and other health problems. 2. Identification and quantification of circulating microorganisms, possibly in small numbers or too small to see under the microscope, e.g. infecting viruses. 3. To target and identify, neutralise or remove unwanted substances from the body, especially the blood plasma. Examples might include drugs taken in overdose. 4. If a particular type of cancer with cells widespread in the body can be shown to have different antigens than ordinary cells then it may be possible to treat cancer by targeting only the unwanted cells in the body. 5. Reducing the possibility of rejection in transplants by more accurate tissue typing between recipient and possible donors. 6. Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic use of antibodies is the diagnosis of AIDS by the ELISA test. 7. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells. 8. Monoclonal antibodies can be used to treat viral diseases, traditionally considered untreatable. 9. Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria gonorrhoeae can be typed using monoclonal antibodies.

10.9 ACTIVE AND PASSIVE IMMUNITY

10.9 ACTIVE AND PASSIVE IMMUNITY

10.9.1 Naturally Acquired Active Immunity Active immunity develops after antigens enter the body and the individuals immune system responds with antibodies. The exposure to antigens may be unintentional or intentional. When former, the immunity that develops is called naturally acquired active immunity.

10.9.1 Naturally Acquired Active Immunity Active immunity develops after antigens enter the body and the individuals immune system responds with antibodies. The exposure to antigens may be unintentional or intentional. When former, the immunity that develops is called naturally acquired active immunity.

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Naturally acquired active immunity usually follows a bout of illness . However, this may not be always and subclinical diseases may also induce this immunity. For example, many have acquired immunity from subclinical cases of mumps (viral) or cryptococcosis (fungal). Memory cells in the lymphoid tissues are responsible for producing antibodies that yield this immunity. The cells remain active for many years and produce IgG immediately upon later entry of the parasite (antigen) to the host. Such an antibody response is also called secondary anamnestic response.

Naturally acquired active immunity usually follows a bout of illness . However, this may not be always and subclinical diseases may also induce this immunity. For example, many have acquired immunity from subclinical cases of mumps (viral) or cryptococcosis (fungal). Memory cells in the lymphoid tissues are responsible for producing antibodies that yield this immunity. The cells remain active for many years and produce IgG immediately upon later entry of the parasite (antigen) to the host. Such an antibody response is also called secondary anamnestic response.

10.9.2 Artificially Acquired Active Immunity Artificially active immunity in induced by the vaccination. e.g., smallpox vaccine, BCG vaccine, rabies, hepatitis B, AIDS vaccine or HIV vaccine tetanus vaccine etc.

10.9.2 Artificially Acquired Active Immunity Artificially active immunity in induced by the vaccination. e.g., smallpox vaccine, BCG vaccine, rabies, hepatitis B, AIDS vaccine or HIV vaccine tetanus vaccine etc.

10.9.3 Naturally Acquired Passive Immunity Passive immunity develops when antibodies enter the body from an outside source. For example, IgG immunoglobulins are an example of such type of antibodies which is transferred from mother to baby during the development and protect the foetus. The exposure to antibodies may be unintentional or intentional. When unintentional, the immunity that develops is called naturally acquired passive immunity.

10.9.3 Naturally Acquired Passive Immunity Passive immunity develops when antibodies enter the body from an outside source. For example, IgG immunoglobulins are an example of such type of antibodies which is transferred from mother to baby during the development and protect the foetus. The exposure to antibodies may be unintentional or intentional. When unintentional, the immunity that develops is called naturally acquired passive immunity.

10.9.4 Artificially Acquired Passive Immunity Immunity acquired by directly injecting artificial antibodies into the host body is called the artificially acquired passive immunity. This method is still used for viral diseases as Lassa fever, hepatitis, and arthropodborne encephalitis, and for bacterial diseases where toxins are involved. For example, established cases of botulism, diphtheria, and tetanus are treated with serum containing the respective antitoxins. Since the diseases are very dangerous and fatal, no risk is taken for introduction of antigens. Instead already made antibodies are introduced into blood. Various terms are used for the serum that renders artificially acquired passive immunity. Note: Active immunity develops slowly. It is long lasting and often gives protection for many years while passive immunity is short-lived and protection is lost within 2 or 3 weeks.

10.9.4 Artificially Acquired Passive Immunity Immunity acquired by directly injecting artificial antibodies into the host body is called the artificially acquired passive immunity. This method is still used for viral diseases as Lassa fever, hepatitis, and arthropodborne encephalitis, and for bacterial diseases where toxins are involved. For example, established cases of botulism, diphtheria, and tetanus are treated with serum containing the respective antitoxins. Since the diseases are very dangerous and fatal, no risk is taken for introduction of antigens. Instead already made antibodies are introduced into blood. Various terms are used for the serum that renders artificially acquired passive immunity. Note: Active immunity develops slowly. It is long lasting and often gives protection for many years while passive immunity is short-lived and protection is lost within 2 or 3 weeks.

10.10 CELLULAR AND HUMORAL IMMUNITY

10.10 CELLULAR AND HUMORAL IMMUNITY

The humoral, immune response is based on antibodies; it can be conferred on non-immune individuals by administration of serum antibodies from an immune individual. Antibodies act as the effector of humoral response.

The humoral, immune response is based on antibodies; it can be conferred on non-immune individuals by administration of serum antibodies from an immune individual. Antibodies act as the effector of humoral response.

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Naturally acquired active immunity usually follows a bout of illness . However, this may not be always and subclinical diseases may also induce this immunity. For example, many have acquired immunity from subclinical cases of mumps (viral) or cryptococcosis (fungal). Memory cells in the lymphoid tissues are responsible for producing antibodies that yield this immunity. The cells remain active for many years and produce IgG immediately upon later entry of the parasite (antigen) to the host. Such an antibody response is also called secondary anamnestic response.

Naturally acquired active immunity usually follows a bout of illness . However, this may not be always and subclinical diseases may also induce this immunity. For example, many have acquired immunity from subclinical cases of mumps (viral) or cryptococcosis (fungal). Memory cells in the lymphoid tissues are responsible for producing antibodies that yield this immunity. The cells remain active for many years and produce IgG immediately upon later entry of the parasite (antigen) to the host. Such an antibody response is also called secondary anamnestic response.

10.9.2 Artificially Acquired Active Immunity Artificially active immunity in induced by the vaccination. e.g., smallpox vaccine, BCG vaccine, rabies, hepatitis B, AIDS vaccine or HIV vaccine tetanus vaccine etc.

10.9.2 Artificially Acquired Active Immunity Artificially active immunity in induced by the vaccination. e.g., smallpox vaccine, BCG vaccine, rabies, hepatitis B, AIDS vaccine or HIV vaccine tetanus vaccine etc.

10.9.3 Naturally Acquired Passive Immunity Passive immunity develops when antibodies enter the body from an outside source. For example, IgG immunoglobulins are an example of such type of antibodies which is transferred from mother to baby during the development and protect the foetus. The exposure to antibodies may be unintentional or intentional. When unintentional, the immunity that develops is called naturally acquired passive immunity.

10.9.3 Naturally Acquired Passive Immunity Passive immunity develops when antibodies enter the body from an outside source. For example, IgG immunoglobulins are an example of such type of antibodies which is transferred from mother to baby during the development and protect the foetus. The exposure to antibodies may be unintentional or intentional. When unintentional, the immunity that develops is called naturally acquired passive immunity.

10.9.4 Artificially Acquired Passive Immunity Immunity acquired by directly injecting artificial antibodies into the host body is called the artificially acquired passive immunity. This method is still used for viral diseases as Lassa fever, hepatitis, and arthropodborne encephalitis, and for bacterial diseases where toxins are involved. For example, established cases of botulism, diphtheria, and tetanus are treated with serum containing the respective antitoxins. Since the diseases are very dangerous and fatal, no risk is taken for introduction of antigens. Instead already made antibodies are introduced into blood. Various terms are used for the serum that renders artificially acquired passive immunity. Note: Active immunity develops slowly. It is long lasting and often gives protection for many years while passive immunity is short-lived and protection is lost within 2 or 3 weeks.

10.9.4 Artificially Acquired Passive Immunity Immunity acquired by directly injecting artificial antibodies into the host body is called the artificially acquired passive immunity. This method is still used for viral diseases as Lassa fever, hepatitis, and arthropodborne encephalitis, and for bacterial diseases where toxins are involved. For example, established cases of botulism, diphtheria, and tetanus are treated with serum containing the respective antitoxins. Since the diseases are very dangerous and fatal, no risk is taken for introduction of antigens. Instead already made antibodies are introduced into blood. Various terms are used for the serum that renders artificially acquired passive immunity. Note: Active immunity develops slowly. It is long lasting and often gives protection for many years while passive immunity is short-lived and protection is lost within 2 or 3 weeks.

10.10 CELLULAR AND HUMORAL IMMUNITY

10.10 CELLULAR AND HUMORAL IMMUNITY

The humoral, immune response is based on antibodies; it can be conferred on non-immune individuals by administration of serum antibodies from an immune individual. Antibodies act as the effector of humoral response.

The humoral, immune response is based on antibodies; it can be conferred on non-immune individuals by administration of serum antibodies from an immune individual. Antibodies act as the effector of humoral response.

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154 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

They bind to the antigens and, thereby, facilitate their elimination, e.g. by forming clusters (1) rough cross-linking of antigen molecules, which are readily ingested by phagocytic cells. Binding of antibodies to a microorganism can activate the complement system, which lyses the microorganism. Antibodies bind to toxins and viral particles, and prevent their subsequent, binding to host cells. In contrast, cell-mediated immunity is based on T cells, which are a type of lymphocyte. T cells are of two types: T helper (TH) and T cytotoxic (Tc) cells. When a TH cell interacts with an antigen-MHC II molecule complex present on an APC, it becomes activated and begins to secrete cytokines. These cytokines activate B cells, Tc cells, and various phagocytic cells. Activated phagocytic cells are able to kill microorganisms like bacteria and protozoa more effectively. When a Tc cell interacts with an antigen-MHC I complex, the Tc cell proliferates under the influence of cytokines produced by activated TH cells. These Tc cells differentiate into cytotoxic T lymphocytes (CTLs). The CTLs kill all such cells that display foreign antigens complexed with MHC I molecules. Such cells are called altered self-cells; they are usually virus-infected cells, tumour cells and foreign tissue cells. Thus TH cells and CTLs are effectors of the cell-mediated immune response. Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells play a major role in the rejection of tumours and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis.

They bind to the antigens and, thereby, facilitate their elimination, e.g. by forming clusters (1) rough cross-linking of antigen molecules, which are readily ingested by phagocytic cells. Binding of antibodies to a microorganism can activate the complement system, which lyses the microorganism. Antibodies bind to toxins and viral particles, and prevent their subsequent, binding to host cells. In contrast, cell-mediated immunity is based on T cells, which are a type of lymphocyte. T cells are of two types: T helper (TH) and T cytotoxic (Tc) cells. When a TH cell interacts with an antigen-MHC II molecule complex present on an APC, it becomes activated and begins to secrete cytokines. These cytokines activate B cells, Tc cells, and various phagocytic cells. Activated phagocytic cells are able to kill microorganisms like bacteria and protozoa more effectively. When a Tc cell interacts with an antigen-MHC I complex, the Tc cell proliferates under the influence of cytokines produced by activated TH cells. These Tc cells differentiate into cytotoxic T lymphocytes (CTLs). The CTLs kill all such cells that display foreign antigens complexed with MHC I molecules. Such cells are called altered self-cells; they are usually virus-infected cells, tumour cells and foreign tissue cells. Thus TH cells and CTLs are effectors of the cell-mediated immune response. Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells play a major role in the rejection of tumours and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis.

10.11 ANTIGEN-ANTIBODY REACTION

10.11 ANTIGEN-ANTIBODY REACTION

1. The antigen antibody reaction is bimolecular association similar to an enzyme-substrate interaction, with an important distinction is, it is not irreversible. 2. When antigen and antibody molecules are brought together in solution, linkage is formed between Fab portion of the antibody and epitopes present on the surface of antigens. 3. This Ag-Ab interaction forms the basis of the humoral response of the immune system. 4. These reactions can be visualized by allowing the Ag and Ab to combine under controlled in vitro conditions. 5. The association between an antibody and an antigen involves various non-covalent interactions that form the basis of Ag-Ab binding, includes: i) Ionic bonds ii) Hydrogen bonds iii) Hydrophobic interaction iv) Van der waals interaction

1. The antigen antibody reaction is bimolecular association similar to an enzyme-substrate interaction, with an important distinction is, it is not irreversible. 2. When antigen and antibody molecules are brought together in solution, linkage is formed between Fab portion of the antibody and epitopes present on the surface of antigens. 3. This Ag-Ab interaction forms the basis of the humoral response of the immune system. 4. These reactions can be visualized by allowing the Ag and Ab to combine under controlled in vitro conditions. 5. The association between an antibody and an antigen involves various non-covalent interactions that form the basis of Ag-Ab binding, includes: i) Ionic bonds ii) Hydrogen bonds iii) Hydrophobic interaction iv) Van der waals interaction

154 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

154 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

They bind to the antigens and, thereby, facilitate their elimination, e.g. by forming clusters (1) rough cross-linking of antigen molecules, which are readily ingested by phagocytic cells. Binding of antibodies to a microorganism can activate the complement system, which lyses the microorganism. Antibodies bind to toxins and viral particles, and prevent their subsequent, binding to host cells. In contrast, cell-mediated immunity is based on T cells, which are a type of lymphocyte. T cells are of two types: T helper (TH) and T cytotoxic (Tc) cells. When a TH cell interacts with an antigen-MHC II molecule complex present on an APC, it becomes activated and begins to secrete cytokines. These cytokines activate B cells, Tc cells, and various phagocytic cells. Activated phagocytic cells are able to kill microorganisms like bacteria and protozoa more effectively. When a Tc cell interacts with an antigen-MHC I complex, the Tc cell proliferates under the influence of cytokines produced by activated TH cells. These Tc cells differentiate into cytotoxic T lymphocytes (CTLs). The CTLs kill all such cells that display foreign antigens complexed with MHC I molecules. Such cells are called altered self-cells; they are usually virus-infected cells, tumour cells and foreign tissue cells. Thus TH cells and CTLs are effectors of the cell-mediated immune response. Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells play a major role in the rejection of tumours and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis.

They bind to the antigens and, thereby, facilitate their elimination, e.g. by forming clusters (1) rough cross-linking of antigen molecules, which are readily ingested by phagocytic cells. Binding of antibodies to a microorganism can activate the complement system, which lyses the microorganism. Antibodies bind to toxins and viral particles, and prevent their subsequent, binding to host cells. In contrast, cell-mediated immunity is based on T cells, which are a type of lymphocyte. T cells are of two types: T helper (TH) and T cytotoxic (Tc) cells. When a TH cell interacts with an antigen-MHC II molecule complex present on an APC, it becomes activated and begins to secrete cytokines. These cytokines activate B cells, Tc cells, and various phagocytic cells. Activated phagocytic cells are able to kill microorganisms like bacteria and protozoa more effectively. When a Tc cell interacts with an antigen-MHC I complex, the Tc cell proliferates under the influence of cytokines produced by activated TH cells. These Tc cells differentiate into cytotoxic T lymphocytes (CTLs). The CTLs kill all such cells that display foreign antigens complexed with MHC I molecules. Such cells are called altered self-cells; they are usually virus-infected cells, tumour cells and foreign tissue cells. Thus TH cells and CTLs are effectors of the cell-mediated immune response. Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells play a major role in the rejection of tumours and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis.

10.11 ANTIGEN-ANTIBODY REACTION

10.11 ANTIGEN-ANTIBODY REACTION

1. The antigen antibody reaction is bimolecular association similar to an enzyme-substrate interaction, with an important distinction is, it is not irreversible. 2. When antigen and antibody molecules are brought together in solution, linkage is formed between Fab portion of the antibody and epitopes present on the surface of antigens. 3. This Ag-Ab interaction forms the basis of the humoral response of the immune system. 4. These reactions can be visualized by allowing the Ag and Ab to combine under controlled in vitro conditions. 5. The association between an antibody and an antigen involves various non-covalent interactions that form the basis of Ag-Ab binding, includes: i) Ionic bonds ii) Hydrogen bonds iii) Hydrophobic interaction iv) Van der waals interaction

1. The antigen antibody reaction is bimolecular association similar to an enzyme-substrate interaction, with an important distinction is, it is not irreversible. 2. When antigen and antibody molecules are brought together in solution, linkage is formed between Fab portion of the antibody and epitopes present on the surface of antigens. 3. This Ag-Ab interaction forms the basis of the humoral response of the immune system. 4. These reactions can be visualized by allowing the Ag and Ab to combine under controlled in vitro conditions. 5. The association between an antibody and an antigen involves various non-covalent interactions that form the basis of Ag-Ab binding, includes: i) Ionic bonds ii) Hydrogen bonds iii) Hydrophobic interaction iv) Van der waals interaction

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Because these interactions are weak individually so a large number of such interactions are required to form a strong Ag-Ab interaction.

Because these interactions are weak individually so a large number of such interactions are required to form a strong Ag-Ab interaction.

Affinity

Affinity

1. Ab affinity is a quantitative measure of binding strength. 2. The combined strength of the non-covalent interaction between a single Ag-binding site on an Ab and a single epitope is the affinity of the Ab for that epitope.

1. Ab affinity is a quantitative measure of binding strength. 2. The combined strength of the non-covalent interaction between a single Ag-binding site on an Ab and a single epitope is the affinity of the Ab for that epitope.

Fig. 10.10. (a) accurate binding between antigen and antibody and showing the higher affinity while (b) showing low affinity because no accurate binding between antigen-antibody takes place.

Fig. 10.10. (a) accurate binding between antigen and antibody and showing the higher affinity while (b) showing low affinity because no accurate binding between antigen-antibody takes place.

3. Low affinity Ab binds with Ag weakly and the high affinity Ab for that epitope. Affinity is the equilibrium constant that describes the Ag-Ab reaction.

3. Low affinity Ab binds with Ag weakly and the high affinity Ab for that epitope. Affinity is the equilibrium constant that describes the Ag-Ab reaction.

Avidity

Avidity

1. Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. 2. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.

1. Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. 2. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.

Specificity

Specificity

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant (or the ability of a population of antibody molecules to react with only one antigen). In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in: 1) The primary structure of an antigen, 2) Isomeric forms of an antigen, and 3) Secondary and tertiary structure of an antigen.

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant (or the ability of a population of antibody molecules to react with only one antigen). In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in: 1) The primary structure of an antigen, 2) Isomeric forms of an antigen, and 3) Secondary and tertiary structure of an antigen.

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Because these interactions are weak individually so a large number of such interactions are required to form a strong Ag-Ab interaction.

Because these interactions are weak individually so a large number of such interactions are required to form a strong Ag-Ab interaction.

Affinity

Affinity

1. Ab affinity is a quantitative measure of binding strength. 2. The combined strength of the non-covalent interaction between a single Ag-binding site on an Ab and a single epitope is the affinity of the Ab for that epitope.

1. Ab affinity is a quantitative measure of binding strength. 2. The combined strength of the non-covalent interaction between a single Ag-binding site on an Ab and a single epitope is the affinity of the Ab for that epitope.

Fig. 10.10. (a) accurate binding between antigen and antibody and showing the higher affinity while (b) showing low affinity because no accurate binding between antigen-antibody takes place.

Fig. 10.10. (a) accurate binding between antigen and antibody and showing the higher affinity while (b) showing low affinity because no accurate binding between antigen-antibody takes place.

3. Low affinity Ab binds with Ag weakly and the high affinity Ab for that epitope. Affinity is the equilibrium constant that describes the Ag-Ab reaction.

3. Low affinity Ab binds with Ag weakly and the high affinity Ab for that epitope. Affinity is the equilibrium constant that describes the Ag-Ab reaction.

Avidity 1. Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. 2. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.

Avidity 1. Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. 2. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.

Specificity

Specificity

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant (or the ability of a population of antibody molecules to react with only one antigen). In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in: 1) The primary structure of an antigen, 2) Isomeric forms of an antigen, and 3) Secondary and tertiary structure of an antigen.

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant (or the ability of a population of antibody molecules to react with only one antigen). In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in: 1) The primary structure of an antigen, 2) Isomeric forms of an antigen, and 3) Secondary and tertiary structure of an antigen.

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Fig. 10.11. (a) Showing the specific binding between Ag and Ab while (b) showing the cross reactivity of antibody with similar type of antigen (this is not accurate binding).

156 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.11. (a) Showing the specific binding between Ag and Ab while (b) showing the cross reactivity of antibody with similar type of antigen (this is not accurate binding).

Cross reactivity

Cross reactivity

Although Ag-Ab are highly specific, in some case Ab elicited by one Ag can cross react with an unrelated Ag. Such cross reactivity occurs if two differents Ags have an identical or very similar epitopes. But in second case the Ab affinity is less than the original epitope. Antibody-antigen interactions are of different types:

Although Ag-Ab are highly specific, in some case Ab elicited by one Ag can cross react with an unrelated Ag. Such cross reactivity occurs if two differents Ags have an identical or very similar epitopes. But in second case the Ab affinity is less than the original epitope. Antibody-antigen interactions are of different types:

Fig. 10.12. Binding of polyclonal and monoclonal antibody with the antigens and form a complex which is precipitated.

Fig. 10.12. Binding of polyclonal and monoclonal antibody with the antigens and form a complex which is precipitated.

I. Precipitation Reaction: This reaction involves thousands of antigen and antibody molecules cross linked at multiple sites to form a structure called a lattice. These become so large that precipitate form can be easily seen. Precipitation tests are performed either in fluid-fluid precipitation, or gelgel precipitation. In the former, the antibody and antigen solutions are layered over each other in a thin tube. In the latter, the diffusion of antigen and antibody takes place through a semisolid gel as agarose.

156 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.11. (a) Showing the specific binding between Ag and Ab while (b) showing the cross reactivity of antibody with similar type of antigen (this is not accurate binding).

I. Precipitation Reaction: This reaction involves thousands of antigen and antibody molecules cross linked at multiple sites to form a structure called a lattice. These become so large that precipitate form can be easily seen. Precipitation tests are performed either in fluid-fluid precipitation, or gelgel precipitation. In the former, the antibody and antigen solutions are layered over each other in a thin tube. In the latter, the diffusion of antigen and antibody takes place through a semisolid gel as agarose.

156 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.11. (a) Showing the specific binding between Ag and Ab while (b) showing the cross reactivity of antibody with similar type of antigen (this is not accurate binding).

Cross reactivity

Cross reactivity

Although Ag-Ab are highly specific, in some case Ab elicited by one Ag can cross react with an unrelated Ag. Such cross reactivity occurs if two differents Ags have an identical or very similar epitopes. But in second case the Ab affinity is less than the original epitope. Antibody-antigen interactions are of different types:

Although Ag-Ab are highly specific, in some case Ab elicited by one Ag can cross react with an unrelated Ag. Such cross reactivity occurs if two differents Ags have an identical or very similar epitopes. But in second case the Ab affinity is less than the original epitope. Antibody-antigen interactions are of different types:

Fig. 10.12. Binding of polyclonal and monoclonal antibody with the antigens and form a complex which is precipitated.

Fig. 10.12. Binding of polyclonal and monoclonal antibody with the antigens and form a complex which is precipitated.

I. Precipitation Reaction: This reaction involves thousands of antigen and antibody molecules cross linked at multiple sites to form a structure called a lattice. These become so large that precipitate form can be easily seen. Precipitation tests are performed either in fluid-fluid precipitation, or gelgel precipitation. In the former, the antibody and antigen solutions are layered over each other in a thin tube. In the latter, the diffusion of antigen and antibody takes place through a semisolid gel as agarose.

I. Precipitation Reaction: This reaction involves thousands of antigen and antibody molecules cross linked at multiple sites to form a structure called a lattice. These become so large that precipitate form can be easily seen. Precipitation tests are performed either in fluid-fluid precipitation, or gelgel precipitation. In the former, the antibody and antigen solutions are layered over each other in a thin tube. In the latter, the diffusion of antigen and antibody takes place through a semisolid gel as agarose.

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There are used the Oudin tube technique or Ouchterlony plate technique. In a procedure, called immunoelectrophoresis, the two techniques of electrophoresis and diffusion are combined for detection of antigens. Agglutination Reaction: In this reaction antibodies react with antigens on the surface of particulate objects and cause the objects to clump together, or agglutinate. These reactions were the earliest to be adapted to diagnostic laboratory. Widal test is used for diagnosis of typhoid fever. This test, developed by Georges Fernand I. Widal (French physician) in 1896, is now supplemented by more sophisticated procedures. A polyvalent serum (a serum containing a mixture of antibodies) is prepared. Passive agglutination is a modern approach in this procedure, where antigens are adsorbed onto the surface of latex spheres, polystyrene particles, red blood cells, bacteria or other carriers. Serum antibodies are then detected by observing agglutination of the carrier particle. Haemagglutination is the agglutination of red blood cells. This process is very important in the determination of blood types before transfusion process. Some viruses as those of mumps and measles that agglutinate red blood cells may be detected in patients serum by this test. Neutralization reaction: Neutralization is a reaction in which antigens and antibodies neutralize each other. The reaction is used to identify toxins and antitoxins, as well as viruses and viral antibodies. An example is the detection of botulism toxin in food. Complement Fixation Test: Complement fixation test was developed by Jules Bordet Octave Gengou in 1907. It was later adopted for syphilis by August von Wassermann in 1906 and for about 75 years it was a mainstay for syphilis diagnosis. These days, technologists use it for detection of antibodies against a variety of viruses, bacteria and fungi. The test is done in two parts: (i) the test system,which utilises the patients serum, a preparation of antigen and complement derived from guinea pigs (ii) the indicator system, requires sheep red blood cells and haemolysin (antibodies against sheep red blood cells). Haemolysins cause lysis of red blood cells in the presence of complement. Flocculation: This test combines the principles of precipitation and agglutination. The antigen exists in a non-cellular particulate form that reacts with antibodies to form large, visible aggregates. An example of this test is Veneral Disease Research Laboratory (VDRL) test used for the rapid screening of patients to detect syphilis. The antigen is alcoholic extract of beef heart called cardiolipin. This reacts with syphilis antibodies in patient serum to form aggregates. Fluorescent Antibody Technique: Fluorescent antibody technique is a slide test performed by combining particles containing antigens with antibodies and a fluorescent dye. When these three complements react, the dye causes the complex to glow an illumination with UV light under

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There are used the Oudin tube technique or Ouchterlony plate technique. In a procedure, called immunoelectrophoresis, the two techniques of electrophoresis and diffusion are combined for detection of antigens. Agglutination Reaction: In this reaction antibodies react with antigens on the surface of particulate objects and cause the objects to clump together, or agglutinate. These reactions were the earliest to be adapted to diagnostic laboratory. Widal test is used for diagnosis of typhoid fever. This test, developed by Georges Fernand I. Widal (French physician) in 1896, is now supplemented by more sophisticated procedures. A polyvalent serum (a serum containing a mixture of antibodies) is prepared. Passive agglutination is a modern approach in this procedure, where antigens are adsorbed onto the surface of latex spheres, polystyrene particles, red blood cells, bacteria or other carriers. Serum antibodies are then detected by observing agglutination of the carrier particle. Haemagglutination is the agglutination of red blood cells. This process is very important in the determination of blood types before transfusion process. Some viruses as those of mumps and measles that agglutinate red blood cells may be detected in patients serum by this test. Neutralization reaction: Neutralization is a reaction in which antigens and antibodies neutralize each other. The reaction is used to identify toxins and antitoxins, as well as viruses and viral antibodies. An example is the detection of botulism toxin in food. Complement Fixation Test: Complement fixation test was developed by Jules Bordet Octave Gengou in 1907. It was later adopted for syphilis by August von Wassermann in 1906 and for about 75 years it was a mainstay for syphilis diagnosis. These days, technologists use it for detection of antibodies against a variety of viruses, bacteria and fungi. The test is done in two parts: (i) the test system,which utilises the patients serum, a preparation of antigen and complement derived from guinea pigs (ii) the indicator system, requires sheep red blood cells and haemolysin (antibodies against sheep red blood cells). Haemolysins cause lysis of red blood cells in the presence of complement. Flocculation: This test combines the principles of precipitation and agglutination. The antigen exists in a non-cellular particulate form that reacts with antibodies to form large, visible aggregates. An example of this test is Veneral Disease Research Laboratory (VDRL) test used for the rapid screening of patients to detect syphilis. The antigen is alcoholic extract of beef heart called cardiolipin. This reacts with syphilis antibodies in patient serum to form aggregates. Fluorescent Antibody Technique: Fluorescent antibody technique is a slide test performed by combining particles containing antigens with antibodies and a fluorescent dye. When these three complements react, the dye causes the complex to glow an illumination with UV light under

There are used the Oudin tube technique or Ouchterlony plate technique. In a procedure, called immunoelectrophoresis, the two techniques of electrophoresis and diffusion are combined for detection of antigens. Agglutination Reaction: In this reaction antibodies react with antigens on the surface of particulate objects and cause the objects to clump together, or agglutinate. These reactions were the earliest to be adapted to diagnostic laboratory. Widal test is used for diagnosis of typhoid fever. This test, developed by Georges Fernand I. Widal (French physician) in 1896, is now supplemented by more sophisticated procedures. A polyvalent serum (a serum containing a mixture of antibodies) is prepared. Passive agglutination is a modern approach in this procedure, where antigens are adsorbed onto the surface of latex spheres, polystyrene particles, red blood cells, bacteria or other carriers. Serum antibodies are then detected by observing agglutination of the carrier particle. Haemagglutination is the agglutination of red blood cells. This process is very important in the determination of blood types before transfusion process. Some viruses as those of mumps and measles that agglutinate red blood cells may be detected in patients serum by this test. Neutralization reaction: Neutralization is a reaction in which antigens and antibodies neutralize each other. The reaction is used to identify toxins and antitoxins, as well as viruses and viral antibodies. An example is the detection of botulism toxin in food. Complement Fixation Test: Complement fixation test was developed by Jules Bordet Octave Gengou in 1907. It was later adopted for syphilis by August von Wassermann in 1906 and for about 75 years it was a mainstay for syphilis diagnosis. These days, technologists use it for detection of antibodies against a variety of viruses, bacteria and fungi. The test is done in two parts: (i) the test system,which utilises the patients serum, a preparation of antigen and complement derived from guinea pigs (ii) the indicator system, requires sheep red blood cells and haemolysin (antibodies against sheep red blood cells). Haemolysins cause lysis of red blood cells in the presence of complement. Flocculation: This test combines the principles of precipitation and agglutination. The antigen exists in a non-cellular particulate form that reacts with antibodies to form large, visible aggregates. An example of this test is Veneral Disease Research Laboratory (VDRL) test used for the rapid screening of patients to detect syphilis. The antigen is alcoholic extract of beef heart called cardiolipin. This reacts with syphilis antibodies in patient serum to form aggregates. Fluorescent Antibody Technique: Fluorescent antibody technique is a slide test performed by combining particles containing antigens with antibodies and a fluorescent dye. When these three complements react, the dye causes the complex to glow an illumination with UV light under

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157

There are used the Oudin tube technique or Ouchterlony plate technique. In a procedure, called immunoelectrophoresis, the two techniques of electrophoresis and diffusion are combined for detection of antigens. Agglutination Reaction: In this reaction antibodies react with antigens on the surface of particulate objects and cause the objects to clump together, or agglutinate. These reactions were the earliest to be adapted to diagnostic laboratory. Widal test is used for diagnosis of typhoid fever. This test, developed by Georges Fernand I. Widal (French physician) in 1896, is now supplemented by more sophisticated procedures. A polyvalent serum (a serum containing a mixture of antibodies) is prepared. Passive agglutination is a modern approach in this procedure, where antigens are adsorbed onto the surface of latex spheres, polystyrene particles, red blood cells, bacteria or other carriers. Serum antibodies are then detected by observing agglutination of the carrier particle. Haemagglutination is the agglutination of red blood cells. This process is very important in the determination of blood types before transfusion process. Some viruses as those of mumps and measles that agglutinate red blood cells may be detected in patients serum by this test. Neutralization reaction: Neutralization is a reaction in which antigens and antibodies neutralize each other. The reaction is used to identify toxins and antitoxins, as well as viruses and viral antibodies. An example is the detection of botulism toxin in food. Complement Fixation Test: Complement fixation test was developed by Jules Bordet Octave Gengou in 1907. It was later adopted for syphilis by August von Wassermann in 1906 and for about 75 years it was a mainstay for syphilis diagnosis. These days, technologists use it for detection of antibodies against a variety of viruses, bacteria and fungi. The test is done in two parts: (i) the test system,which utilises the patients serum, a preparation of antigen and complement derived from guinea pigs (ii) the indicator system, requires sheep red blood cells and haemolysin (antibodies against sheep red blood cells). Haemolysins cause lysis of red blood cells in the presence of complement. Flocculation: This test combines the principles of precipitation and agglutination. The antigen exists in a non-cellular particulate form that reacts with antibodies to form large, visible aggregates. An example of this test is Veneral Disease Research Laboratory (VDRL) test used for the rapid screening of patients to detect syphilis. The antigen is alcoholic extract of beef heart called cardiolipin. This reacts with syphilis antibodies in patient serum to form aggregates. Fluorescent Antibody Technique: Fluorescent antibody technique is a slide test performed by combining particles containing antigens with antibodies and a fluorescent dye. When these three complements react, the dye causes the complex to glow an illumination with UV light under

158 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

158 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

a fluorescent microscope. Two commonly used dyes are fluorescein, which emits an apple green glow, and rhodamine that gives off orange red light. These techniques may be direct or indirect. In direct method, the dye is linked to known antibody molecules. The antibodies are then combined with particles (may be bacteria) that may contain complementary antigens. The tagged antibodies accumulate on the particle surface and the particle glows under the microscope. The indirect method is illustrated by the Ff A-ABS diagnostic procedure used for syphilis antibodies in the blood of patient. VII. Radioimmunoassay (RIA): Radioimmunoassay is an extremely sensitive serological method used to measure the concentration of low molecular weight antigens, like haptens. It was developed in 1960s and since then has been adapted for quantification of hepatitis antigens, reproductive hormones, insulin and some drugs. This test also is useful for detection of tumour viruses in the body before the appearance of tumours. This test can detect trillionths of a gram of substances.

a fluorescent microscope. Two commonly used dyes are fluorescein, which emits an apple green glow, and rhodamine that gives off orange red light. These techniques may be direct or indirect. In direct method, the dye is linked to known antibody molecules. The antibodies are then combined with particles (may be bacteria) that may contain complementary antigens. The tagged antibodies accumulate on the particle surface and the particle glows under the microscope. The indirect method is illustrated by the Ff A-ABS diagnostic procedure used for syphilis antibodies in the blood of patient. VII. Radioimmunoassay (RIA): Radioimmunoassay is an extremely sensitive serological method used to measure the concentration of low molecular weight antigens, like haptens. It was developed in 1960s and since then has been adapted for quantification of hepatitis antigens, reproductive hormones, insulin and some drugs. This test also is useful for detection of tumour viruses in the body before the appearance of tumours. This test can detect trillionths of a gram of substances.

Fig. 10.13. RIA is a highly sensitive technique for measuring antigen or antibody that involves competitive binding of radio-labelled Ag or Ab.

Fig. 10.13. RIA is a highly sensitive technique for measuring antigen or antibody that involves competitive binding of radio-labelled Ag or Ab.

158 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

158 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

a fluorescent microscope. Two commonly used dyes are fluorescein, which emits an apple green glow, and rhodamine that gives off orange red light. These techniques may be direct or indirect. In direct method, the dye is linked to known antibody molecules. The antibodies are then combined with particles (may be bacteria) that may contain complementary antigens. The tagged antibodies accumulate on the particle surface and the particle glows under the microscope. The indirect method is illustrated by the Ff A-ABS diagnostic procedure used for syphilis antibodies in the blood of patient. VII. Radioimmunoassay (RIA): Radioimmunoassay is an extremely sensitive serological method used to measure the concentration of low molecular weight antigens, like haptens. It was developed in 1960s and since then has been adapted for quantification of hepatitis antigens, reproductive hormones, insulin and some drugs. This test also is useful for detection of tumour viruses in the body before the appearance of tumours. This test can detect trillionths of a gram of substances.

a fluorescent microscope. Two commonly used dyes are fluorescein, which emits an apple green glow, and rhodamine that gives off orange red light. These techniques may be direct or indirect. In direct method, the dye is linked to known antibody molecules. The antibodies are then combined with particles (may be bacteria) that may contain complementary antigens. The tagged antibodies accumulate on the particle surface and the particle glows under the microscope. The indirect method is illustrated by the Ff A-ABS diagnostic procedure used for syphilis antibodies in the blood of patient. VII. Radioimmunoassay (RIA): Radioimmunoassay is an extremely sensitive serological method used to measure the concentration of low molecular weight antigens, like haptens. It was developed in 1960s and since then has been adapted for quantification of hepatitis antigens, reproductive hormones, insulin and some drugs. This test also is useful for detection of tumour viruses in the body before the appearance of tumours. This test can detect trillionths of a gram of substances.

Fig. 10.13. RIA is a highly sensitive technique for measuring antigen or antibody that involves competitive binding of radio-labelled Ag or Ab.

Fig. 10.13. RIA is a highly sensitive technique for measuring antigen or antibody that involves competitive binding of radio-labelled Ag or Ab.

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VIII. Radioallergosorbent Test (RAST): This test is an extension of the radioimmunoassay. This may be used to detect IgE or other antibodies as well as a variety of small antigens. RAST is commonly known as “sandwich” teachnique. To detect IgE, specific antigens for this antibody are attached to a matrix particle. Serum suspected to contain IgE is then added. Antibody, if present combines on the surface of the particle. Now another antibody, one that reacts with human antibodies, is added. This antiglobulin antibody carries a radioactive label. The entire complex will, therefore, radioactive if the antiglobulin antibody combines with the IgE. If, IgE is not present, the particles will not show radioactivity. IX. Enzyme Linked Immunosorbent Assay (ELISA): ELISA has actually the same sensitivity as RAST and RIA. However, it does not require radioactivity or expensive equipment. Antigens or antibodies are attached to a solid surface, and the coated surfaces are combined (immunosorbed) with the test material. An enzyme system is then linked to the complex, the remaining enzyme system is washed away and the extent of enzyme activity measured. This indicates the presence of antigens or antibodies in the material. An application of ELISA is found in the Gonozyme test used to detect gonorrhea agent (Neisseria gonorrhoeae) in the patient. ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

VIII. Radioallergosorbent Test (RAST): This test is an extension of the radioimmunoassay. This may be used to detect IgE or other antibodies as well as a variety of small antigens. RAST is commonly known as “sandwich” teachnique. To detect IgE, specific antigens for this antibody are attached to a matrix particle. Serum suspected to contain IgE is then added. Antibody, if present combines on the surface of the particle. Now another antibody, one that reacts with human antibodies, is added. This antiglobulin antibody carries a radioactive label. The entire complex will, therefore, radioactive if the antiglobulin antibody combines with the IgE. If, IgE is not present, the particles will not show radioactivity. IX. Enzyme Linked Immunosorbent Assay (ELISA): ELISA has actually the same sensitivity as RAST and RIA. However, it does not require radioactivity or expensive equipment. Antigens or antibodies are attached to a solid surface, and the coated surfaces are combined (immunosorbed) with the test material. An enzyme system is then linked to the complex, the remaining enzyme system is washed away and the extent of enzyme activity measured. This indicates the presence of antigens or antibodies in the material. An application of ELISA is found in the Gonozyme test used to detect gonorrhea agent (Neisseria gonorrhoeae) in the patient. ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

10.12 IMMUNOLOGICAL TOLERANCE

10.12 IMMUNOLOGICAL TOLERANCE

The administration of antigenic material does not always stimulate an immunological response, a condition termed ‘tolerance’. The classic example of this is the exposure of the immature lymphoid system of neonates to antigen, inducing a state of unresponsiveness to later challenge by the same antigen after the animal has reached immunological maturity. This could be the means whereby, during gestation, the body becomes unresponsive to its own constituents enabling the mature lymphoid system to distinguish in later life between ‘self and ‘non-self’. Tolerance can also be induced in adults, but higher doses of the antigen are required where it has been shown that both T and B cells are made unresponsive. As most antibody responses are T-dependent it is likely that it is these cells which are the affected ones. In order to maintain this state of tolerance it is necessary for the antigen to persist in the animal, as in its absence immunecompetent cells which are being produced throughout life are not being rendered tolerant.

The administration of antigenic material does not always stimulate an immunological response, a condition termed ‘tolerance’. The classic example of this is the exposure of the immature lymphoid system of neonates to antigen, inducing a state of unresponsiveness to later challenge by the same antigen after the animal has reached immunological maturity. This could be the means whereby, during gestation, the body becomes unresponsive to its own constituents enabling the mature lymphoid system to distinguish in later life between ‘self and ‘non-self’. Tolerance can also be induced in adults, but higher doses of the antigen are required where it has been shown that both T and B cells are made unresponsive. As most antibody responses are T-dependent it is likely that it is these cells which are the affected ones. In order to maintain this state of tolerance it is necessary for the antigen to persist in the animal, as in its absence immunecompetent cells which are being produced throughout life are not being rendered tolerant.

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VIII. Radioallergosorbent Test (RAST): This test is an extension of the radioimmunoassay. This may be used to detect IgE or other antibodies as well as a variety of small antigens. RAST is commonly known as “sandwich” teachnique. To detect IgE, specific antigens for this antibody are attached to a matrix particle. Serum suspected to contain IgE is then added. Antibody, if present combines on the surface of the particle. Now another antibody, one that reacts with human antibodies, is added. This antiglobulin antibody carries a radioactive label. The entire complex will, therefore, radioactive if the antiglobulin antibody combines with the IgE. If, IgE is not present, the particles will not show radioactivity. IX. Enzyme Linked Immunosorbent Assay (ELISA): ELISA has actually the same sensitivity as RAST and RIA. However, it does not require radioactivity or expensive equipment. Antigens or antibodies are attached to a solid surface, and the coated surfaces are combined (immunosorbed) with the test material. An enzyme system is then linked to the complex, the remaining enzyme system is washed away and the extent of enzyme activity measured. This indicates the presence of antigens or antibodies in the material. An application of ELISA is found in the Gonozyme test used to detect gonorrhea agent (Neisseria gonorrhoeae) in the patient. ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

VIII. Radioallergosorbent Test (RAST): This test is an extension of the radioimmunoassay. This may be used to detect IgE or other antibodies as well as a variety of small antigens. RAST is commonly known as “sandwich” teachnique. To detect IgE, specific antigens for this antibody are attached to a matrix particle. Serum suspected to contain IgE is then added. Antibody, if present combines on the surface of the particle. Now another antibody, one that reacts with human antibodies, is added. This antiglobulin antibody carries a radioactive label. The entire complex will, therefore, radioactive if the antiglobulin antibody combines with the IgE. If, IgE is not present, the particles will not show radioactivity. IX. Enzyme Linked Immunosorbent Assay (ELISA): ELISA has actually the same sensitivity as RAST and RIA. However, it does not require radioactivity or expensive equipment. Antigens or antibodies are attached to a solid surface, and the coated surfaces are combined (immunosorbed) with the test material. An enzyme system is then linked to the complex, the remaining enzyme system is washed away and the extent of enzyme activity measured. This indicates the presence of antigens or antibodies in the material. An application of ELISA is found in the Gonozyme test used to detect gonorrhea agent (Neisseria gonorrhoeae) in the patient. ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

10.12 IMMUNOLOGICAL TOLERANCE

10.12 IMMUNOLOGICAL TOLERANCE

The administration of antigenic material does not always stimulate an immunological response, a condition termed ‘tolerance’. The classic example of this is the exposure of the immature lymphoid system of neonates to antigen, inducing a state of unresponsiveness to later challenge by the same antigen after the animal has reached immunological maturity. This could be the means whereby, during gestation, the body becomes unresponsive to its own constituents enabling the mature lymphoid system to distinguish in later life between ‘self and ‘non-self’. Tolerance can also be induced in adults, but higher doses of the antigen are required where it has been shown that both T and B cells are made unresponsive. As most antibody responses are T-dependent it is likely that it is these cells which are the affected ones. In order to maintain this state of tolerance it is necessary for the antigen to persist in the animal, as in its absence immunecompetent cells which are being produced throughout life are not being rendered tolerant.

The administration of antigenic material does not always stimulate an immunological response, a condition termed ‘tolerance’. The classic example of this is the exposure of the immature lymphoid system of neonates to antigen, inducing a state of unresponsiveness to later challenge by the same antigen after the animal has reached immunological maturity. This could be the means whereby, during gestation, the body becomes unresponsive to its own constituents enabling the mature lymphoid system to distinguish in later life between ‘self and ‘non-self’. Tolerance can also be induced in adults, but higher doses of the antigen are required where it has been shown that both T and B cells are made unresponsive. As most antibody responses are T-dependent it is likely that it is these cells which are the affected ones. In order to maintain this state of tolerance it is necessary for the antigen to persist in the animal, as in its absence immunecompetent cells which are being produced throughout life are not being rendered tolerant.

160 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Tolerance can occur in several ways: 1. Genetic unresponsiveness. If the animal lacks the necessary genetic ability to recognize antigenic material it will be ‘immunologically’ silent. 2. T-supperssion. Ts cells may be activated more effectively than TH cells, thereby suppressing the immune response. 3. Helplessness. T cells are more readily tolerated than B cells and if they are unable to activate the B cells these cells could be described as ‘helpless’. 4. Clonal deletion. Contact with antigen in the neonate results in death or permanent inactivation of the developing lymphocytes.

160 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Tolerance can occur in several ways: 1. Genetic unresponsiveness. If the animal lacks the necessary genetic ability to recognize antigenic material it will be ‘immunologically’ silent. 2. T-supperssion. Ts cells may be activated more effectively than TH cells, thereby suppressing the immune response. 3. Helplessness. T cells are more readily tolerated than B cells and if they are unable to activate the B cells these cells could be described as ‘helpless’. 4. Clonal deletion. Contact with antigen in the neonate results in death or permanent inactivation of the developing lymphocytes.

10.13 AUTOIMMUNITY

10.13 AUTOIMMUNITY

One fundamental property of an animal’s immune system is that it does not normally react against its own body constituents, i.e. it exhibits tolerance. However, clinical and experimental evidence shows that certain diseases exist in which the patient apparently destroys his/her own cells. The reactions could involve Tc cells, B cells or NK cells, and the result of the reaction with antigen may result in a pathological condition arising (autoimmune disease). Autoimmunity is the mirror-image of tolerance and reflects the loss of tolerance to self. Autoimmunity can arise by the following. 1. Evasion of tolerance to self-antigens. Hidden or sequestered antigens do exist, for instance spermatozoa and eye-lens tissue. These are confined to anatomical sites which do not have access to lymphoid tissue, and exposure of the above to lymphoid cells as a result of surgery or accident results in the production of the corresponding antibodies. Drugs frequently bind to blood elements directly (e.g. penicillin to erythrocytes) and the antibodies to the resultant complex react with, and damage, cells coated with the drug. Viruses, especially those that bud, become associated with the host cell surface antigens with the resultant generation of Tc cells. 2. Break down of tolerance mechanisms: There are at least two mechanisms for maintaining unresponsiveness to self. The first is by specific deletion of self-reactive clones and the second by suppression. A failure of either of these two may result in an autoimmune disease. In normal, healthy individuals, antigen-binding, self-reactive B cells and the resultant low titres of autoantibodies are not uncommon. The origin of the self-reactive B cells is not clear, but there are four ways in which they may become activated. (a) Polyclonal activation. High concentrations of polyclonal activators, such as LPS and high molecular weight dextrans, activate B cells irrespective of the immunoglobulin receptor on the B cell surface.

One fundamental property of an animal’s immune system is that it does not normally react against its own body constituents, i.e. it exhibits tolerance. However, clinical and experimental evidence shows that certain diseases exist in which the patient apparently destroys his/her own cells. The reactions could involve Tc cells, B cells or NK cells, and the result of the reaction with antigen may result in a pathological condition arising (autoimmune disease). Autoimmunity is the mirror-image of tolerance and reflects the loss of tolerance to self. Autoimmunity can arise by the following. 1. Evasion of tolerance to self-antigens. Hidden or sequestered antigens do exist, for instance spermatozoa and eye-lens tissue. These are confined to anatomical sites which do not have access to lymphoid tissue, and exposure of the above to lymphoid cells as a result of surgery or accident results in the production of the corresponding antibodies. Drugs frequently bind to blood elements directly (e.g. penicillin to erythrocytes) and the antibodies to the resultant complex react with, and damage, cells coated with the drug. Viruses, especially those that bud, become associated with the host cell surface antigens with the resultant generation of Tc cells. 2. Break down of tolerance mechanisms: There are at least two mechanisms for maintaining unresponsiveness to self. The first is by specific deletion of self-reactive clones and the second by suppression. A failure of either of these two may result in an autoimmune disease. In normal, healthy individuals, antigen-binding, self-reactive B cells and the resultant low titres of autoantibodies are not uncommon. The origin of the self-reactive B cells is not clear, but there are four ways in which they may become activated. (a) Polyclonal activation. High concentrations of polyclonal activators, such as LPS and high molecular weight dextrans, activate B cells irrespective of the immunoglobulin receptor on the B cell surface.

160 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

160 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Tolerance can occur in several ways: 1. Genetic unresponsiveness. If the animal lacks the necessary genetic ability to recognize antigenic material it will be ‘immunologically’ silent. 2. T-supperssion. Ts cells may be activated more effectively than TH cells, thereby suppressing the immune response. 3. Helplessness. T cells are more readily tolerated than B cells and if they are unable to activate the B cells these cells could be described as ‘helpless’. 4. Clonal deletion. Contact with antigen in the neonate results in death or permanent inactivation of the developing lymphocytes.

Tolerance can occur in several ways: 1. Genetic unresponsiveness. If the animal lacks the necessary genetic ability to recognize antigenic material it will be ‘immunologically’ silent. 2. T-supperssion. Ts cells may be activated more effectively than TH cells, thereby suppressing the immune response. 3. Helplessness. T cells are more readily tolerated than B cells and if they are unable to activate the B cells these cells could be described as ‘helpless’. 4. Clonal deletion. Contact with antigen in the neonate results in death or permanent inactivation of the developing lymphocytes.

10.13 AUTOIMMUNITY

10.13 AUTOIMMUNITY

One fundamental property of an animal’s immune system is that it does not normally react against its own body constituents, i.e. it exhibits tolerance. However, clinical and experimental evidence shows that certain diseases exist in which the patient apparently destroys his/her own cells. The reactions could involve Tc cells, B cells or NK cells, and the result of the reaction with antigen may result in a pathological condition arising (autoimmune disease). Autoimmunity is the mirror-image of tolerance and reflects the loss of tolerance to self. Autoimmunity can arise by the following. 1. Evasion of tolerance to self-antigens. Hidden or sequestered antigens do exist, for instance spermatozoa and eye-lens tissue. These are confined to anatomical sites which do not have access to lymphoid tissue, and exposure of the above to lymphoid cells as a result of surgery or accident results in the production of the corresponding antibodies. Drugs frequently bind to blood elements directly (e.g. penicillin to erythrocytes) and the antibodies to the resultant complex react with, and damage, cells coated with the drug. Viruses, especially those that bud, become associated with the host cell surface antigens with the resultant generation of Tc cells. 2. Break down of tolerance mechanisms: There are at least two mechanisms for maintaining unresponsiveness to self. The first is by specific deletion of self-reactive clones and the second by suppression. A failure of either of these two may result in an autoimmune disease. In normal, healthy individuals, antigen-binding, self-reactive B cells and the resultant low titres of autoantibodies are not uncommon. The origin of the self-reactive B cells is not clear, but there are four ways in which they may become activated. (a) Polyclonal activation. High concentrations of polyclonal activators, such as LPS and high molecular weight dextrans, activate B cells irrespective of the immunoglobulin receptor on the B cell surface.

One fundamental property of an animal’s immune system is that it does not normally react against its own body constituents, i.e. it exhibits tolerance. However, clinical and experimental evidence shows that certain diseases exist in which the patient apparently destroys his/her own cells. The reactions could involve Tc cells, B cells or NK cells, and the result of the reaction with antigen may result in a pathological condition arising (autoimmune disease). Autoimmunity is the mirror-image of tolerance and reflects the loss of tolerance to self. Autoimmunity can arise by the following. 1. Evasion of tolerance to self-antigens. Hidden or sequestered antigens do exist, for instance spermatozoa and eye-lens tissue. These are confined to anatomical sites which do not have access to lymphoid tissue, and exposure of the above to lymphoid cells as a result of surgery or accident results in the production of the corresponding antibodies. Drugs frequently bind to blood elements directly (e.g. penicillin to erythrocytes) and the antibodies to the resultant complex react with, and damage, cells coated with the drug. Viruses, especially those that bud, become associated with the host cell surface antigens with the resultant generation of Tc cells. 2. Break down of tolerance mechanisms: There are at least two mechanisms for maintaining unresponsiveness to self. The first is by specific deletion of self-reactive clones and the second by suppression. A failure of either of these two may result in an autoimmune disease. In normal, healthy individuals, antigen-binding, self-reactive B cells and the resultant low titres of autoantibodies are not uncommon. The origin of the self-reactive B cells is not clear, but there are four ways in which they may become activated. (a) Polyclonal activation. High concentrations of polyclonal activators, such as LPS and high molecular weight dextrans, activate B cells irrespective of the immunoglobulin receptor on the B cell surface.

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Polyclonal activation occurs in parasitic infections and in certain viral infections with the production of a wide spectrum of autoantibodies. (b) Non-specific helper factors. T-cell activation results in the production of a variety of lymphokines which can activate these B cells. (c) Cross-reactive antigens. These are shared by the host and microorganism and this cross-reaction can activate autoreactive B cells. (d) Absence of T-cell suppression. The sudden depletion or elimination of Ts cells can lead to the spontaneous development of autoantibodies due to the maturation of the autoreactive B cells.

Polyclonal activation occurs in parasitic infections and in certain viral infections with the production of a wide spectrum of autoantibodies. (b) Non-specific helper factors. T-cell activation results in the production of a variety of lymphokines which can activate these B cells. (c) Cross-reactive antigens. These are shared by the host and microorganism and this cross-reaction can activate autoreactive B cells. (d) Absence of T-cell suppression. The sudden depletion or elimination of Ts cells can lead to the spontaneous development of autoantibodies due to the maturation of the autoreactive B cells.

Types of autoimmune diseases vary widely, from ‘organ-specific’ diseases such as thyroiditis where there may be stimulation (thyrotoxicosis) by the antibody against the receptor for pituitary thyroid-stimulating hormone (TSH) or inhibition (myxoedema) by cell destruction probably mediated by NK cells and autoantibody, through to ‘non-organ-specific’ diseases such as systemic lupus erythematosus (SLE), where both lesions and autoantibodies are not confined to any one organ. In SLE, antibodies have been detected to DNA, erythrocytes and platelets, and cytotoxic antibodies to T lymphocytes have also been demonstrated. A strong case can be made for rheumatoid arthritis resulting from an autoimmune response to the Fc portion of IgG which gives rise to complexes which are ultimately responsible for the pathological changes, characteristic of the rheumatoid joint.

Types of autoimmune diseases vary widely, from ‘organ-specific’ diseases such as thyroiditis where there may be stimulation (thyrotoxicosis) by the antibody against the receptor for pituitary thyroid-stimulating hormone (TSH) or inhibition (myxoedema) by cell destruction probably mediated by NK cells and autoantibody, through to ‘non-organ-specific’ diseases such as systemic lupus erythematosus (SLE), where both lesions and autoantibodies are not confined to any one organ. In SLE, antibodies have been detected to DNA, erythrocytes and platelets, and cytotoxic antibodies to T lymphocytes have also been demonstrated. A strong case can be made for rheumatoid arthritis resulting from an autoimmune response to the Fc portion of IgG which gives rise to complexes which are ultimately responsible for the pathological changes, characteristic of the rheumatoid joint.

10.14 HYPERSENSITIVITY REACTION

10.14 HYPERSENSITIVITY REACTION

Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. Gell and Coombs Chart of Classification of Hypersensitivity: 1. Type 1—IgE mediated or atopic, or anaphylactic or immidiate hypersensitivity 2. Type 2—IgG mediated or antibody-dependent hypersensitivity 3. Type 3—Immune complex mediated hypersensitivity 4. Type 4—Cell-mediated hypersensitivity or delayed-type hypersensitivity, DTH

Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. Gell and Coombs Chart of Classification of Hypersensitivity: 1. Type 1—IgE mediated or atopic, or anaphylactic or immidiate hypersensitivity 2. Type 2—IgG mediated or antibody-dependent hypersensitivity 3. Type 3—Immune complex mediated hypersensitivity 4. Type 4—Cell-mediated hypersensitivity or delayed-type hypersensitivity, DTH

1. Type 1 — IgE mediated or atopic, or anaphylactic or immidiate Hypersensitivity Type 1 hypersensitivity is an allergic reaction provoked by re-exposure to a specific type of antigen referred to as an allergen. Exposure may be by ingestion,

1. Type 1 — IgE mediated or atopic, or anaphylactic or immidiate Hypersensitivity Type 1 hypersensitivity is an allergic reaction provoked by re-exposure to a specific type of antigen referred to as an allergen. Exposure may be by ingestion,

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Polyclonal activation occurs in parasitic infections and in certain viral infections with the production of a wide spectrum of autoantibodies. (b) Non-specific helper factors. T-cell activation results in the production of a variety of lymphokines which can activate these B cells. (c) Cross-reactive antigens. These are shared by the host and microorganism and this cross-reaction can activate autoreactive B cells. (d) Absence of T-cell suppression. The sudden depletion or elimination of Ts cells can lead to the spontaneous development of autoantibodies due to the maturation of the autoreactive B cells.

Polyclonal activation occurs in parasitic infections and in certain viral infections with the production of a wide spectrum of autoantibodies. (b) Non-specific helper factors. T-cell activation results in the production of a variety of lymphokines which can activate these B cells. (c) Cross-reactive antigens. These are shared by the host and microorganism and this cross-reaction can activate autoreactive B cells. (d) Absence of T-cell suppression. The sudden depletion or elimination of Ts cells can lead to the spontaneous development of autoantibodies due to the maturation of the autoreactive B cells.

Types of autoimmune diseases vary widely, from ‘organ-specific’ diseases such as thyroiditis where there may be stimulation (thyrotoxicosis) by the antibody against the receptor for pituitary thyroid-stimulating hormone (TSH) or inhibition (myxoedema) by cell destruction probably mediated by NK cells and autoantibody, through to ‘non-organ-specific’ diseases such as systemic lupus erythematosus (SLE), where both lesions and autoantibodies are not confined to any one organ. In SLE, antibodies have been detected to DNA, erythrocytes and platelets, and cytotoxic antibodies to T lymphocytes have also been demonstrated. A strong case can be made for rheumatoid arthritis resulting from an autoimmune response to the Fc portion of IgG which gives rise to complexes which are ultimately responsible for the pathological changes, characteristic of the rheumatoid joint.

Types of autoimmune diseases vary widely, from ‘organ-specific’ diseases such as thyroiditis where there may be stimulation (thyrotoxicosis) by the antibody against the receptor for pituitary thyroid-stimulating hormone (TSH) or inhibition (myxoedema) by cell destruction probably mediated by NK cells and autoantibody, through to ‘non-organ-specific’ diseases such as systemic lupus erythematosus (SLE), where both lesions and autoantibodies are not confined to any one organ. In SLE, antibodies have been detected to DNA, erythrocytes and platelets, and cytotoxic antibodies to T lymphocytes have also been demonstrated. A strong case can be made for rheumatoid arthritis resulting from an autoimmune response to the Fc portion of IgG which gives rise to complexes which are ultimately responsible for the pathological changes, characteristic of the rheumatoid joint.

10.14 HYPERSENSITIVITY REACTION

10.14 HYPERSENSITIVITY REACTION

Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. Gell and Coombs Chart of Classification of Hypersensitivity: 1. Type 1—IgE mediated or atopic, or anaphylactic or immidiate hypersensitivity 2. Type 2—IgG mediated or antibody-dependent hypersensitivity 3. Type 3—Immune complex mediated hypersensitivity 4. Type 4—Cell-mediated hypersensitivity or delayed-type hypersensitivity, DTH

Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. Gell and Coombs Chart of Classification of Hypersensitivity: 1. Type 1—IgE mediated or atopic, or anaphylactic or immidiate hypersensitivity 2. Type 2—IgG mediated or antibody-dependent hypersensitivity 3. Type 3—Immune complex mediated hypersensitivity 4. Type 4—Cell-mediated hypersensitivity or delayed-type hypersensitivity, DTH

1. Type 1 — IgE mediated or atopic, or anaphylactic or immidiate Hypersensitivity Type 1 hypersensitivity is an allergic reaction provoked by re-exposure to a specific type of antigen referred to as an allergen. Exposure may be by ingestion,

1. Type 1 — IgE mediated or atopic, or anaphylactic or immidiate Hypersensitivity Type 1 hypersensitivity is an allergic reaction provoked by re-exposure to a specific type of antigen referred to as an allergen. Exposure may be by ingestion,

162 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

162 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.14. General mechanism underlying a type 1 hypersensitivity reaction. Exposure to an allergen activates B cells to form IgE-secreting plasma cells. The secreting IgE molecules bind to IgE-specific Fc receptors on mast cells and blood basophils. Second exposure to an allergen leads to cross-linking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, from mast cells and basophils. The mediators cause smooth muscle contraction, increased vascular permeability and vasodilation.

Fig. 10.14. General mechanism underlying a type 1 hypersensitivity reaction. Exposure to an allergen activates B cells to form IgE-secreting plasma cells. The secreting IgE molecules bind to IgE-specific Fc receptors on mast cells and blood basophils. Second exposure to an allergen leads to cross-linking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, from mast cells and basophils. The mediators cause smooth muscle contraction, increased vascular permeability and vasodilation.

inhalation, injection, or direct contact. The difference between a normal immune response and a type I hypersensitive response is that plasma cells secrete IgE. This class of antibodies binds to Fc receptors on the surface of tissue mast cells and blood basophils. Mast cells and basophils coated by IgE are “sensitized”. Later exposure to the same allergen cross-links the bound IgE on sensitized cells resulting in degranulation and the secretion of pharmacologically active mediators such as histamine, leukotriene, and prostaglandin that act on the surrounding tissues. The principal effects of these products are vasodilation and smooth-muscle contraction. The reaction may be either local or systemic. Symptoms vary from mild irritation to sudden death from anaphylactic shock. Treatment usually involves epinephrine, antihistamines, and corticosteroids. Some examples: Allergic asthma, allergic conjunctivitis, allergic rhinitis (hay fever), anaphylaxis, angioedema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, penicillin.

inhalation, injection, or direct contact. The difference between a normal immune response and a type I hypersensitive response is that plasma cells secrete IgE. This class of antibodies binds to Fc receptors on the surface of tissue mast cells and blood basophils. Mast cells and basophils coated by IgE are “sensitized”. Later exposure to the same allergen cross-links the bound IgE on sensitized cells resulting in degranulation and the secretion of pharmacologically active mediators such as histamine, leukotriene, and prostaglandin that act on the surrounding tissues. The principal effects of these products are vasodilation and smooth-muscle contraction. The reaction may be either local or systemic. Symptoms vary from mild irritation to sudden death from anaphylactic shock. Treatment usually involves epinephrine, antihistamines, and corticosteroids. Some examples: Allergic asthma, allergic conjunctivitis, allergic rhinitis (hay fever), anaphylaxis, angioedema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, penicillin.

2. Type 2 — IgG mediated or antibody-dependent hypersensitivity In type 2 hypersensitivity, the antibodies produced by the immune response bind to antigens on the patient’s own cell surfaces. The antigens recognized in

2. Type 2 — IgG mediated or antibody-dependent hypersensitivity In type 2 hypersensitivity, the antibodies produced by the immune response bind to antigens on the patient’s own cell surfaces. The antigens recognized in

162 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

162 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.14. General mechanism underlying a type 1 hypersensitivity reaction. Exposure to an allergen activates B cells to form IgE-secreting plasma cells. The secreting IgE molecules bind to IgE-specific Fc receptors on mast cells and blood basophils. Second exposure to an allergen leads to cross-linking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, from mast cells and basophils. The mediators cause smooth muscle contraction, increased vascular permeability and vasodilation.

Fig. 10.14. General mechanism underlying a type 1 hypersensitivity reaction. Exposure to an allergen activates B cells to form IgE-secreting plasma cells. The secreting IgE molecules bind to IgE-specific Fc receptors on mast cells and blood basophils. Second exposure to an allergen leads to cross-linking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, from mast cells and basophils. The mediators cause smooth muscle contraction, increased vascular permeability and vasodilation.

inhalation, injection, or direct contact. The difference between a normal immune response and a type I hypersensitive response is that plasma cells secrete IgE. This class of antibodies binds to Fc receptors on the surface of tissue mast cells and blood basophils. Mast cells and basophils coated by IgE are “sensitized”. Later exposure to the same allergen cross-links the bound IgE on sensitized cells resulting in degranulation and the secretion of pharmacologically active mediators such as histamine, leukotriene, and prostaglandin that act on the surrounding tissues. The principal effects of these products are vasodilation and smooth-muscle contraction. The reaction may be either local or systemic. Symptoms vary from mild irritation to sudden death from anaphylactic shock. Treatment usually involves epinephrine, antihistamines, and corticosteroids. Some examples: Allergic asthma, allergic conjunctivitis, allergic rhinitis (hay fever), anaphylaxis, angioedema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, penicillin.

inhalation, injection, or direct contact. The difference between a normal immune response and a type I hypersensitive response is that plasma cells secrete IgE. This class of antibodies binds to Fc receptors on the surface of tissue mast cells and blood basophils. Mast cells and basophils coated by IgE are “sensitized”. Later exposure to the same allergen cross-links the bound IgE on sensitized cells resulting in degranulation and the secretion of pharmacologically active mediators such as histamine, leukotriene, and prostaglandin that act on the surrounding tissues. The principal effects of these products are vasodilation and smooth-muscle contraction. The reaction may be either local or systemic. Symptoms vary from mild irritation to sudden death from anaphylactic shock. Treatment usually involves epinephrine, antihistamines, and corticosteroids. Some examples: Allergic asthma, allergic conjunctivitis, allergic rhinitis (hay fever), anaphylaxis, angioedema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, penicillin.

2. Type 2 — IgG mediated or antibody-dependent hypersensitivity In type 2 hypersensitivity, the antibodies produced by the immune response bind to antigens on the patient’s own cell surfaces. The antigens recognized in

2. Type 2 — IgG mediated or antibody-dependent hypersensitivity In type 2 hypersensitivity, the antibodies produced by the immune response bind to antigens on the patient’s own cell surfaces. The antigens recognized in

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this way may either be intrinsic (“self” antigen, innate part of the patient’s cells) or extrinsic (absorbed onto the cells during exposure to some foreign antigen, possibly as part of infection with a pathogen). These cells are recognised by macrophages or dendritic cells which act as antigen presenting cells, this causes a B cell response, where antibodies are produced against the foreign antigen. An example here is the reaction to penicillin where the drug can bind to red blood cells causing them to be recognised as different, B cell proliferation will take place and antibodies to the drug are produced. IgG and IgM antibodies bind to these antigens to form complexes that activate the classical pathway of complement activation for eliminating cells presenting foreign antigens (which are usually, but not in this case, pathogens). That is, mediators of acute inflammation are generated at the site and membrane attack complexes cause cell lysis and death. The reaction takes hours to a day. Some clinical examples: Autoimmune hemolytic anemia, Goodpasture’s syndrome, erythroblastosis, fetalis, pemphigus, pernicious anemia (if autoimmune), immune thrombocytopenia, transfusion reactions, Hashimoto’s thyroiditis, Graves’ disease, Myasthenia gravis, rheumatic fever Hemolytic disease of the newborn

this way may either be intrinsic (“self” antigen, innate part of the patient’s cells) or extrinsic (absorbed onto the cells during exposure to some foreign antigen, possibly as part of infection with a pathogen). These cells are recognised by macrophages or dendritic cells which act as antigen presenting cells, this causes a B cell response, where antibodies are produced against the foreign antigen. An example here is the reaction to penicillin where the drug can bind to red blood cells causing them to be recognised as different, B cell proliferation will take place and antibodies to the drug are produced. IgG and IgM antibodies bind to these antigens to form complexes that activate the classical pathway of complement activation for eliminating cells presenting foreign antigens (which are usually, but not in this case, pathogens). That is, mediators of acute inflammation are generated at the site and membrane attack complexes cause cell lysis and death. The reaction takes hours to a day. Some clinical examples: Autoimmune hemolytic anemia, Goodpasture’s syndrome, erythroblastosis, fetalis, pemphigus, pernicious anemia (if autoimmune), immune thrombocytopenia, transfusion reactions, Hashimoto’s thyroiditis, Graves’ disease, Myasthenia gravis, rheumatic fever Hemolytic disease of the newborn

3. Type 3 — Immune Complex In type 3 hypersensitivity, soluble immune complexes are (aggregations of antigens and IgG and IgM antibodies) formed in the blood and are deposited in various tissues (typically the skin, kidney and joints) where they may trigger an immune response according to the classical pathway of complement activation (Fig. 10.12). There are two stages relating to the development of the complexes, firstly the complex forms when IgG and IgM antibodies are bound to an antigen, after this, the complexes can form larger ones which can be cleared by the body. It is at the first stage of this formation where clearance is not possible and the antigen-antibody complex will spread and deposit as stated above. The reaction takes hours to days to develop. Some clinical examples: Immune complex glomerulonephritis, rheumatoid arthritis, Serum sickness, subacute bacterial, endocarditis, symptoms of malaria, systemic lupus erythematosus, Arthus reaction, Farmer’s Lung (Arthus-type reaction).

3. Type 3 — Immune Complex In type 3 hypersensitivity, soluble immune complexes are (aggregations of antigens and IgG and IgM antibodies) formed in the blood and are deposited in various tissues (typically the skin, kidney and joints) where they may trigger an immune response according to the classical pathway of complement activation (Fig. 10.12). There are two stages relating to the development of the complexes, firstly the complex forms when IgG and IgM antibodies are bound to an antigen, after this, the complexes can form larger ones which can be cleared by the body. It is at the first stage of this formation where clearance is not possible and the antigen-antibody complex will spread and deposit as stated above. The reaction takes hours to days to develop. Some clinical examples: Immune complex glomerulonephritis, rheumatoid arthritis, Serum sickness, subacute bacterial, endocarditis, symptoms of malaria, systemic lupus erythematosus, Arthus reaction, Farmer’s Lung (Arthus-type reaction).

4. Type 4 — Cell-mediated (Delayed-Type Hypersensitivity, DTH) Cell mediated immunity or Type 4 hypersensitivity is often called delayed type as the reaction takes two to three days to develop. Unlike the other types, it is not antibody mediated but rather is a type of cell-mediated response. CD8+ cytotoxic T cells recognize antigen in a complex with MHC-1 (Type 1 major histocompatibility complex) and CD4+ helper T cells recognize antigen in a complex with MHC-ll (Type 2 major histocompatibility complex. MHC-1

4. Type 4 — Cell-mediated (Delayed-Type Hypersensitivity, DTH) Cell mediated immunity or Type 4 hypersensitivity is often called delayed type as the reaction takes two to three days to develop. Unlike the other types, it is not antibody mediated but rather is a type of cell-mediated response. CD8+ cytotoxic T cells recognize antigen in a complex with MHC-1 (Type 1 major histocompatibility complex) and CD4+ helper T cells recognize antigen in a complex with MHC-ll (Type 2 major histocompatibility complex. MHC-1

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this way may either be intrinsic (“self” antigen, innate part of the patient’s cells) or extrinsic (absorbed onto the cells during exposure to some foreign antigen, possibly as part of infection with a pathogen). These cells are recognised by macrophages or dendritic cells which act as antigen presenting cells, this causes a B cell response, where antibodies are produced against the foreign antigen. An example here is the reaction to penicillin where the drug can bind to red blood cells causing them to be recognised as different, B cell proliferation will take place and antibodies to the drug are produced. IgG and IgM antibodies bind to these antigens to form complexes that activate the classical pathway of complement activation for eliminating cells presenting foreign antigens (which are usually, but not in this case, pathogens). That is, mediators of acute inflammation are generated at the site and membrane attack complexes cause cell lysis and death. The reaction takes hours to a day. Some clinical examples: Autoimmune hemolytic anemia, Goodpasture’s syndrome, erythroblastosis, fetalis, pemphigus, pernicious anemia (if autoimmune), immune thrombocytopenia, transfusion reactions, Hashimoto’s thyroiditis, Graves’ disease, Myasthenia gravis, rheumatic fever Hemolytic disease of the newborn

this way may either be intrinsic (“self” antigen, innate part of the patient’s cells) or extrinsic (absorbed onto the cells during exposure to some foreign antigen, possibly as part of infection with a pathogen). These cells are recognised by macrophages or dendritic cells which act as antigen presenting cells, this causes a B cell response, where antibodies are produced against the foreign antigen. An example here is the reaction to penicillin where the drug can bind to red blood cells causing them to be recognised as different, B cell proliferation will take place and antibodies to the drug are produced. IgG and IgM antibodies bind to these antigens to form complexes that activate the classical pathway of complement activation for eliminating cells presenting foreign antigens (which are usually, but not in this case, pathogens). That is, mediators of acute inflammation are generated at the site and membrane attack complexes cause cell lysis and death. The reaction takes hours to a day. Some clinical examples: Autoimmune hemolytic anemia, Goodpasture’s syndrome, erythroblastosis, fetalis, pemphigus, pernicious anemia (if autoimmune), immune thrombocytopenia, transfusion reactions, Hashimoto’s thyroiditis, Graves’ disease, Myasthenia gravis, rheumatic fever Hemolytic disease of the newborn

3. Type 3 — Immune Complex In type 3 hypersensitivity, soluble immune complexes are (aggregations of antigens and IgG and IgM antibodies) formed in the blood and are deposited in various tissues (typically the skin, kidney and joints) where they may trigger an immune response according to the classical pathway of complement activation (Fig. 10.12). There are two stages relating to the development of the complexes, firstly the complex forms when IgG and IgM antibodies are bound to an antigen, after this, the complexes can form larger ones which can be cleared by the body. It is at the first stage of this formation where clearance is not possible and the antigen-antibody complex will spread and deposit as stated above. The reaction takes hours to days to develop. Some clinical examples: Immune complex glomerulonephritis, rheumatoid arthritis, Serum sickness, subacute bacterial, endocarditis, symptoms of malaria, systemic lupus erythematosus, Arthus reaction, Farmer’s Lung (Arthus-type reaction).

3. Type 3 — Immune Complex In type 3 hypersensitivity, soluble immune complexes are (aggregations of antigens and IgG and IgM antibodies) formed in the blood and are deposited in various tissues (typically the skin, kidney and joints) where they may trigger an immune response according to the classical pathway of complement activation (Fig. 10.12). There are two stages relating to the development of the complexes, firstly the complex forms when IgG and IgM antibodies are bound to an antigen, after this, the complexes can form larger ones which can be cleared by the body. It is at the first stage of this formation where clearance is not possible and the antigen-antibody complex will spread and deposit as stated above. The reaction takes hours to days to develop. Some clinical examples: Immune complex glomerulonephritis, rheumatoid arthritis, Serum sickness, subacute bacterial, endocarditis, symptoms of malaria, systemic lupus erythematosus, Arthus reaction, Farmer’s Lung (Arthus-type reaction).

4. Type 4 — Cell-mediated (Delayed-Type Hypersensitivity, DTH) Cell mediated immunity or Type 4 hypersensitivity is often called delayed type as the reaction takes two to three days to develop. Unlike the other types, it is not antibody mediated but rather is a type of cell-mediated response. CD8+ cytotoxic T cells recognize antigen in a complex with MHC-1 (Type 1 major histocompatibility complex) and CD4+ helper T cells recognize antigen in a complex with MHC-ll (Type 2 major histocompatibility complex. MHC-1

4. Type 4 — Cell-mediated (Delayed-Type Hypersensitivity, DTH) Cell mediated immunity or Type 4 hypersensitivity is often called delayed type as the reaction takes two to three days to develop. Unlike the other types, it is not antibody mediated but rather is a type of cell-mediated response. CD8+ cytotoxic T cells recognize antigen in a complex with MHC-1 (Type 1 major histocompatibility complex) and CD4+ helper T cells recognize antigen in a complex with MHC-ll (Type 2 major histocompatibility complex. MHC-1

164 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.15. Certain types of antigen when react with an antibody they form a Ag-Ab complex which causes the activation of complement system and releases some proteins (C3a, C4a, C5a). These activated proteins cause the degranulation of mast cell which causes the release of histamines. Histamines increase the vascular permeability and the neutrophils come out from the blood vessels where they produce some lytic enzymes and phagocytose the immune complex.

164 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.15. Certain types of antigen when react with an antibody they form a Ag-Ab complex which causes the activation of complement system and releases some proteins (C3a, C4a, C5a). These activated proteins cause the degranulation of mast cell which causes the release of histamines. Histamines increase the vascular permeability and the neutrophils come out from the blood vessels where they produce some lytic enzymes and phagocytose the immune complex.

164 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.15. Certain types of antigen when react with an antibody they form a Ag-Ab complex which causes the activation of complement system and releases some proteins (C3a, C4a, C5a). These activated proteins cause the degranulation of mast cell which causes the release of histamines. Histamines increase the vascular permeability and the neutrophils come out from the blood vessels where they produce some lytic enzymes and phagocytose the immune complex.

164 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 10.15. Certain types of antigen when react with an antibody they form a Ag-Ab complex which causes the activation of complement system and releases some proteins (C3a, C4a, C5a). These activated proteins cause the degranulation of mast cell which causes the release of histamines. Histamines increase the vascular permeability and the neutrophils come out from the blood vessels where they produce some lytic enzymes and phagocytose the immune complex.

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Fig. 10.16. Diagrammatic representation of Type lV hypersensitivity reaction.

Fig. 10.16. Diagrammatic representation of Type lV hypersensitivity reaction.

mainly present on the surface of antigen presenting cells i.e. macrophages, dendritic cells and B-cells. The antigen-presenting cells in this case are macrophages which secrete IL-12, which stimulates the proliferation and differentiation of CD4+ T cells into TH1 cells. TH1 cells secret cytokines and chemokines which activate the resting macrophages thus mediating the immune response. Activated CD8+ T cells (Cytotoxic T cell ) destroy target cells or infected cells on contact while activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, destroy them and eliminate from the body. Some clinical examples: Contact dermatitis (poison ivy rash, for example); temporal arteritis; symptoms of leprosy; symptoms of tuberculosis, transplant rejection, coeliac disease, measles.

mainly present on the surface of antigen presenting cells i.e. macrophages, dendritic cells and B-cells. The antigen-presenting cells in this case are macrophages which secrete IL-12, which stimulates the proliferation and differentiation of CD4+ T cells into TH1 cells. TH1 cells secret cytokines and chemokines which activate the resting macrophages thus mediating the immune response. Activated CD8+ T cells (Cytotoxic T cell ) destroy target cells or infected cells on contact while activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, destroy them and eliminate from the body. Some clinical examples: Contact dermatitis (poison ivy rash, for example); temporal arteritis; symptoms of leprosy; symptoms of tuberculosis, transplant rejection, coeliac disease, measles.

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Fig. 10.16. Diagrammatic representation of Type lV hypersensitivity reaction.

Fig. 10.16. Diagrammatic representation of Type lV hypersensitivity reaction.

mainly present on the surface of antigen presenting cells i.e. macrophages, dendritic cells and B-cells. The antigen-presenting cells in this case are macrophages which secrete IL-12, which stimulates the proliferation and differentiation of CD4+ T cells into TH1 cells. TH1 cells secret cytokines and chemokines which activate the resting macrophages thus mediating the immune response. Activated CD8+ T cells (Cytotoxic T cell ) destroy target cells or infected cells on contact while activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, destroy them and eliminate from the body. Some clinical examples: Contact dermatitis (poison ivy rash, for example); temporal arteritis; symptoms of leprosy; symptoms of tuberculosis, transplant rejection, coeliac disease, measles.

mainly present on the surface of antigen presenting cells i.e. macrophages, dendritic cells and B-cells. The antigen-presenting cells in this case are macrophages which secrete IL-12, which stimulates the proliferation and differentiation of CD4+ T cells into TH1 cells. TH1 cells secret cytokines and chemokines which activate the resting macrophages thus mediating the immune response. Activated CD8+ T cells (Cytotoxic T cell ) destroy target cells or infected cells on contact while activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, destroy them and eliminate from the body. Some clinical examples: Contact dermatitis (poison ivy rash, for example); temporal arteritis; symptoms of leprosy; symptoms of tuberculosis, transplant rejection, coeliac disease, measles.

11

11

Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids

Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids

11.1 ANTIMICROBIAL ASSAY OF ANTIBIOTICS

11.1 ANTIMICROBIAL ASSAY OF ANTIBIOTICS

1. By this assay method we check the potency of the antibiotic agents against microorganisms. 2. The microbiological assay is based upon the inhibition of growth of microorganisms by measured concentration of the antibiotics. 3. Generally two methods are employed for microbiological assay: (a) Cylinder plate method. (b) Tube dilution or turbidimetric method.

1. By this assay method we check the potency of the antibiotic agents against microorganisms. 2. The microbiological assay is based upon the inhibition of growth of microorganisms by measured concentration of the antibiotics. 3. Generally two methods are employed for microbiological assay: (a) Cylinder plate method. (b) Tube dilution or turbidimetric method.

Media used for microbial assay: Different types of liquid and solid media are used, i.e., nutrient broth, nutrient agar L.B. broth, L.B. agar media etc.

Media used for microbial assay: Different types of liquid and solid media are used, i.e., nutrient broth, nutrient agar L.B. broth, L.B. agar media etc.

Table 1: List of mainly used media in lab for antibiotic assay

Table 1: List of mainly used media in lab for antibiotic assay

Nutrient broth

Nutrient agar media

L.B.Broth

L.B. agar media

Nutrient broth

Nutrient agar media

L.B.Broth

L.B. agar media

Beef extract 3 gm

Beef extract 3 gm

Tryptone 10 gm

Tryptone 10 gm

Beef extract 3 gm

Beef extract 3 gm

Tryptone 10 gm

Tryptone 10 gm

Peptone 5 gm

Peptone 5 gm

Yeast extract 5 gm

Yeast extract 5 gm

Peptone 5 gm

Peptone 5 gm

Yeast extract 5 gm

Yeast extract 5 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Water 1000 mL

Water 1000 mL

Out of the above the following media can also be used.

Out of the above the following media can also be used.

Table 2: List of media other than table 1 used for antibiotic assay Ingredient

A

Pepton 6.0 Pancreatic digest of casien 4.0 Yeast extract 3.0 Beef extract 1.5

Water 1000 mL

Table 2: List of media other than table 1 used for antibiotic assay

B

C

D

E

F

G

H

I

J

6.0 3.0 1.5

5.0 1.5 1.5

6.0 4.0 3.0 1.5

6.0 3.0 1.5

6.0 3.0 1.5

9.4 4.7 2.4

17.0 -

10.0 10.0

15.0 (Contd.)

Ingredient

A

Pepton 6.0 Pancreatic digest of casien 4.0 Yeast extract 3.0 Beef extract 1.5

B

C

D

E

F

G

H

I

J

6.0 3.0 1.5

5.0 1.5 1.5

6.0 4.0 3.0 1.5

6.0 3.0 1.5

6.0 3.0 1.5

9.4 4.7 2.4

17.0 -

10.0 10.0

15.0 (Contd.)

11

11

Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids

Antimicrobial Assay of Antibiotics, Vitamins and Amino Acids

11.1 ANTIMICROBIAL ASSAY OF ANTIBIOTICS

11.1 ANTIMICROBIAL ASSAY OF ANTIBIOTICS

1. By this assay method we check the potency of the antibiotic agents against microorganisms. 2. The microbiological assay is based upon the inhibition of growth of microorganisms by measured concentration of the antibiotics. 3. Generally two methods are employed for microbiological assay: (a) Cylinder plate method. (b) Tube dilution or turbidimetric method.

1. By this assay method we check the potency of the antibiotic agents against microorganisms. 2. The microbiological assay is based upon the inhibition of growth of microorganisms by measured concentration of the antibiotics. 3. Generally two methods are employed for microbiological assay: (a) Cylinder plate method. (b) Tube dilution or turbidimetric method.

Media used for microbial assay: Different types of liquid and solid media are used, i.e., nutrient broth, nutrient agar L.B. broth, L.B. agar media etc.

Media used for microbial assay: Different types of liquid and solid media are used, i.e., nutrient broth, nutrient agar L.B. broth, L.B. agar media etc.

Table 1: List of mainly used media in lab for antibiotic assay

Table 1: List of mainly used media in lab for antibiotic assay

Nutrient broth

Nutrient agar media

L.B.Broth

L.B. agar media

Nutrient broth

Nutrient agar media

L.B.Broth

L.B. agar media

Beef extract 3 gm

Beef extract 3 gm

Tryptone 10 gm

Tryptone 10 gm

Beef extract 3 gm

Beef extract 3 gm

Tryptone 10 gm

Tryptone 10 gm

Peptone 5 gm

Peptone 5 gm

Yeast extract 5 gm

Yeast extract 5 gm

Peptone 5 gm

Peptone 5 gm

Yeast extract 5 gm

Yeast extract 5 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Agar 15 gm

Water 1000 mL

Water 1000 mL

Water 1000 mL

Out of the above the following media can also be used.

Out of the above the following media can also be used.

Table 2: List of media other than table 1 used for antibiotic assay Ingredient

A

Pepton 6.0 Pancreatic digest of casien 4.0 Yeast extract 3.0 Beef extract 1.5

Water 1000 mL

Table 2: List of media other than table 1 used for antibiotic assay

B

C

D

E

F

G

H

I

6.0 3.0 1.5

5.0 1.5 1.5

6.0 4.0 3.0 1.5

6.0 3.0 1.5

6.0 3.0 1.5

9.4 4.7 2.4

17.0 -

10.0 10.0

J 15.0 (Contd.)

Ingredient

A

Pepton 6.0 Pancreatic digest of casien 4.0 Yeast extract 3.0 Beef extract 1.5

B

C

D

E

F

G

H

I

6.0 3.0 1.5

5.0 1.5 1.5

6.0 4.0 3.0 1.5

6.0 3.0 1.5

6.0 3.0 1.5

9.4 4.7 2.4

17.0 -

10.0 10.0

J 15.0 (Contd.)

168 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

168 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Table 2: Contd.

Table 2: Contd.

Ingredient

A

B

C

D

E

F

G

H

I

J

Ingredient

A

B

C

D

E

F

G

H

I

J

Dextrose Papaic digest of soy bean Agar Glycerin Polysorbate 80 Sodium chloride Dipotassium hydrogen phosphate Final pH (after sterilisation)

1.0 15.0 -

15.0 -

1.0 -

1.0 15.0 -

15.0 -

15.0 -

10.0 23.5 10.0

2.5 3.0 12.0 10.0* 5.0

17.0 10.0 3.0

5.0 15.0 5.0

1.0 15.0 -

15.0 -

1.0 -

1.0 15.0 -

15.0 -

15.0 -

10.0 23.5 10.0

2.5 3.0 12.0 10.0* 5.0

17.0 10.0 3.0

5.0 15.0 5.0

6.5– 6.6

6.5– 6.6

3.68 6.95– 7.8– 7.05 8.0

7.8– 8.0

5.8– 6.0

6.0– 6.2

2.5 7.1– 7.3

6.9– 7.1

7.2– 7.4

Dextrose Papaic digest of soy bean Agar Glycerin Polysorbate 80 Sodium chloride Dipotassium hydrogen phosphate Final pH (after sterilisation)

6.5– 6.6

6.5– 6.6

3.68 6.95– 7.8– 7.05 8.0

7.8– 8.0

5.8– 6.0

6.0– 6.2

2.5 7.1– 7.3

6.9– 7.1

7.2– 7.4

3.5

3.5

Preparation of standard solution of antibiotics: To prepare stalk solution, weigh accurately the given amount of antibiotics in a suitable solvent which are listed (Table 3) below. For the final use dilute the stock solution as required and keep under proper temperature for the further use.

Preparation of standard solution of antibiotics: To prepare stalk solution, weigh accurately the given amount of antibiotics in a suitable solvent which are listed (Table 3) below. For the final use dilute the stock solution as required and keep under proper temperature for the further use.

Table 3: Stock solutions and test dilutions of standard preparation

Table 3: Stock solutions and test dilutions of standard preparation

S. No. Antibiotic

Initial solvent

Final stock concentration per ml

Median dose zg or units per ml

Incubation temp. (*C)

S. No. Antibiotic

Initial solvent

Final stock concentration per ml

Median dose zg or units per ml

Incubation temp. (*C)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Water DMF7 0.01M HCl 0.1M HCl Methanol Methanol Methanol Methanol Water Methanol Water Water

1 mg 1 mg 100 units 1 mg 1 mg 1 mg 800 units 1 mg 10,000 units 1 mg 1 mg 1 mg

10 zg 1.0 zg 1.0 unit 0.1 zg 1.0 zg 0.1 zg 0.8 unit 1.0 zg 10 unit 5.0 zg 1.0 zg 2.5 zg

32 29 32 35 35 36 37 36 35 29 32 32

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Water DMF7 0.01M HCl 0.1M HCl Methanol Methanol Methanol Methanol Water Methanol Water Water

1 mg 1 mg 100 units 1 mg 1 mg 1 mg 800 units 1 mg 10,000 units 1 mg 1 mg 1 mg

10 zg 1.0 zg 1.0 unit 0.1 zg 1.0 zg 0.1 zg 0.8 unit 1.0 zg 10 unit 5.0 zg 1.0 zg 2.5 zg

32 29 32 35 35 36 37 36 35 29 32 32

Amikacin Amphotericin B Bacitracin Doxycycline Erythromycin Gentamicin Kanamycin Neomycin Polymyxin B Rifampicin Streptomycin Tetracycline

- 35 - 31 - 35 - 37 - 37 - 37.5 –39 - 37.5 - 39 – 31 - 35 - 35

Amikacin Amphotericin B Bacitracin Doxycycline Erythromycin Gentamicin Kanamycin Neomycin Polymyxin B Rifampicin Streptomycin Tetracycline

- 35 - 31 - 35 - 37 - 37 - 37.5 –39 - 37.5 - 39 – 31 - 35 - 35

Test organisms

Test organisms

There are different types of test organisms that used for antibiotic assay, i.e., Bacillus cereus var. mycoides, Bacillus pumilus, Bacillus subtilis, E. coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Saccgarinces cerevisiae, Staphylococcus aureus.

There are different types of test organisms that used for antibiotic assay, i.e., Bacillus cereus var. mycoides, Bacillus pumilus, Bacillus subtilis, E. coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Saccgarinces cerevisiae, Staphylococcus aureus.

Preparation of inoculum

Preparation of inoculum

Take a loopfull culture of bacteria from the Petri plate and inoculate into the test tube and keep for incubation overnight for specified time and temperature in a shaker incubator.

Take a loopfull culture of bacteria from the Petri plate and inoculate into the test tube and keep for incubation overnight for specified time and temperature in a shaker incubator.

168 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

168 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Table 2: Contd.

Table 2: Contd.

Ingredient

A

B

C

D

E

F

G

H

I

J

Ingredient

A

B

C

D

E

F

G

H

I

J

Dextrose Papaic digest of soy bean Agar Glycerin Polysorbate 80 Sodium chloride Dipotassium hydrogen phosphate Final pH (after sterilisation)

1.0 15.0 -

15.0 -

1.0 -

1.0 15.0 -

15.0 -

15.0 -

10.0 23.5 10.0

2.5 3.0 12.0 10.0* 5.0

17.0 10.0 3.0

5.0 15.0 5.0

1.0 15.0 -

15.0 -

1.0 -

1.0 15.0 -

15.0 -

15.0 -

10.0 23.5 10.0

2.5 3.0 12.0 10.0* 5.0

17.0 10.0 3.0

5.0 15.0 5.0

6.5– 6.6

6.5– 6.6

3.68 6.95– 7.8– 7.05 8.0

7.8– 8.0

5.8– 6.0

6.0– 6.2

2.5 7.1– 7.3

6.9– 7.1

7.2– 7.4

Dextrose Papaic digest of soy bean Agar Glycerin Polysorbate 80 Sodium chloride Dipotassium hydrogen phosphate Final pH (after sterilisation)

6.5– 6.6

6.5– 6.6

3.68 6.95– 7.8– 7.05 8.0

7.8– 8.0

5.8– 6.0

6.0– 6.2

2.5 7.1– 7.3

6.9– 7.1

7.2– 7.4

3.5

3.5

Preparation of standard solution of antibiotics: To prepare stalk solution, weigh accurately the given amount of antibiotics in a suitable solvent which are listed (Table 3) below. For the final use dilute the stock solution as required and keep under proper temperature for the further use.

Preparation of standard solution of antibiotics: To prepare stalk solution, weigh accurately the given amount of antibiotics in a suitable solvent which are listed (Table 3) below. For the final use dilute the stock solution as required and keep under proper temperature for the further use.

Table 3: Stock solutions and test dilutions of standard preparation

Table 3: Stock solutions and test dilutions of standard preparation

S. No. Antibiotic

Initial solvent

Final stock concentration per ml

Median dose zg or units per ml

Incubation temp. (*C)

S. No. Antibiotic

Initial solvent

Final stock concentration per ml

Median dose zg or units per ml

Incubation temp. (*C)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Water DMF7 0.01M HCl 0.1M HCl Methanol Methanol Methanol Methanol Water Methanol Water Water

1 mg 1 mg 100 units 1 mg 1 mg 1 mg 800 units 1 mg 10,000 units 1 mg 1 mg 1 mg

10 zg 1.0 zg 1.0 unit 0.1 zg 1.0 zg 0.1 zg 0.8 unit 1.0 zg 10 unit 5.0 zg 1.0 zg 2.5 zg

32 29 32 35 35 36 37 36 35 29 32 32

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Water DMF7 0.01M HCl 0.1M HCl Methanol Methanol Methanol Methanol Water Methanol Water Water

1 mg 1 mg 100 units 1 mg 1 mg 1 mg 800 units 1 mg 10,000 units 1 mg 1 mg 1 mg

10 zg 1.0 zg 1.0 unit 0.1 zg 1.0 zg 0.1 zg 0.8 unit 1.0 zg 10 unit 5.0 zg 1.0 zg 2.5 zg

32 29 32 35 35 36 37 36 35 29 32 32

Amikacin Amphotericin B Bacitracin Doxycycline Erythromycin Gentamicin Kanamycin Neomycin Polymyxin B Rifampicin Streptomycin Tetracycline

- 35 - 31 - 35 - 37 - 37 - 37.5 –39 - 37.5 - 39 – 31 - 35 - 35

Amikacin Amphotericin B Bacitracin Doxycycline Erythromycin Gentamicin Kanamycin Neomycin Polymyxin B Rifampicin Streptomycin Tetracycline

- 35 - 31 - 35 - 37 - 37 - 37.5 –39 - 37.5 - 39 – 31 - 35 - 35

Test organisms

Test organisms

There are different types of test organisms that used for antibiotic assay, i.e., Bacillus cereus var. mycoides, Bacillus pumilus, Bacillus subtilis, E. coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Saccgarinces cerevisiae, Staphylococcus aureus.

There are different types of test organisms that used for antibiotic assay, i.e., Bacillus cereus var. mycoides, Bacillus pumilus, Bacillus subtilis, E. coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Saccgarinces cerevisiae, Staphylococcus aureus.

Preparation of inoculum

Preparation of inoculum

Take a loopfull culture of bacteria from the Petri plate and inoculate into the test tube and keep for incubation overnight for specified time and temperature in a shaker incubator.

Take a loopfull culture of bacteria from the Petri plate and inoculate into the test tube and keep for incubation overnight for specified time and temperature in a shaker incubator.

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

169

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

169

Next day when proper growth of microorganisms occurs dilute the culture media for cup-plate method and pour the measured amount of culture media into the Petri plate. While for tube assay method take a loopfull of culture with the help of inoculum loop, and transfer it into another test tube having antibiotics.

Next day when proper growth of microorganisms occurs dilute the culture media for cup-plate method and pour the measured amount of culture media into the Petri plate. While for tube assay method take a loopfull of culture with the help of inoculum loop, and transfer it into another test tube having antibiotics.

Methods

Methods

I. Cylinder method Principle: The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic and form zone of inhibition. This method is mainly done by two methods: a) Cup-plate method: In this method make a bore of 5 to 8 mm in diameter with the help of sterile borer in Petri plate and the small quantity of antibiotics are added in these bores.

I. Cylinder method Principle: The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic and form zone of inhibition. This method is mainly done by two methods: a) Cup-plate method: In this method make a bore of 5 to 8 mm in diameter with the help of sterile borer in Petri plate and the small quantity of antibiotics are added in these bores.

Procedure

Procedure

1. Prepare the media with the help of Table 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and left for solidifying. 4. When the agar plates solidify add 3 to 5 mL of liquid media containing microorganisms, spread on the plate properly and leave for 5 minute. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make a bore size of 5 to 8 mm with the help of sterile borer and add the suitable amount of antibiotics and keep it into the incubator for temperature as mentioned in Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible to antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

1. Prepare the media with the help of Table 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and left for solidifying. 4. When the agar plates solidify add 3 to 5 mL of liquid media containing microorganisms, spread on the plate properly and leave for 5 minute. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make a bore size of 5 to 8 mm with the help of sterile borer and add the suitable amount of antibiotics and keep it into the incubator for temperature as mentioned in Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible to antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

Paper disk-plate method: The paper disk-plate method is the most commonly used technique for determining susceptibility of microorganisms to antibiotic agents. Small paper disks impregnated with known amount of antibiotic agents are placed upon the surface of an inoculated plate. After incubation the plates are observed for any zone of inhibition (a clear area) around the disk indicates that the organism was inhibited by the drug which diffused into the agar from the disk.

Paper disk-plate method: The paper disk-plate method is the most commonly used technique for determining susceptibility of microorganisms to antibiotic agents. Small paper disks impregnated with known amount of antibiotic agents are placed upon the surface of an inoculated plate. After incubation the plates are observed for any zone of inhibition (a clear area) around the disk indicates that the organism was inhibited by the drug which diffused into the agar from the disk.

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

169

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

169

Next day when proper growth of microorganisms occurs dilute the culture media for cup-plate method and pour the measured amount of culture media into the Petri plate. While for tube assay method take a loopfull of culture with the help of inoculum loop, and transfer it into another test tube having antibiotics.

Next day when proper growth of microorganisms occurs dilute the culture media for cup-plate method and pour the measured amount of culture media into the Petri plate. While for tube assay method take a loopfull of culture with the help of inoculum loop, and transfer it into another test tube having antibiotics.

Methods

Methods

I. Cylinder method Principle: The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic and form zone of inhibition. This method is mainly done by two methods: a) Cup-plate method: In this method make a bore of 5 to 8 mm in diameter with the help of sterile borer in Petri plate and the small quantity of antibiotics are added in these bores.

I. Cylinder method Principle: The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic and form zone of inhibition. This method is mainly done by two methods: a) Cup-plate method: In this method make a bore of 5 to 8 mm in diameter with the help of sterile borer in Petri plate and the small quantity of antibiotics are added in these bores.

Procedure

Procedure

1. Prepare the media with the help of Table 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and left for solidifying. 4. When the agar plates solidify add 3 to 5 mL of liquid media containing microorganisms, spread on the plate properly and leave for 5 minute. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make a bore size of 5 to 8 mm with the help of sterile borer and add the suitable amount of antibiotics and keep it into the incubator for temperature as mentioned in Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible to antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

1. Prepare the media with the help of Table 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and left for solidifying. 4. When the agar plates solidify add 3 to 5 mL of liquid media containing microorganisms, spread on the plate properly and leave for 5 minute. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make a bore size of 5 to 8 mm with the help of sterile borer and add the suitable amount of antibiotics and keep it into the incubator for temperature as mentioned in Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible to antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

Paper disk-plate method: The paper disk-plate method is the most commonly used technique for determining susceptibility of microorganisms to antibiotic agents. Small paper disks impregnated with known amount of antibiotic agents are placed upon the surface of an inoculated plate. After incubation the plates are observed for any zone of inhibition (a clear area) around the disk indicates that the organism was inhibited by the drug which diffused into the agar from the disk.

Paper disk-plate method: The paper disk-plate method is the most commonly used technique for determining susceptibility of microorganisms to antibiotic agents. Small paper disks impregnated with known amount of antibiotic agents are placed upon the surface of an inoculated plate. After incubation the plates are observed for any zone of inhibition (a clear area) around the disk indicates that the organism was inhibited by the drug which diffused into the agar from the disk.

170 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 11.1. Paper disk plate method showing zone of inhibition (antibiotic disks used are Polymyxin (Pb); Neomycin (N); Ceftazidime (Ca); Tetracycline (T30); Cloxacillin (Cx); Ampicillin (A); Chloramphenicol (C); Ciprofloxacin (Cf); Vancomycin (Va).

Procedure

170 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 11.1. Paper disk plate method showing zone of inhibition (antibiotic disks used are Polymyxin (Pb); Neomycin (N); Ceftazidime (Ca); Tetracycline (T30); Cloxacillin (Cx); Ampicillin (A); Chloramphenicol (C); Ciprofloxacin (Cf); Vancomycin (Va).

Procedure

1. Prepare the media with the help of Tables 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and leave for solidifying. 4. When the agar plates solidify add 3 to 5 mL of culture media containing microorganisms, spread on the plate properly and leave for 5 minutes. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make small disk of filter paper and dip into the antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator for proper temperature as mentioned in the Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible for antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

1. Prepare the media with the help of Tables 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and leave for solidifying. 4. When the agar plates solidify add 3 to 5 mL of culture media containing microorganisms, spread on the plate properly and leave for 5 minutes. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make small disk of filter paper and dip into the antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator for proper temperature as mentioned in the Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible for antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

Estimation of potency of antibiotics: Suppose we have 5 different concentrations of antibiotics which are using in the bore of Petri plate and their concentrations are a, b, c, d and e according to their increasing concentration. If L is the zone diameter of lowest concentration and H is the zone diameter of highest concentration. Then

Estimation of potency of antibiotics: Suppose we have 5 different concentrations of antibiotics which are using in the bore of Petri plate and their concentrations are a, b, c, d and e according to their increasing concentration. If L is the zone diameter of lowest concentration and H is the zone diameter of highest concentration. Then

170 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

170 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 11.1. Paper disk plate method showing zone of inhibition (antibiotic disks used are Polymyxin (Pb); Neomycin (N); Ceftazidime (Ca); Tetracycline (T30); Cloxacillin (Cx); Ampicillin (A); Chloramphenicol (C); Ciprofloxacin (Cf); Vancomycin (Va).

Procedure

Fig. 11.1. Paper disk plate method showing zone of inhibition (antibiotic disks used are Polymyxin (Pb); Neomycin (N); Ceftazidime (Ca); Tetracycline (T30); Cloxacillin (Cx); Ampicillin (A); Chloramphenicol (C); Ciprofloxacin (Cf); Vancomycin (Va).

Procedure

1. Prepare the media with the help of Tables 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and leave for solidifying. 4. When the agar plates solidify add 3 to 5 mL of culture media containing microorganisms, spread on the plate properly and leave for 5 minutes. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make small disk of filter paper and dip into the antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator for proper temperature as mentioned in the Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible for antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

1. Prepare the media with the help of Tables 1 and 2. 2. Autoclave the media properly. 3. Pour the media into the Petri plate in the aseptic cabin or in laminar hood and leave for solidifying. 4. When the agar plates solidify add 3 to 5 mL of culture media containing microorganisms, spread on the plate properly and leave for 5 minutes. After that tilt the plate and remove the remaining watery fluid and leave for some time. 5. Make small disk of filter paper and dip into the antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator for proper temperature as mentioned in the Table 3 for overnight. 6. After overnight incubation if the microorganism is susceptible for antibiotic agents, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 7. If the microorganisms are resistant to antibiotic agents there is no zone of inhibition.

Estimation of potency of antibiotics: Suppose we have 5 different concentrations of antibiotics which are using in the bore of Petri plate and their concentrations are a, b, c, d and e according to their increasing concentration. If L is the zone diameter of lowest concentration and H is the zone diameter of highest concentration. Then

Estimation of potency of antibiotics: Suppose we have 5 different concentrations of antibiotics which are using in the bore of Petri plate and their concentrations are a, b, c, d and e according to their increasing concentration. If L is the zone diameter of lowest concentration and H is the zone diameter of highest concentration. Then

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

L = 3a + 2b + c – e ; 5

171

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

H = 3e + 2d + c- a 5

L = 3a + 2b + c – e ; 5

171

H = 3e + 2d + c- a 5

II. Tube assay method or turbidimetric method Principle: The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic. By the tube dilution technique, one can determine the smallest amount of antibiotic or chemotherapeutic agent required to inhibit the growth of microorganism. This amount is referred to as the MIC (minimum inhibitory concentration). A set of tubes with different concentrations of a particular antibiotic is prepared. The tubes are inoculated with the test organism, incubated, and examined for growth of bacteria. Growth is seen to diminish as the concentration of antibiotic increases, and eventually an antibiotic concentration may be observed at which growth fails to occur. This is the minimum inhibitory concentration (MIC).

II. Tube assay method or turbidimetric method Principle: The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic. By the tube dilution technique, one can determine the smallest amount of antibiotic or chemotherapeutic agent required to inhibit the growth of microorganism. This amount is referred to as the MIC (minimum inhibitory concentration). A set of tubes with different concentrations of a particular antibiotic is prepared. The tubes are inoculated with the test organism, incubated, and examined for growth of bacteria. Growth is seen to diminish as the concentration of antibiotic increases, and eventually an antibiotic concentration may be observed at which growth fails to occur. This is the minimum inhibitory concentration (MIC).

Fig. 11.2. In all tubes was added the antimicrobial agent in increasing concentration. Test tube no. 6 shows the MIC.

Fig. 11.2. In all tubes was added the antimicrobial agent in increasing concentration. Test tube no. 6 shows the MIC.

Procedure

Procedure

1. Prepare the broth and take 5 mL into different test tubes. 2. Autoclave all the test tubes properly and when the temperature reaches 40-45°C the different concentrations of antibiotics are added into the test tubes as mentioned in Table 3.

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

L = 3a + 2b + c – e ; 5

171

1. Prepare the broth and take 5 mL into different test tubes. 2. Autoclave all the test tubes properly and when the temperature reaches 40-45°C the different concentrations of antibiotics are added into the test tubes as mentioned in Table 3.

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

H = 3e + 2d + c- a 5

L = 3a + 2b + c – e ; 5

171

H = 3e + 2d + c- a 5

II. Tube assay method or turbidimetric method Principle: The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic. By the tube dilution technique, one can determine the smallest amount of antibiotic or chemotherapeutic agent required to inhibit the growth of microorganism. This amount is referred to as the MIC (minimum inhibitory concentration). A set of tubes with different concentrations of a particular antibiotic is prepared. The tubes are inoculated with the test organism, incubated, and examined for growth of bacteria. Growth is seen to diminish as the concentration of antibiotic increases, and eventually an antibiotic concentration may be observed at which growth fails to occur. This is the minimum inhibitory concentration (MIC).

II. Tube assay method or turbidimetric method Principle: The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic. By the tube dilution technique, one can determine the smallest amount of antibiotic or chemotherapeutic agent required to inhibit the growth of microorganism. This amount is referred to as the MIC (minimum inhibitory concentration). A set of tubes with different concentrations of a particular antibiotic is prepared. The tubes are inoculated with the test organism, incubated, and examined for growth of bacteria. Growth is seen to diminish as the concentration of antibiotic increases, and eventually an antibiotic concentration may be observed at which growth fails to occur. This is the minimum inhibitory concentration (MIC).

Fig. 11.2. In all tubes was added the antimicrobial agent in increasing concentration. Test tube no. 6 shows the MIC.

Fig. 11.2. In all tubes was added the antimicrobial agent in increasing concentration. Test tube no. 6 shows the MIC.

Procedure 1. Prepare the broth and take 5 mL into different test tubes. 2. Autoclave all the test tubes properly and when the temperature reaches 40-45°C the different concentrations of antibiotics are added into the test tubes as mentioned in Table 3.

Procedure 1. Prepare the broth and take 5 mL into different test tubes. 2. Autoclave all the test tubes properly and when the temperature reaches 40-45°C the different concentrations of antibiotics are added into the test tubes as mentioned in Table 3.

172 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 3. Now keep 2 or 3 test tubes blank without microorganisms and in all other tubes put the microbial culture with the help of inoculum loop for overnight culture. 4. Keep all the test tubes in the incubator and check the growth of bacteria after overnight incubation at temperature given in Table 3. 5. Compare all the test tubes with blank and differentiate with turbidity. If turbidity occurs, growth of microorganisms takes place. 6. The minimum concentration at which the bacterial growth does not occur is called MIC for the antibiotic.

172 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 3. Now keep 2 or 3 test tubes blank without microorganisms and in all other tubes put the microbial culture with the help of inoculum loop for overnight culture. 4. Keep all the test tubes in the incubator and check the growth of bacteria after overnight incubation at temperature given in Table 3. 5. Compare all the test tubes with blank and differentiate with turbidity. If turbidity occurs, growth of microorganisms takes place. 6. The minimum concentration at which the bacterial growth does not occur is called MIC for the antibiotic.

11.2 ASSAY OF VITAMINS

11.2 ASSAY OF VITAMINS

For the growth of microorganisms various amino acids and vitamins are required in very small amounts. Various microorganisms are used for the assay of vitamins and amino acids. For the assay of vitamins two types of media are used: 1. Nutritionally complete media without adding amino acids and vitamins where minimal growth of microorganisms takes place which is used as a control. 2. Nutritionally complete media containing amino acids or vitamins and growth of test organisms is observed. The growth of the microorganisms depends upon the amount or dose of amino acid or vitamins added to the medium.

For the growth of microorganisms various amino acids and vitamins are required in very small amounts. Various microorganisms are used for the assay of vitamins and amino acids. For the assay of vitamins two types of media are used: 1. Nutritionally complete media without adding amino acids and vitamins where minimal growth of microorganisms takes place which is used as a control. 2. Nutritionally complete media containing amino acids or vitamins and growth of test organisms is observed. The growth of the microorganisms depends upon the amount or dose of amino acid or vitamins added to the medium.

11.2.1 Microbial Assay of Vitamin A 1. Determination of the potency of vitamin A is based on the measurement of the UV spectrum of the vitamin at the specified wavelength. 2. The potency of vitamin A expressed in terms of the unit, which is 0.344 —gm of all-trans-vitamin A acetate, equivalent to 0.30 —gm of all-transvitamin A alcohol. 3. Vitamin A contains some other substance which is also absorbed during the UV measurement so some preliminary treatment is done by chemical or physical methods or by combination of these methods may be necessary before following the spectrophotometric methods are used. 4. It is also essential that the wavelength scale of the spectrophotometer is checked immediately before the assay. 5. The mercury lines at 313.16 —m and 334.15 —m provide suitable points and for convenience the setting of the instruments on these lines may be related to its setting on the hydrogen lines at 379.7 —m and 496.2 —m. 6. Therefore the absorbance measurement sequence special care. There are two methods for the assay of vitamin A.

11.2.1 Microbial Assay of Vitamin A 1. Determination of the potency of vitamin A is based on the measurement of the UV spectrum of the vitamin at the specified wavelength. 2. The potency of vitamin A expressed in terms of the unit, which is 0.344 —gm of all-trans-vitamin A acetate, equivalent to 0.30 —gm of all-transvitamin A alcohol. 3. Vitamin A contains some other substance which is also absorbed during the UV measurement so some preliminary treatment is done by chemical or physical methods or by combination of these methods may be necessary before following the spectrophotometric methods are used. 4. It is also essential that the wavelength scale of the spectrophotometer is checked immediately before the assay. 5. The mercury lines at 313.16 —m and 334.15 —m provide suitable points and for convenience the setting of the instruments on these lines may be related to its setting on the hydrogen lines at 379.7 —m and 496.2 —m. 6. Therefore the absorbance measurement sequence special care. There are two methods for the assay of vitamin A.

Method A: This method is suitable for preparations which contain vitamin A as an ester and in a form directly soluble in cyclohexane.

Method A: This method is suitable for preparations which contain vitamin A as an ester and in a form directly soluble in cyclohexane.

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3. Now keep 2 or 3 test tubes blank without microorganisms and in all other tubes put the microbial culture with the help of inoculum loop for overnight culture. 4. Keep all the test tubes in the incubator and check the growth of bacteria after overnight incubation at temperature given in Table 3. 5. Compare all the test tubes with blank and differentiate with turbidity. If turbidity occurs, growth of microorganisms takes place. 6. The minimum concentration at which the bacterial growth does not occur is called MIC for the antibiotic.

3. Now keep 2 or 3 test tubes blank without microorganisms and in all other tubes put the microbial culture with the help of inoculum loop for overnight culture. 4. Keep all the test tubes in the incubator and check the growth of bacteria after overnight incubation at temperature given in Table 3. 5. Compare all the test tubes with blank and differentiate with turbidity. If turbidity occurs, growth of microorganisms takes place. 6. The minimum concentration at which the bacterial growth does not occur is called MIC for the antibiotic.

11.2 ASSAY OF VITAMINS

11.2 ASSAY OF VITAMINS

For the growth of microorganisms various amino acids and vitamins are required in very small amounts. Various microorganisms are used for the assay of vitamins and amino acids. For the assay of vitamins two types of media are used: 1. Nutritionally complete media without adding amino acids and vitamins where minimal growth of microorganisms takes place which is used as a control. 2. Nutritionally complete media containing amino acids or vitamins and growth of test organisms is observed. The growth of the microorganisms depends upon the amount or dose of amino acid or vitamins added to the medium.

For the growth of microorganisms various amino acids and vitamins are required in very small amounts. Various microorganisms are used for the assay of vitamins and amino acids. For the assay of vitamins two types of media are used: 1. Nutritionally complete media without adding amino acids and vitamins where minimal growth of microorganisms takes place which is used as a control. 2. Nutritionally complete media containing amino acids or vitamins and growth of test organisms is observed. The growth of the microorganisms depends upon the amount or dose of amino acid or vitamins added to the medium.

11.2.1 Microbial Assay of Vitamin A 1. Determination of the potency of vitamin A is based on the measurement of the UV spectrum of the vitamin at the specified wavelength. 2. The potency of vitamin A expressed in terms of the unit, which is 0.344 —gm of all-trans-vitamin A acetate, equivalent to 0.30 —gm of all-transvitamin A alcohol. 3. Vitamin A contains some other substance which is also absorbed during the UV measurement so some preliminary treatment is done by chemical or physical methods or by combination of these methods may be necessary before following the spectrophotometric methods are used. 4. It is also essential that the wavelength scale of the spectrophotometer is checked immediately before the assay. 5. The mercury lines at 313.16 —m and 334.15 —m provide suitable points and for convenience the setting of the instruments on these lines may be related to its setting on the hydrogen lines at 379.7 —m and 496.2 —m. 6. Therefore the absorbance measurement sequence special care. There are two methods for the assay of vitamin A.

11.2.1 Microbial Assay of Vitamin A 1. Determination of the potency of vitamin A is based on the measurement of the UV spectrum of the vitamin at the specified wavelength. 2. The potency of vitamin A expressed in terms of the unit, which is 0.344 —gm of all-trans-vitamin A acetate, equivalent to 0.30 —gm of all-transvitamin A alcohol. 3. Vitamin A contains some other substance which is also absorbed during the UV measurement so some preliminary treatment is done by chemical or physical methods or by combination of these methods may be necessary before following the spectrophotometric methods are used. 4. It is also essential that the wavelength scale of the spectrophotometer is checked immediately before the assay. 5. The mercury lines at 313.16 —m and 334.15 —m provide suitable points and for convenience the setting of the instruments on these lines may be related to its setting on the hydrogen lines at 379.7 —m and 496.2 —m. 6. Therefore the absorbance measurement sequence special care. There are two methods for the assay of vitamin A.

Method A: This method is suitable for preparations which contain vitamin A as an ester and in a form directly soluble in cyclohexane.

Method A: This method is suitable for preparations which contain vitamin A as an ester and in a form directly soluble in cyclohexane.

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Procedure

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Procedure

1. Weigh accurate amount of vitamin A and dissolve it in cyclohexane (9 to 15 units of vitamin A mL). 2. Determine the wavelength of maximum absorption. 3. Measure the absorbance of the solution against the cyclohexane at the wavelength in given Table 4. Table 4:

1. Weigh accurate amount of vitamin A and dissolve it in cyclohexane (9 to 15 units of vitamin A mL). 2. Determine the wavelength of maximum absorption. 3. Measure the absorbance of the solution against the cyclohexane at the wavelength in given Table 4. Table 4:

S.No.

Wavelength

Relative absorbance

S.No.

Wavelength

Relative absorbance

1. 2. 3. 4. 5.

300 316 328 340 360

0.555 0.907 1.000 0.811 0.299

1. 2. 3. 4. 5.

300 316 328 340 360

0.555 0.907 1.000 0.811 0.299

4. Calculate the absorbance at the wavelength specified as fraction relative to that at 328 —m. Calculate also the absorbance at 328 —m in terms of specific absorbance for the sample. 5. If the wavelength of maximum absorbance lies between 326 and 329 —m and the relative absorbance within 0.02 of those in Table 4, calculate the vitamin A potency of the sample from the following equation:

4. Calculate the absorbance at the wavelength specified as fraction relative to that at 328 —m. Calculate also the absorbance at 328 —m in terms of specific absorbance for the sample. 5. If the wavelength of maximum absorbance lies between 326 and 329 —m and the relative absorbance within 0.02 of those in Table 4, calculate the vitamin A potency of the sample from the following equation:

A328 (1%, 1cm) D 1900 = vitamin A potency in units/gm.

A328 (1%, 1cm) D 1900 = vitamin A potency in units/gm.

6. If the wavelength of maximum absorption lies between 326 and 328 —m but the relative absorbance not within 0.02 of those in the Table 4, calculate a corrected absorbance at 328 —m applying the observed values to the equation.

6. If the wavelength of maximum absorption lies between 326 and 328 —m but the relative absorbance not within 0.02 of those in the Table 4, calculate a corrected absorbance at 328 —m applying the observed values to the equation.

A328 (corr.) = 3.52 (2A328-A316-A340)

A328 (corr.) = 3.52 (2A328-A316-A340)

Method B

Method B

1. This method is used for all trans-vitamin A acetate. This is white to very yellow, free flowing crystals. 2. Very soluble in ethanol (95%), chloroform ether light petroleum fats and fixed oils insoluble in water. 3. The light absorbance of 0.0003% w/v solution in 2-propanol at about 325 —m, not less than 0.458, in cyclohexane at about 327.5 not less than 0.455 and in ethanol at about 326 —m not less than 0.464.

1. This method is used for all trans-vitamin A acetate. This is white to very yellow, free flowing crystals. 2. Very soluble in ethanol (95%), chloroform ether light petroleum fats and fixed oils insoluble in water. 3. The light absorbance of 0.0003% w/v solution in 2-propanol at about 325 —m, not less than 0.458, in cyclohexane at about 327.5 not less than 0.455 and in ethanol at about 326 —m not less than 0.464.

Relative absorbance: Measure the absorbance of a 0.0003% w/v solution cyclohexane at about 327.5 —m and at the following wavelengths. The relative absorbances calculated with reference to the absorbance at about 327.5 —m are within the limits as stated in Table 5.

Relative absorbance: Measure the absorbance of a 0.0003% w/v solution cyclohexane at about 327.5 —m and at the following wavelengths. The relative absorbances calculated with reference to the absorbance at about 327.5 —m are within the limits as stated in Table 5.

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Procedure

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Procedure

1. Weigh accurate amount of vitamin A and dissolve it in cyclohexane (9 to 15 units of vitamin A mL). 2. Determine the wavelength of maximum absorption. 3. Measure the absorbance of the solution against the cyclohexane at the wavelength in given Table 4. Table 4:

1. Weigh accurate amount of vitamin A and dissolve it in cyclohexane (9 to 15 units of vitamin A mL). 2. Determine the wavelength of maximum absorption. 3. Measure the absorbance of the solution against the cyclohexane at the wavelength in given Table 4. Table 4:

S.No.

Wavelength

Relative absorbance

S.No.

Wavelength

Relative absorbance

1. 2. 3. 4. 5.

300 316 328 340 360

0.555 0.907 1.000 0.811 0.299

1. 2. 3. 4. 5.

300 316 328 340 360

0.555 0.907 1.000 0.811 0.299

4. Calculate the absorbance at the wavelength specified as fraction relative to that at 328 —m. Calculate also the absorbance at 328 —m in terms of specific absorbance for the sample. 5. If the wavelength of maximum absorbance lies between 326 and 329 —m and the relative absorbance within 0.02 of those in Table 4, calculate the vitamin A potency of the sample from the following equation: A328 (1%, 1cm) D 1900 = vitamin A potency in units/gm. 6. If the wavelength of maximum absorption lies between 326 and 328 —m but the relative absorbance not within 0.02 of those in the Table 4, calculate a corrected absorbance at 328 —m applying the observed values to the equation.

4. Calculate the absorbance at the wavelength specified as fraction relative to that at 328 —m. Calculate also the absorbance at 328 —m in terms of specific absorbance for the sample. 5. If the wavelength of maximum absorbance lies between 326 and 329 —m and the relative absorbance within 0.02 of those in Table 4, calculate the vitamin A potency of the sample from the following equation: A328 (1%, 1cm) D 1900 = vitamin A potency in units/gm. 6. If the wavelength of maximum absorption lies between 326 and 328 —m but the relative absorbance not within 0.02 of those in the Table 4, calculate a corrected absorbance at 328 —m applying the observed values to the equation.

A328 (corr.) = 3.52 (2A328-A316-A340)

Method B

A328 (corr.) = 3.52 (2A328-A316-A340)

Method B

1. This method is used for all trans-vitamin A acetate. This is white to very yellow, free flowing crystals. 2. Very soluble in ethanol (95%), chloroform ether light petroleum fats and fixed oils insoluble in water. 3. The light absorbance of 0.0003% w/v solution in 2-propanol at about 325 —m, not less than 0.458, in cyclohexane at about 327.5 not less than 0.455 and in ethanol at about 326 —m not less than 0.464.

1. This method is used for all trans-vitamin A acetate. This is white to very yellow, free flowing crystals. 2. Very soluble in ethanol (95%), chloroform ether light petroleum fats and fixed oils insoluble in water. 3. The light absorbance of 0.0003% w/v solution in 2-propanol at about 325 —m, not less than 0.458, in cyclohexane at about 327.5 not less than 0.455 and in ethanol at about 326 —m not less than 0.464.

Relative absorbance: Measure the absorbance of a 0.0003% w/v solution cyclohexane at about 327.5 —m and at the following wavelengths. The relative absorbances calculated with reference to the absorbance at about 327.5 —m are within the limits as stated in Table 5.

Relative absorbance: Measure the absorbance of a 0.0003% w/v solution cyclohexane at about 327.5 —m and at the following wavelengths. The relative absorbances calculated with reference to the absorbance at about 327.5 —m are within the limits as stated in Table 5.

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Table 5:

Table 5:

S.No.

Wavelength

Relative absorbance

S.No.

Wavelength

Relative absorbance

1. 2. 3. 4. 5.

300 312.5 337.5 345 360

0.545 0.845 0.845 0.685 0.290

1. 2. 3. 4. 5.

300 312.5 337.5 345 360

0.545 0.845 0.845 0.685 0.290

– – – – –

0.565 0.865 0.865 0.705 0.310

Procedure

– – – – –

0.565 0.865 0.865 0.705 0.310

Procedure

1. Weigh accurate quantity of substance containing not less than 500 units of vitamin A and not more than 2 gm of fat. 2. Add 50 mg of hydroquinone, 30 mL of ethanol and 3 mL of 50% w/v solution of KOH. 3. Boil gently under a reflux condenser in a stream of oxygen free nitrogen for 30 minutes. Cool the solution rapidly and add 30 mL of water. 4. Transfer to a separator with the aid of ether and extract the vitamin A by shaking for 1 minute. After complete separation discard the aqueous layer and wash the extract with four quantities each of 50 mL of water, mixing properly to avoid the formation of emulsions. 5. Evaporate the separated extract to about 5 mL and remove the remaining solvent in a stream of oxygen-free nitrogen without the application of heat. 6. Dissolve the residue in sufficient quantity of 2-propanol to produce a solution containing 9 to 15 units of vitamin A per mL and measure the absorbance at about 300, 310, 325 and 334 Pm determine the wavelength of maximum absorption. 7. If the wavelength of maximum absorption lies between 323 and 327 Pm and the absorbance at about 300 Pm relative to that at about 325 Pm do not exceed 0.73, a corrected absorbance is derived from the equation.

1. Weigh accurate quantity of substance containing not less than 500 units of vitamin A and not more than 2 gm of fat. 2. Add 50 mg of hydroquinone, 30 mL of ethanol and 3 mL of 50% w/v solution of KOH. 3. Boil gently under a reflux condenser in a stream of oxygen free nitrogen for 30 minutes. Cool the solution rapidly and add 30 mL of water. 4. Transfer to a separator with the aid of ether and extract the vitamin A by shaking for 1 minute. After complete separation discard the aqueous layer and wash the extract with four quantities each of 50 mL of water, mixing properly to avoid the formation of emulsions. 5. Evaporate the separated extract to about 5 mL and remove the remaining solvent in a stream of oxygen-free nitrogen without the application of heat. 6. Dissolve the residue in sufficient quantity of 2-propanol to produce a solution containing 9 to 15 units of vitamin A per mL and measure the absorbance at about 300, 310, 325 and 334 Pm determine the wavelength of maximum absorption. 7. If the wavelength of maximum absorption lies between 323 and 327 Pm and the absorbance at about 300 Pm relative to that at about 325 Pm do not exceed 0.73, a corrected absorbance is derived from the equation.

A325 (corr.) = 6.815A325– 2.555A300– 4.260A334 Calculate the potency of the sample from the expression. A325 (1%, 1cm á1830 = vitamin A potency in units per gm

A325 (corr.) = 6.815A325– 2.555A300– 4.260A334 Calculate the potency of the sample from the expression. A325 (1%, 1cm á1830 = vitamin A potency in units per gm

If the corrected absorbance lies within ±3% of the uncorrected absorbance, ignore the corrected absorbance and calculate the potency from the uncorrected absorbance. If the wavelength of maximum absorbance lies outside the range 323 to 327 Pm or if the relative absorbance at about 300 Pm exceeds 0.73, the unsaponifiable of the sample must be further purified by chromatography.

If the corrected absorbance lies within ±3% of the uncorrected absorbance, ignore the corrected absorbance and calculate the potency from the uncorrected absorbance. If the wavelength of maximum absorbance lies outside the range 323 to 327 Pm or if the relative absorbance at about 300 Pm exceeds 0.73, the unsaponifiable of the sample must be further purified by chromatography.

11.2.2 Microbial Assay of Vitamins D The assay of vitamin D is done in the absence or minimum exposure of light and air by the use of inert gas and low actinic glassware.

11.2.2 Microbial Assay of Vitamins D The assay of vitamin D is done in the absence or minimum exposure of light and air by the use of inert gas and low actinic glassware.

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Table 5:

Table 5:

S.No.

Wavelength

Relative absorbance

S.No.

Wavelength

Relative absorbance

1. 2. 3. 4. 5.

300 312.5 337.5 345 360

0.545 0.845 0.845 0.685 0.290

1. 2. 3. 4. 5.

300 312.5 337.5 345 360

0.545 0.845 0.845 0.685 0.290

– – – – –

0.565 0.865 0.865 0.705 0.310

Procedure 1. Weigh accurate quantity of substance containing not less than 500 units of vitamin A and not more than 2 gm of fat. 2. Add 50 mg of hydroquinone, 30 mL of ethanol and 3 mL of 50% w/v solution of KOH. 3. Boil gently under a reflux condenser in a stream of oxygen free nitrogen for 30 minutes. Cool the solution rapidly and add 30 mL of water. 4. Transfer to a separator with the aid of ether and extract the vitamin A by shaking for 1 minute. After complete separation discard the aqueous layer and wash the extract with four quantities each of 50 mL of water, mixing properly to avoid the formation of emulsions. 5. Evaporate the separated extract to about 5 mL and remove the remaining solvent in a stream of oxygen-free nitrogen without the application of heat. 6. Dissolve the residue in sufficient quantity of 2-propanol to produce a solution containing 9 to 15 units of vitamin A per mL and measure the absorbance at about 300, 310, 325 and 334 Pm determine the wavelength of maximum absorption. 7. If the wavelength of maximum absorption lies between 323 and 327 Pm and the absorbance at about 300 Pm relative to that at about 325 Pm do not exceed 0.73, a corrected absorbance is derived from the equation.

– – – – –

0.565 0.865 0.865 0.705 0.310

Procedure 1. Weigh accurate quantity of substance containing not less than 500 units of vitamin A and not more than 2 gm of fat. 2. Add 50 mg of hydroquinone, 30 mL of ethanol and 3 mL of 50% w/v solution of KOH. 3. Boil gently under a reflux condenser in a stream of oxygen free nitrogen for 30 minutes. Cool the solution rapidly and add 30 mL of water. 4. Transfer to a separator with the aid of ether and extract the vitamin A by shaking for 1 minute. After complete separation discard the aqueous layer and wash the extract with four quantities each of 50 mL of water, mixing properly to avoid the formation of emulsions. 5. Evaporate the separated extract to about 5 mL and remove the remaining solvent in a stream of oxygen-free nitrogen without the application of heat. 6. Dissolve the residue in sufficient quantity of 2-propanol to produce a solution containing 9 to 15 units of vitamin A per mL and measure the absorbance at about 300, 310, 325 and 334 Pm determine the wavelength of maximum absorption. 7. If the wavelength of maximum absorption lies between 323 and 327 Pm and the absorbance at about 300 Pm relative to that at about 325 Pm do not exceed 0.73, a corrected absorbance is derived from the equation.

A325 (corr.) = 6.815A325– 2.555A300– 4.260A334 Calculate the potency of the sample from the expression. A325 (1%, 1cm á1830 = vitamin A potency in units per gm

A325 (corr.) = 6.815A325– 2.555A300– 4.260A334 Calculate the potency of the sample from the expression. A325 (1%, 1cm á1830 = vitamin A potency in units per gm

If the corrected absorbance lies within ±3% of the uncorrected absorbance, ignore the corrected absorbance and calculate the potency from the uncorrected absorbance. If the wavelength of maximum absorbance lies outside the range 323 to 327 Pm or if the relative absorbance at about 300 Pm exceeds 0.73, the unsaponifiable of the sample must be further purified by chromatography.

If the corrected absorbance lies within ±3% of the uncorrected absorbance, ignore the corrected absorbance and calculate the potency from the uncorrected absorbance. If the wavelength of maximum absorbance lies outside the range 323 to 327 Pm or if the relative absorbance at about 300 Pm exceeds 0.73, the unsaponifiable of the sample must be further purified by chromatography.

11.2.2 Microbial Assay of Vitamins D The assay of vitamin D is done in the absence or minimum exposure of light and air by the use of inert gas and low actinic glassware.

11.2.2 Microbial Assay of Vitamins D The assay of vitamin D is done in the absence or minimum exposure of light and air by the use of inert gas and low actinic glassware.

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Preparation of standard vitamin D solution: Dissolve 0.01 gm of cholecalciferol or ergocalciferol in sufficient purified 1, 2-dischloroethane to give a solution containing 10—gm of vitamin D per mL. Procedure: Weigh accurate quantity of the substance under examination equivalent to about 400 units of vitamin D. For capsules, the mixed contents of 20 capsules may be used as the sample. Add 10 mL of freshly prepared 0.01% w/v solution of butylated hydroxytoluene in ethanol 95%, 15 mL of a 50% w/ v solution of KOH and 5 mL ethanol 95%. Reflux on a water bath for 30 minutes, cool and transfer the solution to a separator with the aid of 50 mL of water. Add 75 mL ether and shake vigorously. Allow to separate transfer an aqueous layer to a second separator and extract with three successive quantities each of 30 mL, of ether, adding each ethereal extract to the liquid in the first separation and finally discarding the aqueous solution. In each pour 100 mL of water, through the ethereal solution without shaking and discard the aqueous layers. Add 10 mL of water in each ethereal solution, agitate gently each time and discard the aqueous extracts. Continue the process until the aqueous extracts are neutral to phenolphthalein solution. Dry the ethereal solution by stirring with anhydrous sodium sulphate, decant the ethereal solution. Wash the residue with successive small portion of ether and evaporate the combined solution by washing on a water bath to a volume of 5 mL. Cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 5 mL of trimethyl pentane to obtain the sample preparation. Transfer the sample to the top of chromatographic column no. 1 with the aid of 5 mL of 2, 2, 4-trimethylpentane, elute the column with 2, 2,4trimethylpentane. Transfer the elute to the top of chromatographic column 2, allow the liquid to flow. Add 10 mL of trimethylpentane and discard the eluate. Now elute the column with 50 mL of benzene, evaporate the eluate on a water bath to a volume of about 5 mL, cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 4 ml of purified 1,2-dichloroethane to obtain the sample solution (solution A). Add 1 mL of solution A to each of 3 tubes. In the first tube add 1 mL of a mixture of equal volume of purified 1, 2-dichloroethane and acetic anhydride. In the second tube add 1 mL of purified 1, 2-dichloroethane and in the third tube add 1 mL of the standard preparation of vitamin D. Add 10 mL of antimony trichloride of each solution exactly 1 minute after the addition of the antimony trichloride at the maximum at about 500 Pm using purified 1, 2-dichloroethane as the blank. The amount of vitamin D in mg, in the weight of the sample taken is given by:

Preparation of standard vitamin D solution: Dissolve 0.01 gm of cholecalciferol or ergocalciferol in sufficient purified 1, 2-dischloroethane to give a solution containing 10—gm of vitamin D per mL. Procedure: Weigh accurate quantity of the substance under examination equivalent to about 400 units of vitamin D. For capsules, the mixed contents of 20 capsules may be used as the sample. Add 10 mL of freshly prepared 0.01% w/v solution of butylated hydroxytoluene in ethanol 95%, 15 mL of a 50% w/ v solution of KOH and 5 mL ethanol 95%. Reflux on a water bath for 30 minutes, cool and transfer the solution to a separator with the aid of 50 mL of water. Add 75 mL ether and shake vigorously. Allow to separate transfer an aqueous layer to a second separator and extract with three successive quantities each of 30 mL, of ether, adding each ethereal extract to the liquid in the first separation and finally discarding the aqueous solution. In each pour 100 mL of water, through the ethereal solution without shaking and discard the aqueous layers. Add 10 mL of water in each ethereal solution, agitate gently each time and discard the aqueous extracts. Continue the process until the aqueous extracts are neutral to phenolphthalein solution. Dry the ethereal solution by stirring with anhydrous sodium sulphate, decant the ethereal solution. Wash the residue with successive small portion of ether and evaporate the combined solution by washing on a water bath to a volume of 5 mL. Cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 5 mL of trimethyl pentane to obtain the sample preparation. Transfer the sample to the top of chromatographic column no. 1 with the aid of 5 mL of 2, 2, 4-trimethylpentane, elute the column with 2, 2,4trimethylpentane. Transfer the elute to the top of chromatographic column 2, allow the liquid to flow. Add 10 mL of trimethylpentane and discard the eluate. Now elute the column with 50 mL of benzene, evaporate the eluate on a water bath to a volume of about 5 mL, cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 4 ml of purified 1,2-dichloroethane to obtain the sample solution (solution A). Add 1 mL of solution A to each of 3 tubes. In the first tube add 1 mL of a mixture of equal volume of purified 1, 2-dichloroethane and acetic anhydride. In the second tube add 1 mL of purified 1, 2-dichloroethane and in the third tube add 1 mL of the standard preparation of vitamin D. Add 10 mL of antimony trichloride of each solution exactly 1 minute after the addition of the antimony trichloride at the maximum at about 500 Pm using purified 1, 2-dichloroethane as the blank. The amount of vitamin D in mg, in the weight of the sample taken is given by:

Where A1 = absorbance of solution of first tube, A2 = absorbance of solution of second tube, A3 = absorbance of solution of third tube.

Where A1 = absorbance of solution of first tube, A2 = absorbance of solution of second tube, A3 = absorbance of solution of third tube.

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Preparation of standard vitamin D solution: Dissolve 0.01 gm of cholecalciferol or ergocalciferol in sufficient purified 1, 2-dischloroethane to give a solution containing 10—gm of vitamin D per mL. Procedure: Weigh accurate quantity of the substance under examination equivalent to about 400 units of vitamin D. For capsules, the mixed contents of 20 capsules may be used as the sample. Add 10 mL of freshly prepared 0.01% w/v solution of butylated hydroxytoluene in ethanol 95%, 15 mL of a 50% w/ v solution of KOH and 5 mL ethanol 95%. Reflux on a water bath for 30 minutes, cool and transfer the solution to a separator with the aid of 50 mL of water. Add 75 mL ether and shake vigorously. Allow to separate transfer an aqueous layer to a second separator and extract with three successive quantities each of 30 mL, of ether, adding each ethereal extract to the liquid in the first separation and finally discarding the aqueous solution. In each pour 100 mL of water, through the ethereal solution without shaking and discard the aqueous layers. Add 10 mL of water in each ethereal solution, agitate gently each time and discard the aqueous extracts. Continue the process until the aqueous extracts are neutral to phenolphthalein solution. Dry the ethereal solution by stirring with anhydrous sodium sulphate, decant the ethereal solution. Wash the residue with successive small portion of ether and evaporate the combined solution by washing on a water bath to a volume of 5 mL. Cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 5 mL of trimethyl pentane to obtain the sample preparation. Transfer the sample to the top of chromatographic column no. 1 with the aid of 5 mL of 2, 2, 4-trimethylpentane, elute the column with 2, 2,4trimethylpentane. Transfer the elute to the top of chromatographic column 2, allow the liquid to flow. Add 10 mL of trimethylpentane and discard the eluate. Now elute the column with 50 mL of benzene, evaporate the eluate on a water bath to a volume of about 5 mL, cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 4 ml of purified 1,2-dichloroethane to obtain the sample solution (solution A). Add 1 mL of solution A to each of 3 tubes. In the first tube add 1 mL of a mixture of equal volume of purified 1, 2-dichloroethane and acetic anhydride. In the second tube add 1 mL of purified 1, 2-dichloroethane and in the third tube add 1 mL of the standard preparation of vitamin D. Add 10 mL of antimony trichloride of each solution exactly 1 minute after the addition of the antimony trichloride at the maximum at about 500 Pm using purified 1, 2-dichloroethane as the blank. The amount of vitamin D in mg, in the weight of the sample taken is given by:

Preparation of standard vitamin D solution: Dissolve 0.01 gm of cholecalciferol or ergocalciferol in sufficient purified 1, 2-dischloroethane to give a solution containing 10—gm of vitamin D per mL. Procedure: Weigh accurate quantity of the substance under examination equivalent to about 400 units of vitamin D. For capsules, the mixed contents of 20 capsules may be used as the sample. Add 10 mL of freshly prepared 0.01% w/v solution of butylated hydroxytoluene in ethanol 95%, 15 mL of a 50% w/ v solution of KOH and 5 mL ethanol 95%. Reflux on a water bath for 30 minutes, cool and transfer the solution to a separator with the aid of 50 mL of water. Add 75 mL ether and shake vigorously. Allow to separate transfer an aqueous layer to a second separator and extract with three successive quantities each of 30 mL, of ether, adding each ethereal extract to the liquid in the first separation and finally discarding the aqueous solution. In each pour 100 mL of water, through the ethereal solution without shaking and discard the aqueous layers. Add 10 mL of water in each ethereal solution, agitate gently each time and discard the aqueous extracts. Continue the process until the aqueous extracts are neutral to phenolphthalein solution. Dry the ethereal solution by stirring with anhydrous sodium sulphate, decant the ethereal solution. Wash the residue with successive small portion of ether and evaporate the combined solution by washing on a water bath to a volume of 5 mL. Cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 5 mL of trimethyl pentane to obtain the sample preparation. Transfer the sample to the top of chromatographic column no. 1 with the aid of 5 mL of 2, 2, 4-trimethylpentane, elute the column with 2, 2,4trimethylpentane. Transfer the elute to the top of chromatographic column 2, allow the liquid to flow. Add 10 mL of trimethylpentane and discard the eluate. Now elute the column with 50 mL of benzene, evaporate the eluate on a water bath to a volume of about 5 mL, cool and evaporate to dryness in a current of nitrogen and dissolve the residue in 4 ml of purified 1,2-dichloroethane to obtain the sample solution (solution A). Add 1 mL of solution A to each of 3 tubes. In the first tube add 1 mL of a mixture of equal volume of purified 1, 2-dichloroethane and acetic anhydride. In the second tube add 1 mL of purified 1, 2-dichloroethane and in the third tube add 1 mL of the standard preparation of vitamin D. Add 10 mL of antimony trichloride of each solution exactly 1 minute after the addition of the antimony trichloride at the maximum at about 500 Pm using purified 1, 2-dichloroethane as the blank. The amount of vitamin D in mg, in the weight of the sample taken is given by:

Where A1 = absorbance of solution of first tube, A2 = absorbance of solution of second tube, A3 = absorbance of solution of third tube.

Where A1 = absorbance of solution of first tube, A2 = absorbance of solution of second tube, A3 = absorbance of solution of third tube.

176 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

176 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

1 mg of cholecalciferol or ergocalciferol is equivalent to 40,000 units of vitamins of vitamin D.

1 mg of cholecalciferol or ergocalciferol is equivalent to 40,000 units of vitamins of vitamin D.

11.2.3 Microbial Assay of Calcium Pantothenate The microbial assay of calcium pantothenate is done by following method. 1. Standard stock solution of pantothenate: The stock solution is prepared by dissolving 50 mg of calcium pantothenate reference standard in about 500 mL of water, 10 mL of 0.2N acetic acid and 100 mL of a 1.6% w/v solution of sodium acetate are added and volume is made up to 1000 mL with water. This solution is stored under toluene in a refrigerator. Each mL of this solution contains 50 —g of calcium pantothenate. 2. Standard solution: Standard solution is prepared by diluting the stock solution with water. Standard solution contains 0.04 —g of calcium pantothenate per mL. 3. Test solution: It contains approximately the equivalent of the calcium pantothenate concentration in the standard solution prepared as solution in water.

11.2.3 Microbial Assay of Calcium Pantothenate The microbial assay of calcium pantothenate is done by following method. 1. Standard stock solution of pantothenate: The stock solution is prepared by dissolving 50 mg of calcium pantothenate reference standard in about 500 mL of water, 10 mL of 0.2N acetic acid and 100 mL of a 1.6% w/v solution of sodium acetate are added and volume is made up to 1000 mL with water. This solution is stored under toluene in a refrigerator. Each mL of this solution contains 50 —g of calcium pantothenate. 2. Standard solution: Standard solution is prepared by diluting the stock solution with water. Standard solution contains 0.04 —g of calcium pantothenate per mL. 3. Test solution: It contains approximately the equivalent of the calcium pantothenate concentration in the standard solution prepared as solution in water.

Medium: Media contains:Anhydrous dextrose Anhydrous sodium acetate Acid hydrolysed casein solution Cystine tryptophane solution Polysorbate-80 solution Adenine-guanine-uracil solution Riboflavin-thiamine HCl-biotin solution P-amino benzoic acid-niacin-pyridoxine HCl solution Salt solution A Salt solution B

Medium: Media contains:Anhydrous dextrose Anhydrous sodium acetate Acid hydrolysed casein solution Cystine tryptophane solution Polysorbate-80 solution Adenine-guanine-uracil solution Riboflavin-thiamine HCl-biotin solution P-amino benzoic acid-niacin-pyridoxine HCl solution Salt solution A Salt solution B

10 gm 5 gm 25 mL 25 mL 0.25 mL 5 mL 5 mL 5 mL 5 mL 5 mL

10 gm 5 gm 25 mL 25 mL 0.25 mL 5 mL 5 mL 5 mL 5 mL 5 mL

It is prepared by dissolving the anhydrous dextrose and anhydrous sodium acetate in previously mixed solutions and the pH is adjusted with 1N NaOH to a value of 6.8. Finally it is diluted to 250 mL with water and mixed. Stock culture of organism: Lactobacillus plantarum is used for the assay of calcium pantothenate. Media is prepared by adding 2 gm yeast extract, 500 mg anhydrous dextrose, 500 mg anhydrous sodium acetate and 1.5 gm agar in 100 mL of water. The media is autoclaved properly at 121°C for 15 minutes at pressure 15 pounds. Stub culture is prepared in 3 tubes using Lactobacillus plantarum, incubated at 35°C for 20-22 hours and stored in a refrigerator. Preparation of inoculums: The cells from the stock culture of organism are transferred to a sterile tube containing 10 ml of culture medium. It is incubated at 37°C for 20-22 hours.

It is prepared by dissolving the anhydrous dextrose and anhydrous sodium acetate in previously mixed solutions and the pH is adjusted with 1N NaOH to a value of 6.8. Finally it is diluted to 250 mL with water and mixed. Stock culture of organism: Lactobacillus plantarum is used for the assay of calcium pantothenate. Media is prepared by adding 2 gm yeast extract, 500 mg anhydrous dextrose, 500 mg anhydrous sodium acetate and 1.5 gm agar in 100 mL of water. The media is autoclaved properly at 121°C for 15 minutes at pressure 15 pounds. Stub culture is prepared in 3 tubes using Lactobacillus plantarum, incubated at 35°C for 20-22 hours and stored in a refrigerator. Preparation of inoculums: The cells from the stock culture of organism are transferred to a sterile tube containing 10 ml of culture medium. It is incubated at 37°C for 20-22 hours.

176 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

176 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

1 mg of cholecalciferol or ergocalciferol is equivalent to 40,000 units of vitamins of vitamin D.

1 mg of cholecalciferol or ergocalciferol is equivalent to 40,000 units of vitamins of vitamin D.

11.2.3 Microbial Assay of Calcium Pantothenate The microbial assay of calcium pantothenate is done by following method. 1. Standard stock solution of pantothenate: The stock solution is prepared by dissolving 50 mg of calcium pantothenate reference standard in about 500 mL of water, 10 mL of 0.2N acetic acid and 100 mL of a 1.6% w/v solution of sodium acetate are added and volume is made up to 1000 mL with water. This solution is stored under toluene in a refrigerator. Each mL of this solution contains 50 —g of calcium pantothenate. 2. Standard solution: Standard solution is prepared by diluting the stock solution with water. Standard solution contains 0.04 —g of calcium pantothenate per mL. 3. Test solution: It contains approximately the equivalent of the calcium pantothenate concentration in the standard solution prepared as solution in water.

11.2.3 Microbial Assay of Calcium Pantothenate The microbial assay of calcium pantothenate is done by following method. 1. Standard stock solution of pantothenate: The stock solution is prepared by dissolving 50 mg of calcium pantothenate reference standard in about 500 mL of water, 10 mL of 0.2N acetic acid and 100 mL of a 1.6% w/v solution of sodium acetate are added and volume is made up to 1000 mL with water. This solution is stored under toluene in a refrigerator. Each mL of this solution contains 50 —g of calcium pantothenate. 2. Standard solution: Standard solution is prepared by diluting the stock solution with water. Standard solution contains 0.04 —g of calcium pantothenate per mL. 3. Test solution: It contains approximately the equivalent of the calcium pantothenate concentration in the standard solution prepared as solution in water.

Medium: Media contains:Anhydrous dextrose Anhydrous sodium acetate Acid hydrolysed casein solution Cystine tryptophane solution Polysorbate-80 solution Adenine-guanine-uracil solution Riboflavin-thiamine HCl-biotin solution P-amino benzoic acid-niacin-pyridoxine HCl solution Salt solution A Salt solution B

Medium: Media contains:Anhydrous dextrose Anhydrous sodium acetate Acid hydrolysed casein solution Cystine tryptophane solution Polysorbate-80 solution Adenine-guanine-uracil solution Riboflavin-thiamine HCl-biotin solution P-amino benzoic acid-niacin-pyridoxine HCl solution Salt solution A Salt solution B

10 gm 5 gm 25 mL 25 mL 0.25 mL 5 mL 5 mL 5 mL 5 mL 5 mL

It is prepared by dissolving the anhydrous dextrose and anhydrous sodium acetate in previously mixed solutions and the pH is adjusted with 1N NaOH to a value of 6.8. Finally it is diluted to 250 mL with water and mixed. Stock culture of organism: Lactobacillus plantarum is used for the assay of calcium pantothenate. Media is prepared by adding 2 gm yeast extract, 500 mg anhydrous dextrose, 500 mg anhydrous sodium acetate and 1.5 gm agar in 100 mL of water. The media is autoclaved properly at 121°C for 15 minutes at pressure 15 pounds. Stub culture is prepared in 3 tubes using Lactobacillus plantarum, incubated at 35°C for 20-22 hours and stored in a refrigerator. Preparation of inoculums: The cells from the stock culture of organism are transferred to a sterile tube containing 10 ml of culture medium. It is incubated at 37°C for 20-22 hours.

10 gm 5 gm 25 mL 25 mL 0.25 mL 5 mL 5 mL 5 mL 5 mL 5 mL

It is prepared by dissolving the anhydrous dextrose and anhydrous sodium acetate in previously mixed solutions and the pH is adjusted with 1N NaOH to a value of 6.8. Finally it is diluted to 250 mL with water and mixed. Stock culture of organism: Lactobacillus plantarum is used for the assay of calcium pantothenate. Media is prepared by adding 2 gm yeast extract, 500 mg anhydrous dextrose, 500 mg anhydrous sodium acetate and 1.5 gm agar in 100 mL of water. The media is autoclaved properly at 121°C for 15 minutes at pressure 15 pounds. Stub culture is prepared in 3 tubes using Lactobacillus plantarum, incubated at 35°C for 20-22 hours and stored in a refrigerator. Preparation of inoculums: The cells from the stock culture of organism are transferred to a sterile tube containing 10 ml of culture medium. It is incubated at 37°C for 20-22 hours.

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

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ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

177

Method: In 5 test tubes add 1, 2, 3, 4, and 5 mL of standard solution respectively and in other 5 tubes no standard solution, 5 mL of medium solution is added. To each standard solution tubes add water and make up the volume up to 5 mL. All the test tubes are autoclaved at 121°C and after cooling the media, add 1 drop of bacterial culture or inoculum except 2 tubes (blanks) in which no standard solution is present and mixed properly. Incubate all the test tubes in incubator at 37°C for 20 to 22 hours. The transmittance of the tubes is measured in a spectrophotometer at a specific wavelength at 660 Pm.

Method: In 5 test tubes add 1, 2, 3, 4, and 5 mL of standard solution respectively and in other 5 tubes no standard solution, 5 mL of medium solution is added. To each standard solution tubes add water and make up the volume up to 5 mL. All the test tubes are autoclaved at 121°C and after cooling the media, add 1 drop of bacterial culture or inoculum except 2 tubes (blanks) in which no standard solution is present and mixed properly. Incubate all the test tubes in incubator at 37°C for 20 to 22 hours. The transmittance of the tubes is measured in a spectrophotometer at a specific wavelength at 660 Pm.

11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) The basic medium used for the assay of vitamin B12 activity is quite complex and contains a variety of essential components as a mixture in solution. One set of tubes contains measured amounts of a standard cynocobalamin solution and graded volumes of the test sample are added to another corresponding set of tubes. All the tubes are inoculated with a small amount of culture of Lactobacillus leichmannii and then incubated. The growth of organism is measured by light transmittance in a spectrophotometer. The amount of vitamin B12 present in the test sample is calculated from the standard curve by interpolation.

11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) The basic medium used for the assay of vitamin B12 activity is quite complex and contains a variety of essential components as a mixture in solution. One set of tubes contains measured amounts of a standard cynocobalamin solution and graded volumes of the test sample are added to another corresponding set of tubes. All the tubes are inoculated with a small amount of culture of Lactobacillus leichmannii and then incubated. The growth of organism is measured by light transmittance in a spectrophotometer. The amount of vitamin B12 present in the test sample is calculated from the standard curve by interpolation.

11.3 MICROBIAL ASSAY OF AMINO ACIDS

11.3 MICROBIAL ASSAY OF AMINO ACIDS

There are 20 different types of amino acid present which help in the synthesis of proteins. Various types of microorganisms (bacteria and fungi) are used for the assay of amino acids. For example, Leuconostoc citrovorum was used for alanine, and Streptococcus faecalis for arginine, methionine, threonine and tryptophan. For all other amino acids Leuconostoc mesenteroides was used. These included aspartic acid, cystine, glycine, histidine, lysine, phenylalanine, proline and serine. Microorganisms are grown on their specific media and add a freshly prepared amino acid solution on the same day a standard solution containing 200 —g per mL. With an appropriately diluted solution, duplicate tubes were prepared using from 0 to 1.0 mL per tube in gradations of 0.1 mL. Two tubes were also prepared using 1.0 mL of the original concentrated standard amino acid solution. Duplicate assay tubes were prepared at levels of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of amino acids. Add the amino acid into the media and autoclave it. When the media becomes cool, inoculate all the test tubes with microorganisms and leave some tubes for blank. Incubate all test tubes at 37°C for 15-20 hours. After incubation compare all the test tubes with blank having no microorganisms. Finally determine the potency of the amino acid.

There are 20 different types of amino acid present which help in the synthesis of proteins. Various types of microorganisms (bacteria and fungi) are used for the assay of amino acids. For example, Leuconostoc citrovorum was used for alanine, and Streptococcus faecalis for arginine, methionine, threonine and tryptophan. For all other amino acids Leuconostoc mesenteroides was used. These included aspartic acid, cystine, glycine, histidine, lysine, phenylalanine, proline and serine. Microorganisms are grown on their specific media and add a freshly prepared amino acid solution on the same day a standard solution containing 200 —g per mL. With an appropriately diluted solution, duplicate tubes were prepared using from 0 to 1.0 mL per tube in gradations of 0.1 mL. Two tubes were also prepared using 1.0 mL of the original concentrated standard amino acid solution. Duplicate assay tubes were prepared at levels of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of amino acids. Add the amino acid into the media and autoclave it. When the media becomes cool, inoculate all the test tubes with microorganisms and leave some tubes for blank. Incubate all test tubes at 37°C for 15-20 hours. After incubation compare all the test tubes with blank having no microorganisms. Finally determine the potency of the amino acid.

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

177

ANTIMICROBIAL ASSAY OF ANTIBIOTICS, VITAMINS AND AMINO ACIDS

177

Method: In 5 test tubes add 1, 2, 3, 4, and 5 mL of standard solution respectively and in other 5 tubes no standard solution, 5 mL of medium solution is added. To each standard solution tubes add water and make up the volume up to 5 mL. All the test tubes are autoclaved at 121°C and after cooling the media, add 1 drop of bacterial culture or inoculum except 2 tubes (blanks) in which no standard solution is present and mixed properly. Incubate all the test tubes in incubator at 37°C for 20 to 22 hours. The transmittance of the tubes is measured in a spectrophotometer at a specific wavelength at 660 Pm.

Method: In 5 test tubes add 1, 2, 3, 4, and 5 mL of standard solution respectively and in other 5 tubes no standard solution, 5 mL of medium solution is added. To each standard solution tubes add water and make up the volume up to 5 mL. All the test tubes are autoclaved at 121°C and after cooling the media, add 1 drop of bacterial culture or inoculum except 2 tubes (blanks) in which no standard solution is present and mixed properly. Incubate all the test tubes in incubator at 37°C for 20 to 22 hours. The transmittance of the tubes is measured in a spectrophotometer at a specific wavelength at 660 Pm.

11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) The basic medium used for the assay of vitamin B12 activity is quite complex and contains a variety of essential components as a mixture in solution. One set of tubes contains measured amounts of a standard cynocobalamin solution and graded volumes of the test sample are added to another corresponding set of tubes. All the tubes are inoculated with a small amount of culture of Lactobacillus leichmannii and then incubated. The growth of organism is measured by light transmittance in a spectrophotometer. The amount of vitamin B12 present in the test sample is calculated from the standard curve by interpolation.

11.2.4 Microbial Assay of Vitamin B12 (cyanocobalamin) The basic medium used for the assay of vitamin B12 activity is quite complex and contains a variety of essential components as a mixture in solution. One set of tubes contains measured amounts of a standard cynocobalamin solution and graded volumes of the test sample are added to another corresponding set of tubes. All the tubes are inoculated with a small amount of culture of Lactobacillus leichmannii and then incubated. The growth of organism is measured by light transmittance in a spectrophotometer. The amount of vitamin B12 present in the test sample is calculated from the standard curve by interpolation.

11.3 MICROBIAL ASSAY OF AMINO ACIDS

11.3 MICROBIAL ASSAY OF AMINO ACIDS

There are 20 different types of amino acid present which help in the synthesis of proteins. Various types of microorganisms (bacteria and fungi) are used for the assay of amino acids. For example, Leuconostoc citrovorum was used for alanine, and Streptococcus faecalis for arginine, methionine, threonine and tryptophan. For all other amino acids Leuconostoc mesenteroides was used. These included aspartic acid, cystine, glycine, histidine, lysine, phenylalanine, proline and serine. Microorganisms are grown on their specific media and add a freshly prepared amino acid solution on the same day a standard solution containing 200 —g per mL. With an appropriately diluted solution, duplicate tubes were prepared using from 0 to 1.0 mL per tube in gradations of 0.1 mL. Two tubes were also prepared using 1.0 mL of the original concentrated standard amino acid solution. Duplicate assay tubes were prepared at levels of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of amino acids. Add the amino acid into the media and autoclave it. When the media becomes cool, inoculate all the test tubes with microorganisms and leave some tubes for blank. Incubate all test tubes at 37°C for 15-20 hours. After incubation compare all the test tubes with blank having no microorganisms. Finally determine the potency of the amino acid.

There are 20 different types of amino acid present which help in the synthesis of proteins. Various types of microorganisms (bacteria and fungi) are used for the assay of amino acids. For example, Leuconostoc citrovorum was used for alanine, and Streptococcus faecalis for arginine, methionine, threonine and tryptophan. For all other amino acids Leuconostoc mesenteroides was used. These included aspartic acid, cystine, glycine, histidine, lysine, phenylalanine, proline and serine. Microorganisms are grown on their specific media and add a freshly prepared amino acid solution on the same day a standard solution containing 200 —g per mL. With an appropriately diluted solution, duplicate tubes were prepared using from 0 to 1.0 mL per tube in gradations of 0.1 mL. Two tubes were also prepared using 1.0 mL of the original concentrated standard amino acid solution. Duplicate assay tubes were prepared at levels of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of amino acids. Add the amino acid into the media and autoclave it. When the media becomes cool, inoculate all the test tubes with microorganisms and leave some tubes for blank. Incubate all test tubes at 37°C for 15-20 hours. After incubation compare all the test tubes with blank having no microorganisms. Finally determine the potency of the amino acid.

SECTION E

SECTION E

SECTION E

SECTION E

12

12

Experiments

Experiments

MICROBIOLOGY LABORATORY SAFETY RULES AND PROCEDURES

MICROBIOLOGY LABORATORY SAFETY RULES AND PROCEDURES

1. No food or drinks are permitted in the laboratory at any time. 2. Only closed-toe shoes are to be worn in the laboratory. Sandals are not permitted. 3. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The application of cosmetics in the laboratory is prohibited in the laboratory. 4. Work areas/surfaces must be disinfected before and after use. 5. Laboratory coats must be worn and buttoned while in the laboratory. Laboratory coats should not be worn outside the laboratory. 6. Protective eyewear must be worn when performing any exercise or procedure in the laboratory. 7. Long hair should be secured behind your head. 8. Hands must be washed before leaving the laboratory. 9. All unnecessary books, purses, briefcases, etc., must be kept off the countertops. 10. Never pipette anything by mouth (including water). Always use pipetting devices. 11. Label all materials with your name, date, and any other applicable information (e.g., media, organism, etc.). 12. Dispose of wastes in their proper containers. 13. When handling chemicals, note the hazard code on the bottle and take the appropriate precautions indicated. 14. Do not pour chemicals down the sink. 15. Return all chemicals, reagents, cultures, and glassware to their appropriate places.

1. No food or drinks are permitted in the laboratory at any time. 2. Only closed-toe shoes are to be worn in the laboratory. Sandals are not permitted. 3. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The application of cosmetics in the laboratory is prohibited in the laboratory. 4. Work areas/surfaces must be disinfected before and after use. 5. Laboratory coats must be worn and buttoned while in the laboratory. Laboratory coats should not be worn outside the laboratory. 6. Protective eyewear must be worn when performing any exercise or procedure in the laboratory. 7. Long hair should be secured behind your head. 8. Hands must be washed before leaving the laboratory. 9. All unnecessary books, purses, briefcases, etc., must be kept off the countertops. 10. Never pipette anything by mouth (including water). Always use pipetting devices. 11. Label all materials with your name, date, and any other applicable information (e.g., media, organism, etc.). 12. Dispose of wastes in their proper containers. 13. When handling chemicals, note the hazard code on the bottle and take the appropriate precautions indicated. 14. Do not pour chemicals down the sink. 15. Return all chemicals, reagents, cultures, and glassware to their appropriate places.

12

12

Experiments

Experiments

MICROBIOLOGY LABORATORY SAFETY RULES AND PROCEDURES 1. No food or drinks are permitted in the laboratory at any time. 2. Only closed-toe shoes are to be worn in the laboratory. Sandals are not permitted. 3. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The application of cosmetics in the laboratory is prohibited in the laboratory. 4. Work areas/surfaces must be disinfected before and after use. 5. Laboratory coats must be worn and buttoned while in the laboratory. Laboratory coats should not be worn outside the laboratory. 6. Protective eyewear must be worn when performing any exercise or procedure in the laboratory. 7. Long hair should be secured behind your head. 8. Hands must be washed before leaving the laboratory. 9. All unnecessary books, purses, briefcases, etc., must be kept off the countertops. 10. Never pipette anything by mouth (including water). Always use pipetting devices. 11. Label all materials with your name, date, and any other applicable information (e.g., media, organism, etc.). 12. Dispose of wastes in their proper containers. 13. When handling chemicals, note the hazard code on the bottle and take the appropriate precautions indicated. 14. Do not pour chemicals down the sink. 15. Return all chemicals, reagents, cultures, and glassware to their appropriate places.

MICROBIOLOGY LABORATORY SAFETY RULES AND PROCEDURES 1. No food or drinks are permitted in the laboratory at any time. 2. Only closed-toe shoes are to be worn in the laboratory. Sandals are not permitted. 3. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The application of cosmetics in the laboratory is prohibited in the laboratory. 4. Work areas/surfaces must be disinfected before and after use. 5. Laboratory coats must be worn and buttoned while in the laboratory. Laboratory coats should not be worn outside the laboratory. 6. Protective eyewear must be worn when performing any exercise or procedure in the laboratory. 7. Long hair should be secured behind your head. 8. Hands must be washed before leaving the laboratory. 9. All unnecessary books, purses, briefcases, etc., must be kept off the countertops. 10. Never pipette anything by mouth (including water). Always use pipetting devices. 11. Label all materials with your name, date, and any other applicable information (e.g., media, organism, etc.). 12. Dispose of wastes in their proper containers. 13. When handling chemicals, note the hazard code on the bottle and take the appropriate precautions indicated. 14. Do not pour chemicals down the sink. 15. Return all chemicals, reagents, cultures, and glassware to their appropriate places.

182 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 16. Do not pour biohazardous fluids down the sink. 17. Glassware should be washed with soap and water, and then rinsed with distilled water. 18. Flame transfer loops, wires, or needles before and immediately after use to transfer biological material. 19. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing infectious material. 20. Be careful around Bunsen burners. Flames cannot always been seen. 21. Turn off incinerators before leaving the laboratory. 22. Immediately, report any broken glass, especially those containing infectious materials. 23. If you are injured in the laboratory, immediately contact your course instructor. 24. Any chemical or biological fluid spills must be immediately reported to your course instructor. 25. Follow all instructors given by your course instructor for cleaning up any spills or broken glass. 26. Familiarize yourself with safety equipment in the laboratory and emergency escape routes. 27. Always wipe and clean the lenses of your microscope before putting it away. Use the appropriate tissue paper and cleaning solution for this purpose. 28. Use appropriate universal precautions with all biological fluids. 29. Do not remove any materials from the laboratory without the written permission of the course instructor. 30. Before leaving the lab hands should be washed properly with soap or wiped with ethyl alcohol. 31. Ultraviolet Light. Exposure to ultraviolet (UV) light can cause acute eye irritation. Since the retina cannot detect UV light, you can have serious eye damage and not realize it until 30 minutes to 24 hours after exposure. Therefore, always wear appropriate eye protection when using UV lamps.

182 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 16. Do not pour biohazardous fluids down the sink. 17. Glassware should be washed with soap and water, and then rinsed with distilled water. 18. Flame transfer loops, wires, or needles before and immediately after use to transfer biological material. 19. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing infectious material. 20. Be careful around Bunsen burners. Flames cannot always been seen. 21. Turn off incinerators before leaving the laboratory. 22. Immediately, report any broken glass, especially those containing infectious materials. 23. If you are injured in the laboratory, immediately contact your course instructor. 24. Any chemical or biological fluid spills must be immediately reported to your course instructor. 25. Follow all instructors given by your course instructor for cleaning up any spills or broken glass. 26. Familiarize yourself with safety equipment in the laboratory and emergency escape routes. 27. Always wipe and clean the lenses of your microscope before putting it away. Use the appropriate tissue paper and cleaning solution for this purpose. 28. Use appropriate universal precautions with all biological fluids. 29. Do not remove any materials from the laboratory without the written permission of the course instructor. 30. Before leaving the lab hands should be washed properly with soap or wiped with ethyl alcohol. 31. Ultraviolet Light. Exposure to ultraviolet (UV) light can cause acute eye irritation. Since the retina cannot detect UV light, you can have serious eye damage and not realize it until 30 minutes to 24 hours after exposure. Therefore, always wear appropriate eye protection when using UV lamps.

182 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 16. Do not pour biohazardous fluids down the sink. 17. Glassware should be washed with soap and water, and then rinsed with distilled water. 18. Flame transfer loops, wires, or needles before and immediately after use to transfer biological material. 19. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing infectious material. 20. Be careful around Bunsen burners. Flames cannot always been seen. 21. Turn off incinerators before leaving the laboratory. 22. Immediately, report any broken glass, especially those containing infectious materials. 23. If you are injured in the laboratory, immediately contact your course instructor. 24. Any chemical or biological fluid spills must be immediately reported to your course instructor. 25. Follow all instructors given by your course instructor for cleaning up any spills or broken glass. 26. Familiarize yourself with safety equipment in the laboratory and emergency escape routes. 27. Always wipe and clean the lenses of your microscope before putting it away. Use the appropriate tissue paper and cleaning solution for this purpose. 28. Use appropriate universal precautions with all biological fluids. 29. Do not remove any materials from the laboratory without the written permission of the course instructor. 30. Before leaving the lab hands should be washed properly with soap or wiped with ethyl alcohol. 31. Ultraviolet Light. Exposure to ultraviolet (UV) light can cause acute eye irritation. Since the retina cannot detect UV light, you can have serious eye damage and not realize it until 30 minutes to 24 hours after exposure. Therefore, always wear appropriate eye protection when using UV lamps.

182 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY 16. Do not pour biohazardous fluids down the sink. 17. Glassware should be washed with soap and water, and then rinsed with distilled water. 18. Flame transfer loops, wires, or needles before and immediately after use to transfer biological material. 19. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing infectious material. 20. Be careful around Bunsen burners. Flames cannot always been seen. 21. Turn off incinerators before leaving the laboratory. 22. Immediately, report any broken glass, especially those containing infectious materials. 23. If you are injured in the laboratory, immediately contact your course instructor. 24. Any chemical or biological fluid spills must be immediately reported to your course instructor. 25. Follow all instructors given by your course instructor for cleaning up any spills or broken glass. 26. Familiarize yourself with safety equipment in the laboratory and emergency escape routes. 27. Always wipe and clean the lenses of your microscope before putting it away. Use the appropriate tissue paper and cleaning solution for this purpose. 28. Use appropriate universal precautions with all biological fluids. 29. Do not remove any materials from the laboratory without the written permission of the course instructor. 30. Before leaving the lab hands should be washed properly with soap or wiped with ethyl alcohol. 31. Ultraviolet Light. Exposure to ultraviolet (UV) light can cause acute eye irritation. Since the retina cannot detect UV light, you can have serious eye damage and not realize it until 30 minutes to 24 hours after exposure. Therefore, always wear appropriate eye protection when using UV lamps.

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EXPERIMENT NO. 1

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EXPERIMENT NO. 1

Aim: To study the light (or compound) and electron microscope.

Aim: To study the light (or compound) and electron microscope.

Requirement: Light microscope and electron microscope.

Requirement: Light microscope and electron microscope.

Theory

Theory

Microscope is an instrument which is used to see the microscope objects or particles which are not seen by the necked eyes. e.g. Bacteria – 0.5-10 Pm, Fungi –5-10 Pm, Algae-1 Pm to many feet, Protozoa – 2-200 Pm. Microscope is based on the principle of magnification. Magnification is the property of microscope in which magnify the objects like microorganisms, plant cells, animal cells, etc.

Microscope is an instrument which is used to see the microscope objects or particles which are not seen by the necked eyes. e.g. Bacteria – 0.5-10 Pm, Fungi –5-10 Pm, Algae-1 Pm to many feet, Protozoa – 2-200 Pm. Microscope is based on the principle of magnification. Magnification is the property of microscope in which magnify the objects like microorganisms, plant cells, animal cells, etc.

Types of Microscopes

Types of Microscopes

A. Light or optical or compound microscope. B. Electron microscope.

A. Light or optical or compound microscope. B. Electron microscope.

A. Light or compound microscope: Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

A. Light or compound microscope: Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

Bright Field Microscopy

Bright Field Microscopy

In this, the microscopic field (the area where slide is placed) is brightly lighted and the micro-organisms appear dark because they absorb some of the light. Generally, microorganisms do not absorb much light but staining them with a dye greatly increases their light absorbing ability. Magnification: The total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of eye piece. Resolving power: It is the ability to separate the two or more adjacent objects or points. R.P = Wave length of light in (Pm) / 2 X numerical aperture of the objective lens. Numerical Aperture: The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

In this, the microscopic field (the area where slide is placed) is brightly lighted and the micro-organisms appear dark because they absorb some of the light. Generally, microorganisms do not absorb much light but staining them with a dye greatly increases their light absorbing ability. Magnification: The total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of eye piece. Resolving power: It is the ability to separate the two or more adjacent objects or points. R.P = Wave length of light in (Pm) / 2 X numerical aperture of the objective lens. Numerical Aperture: The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

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EXPERIMENT NO. 1

183

EXPERIMENT NO. 1

Aim: To study the light (or compound) and electron microscope.

Aim: To study the light (or compound) and electron microscope.

Requirement: Light microscope and electron microscope.

Requirement: Light microscope and electron microscope.

Theory

Theory

Microscope is an instrument which is used to see the microscope objects or particles which are not seen by the necked eyes. e.g. Bacteria – 0.5-10 Pm, Fungi –5-10 Pm, Algae-1 Pm to many feet, Protozoa – 2-200 Pm. Microscope is based on the principle of magnification. Magnification is the property of microscope in which magnify the objects like microorganisms, plant cells, animal cells, etc.

Microscope is an instrument which is used to see the microscope objects or particles which are not seen by the necked eyes. e.g. Bacteria – 0.5-10 Pm, Fungi –5-10 Pm, Algae-1 Pm to many feet, Protozoa – 2-200 Pm. Microscope is based on the principle of magnification. Magnification is the property of microscope in which magnify the objects like microorganisms, plant cells, animal cells, etc.

Types of Microscopes

Types of Microscopes

A. Light or optical or compound microscope. B. Electron microscope.

A. Light or optical or compound microscope. B. Electron microscope.

A. Light or compound microscope: Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

A. Light or compound microscope: Based on the principle of magnification (means small object appear enlarge) light microscopy are different types: 1. Bright field microscopy 2. Dark field microscopy 3. Fluorescence microscopy 4. Phase contrast microscopy.

Bright Field Microscopy

Bright Field Microscopy

In this, the microscopic field (the area where slide is placed) is brightly lighted and the micro-organisms appear dark because they absorb some of the light. Generally, microorganisms do not absorb much light but staining them with a dye greatly increases their light absorbing ability. Magnification: The total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of eye piece. Resolving power: It is the ability to separate the two or more adjacent objects or points. R.P = Wave length of light in (Pm) / 2 X numerical aperture of the objective lens. Numerical Aperture: The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

In this, the microscopic field (the area where slide is placed) is brightly lighted and the micro-organisms appear dark because they absorb some of the light. Generally, microorganisms do not absorb much light but staining them with a dye greatly increases their light absorbing ability. Magnification: The total magnification of the microscope is determined as magnifying power of the objective lens multiplying by the magnifying power of eye piece. Resolving power: It is the ability to separate the two or more adjacent objects or points. R.P = Wave length of light in (Pm) / 2 X numerical aperture of the objective lens. Numerical Aperture: The angle T subtended by the optical axis and outermost rays still covered by the objective is to measure the aperture of objective, it is the half aperture angle. The magnitude is expressed as sine value. The numerical aperture is calculated as: refractive index of the medium is multiplied by the sine value of half aperture angle.

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184 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 12.1. Structure of microscope.

Fig. 12.1. Structure of microscope.

N.A = n sinT

N.A = n sinT

Where n = refractive index For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.58. Limit of resolution: It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture.

Where n = refractive index For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.58. Limit of resolution: It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture.

B. Electron microscope: A beam of electrons are used in electron microscope in place of light wave to produce the image specimen. It can be of two types: Transmission electron microscope and scanning electron microscope. a. Transmission electron microscope (TEM): The electron microscope provides tremendous useful magnification because of the much higher resolution obtainable with the extremely short wavelength of the electron beam used to magnify the specimen. In electron microscopy electron beams and magnetic field are used to produce the images. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to X 400,000.

B. Electron microscope: A beam of electrons are used in electron microscope in place of light wave to produce the image specimen. It can be of two types: Transmission electron microscope and scanning electron microscope. a. Transmission electron microscope (TEM): The electron microscope provides tremendous useful magnification because of the much higher resolution obtainable with the extremely short wavelength of the electron beam used to magnify the specimen. In electron microscopy electron beams and magnetic field are used to produce the images. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to X 400,000.

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184 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 12.1. Structure of microscope.

Fig. 12.1. Structure of microscope.

N.A = n sinT

N.A = n sinT

Where n = refractive index For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.58. Limit of resolution: It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture.

Where n = refractive index For the dry objectives the value of n is 1, since 1 is refractive index of air. When immersion oil is used as the medium n is 1.58. Limit of resolution: It is the smallest distance by which two objects can be separated and can be distinguished as two separate objects. The greatest resolution in light microscope is obtained with the shortest wavelength of visible light and an objective with maximum numerical aperture.

B. Electron microscope: A beam of electrons are used in electron microscope in place of light wave to produce the image specimen. It can be of two types: Transmission electron microscope and scanning electron microscope. a. Transmission electron microscope (TEM): The electron microscope provides tremendous useful magnification because of the much higher resolution obtainable with the extremely short wavelength of the electron beam used to magnify the specimen. In electron microscopy electron beams and magnetic field are used to produce the images. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to X 400,000.

B. Electron microscope: A beam of electrons are used in electron microscope in place of light wave to produce the image specimen. It can be of two types: Transmission electron microscope and scanning electron microscope. a. Transmission electron microscope (TEM): The electron microscope provides tremendous useful magnification because of the much higher resolution obtainable with the extremely short wavelength of the electron beam used to magnify the specimen. In electron microscopy electron beams and magnetic field are used to produce the images. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to X 400,000.

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b. Scanning electron microscope (SEM): In this type of microscope the narrow beam of electrons rapidly move over the surface of the specimen. This cause the release of a shower of secondary electrons and other type of radiations over the surface of specimen. The intensity of these secondary electrons depends upon the chemical composition of the irradiate objects. The secondary electrons are collected by a detector which generates an electronic signal. These signals are than scanned in the manner of a television system to produce an image on a cathode ray tube.

185

b. Scanning electron microscope (SEM): In this type of microscope the narrow beam of electrons rapidly move over the surface of the specimen. This cause the release of a shower of secondary electrons and other type of radiations over the surface of specimen. The intensity of these secondary electrons depends upon the chemical composition of the irradiate objects. The secondary electrons are collected by a detector which generates an electronic signal. These signals are than scanned in the manner of a television system to produce an image on a cathode ray tube.

Difference between light and electron microscope:

Difference between light and electron microscope:

S.No.

Light Microscope

Electron Microscope

S.No.

Light Microscope

Electron Microscope

1

It is used to study the general structure of cells.

It is used to study the ultra-structure of cells.

1

It is used to study the general structure of cells.

It is used to study the ultra-structure of cells.

2.

Source of illumination is visible light

Source of illumination is narrow electron beams.

2.

Source of illumination is visible light

Source of illumination is narrow electron beams.

3.

Glass lens are used for magnification.

High power electromagnets are used.

3.

Glass lens are used for magnification.

High power electromagnets are used.

4.

Living and dead organisms can be observed directly by human eye.

Only dead organisms are examined. Images cannot be seen by human eyes directly.

4.

Living and dead organisms can be observed directly by human eye.

Only dead organisms are examined. Images cannot be seen by human eyes directly.

5.

Resolving power is 2500Å

Resolving power is 10Å

5.

Resolving power is 2500Å

Resolving power is 10Å

6.

It can magnify objects up to 1000 times. It can magnify objects up to 3,00,000 times.

6.

It can magnify objects up to 1000 times. It can magnify objects up to 3,00,000 times.

Note: For detail study, refer the chapter 4 under section microscopy.

EXPERIMENTS

Note: For detail study, refer the chapter 4 under section microscopy.

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EXPERIMENTS

b. Scanning electron microscope (SEM): In this type of microscope the narrow beam of electrons rapidly move over the surface of the specimen. This cause the release of a shower of secondary electrons and other type of radiations over the surface of specimen. The intensity of these secondary electrons depends upon the chemical composition of the irradiate objects. The secondary electrons are collected by a detector which generates an electronic signal. These signals are than scanned in the manner of a television system to produce an image on a cathode ray tube.

185

b. Scanning electron microscope (SEM): In this type of microscope the narrow beam of electrons rapidly move over the surface of the specimen. This cause the release of a shower of secondary electrons and other type of radiations over the surface of specimen. The intensity of these secondary electrons depends upon the chemical composition of the irradiate objects. The secondary electrons are collected by a detector which generates an electronic signal. These signals are than scanned in the manner of a television system to produce an image on a cathode ray tube.

Difference between light and electron microscope:

Difference between light and electron microscope:

S.No.

Light Microscope

Electron Microscope

S.No.

Light Microscope

Electron Microscope

1

It is used to study the general structure of cells.

It is used to study the ultra-structure of cells.

1

It is used to study the general structure of cells.

It is used to study the ultra-structure of cells.

2.

Source of illumination is visible light

Source of illumination is narrow electron beams.

2.

Source of illumination is visible light

Source of illumination is narrow electron beams.

3.

Glass lens are used for magnification.

High power electromagnets are used.

3.

Glass lens are used for magnification.

High power electromagnets are used.

4.

Living and dead organisms can be observed directly by human eye.

Only dead organisms are examined. Images cannot be seen by human eyes directly.

4.

Living and dead organisms can be observed directly by human eye.

Only dead organisms are examined. Images cannot be seen by human eyes directly.

5.

Resolving power is 2500Å

Resolving power is 10Å

5.

Resolving power is 2500Å

Resolving power is 10Å

6.

It can magnify objects up to 1000 times. It can magnify objects up to 3,00,000 times.

6.

It can magnify objects up to 1000 times. It can magnify objects up to 3,00,000 times.

Note: For detail study, refer the chapter 4 under section microscopy.

Note: For detail study, refer the chapter 4 under section microscopy.

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EXPERIMENT NO. 2

186 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 2

Aim: To study the different instruments used in microbiology lab.

Aim: To study the different instruments used in microbiology lab.

Requirements: Inoculation needle and inoculation loop, Bunsen burner (Spirit lamp), water bath, autoclave, laminar air flow, incubator, hot air oven, quebec colony counter, pH meter, spectrophotometer, centrifuge, microscope etc.

Requirements: Inoculation needle and inoculation loop, Bunsen burner (Spirit lamp), water bath, autoclave, laminar air flow, incubator, hot air oven, quebec colony counter, pH meter, spectrophotometer, centrifuge, microscope etc.

Inoculation needle and inoculation loop

Inoculation needle and inoculation loop

It is made up of a long platinum wire fixed into a metallic rod. The wire loop has a handle with steel screw shaft in which platinum wire is to be fitted. At the top of the needle there is a small circular loop which is helping in transferring the culture media. Both are sterilized by using Bunsen burner/spirit lamp till the needle or loop becomes red hot. The loop and wire also used for picking small quantities of solid material from a microbial colony, and can be used to inoculate either a liquid or solid medium.

It is made up of a long platinum wire fixed into a metallic rod. The wire loop has a handle with steel screw shaft in which platinum wire is to be fitted. At the top of the needle there is a small circular loop which is helping in transferring the culture media. Both are sterilized by using Bunsen burner/spirit lamp till the needle or loop becomes red hot. The loop and wire also used for picking small quantities of solid material from a microbial colony, and can be used to inoculate either a liquid or solid medium.

Fig. 12.2. Structure of inoculum needle and loops.

Fig. 12.2. Structure of inoculum needle and loops.

Bunsen burner or spirit lamp

Bunsen burner or spirit lamp

The basic device for providing heat in the laboratory experiment is the Bunsen burner. It is also called spirit lamp. The gas enters the burner at the base, and its supply is regulated externally by the gas cock, as the gas streams upwards through a get inside the base, air is pulled in through the air-intake holes just above the base. The amount of air can be controlled by rotating a sleeve that fits over holes in the body of the burner. When the air supply to the burner is increased, the flame blows out or detaches itself from the burner tip. It is also used during transfer and purification of microbial cultures. Sterilization of tools by using spirit lamp is called incineration.

The basic device for providing heat in the laboratory experiment is the Bunsen burner. It is also called spirit lamp. The gas enters the burner at the base, and its supply is regulated externally by the gas cock, as the gas streams upwards through a get inside the base, air is pulled in through the air-intake holes just above the base. The amount of air can be controlled by rotating a sleeve that fits over holes in the body of the burner. When the air supply to the burner is increased, the flame blows out or detaches itself from the burner tip. It is also used during transfer and purification of microbial cultures. Sterilization of tools by using spirit lamp is called incineration.

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186 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 2

EXPERIMENT NO. 2

Aim: To study the different instruments used in microbiology lab.

Aim: To study the different instruments used in microbiology lab.

Requirements: Inoculation needle and inoculation loop, Bunsen burner (Spirit lamp), water bath, autoclave, laminar air flow, incubator, hot air oven, quebec colony counter, pH meter, spectrophotometer, centrifuge, microscope etc.

Requirements: Inoculation needle and inoculation loop, Bunsen burner (Spirit lamp), water bath, autoclave, laminar air flow, incubator, hot air oven, quebec colony counter, pH meter, spectrophotometer, centrifuge, microscope etc.

Inoculation needle and inoculation loop

Inoculation needle and inoculation loop

It is made up of a long platinum wire fixed into a metallic rod. The wire loop has a handle with steel screw shaft in which platinum wire is to be fitted. At the top of the needle there is a small circular loop which is helping in transferring the culture media. Both are sterilized by using Bunsen burner/spirit lamp till the needle or loop becomes red hot. The loop and wire also used for picking small quantities of solid material from a microbial colony, and can be used to inoculate either a liquid or solid medium.

It is made up of a long platinum wire fixed into a metallic rod. The wire loop has a handle with steel screw shaft in which platinum wire is to be fitted. At the top of the needle there is a small circular loop which is helping in transferring the culture media. Both are sterilized by using Bunsen burner/spirit lamp till the needle or loop becomes red hot. The loop and wire also used for picking small quantities of solid material from a microbial colony, and can be used to inoculate either a liquid or solid medium.

Fig. 12.2. Structure of inoculum needle and loops.

Fig. 12.2. Structure of inoculum needle and loops.

Bunsen burner or spirit lamp

Bunsen burner or spirit lamp

The basic device for providing heat in the laboratory experiment is the Bunsen burner. It is also called spirit lamp. The gas enters the burner at the base, and its supply is regulated externally by the gas cock, as the gas streams upwards through a get inside the base, air is pulled in through the air-intake holes just above the base. The amount of air can be controlled by rotating a sleeve that fits over holes in the body of the burner. When the air supply to the burner is increased, the flame blows out or detaches itself from the burner tip. It is also used during transfer and purification of microbial cultures. Sterilization of tools by using spirit lamp is called incineration.

The basic device for providing heat in the laboratory experiment is the Bunsen burner. It is also called spirit lamp. The gas enters the burner at the base, and its supply is regulated externally by the gas cock, as the gas streams upwards through a get inside the base, air is pulled in through the air-intake holes just above the base. The amount of air can be controlled by rotating a sleeve that fits over holes in the body of the burner. When the air supply to the burner is increased, the flame blows out or detaches itself from the burner tip. It is also used during transfer and purification of microbial cultures. Sterilization of tools by using spirit lamp is called incineration.

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Water bath

Water bath

It is an instrument that is used to provide constant temperature to a sample. It consists of an insulating box made of steel fitted with electrode heating coil. The temperature is controlled by thermostat. In water bath the plate form rotates, then it is called water bath shaker.

It is an instrument that is used to provide constant temperature to a sample. It consists of an insulating box made of steel fitted with electrode heating coil. The temperature is controlled by thermostat. In water bath the plate form rotates, then it is called water bath shaker.

Autoclave

Autoclave

Fig. 12.3. Structure of autoclave.

Fig. 12.3. Structure of autoclave.

Autoclave/pressure cooker is made up of gun metal sheets which are supported in an iron case. The steam passes from below at the base. The side walls are heated by the steam jacket. The pressure is regulated by pressure meter. It consists of safety valve that guard against the accidents. It is usually operated at 15 lb/inch2 steam pressure for 20 minutes at 120°C in order to kill the spores. The relationship between temperature and pressure is as:

Autoclave/pressure cooker is made up of gun metal sheets which are supported in an iron case. The steam passes from below at the base. The side walls are heated by the steam jacket. The pressure is regulated by pressure meter. It consists of safety valve that guard against the accidents. It is usually operated at 15 lb/inch2 steam pressure for 20 minutes at 120°C in order to kill the spores. The relationship between temperature and pressure is as:

EXPERIMENTS

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EXPERIMENTS

187

Water bath

Water bath

It is an instrument that is used to provide constant temperature to a sample. It consists of an insulating box made of steel fitted with electrode heating coil. The temperature is controlled by thermostat. In water bath the plate form rotates, then it is called water bath shaker.

It is an instrument that is used to provide constant temperature to a sample. It consists of an insulating box made of steel fitted with electrode heating coil. The temperature is controlled by thermostat. In water bath the plate form rotates, then it is called water bath shaker.

Autoclave

Autoclave

Fig. 12.3. Structure of autoclave.

Fig. 12.3. Structure of autoclave.

Autoclave/pressure cooker is made up of gun metal sheets which are supported in an iron case. The steam passes from below at the base. The side walls are heated by the steam jacket. The pressure is regulated by pressure meter. It consists of safety valve that guard against the accidents. It is usually operated at 15 lb/inch2 steam pressure for 20 minutes at 120°C in order to kill the spores. The relationship between temperature and pressure is as:

Autoclave/pressure cooker is made up of gun metal sheets which are supported in an iron case. The steam passes from below at the base. The side walls are heated by the steam jacket. The pressure is regulated by pressure meter. It consists of safety valve that guard against the accidents. It is usually operated at 15 lb/inch2 steam pressure for 20 minutes at 120°C in order to kill the spores. The relationship between temperature and pressure is as:

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188 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Pressure (lb/inch2)

Temperature (°C)

Pressure (lb/inch2)

Temperature (°C)

Pressure (lb/inch2)

Temperature (°C)

Pressure (lb/inch2)

Temperature (°C)

00 05 10 15

100 109 115.5 121.5

20 25 30 40

126.5 130.5 135.5 141.5

00 05 10 15

100 109 115.5 121.5

20 25 30 40

126.5 130.5 135.5 141.5

Precautions: The level of water in the autoclave should be checked before operating. The water level should be above the heating rod or wire. Air should be completely evacuated and the steam must have access to the materials to be sterilized. The heat sensitive material should not be sterilized by autoclave.

Precautions: The level of water in the autoclave should be checked before operating. The water level should be above the heating rod or wire. Air should be completely evacuated and the steam must have access to the materials to be sterilized. The heat sensitive material should not be sterilized by autoclave.

Laminar Air Flow

Laminar Air Flow

It is an apparatus consists of an air blower in the rear side of the chamber which can produce air flow with uniform velocity along parallel flow lines. It constitutes HEPA filters which mainly remove particles as small as 0.20 —m. It is based on flow of air current of uniform velocity along parallel flow lines which help in transferring microbial cultures in aseptic conditions. Air is passed through the filters into enclosure and the filter does not allow any kind of microbe to enter into the system. Two tubes, one fluorescent and UV tubes are

It is an apparatus consists of an air blower in the rear side of the chamber which can produce air flow with uniform velocity along parallel flow lines. It constitutes HEPA filters which mainly remove particles as small as 0.20 —m. It is based on flow of air current of uniform velocity along parallel flow lines which help in transferring microbial cultures in aseptic conditions. Air is passed through the filters into enclosure and the filter does not allow any kind of microbe to enter into the system. Two tubes, one fluorescent and UV tubes are

Fig. 12.4. Structure of horizontal laminar air flow.

Fig. 12.4. Structure of horizontal laminar air flow.

188 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

188 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Pressure (lb/inch2)

Temperature (°C)

Pressure (lb/inch2)

Temperature (°C)

Pressure (lb/inch2)

Temperature (°C)

Pressure (lb/inch2)

Temperature (°C)

00 05 10 15

100 109 115.5 121.5

20 25 30 40

126.5 130.5 135.5 141.5

00 05 10 15

100 109 115.5 121.5

20 25 30 40

126.5 130.5 135.5 141.5

Precautions: The level of water in the autoclave should be checked before operating. The water level should be above the heating rod or wire. Air should be completely evacuated and the steam must have access to the materials to be sterilized. The heat sensitive material should not be sterilized by autoclave.

Precautions: The level of water in the autoclave should be checked before operating. The water level should be above the heating rod or wire. Air should be completely evacuated and the steam must have access to the materials to be sterilized. The heat sensitive material should not be sterilized by autoclave.

Laminar Air Flow

Laminar Air Flow

It is an apparatus consists of an air blower in the rear side of the chamber which can produce air flow with uniform velocity along parallel flow lines. It constitutes HEPA filters which mainly remove particles as small as 0.20 —m. It is based on flow of air current of uniform velocity along parallel flow lines which help in transferring microbial cultures in aseptic conditions. Air is passed through the filters into enclosure and the filter does not allow any kind of microbe to enter into the system. Two tubes, one fluorescent and UV tubes are

It is an apparatus consists of an air blower in the rear side of the chamber which can produce air flow with uniform velocity along parallel flow lines. It constitutes HEPA filters which mainly remove particles as small as 0.20 —m. It is based on flow of air current of uniform velocity along parallel flow lines which help in transferring microbial cultures in aseptic conditions. Air is passed through the filters into enclosure and the filter does not allow any kind of microbe to enter into the system. Two tubes, one fluorescent and UV tubes are

Fig. 12.4. Structure of horizontal laminar air flow.

Fig. 12.4. Structure of horizontal laminar air flow.

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fitted, two switch for these tubes and a separate switch for regulation of air the air flow are fitted outside the apparatus. Due to uniform velocity and parallel flow of air current, pouring of media, plating, slant preparations, streaking etc. without any kind of contamination are performed. Initially dust particles are removed with the help of smooth cloth or cotton containing alcohol, switch on the UV light for the period of 15 minutes, so as to kill the germs in the working place. The front cover sheet of laminar flow is opened to keep the desired material inside. Sit properly in the front of chamber again and wipe the working place with alcohol to reduce contamination. All the work related to pouring, plating, streaking etc. are to be carried out in the flame zone of the spirit lamp.

fitted, two switch for these tubes and a separate switch for regulation of air the air flow are fitted outside the apparatus. Due to uniform velocity and parallel flow of air current, pouring of media, plating, slant preparations, streaking etc. without any kind of contamination are performed. Initially dust particles are removed with the help of smooth cloth or cotton containing alcohol, switch on the UV light for the period of 15 minutes, so as to kill the germs in the working place. The front cover sheet of laminar flow is opened to keep the desired material inside. Sit properly in the front of chamber again and wipe the working place with alcohol to reduce contamination. All the work related to pouring, plating, streaking etc. are to be carried out in the flame zone of the spirit lamp.

Precautions

Precautions

1. Put off the shoes before entering to operate the apparatus. 2. Wash the hands with detergent. 3. Should not talk and sneeze inside the chamber while performing the microbial culture transfer. 4. Switch on the U.V. light only for 10-15 minutes before starting the work. U.V. light may cause dangerous effects on your body.

Incubator

1. Put off the shoes before entering to operate the apparatus. 2. Wash the hands with detergent. 3. Should not talk and sneeze inside the chamber while performing the microbial culture transfer. 4. Switch on the U.V. light only for 10-15 minutes before starting the work. U.V. light may cause dangerous effects on your body.

Incubator

Fig. 12.5. Structure of incubator.

Fig. 12.5. Structure of incubator.

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fitted, two switch for these tubes and a separate switch for regulation of air the air flow are fitted outside the apparatus. Due to uniform velocity and parallel flow of air current, pouring of media, plating, slant preparations, streaking etc. without any kind of contamination are performed. Initially dust particles are removed with the help of smooth cloth or cotton containing alcohol, switch on the UV light for the period of 15 minutes, so as to kill the germs in the working place. The front cover sheet of laminar flow is opened to keep the desired material inside. Sit properly in the front of chamber again and wipe the working place with alcohol to reduce contamination. All the work related to pouring, plating, streaking etc. are to be carried out in the flame zone of the spirit lamp.

fitted, two switch for these tubes and a separate switch for regulation of air the air flow are fitted outside the apparatus. Due to uniform velocity and parallel flow of air current, pouring of media, plating, slant preparations, streaking etc. without any kind of contamination are performed. Initially dust particles are removed with the help of smooth cloth or cotton containing alcohol, switch on the UV light for the period of 15 minutes, so as to kill the germs in the working place. The front cover sheet of laminar flow is opened to keep the desired material inside. Sit properly in the front of chamber again and wipe the working place with alcohol to reduce contamination. All the work related to pouring, plating, streaking etc. are to be carried out in the flame zone of the spirit lamp.

Precautions

Precautions

1. Put off the shoes before entering to operate the apparatus. 2. Wash the hands with detergent. 3. Should not talk and sneeze inside the chamber while performing the microbial culture transfer. 4. Switch on the U.V. light only for 10-15 minutes before starting the work. U.V. light may cause dangerous effects on your body.

Incubator

1. Put off the shoes before entering to operate the apparatus. 2. Wash the hands with detergent. 3. Should not talk and sneeze inside the chamber while performing the microbial culture transfer. 4. Switch on the U.V. light only for 10-15 minutes before starting the work. U.V. light may cause dangerous effects on your body.

Incubator

Fig. 12.5. Structure of incubator.

Fig. 12.5. Structure of incubator.

190 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

190 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

An incubator is an instrument that consists of steel/copper chamber around which warm water or air is circulated by electric current. The temperature of the incubator is constant due to its control by using thermostat. It is made up of double walled chamber adjacent to desired temperature. It is done by using an external knob controlling the thermostat system. Temperature greatly influenced the microbial growth. Therefore instrument is generally designed that can allow the desired microorganisms to grow at a particular temperature. Small square type incubators are better than large ones.

An incubator is an instrument that consists of steel/copper chamber around which warm water or air is circulated by electric current. The temperature of the incubator is constant due to its control by using thermostat. It is made up of double walled chamber adjacent to desired temperature. It is done by using an external knob controlling the thermostat system. Temperature greatly influenced the microbial growth. Therefore instrument is generally designed that can allow the desired microorganisms to grow at a particular temperature. Small square type incubators are better than large ones.

Precaution

Precaution

The door of the incubator should be opened only when necessary.

The door of the incubator should be opened only when necessary.

Fig. 12.6. Structure of (a) closed position and (b)open position hot air oven.

Fig. 12.6. Structure of (a) closed position and (b)open position hot air oven.

Hot air oven

Hot air oven

It consists of double-walled chamber; the gap between two walls is insulated. It is heated from below by using the electric current and heating elements. Hot air oven is generally used for sterilization of glassware, metal devices and other articles which are spoiled by autoclaving. For such purpose dry heat sterilization is used. It works on the principle of killing the microbes by oxidation. For sterilization the holding time depends upon the temperature and 160 degrees for 1 hour is considered to be suitable. The air inside is circulated by fan to ensure that all parts are kept at required temperature.

It consists of double-walled chamber; the gap between two walls is insulated. It is heated from below by using the electric current and heating elements. Hot air oven is generally used for sterilization of glassware, metal devices and other articles which are spoiled by autoclaving. For such purpose dry heat sterilization is used. It works on the principle of killing the microbes by oxidation. For sterilization the holding time depends upon the temperature and 160 degrees for 1 hour is considered to be suitable. The air inside is circulated by fan to ensure that all parts are kept at required temperature.

Quebec colony counter

Quebec colony counter

It is a device for counting the small or closely growing colonies of bacteria or other microorganisms on the surface of media. Lens is fitted on the box and

It is a device for counting the small or closely growing colonies of bacteria or other microorganisms on the surface of media. Lens is fitted on the box and

190 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

190 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

An incubator is an instrument that consists of steel/copper chamber around which warm water or air is circulated by electric current. The temperature of the incubator is constant due to its control by using thermostat. It is made up of double walled chamber adjacent to desired temperature. It is done by using an external knob controlling the thermostat system. Temperature greatly influenced the microbial growth. Therefore instrument is generally designed that can allow the desired microorganisms to grow at a particular temperature. Small square type incubators are better than large ones.

An incubator is an instrument that consists of steel/copper chamber around which warm water or air is circulated by electric current. The temperature of the incubator is constant due to its control by using thermostat. It is made up of double walled chamber adjacent to desired temperature. It is done by using an external knob controlling the thermostat system. Temperature greatly influenced the microbial growth. Therefore instrument is generally designed that can allow the desired microorganisms to grow at a particular temperature. Small square type incubators are better than large ones.

Precaution

Precaution

The door of the incubator should be opened only when necessary.

The door of the incubator should be opened only when necessary.

Fig. 12.6. Structure of (a) closed position and (b)open position hot air oven.

Fig. 12.6. Structure of (a) closed position and (b)open position hot air oven.

Hot air oven

Hot air oven

It consists of double-walled chamber; the gap between two walls is insulated. It is heated from below by using the electric current and heating elements. Hot air oven is generally used for sterilization of glassware, metal devices and other articles which are spoiled by autoclaving. For such purpose dry heat sterilization is used. It works on the principle of killing the microbes by oxidation. For sterilization the holding time depends upon the temperature and 160 degrees for 1 hour is considered to be suitable. The air inside is circulated by fan to ensure that all parts are kept at required temperature.

It consists of double-walled chamber; the gap between two walls is insulated. It is heated from below by using the electric current and heating elements. Hot air oven is generally used for sterilization of glassware, metal devices and other articles which are spoiled by autoclaving. For such purpose dry heat sterilization is used. It works on the principle of killing the microbes by oxidation. For sterilization the holding time depends upon the temperature and 160 degrees for 1 hour is considered to be suitable. The air inside is circulated by fan to ensure that all parts are kept at required temperature.

Quebec colony counter

Quebec colony counter

It is a device for counting the small or closely growing colonies of bacteria or other microorganisms on the surface of media. Lens is fitted on the box and

It is a device for counting the small or closely growing colonies of bacteria or other microorganisms on the surface of media. Lens is fitted on the box and

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can be adjusted to see the colonies. Petri plate is kept on a slanting platform meant for it and illuminated with the help of light source from beneath.

can be adjusted to see the colonies. Petri plate is kept on a slanting platform meant for it and illuminated with the help of light source from beneath.

pH meter

pH meter

Fig. 12.7. Structure of pH meter.

Fig. 12.7. Structure of pH meter.

pH can be defined as a negative log of hydrogen ions concentration. It is the degree of acidity and alkalinity of a solution on a scale 1 to 14. pH values 1-7 show the acid values, pH -7 neutrality and pH 7-14 alkalinity. pH of water is 7 at 25 degrees. The acid is proton donor and base is the proton acceptor i.e. acid dissociates and protects H+ concentration. A normal pH meter consists of a glass electrode made up of thin glass membrane which is selectively permeable to H+ concentration.

pH can be defined as a negative log of hydrogen ions concentration. It is the degree of acidity and alkalinity of a solution on a scale 1 to 14. pH values 1-7 show the acid values, pH -7 neutrality and pH 7-14 alkalinity. pH of water is 7 at 25 degrees. The acid is proton donor and base is the proton acceptor i.e. acid dissociates and protects H+ concentration. A normal pH meter consists of a glass electrode made up of thin glass membrane which is selectively permeable to H+ concentration.

Balance

Balance

Accurate amount of chemical can be weighed by using a balance. There are several types of balances for weighing such as single pan, chemical or analytical. Electrical balances are mainly used which works in the presence of electricity that shows digital display of weights. The material to be weighed is placed on the pan of balance and required counter weights are removed by using knobs and the digital scale moves down and up.

Accurate amount of chemical can be weighed by using a balance. There are several types of balances for weighing such as single pan, chemical or analytical. Electrical balances are mainly used which works in the presence of electricity that shows digital display of weights. The material to be weighed is placed on the pan of balance and required counter weights are removed by using knobs and the digital scale moves down and up.

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can be adjusted to see the colonies. Petri plate is kept on a slanting platform meant for it and illuminated with the help of light source from beneath.

can be adjusted to see the colonies. Petri plate is kept on a slanting platform meant for it and illuminated with the help of light source from beneath.

pH meter

pH meter

Fig. 12.7. Structure of pH meter.

Fig. 12.7. Structure of pH meter.

pH can be defined as a negative log of hydrogen ions concentration. It is the degree of acidity and alkalinity of a solution on a scale 1 to 14. pH values 1-7 show the acid values, pH -7 neutrality and pH 7-14 alkalinity. pH of water is 7 at 25 degrees. The acid is proton donor and base is the proton acceptor i.e. acid dissociates and protects H+ concentration. A normal pH meter consists of a glass electrode made up of thin glass membrane which is selectively permeable to H+ concentration.

pH can be defined as a negative log of hydrogen ions concentration. It is the degree of acidity and alkalinity of a solution on a scale 1 to 14. pH values 1-7 show the acid values, pH -7 neutrality and pH 7-14 alkalinity. pH of water is 7 at 25 degrees. The acid is proton donor and base is the proton acceptor i.e. acid dissociates and protects H+ concentration. A normal pH meter consists of a glass electrode made up of thin glass membrane which is selectively permeable to H+ concentration.

Balance

Balance

Accurate amount of chemical can be weighed by using a balance. There are several types of balances for weighing such as single pan, chemical or analytical. Electrical balances are mainly used which works in the presence of electricity that shows digital display of weights. The material to be weighed is placed on the pan of balance and required counter weights are removed by using knobs and the digital scale moves down and up.

Accurate amount of chemical can be weighed by using a balance. There are several types of balances for weighing such as single pan, chemical or analytical. Electrical balances are mainly used which works in the presence of electricity that shows digital display of weights. The material to be weighed is placed on the pan of balance and required counter weights are removed by using knobs and the digital scale moves down and up.

192 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

192 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 12.8. Structure of balance.

Fig. 12.8. Structure of balance.

Spectrophotometer

Spectrophotometer

It is an instrument that utilizes light as a source of radiation and measures changes in optical density or absorbance.

It is an instrument that utilizes light as a source of radiation and measures changes in optical density or absorbance.

Fig. 12.9. Structure of UV-Visible spectrophotometer.

Fig. 12.9. Structure of UV-Visible spectrophotometer.

192 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

192 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 12.8. Structure of balance.

Fig. 12.8. Structure of balance.

Spectrophotometer

Spectrophotometer

It is an instrument that utilizes light as a source of radiation and measures changes in optical density or absorbance.

It is an instrument that utilizes light as a source of radiation and measures changes in optical density or absorbance.

Fig. 12.9. Structure of UV-Visible spectrophotometer.

Fig. 12.9. Structure of UV-Visible spectrophotometer.

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It has three basic principles: (a) Sources of radiation, (b) A unit for dispensing radiation at different wavelengths. (c) A device that detect the amount of radiation at different wavelengths. It uses monochromatic light. It is of many types: a. Visible range spectrophotometer (ranging from 400 Pm to 1000 Pm). b. UV visible spectrophotometer – it utilizes 200 to 1000 Pm wavelength of light. c. IR spectrophotometer: It’s ranging from 1000 Pm – 3000 Pm

It has three basic principles: (a) Sources of radiation, (b) A unit for dispensing radiation at different wavelengths. (c) A device that detect the amount of radiation at different wavelengths. It uses monochromatic light. It is of many types: a. Visible range spectrophotometer (ranging from 400 Pm to 1000 Pm). b. UV visible spectrophotometer – it utilizes 200 to 1000 Pm wavelength of light. c. IR spectrophotometer: It’s ranging from 1000 Pm – 3000 Pm

Centrifuge

Centrifuge

Fig. 12.10. Structure of small centrifuge.

Fig. 12.10. Structure of small centrifuge.

A centrifuge is a piece of equipment, generally driven by an electric motor that puts an object in rotation around a fixed axis applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes more dense substances to separate out along the radial direction (the bottom of the tube) and the lighter objects will tend to move to the top of the tube in the rotating picture. In the picture shown, the rotating unit, called the rotor, has fixed holes drilled at an angle (to the vertical). Test tubes are placed in these slots and the rotor is spun. As the centrifugal force is in the horizontal plane and the tubes are fixed at an angle, the particles have to travel only a little distance before they hit the wall and drop down to the bottom. These angle rotors are very popular in the lab for routine use.

A centrifuge is a piece of equipment, generally driven by an electric motor that puts an object in rotation around a fixed axis applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes more dense substances to separate out along the radial direction (the bottom of the tube) and the lighter objects will tend to move to the top of the tube in the rotating picture. In the picture shown, the rotating unit, called the rotor, has fixed holes drilled at an angle (to the vertical). Test tubes are placed in these slots and the rotor is spun. As the centrifugal force is in the horizontal plane and the tubes are fixed at an angle, the particles have to travel only a little distance before they hit the wall and drop down to the bottom. These angle rotors are very popular in the lab for routine use.

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It has three basic principles: (a) Sources of radiation, (b) A unit for dispensing radiation at different wavelengths. (c) A device that detect the amount of radiation at different wavelengths. It uses monochromatic light. It is of many types: a. Visible range spectrophotometer (ranging from 400 Pm to 1000 Pm). b. UV visible spectrophotometer – it utilizes 200 to 1000 Pm wavelength of light. c. IR spectrophotometer: It’s ranging from 1000 Pm – 3000 Pm

It has three basic principles: (a) Sources of radiation, (b) A unit for dispensing radiation at different wavelengths. (c) A device that detect the amount of radiation at different wavelengths. It uses monochromatic light. It is of many types: a. Visible range spectrophotometer (ranging from 400 Pm to 1000 Pm). b. UV visible spectrophotometer – it utilizes 200 to 1000 Pm wavelength of light. c. IR spectrophotometer: It’s ranging from 1000 Pm – 3000 Pm

Centrifuge

Centrifuge

Fig. 12.10. Structure of small centrifuge.

Fig. 12.10. Structure of small centrifuge.

A centrifuge is a piece of equipment, generally driven by an electric motor that puts an object in rotation around a fixed axis applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes more dense substances to separate out along the radial direction (the bottom of the tube) and the lighter objects will tend to move to the top of the tube in the rotating picture. In the picture shown, the rotating unit, called the rotor, has fixed holes drilled at an angle (to the vertical). Test tubes are placed in these slots and the rotor is spun. As the centrifugal force is in the horizontal plane and the tubes are fixed at an angle, the particles have to travel only a little distance before they hit the wall and drop down to the bottom. These angle rotors are very popular in the lab for routine use.

A centrifuge is a piece of equipment, generally driven by an electric motor that puts an object in rotation around a fixed axis applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes more dense substances to separate out along the radial direction (the bottom of the tube) and the lighter objects will tend to move to the top of the tube in the rotating picture. In the picture shown, the rotating unit, called the rotor, has fixed holes drilled at an angle (to the vertical). Test tubes are placed in these slots and the rotor is spun. As the centrifugal force is in the horizontal plane and the tubes are fixed at an angle, the particles have to travel only a little distance before they hit the wall and drop down to the bottom. These angle rotors are very popular in the lab for routine use.

194 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY There are many types of centrifuge: 1. Small bench centrifuge: the speed of small bench centrifuge varies from 4000-6000 rpm. 2. Refrigerated centrifuge: the speed of refrigerated centrifuge comprises of 6000 rpm. 3. High speed refrigerated centrifuge: Speed of this centrifuge is 25,000 rpm. 4. Ultracentrifuge: It’s speed comprise of 80,000 rpm.

194 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY There are many types of centrifuge: 1. Small bench centrifuge: the speed of small bench centrifuge varies from 4000-6000 rpm. 2. Refrigerated centrifuge: the speed of refrigerated centrifuge comprises of 6000 rpm. 3. High speed refrigerated centrifuge: Speed of this centrifuge is 25,000 rpm. 4. Ultracentrifuge: It’s speed comprise of 80,000 rpm.

194 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY There are many types of centrifuge: 1. Small bench centrifuge: the speed of small bench centrifuge varies from 4000-6000 rpm. 2. Refrigerated centrifuge: the speed of refrigerated centrifuge comprises of 6000 rpm. 3. High speed refrigerated centrifuge: Speed of this centrifuge is 25,000 rpm. 4. Ultracentrifuge: It’s speed comprise of 80,000 rpm.

194 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY There are many types of centrifuge: 1. Small bench centrifuge: the speed of small bench centrifuge varies from 4000-6000 rpm. 2. Refrigerated centrifuge: the speed of refrigerated centrifuge comprises of 6000 rpm. 3. High speed refrigerated centrifuge: Speed of this centrifuge is 25,000 rpm. 4. Ultracentrifuge: It’s speed comprise of 80,000 rpm.

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EXPERIMENT NO. 3

EXPERIMENT NO. 3

Aim: To study the detection of movement of bacteria with the help of hanging drop Method.

Aim: To study the detection of movement of bacteria with the help of hanging drop Method.

Requirements: 24-hour broth culture of bacteria, 2 hollow-ground slides (slide having cavity at the centre), Several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, vaseline or petroleum jelly, microscope.

Requirements: 24-hour broth culture of bacteria, 2 hollow-ground slides (slide having cavity at the centre), Several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, vaseline or petroleum jelly, microscope.

Theory

Theory

This technique is meant for microscopic observation of living bacteria. The simplest method for examining living microorganisms is to suspend them in a fluid (water, saline, or broth) and prepare either a “hanging drop” or a simple “wet mount.” The slide for a hanging drop method includes a concave well in the centre; the cover glass holds a drop of the suspension. When the cover glass is inverted over the well of the slide, the drop hangs from the glass in the hollow concavity of the slide (fig. 12.10, step 4). Microscopic study of such a wet preparation can provide useful information. Primarily, the method is used to determine whether or not an organism is motile, but it also permits an undistorted view of natural patterns of cell groupings and of individual cell shape.

This technique is meant for microscopic observation of living bacteria. The simplest method for examining living microorganisms is to suspend them in a fluid (water, saline, or broth) and prepare either a “hanging drop” or a simple “wet mount.” The slide for a hanging drop method includes a concave well in the centre; the cover glass holds a drop of the suspension. When the cover glass is inverted over the well of the slide, the drop hangs from the glass in the hollow concavity of the slide (fig. 12.10, step 4). Microscopic study of such a wet preparation can provide useful information. Primarily, the method is used to determine whether or not an organism is motile, but it also permits an undistorted view of natural patterns of cell groupings and of individual cell shape.

Fig. 12.11. Hanging droplet method.

Fig. 12.11. Hanging droplet method.

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EXPERIMENT NO. 3

EXPERIMENT NO. 3

Aim: To study the detection of movement of bacteria with the help of hanging drop Method.

Aim: To study the detection of movement of bacteria with the help of hanging drop Method.

Requirements: 24-hour broth culture of bacteria, 2 hollow-ground slides (slide having cavity at the centre), Several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, vaseline or petroleum jelly, microscope.

Requirements: 24-hour broth culture of bacteria, 2 hollow-ground slides (slide having cavity at the centre), Several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, vaseline or petroleum jelly, microscope.

Theory

Theory

This technique is meant for microscopic observation of living bacteria. The simplest method for examining living microorganisms is to suspend them in a fluid (water, saline, or broth) and prepare either a “hanging drop” or a simple “wet mount.” The slide for a hanging drop method includes a concave well in the centre; the cover glass holds a drop of the suspension. When the cover glass is inverted over the well of the slide, the drop hangs from the glass in the hollow concavity of the slide (fig. 12.10, step 4). Microscopic study of such a wet preparation can provide useful information. Primarily, the method is used to determine whether or not an organism is motile, but it also permits an undistorted view of natural patterns of cell groupings and of individual cell shape.

This technique is meant for microscopic observation of living bacteria. The simplest method for examining living microorganisms is to suspend them in a fluid (water, saline, or broth) and prepare either a “hanging drop” or a simple “wet mount.” The slide for a hanging drop method includes a concave well in the centre; the cover glass holds a drop of the suspension. When the cover glass is inverted over the well of the slide, the drop hangs from the glass in the hollow concavity of the slide (fig. 12.10, step 4). Microscopic study of such a wet preparation can provide useful information. Primarily, the method is used to determine whether or not an organism is motile, but it also permits an undistorted view of natural patterns of cell groupings and of individual cell shape.

Fig. 12.11. Hanging droplet method.

Fig. 12.11. Hanging droplet method.

196 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

196 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Procedure

Procedure

1. With a toothpick, make a small ring of vaseline around the cavity of a glass slide (fig. 12.11, step1). Do not use too much vaseline. 2. Aseptically place the loopful of culture of bacteria in the centre of the cover glass with the help of sterilized inoculum loop as in (fig. 12.11, step 2) (do not spread it around). 3. Hold the hollow-ground slide inverted with the well down over the cover glass (fig. 12.11, step 3), then press it down gently so that the vaseline adheres to the cover glass. Now turn the slide over (fig. 12.11, step 4). 4. Place the slide on the microscope stage, cover glass up. Examine the drop by first locating its edge under low power and focusing on the drop. Switch to the high-dry objective and then, using immersion oil (be very careful not to break the cover glass with the latter). In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast. Note bacterial shape, size, arrangement, and motility. Be careful to distinguish between motility and Brownian movement. 5. Record your observations of the size, shape, cell groupings, and motility of bacteria. Discard your cover slips and any contaminated slides in a container with disinfectant solution.

Hints and precautions

1. With a toothpick, make a small ring of vaseline around the cavity of a glass slide (fig. 12.11, step1). Do not use too much vaseline. 2. Aseptically place the loopful of culture of bacteria in the centre of the cover glass with the help of sterilized inoculum loop as in (fig. 12.11, step 2) (do not spread it around). 3. Hold the hollow-ground slide inverted with the well down over the cover glass (fig. 12.11, step 3), then press it down gently so that the vaseline adheres to the cover glass. Now turn the slide over (fig. 12.11, step 4). 4. Place the slide on the microscope stage, cover glass up. Examine the drop by first locating its edge under low power and focusing on the drop. Switch to the high-dry objective and then, using immersion oil (be very careful not to break the cover glass with the latter). In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast. Note bacterial shape, size, arrangement, and motility. Be careful to distinguish between motility and Brownian movement. 5. Record your observations of the size, shape, cell groupings, and motility of bacteria. Discard your cover slips and any contaminated slides in a container with disinfectant solution.

Hints and precautions

1. Always make sure the specimen is on the top side of the slide. 2. Particular care must be taken to avoid breaking the cover slip since it is more vulnerable when supported only around its edges. 3. With depression slides, the added thickness of the slide and cover slip may preclude the use of the oil immersion objective with some microscopes. 4. If your microscope is equipped with an automatic stop, it may be necessary to bring the image into focus by using the coarse adjustment knob.

1. Always make sure the specimen is on the top side of the slide. 2. Particular care must be taken to avoid breaking the cover slip since it is more vulnerable when supported only around its edges. 3. With depression slides, the added thickness of the slide and cover slip may preclude the use of the oil immersion objective with some microscopes. 4. If your microscope is equipped with an automatic stop, it may be necessary to bring the image into focus by using the coarse adjustment knob.

Result: Examine the microorganisms in the concavity slide and draw the structure.

Result: Examine the microorganisms in the concavity slide and draw the structure.

196 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

196 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Procedure

Procedure

1. With a toothpick, make a small ring of vaseline around the cavity of a glass slide (fig. 12.11, step1). Do not use too much vaseline. 2. Aseptically place the loopful of culture of bacteria in the centre of the cover glass with the help of sterilized inoculum loop as in (fig. 12.11, step 2) (do not spread it around). 3. Hold the hollow-ground slide inverted with the well down over the cover glass (fig. 12.11, step 3), then press it down gently so that the vaseline adheres to the cover glass. Now turn the slide over (fig. 12.11, step 4). 4. Place the slide on the microscope stage, cover glass up. Examine the drop by first locating its edge under low power and focusing on the drop. Switch to the high-dry objective and then, using immersion oil (be very careful not to break the cover glass with the latter). In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast. Note bacterial shape, size, arrangement, and motility. Be careful to distinguish between motility and Brownian movement. 5. Record your observations of the size, shape, cell groupings, and motility of bacteria. Discard your cover slips and any contaminated slides in a container with disinfectant solution.

Hints and precautions

1. With a toothpick, make a small ring of vaseline around the cavity of a glass slide (fig. 12.11, step1). Do not use too much vaseline. 2. Aseptically place the loopful of culture of bacteria in the centre of the cover glass with the help of sterilized inoculum loop as in (fig. 12.11, step 2) (do not spread it around). 3. Hold the hollow-ground slide inverted with the well down over the cover glass (fig. 12.11, step 3), then press it down gently so that the vaseline adheres to the cover glass. Now turn the slide over (fig. 12.11, step 4). 4. Place the slide on the microscope stage, cover glass up. Examine the drop by first locating its edge under low power and focusing on the drop. Switch to the high-dry objective and then, using immersion oil (be very careful not to break the cover glass with the latter). In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast. Note bacterial shape, size, arrangement, and motility. Be careful to distinguish between motility and Brownian movement. 5. Record your observations of the size, shape, cell groupings, and motility of bacteria. Discard your cover slips and any contaminated slides in a container with disinfectant solution.

Hints and precautions

1. Always make sure the specimen is on the top side of the slide. 2. Particular care must be taken to avoid breaking the cover slip since it is more vulnerable when supported only around its edges. 3. With depression slides, the added thickness of the slide and cover slip may preclude the use of the oil immersion objective with some microscopes. 4. If your microscope is equipped with an automatic stop, it may be necessary to bring the image into focus by using the coarse adjustment knob.

1. Always make sure the specimen is on the top side of the slide. 2. Particular care must be taken to avoid breaking the cover slip since it is more vulnerable when supported only around its edges. 3. With depression slides, the added thickness of the slide and cover slip may preclude the use of the oil immersion objective with some microscopes. 4. If your microscope is equipped with an automatic stop, it may be necessary to bring the image into focus by using the coarse adjustment knob.

Result: Examine the microorganisms in the concavity slide and draw the structure.

Result: Examine the microorganisms in the concavity slide and draw the structure.

EXPERIMENTS

197

EXPERIMENTS

197

EXPERIMENT NO. 4

EXPERIMENT NO. 4

Aim: To study the detection of movement of bacteria with the help of wet mount Method.

Aim: To study the detection of movement of bacteria with the help of wet mount Method.

Requirements: 24-hour broth culture of bacteria, glass slides, several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, sealing wax or nail polish, microscope.

Requirements: 24-hour broth culture of bacteria, glass slides, several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, sealing wax or nail polish, microscope.

Theory

Theory

In wet-mount method motility of strictly aerobic bacteria is observed. The fluid film is thinner than that of hanging-drop preparations and therefore the preparation tends to dry up more quickly, even when sealed. Although the hanging drop is the classical method for viewing unstained microorganisms, the wet mount is easier to perform and usually provides sufficient information.

In wet-mount method motility of strictly aerobic bacteria is observed. The fluid film is thinner than that of hanging-drop preparations and therefore the preparation tends to dry up more quickly, even when sealed. Although the hanging drop is the classical method for viewing unstained microorganisms, the wet mount is easier to perform and usually provides sufficient information.

Procedure

Procedure

1. Place a loopful or 1 drop of sample on a clean slide and cover it with cover slip. 2. Seal the edge of cover slip with wax or nail polish. The motility of strictly aerobic bacteria can be observed only for a brief period because of lack of oxygen. The inclusions of small air bubbles prolongs the activity of microbes. Generally phase contrast microscopy is recommended. 3. Examine the slide under the microscope.

1. Place a loopful or 1 drop of sample on a clean slide and cover it with cover slip. 2. Seal the edge of cover slip with wax or nail polish. The motility of strictly aerobic bacteria can be observed only for a brief period because of lack of oxygen. The inclusions of small air bubbles prolongs the activity of microbes. Generally phase contrast microscopy is recommended. 3. Examine the slide under the microscope.

Result: Examine and draw the structure of microorganisms in your note book.

Result: Examine and draw the structure of microorganisms in your note book.

EXPERIMENTS

197

EXPERIMENTS

197

EXPERIMENT NO. 4

EXPERIMENT NO. 4

Aim: To study the detection of movement of bacteria with the help of wet mount Method.

Aim: To study the detection of movement of bacteria with the help of wet mount Method.

Requirements: 24-hour broth culture of bacteria, glass slides, several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, sealing wax or nail polish, microscope.

Requirements: 24-hour broth culture of bacteria, glass slides, several cover glasses, wire inoculating loop, Bunsen burner or spirit lamp, permanent marking pen, sealing wax or nail polish, microscope.

Theory

Theory

In wet-mount method motility of strictly aerobic bacteria is observed. The fluid film is thinner than that of hanging-drop preparations and therefore the preparation tends to dry up more quickly, even when sealed. Although the hanging drop is the classical method for viewing unstained microorganisms, the wet mount is easier to perform and usually provides sufficient information.

In wet-mount method motility of strictly aerobic bacteria is observed. The fluid film is thinner than that of hanging-drop preparations and therefore the preparation tends to dry up more quickly, even when sealed. Although the hanging drop is the classical method for viewing unstained microorganisms, the wet mount is easier to perform and usually provides sufficient information.

Procedure

Procedure

1. Place a loopful or 1 drop of sample on a clean slide and cover it with cover slip. 2. Seal the edge of cover slip with wax or nail polish. The motility of strictly aerobic bacteria can be observed only for a brief period because of lack of oxygen. The inclusions of small air bubbles prolongs the activity of microbes. Generally phase contrast microscopy is recommended. 3. Examine the slide under the microscope.

1. Place a loopful or 1 drop of sample on a clean slide and cover it with cover slip. 2. Seal the edge of cover slip with wax or nail polish. The motility of strictly aerobic bacteria can be observed only for a brief period because of lack of oxygen. The inclusions of small air bubbles prolongs the activity of microbes. Generally phase contrast microscopy is recommended. 3. Examine the slide under the microscope.

Result: Examine and draw the structure of microorganisms in your note book.

Result: Examine and draw the structure of microorganisms in your note book.

198 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

198 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 5

EXPERIMENT NO. 5

Aim: To study the identification of bacteria with the help of simple staining.

Aim: To study the identification of bacteria with the help of simple staining.

Requirements: Glass slide, inoculating loop, spirit lamp, crystal violet, immersion oil, microscope, distilled water,.

Requirements: Glass slide, inoculating loop, spirit lamp, crystal violet, immersion oil, microscope, distilled water,.

Theory

Theory

From the experiment 3 and 4 we cannot study the bacterial shape and size because in fluid suspensions bacteria bounce by Brownian movement or true motility and difficult to visualize sharply. By the staining techniques we can easily study the complete morphology of bacteria. This technique is recommended to study the morphology and arrangement of bacterial cells. In simple staining single dye is used so it is also called monochromatic staining. By this you cannot differentiate gram positive and gram negative bacteria. Simple staining is performed by using basic dyes. e.g. crystal violet, methylene blue, carbolfuchsin etc. These dyes penetrate into the bacterial cell wall and give the color of dye using.

From the experiment 3 and 4 we cannot study the bacterial shape and size because in fluid suspensions bacteria bounce by Brownian movement or true motility and difficult to visualize sharply. By the staining techniques we can easily study the complete morphology of bacteria. This technique is recommended to study the morphology and arrangement of bacterial cells. In simple staining single dye is used so it is also called monochromatic staining. By this you cannot differentiate gram positive and gram negative bacteria. Simple staining is performed by using basic dyes. e.g. crystal violet, methylene blue, carbolfuchsin etc. These dyes penetrate into the bacterial cell wall and give the color of dye using.

Procedure

Procedure

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly i.e., make a thin bacterial smear. 4. Air dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air dries the slide and examine under the microscope using immersion oil.

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly i.e., make a thin bacterial smear. 4. Air dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air dries the slide and examine under the microscope using immersion oil.

Result: The stained bacteria are deep violet in color. The color depends upon the stain used.

Result: The stained bacteria are deep violet in color. The color depends upon the stain used.

Precaution

Precaution

1. All the dyes are carcinogenic in nature so avoid them to intimate contact. 2. Hold the slide from one end during the experiment.

198 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

1. All the dyes are carcinogenic in nature so avoid them to intimate contact. 2. Hold the slide from one end during the experiment.

198 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 5

EXPERIMENT NO. 5

Aim: To study the identification of bacteria with the help of simple staining.

Aim: To study the identification of bacteria with the help of simple staining.

Requirements: Glass slide, inoculating loop, spirit lamp, crystal violet, immersion oil, microscope, distilled water,.

Requirements: Glass slide, inoculating loop, spirit lamp, crystal violet, immersion oil, microscope, distilled water,.

Theory

Theory

From the experiment 3 and 4 we cannot study the bacterial shape and size because in fluid suspensions bacteria bounce by Brownian movement or true motility and difficult to visualize sharply. By the staining techniques we can easily study the complete morphology of bacteria. This technique is recommended to study the morphology and arrangement of bacterial cells. In simple staining single dye is used so it is also called monochromatic staining. By this you cannot differentiate gram positive and gram negative bacteria. Simple staining is performed by using basic dyes. e.g. crystal violet, methylene blue, carbolfuchsin etc. These dyes penetrate into the bacterial cell wall and give the color of dye using.

From the experiment 3 and 4 we cannot study the bacterial shape and size because in fluid suspensions bacteria bounce by Brownian movement or true motility and difficult to visualize sharply. By the staining techniques we can easily study the complete morphology of bacteria. This technique is recommended to study the morphology and arrangement of bacterial cells. In simple staining single dye is used so it is also called monochromatic staining. By this you cannot differentiate gram positive and gram negative bacteria. Simple staining is performed by using basic dyes. e.g. crystal violet, methylene blue, carbolfuchsin etc. These dyes penetrate into the bacterial cell wall and give the color of dye using.

Procedure

Procedure

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly i.e., make a thin bacterial smear. 4. Air dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air dries the slide and examine under the microscope using immersion oil.

1. Take a glass slide, wash and dry it properly. 2. The glass slide should be grease free i.e., free from oily substances. 3. Take a drop of bacterial culture and spread it properly i.e., make a thin bacterial smear. 4. Air dry the side followed by heat fixes. 5. Add the drop of crystal violet and keep it for 30 to 45 seconds. 6. Wash the smear gently with slowly running tap water. 7. Air dries the slide and examine under the microscope using immersion oil.

Result: The stained bacteria are deep violet in color. The color depends upon the stain used.

Result: The stained bacteria are deep violet in color. The color depends upon the stain used.

Precaution

Precaution

1. All the dyes are carcinogenic in nature so avoid them to intimate contact. 2. Hold the slide from one end during the experiment.

1. All the dyes are carcinogenic in nature so avoid them to intimate contact. 2. Hold the slide from one end during the experiment.

EXPERIMENTS

199

EXPERIMENTS

199

EXPERIMENT NO. 6

EXPERIMENT NO. 6

Aim: Three suspensions of microorganisms are marked as A, B and C are provided. Prepare the smears and stain them by gram staining and identify the microorganisms.

Aim: Three suspensions of microorganisms are marked as A, B and C are provided. Prepare the smears and stain them by gram staining and identify the microorganisms.

Requirements: Inoculating loop, glass slide, spirit lamp, crystal violet, iodine solution, ethyl alcohol, safranin, immersion oil, microscope, distilled water.

Requirements: Inoculating loop, glass slide, spirit lamp, crystal violet, iodine solution, ethyl alcohol, safranin, immersion oil, microscope, distilled water.

Procedure

Procedure

1. Wash 3 glass slides properly and air dry. 2. Take a loopful of bacterial culture and spread it properly i.e. make a thin bacterial smear. 3. Air dry the slides and heat fix it. 4. Cool the slides and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash all the slides with distilled water for few seconds, using wash bottle. 6. Cover each slide with gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more color flows from the smear. Note: gram positive bacteria are not affected while gram negative bacteria are decolourized completely. 8. Wash the slides with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slides with distilled water and dry it properly. 10. Examine all the slides under microscope using immersion oil.

1. Wash 3 glass slides properly and air dry. 2. Take a loopful of bacterial culture and spread it properly i.e. make a thin bacterial smear. 3. Air dry the slides and heat fix it. 4. Cool the slides and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash all the slides with distilled water for few seconds, using wash bottle. 6. Cover each slide with gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more color flows from the smear. Note: gram positive bacteria are not affected while gram negative bacteria are decolourized completely. 8. Wash the slides with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slides with distilled water and dry it properly. 10. Examine all the slides under microscope using immersion oil.

Result: A Gram positive bacterium shows violet or deep blue color while a gram negative bacterium shows purple or red colour. If the shape of bacteria is spherical they are cocci and if they are rod-shaped than bacilli. (refer topic 2.3 shape of bacterial cell) and you can identify your sample. Precaution: Same as experiment no.5 Note: For the experiment Number 7, 8, 9 and 10 refer the chapter 4th staining techniques.

Result: A Gram positive bacterium shows violet or deep blue color while a gram negative bacterium shows purple or red colour. If the shape of bacteria is spherical they are cocci and if they are rod-shaped than bacilli. (refer topic 2.3 shape of bacterial cell) and you can identify your sample. Precaution: Same as experiment no.5 Note: For the experiment Number 7, 8, 9 and 10 refer the chapter 4th staining techniques.

Experiment No. 7: To study the identification of bacteria help of negative staining. Experiment No. 8: To study the identification of bacteria help of endospore staining. Experiment No. 9: To study the identification of bacteria help of flagella staining staining. Experiment No. 10: To study the identification of bacteria help of capsule staining.

with the with the with the with the

EXPERIMENTS

Experiment No. 7: To study the identification of bacteria help of negative staining. Experiment No. 8: To study the identification of bacteria help of endospore staining. Experiment No. 9: To study the identification of bacteria help of flagella staining staining. Experiment No. 10: To study the identification of bacteria help of capsule staining.

199

with the with the with the with the

EXPERIMENTS

199

EXPERIMENT NO. 6

EXPERIMENT NO. 6

Aim: Three suspensions of microorganisms are marked as A, B and C are provided. Prepare the smears and stain them by gram staining and identify the microorganisms.

Aim: Three suspensions of microorganisms are marked as A, B and C are provided. Prepare the smears and stain them by gram staining and identify the microorganisms.

Requirements: Inoculating loop, glass slide, spirit lamp, crystal violet, iodine solution, ethyl alcohol, safranin, immersion oil, microscope, distilled water.

Requirements: Inoculating loop, glass slide, spirit lamp, crystal violet, iodine solution, ethyl alcohol, safranin, immersion oil, microscope, distilled water.

Procedure

Procedure

1. Wash 3 glass slides properly and air dry. 2. Take a loopful of bacterial culture and spread it properly i.e. make a thin bacterial smear. 3. Air dry the slides and heat fix it. 4. Cool the slides and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash all the slides with distilled water for few seconds, using wash bottle. 6. Cover each slide with gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more color flows from the smear. Note: gram positive bacteria are not affected while gram negative bacteria are decolourized completely. 8. Wash the slides with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slides with distilled water and dry it properly. 10. Examine all the slides under microscope using immersion oil.

1. Wash 3 glass slides properly and air dry. 2. Take a loopful of bacterial culture and spread it properly i.e. make a thin bacterial smear. 3. Air dry the slides and heat fix it. 4. Cool the slides and add a drop of crystal violet and spread it over the smear and keep it for 30 seconds. 5. Wash all the slides with distilled water for few seconds, using wash bottle. 6. Cover each slide with gram’s iodine solution for 60 seconds. 7. Wash off the iodine solution with 95% ethyl alcohol. Add the alcohol drop by drop until no more color flows from the smear. Note: gram positive bacteria are not affected while gram negative bacteria are decolourized completely. 8. Wash the slides with distilled water and apply safranin for 30 seconds (counter staining). 9. Wash the slides with distilled water and dry it properly. 10. Examine all the slides under microscope using immersion oil.

Result: A Gram positive bacterium shows violet or deep blue color while a gram negative bacterium shows purple or red colour. If the shape of bacteria is spherical they are cocci and if they are rod-shaped than bacilli. (refer topic 2.3 shape of bacterial cell) and you can identify your sample. Precaution: Same as experiment no.5 Note: For the experiment Number 7, 8, 9 and 10 refer the chapter 4th staining techniques.

Result: A Gram positive bacterium shows violet or deep blue color while a gram negative bacterium shows purple or red colour. If the shape of bacteria is spherical they are cocci and if they are rod-shaped than bacilli. (refer topic 2.3 shape of bacterial cell) and you can identify your sample. Precaution: Same as experiment no.5 Note: For the experiment Number 7, 8, 9 and 10 refer the chapter 4th staining techniques.

Experiment No. 7: To study the identification of bacteria help of negative staining. Experiment No. 8: To study the identification of bacteria help of endospore staining. Experiment No. 9: To study the identification of bacteria help of flagella staining staining. Experiment No. 10: To study the identification of bacteria help of capsule staining.

with the with the with the with the

Experiment No. 7: To study the identification of bacteria help of negative staining. Experiment No. 8: To study the identification of bacteria help of endospore staining. Experiment No. 9: To study the identification of bacteria help of flagella staining staining. Experiment No. 10: To study the identification of bacteria help of capsule staining.

with the with the with the with the

200 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 11

200 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 11

Aim: To study the sterilization techniques.

Aim: To study the sterilization techniques.

Note: For the detail study refer the chapter 9 under the topic “sterilization methods” (9.4). Read carefully different types of sterilization methods and their applications.

Note: For the detail study refer the chapter 9 under the topic “sterilization methods” (9.4). Read carefully different types of sterilization methods and their applications.

Fig. 12.12. Structure of autoclave.

Fig. 12.12. Structure of autoclave.

200 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 11

200 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 11

Aim: To study the sterilization techniques.

Aim: To study the sterilization techniques.

Note: For the detail study refer the chapter 9 under the topic “sterilization methods” (9.4). Read carefully different types of sterilization methods and their applications.

Note: For the detail study refer the chapter 9 under the topic “sterilization methods” (9.4). Read carefully different types of sterilization methods and their applications.

Fig. 12.12. Structure of autoclave.

Fig. 12.12. Structure of autoclave.

EXPERIMENTS

201

EXPERIMENTS

EXPERIMENT NO. 12

201

EXPERIMENT NO. 12

Aim: To study the preparation of different types of culture media.

Aim: To study the preparation of different types of culture media.

Theory

Theory

The food supplements which support the growth of microorganism is called culture medium. Generally, the common media contain both organic and inorganic nutrients but for the cultivation of many microorganisms specialized media are prepared. If the media is in liquid form it is called broth media and when agar is added for solidification it is called solid media. Agar does not provide any nutritional supplement, it helps only in solidification. Basically, the culture media are of three types: Natural, semi-synthetic and synthetic media. 1. Natural medium: The natural medium is that which contains the natural products such as diluted blood, milk, urine, vegetable juices, animal cells/ tissue/organs and peptones, etc. In such medium the exact chemical composition is not known. 2. Semi-synthetic medium: The semi-synthetic medium is that in which the chemical substances are partially added. For example, nutrient agar media, L.B. agar media, potato dextrose agar media, etc. in other words, media containing agar is called semi-synthetic media. 3. Synthetic medium: The medium in which chemical substances are present in known concentration is called synthetic medium. Such types of media are help in isolation of microorganisms. Synthetic media are different types; a. General-purpose media: For the routine microbiological work. b. Differential media: Help in, to differentiate the group of microorganisms. c. Selective media: By using certain agents help in the isolation of particular type of bacteria. For e.g. Brilliant green agar media is used for the isolation of Salmonella from faces. d. Assay media: Such type of media is used for the assay of antibiotics, amino acids, vitamins etc.

The food supplements which support the growth of microorganism is called culture medium. Generally, the common media contain both organic and inorganic nutrients but for the cultivation of many microorganisms specialized media are prepared. If the media is in liquid form it is called broth media and when agar is added for solidification it is called solid media. Agar does not provide any nutritional supplement, it helps only in solidification. Basically, the culture media are of three types: Natural, semi-synthetic and synthetic media. 1. Natural medium: The natural medium is that which contains the natural products such as diluted blood, milk, urine, vegetable juices, animal cells/ tissue/organs and peptones, etc. In such medium the exact chemical composition is not known. 2. Semi-synthetic medium: The semi-synthetic medium is that in which the chemical substances are partially added. For example, nutrient agar media, L.B. agar media, potato dextrose agar media, etc. in other words, media containing agar is called semi-synthetic media. 3. Synthetic medium: The medium in which chemical substances are present in known concentration is called synthetic medium. Such types of media are help in isolation of microorganisms. Synthetic media are different types; a. General-purpose media: For the routine microbiological work. b. Differential media: Help in, to differentiate the group of microorganisms. c. Selective media: By using certain agents help in the isolation of particular type of bacteria. For e.g. Brilliant green agar media is used for the isolation of Salmonella from faces. d. Assay media: Such type of media is used for the assay of antibiotics, amino acids, vitamins etc.

Preparation of media

Preparation of media

1. Preparation of broth (or liquid) media: This type of media is commonly used in the cultivation of bacteria and fungi. For e.g., Nutrient broth media and L.B. broth media. Composition of nutrient broth media: Beef extract 0.30% (or 0.3 gm) Peptone 0.50% (or 0.5 gm) Distilled water 100 mL pH 7.0

EXPERIMENTS

1. Preparation of broth (or liquid) media: This type of media is commonly used in the cultivation of bacteria and fungi. For e.g., Nutrient broth media and L.B. broth media. Composition of nutrient broth media: Beef extract 0.30% (or 0.3 gm) Peptone 0.50% (or 0.5 gm) Distilled water 100 mL pH 7.0

201

EXPERIMENTS

EXPERIMENT NO. 12

201

EXPERIMENT NO. 12

Aim: To study the preparation of different types of culture media.

Aim: To study the preparation of different types of culture media.

Theory

Theory

The food supplements which support the growth of microorganism is called culture medium. Generally, the common media contain both organic and inorganic nutrients but for the cultivation of many microorganisms specialized media are prepared. If the media is in liquid form it is called broth media and when agar is added for solidification it is called solid media. Agar does not provide any nutritional supplement, it helps only in solidification. Basically, the culture media are of three types: Natural, semi-synthetic and synthetic media. 1. Natural medium: The natural medium is that which contains the natural products such as diluted blood, milk, urine, vegetable juices, animal cells/ tissue/organs and peptones, etc. In such medium the exact chemical composition is not known. 2. Semi-synthetic medium: The semi-synthetic medium is that in which the chemical substances are partially added. For example, nutrient agar media, L.B. agar media, potato dextrose agar media, etc. in other words, media containing agar is called semi-synthetic media. 3. Synthetic medium: The medium in which chemical substances are present in known concentration is called synthetic medium. Such types of media are help in isolation of microorganisms. Synthetic media are different types; a. General-purpose media: For the routine microbiological work. b. Differential media: Help in, to differentiate the group of microorganisms. c. Selective media: By using certain agents help in the isolation of particular type of bacteria. For e.g. Brilliant green agar media is used for the isolation of Salmonella from faces. d. Assay media: Such type of media is used for the assay of antibiotics, amino acids, vitamins etc.

The food supplements which support the growth of microorganism is called culture medium. Generally, the common media contain both organic and inorganic nutrients but for the cultivation of many microorganisms specialized media are prepared. If the media is in liquid form it is called broth media and when agar is added for solidification it is called solid media. Agar does not provide any nutritional supplement, it helps only in solidification. Basically, the culture media are of three types: Natural, semi-synthetic and synthetic media. 1. Natural medium: The natural medium is that which contains the natural products such as diluted blood, milk, urine, vegetable juices, animal cells/ tissue/organs and peptones, etc. In such medium the exact chemical composition is not known. 2. Semi-synthetic medium: The semi-synthetic medium is that in which the chemical substances are partially added. For example, nutrient agar media, L.B. agar media, potato dextrose agar media, etc. in other words, media containing agar is called semi-synthetic media. 3. Synthetic medium: The medium in which chemical substances are present in known concentration is called synthetic medium. Such types of media are help in isolation of microorganisms. Synthetic media are different types; a. General-purpose media: For the routine microbiological work. b. Differential media: Help in, to differentiate the group of microorganisms. c. Selective media: By using certain agents help in the isolation of particular type of bacteria. For e.g. Brilliant green agar media is used for the isolation of Salmonella from faces. d. Assay media: Such type of media is used for the assay of antibiotics, amino acids, vitamins etc.

Preparation of media

Preparation of media

1. Preparation of broth (or liquid) media: This type of media is commonly used in the cultivation of bacteria and fungi. For e.g., Nutrient broth media and L.B. broth media. Composition of nutrient broth media: Beef extract 0.30% (or 0.3 gm) Peptone 0.50% (or 0.5 gm) Distilled water 100 mL pH 7.0

1. Preparation of broth (or liquid) media: This type of media is commonly used in the cultivation of bacteria and fungi. For e.g., Nutrient broth media and L.B. broth media. Composition of nutrient broth media: Beef extract 0.30% (or 0.3 gm) Peptone 0.50% (or 0.5 gm) Distilled water 100 mL pH 7.0

202 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Composition of L.B. broth media: Tryptone 1.00% (or 1 gm) Yeast extract 0.50% (or 0.5 gm) NaCl 1.00% (or 1 gm) Distilled water 100 mL pH 7.0

Procedure

202 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Composition of L.B. broth media: Tryptone 1.00% (or 1 gm) Yeast extract 0.50% (or 0.5 gm) NaCl 1.00% (or 1 gm) Distilled water 100 mL pH 7.0

Procedure

1. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 2. Add the more distilled water to make up the volume up to 100 mL. 3. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 4. Dispense 5-8 mL of broth into each culture tubes. 5. Prepare the cotton plug and apply them into the mouth of broth tubes. 6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Transfer all the broth tubes into a test tube stand or beaker or iron basket. 8. Place the basket inside the autoclave/pressure cooker and sterilize at 121°C for 20 minutes. 9. When temperature cool down take out the broth tubes. 10. Use the broth tube when required or store at room temperature for further use.

1. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 2. Add the more distilled water to make up the volume up to 100 mL. 3. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 4. Dispense 5-8 mL of broth into each culture tubes. 5. Prepare the cotton plug and apply them into the mouth of broth tubes. 6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Transfer all the broth tubes into a test tube stand or beaker or iron basket. 8. Place the basket inside the autoclave/pressure cooker and sterilize at 121°C for 20 minutes. 9. When temperature cool down take out the broth tubes. 10. Use the broth tube when required or store at room temperature for further use.

2. Preparation of solid media: When the agar is added into the above liquid media, it is called solid media. For e.g., nutrient agar media, L.B agar media, potato dextrose agar media, etc. For the preparation of solid media use 1.50% or 1.5 gm of agar into the nutrient broth/L.B broth media.

2. Preparation of solid media: When the agar is added into the above liquid media, it is called solid media. For e.g., nutrient agar media, L.B agar media, potato dextrose agar media, etc. For the preparation of solid media use 1.50% or 1.5 gm of agar into the nutrient broth/L.B broth media.

You can prepare the nutrient agar or L.B agar media by adding the 1.5 gm of agar into the above given composition of nutrient broth/L.B broth media. Composition of potato dextrose agar (PDA) media: Potato tuber 20.0% or 20 gm Dextrose 2.0% or 2 gm Agar 1.5% or 1.5 gm Distilled water 100 mL pH 5.6

You can prepare the nutrient agar or L.B agar media by adding the 1.5 gm of agar into the above given composition of nutrient broth/L.B broth media. Composition of potato dextrose agar (PDA) media: Potato tuber 20.0% or 20 gm Dextrose 2.0% or 2 gm Agar 1.5% or 1.5 gm Distilled water 100 mL pH 5.6

Procedure

Procedure

1. Take potato tubers, peel off and weigh 20 gm. 2. Chop the tubers into small pieces with the help of knife.

202 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Composition of L.B. broth media: Tryptone 1.00% (or 1 gm) Yeast extract 0.50% (or 0.5 gm) NaCl 1.00% (or 1 gm) Distilled water 100 mL pH 7.0

Procedure

1. Take potato tubers, peel off and weigh 20 gm. 2. Chop the tubers into small pieces with the help of knife.

202 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY Composition of L.B. broth media: Tryptone 1.00% (or 1 gm) Yeast extract 0.50% (or 0.5 gm) NaCl 1.00% (or 1 gm) Distilled water 100 mL pH 7.0

Procedure

1. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 2. Add the more distilled water to make up the volume up to 100 mL. 3. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 4. Dispense 5-8 mL of broth into each culture tubes. 5. Prepare the cotton plug and apply them into the mouth of broth tubes. 6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Transfer all the broth tubes into a test tube stand or beaker or iron basket. 8. Place the basket inside the autoclave/pressure cooker and sterilize at 121°C for 20 minutes. 9. When temperature cool down take out the broth tubes. 10. Use the broth tube when required or store at room temperature for further use.

1. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 2. Add the more distilled water to make up the volume up to 100 mL. 3. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 4. Dispense 5-8 mL of broth into each culture tubes. 5. Prepare the cotton plug and apply them into the mouth of broth tubes. 6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Transfer all the broth tubes into a test tube stand or beaker or iron basket. 8. Place the basket inside the autoclave/pressure cooker and sterilize at 121°C for 20 minutes. 9. When temperature cool down take out the broth tubes. 10. Use the broth tube when required or store at room temperature for further use.

2. Preparation of solid media: When the agar is added into the above liquid media, it is called solid media. For e.g., nutrient agar media, L.B agar media, potato dextrose agar media, etc. For the preparation of solid media use 1.50% or 1.5 gm of agar into the nutrient broth/L.B broth media.

2. Preparation of solid media: When the agar is added into the above liquid media, it is called solid media. For e.g., nutrient agar media, L.B agar media, potato dextrose agar media, etc. For the preparation of solid media use 1.50% or 1.5 gm of agar into the nutrient broth/L.B broth media.

You can prepare the nutrient agar or L.B agar media by adding the 1.5 gm of agar into the above given composition of nutrient broth/L.B broth media. Composition of potato dextrose agar (PDA) media: Potato tuber 20.0% or 20 gm Dextrose 2.0% or 2 gm Agar 1.5% or 1.5 gm Distilled water 100 mL pH 5.6

You can prepare the nutrient agar or L.B agar media by adding the 1.5 gm of agar into the above given composition of nutrient broth/L.B broth media. Composition of potato dextrose agar (PDA) media: Potato tuber 20.0% or 20 gm Dextrose 2.0% or 2 gm Agar 1.5% or 1.5 gm Distilled water 100 mL pH 5.6

Procedure

Procedure

1. Take potato tubers, peel off and weigh 20 gm. 2. Chop the tubers into small pieces with the help of knife.

1. Take potato tubers, peel off and weigh 20 gm. 2. Chop the tubers into small pieces with the help of knife.

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3. Transfer the chopped potatoes into a beaker containing about 50 mL of distilled water. 4. Boil the contents with the help of heater for about 20 minutes. 5. Decant supernatant, filter with four folds of the muslin cloth and collect the filtrate into a beaker. This filtrate is called potato extract. 6. Transfer dextrose 2 gm and agar 1.5 gm into the extract and gently heat and shake to dissolve the ingredients. 7. Finally, transfer this medium into a measuring cylinder of 100 mL and make up the volume upto 100 mL with distilled water. 8. Measure the pH of the medium and adjust to 5.6 by using 1N HCl or NaOH drop-wise. 9. Pour the medium into the conical flask, put cotton plug, cover the plug with aluminium foil/paper and autoclave at 121°C for 20 minutes. 10. When temperature cools down take out the flasks and pour it into the petri plates inside the laminar air hood, nearly 20 mL of medium into 1 plate. 11. When the agar plates are solidified close the lid and use as required.

3. Transfer the chopped potatoes into a beaker containing about 50 mL of distilled water. 4. Boil the contents with the help of heater for about 20 minutes. 5. Decant supernatant, filter with four folds of the muslin cloth and collect the filtrate into a beaker. This filtrate is called potato extract. 6. Transfer dextrose 2 gm and agar 1.5 gm into the extract and gently heat and shake to dissolve the ingredients. 7. Finally, transfer this medium into a measuring cylinder of 100 mL and make up the volume upto 100 mL with distilled water. 8. Measure the pH of the medium and adjust to 5.6 by using 1N HCl or NaOH drop-wise. 9. Pour the medium into the conical flask, put cotton plug, cover the plug with aluminium foil/paper and autoclave at 121°C for 20 minutes. 10. When temperature cools down take out the flasks and pour it into the petri plates inside the laminar air hood, nearly 20 mL of medium into 1 plate. 11. When the agar plates are solidified close the lid and use as required.

Note: This procedure for 100 mL, you can calculate the formula as your required volume.

Note: This procedure for 100 mL, you can calculate the formula as your required volume.

Fig. 12.13. Pouring of plate inside the laminar hood.

EXPERIMENTS

Fig. 12.13. Pouring of plate inside the laminar hood.

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3. Transfer the chopped potatoes into a beaker containing about 50 mL of distilled water. 4. Boil the contents with the help of heater for about 20 minutes. 5. Decant supernatant, filter with four folds of the muslin cloth and collect the filtrate into a beaker. This filtrate is called potato extract. 6. Transfer dextrose 2 gm and agar 1.5 gm into the extract and gently heat and shake to dissolve the ingredients. 7. Finally, transfer this medium into a measuring cylinder of 100 mL and make up the volume upto 100 mL with distilled water. 8. Measure the pH of the medium and adjust to 5.6 by using 1N HCl or NaOH drop-wise. 9. Pour the medium into the conical flask, put cotton plug, cover the plug with aluminium foil/paper and autoclave at 121°C for 20 minutes. 10. When temperature cools down take out the flasks and pour it into the petri plates inside the laminar air hood, nearly 20 mL of medium into 1 plate. 11. When the agar plates are solidified close the lid and use as required.

3. Transfer the chopped potatoes into a beaker containing about 50 mL of distilled water. 4. Boil the contents with the help of heater for about 20 minutes. 5. Decant supernatant, filter with four folds of the muslin cloth and collect the filtrate into a beaker. This filtrate is called potato extract. 6. Transfer dextrose 2 gm and agar 1.5 gm into the extract and gently heat and shake to dissolve the ingredients. 7. Finally, transfer this medium into a measuring cylinder of 100 mL and make up the volume upto 100 mL with distilled water. 8. Measure the pH of the medium and adjust to 5.6 by using 1N HCl or NaOH drop-wise. 9. Pour the medium into the conical flask, put cotton plug, cover the plug with aluminium foil/paper and autoclave at 121°C for 20 minutes. 10. When temperature cools down take out the flasks and pour it into the petri plates inside the laminar air hood, nearly 20 mL of medium into 1 plate. 11. When the agar plates are solidified close the lid and use as required.

Note: This procedure for 100 mL, you can calculate the formula as your required volume.

Note: This procedure for 100 mL, you can calculate the formula as your required volume.

Fig. 12.13. Pouring of plate inside the laminar hood.

Fig. 12.13. Pouring of plate inside the laminar hood.

204 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

204 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

3. Preparation of agar slants in culture tubes: After autoclaving the solid media transfer into the culture tubes and placed in slanting direction, it gives agar slants after solidification.

Procedure

3. Preparation of agar slants in culture tubes: After autoclaving the solid media transfer into the culture tubes and placed in slanting direction, it gives agar slants after solidification.

Procedure

1. Prepare the any one agar media as described above and autoclaved it. After autoclaving dispense 5-8 mL of agar media into the culture tube and placed in slanting position by giving a support.

1. Prepare the any one agar media as described above and autoclaved it. After autoclaving dispense 5-8 mL of agar media into the culture tube and placed in slanting position by giving a support.

Fig. 12.14. Structure of agar slant.

Fig. 12.14. Structure of agar slant.

1. Wight for 30 minutes for solidification agar media. 2. Use the slants for culture transfer if required or store them for further use.

1. Wight for 30 minutes for solidification agar media. 2. Use the slants for culture transfer if required or store them for further use.

4. Preparation of Nutrient agar plate/L.B. agar plates: Composition for Nutrient agar/L.B. agar media:

4. Preparation of Nutrient agar plate/L.B. agar plates: Composition for Nutrient agar/L.B. agar media:

Nutrient agar media Beef extract Peptone Agar Distilled water

L.B. agar media 0.3% 0.5% 1.5% 100 mL

Tryptone Yeast extract NaCl Agar Distilled water

Nutrient agar media 1.0% 0.5% 1.0% 1.5% 100 mL

Procedure

Beef extract Peptone Agar Distilled water

L.B. agar media 0.3% 0.5% 1.5% 100 mL

Tryptone Yeast extract NaCl Agar Distilled water

1.0% 0.5% 1.0% 1.5% 100 mL

Procedure

1. Wash the petri plates and dry into the hot air oven. Packed into the aluminium foil/paper and tie with a rubber band or thread. 2. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 3. Add the more distilled water to make up the volume up to 100 mL. 4. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 5. Prepare the cotton plug and apply them into the mouth of conical flask.

204 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

1. Wash the petri plates and dry into the hot air oven. Packed into the aluminium foil/paper and tie with a rubber band or thread. 2. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 3. Add the more distilled water to make up the volume up to 100 mL. 4. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 5. Prepare the cotton plug and apply them into the mouth of conical flask.

204 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

3. Preparation of agar slants in culture tubes: After autoclaving the solid media transfer into the culture tubes and placed in slanting direction, it gives agar slants after solidification.

Procedure

3. Preparation of agar slants in culture tubes: After autoclaving the solid media transfer into the culture tubes and placed in slanting direction, it gives agar slants after solidification.

Procedure

1. Prepare the any one agar media as described above and autoclaved it. After autoclaving dispense 5-8 mL of agar media into the culture tube and placed in slanting position by giving a support.

1. Prepare the any one agar media as described above and autoclaved it. After autoclaving dispense 5-8 mL of agar media into the culture tube and placed in slanting position by giving a support.

Fig. 12.14. Structure of agar slant.

Fig. 12.14. Structure of agar slant.

1. Wight for 30 minutes for solidification agar media. 2. Use the slants for culture transfer if required or store them for further use.

1. Wight for 30 minutes for solidification agar media. 2. Use the slants for culture transfer if required or store them for further use.

4. Preparation of Nutrient agar plate/L.B. agar plates: Composition for Nutrient agar/L.B. agar media:

4. Preparation of Nutrient agar plate/L.B. agar plates: Composition for Nutrient agar/L.B. agar media:

Nutrient agar media Beef extract Peptone Agar Distilled water

L.B. agar media 0.3% 0.5% 1.5% 100 mL

Tryptone Yeast extract NaCl Agar Distilled water

Nutrient agar media 1.0% 0.5% 1.0% 1.5% 100 mL

Procedure 1. Wash the petri plates and dry into the hot air oven. Packed into the aluminium foil/paper and tie with a rubber band or thread. 2. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 3. Add the more distilled water to make up the volume up to 100 mL. 4. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 5. Prepare the cotton plug and apply them into the mouth of conical flask.

Beef extract Peptone Agar Distilled water

L.B. agar media 0.3% 0.5% 1.5% 100 mL

Tryptone Yeast extract NaCl Agar Distilled water

1.0% 0.5% 1.0% 1.5% 100 mL

Procedure 1. Wash the petri plates and dry into the hot air oven. Packed into the aluminium foil/paper and tie with a rubber band or thread. 2. Accurately weigh the chemical ingredients and transfer into the conical flask containing 50 mL of distilled water. Dissolve the ingredient by agitation or by slight heating. 3. Add the more distilled water to make up the volume up to 100 mL. 4. Measure the pH of the media by using pH meter and adjust the pH to 7.0 by adding drop of either HCl or NaOH solution. 5. Prepare the cotton plug and apply them into the mouth of conical flask.

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6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Place the petri plates and conical flask inside the autoclave/pressure cooker and sterilize at 1210C for 20 minutes. 8. When temperature cool down take out the petri plates and conical flask. 9. Pour the 20 mL of agar media into each petri plates inside the laminar air hood when the temperature of conical flask reach near about 40%. 10. Wait for 30 minutes and when the media are solidify close the lid of the petri plates. 11. Use the plates when required or store at room temperature for further use. Note: Use the agar plates immediately or within 2 or 3 days.

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6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Place the petri plates and conical flask inside the autoclave/pressure cooker and sterilize at 1210C for 20 minutes. 8. When temperature cool down take out the petri plates and conical flask. 9. Pour the 20 mL of agar media into each petri plates inside the laminar air hood when the temperature of conical flask reach near about 40%. 10. Wait for 30 minutes and when the media are solidify close the lid of the petri plates. 11. Use the plates when required or store at room temperature for further use. Note: Use the agar plates immediately or within 2 or 3 days.

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6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Place the petri plates and conical flask inside the autoclave/pressure cooker and sterilize at 1210C for 20 minutes. 8. When temperature cool down take out the petri plates and conical flask. 9. Pour the 20 mL of agar media into each petri plates inside the laminar air hood when the temperature of conical flask reach near about 40%. 10. Wait for 30 minutes and when the media are solidify close the lid of the petri plates. 11. Use the plates when required or store at room temperature for further use. Note: Use the agar plates immediately or within 2 or 3 days.

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6. Tightly cover the mouth of cotton plugs with aluminium foils or a paper and tie with a rubber band or thread. 7. Place the petri plates and conical flask inside the autoclave/pressure cooker and sterilize at 1210C for 20 minutes. 8. When temperature cool down take out the petri plates and conical flask. 9. Pour the 20 mL of agar media into each petri plates inside the laminar air hood when the temperature of conical flask reach near about 40%. 10. Wait for 30 minutes and when the media are solidify close the lid of the petri plates. 11. Use the plates when required or store at room temperature for further use. Note: Use the agar plates immediately or within 2 or 3 days.

206 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 13

206 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 13

Aim: To study the isolation of pure culture from mixed culture.

Aim: To study the isolation of pure culture from mixed culture.

Requirements: Inoculating loop, spirit lamp, nutrient agar plate/L.B agar plate. Theory: Generally, bacteria exit in mixed population. It is very rare to get a single and pure form. For studying the morphology and physiological characters of an individual species it is essential to separate them from the others to get pure culture. There are many important methods for isolating pure culture from the mixed culture. 1. Streak plate method 2. Pour plate method 3. Spread plate method 4. Serial dilution method.

Requirements: Inoculating loop, spirit lamp, nutrient agar plate/L.B agar plate. Theory: Generally, bacteria exit in mixed population. It is very rare to get a single and pure form. For studying the morphology and physiological characters of an individual species it is essential to separate them from the others to get pure culture. There are many important methods for isolating pure culture from the mixed culture. 1. Streak plate method 2. Pour plate method 3. Spread plate method 4. Serial dilution method.

Streak plate method

Streak plate method

In this method, when the mixed population of bacteria is obtained from the first plate, pick up the single colony from this plate and streak the new fresh agar plate. Again pick up the single colony from the second plate and streak the third plate. By repeating this way, you will finally get the pure colony.

In this method, when the mixed population of bacteria is obtained from the first plate, pick up the single colony from this plate and streak the new fresh agar plate. Again pick up the single colony from the second plate and streak the third plate. By repeating this way, you will finally get the pure colony.

Fig. 12.15. Isolation of pure culture by streak plate method. Plate D showing the isolated colony of E. coli. Even plate C showing two types of colony.

Fig. 12.15. Isolation of pure culture by streak plate method. Plate D showing the isolated colony of E. coli. Even plate C showing two types of colony.

206 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 13

206 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 13

Aim: To study the isolation of pure culture from mixed culture.

Aim: To study the isolation of pure culture from mixed culture.

Requirements: Inoculating loop, spirit lamp, nutrient agar plate/L.B agar plate. Theory: Generally, bacteria exit in mixed population. It is very rare to get a single and pure form. For studying the morphology and physiological characters of an individual species it is essential to separate them from the others to get pure culture. There are many important methods for isolating pure culture from the mixed culture. 1. Streak plate method 2. Pour plate method 3. Spread plate method 4. Serial dilution method.

Requirements: Inoculating loop, spirit lamp, nutrient agar plate/L.B agar plate. Theory: Generally, bacteria exit in mixed population. It is very rare to get a single and pure form. For studying the morphology and physiological characters of an individual species it is essential to separate them from the others to get pure culture. There are many important methods for isolating pure culture from the mixed culture. 1. Streak plate method 2. Pour plate method 3. Spread plate method 4. Serial dilution method.

Streak plate method

Streak plate method

In this method, when the mixed population of bacteria is obtained from the first plate, pick up the single colony from this plate and streak the new fresh agar plate. Again pick up the single colony from the second plate and streak the third plate. By repeating this way, you will finally get the pure colony.

In this method, when the mixed population of bacteria is obtained from the first plate, pick up the single colony from this plate and streak the new fresh agar plate. Again pick up the single colony from the second plate and streak the third plate. By repeating this way, you will finally get the pure colony.

Fig. 12.15. Isolation of pure culture by streak plate method. Plate D showing the isolated colony of E. coli. Even plate C showing two types of colony.

Fig. 12.15. Isolation of pure culture by streak plate method. Plate D showing the isolated colony of E. coli. Even plate C showing two types of colony.

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Procedure

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Procedure

1. Prepare the L.B. agar plates or nutrients agar plates according to experiment number 12. 2. Take sample in beaker. Sample may be pond water or your drinking water or tap water or take small amount of soil and add water, etc. 3. Wipe the laminar hood properly with alcohol and switch on the UV light for 15 minutes. Keep only necessary things inside the laminar hood. 4. Take the inoculum loop in right hand and heat it for sterilization, cool it and take a sample by just dipping the inoculum loop into the sample. 5. Open the lid of petri plate near the spirit lamp and streak the plate in the above manner as given in the figure. 6. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 7. When the bacterial colonies are arise. These are mixed in population. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 8. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 9. When the bacterial colonies are arise after second time streaking, this may contain some pure colony with other mixed colony. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 10. Again keep this streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 11. By repeating this way you will finally get the pure colony of bacteria after 4th or 5th streaking.

1. Prepare the L.B. agar plates or nutrients agar plates according to experiment number 12. 2. Take sample in beaker. Sample may be pond water or your drinking water or tap water or take small amount of soil and add water, etc. 3. Wipe the laminar hood properly with alcohol and switch on the UV light for 15 minutes. Keep only necessary things inside the laminar hood. 4. Take the inoculum loop in right hand and heat it for sterilization, cool it and take a sample by just dipping the inoculum loop into the sample. 5. Open the lid of petri plate near the spirit lamp and streak the plate in the above manner as given in the figure. 6. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 7. When the bacterial colonies are arise. These are mixed in population. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 8. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 9. When the bacterial colonies are arise after second time streaking, this may contain some pure colony with other mixed colony. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 10. Again keep this streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 11. By repeating this way you will finally get the pure colony of bacteria after 4th or 5th streaking.

Note: Keep the plate in inverted position or upside down position in the incubator otherwise vapour arise from the plate drops down on the colonies and the colonies will mix up.

Note: Keep the plate in inverted position or upside down position in the incubator otherwise vapour arise from the plate drops down on the colonies and the colonies will mix up.

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Procedure

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Procedure

1. Prepare the L.B. agar plates or nutrients agar plates according to experiment number 12. 2. Take sample in beaker. Sample may be pond water or your drinking water or tap water or take small amount of soil and add water, etc. 3. Wipe the laminar hood properly with alcohol and switch on the UV light for 15 minutes. Keep only necessary things inside the laminar hood. 4. Take the inoculum loop in right hand and heat it for sterilization, cool it and take a sample by just dipping the inoculum loop into the sample. 5. Open the lid of petri plate near the spirit lamp and streak the plate in the above manner as given in the figure. 6. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 7. When the bacterial colonies are arise. These are mixed in population. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 8. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 9. When the bacterial colonies are arise after second time streaking, this may contain some pure colony with other mixed colony. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 10. Again keep this streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 11. By repeating this way you will finally get the pure colony of bacteria after 4th or 5th streaking.

1. Prepare the L.B. agar plates or nutrients agar plates according to experiment number 12. 2. Take sample in beaker. Sample may be pond water or your drinking water or tap water or take small amount of soil and add water, etc. 3. Wipe the laminar hood properly with alcohol and switch on the UV light for 15 minutes. Keep only necessary things inside the laminar hood. 4. Take the inoculum loop in right hand and heat it for sterilization, cool it and take a sample by just dipping the inoculum loop into the sample. 5. Open the lid of petri plate near the spirit lamp and streak the plate in the above manner as given in the figure. 6. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 7. When the bacterial colonies are arise. These are mixed in population. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 8. Keep the streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 9. When the bacterial colonies are arise after second time streaking, this may contain some pure colony with other mixed colony. To get a pure culture pick up single colonies from this plate and streak the new fresh agar plate inside the laminar hood near the spirit lamp. 10. Again keep this streaked plate in inverted position or upside down position at 25°C-30°C for 24 to 48 hours in an incubator. 11. By repeating this way you will finally get the pure colony of bacteria after 4th or 5th streaking.

Note: Keep the plate in inverted position or upside down position in the incubator otherwise vapour arise from the plate drops down on the colonies and the colonies will mix up.

Note: Keep the plate in inverted position or upside down position in the incubator otherwise vapour arise from the plate drops down on the colonies and the colonies will mix up.

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208 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 14

EXPERIMENT NO. 14

Aim: To Determine the antibiotic sensitivity of bacteria in nutrient agar plate.

Aim: To Determine the antibiotic sensitivity of bacteria in nutrient agar plate.

Requirements: Nutrient agar plate, Over night bacterial culture, Metronidazole, ampicillin, Plant extract, Spirit lamp, forecep.

Requirements: Nutrient agar plate, Over night bacterial culture, Metronidazole, ampicillin, Plant extract, Spirit lamp, forecep.

Theory

Theory

With the help of this experiment students can check the antimicrobial activity of any plant extract. To check the antimicrobial potency of any plant, we use the plant extract and one or more antibiotics like ampicillin or tetracycline as standard. The antimicrobial activity of the plant is done by various methods like disc diffusion method, cup plate method, etc. In disc diffusion method make small disc of filter paper, dip some disc into the plant extract or sample and some in standard drug and keep into the agar at proper distance as shown in the figure. In case of cup plate method make a small hole into the agar plate and add small amount of sample and standard drug into these holes. Keep the plate in an incubator upside down position and after 24-48 hours check the zone of inhibition around the disc. Measure the zone of inhibition and compare with the standard drug.

With the help of this experiment students can check the antimicrobial activity of any plant extract. To check the antimicrobial potency of any plant, we use the plant extract and one or more antibiotics like ampicillin or tetracycline as standard. The antimicrobial activity of the plant is done by various methods like disc diffusion method, cup plate method, etc. In disc diffusion method make small disc of filter paper, dip some disc into the plant extract or sample and some in standard drug and keep into the agar at proper distance as shown in the figure. In case of cup plate method make a small hole into the agar plate and add small amount of sample and standard drug into these holes. Keep the plate in an incubator upside down position and after 24-48 hours check the zone of inhibition around the disc. Measure the zone of inhibition and compare with the standard drug.

Fig.12.16. Zone of inhibition (1) Plant extract, (2) Metronidazole, (3) Ampicillin. Ampicillin and metronidazole used as standard. (Concentration of plant extract is 50 zgm/mL or 100 zgm/ml, for concentration of antibiotics check the Table 3 in Chapter 11.

Fig.12.16. Zone of inhibition (1) Plant extract, (2) Metronidazole, (3) Ampicillin. Ampicillin and metronidazole used as standard. (Concentration of plant extract is 50 zgm/mL or 100 zgm/ml, for concentration of antibiotics check the Table 3 in Chapter 11.

Procedure

Procedure

1. Prepare the nutrient agar or L.B. agar media in the conical flask and properly sterilized into the autoclave. 2. Pour the media into the petri plate in the aseptic cabin or in laminar hood and left for solidifying.

208 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

1. Prepare the nutrient agar or L.B. agar media in the conical flask and properly sterilized into the autoclave. 2. Pour the media into the petri plate in the aseptic cabin or in laminar hood and left for solidifying.

208 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 14

EXPERIMENT NO. 14

Aim: To Determine the antibiotic sensitivity of bacteria in nutrient agar plate.

Aim: To Determine the antibiotic sensitivity of bacteria in nutrient agar plate.

Requirements: Nutrient agar plate, Over night bacterial culture, Metronidazole, ampicillin, Plant extract, Spirit lamp, forecep.

Requirements: Nutrient agar plate, Over night bacterial culture, Metronidazole, ampicillin, Plant extract, Spirit lamp, forecep.

Theory

Theory

With the help of this experiment students can check the antimicrobial activity of any plant extract. To check the antimicrobial potency of any plant, we use the plant extract and one or more antibiotics like ampicillin or tetracycline as standard. The antimicrobial activity of the plant is done by various methods like disc diffusion method, cup plate method, etc. In disc diffusion method make small disc of filter paper, dip some disc into the plant extract or sample and some in standard drug and keep into the agar at proper distance as shown in the figure. In case of cup plate method make a small hole into the agar plate and add small amount of sample and standard drug into these holes. Keep the plate in an incubator upside down position and after 24-48 hours check the zone of inhibition around the disc. Measure the zone of inhibition and compare with the standard drug.

With the help of this experiment students can check the antimicrobial activity of any plant extract. To check the antimicrobial potency of any plant, we use the plant extract and one or more antibiotics like ampicillin or tetracycline as standard. The antimicrobial activity of the plant is done by various methods like disc diffusion method, cup plate method, etc. In disc diffusion method make small disc of filter paper, dip some disc into the plant extract or sample and some in standard drug and keep into the agar at proper distance as shown in the figure. In case of cup plate method make a small hole into the agar plate and add small amount of sample and standard drug into these holes. Keep the plate in an incubator upside down position and after 24-48 hours check the zone of inhibition around the disc. Measure the zone of inhibition and compare with the standard drug.

Fig.12.16. Zone of inhibition (1) Plant extract, (2) Metronidazole, (3) Ampicillin. Ampicillin and metronidazole used as standard. (Concentration of plant extract is 50 zgm/mL or 100 zgm/ml, for concentration of antibiotics check the Table 3 in Chapter 11.

Fig.12.16. Zone of inhibition (1) Plant extract, (2) Metronidazole, (3) Ampicillin. Ampicillin and metronidazole used as standard. (Concentration of plant extract is 50 zgm/mL or 100 zgm/ml, for concentration of antibiotics check the Table 3 in Chapter 11.

Procedure 1. Prepare the nutrient agar or L.B. agar media in the conical flask and properly sterilized into the autoclave. 2. Pour the media into the petri plate in the aseptic cabin or in laminar hood and left for solidifying.

Procedure 1. Prepare the nutrient agar or L.B. agar media in the conical flask and properly sterilized into the autoclave. 2. Pour the media into the petri plate in the aseptic cabin or in laminar hood and left for solidifying.

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3. When the agar plates are solidify add 3 to 5 mL of bacterial culture, spread on the plate properly and left for 5 minute. After then tilt the plate and remove the remaining watery fluid with the help of micropipette and left for some time. 4. Make small disk of filter paper and dip into the plant extract and antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator at 300C to 350C. 5. After 24 to 48 hours of incubation, if the microorganism is susceptible for plant extract or antibiotic, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 6. If the microorganisms are resistant to antibiotic there is no zone of inhibition.

3. When the agar plates are solidify add 3 to 5 mL of bacterial culture, spread on the plate properly and left for 5 minute. After then tilt the plate and remove the remaining watery fluid with the help of micropipette and left for some time. 4. Make small disk of filter paper and dip into the plant extract and antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator at 300C to 350C. 5. After 24 to 48 hours of incubation, if the microorganism is susceptible for plant extract or antibiotic, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 6. If the microorganisms are resistant to antibiotic there is no zone of inhibition.

Result: The bacteria showing the susceptibility with plant extract (represented by 1) and ampicillin (represented by 3) while resistant with metronidazole (represented by 2) as shown in the figure.

Result: The bacteria showing the susceptibility with plant extract (represented by 1) and ampicillin (represented by 3) while resistant with metronidazole (represented by 2) as shown in the figure.

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3. When the agar plates are solidify add 3 to 5 mL of bacterial culture, spread on the plate properly and left for 5 minute. After then tilt the plate and remove the remaining watery fluid with the help of micropipette and left for some time. 4. Make small disk of filter paper and dip into the plant extract and antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator at 300C to 350C. 5. After 24 to 48 hours of incubation, if the microorganism is susceptible for plant extract or antibiotic, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 6. If the microorganisms are resistant to antibiotic there is no zone of inhibition.

3. When the agar plates are solidify add 3 to 5 mL of bacterial culture, spread on the plate properly and left for 5 minute. After then tilt the plate and remove the remaining watery fluid with the help of micropipette and left for some time. 4. Make small disk of filter paper and dip into the plant extract and antibiotic solution and keep into the surface of agar plate in a proper distance and keep the plate into the incubator at 300C to 350C. 5. After 24 to 48 hours of incubation, if the microorganism is susceptible for plant extract or antibiotic, zone of inhibition or clear zone is visible. Measure the diameter of the clear zone. 6. If the microorganisms are resistant to antibiotic there is no zone of inhibition.

Result: The bacteria showing the susceptibility with plant extract (represented by 1) and ampicillin (represented by 3) while resistant with metronidazole (represented by 2) as shown in the figure.

Result: The bacteria showing the susceptibility with plant extract (represented by 1) and ampicillin (represented by 3) while resistant with metronidazole (represented by 2) as shown in the figure.

210 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

210 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 15

EXPERIMENT NO. 15

Aim: To identify the patient suspected with pulmonary tuberculosis in the given sputum sample.

Aim: To identify the patient suspected with pulmonary tuberculosis in the given sputum sample.

Note: Tubercle bacilli are stained by acid fast staining because higher amount of mycolic acid are present in their cell wall.

Note: Tubercle bacilli are stained by acid fast staining because higher amount of mycolic acid are present in their cell wall.

Requirements: Glass slide, Spirit lamp, Sticking loop, overnight bacterial culture, carbolfuchsin, acid alcohol, methylene blue, immersion oil, distilled water, microscope.

Requirements: Glass slide, Spirit lamp, Sticking loop, overnight bacterial culture, carbolfuchsin, acid alcohol, methylene blue, immersion oil, distilled water, microscope.

Procedure

Procedure

1. Wash the glass slide properly and air dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear, air dry and heat fix the smear. 3. Flood the bacterial smear with carbolfuchsin and keep for 3 to 5 minutes above the water bath for steaming. 4. Cool the slide and wash with distilled water. 5. Decolorize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 6. Add the methylene blue and keep for 1 to 2 minutes. 7. Wash the slide with distilled water. 8. Air dries the slide and examine under the microscope using immersion oil.

1. Wash the glass slide properly and air dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear, air dry and heat fix the smear. 3. Flood the bacterial smear with carbolfuchsin and keep for 3 to 5 minutes above the water bath for steaming. 4. Cool the slide and wash with distilled water. 5. Decolorize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 6. Add the methylene blue and keep for 1 to 2 minutes. 7. Wash the slide with distilled water. 8. Air dries the slide and examine under the microscope using immersion oil.

Result and interpretation

Result and interpretation

Smear shows thin bright red, slightly curved bacilli with beaded appearance showing palisade arrangement. However, all other materials will take on the colour of counterstain (methylene blue), i.e., blue. Thin bright red slightly curved beaded appearance are the feature of human tubercle bacilli.

Smear shows thin bright red, slightly curved bacilli with beaded appearance showing palisade arrangement. However, all other materials will take on the colour of counterstain (methylene blue), i.e., blue. Thin bright red slightly curved beaded appearance are the feature of human tubercle bacilli.

210 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

210 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

EXPERIMENT NO. 15

EXPERIMENT NO. 15

Aim: To identify the patient suspected with pulmonary tuberculosis in the given sputum sample.

Aim: To identify the patient suspected with pulmonary tuberculosis in the given sputum sample.

Note: Tubercle bacilli are stained by acid fast staining because higher amount of mycolic acid are present in their cell wall.

Note: Tubercle bacilli are stained by acid fast staining because higher amount of mycolic acid are present in their cell wall.

Requirements: Glass slide, Spirit lamp, Sticking loop, overnight bacterial culture, carbolfuchsin, acid alcohol, methylene blue, immersion oil, distilled water, microscope.

Requirements: Glass slide, Spirit lamp, Sticking loop, overnight bacterial culture, carbolfuchsin, acid alcohol, methylene blue, immersion oil, distilled water, microscope.

Procedure

Procedure

1. Wash the glass slide properly and air dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear, air dry and heat fix the smear. 3. Flood the bacterial smear with carbolfuchsin and keep for 3 to 5 minutes above the water bath for steaming. 4. Cool the slide and wash with distilled water. 5. Decolorize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 6. Add the methylene blue and keep for 1 to 2 minutes. 7. Wash the slide with distilled water. 8. Air dries the slide and examine under the microscope using immersion oil.

1. Wash the glass slide properly and air dry. 2. Take a loopful of bacterial culture and spread it properly, i.e., make a thin bacterial smear, air dry and heat fix the smear. 3. Flood the bacterial smear with carbolfuchsin and keep for 3 to 5 minutes above the water bath for steaming. 4. Cool the slide and wash with distilled water. 5. Decolorize the smear with acid alcohol for 10 to 15 seconds or until the smear is a faint pink colour and wash the slide with distilled water. 6. Add the methylene blue and keep for 1 to 2 minutes. 7. Wash the slide with distilled water. 8. Air dries the slide and examine under the microscope using immersion oil.

Result and interpretation

Result and interpretation

Smear shows thin bright red, slightly curved bacilli with beaded appearance showing palisade arrangement. However, all other materials will take on the colour of counterstain (methylene blue), i.e., blue. Thin bright red slightly curved beaded appearance are the feature of human tubercle bacilli.

Smear shows thin bright red, slightly curved bacilli with beaded appearance showing palisade arrangement. However, all other materials will take on the colour of counterstain (methylene blue), i.e., blue. Thin bright red slightly curved beaded appearance are the feature of human tubercle bacilli.

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EXPERIMENT NO. 16

EXPERIMENT NO. 16

Aim: To study the transfer of microorganisms by aseptic technique. (a) Transfer of bacterial culture from one test tube to another fresh test tube. (b) Transfer of single bacterial colony from one petri plate to another fresh agar plate.

Aim: To study the transfer of microorganisms by aseptic technique. (a) Transfer of bacterial culture from one test tube to another fresh test tube. (b) Transfer of single bacterial colony from one petri plate to another fresh agar plate.

Requirements: Laminar air hood, Test tube/petri plate containing overnight bacterial culture, Fresh test tube/petri plate, Inoculating loop, Spirit lamp, etc.

Requirements: Laminar air hood, Test tube/petri plate containing overnight bacterial culture, Fresh test tube/petri plate, Inoculating loop, Spirit lamp, etc.

Theory

Theory

In natural environment, microorganisms usually exist as mixed population. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism. In working with microorganisms, we must have a sterile nutrient medium in which microorganisms to grow. Anything in or on which we grow microorganisms is termed a medium. A sterile medium is one that is free of all life forms (means free from microorganism). It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed. Finally, in working with microorganisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.

In natural environment, microorganisms usually exist as mixed population. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism. In working with microorganisms, we must have a sterile nutrient medium in which microorganisms to grow. Anything in or on which we grow microorganisms is termed a medium. A sterile medium is one that is free of all life forms (means free from microorganism). It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed. Finally, in working with microorganisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.

Forms of Culture Media

Forms of Culture Media

1. Broth tubes are tubes containing a liquid medium, such as nutrient broth media contains beef extract, peptone and sodium chloride and distilled water. It does not contain agar. After incubation, growth of bacteria (development of many cells from a few cells) may be observed as 1 or a combination of 3 forms: a. Pellicle: A mass of organisms is floating on top of the broth. b. Turbidity: The organisms appear as a general cloudiness throughout the broth. c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube broth 2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface. 3. Stab tubes (deeps) are tubes of hardened agar medium that are inoculated by “stabbing” the inoculum into the agar.

EXPERIMENTS

1. Broth tubes are tubes containing a liquid medium, such as nutrient broth media contains beef extract, peptone and sodium chloride and distilled water. It does not contain agar. After incubation, growth of bacteria (development of many cells from a few cells) may be observed as 1 or a combination of 3 forms: a. Pellicle: A mass of organisms is floating on top of the broth. b. Turbidity: The organisms appear as a general cloudiness throughout the broth. c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube broth 2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface. 3. Stab tubes (deeps) are tubes of hardened agar medium that are inoculated by “stabbing” the inoculum into the agar.

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EXPERIMENT NO. 16

EXPERIMENT NO. 16

Aim: To study the transfer of microorganisms by aseptic technique. (a) Transfer of bacterial culture from one test tube to another fresh test tube. (b) Transfer of single bacterial colony from one petri plate to another fresh agar plate.

Aim: To study the transfer of microorganisms by aseptic technique. (a) Transfer of bacterial culture from one test tube to another fresh test tube. (b) Transfer of single bacterial colony from one petri plate to another fresh agar plate.

Requirements: Laminar air hood, Test tube/petri plate containing overnight bacterial culture, Fresh test tube/petri plate, Inoculating loop, Spirit lamp, etc.

Requirements: Laminar air hood, Test tube/petri plate containing overnight bacterial culture, Fresh test tube/petri plate, Inoculating loop, Spirit lamp, etc.

Theory

Theory

In natural environment, microorganisms usually exist as mixed population. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism. In working with microorganisms, we must have a sterile nutrient medium in which microorganisms to grow. Anything in or on which we grow microorganisms is termed a medium. A sterile medium is one that is free of all life forms (means free from microorganism). It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed. Finally, in working with microorganisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.

In natural environment, microorganisms usually exist as mixed population. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism. In working with microorganisms, we must have a sterile nutrient medium in which microorganisms to grow. Anything in or on which we grow microorganisms is termed a medium. A sterile medium is one that is free of all life forms (means free from microorganism). It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed. Finally, in working with microorganisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.

Forms of Culture Media

Forms of Culture Media

1. Broth tubes are tubes containing a liquid medium, such as nutrient broth media contains beef extract, peptone and sodium chloride and distilled water. It does not contain agar. After incubation, growth of bacteria (development of many cells from a few cells) may be observed as 1 or a combination of 3 forms: a. Pellicle: A mass of organisms is floating on top of the broth. b. Turbidity: The organisms appear as a general cloudiness throughout the broth. c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube broth 2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface. 3. Stab tubes (deeps) are tubes of hardened agar medium that are inoculated by “stabbing” the inoculum into the agar.

1. Broth tubes are tubes containing a liquid medium, such as nutrient broth media contains beef extract, peptone and sodium chloride and distilled water. It does not contain agar. After incubation, growth of bacteria (development of many cells from a few cells) may be observed as 1 or a combination of 3 forms: a. Pellicle: A mass of organisms is floating on top of the broth. b. Turbidity: The organisms appear as a general cloudiness throughout the broth. c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube broth 2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface. 3. Stab tubes (deeps) are tubes of hardened agar medium that are inoculated by “stabbing” the inoculum into the agar.

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Fig. 12.17. Test tube showing turbidity and sedimentation.

4. Agar plates are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms.

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Fig. 12.17. Test tube showing turbidity and sedimentation.

4. Agar plates are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms.

Aseptic Technique

Aseptic Technique

Note: Make a fresh test tubes and agar plates as given in previous experiments. Give the bacterial culture into the test tube and streak the agar plate. Keep them into the incubator for the overnight growth. Next day when growth of bacteria is visible after overnight incubation ready for the transfer of bacterial culture into fresh test tube/petri plate. The procedure for aseptically transferring microorganisms is as followsSterilize the inoculating loop. The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way, all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum. A. Transfer of inoculum from one test tube containing bacterial culture to another fresh test tube having broth. (Broth means liquid medium) i. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil. ii. Remove the cap of the pure culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.

Note: Make a fresh test tubes and agar plates as given in previous experiments. Give the bacterial culture into the test tube and streak the agar plate. Keep them into the incubator for the overnight growth. Next day when growth of bacteria is visible after overnight incubation ready for the transfer of bacterial culture into fresh test tube/petri plate. The procedure for aseptically transferring microorganisms is as followsSterilize the inoculating loop. The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way, all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum. A. Transfer of inoculum from one test tube containing bacterial culture to another fresh test tube having broth. (Broth means liquid medium) i. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil. ii. Remove the cap of the pure culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.

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212 A TEXTBOOK OF PHARMACEUTICAL MICROBIOLOGY

Fig. 12.17. Test tube showing turbidity and sedimentation.

4. Agar plates are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms.

Fig. 12.17. Test tube showing turbidity and sedimentation.

4. Agar plates are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms.

Aseptic Technique

Aseptic Technique

Note: Make a fresh test tubes and agar plates as given in previous experiments. Give the bacterial culture into the test tube and streak the agar plate. Keep them into the incubator for the overnight growth. Next day when growth of bacteria is visible after overnight incubation ready for the transfer of bacterial culture into fresh test tube/petri plate. The procedure for aseptically transferring microorganisms is as followsSterilize the inoculating loop. The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way, all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum. A. Transfer of inoculum from one test tube containing bacterial culture to another fresh test tube having broth. (Broth means liquid medium) i. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil. ii. Remove the cap of the pure culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.

Note: Make a fresh test tubes and agar plates as given in previous experiments. Give the bacterial culture into the test tube and streak the agar plate. Keep them into the incubator for the overnight growth. Next day when growth of bacteria is visible after overnight incubation ready for the transfer of bacterial culture into fresh test tube/petri plate. The procedure for aseptically transferring microorganisms is as followsSterilize the inoculating loop. The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way, all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum. A. Transfer of inoculum from one test tube containing bacterial culture to another fresh test tube having broth. (Broth means liquid medium) i. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil. ii. Remove the cap of the pure culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.

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iii. Very briefly hold a flame to the lip of the culture tube. This creates a convection current that forces air out of the tube and preventing airborne contaminants from entering the tube. The heat of the gas burner also causes the air around your work area to rise, and this also reduces the chance of airborne microorganisms contaminating your cultures. iv. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculums. v. Again hold a flame the lip of the culture tube. Replace the cap and keep the test tube in the test tube holder. vi. Take the next test tube where you have to transfer the culture. Place the loopful of inoculum into this broth tube, and withdraw the loop. Do not lay the loop down. vii. Again hold a flame the lip of the culture tube and replace the cap. viii. Resterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again. (B) Transfer the bacterial colony from one plate (organisms growing on an agar surface in a petri plate) to another fresh agar plate: i. Sterilize the inoculating loop in the flame of a gas burner.

Fig. 12.18. Transfer of bacterial colony from one petri plate to another.

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iii. Very briefly hold a flame to the lip of the culture tube. This creates a convection current that forces air out of the tube and preventing airborne contaminants from entering the tube. The heat of the gas burner also causes the air around your work area to rise, and this also reduces the chance of airborne microorganisms contaminating your cultures. iv. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculums. v. Again hold a flame the lip of the culture tube. Replace the cap and keep the test tube in the test tube holder. vi. Take the next test tube where you have to transfer the culture. Place the loopful of inoculum into this broth tube, and withdraw the loop. Do not lay the loop down. vii. Again hold a flame the lip of the culture tube and replace the cap. viii. Resterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again. (B) Transfer the bacterial colony from one plate (organisms growing on an agar surface in a petri plate) to another fresh agar plate: i. Sterilize the inoculating loop in the flame of a gas burner.

Fig. 12.18. Transfer of bacterial colony from one petri plate to another.

ii. Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop. iii. Scrape off a small amount of the organisms as shown in the above figure and close the lid. iv. Take another fresh agar petri plate and streak the loop across the surface of the agar medium. Which allow you to obtain single isolated bacterial colonies originating from a single bacterium. v. Remove the loop and close the lid and keep into the incubator for overnight. vi. Resterilize the inoculating loop.

ii. Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop. iii. Scrape off a small amount of the organisms as shown in the above figure and close the lid. iv. Take another fresh agar petri plate and streak the loop across the surface of the agar medium. Which allow you to obtain single isolated bacterial colonies originating from a single bacterium. v. Remove the loop and close the lid and keep into the incubator for overnight. vi. Resterilize the inoculating loop.

In the future, every procedure in the lab will be done using similar aseptic technique.

In the future, every procedure in the lab will be done using similar aseptic technique.

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iii. Very briefly hold a flame to the lip of the culture tube. This creates a convection current that forces air out of the tube and preventing airborne contaminants from entering the tube. The heat of the gas burner also causes the air around your work area to rise, and this also reduces the chance of airborne microorganisms contaminating your cultures. iv. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculums. v. Again hold a flame the lip of the culture tube. Replace the cap and keep the test tube in the test tube holder. vi. Take the next test tube where you have to transfer the culture. Place the loopful of inoculum into this broth tube, and withdraw the loop. Do not lay the loop down. vii. Again hold a flame the lip of the culture tube and replace the cap. viii. Resterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again. (B) Transfer the bacterial colony from one plate (organisms growing on an agar surface in a petri plate) to another fresh agar plate: i. Sterilize the inoculating loop in the flame of a gas burner.

Fig. 12.18. Transfer of bacterial colony from one petri plate to another.

EXPERIMENTS

213

iii. Very briefly hold a flame to the lip of the culture tube. This creates a convection current that forces air out of the tube and preventing airborne contaminants from entering the tube. The heat of the gas burner also causes the air around your work area to rise, and this also reduces the chance of airborne microorganisms contaminating your cultures. iv. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculums. v. Again hold a flame the lip of the culture tube. Replace the cap and keep the test tube in the test tube holder. vi. Take the next test tube where you have to transfer the culture. Place the loopful of inoculum into this broth tube, and withdraw the loop. Do not lay the loop down. vii. Again hold a flame the lip of the culture tube and replace the cap. viii. Resterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again. (B) Transfer the bacterial colony from one plate (organisms growing on an agar surface in a petri plate) to another fresh agar plate: i. Sterilize the inoculating loop in the flame of a gas burner.

Fig. 12.18. Transfer of bacterial colony from one petri plate to another.

ii. Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop. iii. Scrape off a small amount of the organisms as shown in the above figure and close the lid. iv. Take another fresh agar petri plate and streak the loop across the surface of the agar medium. Which allow you to obtain single isolated bacterial colonies originating from a single bacterium. v. Remove the loop and close the lid and keep into the incubator for overnight. vi. Resterilize the inoculating loop.

ii. Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop. iii. Scrape off a small amount of the organisms as shown in the above figure and close the lid. iv. Take another fresh agar petri plate and streak the loop across the surface of the agar medium. Which allow you to obtain single isolated bacterial colonies originating from a single bacterium. v. Remove the loop and close the lid and keep into the incubator for overnight. vi. Resterilize the inoculating loop.

In the future, every procedure in the lab will be done using similar aseptic technique.

In the future, every procedure in the lab will be done using similar aseptic technique.

References

1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13.

14.

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Abrutyn, E., Goldmann, D. and Scheckler, W., eds. Saunders infection control reference service, 2nd edition, Philadelphia, Saunders, 2001. AIDS/TB Committee of the Society of Health Care Epidemiology of America. Management of healthcare workers infected with hepatitis B virus, hepatitis C virus, human immunodeficiency virus or other blood-borne pathogens. Infect Control Hosp Epidemiol, 1997, 18: 347–363. Atlas, R.M., Parks, L.C. and Press C.R.C. Handbook of Microbiological Media, London. 1993. Benacerraf, B. et al. A History of Bacteriology and Immunology, William Heinemann, London, 1980. Bhatia, Rajesh, and Ichhpujani, R.L. Essential of Medical Microbiology, Jaypee Brothers medical Publishers (P) Ltd., 2nd edition, 1999. Blackmore, J.A. and Parry, T.E. Microbiological assay of amino acids in serum: valine, leucine and methionine, J. Clin. Path., 1972, 25: 171-175. Carter, S.J. Cooper and Gunn’s Tutorial Pharmacy, CBS Publishers and Distributors, New Delhi, 6th edition, reprint 2006. CDC guidelines for infection control in hospital personnel. Am. J. Infect. Control, 1998, 26: 289-354 or Infect Control Hosp Epidemiol 1996. 17: 438-473. Chattergoon, M. et al. Genetic Immunization: A New Era in Vaccines and Immune Therapies, FASEB J. 11: 754-60, 1997. Dassa, E. ABC Transport : In : Encyclopedia of Microbiology, 2nd edition, Vol. 1, Lederberg J, Ed-in-Chief, 1-12, Academic Press, San Diego, 2000. Denyer, S.P. et al. Filtration Sterilization: In Principles and Practice of Disinfection, Preservation and Sterilization (ed. Russell, A.D. et al.) Blackwell Scientific Publications, Oxford (UK), 1982. Ducel, G., Fabry, J. and Nicolle L. Prevention of hospital-acquired infections, A PRACTICAL GUIDE, 2nd edition, WHO/CDS/CSR/EPH/2002.12. Griffiths, William M. Miller Jeffrey, H. and Suzuki David, T. et al. Genetics and the organism: Introduction, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey H. and Suzuki David, T. et al. Interaction between the alleles of one gene, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Human Genetics, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Bacterial conjugation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al., Bacterial Transformation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000.

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Abrutyn, E., Goldmann, D. and Scheckler, W., eds. Saunders infection control reference service, 2nd edition, Philadelphia, Saunders, 2001. AIDS/TB Committee of the Society of Health Care Epidemiology of America. Management of healthcare workers infected with hepatitis B virus, hepatitis C virus, human immunodeficiency virus or other blood-borne pathogens. Infect Control Hosp Epidemiol, 1997, 18: 347–363. Atlas, R.M., Parks, L.C. and Press C.R.C. Handbook of Microbiological Media, London. 1993. Benacerraf, B. et al. A History of Bacteriology and Immunology, William Heinemann, London, 1980. Bhatia, Rajesh, and Ichhpujani, R.L. Essential of Medical Microbiology, Jaypee Brothers medical Publishers (P) Ltd., 2nd edition, 1999. Blackmore, J.A. and Parry, T.E. Microbiological assay of amino acids in serum: valine, leucine and methionine, J. Clin. Path., 1972, 25: 171-175. Carter, S.J. Cooper and Gunn’s Tutorial Pharmacy, CBS Publishers and Distributors, New Delhi, 6th edition, reprint 2006. CDC guidelines for infection control in hospital personnel. Am. J. Infect. Control, 1998, 26: 289-354 or Infect Control Hosp Epidemiol 1996. 17: 438-473. Chattergoon, M. et al. Genetic Immunization: A New Era in Vaccines and Immune Therapies, FASEB J. 11: 754-60, 1997. Dassa, E. ABC Transport : In : Encyclopedia of Microbiology, 2nd edition, Vol. 1, Lederberg J, Ed-in-Chief, 1-12, Academic Press, San Diego, 2000. Denyer, S.P. et al. Filtration Sterilization: In Principles and Practice of Disinfection, Preservation and Sterilization (ed. Russell, A.D. et al.) Blackwell Scientific Publications, Oxford (UK), 1982. Ducel, G., Fabry, J. and Nicolle L. Prevention of hospital-acquired infections, A PRACTICAL GUIDE, 2nd edition, WHO/CDS/CSR/EPH/2002.12. Griffiths, William M. Miller Jeffrey, H. and Suzuki David, T. et al. Genetics and the organism: Introduction, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey H. and Suzuki David, T. et al. Interaction between the alleles of one gene, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Human Genetics, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Bacterial conjugation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al., Bacterial Transformation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000.

Abrutyn, E., Goldmann, D. and Scheckler, W., eds. Saunders infection control reference service, 2nd edition, Philadelphia, Saunders, 2001. AIDS/TB Committee of the Society of Health Care Epidemiology of America. Management of healthcare workers infected with hepatitis B virus, hepatitis C virus, human immunodeficiency virus or other blood-borne pathogens. Infect Control Hosp Epidemiol, 1997, 18: 347–363. Atlas, R.M., Parks, L.C. and Press C.R.C. Handbook of Microbiological Media, London. 1993. Benacerraf, B. et al. A History of Bacteriology and Immunology, William Heinemann, London, 1980. Bhatia, Rajesh, and Ichhpujani, R.L. Essential of Medical Microbiology, Jaypee Brothers medical Publishers (P) Ltd., 2nd edition, 1999. Blackmore, J.A. and Parry, T.E. Microbiological assay of amino acids in serum: valine, leucine and methionine, J. Clin. Path., 1972, 25: 171-175. Carter, S.J. Cooper and Gunn’s Tutorial Pharmacy, CBS Publishers and Distributors, New Delhi, 6th edition, reprint 2006. CDC guidelines for infection control in hospital personnel. Am. J. Infect. Control, 1998, 26: 289-354 or Infect Control Hosp Epidemiol 1996. 17: 438-473. Chattergoon, M. et al. Genetic Immunization: A New Era in Vaccines and Immune Therapies, FASEB J. 11: 754-60, 1997. Dassa, E. ABC Transport : In : Encyclopedia of Microbiology, 2nd edition, Vol. 1, Lederberg J, Ed-in-Chief, 1-12, Academic Press, San Diego, 2000. Denyer, S.P. et al. Filtration Sterilization: In Principles and Practice of Disinfection, Preservation and Sterilization (ed. Russell, A.D. et al.) Blackwell Scientific Publications, Oxford (UK), 1982. Ducel, G., Fabry, J. and Nicolle L. Prevention of hospital-acquired infections, A PRACTICAL GUIDE, 2nd edition, WHO/CDS/CSR/EPH/2002.12. Griffiths, William M. Miller Jeffrey, H. and Suzuki David, T. et al. Genetics and the organism: Introduction, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey H. and Suzuki David, T. et al. Interaction between the alleles of one gene, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Human Genetics, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Bacterial conjugation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al., Bacterial Transformation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000.

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3. 4. 5. 6. 7. 8. 9. 10. 11.

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Abrutyn, E., Goldmann, D. and Scheckler, W., eds. Saunders infection control reference service, 2nd edition, Philadelphia, Saunders, 2001. AIDS/TB Committee of the Society of Health Care Epidemiology of America. Management of healthcare workers infected with hepatitis B virus, hepatitis C virus, human immunodeficiency virus or other blood-borne pathogens. Infect Control Hosp Epidemiol, 1997, 18: 347–363. Atlas, R.M., Parks, L.C. and Press C.R.C. Handbook of Microbiological Media, London. 1993. Benacerraf, B. et al. A History of Bacteriology and Immunology, William Heinemann, London, 1980. Bhatia, Rajesh, and Ichhpujani, R.L. Essential of Medical Microbiology, Jaypee Brothers medical Publishers (P) Ltd., 2nd edition, 1999. Blackmore, J.A. and Parry, T.E. Microbiological assay of amino acids in serum: valine, leucine and methionine, J. Clin. Path., 1972, 25: 171-175. Carter, S.J. Cooper and Gunn’s Tutorial Pharmacy, CBS Publishers and Distributors, New Delhi, 6th edition, reprint 2006. CDC guidelines for infection control in hospital personnel. Am. J. Infect. Control, 1998, 26: 289-354 or Infect Control Hosp Epidemiol 1996. 17: 438-473. Chattergoon, M. et al. Genetic Immunization: A New Era in Vaccines and Immune Therapies, FASEB J. 11: 754-60, 1997. Dassa, E. ABC Transport : In : Encyclopedia of Microbiology, 2nd edition, Vol. 1, Lederberg J, Ed-in-Chief, 1-12, Academic Press, San Diego, 2000. Denyer, S.P. et al. Filtration Sterilization: In Principles and Practice of Disinfection, Preservation and Sterilization (ed. Russell, A.D. et al.) Blackwell Scientific Publications, Oxford (UK), 1982. Ducel, G., Fabry, J. and Nicolle L. Prevention of hospital-acquired infections, A PRACTICAL GUIDE, 2nd edition, WHO/CDS/CSR/EPH/2002.12. Griffiths, William M. Miller Jeffrey, H. and Suzuki David, T. et al. Genetics and the organism: Introduction, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey H. and Suzuki David, T. et al. Interaction between the alleles of one gene, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Human Genetics, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al. Bacterial conjugation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000. Griffiths, William M., Miller Jeffrey, H. and Suzuki David, T. et al., Bacterial Transformation, An Introduction to Genetic Analysis, New York: W.H. Freeman, 7th edition, 2000.

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Index

A Acid fast staining, 28 Acids, production of, 6 Actinomycetes, 41 classification of, 20 cultivation of, 55 isolation of, 55 nutritional habit of, 55 Active immunity, 152 Alternative pathway, 143 Amino acids, microbial assay of, 177 Animal cell, structure of, 16 Animal inoculation, 59 Antibiotics, antimicrobial assay of, 167 production of, 4 Antibody, structure of, 148 Antibody-dependent hypersensitivity, 162 Antigen, 145 characteristics of, 145 Antigen-antibody reaction, 154 Antigenicity, 145 Antiseptics, 108 Artificially acquired active immunity, 153

Artificially acquired passive immunity, 153 Autoclave, structure of, 200 Autoimmunity, 160 Autotrophic bacteria, 41 B Bacteria, 41 agriculture used in, 7 classification of, 21 cultivation of, 45 dairy products used in, 6 isolation of, 48 Bacterial cell structure, description of, 10 Bacterial cell, shape of, 14 structure of, 9 Bacterial flagella, structure of, 12 Balance, structure of, 192 Biological methods of selection, 50 Bright field microscopy, 32, 183 Bunsen burner, 186 C Calcium pantothenate, microbial assay of, 176

Index

A Acid fast staining, 28 Acids, production of, 6 Actinomycetes, 41 classification of, 20 cultivation of, 55 isolation of, 55 nutritional habit of, 55 Active immunity, 152 Alternative pathway, 143 Amino acids, microbial assay of, 177 Animal cell, structure of, 16 Animal inoculation, 59 Antibiotics, antimicrobial assay of, 167 production of, 4 Antibody, structure of, 148 Antibody-dependent hypersensitivity, 162 Antigen, 145 characteristics of, 145 Antigen-antibody reaction, 154 Antigenicity, 145 Antiseptics, 108 Artificially acquired active immunity, 153

Index

A Acid fast staining, 28 Acids, production of, 6 Actinomycetes, 41 classification of, 20 cultivation of, 55 isolation of, 55 nutritional habit of, 55 Active immunity, 152 Alternative pathway, 143 Amino acids, microbial assay of, 177 Animal cell, structure of, 16 Animal inoculation, 59 Antibiotics, antimicrobial assay of, 167 production of, 4 Antibody, structure of, 148 Antibody-dependent hypersensitivity, 162 Antigen, 145 characteristics of, 145 Antigen-antibody reaction, 154 Antigenicity, 145 Antiseptics, 108 Artificially acquired active immunity, 153

Artificially acquired passive immunity, 153 Autoclave, structure of, 200 Autoimmunity, 160 Autotrophic bacteria, 41 B Bacteria, 41 agriculture used in, 7 classification of, 21 cultivation of, 45 dairy products used in, 6 isolation of, 48 Bacterial cell structure, description of, 10 Bacterial cell, shape of, 14 structure of, 9 Bacterial flagella, structure of, 12 Balance, structure of, 192 Biological methods of selection, 50 Bright field microscopy, 32, 183 Bunsen burner, 186 C Calcium pantothenate, microbial assay of, 176

Artificially acquired passive immunity, 153 Autoclave, structure of, 200 Autoimmunity, 160 Autotrophic bacteria, 41 B Bacteria, 41 agriculture used in, 7 classification of, 21 cultivation of, 45 dairy products used in, 6 isolation of, 48 Bacterial cell structure, description of, 10 Bacterial cell, shape of, 14 structure of, 9 Bacterial flagella, structure of, 12 Balance, structure of, 192 Biological methods of selection, 50 Bright field microscopy, 32, 183 Bunsen burner, 186 C Calcium pantothenate, microbial assay of, 176

Index

A Acid fast staining, 28 Acids, production of, 6 Actinomycetes, 41 classification of, 20 cultivation of, 55 isolation of, 55 nutritional habit of, 55 Active immunity, 152 Alternative pathway, 143 Amino acids, microbial assay of, 177 Animal cell, structure of, 16 Animal inoculation, 59 Antibiotics, antimicrobial assay of, 167 production of, 4 Antibody, structure of, 148 Antibody-dependent hypersensitivity, 162 Antigen, 145 characteristics of, 145 Antigen-antibody reaction, 154 Antigenicity, 145 Antiseptics, 108 Artificially acquired active immunity, 153

Artificially acquired passive immunity, 153 Autoclave, structure of, 200 Autoimmunity, 160 Autotrophic bacteria, 41 B Bacteria, 41 agriculture used in, 7 classification of, 21 cultivation of, 45 dairy products used in, 6 isolation of, 48 Bacterial cell structure, description of, 10 Bacterial cell, shape of, 14 structure of, 9 Bacterial flagella, structure of, 12 Balance, structure of, 192 Biological methods of selection, 50 Bright field microscopy, 32, 183 Bunsen burner, 186 C Calcium pantothenate, microbial assay of, 176

218 INDEX Capacity use-dilution test, 107 Capsule staining, 29 Cell, history of, 9 Cell-mediated, 163 Cellular immunity, 153 Chemical method of selection, 50 Chemoautotrophic bacteria, 42 Chemosynthetic bacteria, 42 Chick embryo, 59 Chick Martin test, 106 Chromosomes, 63 Classical pathway, 141 Clostridium acetobutylicum, 5 Clostridium tetani, 13 Complement system, 140 Compound microscope, 32 Conjugation, 83 Conventional clean room system, 125 Copland’s four-kingdom, classification of, 19 Cross reactivity, 146, 156 Cultivation, 41 Culture media, 92, 201 forms of, 211 Cylindrical, 15 D Dark field microscopy, 34 Delayed-type hypersensitivity (DTH), 163 Deoxyribonucleic acid (DNA), 65 Diluting fluids, 95 Disinfectants, 103 evaluation of, 105 Disinfection process, 104 DNA replication, process of, 76 DNA, amplification of, 87 quantification of, 87

218 INDEX E Electron microscope, 35 Endospore staining, 28 Endotoxin, 143 Enzymes, production of, 6 Epitopes, 146 Eukaryotic cell, 17 structure of, 16 Exotoxin, 143 F Factory hygiene, 124 Filter paper test, 107 Filtration, sterilization by, 120 Five-kingdom classification, 20 Flagella staining, 29 Flagella, function of, 13 Fluorescence microscopy, 34 Fungi, 41 cultivation of, 56 isolation of, 56 nutritional habit of, 56 G Gaseous sterilization, application of, 124 Gene transfer, process of, 82 Gene, 64 Genetic code, 74 Genotype, 74 Gram staining, 26 procedure of, 27 Gram-negative bacteria, 27 cell wall of, 10 Gram-positive bacteria, 27 cell wall of, 10 Growth curve, 44

218 INDEX Capacity use-dilution test, 107 Capsule staining, 29 Cell, history of, 9 Cell-mediated, 163 Cellular immunity, 153 Chemical method of selection, 50 Chemoautotrophic bacteria, 42 Chemosynthetic bacteria, 42 Chick embryo, 59 Chick Martin test, 106 Chromosomes, 63 Classical pathway, 141 Clostridium acetobutylicum, 5 Clostridium tetani, 13 Complement system, 140 Compound microscope, 32 Conjugation, 83 Conventional clean room system, 125 Copland’s four-kingdom, classification of, 19 Cross reactivity, 146, 156 Cultivation, 41 Culture media, 92, 201 forms of, 211 Cylindrical, 15 D Dark field microscopy, 34 Delayed-type hypersensitivity (DTH), 163 Deoxyribonucleic acid (DNA), 65 Diluting fluids, 95 Disinfectants, 103 evaluation of, 105 Disinfection process, 104 DNA replication, process of, 76 DNA, amplification of, 87 quantification of, 87

Capacity use-dilution test, 107 Capsule staining, 29 Cell, history of, 9 Cell-mediated, 163 Cellular immunity, 153 Chemical method of selection, 50 Chemoautotrophic bacteria, 42 Chemosynthetic bacteria, 42 Chick embryo, 59 Chick Martin test, 106 Chromosomes, 63 Classical pathway, 141 Clostridium acetobutylicum, 5 Clostridium tetani, 13 Complement system, 140 Compound microscope, 32 Conjugation, 83 Conventional clean room system, 125 Copland’s four-kingdom, classification of, 19 Cross reactivity, 146, 156 Cultivation, 41 Culture media, 92, 201 forms of, 211 Cylindrical, 15 D Dark field microscopy, 34 Delayed-type hypersensitivity (DTH), 163 Deoxyribonucleic acid (DNA), 65 Diluting fluids, 95 Disinfectants, 103 evaluation of, 105 Disinfection process, 104 DNA replication, process of, 76 DNA, amplification of, 87 quantification of, 87

E Electron microscope, 35 Endospore staining, 28 Endotoxin, 143 Enzymes, production of, 6 Epitopes, 146 Eukaryotic cell, 17 structure of, 16 Exotoxin, 143 F Factory hygiene, 124 Filter paper test, 107 Filtration, sterilization by, 120 Five-kingdom classification, 20 Flagella staining, 29 Flagella, function of, 13 Fluorescence microscopy, 34 Fungi, 41 cultivation of, 56 isolation of, 56 nutritional habit of, 56 G Gaseous sterilization, application of, 124 Gene transfer, process of, 82 Gene, 64 Genetic code, 74 Genotype, 74 Gram staining, 26 procedure of, 27 Gram-negative bacteria, 27 cell wall of, 10 Gram-positive bacteria, 27 cell wall of, 10 Growth curve, 44

218 INDEX E Electron microscope, 35 Endospore staining, 28 Endotoxin, 143 Enzymes, production of, 6 Epitopes, 146 Eukaryotic cell, 17 structure of, 16 Exotoxin, 143 F Factory hygiene, 124 Filter paper test, 107 Filtration, sterilization by, 120 Five-kingdom classification, 20 Flagella staining, 29 Flagella, function of, 13 Fluorescence microscopy, 34 Fungi, 41 cultivation of, 56 isolation of, 56 nutritional habit of, 56 G Gaseous sterilization, application of, 124 Gene transfer, process of, 82 Gene, 64 Genetic code, 74 Genotype, 74 Gram staining, 26 procedure of, 27 Gram-negative bacteria, 27 cell wall of, 10 Gram-positive bacteria, 27 cell wall of, 10 Growth curve, 44

Capacity use-dilution test, 107 Capsule staining, 29 Cell, history of, 9 Cell-mediated, 163 Cellular immunity, 153 Chemical method of selection, 50 Chemoautotrophic bacteria, 42 Chemosynthetic bacteria, 42 Chick embryo, 59 Chick Martin test, 106 Chromosomes, 63 Classical pathway, 141 Clostridium acetobutylicum, 5 Clostridium tetani, 13 Complement system, 140 Compound microscope, 32 Conjugation, 83 Conventional clean room system, 125 Copland’s four-kingdom, classification of, 19 Cross reactivity, 146, 156 Cultivation, 41 Culture media, 92, 201 forms of, 211 Cylindrical, 15 D Dark field microscopy, 34 Delayed-type hypersensitivity (DTH), 163 Deoxyribonucleic acid (DNA), 65 Diluting fluids, 95 Disinfectants, 103 evaluation of, 105 Disinfection process, 104 DNA replication, process of, 76 DNA, amplification of, 87 quantification of, 87

E Electron microscope, 35 Endospore staining, 28 Endotoxin, 143 Enzymes, production of, 6 Epitopes, 146 Eukaryotic cell, 17 structure of, 16 Exotoxin, 143 F Factory hygiene, 124 Filter paper test, 107 Filtration, sterilization by, 120 Five-kingdom classification, 20 Flagella staining, 29 Flagella, function of, 13 Fluorescence microscopy, 34 Fungi, 41 cultivation of, 56 isolation of, 56 nutritional habit of, 56 G Gaseous sterilization, application of, 124 Gene transfer, process of, 82 Gene, 64 Genetic code, 74 Genotype, 74 Gram staining, 26 procedure of, 27 Gram-negative bacteria, 27 cell wall of, 10 Gram-positive bacteria, 27 cell wall of, 10 Growth curve, 44

INDEX

H Hanging droplet method, 195 Heterotrophic bacteria, 43 Horizontal laminar air flow, structure of, 188 Hospital hygiene, 124 Hospital infection, control of, 130 Hot air oven, 190 Human virus, 58 Humoral immunity, 153 Hybridoma technology, 150 Hypersensitivity reaction, 161 I Ideal disinfectant, properties of, 104 IgM antibody, pentameric structure of, 149 Immediate hypersensitivity, 161 Immune complex, 163 Immunogen, 145 Immunoglobulin A, 149 Immunoglobulin classes, 148 Immunoglobulin D, 150 Immunoglobulin E, 150 Immunoglobulin G, 148 Immunoglobulin M, 149 Immunogrenicity, 145 Immunological tolerance, 159 Immunology, history of, 137 Incubator, structure of, 189 Inflammation, 144 Inoculation loop, 186 Inoculation needle, 186 Inoculum, preparation of, 168 Ionizing radiation, sterilization by, 118 Isolation, 41

L Laminar air flow, 126, 188 M Membrane filtration, 95 Messenger RNA (mRNA), 70 Microbes, classification of, 19 identification of, 25 Microbial death kinetics, 112 Microbial genetics, 63 Microbiology laboratory safety rules and procedures, 181 Microbiology, 3 scope of, 4 Microorganisms, role of, 4 Microscopes, structure of, 32, 184 types of, 32, 183 Microscopy, 30 Minimum inhibitory concentration, determination of, 107 Monoclonal antibodies, applications of, 150 preparation of, 150 Mucous membranes, 140 Mutation, molecular basis of, 89 N Naturally acquired active immunity, 152 Naturally acquired passive immunity, 153 Negative staining, 30 Nitrosomonas, 13 Non-specific defence mechanisms, 140 Nosocomial infection, 129 prevention of, 130 route of transmission, 129 Numerical aperture, 33 Nutrition, 41

INDEX

H Hanging droplet method, 195 Heterotrophic bacteria, 43 Horizontal laminar air flow, structure of, 188 Hospital hygiene, 124 Hospital infection, control of, 130 Hot air oven, 190 Human virus, 58 Humoral immunity, 153 Hybridoma technology, 150 Hypersensitivity reaction, 161 I Ideal disinfectant, properties of, 104 IgM antibody, pentameric structure of, 149 Immediate hypersensitivity, 161 Immune complex, 163 Immunogen, 145 Immunoglobulin A, 149 Immunoglobulin classes, 148 Immunoglobulin D, 150 Immunoglobulin E, 150 Immunoglobulin G, 148 Immunoglobulin M, 149 Immunogrenicity, 145 Immunological tolerance, 159 Immunology, history of, 137 Incubator, structure of, 189 Inflammation, 144 Inoculation loop, 186 Inoculation needle, 186 Inoculum, preparation of, 168 Ionizing radiation, sterilization by, 118 Isolation, 41

219

INDEX

H Hanging droplet method, 195 Heterotrophic bacteria, 43 Horizontal laminar air flow, structure of, 188 Hospital hygiene, 124 Hospital infection, control of, 130 Hot air oven, 190 Human virus, 58 Humoral immunity, 153 Hybridoma technology, 150 Hypersensitivity reaction, 161 I Ideal disinfectant, properties of, 104 IgM antibody, pentameric structure of, 149 Immediate hypersensitivity, 161 Immune complex, 163 Immunogen, 145 Immunoglobulin A, 149 Immunoglobulin classes, 148 Immunoglobulin D, 150 Immunoglobulin E, 150 Immunoglobulin G, 148 Immunoglobulin M, 149 Immunogrenicity, 145 Immunological tolerance, 159 Immunology, history of, 137 Incubator, structure of, 189 Inflammation, 144 Inoculation loop, 186 Inoculation needle, 186 Inoculum, preparation of, 168 Ionizing radiation, sterilization by, 118 Isolation, 41

219

L Laminar air flow, 126, 188 M Membrane filtration, 95 Messenger RNA (mRNA), 70 Microbes, classification of, 19 identification of, 25 Microbial death kinetics, 112 Microbial genetics, 63 Microbiology laboratory safety rules and procedures, 181 Microbiology, 3 scope of, 4 Microorganisms, role of, 4 Microscopes, structure of, 32, 184 types of, 32, 183 Microscopy, 30 Minimum inhibitory concentration, determination of, 107 Monoclonal antibodies, applications of, 150 preparation of, 150 Mucous membranes, 140 Mutation, molecular basis of, 89 N Naturally acquired active immunity, 152 Naturally acquired passive immunity, 153 Negative staining, 30 Nitrosomonas, 13 Non-specific defence mechanisms, 140 Nosocomial infection, 129 prevention of, 130 route of transmission, 129 Numerical aperture, 33 Nutrition, 41

L Laminar air flow, 126, 188 M Membrane filtration, 95 Messenger RNA (mRNA), 70 Microbes, classification of, 19 identification of, 25 Microbial death kinetics, 112 Microbial genetics, 63 Microbiology laboratory safety rules and procedures, 181 Microbiology, 3 scope of, 4 Microorganisms, role of, 4 Microscopes, structure of, 32, 184 types of, 32, 183 Microscopy, 30 Minimum inhibitory concentration, determination of, 107 Monoclonal antibodies, applications of, 150 preparation of, 150 Mucous membranes, 140 Mutation, molecular basis of, 89 N Naturally acquired active immunity, 152 Naturally acquired passive immunity, 153 Negative staining, 30 Nitrosomonas, 13 Non-specific defence mechanisms, 140 Nosocomial infection, 129 prevention of, 130 route of transmission, 129 Numerical aperture, 33 Nutrition, 41

INDEX

H Hanging droplet method, 195 Heterotrophic bacteria, 43 Horizontal laminar air flow, structure of, 188 Hospital hygiene, 124 Hospital infection, control of, 130 Hot air oven, 190 Human virus, 58 Humoral immunity, 153 Hybridoma technology, 150 Hypersensitivity reaction, 161 I Ideal disinfectant, properties of, 104 IgM antibody, pentameric structure of, 149 Immediate hypersensitivity, 161 Immune complex, 163 Immunogen, 145 Immunoglobulin A, 149 Immunoglobulin classes, 148 Immunoglobulin D, 150 Immunoglobulin E, 150 Immunoglobulin G, 148 Immunoglobulin M, 149 Immunogrenicity, 145 Immunological tolerance, 159 Immunology, history of, 137 Incubator, structure of, 189 Inflammation, 144 Inoculation loop, 186 Inoculation needle, 186 Inoculum, preparation of, 168 Ionizing radiation, sterilization by, 118 Isolation, 41

219

219

L Laminar air flow, 126, 188 M Membrane filtration, 95 Messenger RNA (mRNA), 70 Microbes, classification of, 19 identification of, 25 Microbial death kinetics, 112 Microbial genetics, 63 Microbiology laboratory safety rules and procedures, 181 Microbiology, 3 scope of, 4 Microorganisms, role of, 4 Microscopes, structure of, 32, 184 types of, 32, 183 Microscopy, 30 Minimum inhibitory concentration, determination of, 107 Monoclonal antibodies, applications of, 150 preparation of, 150 Mucous membranes, 140 Mutation, molecular basis of, 89 N Naturally acquired active immunity, 152 Naturally acquired passive immunity, 153 Negative staining, 30 Nitrosomonas, 13 Non-specific defence mechanisms, 140 Nosocomial infection, 129 prevention of, 130 route of transmission, 129 Numerical aperture, 33 Nutrition, 41

220 INDEX P Paper disk plate method, 170 Passive immunity, 152 Penicillium notatum, 4 pH meter, 191 structure of, 191 Phagocytosis, 140 Phase contrast microscopy, 35 Phenol coefficient test, 105 Phenotype, 74 Photosynthetic bacteria, 41 Physical method of selection, 49 Polymerase chain reaction (PCR), 85 application of, 87 Pour-plate technique, 53 Preservatives, 108 Prokaryotic cell, 17 Protein synthesis, initiation stage of, 79 steps of, 78 termination of, 82 Pseudomonas thiobacillus, 13 Pure culture isolation, 51 Q Quebec colony counter, 190 R Replication, 75 Resistance, expressions of, 113 Resolution, limit of, 33 Resolving power, 33 Ribosomal RNA (rRNA), 72 Rickettsiae, classification of, 22 Rideal Walker test, 105 RNA, biological roles of, 73 structure of, 68 types of, 70 Rod shaped, 15

220 INDEX S Scanning electron microscope, 36 Selection in nature, 51 Serial dilution technique, 54 Simple staining, 26 Skin membranes, 140 Small centrifuge, structure of, 193 Soil fertility, used in, 7 Somatic vs Gametic mutations, 89 Spherical (Cocci), 14 Spirillum serpens, 13 Spirit lamp, 186 Spirochaetes, classification of, 23 Spontaneous vs induced mutations, 89 Spread-plate technique, 53 Sterile products, manufacture of, 127 Sterility assurance, 113 Sterility testing, 91 Sterilization by heat, 115 Sterilization methods, 115 Sterilization, 111 Streak plate method, 206 Streak-plate technique, 51 Streptomycetaceae, 20 T Taxonomy, classification of, 19 Test procedures, 94 Three-kingdom classification, 19 Transcription, 77 Transduction, 84 Transfer RNA (tRNA), 71 Transformation, 82 Transmission electron microscopy, 36 Two-kingdom classification, 19

220 INDEX P Paper disk plate method, 170 Passive immunity, 152 Penicillium notatum, 4 pH meter, 191 structure of, 191 Phagocytosis, 140 Phase contrast microscopy, 35 Phenol coefficient test, 105 Phenotype, 74 Photosynthetic bacteria, 41 Physical method of selection, 49 Polymerase chain reaction (PCR), 85 application of, 87 Pour-plate technique, 53 Preservatives, 108 Prokaryotic cell, 17 Protein synthesis, initiation stage of, 79 steps of, 78 termination of, 82 Pseudomonas thiobacillus, 13 Pure culture isolation, 51 Q Quebec colony counter, 190 R Replication, 75 Resistance, expressions of, 113 Resolution, limit of, 33 Resolving power, 33 Ribosomal RNA (rRNA), 72 Rickettsiae, classification of, 22 Rideal Walker test, 105 RNA, biological roles of, 73 structure of, 68 types of, 70 Rod shaped, 15

P Paper disk plate method, 170 Passive immunity, 152 Penicillium notatum, 4 pH meter, 191 structure of, 191 Phagocytosis, 140 Phase contrast microscopy, 35 Phenol coefficient test, 105 Phenotype, 74 Photosynthetic bacteria, 41 Physical method of selection, 49 Polymerase chain reaction (PCR), 85 application of, 87 Pour-plate technique, 53 Preservatives, 108 Prokaryotic cell, 17 Protein synthesis, initiation stage of, 79 steps of, 78 termination of, 82 Pseudomonas thiobacillus, 13 Pure culture isolation, 51 Q Quebec colony counter, 190 R Replication, 75 Resistance, expressions of, 113 Resolution, limit of, 33 Resolving power, 33 Ribosomal RNA (rRNA), 72 Rickettsiae, classification of, 22 Rideal Walker test, 105 RNA, biological roles of, 73 structure of, 68 types of, 70 Rod shaped, 15

S Scanning electron microscope, 36 Selection in nature, 51 Serial dilution technique, 54 Simple staining, 26 Skin membranes, 140 Small centrifuge, structure of, 193 Soil fertility, used in, 7 Somatic vs Gametic mutations, 89 Spherical (Cocci), 14 Spirillum serpens, 13 Spirit lamp, 186 Spirochaetes, classification of, 23 Spontaneous vs induced mutations, 89 Spread-plate technique, 53 Sterile products, manufacture of, 127 Sterility assurance, 113 Sterility testing, 91 Sterilization by heat, 115 Sterilization methods, 115 Sterilization, 111 Streak plate method, 206 Streak-plate technique, 51 Streptomycetaceae, 20 T Taxonomy, classification of, 19 Test procedures, 94 Three-kingdom classification, 19 Transcription, 77 Transduction, 84 Transfer RNA (tRNA), 71 Transformation, 82 Transmission electron microscopy, 36 Two-kingdom classification, 19

220 INDEX S Scanning electron microscope, 36 Selection in nature, 51 Serial dilution technique, 54 Simple staining, 26 Skin membranes, 140 Small centrifuge, structure of, 193 Soil fertility, used in, 7 Somatic vs Gametic mutations, 89 Spherical (Cocci), 14 Spirillum serpens, 13 Spirit lamp, 186 Spirochaetes, classification of, 23 Spontaneous vs induced mutations, 89 Spread-plate technique, 53 Sterile products, manufacture of, 127 Sterility assurance, 113 Sterility testing, 91 Sterilization by heat, 115 Sterilization methods, 115 Sterilization, 111 Streak plate method, 206 Streak-plate technique, 51 Streptomycetaceae, 20 T Taxonomy, classification of, 19 Test procedures, 94 Three-kingdom classification, 19 Transcription, 77 Transduction, 84 Transfer RNA (tRNA), 71 Transformation, 82 Transmission electron microscopy, 36 Two-kingdom classification, 19

P Paper disk plate method, 170 Passive immunity, 152 Penicillium notatum, 4 pH meter, 191 structure of, 191 Phagocytosis, 140 Phase contrast microscopy, 35 Phenol coefficient test, 105 Phenotype, 74 Photosynthetic bacteria, 41 Physical method of selection, 49 Polymerase chain reaction (PCR), 85 application of, 87 Pour-plate technique, 53 Preservatives, 108 Prokaryotic cell, 17 Protein synthesis, initiation stage of, 79 steps of, 78 termination of, 82 Pseudomonas thiobacillus, 13 Pure culture isolation, 51 Q Quebec colony counter, 190 R Replication, 75 Resistance, expressions of, 113 Resolution, limit of, 33 Resolving power, 33 Ribosomal RNA (rRNA), 72 Rickettsiae, classification of, 22 Rideal Walker test, 105 RNA, biological roles of, 73 structure of, 68 types of, 70 Rod shaped, 15

S Scanning electron microscope, 36 Selection in nature, 51 Serial dilution technique, 54 Simple staining, 26 Skin membranes, 140 Small centrifuge, structure of, 193 Soil fertility, used in, 7 Somatic vs Gametic mutations, 89 Spherical (Cocci), 14 Spirillum serpens, 13 Spirit lamp, 186 Spirochaetes, classification of, 23 Spontaneous vs induced mutations, 89 Spread-plate technique, 53 Sterile products, manufacture of, 127 Sterility assurance, 113 Sterility testing, 91 Sterilization by heat, 115 Sterilization methods, 115 Sterilization, 111 Streak plate method, 206 Streak-plate technique, 51 Streptomycetaceae, 20 T Taxonomy, classification of, 19 Test procedures, 94 Three-kingdom classification, 19 Transcription, 77 Transduction, 84 Transfer RNA (tRNA), 71 Transformation, 82 Transmission electron microscopy, 36 Two-kingdom classification, 19

INDEX

U UV radiation, sterilization by, 119 UV-visible spectrophotometer, structure of, 192 V Variation, 63 Viruses, 41 classification of, 23 nutritional habit of, 58 Vitamin A, microbial assay of, 172 Vitamin B12, microbial assay of, 177 Vitamin D, microbial assay of, 174

Vitamin production, 6 Vitamins, assay of, 172 W Water bath, 187 Watson & Crick’s observations, 68 Yeast, alcohol industry used in, 5 baking industry used in, 6 cosmetics used in, 6 perfumes uses in, 6 vinegar industry used in, 5 Z Zone of inhibition, determination of, 107

INDEX

U UV radiation, sterilization by, 119 UV-visible spectrophotometer, structure of, 192 V Variation, 63 Viruses, 41 classification of, 23 nutritional habit of, 58 Vitamin A, microbial assay of, 172 Vitamin B12, microbial assay of, 177 Vitamin D, microbial assay of, 174

221 U UV radiation, sterilization by, 119 UV-visible spectrophotometer, structure of, 192 V Variation, 63 Viruses, 41 classification of, 23 nutritional habit of, 58 Vitamin A, microbial assay of, 172 Vitamin B12, microbial assay of, 177 Vitamin D, microbial assay of, 174

W Water bath, 187 Watson & Crick’s observations, 68 Yeast, alcohol industry used in, 5 baking industry used in, 6 cosmetics used in, 6 perfumes uses in, 6 vinegar industry used in, 5 Z Zone of inhibition, determination of, 107

W Water bath, 187 Watson & Crick’s observations, 68 Yeast, alcohol industry used in, 5 baking industry used in, 6 cosmetics used in, 6 perfumes uses in, 6 vinegar industry used in, 5

INDEX

U UV radiation, sterilization by, 119 UV-visible spectrophotometer, structure of, 192 V Variation, 63 Viruses, 41 classification of, 23 nutritional habit of, 58 Vitamin A, microbial assay of, 172 Vitamin B12, microbial assay of, 177 Vitamin D, microbial assay of, 174

221

Vitamin production, 6 Vitamins, assay of, 172

221

Vitamin production, 6 Vitamins, assay of, 172

Z Zone of inhibition, determination of, 107

INDEX

221

Vitamin production, 6 Vitamins, assay of, 172 W Water bath, 187 Watson & Crick’s observations, 68 Yeast, alcohol industry used in, 5 baking industry used in, 6 cosmetics used in, 6 perfumes uses in, 6 vinegar industry used in, 5 Z Zone of inhibition, determination of, 107

A Textbook of

TM

Pharmaceutical Microbiology (With Experiments)

Prahlad Singh Mehra Distributed by: