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English Pages XXXIV, 487 [515] Year 2020
Abhishek Chauhan Tanu Jindal
Microbiological Methods for Environment, Food and Pharmaceutical Analysis
Microbiological Methods for Environment, Food and Pharmaceutical Analysis
Abhishek Chauhan • Tanu Jindal
Microbiological Methods for Environment, Food and Pharmaceutical Analysis
Abhishek Chauhan Amity Institute of Environmental Toxicology, Safety and Management Amity University Noida, Uttar Pradesh, India
Tanu Jindal Amity Institute of Environmental Toxicology, Safety and Management Amity University Noida, Uttar Pradesh, India
ISBN 978-3-030-52023-6 ISBN 978-3-030-52024-3 (eBook) https://doi.org/10.1007/978-3-030-52024-3 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface
Discovery of microorganisms and the field microbiology began with the invention of the first microscope in the year 1665, and since then, exponential growth of this science can be seen. In fact, the current world largely depends upon the various aspects of microbes, organisms that are invisible to the naked eye. These tiny organisms demonstrate their presence in both positive and negative facets of life. Microorganisms including bacteria, yeast, molds, etc., are ubiquitous in nature and their applications are as well omnipresent throughout. Right from the air, soil, and water, that is, three main constituents of the Earth, the applicability of microbes can be found in any means. This science is gaining importance globally as far as the safety and quality of various commodities such as food, water and wastewater, air, cosmetics, drugs and pharmaceuticals, and antimicrobial preparation such as paints, emulsions, herbal preparations, and medical devices are concerned. Safety and quality evaluation of these commodities is a foremost requirement in order to provide the best services to mankind and for the betterment of society. Microbes are significant to all our lives in enormous ways. Sometimes, the influence of microorganisms on human life is beneficial, whereas at other times, it is detrimental. For example, microorganisms are required to produce cheese, yogurt, bread, alcohol, antibiotics, vaccines, vitamins, enzymes wine, beer, etc. Several microbial products contribute to public health such as aiding in nutrition, interrupting the spread of disease, and improving the quality of life in the years ahead. Undesirable presence of microorganisms in various commodities such as food, water, drugs, and pharmaceutical products makes it essential to ensure the safety and quality of these products, which is usually achieved by quality evaluation or analysis by carrying out microbiological experiments. The textbook Microbiological Methods for Environment, Food and Pharmaceutical Analysis specifically aims at the ever-demanding thoughtful need of a well-documented compilation of factual details. The book emphasizes specifically on applied aspects of industrial microbiology including water, soil, air, food, and pharmaceuticals. Good Microbiological Laboratory Practices (GMLP) covering all aspects of Good Laboratory Management System is well written in a technical manner so that if applied, it would aid in creating a state-of-the-art laboratory. v
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Special elaborations have been given to management with respect to safety, housekeeping, spillage, disposal, and avoidance of laboratory-acquired infections. Importance of microbiological culture media and their composition has also been given so as to inform the user about ingredients of the media having characteristic behavior during experiment. Knowledge of sterilization and disinfection procedure from the point of view safety is an utmost requirement in laboratory while working with highly contaminated sample and handling pathogenic culture. A concise revelation about major and minor instruments/equipment used in industrial microbiology has been given. Microbiological methods have been written in simple, lucid, and crisp language keeping in view the requirement of industries and research organizations working on quality evaluation of variety of products. A fairly up-to-date “Index” in the textbook will surely enlarge the vision of its readers by giving an easy access of subject-enriched, well-documented text. The book is highly useful for industrialists, researchers, academicians, and students from both undergraduate and postgraduate levels of microbiology, biotechnology, botany, and pharmaceutical sciences. It would have significant contribution in the effective knowledge of scientists/analysts/lab personnel/technical managers/ quality managers working with the industrial aspect of microbiology. The authors are grateful that Springer Nature has enthusiastically agreed to publish this work and appreciates their help and collaboration on this project. Noida, India
Abhishek Chauhan Tanu Jindal
About the Book
The current book has been intended with various microbiological methods pertaining to water, soil, air, food, and pharmaceutical analysis with respect to quality and safety evaluation of products. As per the requirements of different sectors, this textbook has been divided into several chapters of current microbiological research interest. Chapters 1 and 2 introduce a detailed knowledge of good microbiological and management practices, Chap. 3 is designed to provide the essential information pertaining to various types of microbiological culture media and their types and role of ingredients. Chapter 4 describes various methods of sterilization and disinfection. Microbiological experiments required several major and minor equipment; therefore, Chap. 5 has been designed in order to provide the necessary information with respect to all required instrumentations and equipment. Chapter 6 describes methods of sampling for water, soil, air, food, and pharmaceutical products. The Chaps. 7, 8, and 9 demonstrate the standard laboratory procedures for carrying out the analysis of water, soil, air, food, and pharmaceuticals. Methods written in this book are significantly sub-headed to make it more user-friendly with better accuracy for evaluation of the products for betterment of mankind and society. Chapter 10 describes various methods relating to biochemical and molecular characteristics of microorganisms in order to identify the microbe effectively. The script is also equipped with relevant tables, figures, and photographs. The textbook specifically aims at the ever-demanding thoughtful need of a well-documented compilation of factual details. The book is written in simple, lucid, and crisp language keeping in view the requirement of industries and research organizations working on quality evaluation of a variety of products. Abhishek Chauhan Tanu Jindal
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Contents
1 Introductory Analytical Microbiology �������������������������������������������������� 1 1.1 Introduction�������������������������������������������������������������������������������������� 1 1.2 Analytical Microbiology: Various Disciplines���������������������������������� 2 1.3 Food Microbiology �������������������������������������������������������������������������� 3 1.3.1 Microbiological Analysis of Food and Food Products��������� 3 1.3.2 Microbial Contamination of Food and Food Products: Major Factors������������������������������������������������������������������������ 4 1.4 Environmental Microbiology������������������������������������������������������������ 7 1.4.1 Water Microbiology�������������������������������������������������������������� 8 1.4.2 Air Microbiology������������������������������������������������������������������ 9 1.4.3 Soil Microbiology ���������������������������������������������������������������� 9 1.5 Pharmaceutical Microbiology���������������������������������������������������������� 9 1.6 Accreditation for Microbiological Laboratories ������������������������������ 11 1.7 Quality Assessment�������������������������������������������������������������������������� 11 1.7.1 Internal Quality Assessment ������������������������������������������������ 11 1.7.2 External Quality Assessment������������������������������������������������ 12 References�������������������������������������������������������������������������������������������������� 13 2 Good Microbiological Laboratory Practices ���������������������������������������� 15 2.1 Introduction�������������������������������������������������������������������������������������� 15 2.2 Safety Management�������������������������������������������������������������������������� 15 2.2.1 Biosafety Level (BSL)���������������������������������������������������������� 16 2.2.2 Avoidance of Laboratory-Acquired Infections �������������������� 16 2.3 Entry to Microbiology Lab �������������������������������������������������������������� 18 2.4 Housekeeping Management�������������������������������������������������������������� 18 2.5 Spillage Management������������������������������������������������������������������������ 19 2.6 Disposal Management���������������������������������������������������������������������� 19 2.6.1 Containers for Waste Material in Microbiology Lab������������ 20 2.6.2 Discard Bins, Bags and Jars�������������������������������������������������� 20 2.6.3 Good Disposal Practices (GDP)������������������������������������������� 21 References�������������������������������������������������������������������������������������������������� 22 ix
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3 Microbiological Culture Media: Types, Role and Composition���������� 23 3.1 Introduction�������������������������������������������������������������������������������������� 23 3.2 Types of Media��������������������������������������������������������������������������������� 23 3.3 Consistency-Based Classification of Media�������������������������������������� 24 3.3.1 Liquid (Broth) Media������������������������������������������������������������ 24 3.3.2 Semisolid Media ������������������������������������������������������������������ 24 3.3.3 Solid Media�������������������������������������������������������������������������� 24 3.4 Composition-Based Classification of Media������������������������������������ 25 3.4.1 Chemically Defined Media (Synthetic Media) �������������������� 25 3.4.2 Complex Media�������������������������������������������������������������������� 25 3.5 Function-Based Classification of Media������������������������������������������ 26 3.5.1 All Purpose Media���������������������������������������������������������������� 26 3.5.2 Selective/Differential Media ������������������������������������������������ 26 3.5.3 Enrichment Media���������������������������������������������������������������� 26 3.5.4 Reducing Media�������������������������������������������������������������������� 27 3.6 Culture Media: Check Points������������������������������������������������������������ 27 3.7 Culture Media: Role of Ingredients (Table 33.1.1)�������������������������� 28 3.8 Culture Media: Compositions ���������������������������������������������������������� 30 3.8.1 Actinomycetes Isolation Agar���������������������������������������������� 30 3.8.2 Alkaline Peptone Water�������������������������������������������������������� 30 3.8.3 Ammonifying Bacteria Isolation Medium���������������������������� 31 3.8.4 Anaerobic Agar �������������������������������������������������������������������� 31 3.8.5 Antibiotic Assay Medium No-1�������������������������������������������� 31 3.8.6 Antibiotic Assay Medium No-12������������������������������������������ 32 3.8.7 Antibiotic Assay Medium No-19������������������������������������������ 32 3.8.8 Antibiotic Assay Medium No-11������������������������������������������ 33 3.8.9 Baird–Parker Agar Medium�������������������������������������������������� 33 3.8.10 BG-11 Medium �������������������������������������������������������������������� 34 3.8.11 Biotin Assay Medium ���������������������������������������������������������� 34 3.8.12 Bismuth Sulphite Agar (BSA)���������������������������������������������� 35 3.8.13 Brain–Heart Infusion Broth�������������������������������������������������� 35 3.8.14 Blood Agar Medium ������������������������������������������������������������ 36 3.8.15 Baird Parker Agar Medium �������������������������������������������������� 36 3.8.16 Brilliant Green Agar (BGA)������������������������������������������������� 36 3.8.17 Brilliant Green Bile Lactose (BGBL) Broth������������������������ 37 3.8.18 Buffered Peptone Water (BPW)�������������������������������������������� 37 3.8.19 Buffered Sodium Chloride Peptone Solution ���������������������� 38 3.8.20 Cetrimide Agar���������������������������������������������������������������������� 38 3.8.21 Chloramphenicol Yeast Extract Glucose Agar (CYGA) ������ 39 3.8.22 Columbia Agar���������������������������������������������������������������������� 39 3.8.23 Cook Meat Medium (CMM)������������������������������������������������ 40 3.8.24 Deoxycholate Citrate Agar (DCA)��������������������������������������� 40 3.8.25 Differential Reinforced Clostridia Broth������������������������������ 40 3.8.26 Enterobacteria Enrichment Broth ���������������������������������������� 41 3.8.27 Eosin Methylene Blue (EMB) Agar�������������������������������������� 41
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3.8.28 Ethyl Violet Azide Dextrose Agar���������������������������������������� 42 3.8.29 Fluid Thioglycollate Medium (FTM) ���������������������������������� 42 3.8.30 Glucose Agar������������������������������������������������������������������������ 43 3.8.31 Glucose-Salt-Teepol Broth���������������������������������������������������� 43 3.8.32 GN Broth������������������������������������������������������������������������������ 44 3.8.33 Glucose Yeast Extract Agar�������������������������������������������������� 44 3.8.34 Hugh-Leifson Medium��������������������������������������������������������� 44 3.8.35 K-Agar Medium�������������������������������������������������������������������� 45 3.8.36 KG (Kim Goepfert) Agar Base �������������������������������������������� 45 3.8.37 Lactose Broth������������������������������������������������������������������������ 46 3.8.38 MacConkey Broth���������������������������������������������������������������� 46 3.8.39 Mac Conkey Agar (MCA)���������������������������������������������������� 47 3.8.40 Malt Extract Broth���������������������������������������������������������������� 47 3.8.41 Mannitol Salt Agar���������������������������������������������������������������� 48 3.8.42 Mannitol Yeast Polymyxin Agar (MYP Agar)���������������������� 48 3.8.43 Minimal Agar������������������������������������������������������������������������ 48 3.8.44 Motility Test Medium ���������������������������������������������������������� 49 3.8.45 MR-VP Medium (Glucose Phosphate Broth) ���������������������� 49 3.8.46 Mueller Hinton Broth (MHB)���������������������������������������������� 50 3.8.47 Muller Hinton Agar�������������������������������������������������������������� 50 3.8.48 Nitrate Medium�������������������������������������������������������������������� 51 3.8.49 Nutrient Agar������������������������������������������������������������������������ 51 3.8.50 Nutrient Broth ���������������������������������������������������������������������� 51 3.8.51 Osmophilic Agar ������������������������������������������������������������������ 52 3.8.52 Osmophilic Dilution Blanks ������������������������������������������������ 52 3.8.53 Pantothenate Assay Medium������������������������������������������������ 52 3.8.54 Pantothenate Inoculum Broth ���������������������������������������������� 53 3.8.55 Peptone Water ���������������������������������������������������������������������� 53 3.8.56 Plate Count Agar ������������������������������������������������������������������ 54 3.8.57 Rappaport Vassiliadis Medium (RV)������������������������������������ 54 3.8.58 Sabouraud Dextrose Agar ���������������������������������������������������� 55 3.8.59 Sabouraud Dextrose Broth���������������������������������������������������� 55 3.8.60 Selenite Cystine Broth (SCB) ���������������������������������������������� 55 3.8.61 Simmon’s Citrate Agar���������������������������������������������������������� 56 3.8.62 Skim Milk Agar�������������������������������������������������������������������� 56 3.8.63 Soyabean Casein Digest Broth���������������������������������������������� 57 3.8.64 Soybean Casein Digest Agar������������������������������������������������ 57 3.8.65 Starch Agar��������������������������������������������������������������������������� 57 3.8.66 TC-SMAC Agar Medium������������������������������������������������������ 58 3.8.67 Tergitol – 7 Agar (T-7)���������������������������������������������������������� 58 3.8.68 Tetrathioanate Broth (TTB)�������������������������������������������������� 59 3.8.69 Thiosulphate-Citrate-Bile Salts-Sucrose Agar (TCBS)�������� 59 3.8.70 Top Agar�������������������������������������������������������������������������������� 59 3.8.71 Triple Sugar-Iron Agar Medium ������������������������������������������ 60 3.8.72 Urease Broth Medium���������������������������������������������������������� 60
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3.8.73 Veal Infusion Agar���������������������������������������������������������������� 61 3.8.74 Veal Infusion Broth �������������������������������������������������������������� 61 3.8.75 Violet Red Bile Agar������������������������������������������������������������ 61 3.8.76 Vogel-Bonner (VB) Medium (Table 3.76)���������������������������� 62 3.8.77 Willis and Hobb’s Medium with Neomycin (Egg-Yolk Medium)�������������������������������������������������������������������������������� 62 3.8.78 Xylose Lysine Deoxycholate Agar��������������������������������������� 63 3.8.79 Yeast Extract Broth �������������������������������������������������������������� 63 References�������������������������������������������������������������������������������������������������� 64 4 Methods of Sterilization and Disinfection���������������������������������������������� 67 4.1 Introduction�������������������������������������������������������������������������������������� 67 4.2 Methods for Sterilization������������������������������������������������������������������ 67 4.2.1 Heat Sterilization������������������������������������������������������������������ 67 4.2.2 Incineration �������������������������������������������������������������������������� 68 4.2.3 Moist Heat���������������������������������������������������������������������������� 68 4.2.4 Tyndalliztion ������������������������������������������������������������������������ 68 4.2.5 Dry Heat�������������������������������������������������������������������������������� 68 4.2.6 Radiation Sterilization���������������������������������������������������������� 69 4.2.7 Filtration Sterilization ���������������������������������������������������������� 69 4.3 Chemical Sterilization/Disinfection�������������������������������������������������� 70 4.3.1 Phenolics ������������������������������������������������������������������������������ 70 4.3.2 Hypochlorites������������������������������������������������������������������������ 70 4.3.3 Aldehydes ���������������������������������������������������������������������������� 70 4.3.4 Alcohols�������������������������������������������������������������������������������� 71 4.3.5 Quaternary Ammonium Compounds (QAC)������������������������ 71 4.3.6 Iodophores���������������������������������������������������������������������������� 71 References�������������������������������������������������������������������������������������������������� 72 5 Equipments and Instruments for Microbiological Laboratories�������� 73 5.1 Introduction�������������������������������������������������������������������������������������� 73 5.2 Major Equipments: Brief Description���������������������������������������������� 74 5.2.1 Optical Microscope�������������������������������������������������������������� 74 5.2.2 Water Bath���������������������������������������������������������������������������� 74 5.2.3 Refrigerators ������������������������������������������������������������������������ 74 5.2.4 Incubators������������������������������������������������������������������������������ 75 5.2.5 Centrifuge ���������������������������������������������������������������������������� 75 5.2.6 Biosafety Cabinets (BSC) ���������������������������������������������������� 75 5.2.7 Hot-Air Oven������������������������������������������������������������������������ 77 5.2.8 Autoclave������������������������������������������������������������������������������ 77 5.2.9 Homogenizers, Blenders and Mixers������������������������������������ 78 5.2.10 Vortex Mixer ������������������������������������������������������������������������ 78 5.2.11 Weighing Balance ���������������������������������������������������������������� 78 5.2.12 pH Meter ������������������������������������������������������������������������������ 79 5.2.13 Colony Counting Device������������������������������������������������������ 79 5.2.14 Microwave Oven ������������������������������������������������������������������ 79
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5.3 Minor Equipments and Other Requirements������������������������������������ 79 5.3.1 Bunsen Burners�������������������������������������������������������������������� 79 5.3.2 Inoculation Loops ���������������������������������������������������������������� 79 5.3.3 Inoculation Needle���������������������������������������������������������������� 80 5.3.4 Forceps (Stainless Steel) ������������������������������������������������������ 80 5.3.5 Spreaders������������������������������������������������������������������������������ 80 5.3.6 Microscopic Slides and Cover Slips ������������������������������������ 80 5.3.7 Pasteur Pipettes �������������������������������������������������������������������� 80 5.3.8 Graduated Pipettes���������������������������������������������������������������� 81 5.3.9 Glassware and Plasticware���������������������������������������������������� 81 5.3.10 Petri-dishes���������������������������������������������������������������������������� 81 5.3.11 Test Tubes and Bottles���������������������������������������������������������� 82 5.3.12 Conical Flask������������������������������������������������������������������������ 82 5.3.13 Marker Pen���������������������������������������������������������������������������� 82 5.3.14 Personal Protective Equipments (PPE) �������������������������������� 82 5.3.15 Autoclavable/Roasting Bag�������������������������������������������������� 82 5.3.16 Thermometer������������������������������������������������������������������������ 82 5.3.17 Spillage Kit �������������������������������������������������������������������������� 83 5.3.18 Disinfectants ������������������������������������������������������������������������ 83 5.3.19 Ethanol (70% Industrial Methylated Spirit)�������������������������� 83 5.3.20 Autoclave Indicator Tape������������������������������������������������������ 83 5.3.21 Sterilizer Control Tube/Strip������������������������������������������������ 83 5.3.22 Non-absorbent Cotton Wool ������������������������������������������������ 83 5.4 Equipments: Quality Aspects������������������������������������������������������������ 84 References�������������������������������������������������������������������������������������������������� 85 6 Methods of Sampling for Environment, Food and Pharmaceutical Analysis ���������������������������������������������������������������������������������������������������� 87 6.1 Introduction�������������������������������������������������������������������������������������� 87 6.2 Method for Water Sampling�������������������������������������������������������������� 88 6.3 Method for Soil Sampling���������������������������������������������������������������� 88 6.4 Method for Air Sampling������������������������������������������������������������������ 89 6.4.1 Passive Method �������������������������������������������������������������������� 89 6.4.2 Active Method���������������������������������������������������������������������� 89 6.5 Method for Food and Food Products Sampling�������������������������������� 90 6.6 Method for Pharmaceutical Products Sampling ������������������������������ 90 References�������������������������������������������������������������������������������������������������� 91 7 Microbiological Methods for Water, Soil and Air Analysis������������������ 93 7.1 Introduction�������������������������������������������������������������������������������������� 93 7.2 Method for the Determination of Most Probable Number (MPN) of Coliforms in Water Sample���������������������������������������������� 94 7.2.1 Method Overview ���������������������������������������������������������������� 94 7.2.2 Scope of the Method ������������������������������������������������������������ 94 7.2.3 Experimental Requirements (Table 7.1) ������������������������������ 95 7.2.4 Experimental Procedure�������������������������������������������������������� 95
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7.2.5 Interpretation and Expression of Results������������������������������ 97 7.2.6 Quality Control �������������������������������������������������������������������� 97 7.2.7 Precautions���������������������������������������������������������������������������� 103 7.2.8 Disposal�������������������������������������������������������������������������������� 103 7.3 Method for the Detection and Isolation of Escherichia coli in Continuation of MPN Coliform in Water Sample������������������������ 103 7.3.1 Method Overview ���������������������������������������������������������������� 103 7.3.2 Experimental Requirements (Table 7.3) ������������������������������ 104 7.3.3 Experimental Procedure�������������������������������������������������������� 104 7.3.4 Expression of Result ������������������������������������������������������������ 105 7.3.5 Quality Control �������������������������������������������������������������������� 105 7.3.6 Precautions���������������������������������������������������������������������������� 106 7.3.7 Disposal�������������������������������������������������������������������������������� 106 7.4 Method for the Detection of Coliforms Bacteria in Water Sample by Membrane Filtration�������������������������������������������������������������������� 107 7.4.1 Method Overview ���������������������������������������������������������������� 107 7.4.2 Scope of the Method ������������������������������������������������������������ 107 7.4.3 Requirements (Table 7.4)������������������������������������������������������ 107 7.4.4 Experimental Procedure�������������������������������������������������������� 107 7.4.5 Interpretation and Expression of Results������������������������������ 109 7.4.6 Quality Control �������������������������������������������������������������������� 109 7.4.7 Precautions���������������������������������������������������������������������������� 110 7.4.8 Disposal�������������������������������������������������������������������������������� 110 7.5 Method for the Detection and Identification of Escherichia coli in Water Sample by Membrane Filtration���������������������������������������� 110 7.5.1 Experimental Overview�������������������������������������������������������� 110 7.5.2 Scope of the Method ������������������������������������������������������������ 111 7.5.3 Experimental Requirements (Table 7.5) ������������������������������ 111 7.5.4 Experimental Procedure�������������������������������������������������������� 111 7.5.5 Quality Control �������������������������������������������������������������������� 113 7.5.6 Precautions���������������������������������������������������������������������������� 113 7.5.7 Disposal ������������������������������������������������������������������������������� 115 7.6 Method for the Detection and Enumeration of Yeast and Mold in Water Sample by Membrane Filtration���������������������������������������� 117 7.6.1 Method Overview ���������������������������������������������������������������� 117 7.6.2 Scope of the Method ������������������������������������������������������������ 117 7.6.3 Experimental Requirements (Table 7.7) ������������������������������ 117 7.6.4 Experimental Procedure�������������������������������������������������������� 117 7.6.5 Interpretation and Expression of Results������������������������������ 120 7.6.6 Quality Control �������������������������������������������������������������������� 120 7.6.7 Precautions���������������������������������������������������������������������������� 120 7.6.8 Disposal�������������������������������������������������������������������������������� 120 7.7 Method for the Detection of Fecal Streptococci in Water Sample by Membrane Filtration�������������������������������������������������������������������� 121 7.7.1 Method Overview ���������������������������������������������������������������� 121 7.7.2 Scope of the Method ������������������������������������������������������������ 121
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7.7.3 Experimental Requirements (Table 7.8) ������������������������������ 121 7.7.4 Experimental Procedure�������������������������������������������������������� 121 7.7.5 Interpretation and Expression of Results������������������������������ 123 7.7.6 Quality Control �������������������������������������������������������������������� 123 7.7.7 Precautions���������������������������������������������������������������������������� 123 7.7.8 Disposal�������������������������������������������������������������������������������� 124 7.8 Method for the Detection and Identification of Shigella in Water Sample by Membrane Filtration ������������������������������������������������������ 124 7.8.1 Method Overview ���������������������������������������������������������������� 124 7.8.2 Scope of the Method ������������������������������������������������������������ 124 7.8.3 Experimental Requirements (Table 7.9) ������������������������������ 124 7.8.4 Experimental Procedure�������������������������������������������������������� 124 7.8.5 Interpretation and Expression of Result�������������������������������� 127 7.8.6 Quality Control �������������������������������������������������������������������� 127 7.8.7 Precautions���������������������������������������������������������������������������� 127 7.8.8 Disposal�������������������������������������������������������������������������������� 128 7.9 Method for the Detection and Identification of Staphylococcus aureus in Water Sample by Membrane Filtration ���������������������������� 128 7.9.1 Method Overview ���������������������������������������������������������������� 128 7.9.2 Scope of the Method ������������������������������������������������������������ 128 7.9.3 Experimental Requirements (Table 7.11) ���������������������������� 129 7.9.4 Experimental Procedure�������������������������������������������������������� 129 7.9.5 Interpretation and Expression of Results������������������������������ 131 7.9.6 Quality Control �������������������������������������������������������������������� 132 7.9.7 Precautions���������������������������������������������������������������������������� 132 7.9.8 Disposal�������������������������������������������������������������������������������� 132 7.10 Method for the Detection and Confirmation of Sulphite Reducing Anaerobes (SRB) in Water Sample�������������������������������������������������� 132 7.10.1 Method Overview ���������������������������������������������������������������� 132 7.10.2 Scope of the Method ������������������������������������������������������������ 133 7.10.3 Experimental Requirements (Table 7.13) ���������������������������� 133 7.10.4 Experimental Procedure�������������������������������������������������������� 133 7.10.5 Interpretation and Expression of Results������������������������������ 135 7.10.6 Quality Control �������������������������������������������������������������������� 135 7.10.7 Precautions���������������������������������������������������������������������������� 135 7.10.8 Disposal�������������������������������������������������������������������������������� 136 7.11 Method for the Detection and Identification of Vibrio cholerae in Water Sample�������������������������������������������������������������������������������� 136 7.11.1 Method Overview ���������������������������������������������������������������� 136 7.11.2 Scope of the Method ������������������������������������������������������������ 136 7.11.3 Experimental Requirements (Table 7.14) ���������������������������� 137 7.11.4 Experimental Procedure�������������������������������������������������������� 137 7.11.5 Interpretation and Expression of Result�������������������������������� 139 7.11.6 Quality Control �������������������������������������������������������������������� 139 7.11.7 Precautions���������������������������������������������������������������������������� 139 7.11.8 Disposal�������������������������������������������������������������������������������� 140
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7.12 Method for the Detection and Identification of Vibrio parahaemolyticus in Water Sample by Membrane Filtration ���������� 140 7.12.1 Experimental Overview�������������������������������������������������������� 140 7.12.2 Scope of the Method ������������������������������������������������������������ 140 7.12.3 Experimental Requirements (Table 7.16) ���������������������������� 140 7.12.4 Experimental Procedure�������������������������������������������������������� 140 7.12.5 Interpretation and Expression of Results������������������������������ 143 7.12.6 Quality Control �������������������������������������������������������������������� 143 7.12.7 Precautions���������������������������������������������������������������������������� 143 7.12.8 Disposal�������������������������������������������������������������������������������� 144 7.13 Method for the Detection and Identification of Pseudomonas aeruginosa in Water Sample by Membrane Filtration���������������������� 144 7.13.1 Experimental Overview�������������������������������������������������������� 144 7.13.2 Scope of the Method ������������������������������������������������������������ 145 7.13.3 Experimental Requirements (Table 7.18) ���������������������������� 145 7.13.4 Experimental Procedure�������������������������������������������������������� 145 7.13.5 Interpretation and Expression of Results������������������������������ 147 7.13.6 Quality Control �������������������������������������������������������������������� 147 7.13.7 Precautions���������������������������������������������������������������������������� 149 7.13.8 Disposal�������������������������������������������������������������������������������� 149 7.14 Method for the Detection and Identification of Salmonella in Water Sample by Membrane Filtration�������������������������������������������� 150 7.14.1 Method Overview ���������������������������������������������������������������� 150 7.14.2 Scope of the Method ������������������������������������������������������������ 150 7.14.3 Experimental Requirements (Table 7.20) ���������������������������� 150 7.14.4 Experimental Procedure�������������������������������������������������������� 150 7.14.5 Serological Identification������������������������������������������������������ 152 7.14.6 Interpretation and Expression of Results������������������������������ 153 7.14.7 Quality Control �������������������������������������������������������������������� 154 7.14.8 Precautions���������������������������������������������������������������������������� 154 7.14.9 Disposal�������������������������������������������������������������������������������� 155 7.15 Method for the Detection and Identification of Clostridium perfringens in Water Sample by Membrane Filtration �������������������� 155 7.15.1 Experimental Overview�������������������������������������������������������� 155 7.15.2 Scope of the Method ������������������������������������������������������������ 156 7.15.3 Experimental Requirements (Table 7.22) ���������������������������� 156 7.15.4 Experimental Procedure�������������������������������������������������������� 156 7.15.5 Interpretation and Expression of Results������������������������������ 158 7.15.6 Quality Control �������������������������������������������������������������������� 158 7.15.7 Precautions���������������������������������������������������������������������������� 158 7.15.8 Disposal�������������������������������������������������������������������������������� 159 7.16 Method for the Determination of Total Bacterial Count in Water Sample���������������������������������������������������������������������������������������������� 159 7.16.1 Method Overview ���������������������������������������������������������������� 159 7.16.2 Scope of the Method ������������������������������������������������������������ 159
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7.16.3 Experimental Requirements (Table 7.24) ���������������������������� 160 7.16.4 Experimental Procedure�������������������������������������������������������� 160 7.16.5 Calculation���������������������������������������������������������������������������� 161 7.16.6 Expression of Results������������������������������������������������������������ 162 7.16.7 Quality Controls�������������������������������������������������������������������� 163 7.16.8 Precautions���������������������������������������������������������������������������� 164 7.16.9 Disposal�������������������������������������������������������������������������������� 164 7.17 Method for the Isolation and Enumeration of Actinomycetes in Water Sample by a Membrane Filtration�������������������������������������� 165 7.17.1 Method Overview ���������������������������������������������������������������� 165 7.17.2 Scope of the Method ������������������������������������������������������������ 165 7.17.3 Experimental Requirements (Table 7.29) ���������������������������� 165 7.17.4 Experimental Procedure�������������������������������������������������������� 165 7.17.5 Calculation���������������������������������������������������������������������������� 167 7.17.6 Confirmation Tests���������������������������������������������������������������� 167 7.17.7 Expression of Results������������������������������������������������������������ 167 7.17.8 Quality Control �������������������������������������������������������������������� 168 7.17.9 Precautions���������������������������������������������������������������������������� 168 7.17.10 Disposal������������������������������������������������������������������������������ 168 7.18 Method for the Detection and Identification of Blue Green Algae in Water and Soil Sample������������������������������������������������������������������ 169 7.18.1 Method Overview ���������������������������������������������������������������� 169 7.18.2 Scope of the Method ������������������������������������������������������������ 169 7.18.3 Experimental Requirements (Table 7.30) ���������������������������� 169 7.18.4 Experimental Procedure�������������������������������������������������������� 169 7.18.5 Expression of Result ������������������������������������������������������������ 171 7.18.6 Quality Control �������������������������������������������������������������������� 173 7.18.7 Precautions���������������������������������������������������������������������������� 175 7.18.8 Disposal�������������������������������������������������������������������������������� 175 7.19 Method for the Detection and Identification of Ammonifying Bacteria in Water and Soil Sample���������������������������������������������������� 176 7.19.1 Method Overview ���������������������������������������������������������������� 176 7.19.2 Scope of the Method ������������������������������������������������������������ 176 7.19.3 Experimental Requirements (Table 7.31) ���������������������������� 176 7.19.4 Experimental Procedure�������������������������������������������������������� 176 7.19.5 Expression of Result ������������������������������������������������������������ 178 7.19.6 Quality Control �������������������������������������������������������������������� 178 7.19.7 Precautions���������������������������������������������������������������������������� 178 7.19.8 Disposal�������������������������������������������������������������������������������� 178 7.20 Method for the Detection of Nitrifying Bacteria in Soil Sample������ 179 7.20.1 Method Overview ���������������������������������������������������������������� 179 7.20.2 Scope of the Method ������������������������������������������������������������ 179 7.20.3 Experimental Requirements (Table 7.32) ���������������������������� 179 7.20.4 Experimental Procedure�������������������������������������������������������� 179 7.20.5 Interpretation������������������������������������������������������������������������ 181
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7.20.6 Quality Control �������������������������������������������������������������������� 181 7.20.7 Precautions���������������������������������������������������������������������������� 181 7.20.8 Disposal�������������������������������������������������������������������������������� 182 7.21 Method for the Detection and Identification of Bacillus thuringiensis and Microscopic Examination of Delta Endotoxin Crystals in Soil Samples ������������������������������������������������������������������ 182 7.21.1 Experimental Overview�������������������������������������������������������� 182 7.21.2 Scope of the Method ������������������������������������������������������������ 182 7.21.3 Experimental Requirements (Table 7.33) ���������������������������� 182 7.21.4 Experimental Procedure�������������������������������������������������������� 182 7.21.5 Interpretation and Expression of Results������������������������������ 184 7.21.6 Quality Control �������������������������������������������������������������������� 184 7.21.7 Precautions���������������������������������������������������������������������������� 185 7.21.8 Disposal�������������������������������������������������������������������������������� 185 7.22 Method for the Determination of Total Microbial Count (TMC) Total Bacterial Count (TBC) and Total Fungal Count (FC) per m3 of Air�������������������������������������������������������������������������������������� 185 7.22.1 Method Overview ���������������������������������������������������������������� 185 7.22.2 Scope of the Method ������������������������������������������������������������ 186 7.22.3 Experimental Requirements (Table 7.35) ���������������������������� 186 7.22.4 Experimental Procedure�������������������������������������������������������� 186 7.22.5 Interpretation and Expression of Results������������������������������ 188 7.22.6 Quality Control �������������������������������������������������������������������� 188 7.22.7 Precautions���������������������������������������������������������������������������� 189 7.22.8 Disposal�������������������������������������������������������������������������������� 189 7.23 Method for the Microbiological Monitoring of Air by Settle Plate ���������������������������������������������������������������������������������� 189 7.23.1 Method Overview ���������������������������������������������������������������� 189 7.23.2 Scope of the Method ������������������������������������������������������������ 189 7.23.3 Experimental Requirements (Table 7.36) ���������������������������� 190 7.23.4 Experimental Procedure�������������������������������������������������������� 190 7.23.5 Interpretation and Expression of Results������������������������������ 191 7.23.6 Quality Control �������������������������������������������������������������������� 191 7.23.7 Precautions���������������������������������������������������������������������������� 191 7.23.8 Disposal�������������������������������������������������������������������������������� 192 References�������������������������������������������������������������������������������������������������� 192 8 Microbiological Methods for Food Analysis������������������������������������������ 197 8.1 Introduction�������������������������������������������������������������������������������������� 197 8.2 Method for the Determination of Total Bacterial Count in Food and Food Products���������������������������������������������������������������������������� 198 8.2.1 Method Overview ���������������������������������������������������������������� 198 8.2.2 Scope of the Method ������������������������������������������������������������ 198 8.2.3 Experimental Requirements (Table 8.1) ������������������������������ 198 8.2.4 Experimental Procedure�������������������������������������������������������� 198
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8.2.5 Calculation���������������������������������������������������������������������������� 202 8.2.6 Expression of Results������������������������������������������������������������ 203 8.2.7 Quality Controls�������������������������������������������������������������������� 203 8.2.8 Precautions���������������������������������������������������������������������������� 203 8.2.9 Disposal�������������������������������������������������������������������������������� 205 8.3 Method for the Determination of Total Coliform Count in Food and Food Products���������������������������������������������������������������������������� 205 8.3.1 Method Overview ���������������������������������������������������������������� 205 8.3.2 Scope of the Method ������������������������������������������������������������ 205 8.3.3 Experimental Requirements (Table 8.6) ������������������������������ 206 8.3.4 Experimental Procedure�������������������������������������������������������� 206 8.3.5 Calculation���������������������������������������������������������������������������� 208 8.3.6 Expression of Results������������������������������������������������������������ 209 8.3.7 Quality Controls�������������������������������������������������������������������� 210 8.3.8 Precautions���������������������������������������������������������������������������� 211 8.3.9 Disposal�������������������������������������������������������������������������������� 212 8.4 Method for the Determination of Total Yeast and Mould Count in Food and Food Products �������������������������������������������������������������� 212 8.4.1 Method Overview ���������������������������������������������������������������� 212 8.4.2 Scope of the Method ������������������������������������������������������������ 213 8.4.3 Experimental Requirements (Table 8.11) ���������������������������� 213 8.4.4 Experimental Procedure�������������������������������������������������������� 213 8.4.5 Observation �������������������������������������������������������������������������� 215 8.4.6 Calculation���������������������������������������������������������������������������� 217 8.4.7 Expression of Results������������������������������������������������������������ 217 8.4.8 Quality Controls�������������������������������������������������������������������� 217 8.4.9 Precautions���������������������������������������������������������������������������� 217 8.4.10 Disposal�������������������������������������������������������������������������������� 219 8.5 Method for the Enumeration of E. coli in Food and Food Products���������������������������������������������������������������������������� 219 8.5.1 Method Overview ���������������������������������������������������������������� 219 8.5.2 Scope of the Method ������������������������������������������������������������ 220 8.5.3 Experimental Requirements (Table 8.16) ���������������������������� 220 8.5.4 Experimental Procedure�������������������������������������������������������� 220 8.5.5 Preparation of Media������������������������������������������������������������ 220 8.5.6 Observation �������������������������������������������������������������������������� 222 8.5.7 Calculation���������������������������������������������������������������������������� 222 8.5.8 Expression of Results������������������������������������������������������������ 224 8.5.9 Quality Controls�������������������������������������������������������������������� 226 8.5.10 Precautions���������������������������������������������������������������������������� 226 8.5.11 Disposal�������������������������������������������������������������������������������� 226 8.6 Method for the Enumeration of S. aureus in Food and Food Products�������������������������������������������������������������������������������������������� 226 8.6.1 Method Overview ���������������������������������������������������������������� 226 8.6.2 Scope of the Method ������������������������������������������������������������ 227
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8.6.3 Experimental Requirements (Table 8.21) ���������������������������� 227 8.6.4 Experimental Procedure�������������������������������������������������������� 227 8.6.5 Observation �������������������������������������������������������������������������� 229 8.6.6 Calculation���������������������������������������������������������������������������� 230 8.6.7 Expression of Results������������������������������������������������������������ 231 8.6.8 Quality Controls�������������������������������������������������������������������� 231 8.6.9 Precautions���������������������������������������������������������������������������� 232 8.6.10 Disposal�������������������������������������������������������������������������������� 233 8.7 Method for the Enumeration of Bacillus cereus in Food and Food Products�������������������������������������������������������������������������������������������� 233 8.7.1 Method Overview ���������������������������������������������������������������� 233 8.7.2 Scope of the Method ������������������������������������������������������������ 234 8.7.3 Experimental Requirements (Table 8.26) ���������������������������� 234 8.7.4 Experimental Procedure�������������������������������������������������������� 234 8.7.5 Observation �������������������������������������������������������������������������� 236 8.7.6 Calculation���������������������������������������������������������������������������� 237 8.7.7 Expression of Results������������������������������������������������������������ 237 8.7.8 Quality Controls�������������������������������������������������������������������� 240 8.7.9 Precautions���������������������������������������������������������������������������� 240 8.7.10 Disposal�������������������������������������������������������������������������������� 241 8.8 Method for the Detection and Identification of Escherichia coli in Food and Food Products �������������������������������������������������������������� 241 8.8.1 Method Overview ���������������������������������������������������������������� 241 8.8.2 Scope of the Method ������������������������������������������������������������ 241 8.8.3 Experimental Requirements (Table 8.31) ���������������������������� 241 8.8.4 Experimental Procedure�������������������������������������������������������� 241 8.8.5 Molecular Identification�������������������������������������������������������� 244 8.8.6 Interpretation and Expression of Results������������������������������ 244 8.8.7 Quality Control �������������������������������������������������������������������� 244 8.8.8 Precautions���������������������������������������������������������������������������� 244 8.8.9 Disposal�������������������������������������������������������������������������������� 246 8.9 Method for the Detection and Identification of Salmonella in Food and Food Products �������������������������������������������������������������� 247 8.9.1 Method Overview ���������������������������������������������������������������� 247 8.9.2 Experimental Requirements (Table 8.33) ���������������������������� 248 8.9.3 Experimental Procedure�������������������������������������������������������� 248 8.9.4 Observation �������������������������������������������������������������������������� 250 8.9.5 Biochemical Identification���������������������������������������������������� 250 8.9.6 Serological Identification������������������������������������������������������ 250 8.9.7 Molecular Identification�������������������������������������������������������� 251 8.9.8 Interpretation������������������������������������������������������������������������ 251 8.9.9 Quality Control �������������������������������������������������������������������� 251 8.9.10 Precautions���������������������������������������������������������������������������� 251 8.9.11 Disposal�������������������������������������������������������������������������������� 252
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8.10 Method for the Detection and Identification of Staphylococcus aureus in Food and Food Products���������������������������������������������������� 253 8.10.1 Method Overview ���������������������������������������������������������������� 253 8.10.2 Scope of the Method ������������������������������������������������������������ 255 8.10.3 Experimental Requirements (Table 8.35) ���������������������������� 255 8.10.4 Experimental Procedure�������������������������������������������������������� 255 8.10.5 Observation �������������������������������������������������������������������������� 256 8.10.6 Biochemical Identification���������������������������������������������������� 257 8.10.7 Molecular Identification�������������������������������������������������������� 257 8.10.8 Interpretation and Expression of Result�������������������������������� 257 8.10.9 Quality Control �������������������������������������������������������������������� 258 8.10.10 Precautions�������������������������������������������������������������������������� 258 8.10.11 Disposal������������������������������������������������������������������������������ 259 8.11 Method for the Detection and Identification of Shigella in Food and Food Products���������������������������������������������������������������������������� 259 8.11.1 Method Overview ���������������������������������������������������������������� 259 8.11.2 Scope of the Method ������������������������������������������������������������ 260 8.11.3 Experimental Requirements (Table 8.37) ���������������������������� 260 8.11.4 Experimental Procedure�������������������������������������������������������� 260 8.11.5 Observation �������������������������������������������������������������������������� 262 8.11.6 Molecular Identification�������������������������������������������������������� 262 8.11.7 Interpretation and Expression of Result�������������������������������� 263 8.11.8 Quality Control �������������������������������������������������������������������� 264 8.11.9 Precautions���������������������������������������������������������������������������� 264 8.11.10 Disposal������������������������������������������������������������������������������ 264 8.12 Method for the Detection and Identification of Vibrio cholerae in Food and Food Products �������������������������������������������������������������� 264 8.12.1 Method Overview ���������������������������������������������������������������� 264 8.12.2 Scope of the Method ������������������������������������������������������������ 265 8.12.3 Experimental Requirements (Table 8.39) ���������������������������� 265 8.12.4 Experimental Procedure�������������������������������������������������������� 265 8.12.5 Molecular Identification�������������������������������������������������������� 267 8.12.6 Interpretation and Expression of Results������������������������������ 267 8.12.7 Quality Control �������������������������������������������������������������������� 267 8.12.8 Precautions���������������������������������������������������������������������������� 268 8.12.9 Disposal�������������������������������������������������������������������������������� 268 8.13 Method for the Detection and Identification of Vibrio parahaemolyticus in Food and Food Products���������������������������������� 268 8.13.1 Method Overview ���������������������������������������������������������������� 268 8.13.2 Scope of the Method ������������������������������������������������������������ 269 8.13.3 Experimental Requirements (Table 8.41) ���������������������������� 269 8.13.4 Experimental Procedure�������������������������������������������������������� 269 8.13.5 Molecular Identification�������������������������������������������������������� 271 8.13.6 Interpretation and Expression of Result�������������������������������� 271 8.13.7 Quality Control �������������������������������������������������������������������� 272
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8.13.8 Precautions���������������������������������������������������������������������������� 272 8.13.9 Disposal�������������������������������������������������������������������������������� 272 8.14 Method for the Detection and Identification of Clostridium perfringens in Food and Food Products�������������������������������������������� 272 8.14.1 Method Overview ���������������������������������������������������������������� 272 8.14.2 Scope of the Method ������������������������������������������������������������ 273 8.14.3 Experimental Requirements (Table 8.43) ���������������������������� 273 8.14.4 Experimental Procedure�������������������������������������������������������� 273 8.14.5 Molecular Identification�������������������������������������������������������� 275 8.14.6 Interpretation and Expression of Result�������������������������������� 275 8.14.7 Quality Control �������������������������������������������������������������������� 276 8.14.8 Precautions���������������������������������������������������������������������������� 276 8.14.9 Disposal�������������������������������������������������������������������������������� 276 8.15 Method for the Identification of E. coli O157: H7 in Food and Food Products by Tube Agglutination���������������������������������������������� 276 8.15.1 Method Overview ���������������������������������������������������������������� 276 8.15.2 Scope of the Method ������������������������������������������������������������ 277 8.15.3 Experimental Requirements (Table 8.45) ���������������������������� 277 8.15.4 Procedure������������������������������������������������������������������������������ 277 8.15.5 Precautions���������������������������������������������������������������������������� 281 8.15.6 Disposal�������������������������������������������������������������������������������� 281 8.16 Method for the Enumeration of Osmophilic Microorganisms in Food and Food Products �������������������������������������������������������������� 282 8.16.1 Method Overview ���������������������������������������������������������������� 282 8.16.2 Scope of the Method ������������������������������������������������������������ 282 8.16.3 Experimental Requirements (Table 8.46) ���������������������������� 282 8.16.4 Procedure������������������������������������������������������������������������������ 282 8.16.5 Calculation���������������������������������������������������������������������������� 284 8.16.6 Quality Controls�������������������������������������������������������������������� 284 8.16.7 Precautions���������������������������������������������������������������������������� 285 8.16.8 Disposal�������������������������������������������������������������������������������� 285 8.17 Method for the Isolation, Identification and Enumeration of Enterobacter sakazakii in Food and Food Products������������������������� 285 8.17.1 Method Overview ���������������������������������������������������������������� 285 8.17.2 Scope of the Method ������������������������������������������������������������ 286 8.17.3 Requirements (Table 8.47)���������������������������������������������������� 286 8.17.4 Experimental Procedure�������������������������������������������������������� 286 8.17.5 Molecular Identification�������������������������������������������������������� 288 8.17.6 Expression of Result ������������������������������������������������������������ 288 8.17.7 Quality Control �������������������������������������������������������������������� 288 8.17.8 Precautions���������������������������������������������������������������������������� 289 8.17.9 Disposal�������������������������������������������������������������������������������� 290 8.18 Method for the Isolation and Enumeration of Thermophilic Acidophilic Bacteria (TAB) in Food and Food Products������������������ 290 8.18.1 Method Overview ���������������������������������������������������������������� 290 8.18.2 Scope of the Method ������������������������������������������������������������ 290
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8.18.3 Requirements (Table 8.49)���������������������������������������������������� 291 8.18.4 Experimental procedure�������������������������������������������������������� 291 8.19 Method for the Microbiological Monitoring of Surfaces ���������������� 294 8.19.1 Method Overview ���������������������������������������������������������������� 294 8.19.2 Scope of the method������������������������������������������������������������� 294 8.19.3 Experimental Requirements (Table 8.50) ���������������������������� 294 8.19.4 Experimental Procedure�������������������������������������������������������� 294 8.19.5 Sampling Procedures and Calculations�������������������������������� 295 8.19.6 Interpretation������������������������������������������������������������������������ 298 8.19.7 Quality Control �������������������������������������������������������������������� 298 8.19.8 Precautions���������������������������������������������������������������������������� 298 8.19.9 Disposal�������������������������������������������������������������������������������� 299 References�������������������������������������������������������������������������������������������������� 299 9 Microbiological Methods for Pharmaceutical Analysis������������������������ 303 9.1 Introduction�������������������������������������������������������������������������������������� 303 9.2 Method for the Determination of Total Aerobic Microbial Count in Pharmaceutical Products�������������������������������������������������������������� 304 9.2.1 Method Overview ���������������������������������������������������������������� 304 9.2.2 Scope of the Method ������������������������������������������������������������ 304 9.2.3 Experimental Requirements (Table 9.1) ������������������������������ 304 9.2.4 Experimental Procedure�������������������������������������������������������� 304 9.2.5 Calculation���������������������������������������������������������������������������� 307 9.2.6 Expression of Results������������������������������������������������������������ 308 9.2.7 Quality Control �������������������������������������������������������������������� 309 9.2.8 Precautions���������������������������������������������������������������������������� 309 9.2.9 Disposal�������������������������������������������������������������������������������� 310 9.3 Method for the Determination of Total Aerobic Fungal Count in Pharmaceutical Products�������������������������������������������������������������� 310 9.3.1 Method Overview ���������������������������������������������������������������� 310 9.3.2 Scope of the Method ������������������������������������������������������������ 310 9.3.3 Experimental Requirements (Table 9.6) ������������������������������ 311 9.3.4 Experimental Procedure�������������������������������������������������������� 311 9.3.5 Calculation���������������������������������������������������������������������������� 313 9.3.6 Expression of Results������������������������������������������������������������ 314 9.3.7 Quality Control �������������������������������������������������������������������� 315 9.3.8 Precautions���������������������������������������������������������������������������� 315 9.3.9 Disposal�������������������������������������������������������������������������������� 316 9.4 Method for the Detection and Confirmation of Escherichia coli in Pharmaceutical Products�������������������������������������������������������������� 317 9.4.1 Method Overview ���������������������������������������������������������������� 317 9.4.2 Scope of the Method ������������������������������������������������������������ 317 9.4.3 Experimental Requirements (Table 9.11) ���������������������������� 317 9.4.4 Experimental Procedure�������������������������������������������������������� 317 9.4.5 Interpretation and Expression of Result�������������������������������� 320
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9.4.6 Quality Control �������������������������������������������������������������������� 320 9.4.7 Precautions���������������������������������������������������������������������������� 320 9.4.8 Disposal�������������������������������������������������������������������������������� 321 9.5 Method for the Detection and Confirmation of Salmonella in Pharmaceutical Products������������������������������������������������������������������ 321 9.5.1 Method Overview ���������������������������������������������������������������� 321 9.5.2 Scope of the Method ������������������������������������������������������������ 321 9.5.3 Experimental Requirements (Table 9.13) ���������������������������� 322 9.5.4 Experimental Procedure�������������������������������������������������������� 322 9.5.5 Serological Identification������������������������������������������������������ 323 9.5.6 Interpretation and Expression of Results������������������������������ 324 9.5.7 Quality Control �������������������������������������������������������������������� 324 9.5.8 Precautions���������������������������������������������������������������������������� 325 9.5.9 Disposal�������������������������������������������������������������������������������� 325 9.6 Method for Detection and Confirmation of Staphylococcus aureus in Pharmaceutical Products�������������������������������������������������������������� 325 9.6.1 Method Overview ���������������������������������������������������������������� 325 9.6.2 Scope of the Method ������������������������������������������������������������ 326 9.6.3 Experimental Requirements (Table 9.15) ���������������������������� 326 9.6.4 Experimental Procedure�������������������������������������������������������� 326 9.6.5 Expression of Result ������������������������������������������������������������ 328 9.6.6 Quality Control �������������������������������������������������������������������� 328 9.6.7 Precautions���������������������������������������������������������������������������� 328 9.6.8 Disposal�������������������������������������������������������������������������������� 329 9.7 Method for the Detection and Confirmation of Pseudomonas aeruginosa in Pharmaceutical Products�������������������������������������������� 329 9.7.1 Scope of the Method ������������������������������������������������������������ 329 9.7.2 Experimental Requirements (Table 9.17) ���������������������������� 329 9.7.3 Experimental Procedure�������������������������������������������������������� 329 9.7.4 Expression of Result ������������������������������������������������������������ 331 9.7.5 Quality Control �������������������������������������������������������������������� 331 9.7.6 Precautions���������������������������������������������������������������������������� 332 9.7.7 Disposal�������������������������������������������������������������������������������� 333 9.8 Method for the Detection and Confirmation of Shigella in Pharmaceutical Products�������������������������������������������������������������� 333 9.8.1 Method Overview ���������������������������������������������������������������� 333 9.8.2 Scope of the Method ������������������������������������������������������������ 333 9.8.3 Experimental Requirements (Table 9.19) ���������������������������� 333 9.8.4 Experimental Procedure�������������������������������������������������������� 333 9.8.5 Expression of Result ������������������������������������������������������������ 335 9.8.6 Quality Control �������������������������������������������������������������������� 336 9.8.7 Precautions���������������������������������������������������������������������������� 336 9.8.8 Disposal�������������������������������������������������������������������������������� 336 9.9 Method for the Detection and Confirmation of Clostridia in Pharmaceutical Products�������������������������������������������������������������� 336
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9.9.1 Experimental Overview�������������������������������������������������������� 336 9.9.2 Scope of the Method ������������������������������������������������������������ 337 9.9.3 Experimental Requirements (Table 9.21) ���������������������������� 337 9.9.4 Experimental Procedure�������������������������������������������������������� 337 9.9.5 Expression of Result ������������������������������������������������������������ 339 9.9.6 Quality Control �������������������������������������������������������������������� 339 9.9.7 Precautions���������������������������������������������������������������������������� 339 9.9.8 Disposal�������������������������������������������������������������������������������� 340 9.10 Method for the Detection and Confirmation of Candida albicans in Pharmaceutical Products�������������������������������������������������������������� 340 9.10.1 Method Overview ���������������������������������������������������������������� 340 9.10.2 Scope of the Method ������������������������������������������������������������ 340 9.10.3 Experimental Requirements (Table 9.23) ���������������������������� 340 9.10.4 Experimental Procedure�������������������������������������������������������� 340 9.10.5 Interpretation and Expression of Result�������������������������������� 342 9.10.6 Quality Control �������������������������������������������������������������������� 342 9.10.7 Precautions���������������������������������������������������������������������������� 342 9.10.8 Disposal�������������������������������������������������������������������������������� 343 9.11 Method for the Detection and Confirmation of Bile-Tolerant Gram-Negative Bacteria in Pharmaceutical Products���������������������� 343 9.11.1 Method Overview ���������������������������������������������������������������� 343 9.11.2 Scope of the Method ������������������������������������������������������������ 343 9.11.3 Experimental Requirements (Table 9.24) ���������������������������� 344 9.11.4 Experimental Procedure�������������������������������������������������������� 344 9.11.5 Observation �������������������������������������������������������������������������� 345 9.11.6 Interpretation and Expression of Result�������������������������������� 345 9.11.7 Quality Control �������������������������������������������������������������������� 345 9.11.8 Precautions���������������������������������������������������������������������������� 346 9.11.9 Disposal�������������������������������������������������������������������������������� 346 9.12 Method for the Estimation of Antibiotic Potency by Agar Well Diffusion Assay������������������������������������������������������������������������ 346 9.12.1 Method Overview ���������������������������������������������������������������� 346 9.12.2 Scope of the Method ������������������������������������������������������������ 346 9.12.3 Experimental Requirements (Table 9.25) ���������������������������� 347 9.12.4 Experimental Procedure�������������������������������������������������������� 347 9.12.5 Calculation���������������������������������������������������������������������������� 350 9.12.6 Quality Control �������������������������������������������������������������������� 351 9.12.7 Precautions���������������������������������������������������������������������������� 351 9.12.8 Disposal�������������������������������������������������������������������������������� 351 9.13 Method for the Estimation of Vitamin B12 by Agar Well Diffusion Assay�������������������������������������������������������������������������������� 352 9.13.1 Method Overview ���������������������������������������������������������������� 352 9.13.2 Scope of the Method ������������������������������������������������������������ 352 9.13.3 Experimental Requirements (Table 9.27) ���������������������������� 352 9.13.4 Experimental Procedure�������������������������������������������������������� 352
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9.13.5 Calculation���������������������������������������������������������������������������� 355 9.13.6 Quality Control �������������������������������������������������������������������� 356 9.13.7 Precautions���������������������������������������������������������������������������� 356 9.13.8 Disposal�������������������������������������������������������������������������������� 356 9.14 Method for Sterility Testing of Pharmaceutical Products���������������� 356 9.14.1 Method Overview ���������������������������������������������������������������� 356 9.14.2 Scope of the Method ������������������������������������������������������������ 357 9.14.3 Experimental Requirements (Table 9.28) ���������������������������� 357 9.14.4 Experimental Procedure�������������������������������������������������������� 357 9.14.5 Controls�������������������������������������������������������������������������������� 361 9.14.6 Positive Control�������������������������������������������������������������������� 361 9.14.7 Positive Product control�������������������������������������������������������� 361 9.14.8 Negative Control ������������������������������������������������������������������ 362 9.14.9 Media Control and Air Exposure Control���������������������������� 362 9.14.10 Observation ������������������������������������������������������������������������ 362 9.14.11 Expression of Results���������������������������������������������������������� 362 9.14.12 Precautions�������������������������������������������������������������������������� 364 9.14.13 Disposal������������������������������������������������������������������������������ 364 9.15 Method for the Detection of Bacterial Endotoxin in Pharmaceutical Products������������������������������������������������������������������ 364 9.15.1 Method Overview ���������������������������������������������������������������� 364 9.15.2 Scope of the Method ������������������������������������������������������������ 365 9.15.3 Experimental Requirements (Table 9.29) ���������������������������� 365 9.15.4 Experimental Procedure�������������������������������������������������������� 365 9.15.5 Establishment of Endotoxin Limit���������������������������������������� 367 9.15.6 Method���������������������������������������������������������������������������������� 367 9.15.7 Observation �������������������������������������������������������������������������� 368 9.15.8 Expression of Results������������������������������������������������������������ 368 9.15.9 Precautions���������������������������������������������������������������������������� 368 9.15.10 Disposal������������������������������������������������������������������������������ 368 9.16 Method for the Detection and Quantification of Calcium Pantothenate in Drug Samples���������������������������������������������������������� 369 9.16.1 Method Overview ���������������������������������������������������������������� 369 9.16.2 Scope of the Method ������������������������������������������������������������ 369 9.16.3 Experimental Requirements (Table 9.30) ���������������������������� 369 9.16.4 Experimental Procedure�������������������������������������������������������� 369 9.16.5 Calculation���������������������������������������������������������������������������� 372 9.16.6 Precautions���������������������������������������������������������������������������� 372 9.16.7 Disposal�������������������������������������������������������������������������������� 373 9.17 Method for the Detection and Quantification of Biotin in Pharmaceutical Products������������������������������������������������������������������ 373 9.17.1 Method Overview ���������������������������������������������������������������� 373 9.17.2 Scope of the Method ������������������������������������������������������������ 373 9.17.3 Experimental Requirements (Table 9.31) ���������������������������� 373 9.17.4 Experimental Procedure�������������������������������������������������������� 373
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9.17.5 Calculation���������������������������������������������������������������������������� 376 9.17.6 Precautions���������������������������������������������������������������������������� 377 9.17.7 Disposal�������������������������������������������������������������������������������� 377 9.18 Method for the Antimicrobial Effectiveness Testing or Preservative Testing of Pharmaceutical Products ���������������������������� 377 9.18.1 Method Overview ���������������������������������������������������������������� 377 9.18.2 Scope of the Method ������������������������������������������������������������ 378 9.18.3 Experimental Requirements (Table 9.32) ���������������������������� 378 9.18.4 Experimental Procedure�������������������������������������������������������� 378 9.18.5 Precautions���������������������������������������������������������������������������� 381 9.18.6 Disposal�������������������������������������������������������������������������������� 381 9.19 Method for the Determination of Bioburden������������������������������������ 381 9.19.1 Experimental Overview�������������������������������������������������������� 381 9.19.2 Scope of the Method ������������������������������������������������������������ 381 9.19.3 Experimental Requirements (Table 9.33) ���������������������������� 382 9.19.4 Experimental Procedure�������������������������������������������������������� 382 9.19.5 Expression of Results������������������������������������������������������������ 385 9.19.6 Precautions���������������������������������������������������������������������������� 386 9.19.7 Disposal�������������������������������������������������������������������������������� 386 9.20 Method for the Determination of the Level of Lactic Acid Bacillus Organism in Pharmaceutical Preparations�������������������������� 386 9.20.1 Method Overview ���������������������������������������������������������������� 386 9.20.2 Scope of the Method ������������������������������������������������������������ 386 9.20.3 Experimental Requirements (Table 9.34) ���������������������������� 387 9.20.4 Experimental Procedure�������������������������������������������������������� 387 9.20.5 Precautions���������������������������������������������������������������������������� 389 9.20.6 Disposal�������������������������������������������������������������������������������� 390 9.21 Method for the Determination of Mutagenicity of Chemicals by Bacterial Reverse Mutation Assay (Ames Test)�������������������������� 390 9.21.1 Method Overview ���������������������������������������������������������������� 390 9.21.2 Scope of the Method ������������������������������������������������������������ 390 9.21.3 Experimental Requirements (Table 9.35) ���������������������������� 391 9.21.4 Experimental Procedure�������������������������������������������������������� 391 9.21.5 Interpretation and Expression of Results������������������������������ 396 9.21.6 Quality Control �������������������������������������������������������������������� 396 9.21.7 Precautions���������������������������������������������������������������������������� 396 9.21.8 Disposal�������������������������������������������������������������������������������� 396 9.22 Method for the Determination of Antimicrobial Properties of Pharmaceutically Important Compounds������������������������������������������ 397 9.22.1 Method Overview ���������������������������������������������������������������� 397 9.22.2 Scope of the Method ������������������������������������������������������������ 397 9.22.3 Experimental Requirements (Table 9.36) ���������������������������� 397 9.22.4 Experimental Procedure�������������������������������������������������������� 397 9.22.5 Observation and Expression of Results�������������������������������� 400 9.22.6 Quality Control �������������������������������������������������������������������� 400
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9.22.7 Precautions���������������������������������������������������������������������������� 403 9.22.8 Disposal�������������������������������������������������������������������������������� 403 9.23 Method for Determination of Minimum Inhibitory Concentration (MIC) of Pharmaceutically Important Compounds and Extracts������������������������������������������������������������������ 404 9.23.1 Method Overview ���������������������������������������������������������������� 404 9.23.2 Scope of the Method ������������������������������������������������������������ 404 9.23.3 Experimental Requirements (Table 9.37) ���������������������������� 404 9.23.4 Experimental Procedure�������������������������������������������������������� 404 9.23.5 Interpretation and Expression of Results������������������������������ 409 9.23.6 Quality Control �������������������������������������������������������������������� 409 9.23.7 Precautions���������������������������������������������������������������������������� 410 9.23.8 Disposal�������������������������������������������������������������������������������� 412 9.24 Method for the Determination of Multiple Antibiotic Resistance and Susceptibility Pattern of Microorganisms���������������������������������� 412 9.24.1 Method Overview ���������������������������������������������������������������� 412 9.24.2 Experimental Requirements (Table 9.38) ���������������������������� 412 9.24.3 Experimental Procedure�������������������������������������������������������� 412 9.24.4 Observation and Expression of Results�������������������������������� 415 9.24.5 Quality Control �������������������������������������������������������������������� 416 9.24.6 Precautions���������������������������������������������������������������������������� 416 9.24.7 Disposal�������������������������������������������������������������������������������� 419 References�������������������������������������������������������������������������������������������������� 419 10 Biochemical and Molecular Methods for Bacterial Identification������ 425 10.1 Introduction������������������������������������������������������������������������������������ 425 10.2 Gram’s Staining������������������������������������������������������������������������������ 426 10.2.1 Method Overview�������������������������������������������������������������� 426 10.2.2 Major Requirements���������������������������������������������������������� 426 10.2.3 Experimental Procedure���������������������������������������������������� 426 10.2.4 Interpretation and Expression of Result���������������������������� 427 10.2.5 Quality Control������������������������������������������������������������������ 427 10.2.6 Precautions������������������������������������������������������������������������ 427 10.2.7 Disposal ���������������������������������������������������������������������������� 428 10.3 Coagulase Test�������������������������������������������������������������������������������� 428 10.3.1 Method Overview�������������������������������������������������������������� 428 10.3.2 Major Requirements���������������������������������������������������������� 428 10.3.3 Experimental Procedure���������������������������������������������������� 428 10.3.4 Interpretation and Expression of Results�������������������������� 429 10.3.5 Quality Control������������������������������������������������������������������ 429 10.3.6 Precautions������������������������������������������������������������������������ 430 10.3.7 Disposal ���������������������������������������������������������������������������� 430 10.4 Catalase Test ���������������������������������������������������������������������������������� 430 10.4.1 Method Overview�������������������������������������������������������������� 430 10.4.2 Major Requirements���������������������������������������������������������� 430
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10.4.3 Experimental Procedure���������������������������������������������������� 431 10.4.4 Interpretation and Expression of Result���������������������������� 431 10.4.5 Quality Control������������������������������������������������������������������ 431 10.4.6 Precautions������������������������������������������������������������������������ 432 10.4.7 Disposal ���������������������������������������������������������������������������� 432 10.5 Oxidase Test������������������������������������������������������������������������������������ 432 10.5.1 Method Overview�������������������������������������������������������������� 432 10.5.2 Major Requirements���������������������������������������������������������� 432 10.5.3 Experimental Procedure���������������������������������������������������� 432 10.5.4 Interpretation and Expression of Result���������������������������� 433 10.5.5 Quality Control������������������������������������������������������������������ 433 10.5.6 Precautions������������������������������������������������������������������������ 434 10.5.7 Disposal ���������������������������������������������������������������������������� 434 10.6 Nitrate Reduction Test�������������������������������������������������������������������� 434 10.6.1 Method Overview�������������������������������������������������������������� 434 10.6.2 Major Requirements���������������������������������������������������������� 434 10.6.3 Experimental Procedure���������������������������������������������������� 435 10.6.4 Interpretation �������������������������������������������������������������������� 435 10.6.5 Quality Control������������������������������������������������������������������ 436 10.6.6 Precautions������������������������������������������������������������������������ 436 10.6.7 Disposal ���������������������������������������������������������������������������� 436 10.7 Hugh-Leifson (H-L) Test���������������������������������������������������������������� 436 10.7.1 Method Overview�������������������������������������������������������������� 436 10.7.2 Major Requirements���������������������������������������������������������� 436 10.7.3 Test Procedure ������������������������������������������������������������������ 437 10.7.4 Interpretation �������������������������������������������������������������������� 437 10.7.5 Quality Control������������������������������������������������������������������ 438 10.7.6 Precautions������������������������������������������������������������������������ 438 10.7.7 Disposal ���������������������������������������������������������������������������� 438 10.8 Indole Test�������������������������������������������������������������������������������������� 438 10.8.1 Method Overview�������������������������������������������������������������� 438 10.8.2 Major Requirements���������������������������������������������������������� 438 10.8.3 Experimental Procedure���������������������������������������������������� 439 10.8.4 Interpretation �������������������������������������������������������������������� 439 10.8.5 Quality Control������������������������������������������������������������������ 440 10.8.6 Precautions������������������������������������������������������������������������ 440 10.8.7 Disposal ���������������������������������������������������������������������������� 440 10.9 Methyl Red Test������������������������������������������������������������������������������ 440 10.9.1 Method Overview�������������������������������������������������������������� 440 10.9.2 Major Requirements���������������������������������������������������������� 440 10.9.3 Experimental Procedure���������������������������������������������������� 441 10.9.4 Interpretation and Expression of Result���������������������������� 441 10.9.5 Quality Control������������������������������������������������������������������ 441 10.9.6 Precautions������������������������������������������������������������������������ 442 10.9.7 Disposal ���������������������������������������������������������������������������� 442
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10.10 Voges-Proskauer’s Test ������������������������������������������������������������������ 442 10.10.1 Method Overview�������������������������������������������������������������� 442 10.10.2 Major Requirements���������������������������������������������������������� 442 10.10.3 Experimental Procedure���������������������������������������������������� 442 10.10.4 Interpretation and Expression of Result���������������������������� 443 10.10.5 Quality Control������������������������������������������������������������������ 443 10.10.6 Precautions������������������������������������������������������������������������ 444 10.10.7 Disposal ���������������������������������������������������������������������������� 444 10.11 Triple Sugar Iron Agar (TSI) Test �������������������������������������������������� 444 10.11.1 Method Overview�������������������������������������������������������������� 444 10.11.2 Major Requirements���������������������������������������������������������� 444 10.11.3 Experimental Procedure���������������������������������������������������� 445 10.11.4 Interpretation and Expression of Result���������������������������� 445 10.11.5 Quality Control������������������������������������������������������������������ 445 10.11.6 Precautions������������������������������������������������������������������������ 445 10.11.7 Disposal ���������������������������������������������������������������������������� 445 10.12 Urease Test�������������������������������������������������������������������������������������� 446 10.12.1 Method Overview�������������������������������������������������������������� 446 10.12.2 Major Requirements���������������������������������������������������������� 446 10.12.3 Experimental Procedure���������������������������������������������������� 446 10.12.4 Interpretation and Expression of Result���������������������������� 447 10.12.5 Quality Control������������������������������������������������������������������ 447 10.12.6 Precautions������������������������������������������������������������������������ 447 10.12.7 Disposal ���������������������������������������������������������������������������� 447 10.13 Citrate Utilization Test�������������������������������������������������������������������� 448 10.13.1 Method Overview�������������������������������������������������������������� 448 10.13.2 Major Requirements���������������������������������������������������������� 448 10.13.3 Experimental Procedure���������������������������������������������������� 448 10.13.4 Interpretation and Expression of Result���������������������������� 448 10.13.5 Quality Control������������������������������������������������������������������ 448 10.13.6 Precautions������������������������������������������������������������������������ 449 10.13.7 Disposal ���������������������������������������������������������������������������� 449 10.14 Casein Hydrolysis Test�������������������������������������������������������������������� 449 10.14.1 Method Overview�������������������������������������������������������������� 449 10.14.2 Major Requirements���������������������������������������������������������� 450 10.14.3 Experimental Procedure���������������������������������������������������� 450 10.14.4 Interpretation and Expression of Results�������������������������� 450 10.14.5 Quality Control������������������������������������������������������������������ 450 10.14.6 Precautions������������������������������������������������������������������������ 450 10.14.7 Disposal ���������������������������������������������������������������������������� 451 10.15 Gelatin Liquefaction Test���������������������������������������������������������������� 451 10.15.1 Method Overview�������������������������������������������������������������� 451 10.15.2 Major Requirements���������������������������������������������������������� 451 10.15.3 Experimental Procedure���������������������������������������������������� 452 10.15.4 Interpretation and Expression of Result���������������������������� 452
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10.15.5 Quality Control������������������������������������������������������������������ 452 10.15.6 Precautions������������������������������������������������������������������������ 453 10.15.7 Disposal ���������������������������������������������������������������������������� 453 10.16 ONPG Test�������������������������������������������������������������������������������������� 453 10.16.1 Method Overview�������������������������������������������������������������� 453 10.16.2 Major Requirements���������������������������������������������������������� 453 10.16.3 Experimental Procedure���������������������������������������������������� 453 10.16.4 Interpretation and Expression of Result���������������������������� 454 10.16.5 Quality Control������������������������������������������������������������������ 454 10.16.6 Precautions������������������������������������������������������������������������ 454 10.16.7 Disposal ���������������������������������������������������������������������������� 454 10.17 Starch Hydrolysis Test�������������������������������������������������������������������� 455 10.17.1 Method Overview�������������������������������������������������������������� 455 10.17.2 Major Requirements���������������������������������������������������������� 455 10.17.3 Experimental Procedure���������������������������������������������������� 455 10.17.4 Interpretation and Expression of Result���������������������������� 456 10.17.5 Quality Control������������������������������������������������������������������ 456 10.17.6 Precautions������������������������������������������������������������������������ 456 10.17.7 Disposal ���������������������������������������������������������������������������� 456 10.18 Motility Test������������������������������������������������������������������������������������ 457 10.18.1 Method Overview�������������������������������������������������������������� 457 10.18.2 Major Requirements���������������������������������������������������������� 457 10.18.3 Experimental Procedure���������������������������������������������������� 457 10.18.4 Interpretation and Expression of Result���������������������������� 458 10.18.5 Quality Control������������������������������������������������������������������ 458 10.18.6 Precautions������������������������������������������������������������������������ 459 10.18.7 Disposal ���������������������������������������������������������������������������� 459 10.19 Phenyl Pyruvic Acid Test���������������������������������������������������������������� 459 10.19.1 Method Overview�������������������������������������������������������������� 459 10.19.2 Major Requirements���������������������������������������������������������� 459 10.19.3 Experimental Procedure���������������������������������������������������� 460 10.19.4 Interpretation and Expression of Result���������������������������� 460 10.19.5 Quality Control������������������������������������������������������������������ 460 10.19.6 Precautions������������������������������������������������������������������������ 460 10.19.7 Disposal ���������������������������������������������������������������������������� 460 10.20 Molecular Identification of Bacteria ���������������������������������������������� 461 10.20.1 Method Overview�������������������������������������������������������������� 461 10.20.2 Experimental Requirements���������������������������������������������� 461 10.20.3 Experimental Procedure���������������������������������������������������� 462 10.20.4 Sequencing������������������������������������������������������������������������ 465 10.20.5 Identification of Bacteria Using 16s RNA Sequence�������� 466 10.20.6 Interpretation and Expression of Result���������������������������� 466 References�������������������������������������������������������������������������������������������������� 466 Index������������������������������������������������������������������������������������������������������������������ 469
About the Author
Abhishek Chauhan obtained his B.Sc. in Industrial Microbiology as well as M.Sc. and Ph.D. in Microbiology from Gurukul Kangri University, Haridwar, Uttarakhand, India. He is currently working as a Senior Scientist at Amity University, Noida, UP, India, and formerly worked as a Scientist “C” and Head of the Department of Microbiology, Shriram Institute for Industrial Research (SRI), Delhi, India. He is known for his analytical and research mindset and supervised approximately more than 25 industry-sponsored projects. While working at SRI, he has developed and validated several microbiological methods related to food, water, drugs, and pharmaceuticals. Dr. Chauhan has participated in the 35th Indian Scientific Expedition to Antarctica and tenth Indian Southern Ocean Expedition, scientific ventures of Ministry of Earth Science, Govt. of India. He has total 15 years of rich experience and has guided more than 10 postgraduate students on emerging issues of microbiology and biotechnology. Dr. Chauhan has authored a research book on key aspects of “Antibacterial Activity of Cyanobacteria” and edited a book entitled Plants and Microbes: An Innovative Approach. He has delivered various plenary lectures and invited talk at various symposium and conferences and has been a key speaker of teleconferencing talk on “Food and Diet” under EduSAT network, Vigyan Prasar, DST, Govt. of India. Dr. Chauhan has been a reviewer for many peer-reviewed journals and published more than 45 research papers and honored with prestigious awards such as “Young Scientist Award” (2013), STOX-Appreciation Award (2016), NCEEBR-Certificate of Excellence for Oral Presentation, and Best Presentation Award (IIPA-DST Govt. of India) 2012. He has also been certified on various quality certification and accreditations such as ISO: 17025 (Laboratory Quality Management System), ISO: 17043 (Proficiency Testing), ISO: 22000, ISO:9001, and ISO:14001. Dr. Chauhan is an active member of the Indian Science Congress Association, Society for Plant Research, and Association of Microbiologists of India. Tanu Jindal completed her Ph.D. in Ecotoxicology from the Department of Zoology, University of Delhi, in 1999. She is Group Additional Pro Vice Chancellor (R&D) and the Director of Amity Institute for Environmental Toxicology, Safety xxxiii
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and Management (AIETSM) and Amity Institute of Environmental Sciences (AIES) at Amity University, Noida. Prof. Jindal is responsible for Ph.D., M.Sc., and B.Sc. Environmental Science courses to promote environmental research and studies with syllabus covering important and current area of environmental science. She has filed six patents on lysimetric-device, apparatus to estimate the loss of xenobiotics by volatilization and mineralization, natural pesticide, photochemical method to dispose of dilute pesticide waste, and cost-effective water testing kit. Prof. Jindal has completed projects on Yamuna, Hindon, and Ghagghar Rivers and groundwater contamination through pesticides, MoEF&CC; contamination of soil and groundwater through leaching of drains in Delhi, MoES; groundwater contamination of Chlorpyrifos in soils at different pH with CWRDM, DST; environmental monitoring studies at Antarctica – NCAOR, Goa; and impact of electromagnetic radiation, DST. Her recent initiatives are projects on Development of Lysimeter, DST; Air Pollution Monitoring through CAAQMS-UPPCB; and Development of Emission Factors, CPCB. Establishment of the Pesticide Referral Laboratory and MRL fixation of pulses and spices were her research endeavors at ICAR. Her expertise area is ISO-17025, GLP-studies, radio and stable isotope tracer techniques, GCMS and LCMS studies, etc. Prof. Jindal taught Pesticides Chemistry and Toxicology at Delhi University for 7 years to M.Sc. students. She holds membership of eminent scientific societies. She has travelled extensively nationally and internationally presenting papers and has publications in referred journals of high impact factor. Prof. Jindal has published more than 38 papers in reputed journals and has been serving as an editorial board member of repute. She has to her credit Editor of The Year Award 2017 by MTRES, Excellence in Research and Teaching Award 2017 by National Environmental Science Academy, prestigious Scientist of the year Award – 2015, Environmentalist of the Year Award – 2014 by NESA, New Investigator Award presentation at ACS, and DST Young Scientist Award Project. Prof. Jindal has received travel awards from CSIR, DST, and INSA.
Chapter 1
Introductory Analytical Microbiology
1.1 Introduction For the last few decades, comprehension of Microbiology has become vital for personnel in many different disciplines. The importance of the subject in relation to water, air, food, medicine and health is well known. That the growth of microorganisms may produce troubles for engineers, and which may come as a surprise to many of us. Engineers in industry, particularly chemical and biochemical engineers must deal with such problems, and they find intricacy in dealing with them because they lack the understanding of the characteristics of the various organisms concerned. Microbiology simply refers to the ‘study of minute organisms. These organisms are too small to be seen by the unaided human eyes i.e. specific instruments like microscopes are required for their visualization. However, some exceptions like higher molds are considered as microbe despite their macro structure. Microorganisms are ubiquitous (=omnipresent) and they include all life forms other than plants and animals. It is an interesting fact that the human body which consists of about 1013 cells, is inhabited by approximately 1015 microbial cells in and on its various organs like intestine, oral cavity, skin etc. Similarly, only one gram of soil is estimated to have infinite number of microbes of all types. Despite their tiny size and mass, the total microbial biomass is large enough to counter the macroscopic life size. These miniature creations of the God have a vast impact throughout and form the basis for all life on earth. Even though they are very tiny, they have a major influence on several catalytic transformations which are essential for the sustainability of life on the earth. Microbes inhabit a wide range of habitats and they can be found even in those places which are assumed to be completely uninhabitable. Examples of such places include high altitude regions having very low temperature like Arctic and Antarctic, high temperature regions like desserts, hot springs, highly alkaline or acidic
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_1
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environments or even in the atmosphere deprive of oxygen or abundance of carbon-di-oxide. Microorganisms are usually unicellular entities having a single cell body only. Examples include bacteria, single cell yeasts and some algae. Cyanobacteria (also refers as blue-green algae) are characterized by having the filamentous structures in which cells are terminally attached in a row forming a filament. Multi-cellular fungal and algal species are also identified but they are not differentiated into tissues and organs. If one looks inside the cellular structure of microorganisms, they can be classified as prokaryotic and eukaryotic. Prokaryotes like bacteria, cyanobacteria do not have a true nucleus and other internal membrane systems viz. endoplasmic reticulum, mitochondria, Golgi-bodies etc. on the other hand, eukaryotic microbes like fungi (yeasts and molds), algae are having the true nucleus and other membrane bound structures. Biochemically, microbes are having several macromolecules like proteins, nucleic acids (DNA & RNA), polysaccharides, hetero-polymers etc. and this forms the basis of their broad classification. Based on the biochemical structure of the cell wall, bacteria are classified in to two groups i.e. Gram positive and Gram negative. Gram positive bacteria consist of a thick layer of peptidoglycan outside the cell membranes. Examples of such bacteria are. Staphylococcus aureus, Streptococcus, Bacillus, Clostridium etc. On the other hand, The Gram negatives do have a very fine layer of peptidoglycan which is further covered by other membrane composed of lipopolysaccharide (LPS) such as Escherichia coli, Pseudomonas, Salmonella, Shigella, Vibrio cholerae etc.
1.2 Analytical Microbiology: Various Disciplines Microbiology is not only basic science, but it also is having a vast applicability for the society. It has several disciplines having applied aspects of microbes. Based on their effect on the society; bacteria are classified as ‘Good’ and ‘Bad’. Good bacteria are the organisms which in many ways are beneficial for the society, for example, lactic acid bacteria use in a probiotic preparation to enhance the digestive capabilities by the human intestine. Similarly, other organisms present in our body as a normal microflora, regulate many of activities. Several industrial important microorganisms are being used throughout for the production of a number of products like alcoholic beverages, cheese, bread, vinegar, vitamins, antibiotics, citric acid, lactic acid, enzymes and many more. Sometimes, bacteria act as devil and are responsible for a number of ailments in humans and other organisms. Human beings are greatly affected by all kind of microbes (including bacteria and fungi) for various types of diseases like cholera, diarrhea, typhoid, gastrointestinal problems, urinary tract infections, respiratory infections, blood infections etc. These bad bacteria also cause food spoilage and deterioration of other materials, hence, also called as ‘invisible allies’.
1.3 Food Microbiology
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Broadly following disciplines are considered as the fingers of Microbiology Hand.
1.3 Food Microbiology Food Microbiology deals with the food-borne microorganisms in both “Good” and ‘Bad’ manner. Food-borne microorganisms can enter in food chain at any stage from ‘Farm-to-Fork’ and cause contamination and spoilage. Microbiologists are involved both in quality control and quality assurance during manufacturing of drinks and food by following the Hazard Analysis and Critical Control Points (HACCP). In industrial production facilities, technicians are also involved in the maintenance of the microbial culture that is used to start the fermentation of several products and to stop decline of current strains and to grow or improve existing ones. Similarly, Technicians maintain the strains of yeasts and other beneficial microbes and supervising the fermentation. All the microbiological test parameters such as presence and absence of pathogens such as E. coli, Salmonella, Staphylococcus, Listeria, Campylobacter etc. are checked during the quality control and Quality assurance of final products. Mycotoxins such as Aflatoxin, Ochratoxin, T-2 Toxin are the secondary metabolites and are produced by certain fungal species, for example, Aspergillus, Claviceps, Penicillium and Fusarium during their late exponential phase of growth. These proteins are highly heat-stable and responsible for acute or chronic diseases such as several types of cancers, problems associated with gastrointestinal tract, reproductive system etc. when consumed along with the contaminated food.
1.3.1 Microbiological Analysis of Food and Food Products Microbiological analysis of food is important as the growth of microbes occurs in food contaminated with pathogenic bacteria and fungi which critically affects the quality of food and farm products. These pathogenic microorganism in food products has hazardous effects on human health. Microbial contamination of food products results in two types of food-borne illnesses i.e. firstly, due to ingestion of toxins produced by growing microbes in food and secondly, an infection caused by the ingestion of microbes along with contaminated food. The food borne disease incidence have been growing world over, therefore, national and international regulatory agencies are setting standards and updating existing one for ‘Food Quality & Safety’ to overcome the problems of food-borne illnesses. Since, microbiological parameters have been established as the responsible factors for food safety, hence, microbiological evaluation for food safety are the key subjects in the current era. Major objectives of microbiological analysis of food are to evaluate the presence and absence of different types of microbes recommended for analysis and determining their nature in terms of pathogenicity and toxicity. It is also worthwhile to know
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about different categories of levels of contamination of microbes in Food. The method adopted for analysis may vary from product to product based on the level of contamination. Ingredients and water used for food preparation should also be evaluated for microbial testing in addition to final cooked food. Quality of water and ingredients is necessary for determining the suitable system and measures to ensure the safety & quality of finished food products (Table 1.1).
1.3.2 M icrobial Contamination of Food and Food Products: Major Factors In most of the cases of food spoilage, mixed microflora including various types of bacteria, fungi and their metabolic end-products such as toxins are accountable. Several factors may contribute during the process of spoilage of food products leading to microbial growth and the accumulation of harmful products. These factors can be elucidated as follows: 1.3.2.1 Temperature Temperature plays a significant role in multiplication of microbes as it affects the enzyme activity and central dogma i.e. replication, transcription and translation phenomena responsible for reproduction of microorganisms. Based upon the temperature requirement for optimal growth; bacteria are classified into four different categories i.e. a) mesophiles, the category of bacterial population including human pathogens, that grow between 10 to 45 °C; b) psychrophiles, grow optimally between −1 to 10 °C; c) psychrotrophs, grow over a large temperature range of 0 to 45 °C and d) thermophiles, grows at a temperature as high as 55 °C and even more such as hyperthermophiles. There might be the presence of thousands of different bacteria that grow in the food and compete. As a result, only those bacteria that acclimatize and grow faster than others predominate in that kind of food product and are responsible for spoilage at a given temperature. It has also observed that the growth of microbes on a given food greatly depends on the bacterial species that are present on the food before harvesting. To thrive, microorganisms require a temperature between 5 to 65 °C and above 65 °C, survival rate usually decreases significantly. If boiled for as long as 10 minutes, rapid death of microbes occurs. However, upon heating, the microorganisms gradually die off, but not all at the same time, therefore, heating at temperatures lesser than 100 °C must be sustained for a longer period. Bacterial growth is also slowed down at 0 to 5 °C (refrigerator condition), food products may be store for a few additional days at this temperature. At temperature
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Table 1.1 List of Microbiological safety parameters generally recommended for the analysis of different food products Food products/ Commodities Beer and other alcoholic beverages Egg and egg products Fish and sea food
Fruit and fruit products
Gelatin products Herbs spices Infant foods (instant milk) Jam, juices and sauces Meat and meat products
Raw milk
Cheese
Butter Ice cream edible casein Packaged pasteurized milk Malted milk food Nut and nut products
Cereals, grains and cereal based complementary foods Cooked food products Wheat floor
Name microbiological parameter/Test recommended Total plate count, Coliform count, Yeast and Mold count Enumeration of coliform Bacteria, Total plate count, Yeast and Mold count, detection of Salmonella spp. Aerobic plate count, Coliform count, detection of Escherichia coli, Salmonella, Staphylococcus aureus, V. cholerae, V. parahaemolyticus, Shigella,, Listeria monocytogenes Aerobic plate count, Yeast and Mold count, detection of Staphylococcus aureus, Enterobacteriaceae, Escherichia coli, Salmonella spp. Listeria monocytogenes, V. cholerae Total bacterial count, Yeast and Molds count and detection of Escherichia coli, Salmonella spp. Salmonella spp. Total bacterial count, Coliform count, detection of Staphylococcus aureus, Escherichia coli, Salmonella and Shigella Aerobic plate count, Coliform count, detection of Staphylococcus aureus, Escherichia coli, Salmonella and Shigella Aerobic plate count, Yeast and Molds count, detection of Staphylococcus aureus, Enterobacteriaceae detection of, Salmonella spp,, Listeria monocytogenes, V. cholerae Aerobic plate count, coliform count, Yeast and Mould count, detection of Staphylococcus aureus, Escherichia coli, Salmonella, Shigella Total bacterial count, coliform count, Yeast and Mold count, detection of Staphylococcus aureus, Escherichia coli, Salmonella, Shigella, Listeria monocytogenes Coliform count, yeast and Mold count Standard plate count, Total bacterial count, Coliform count and Yeast and Mold count Total plate count and Coliform count Total bacterial count, Coliform count Aerobic plate count, Yeast and Mold count, Staphylococcus aureus, Enterobacteriaceae detection of, Salmonella spp, Listeria monocytogenes, V. cholerae Aerobic plate count, Coliform count, Yeast and Mold count, detection of Escherichia coli, Salmonella and Staphylococcus aureus Total bacterial count, Coliform count, detection of Salmonella and Staphylococcus aureus Total bacterial count, Coliform count
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below 0 °C, bacterial growth is stopped in totally due to inhibition of enzymatic activity, but the microorganisms remain alive and when the temperature rises above 0 °C to optimal they will become active again. Although bacteria multiply faster at ambient temperature, spoilage of food material may occur during refrigeration also. However, refrigeration slower down the metabolism of bacteria, and decreased rate of multiplication, hence, spoilage takes longer time than usual. It has been researched that two major factors of microbial growth are controlled by temperature i.e. lag phase and the growth rate (rate of cell division or generation time). 1.3.2.2 Water and Moisture Water and moisture are also the key factors which affect the quality of products throughout its complete post-harvest period till it reaching the consumer. Moisture is unique sole feature that directly effects the shelf life of the products. The safe moisture level of 8 to 12% is not satisfactory for the growth of most of the grain insects, microbes or mites. Water availability is measured in terms of water activity and is obtain using the following formula:
Water Activity ( a w ) = Vapor Pressure of Food / Vapor Pressure of Water
Microorganisms that tolerate high salt solutions are involved in spoilage of pickling brines, while osmophilic microorganisms are involved in spoilage of products with higher sugar content. Most of the fungal species grow even if a very small amount of moisture is present in the food commodity, hence, these ‘unwanted guests’ are referred to as the most contaminating microflora of moistened food. 1.3.2.3 Degree of Acidity The degree of acidity is measured in terms of pH. On the basis of pH optimal growth requirement, bacteria are classified as acidophiles, neutrophiles and alkalophiles. pH greatly affects the enzymatic activity, hence the growth of microbes as well. It has been observed that bacteria grow best in an environment that is not too acidic. Less acidic products, for example, milk having the pH near to 7, are therefore especially susceptible to bacterial spoilage. Adding acidity to products slows down the process of microbial spoilage. This is the reason that adding salt to pickles and prevents the growth of microbes, hence, act as preservative. Beer, yogurt, wine, vinegar and fruits are some of the examples of acidic foods which are more acidic and less prone to microbial spoilage.
1.4 Environmental Microbiology
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1.3.2.4 Oxygen Microbes are aerobic, anaerobic or microaerophile depending upon their need for oxygen. If there is a shortage of oxygen, it is difficult for certain kinds of microbial species to survive, however, as soon as the oxygen supply is increased, these residual bacteria will again grow and multiply. Some types of microorganisms even thrive in the oxygen-poor environment i.e. anaerobic in nature and responsible for spoilage in absence of oxygen such as in case of canned food products. 1.3.2.5 Packaging Food packaging has a profound impact on shelf life and significantly affects the quality and safety of food products. Materials used for packaging can have tremendous effect on the growth of microorganisms. Advanced technologies such as active packaging or anti-microbial packaging materials (e.g. tetra packs) are available which ensure prevention of microbial growth in the packaged food. 1.3.2.6 Hygiene and Sanitation The best way to battle the food-borne problems is to preserve good hygienic and sanitation situations around the all steps of food processing (storage, packing and processing). Protection should be taken on management the food and farm commodities made from them so that there is no injury to the vessels & contents remain intact and unexposed to the atmosphere. High sanitary conditions are prerequisite for quality and shelf life. Besides this, sterilization of the packaging material and pasteurization of the product/ vacuum packaging are other suitable methods to prevent the food from microbial attack.
1.4 Environmental Microbiology Environmental microbiology is the study of microbes (Bacteria, algae, fungi etc.) in soil, fresh water, the sea, and other habitats. The waste produced by large number of factories are treated using microorganisms. They are also used in production processes like to substitute hazardous chemicals in dye production and leather processing. Bioremediation processes are being developed to remediate water contaminated with toxic heavy metals, pesticides, petroleum products insecticides and phenolic compounds.
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1.4.1 Water Microbiology Safe drinking water is of utmost importance to provide tangible benefits to health and active efforts should be made to achieve the quality of drinking water. Water can be defined as safe if does not cause any significant hazard to health over a lifetime of consumption. Safe drinking water is thus suitable for all purposes, including personal hygiene. Although several definitions and guidelines are available for packaged drinking water as well as water for human consumption, there are several other quality parameters intended to be used for some other purposes like water in food production, in pharmaceutical or other industrial uses. Access to safe and quality water is very difficult to be obtained by several populations throughout the world. According to the WHO, the mortality of water associated diseases exceeds five million people per year. From these, more than 50% are microbial intestinal infections, with cholera standing out in the first place. There are different categories of water such as drinking water (other than natural mineral water), packaged drinking water (other than packaged natural mineral water), natural mineral water, naturally carbonated natural mineral water, non-carbonated natural mineral water, decarbonated natural mineral water, natural mineral water fortified with carbon dioxide from the source, carbonated natural mineral water, packaged natural mineral water. Microbial testing may be done to verify that a specific handling or process has been effective (Table 1.2).
Table 1.2 List of microbiological parameters generally recommended for the analysis of different water samples Categories Drinking water Swimming Pool water Packaged drinking water, natural mineral water
Reverse osmosis (RO) water Dialysis water Water used for manufacturing of food and other industry products Ground water, underground water, effluent and wastewater Lake water, pond water and sea water
Name microbiological parameter/Test recommended Total coliform bacteria and detection of Escherichia coli Total bacterial count (standard plate count) total Coliform Bacteria Detection of Escherichia coli, Salmonella, Staphylococcus aureus, V. cholerae, V. parahaemolyticus, Shigella, Fecal streptococci, Pseudomonas aeruginosa, Sulphite reducing anaerobes, Total coliform bacteria, Yeast and mold count, Total bacterial count at two temperature (22 and 37) Total bacterial count (aerobic plate count) yeast and mold count Bacterial endotoxin test and total bacterial count (aerobic plate count) yeast and mold count Total bacterial count (standard plate count) MPN coliform bacterial detection, proteolytic and lipolytic bacterial count.
MPN coliform bacterial detection, detection of Escherichia coli, Sulphate reducing bacteria Detection of algae and other related microorganisms
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1.4.2 Air Microbiology Air Microbiology is the study of living microorganisms such as bacteria, fungi, and viruses etc. which are suspended in the form of aerosol in the air. These microbial aerosols can travel long distances with the help of wind and precipitation, increasing the widespread occurrence of these microorganisms, hence the diseases caused by them. Therefore, study of air-borne microflora is highly essential in almost all prone areas like operation theaters, manufacturing premises, working zones etc. These microorganisms can also interfere while performing the experiments in the Microbiology laboratory where the samples can get contaminated by the air-borne microbes, for instance, in clean room area, sterile area, sample storage chambers, incubators etc. This section, therefore, pertains to various methods for monitoring indoor air as well as compressed air for microbiological purposes.
1.4.3 Soil Microbiology Soil Microbiology is a discipline of microbiology which concerned with the study of microorganisms that exist is soil environment. The aim of the soil analysis is to know the number of microorganisms that play main roles in the promotion of the growth. The analysis of soil for microbiological contamination is essential in order to produce good quality of water, food and farm products, drugs and pharmaceuticals. The analysis is also playing a significant role when it comes to bioremediation and biodegradation of contaminated sites.
1.5 Pharmaceutical Microbiology Drug and pharmaceutical microbiology is the study of microorganisms (bacteria and fungi) present in or on the drug and pharmaceutical product, during manufacturing of injectable or non- injectable items these microorganisms enter in the preparation hence decrease the self-life of products or contaminate the product which cerate the problems in human being. There are certain microorganisms such as Gram-negative bacteria which contaminate the drug formulation and preparation and during their stationary phase, they produce endotoxin which is a heat stable substance responsible for fever. Thus, microbial contamination of drug and pharmaceuticals products is a matter of great importance to the industry and it can become a major cause of both product and economic losses. Moreover, the contamination of these products can result in them being converted into products hazardous for consumers. In pharmaceutical industry water, raw material, intermediate products and finished products are checked for enumeration and isolation of microorganism. Presence and absence of common human pathogenic organisms such as Escherichia
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coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus etc. are checked as per the monograph written in the pharmacopoeia. Pharmaceutical preparation is also received for other parameter like total aerobic microbial count and total yeast and mould count. However, their limits are also given in the monograph of respective pharmacopoeia and compliance of the products are given on the basis of such monograph. Sterility test of Parenteral and non- parenteral products are also done by using membrane filtration and direct inoculation techniques. Bacterial endotoxin test is done both qualitatively and quantitatively again as per the requirements given in the monograph. In pharmaceutical industry microbiological contamination of all the equipments and person eel are also checked to avoid the further contamination. Moreover, all the control areas (rooms) are monitored for air quality. Overall, in Drug and Pharmaceutical industry the products are manufactured in a controlled way as to produce the quality drugs which intended to provide the relief to mankind and society (Table 1.3).
Table 1.3 List of microbiological safety parameters generally recommended for the analysis of different pharmaceuticals products Pharmaceuticals products/Commodities Parental preparations (e.g. IV and IM injectables), aqueous solutions and suspensions, non-parenteral preparations ophthalmic preparations (e.g. eye-drops, eye-ointments, eye-lotions etc.) oils and oily solutions, ointments and creams, antibiotic solids, bulks and blends, surgical items, catheters, gauze, swabs, cotton balls, blades, needles and scalpel and syringes. Vitamin capsules, tablets and granular powder and syrups/ liquids Water for injection, dialysis water and other injectables Antibiotics, raw materials, oral suspension, water for injection, dialysis water, oral suspension, tablets, syrup, ayurvedic and homeopathic preparations other related products. Surgical blades, medical devices and other related products Raw materials, tablets, probiotics and other related products Raw materials, oral suspension, oral suspension, tablets, syrup, ayurvedic and homeopathic preparations other related products.
Antibiotics: Amikacin, Amphotericin B, Bacitracin, Bleomycin¸ Carbenicillin, Doxycycline, Erythromycin, Framycetin, Gentamicin, Kanamycin Sulphate, Kanamycin B, Neomycin, Novobiocin, Nystatin, Oxytetracycline, Polymyxin B, Rifampicin, Streptomycin, Tetracycline other related antibiotic products
Name microbiological parameter/ Test recommended Sterility testing
Vitamin B12, biotin, calcium pantothenate Bacterial endotoxin test Microbial limit test
Bioburden test Lactic acid bacillus Specific pathogen detection Escherichia coli, Salmonella spp. Staphylococcus aureus, Pseudomonas aeruginosa, Shigella spp. Clostridia spp. Candida albicans Antibiotic assay
1.7 Quality Assessment
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1.6 Accreditation for Microbiological Laboratories It is a valuable component of quality assurance and involves the audit by an external independent agency of an applicant’s organizational and quality assurance program to ascertain the compliance of ISO 17025: 2017. It is a comprehensive system covering all aspects of the laboratory, including organization & administration, staff development & education, facilities & equipment, and policies & procedures. Participation in all relevant quality assessment schemes is a requirement for accreditation. Accreditation bodies like National Accreditation Board for Testing and Calibration Laboratories (NABL) in India signatory to ‘International Laboratory Accreditation Cooperation (ILAC)’ and ‘Asia-Pacific Laboratory Accreditation Cooperation (APLAC)’ provide accreditation to laboratory as per ISO 17025:2017.
1.7 Quality Assessment This is principally based on the postulation that the results obtained should imitate what is occurring in daily practice. It is a management tool in which output achieved should be used correctly in order to ascertain the success and hence, it is necessary to have the truthful and acceptable approach of the participating labs. It is, therefore, utmost important to carry out the evaluation of quality assessment samples by the same staff and with the same methods and reagents as samples received on daily routine. Moreover, the complex diversity of microbiological investigations makes it difficult to produce control materials that cover all areas, and several technical difficulties must be overcome in preparation of quality assessment samples. Quality assessments may be systematizing within one laboratory i.e. internal quality assessment or between the laboratories i.e. external quality assessment.
1.7.1 Internal Quality Assessment Internal quality assessment should be designed by senior officials for the repeated evaluation of original samples (i.e. not simulated ones), thereby enabling direct comparisons with previously obtained results. False identification may be given to repeated samples so that analysts would be unaware of the fact that these samples are meant for quality control. Care should be taken with the use of stored materials because deterioration may prevent the production of previous results.
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1.7.2 External Quality Assessment External quality assessment is usually done on a national basis under the program of ILC (i.e. Inter laboratory comparison) between as many as concerned laboratories. Simulated samples of known but undisclosed content is dispatched to different labs where they examine these samples for the required parameters and report their findings to the organizing or nodal laboratory. The organizer compares the result from all participating laboratories in terms of ‘comparable’ for qualitative analysis and ‘Z-score’ for quantitative analysis which leads to the evaluation of efficacy and accuracy of the concerned lab. During external quality assessment, following points should be considered: (a) Confidentiality is highly required and is achieved by allocating each laboratory a unique identification number known only to the nodal lab. This number is used in all further communications and moreover, individual lab’s results are not revealed to other parties except under previously agreed and strictly defined circumstances. (b) External quality assessment program should include as many as parameters as to reflect the laboratory’s routine capabilities. (c) Nodal lab should also mention basic and comprehensive requirements such as specific standards or guidelines for carrying out the analysis and reporting of results. (d) Samples, sent to the participating labs, should resemble, as closely as possible. Prior to sending the sample for quality assessment, stability and homogeneity studies should be carried out by the nodal lab itself to ensure their comparability. (e) Samples should yield clear-cut results so that participating labs can evaluate their performance by getting to know if their results are incorrect. This is, however, quite difficult, because of the ‘borderline’ results and failure of laboratories to use optimal methods. (f) Quantity of sample should be appropriate according to the tests to be performed. In some cases where enough quantities of samples are not available, artificial specimens can be produced. (g) Samples designed to isolate microorganisms must have approximately same number of organisms in each vial/flask and this can be achieved easily by freeze-drying process. (h) There should not be any undue pressures on analysts to manipulate the results in order to gain the economic viability of the laboratory by providing proof of excellence to customers.
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References 1. Varnam AH, Sutherland JP (1994) Beverages. Springer Science and Business Media LLC, Springer-Verlag US 2. Ashutosh K (2008) Pharmaceutical microbiology. New Age International (P) Ltd, New Delhi 3. Chauhan A, Ranjan A, Basniwal RK, Jindal T (2016) Probiotic, prebiotic and synbiotics in the prevention of lifestyle disorders. Int J Curr Microbiol App Sci 4(2):933–947 4. Goyal P, Chauhan A, Aggarwal ML, Chacko KM (2012) Microbiological aspects of water: key criteria of quality. Curr Res Biol Pharm Sci 1(1):57–66 5. ISO/IEC 17025 2017 General requirements for the competence of testing and calibration laboratories 6. Hilgren JD (2000) Antimicrobial efficacy of a peroxyacetic/octanoic acid mixture in fresh‐ cut‐vegetable process waters. Journal of Food Science, 65(8):1376–1379 7. Kaushik P (2000) Introductory microbiology. Emkay Publications, New Delhi, pp VIII+1–VIII46 8. Kaushik P, Chauhan A (2009) Cyanobacteria: antibacterial activity. New India Publishing, New Delhi 9. Kaushik P, Kaushik K. Microbiology (questions and answers), 5th edn. S. Chand Publishing, Delhi 10. Lemarchand K, Masson L, Brousseau R (2004) Molecular biology and DNA microarray technology for microbial quality monitoring of water. Crit Rev Microbiol 30:145–172 11. Hossain MN, Fakruddin M, Islam MN (2012) Development of fruit Dahi (yoghurt) fortified with strawberry, orange and grapes juice. Am J Food Technol 7:562–570 12. Wahba NM (2010) Antimicrobial effects of pepper, parsley, and dill and their roles in the microbiological quality enhancement of traditional Egyptian Kareish cheese. Foodborne Pathog Dis 4:411–418 13. Pelczar MJ, Chan EC, Kreig MR (1986) Microbiology, 5th edn. McGraw Hill Book Co., New York 14. Prescott LM et al (2004) Microbiology, 6th edn. McGraw Hill-Higher Education, New Delhi 15. Stainer RY et al (1986) The microbial world, 5th edn. Prentice-Hall, Upper Saddle River 16. Kajs TM, Hagenmaier R, Vanderzant C, Mattil KF (1976) Microbiological evaluation of coconut and coconut products. J Food Sci 41(2):352–356
Chapter 2
Good Microbiological Laboratory Practices
2.1 Introduction Good Microbiological Laboratory Practices (GMLPs) are having the major concern at developing proficiency in order to protect operators i.e. students, teachers, technicians and Scientists from the very minor likelihood of infection, any uncontrolled spread of microbes and contamination from external sources. For achieving this, it is significant to organize the workplace judiciously to ensure safe and active operations.
2.2 Safety Management Microorganisms show variability in their ability to cause infections starting from harmless to serious illnesses leading to public and epidemic diseases. They are classified as ‘Hazard Groups’ or ‘Risk Groups’ based on the hazards they can cause to lab personnel and even to the community in case of their ‘escape’ from the lab. Major factors associated while classifying these groups are (a) significance of the disease caused, (b) accessibility of therapeutic agents to cure the disease, (c) routes of infection (possibly by at least four routes i.e. lungs, skin, mouth and eyes), and (d) history of lab infection caused by organisms. On this basis, four hazard groups are formed which are as follows: 1 2 3
Hazard Group 1 Hazard Group 2 Hazard Group 3
Low Risk
Low to individual and to community Moderate to individual, Low to community High to individual, Low to community
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Hazard Group 4
High Risk
Very high to individual and to community
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_2
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2.2.1 Biosafety Level (BSL) Based on four Hazard Groups, following BSLs are designated with respect to lab premises, equipments, techniques and precautions. BSL 1 & 2: Designed to work with Hazard Group 1 & 2 organisms with following properties: (a) Enough space as per requirement of Basic Microbiology Lab (b) Non-absorbent, Smooth, easy to clean and resistant to chemicals walls, ceilings and floors (c) Slip resistant floors (d) Adequate lighting and heating (e) Provision of wash basins and sinks (f) Adequate storage facilities (g) Protective Coverings (h) Safety cabinets BSL 3: Intended to work with Hazard Group 3 organisms with following properties: (a) All requirement pertaining to Basic Microbiology Lab (b) Physical separation of rooms intended for different purposes with lockable doors (c) One-way ventilation usually attained by having a lesser pressure in the lab than adjacent room. (d) Complete exhaustion of air to atmosphere with no recirculation to other parts of lab, usually coupled with Biosafety Cabinet. (e) Personal Protective Equipment (PPEs) BSL 4: Intended to work with Hazard Group 4 organisms with following properties: (a) All requirement pertaining to Level 3 (b) Air-lock entry (c) Shower exit (d) Waste disposal (e) Class III Biosafety Cabinet (f) Pressure gradient (g) Double door autoclave
2.2.2 Avoidance of Laboratory-Acquired Infections To avoid the laboratory-acquired infections, following practices should be considered: 1 . Awareness to the potential hazards of organisms used/isolated during the study 2. Awareness to the possible routes by which pathogen can enter the human body and cause infection
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3 . Awareness to the potential hazards of the materials used during the study 4. Primary Barriers – Prevention of dispersal of organisms into the lab by following means: (i) Separate ‘High Risk’ potentially infected materials (ii) Avoid mouth pipetting while taking anything e.g. rubber tubes, pens, pencils, labels, fingers, chemicals, glassware etc. in or at the mouth (iii) Avoid eating or drinking in the lab (iv) Properly groomed hair and nails (v) Restricted use of needles and sharp glass Pasteur pipettes (vi) Immediately replace the broken glassware (vii) Careful use of equipment such as centrifuge (viii) Proper sterilization of inoculation loops prior and after use (ix) Regular inspection of homogenizers thus to avoid aerosol dispersal (x) Proper use of Biosafety Cabinet (xi) Use of suitable disinfectant at appropriate dilution (xii) Regular disinfection of work surfaces and proper spillage management (xiii) Discarding of micro-tips or other small materials into jars having disinfectant (usually 1:40 Dettol) which should be freshly prepared daily. (xiv) Provision of discard bins and biohazard bags (as per requirement) near the work benches (xv) Daily cleaning and decontamination and autoclaving (if required) of discard jars and bins (xvi) Robust and leak proof sample containers, discard bins etc. (xvii) Proper sterilization of all discarded infectious materials prior to sending it to nodal agency for final disposal 5. Secondary Barriers – Protection of worker in case the primary barriers fail as follows: (i) Proper wearing of Personal Protective Equipment (PPEs) including gloves, mask, cap, hood, lab-coat, shoe cover, safety goggles or visor. These should be removed at the time of leaving the lab and not to be worn outside the lab (ii) Immediate hand washing following the experimental work using appropriate soap/disinfectant. (iii) First aid facility in case of cuts, scratches and abrasions (iv) Careful handling of pathogens using Biosafety cabinets and under trained senior personnel (v) Intermittent health check up and immunization, if required 6. Tertiary Barriers – Additional protection to workers and prevention of escape of organism into the community
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2.3 Entry to Microbiology Lab Microbiology lab is among the highly sophisticated and should not be taken for granted even at the time of entry. Entry to Microbiology lab is first-most and very crucial in terms of safety of lab-personnel with respect to undesirable infections and can be done while considering the following aspects: (a) Only authorized persons should be allowed to enter the lab. (b) List of authorized personnel should be displayed right at the entry to the lab. (c) Other persons can enter the lab with due permission of concerned authority. (d) There should be provision for automatic air curtain placed on the top of the entry gate, primarily to remove surface microflora. This curtain has to be designed in the way so that air flow should be outside. (e) Shoes should be worn off and place in the shoe racks near the entry and put on the lab slipper while in the lab. (f) Lab slippers are not allowed outside the lab. (g) Shoes are allowed in the laboratory provided the shoe covers are used. (h) After wearing lab slippers/shoe covers, hands should be disinfected using proper disinfectant. (i) Laboratory coats should be put on while being in the lab but should be removed before leaving and put in the bucket designated as “Used Garments”. (j) These discarded clothing are either disposed off or laundered by the institution.
2.4 Housekeeping Management 1. Microbiology lab should be cleaned every day by a sweeper with alternate disinfectant 2. Scrubbing of laboratory floor is also recommended on periodic basis 3. Regular cleaning of working tables, chemical shelves, electrical items viz. ceiling fans, tube lights, UV lights, exhaust fans, air conditioners etc. 4. Replacement of damaged tiles of shelves, floor and roof, fused tube lights etc. 5. Regular cleaning of glassware using chromic acid/detergent followed by rinsing with distilled water and drying in air/hot-air oven as required. 6. Check all electrical items and water-taps before leaving the lab in the evening. 7. Use of safety measures (sterilized gloves, facemasks and head caps) while working with bacterial and fungal cultures. 8. Wear clean lab gowns or uniforms coats, chosen for lab use while being in the lab and should be removed before leaving the lab. 9. Permit only authorized persons in ‘clean room’ area when experiments are in progress. 10. Place appropriate fire extinguishers at noticeable sites in the lab. All lab personnel should be trained to operate these extinguishers.
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1 1. Availability and easy accessibility of ‘First-aid Box’. 12. Availability and easy accessibility of ‘Spill Kits’.
2.5 Spillage Management Some infectious materials when fallen can lead to substantial infection upon exposures to the lab personnel. Following spillage management practices should be used in such cases. 1. In case of spillage that might produce aerosols/droplets of an infectious agent, instantly leave the area, close the door, and decontaminate clothing. 2. Permit at least 25–30 min for the droplets to settle and for the aerosol concentration to decline. 3. Report the spillages of cultures immediately to the concerned faculty. 4. Spillage should be dealt very carefully and quickly. 5. Do not touch with unprotected hands spilled cultures and surrounding debris such as glass, cotton wool and plugs 6. Immediately wear PPEs 7. Disinfect the zone by layering the spill with paper towel soaked in a suitable disinfectant such as Dettol or 70% isopropyl alcohol and leave for 15–30 min. 8. Clean the spill from its outer edges and towards the center with paper towel 9. All disposable material should then be transferred to an Autoclavable disposal bag for autoclaving and disposal. 10. In case of broken glassware, it should be cleaned judiciously into an appropriate container, autoclaved and disposed of in a puncture proof container. 11. In case of splashes on clothing and the skin, decontamination should be done immediately using appropriate disinfectant. 12. Washing with soap and hot water should be adequate, but if essential, the skin can be sanitized. 13. Attention should also be taken to avoid generating aerosols during practical work. The risk of spillages happening is lessened by using cultures grown on agar instead of in liquid media whenever possible. 14. The risk is reduced by following to Good Microbiological Laboratory Practices with special attention to the correct use of pipettes. 15. Keeping of a record of all such incidents is recommended.
2.6 Disposal Management Waste generated in the Microbiology lab may contain infectious agents; therefore, it is highly essential to ensure their proper disposal. Lab personnel are advised to transfer all inoculated media, used Petri-dishes & other contaminated stuff directly
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to disposal site to avoid any contamination in the surroundings. The waste should be made ‘safe’ on site before final disposal by (a) autoclaving, (b) chemical disinfection or (c) incineration, depending upon the nature of material. No any infected material shall leave the Laboratory
2.6.1 Containers for Waste Material in Microbiology Lab Following types of containers are used for discarding the waste infected materials from Microbiology Lab: 1. Color-coded containers, as mentioned below, for cultures, samples, wrappers, needles and syringes. (a) Yellow: for incineration (b) Blue: for autoclaving (c) Black: for normal household waste 2. Discard jars for slides, pipettes, micro-tips, small disposable items
2.6.2 Discard Bins, Bags and Jars 1. Bins are should be made up of stainless steel 2. Bags should be supported in discard bins 3. They should have solid bottoms without any leakage to avoid any escape of contaminated materials. 4. They should never be completely filled. Rough handling can cause bursting. 5. Bins should be color coded for easy recognition. 6. Bins should be covered with lids and bags may be tied to avoid contamination. 7. Jars should be deep enough to hold things, robust, unbreakable and autoclavable like polypropylene beakers of 1-litre capacity. 8. Glass jars are usually not recommended as it can be easily broken. 9. Jars should not be overloaded and should contain only 750 ml of diluted disinfectant to provide proper space for displacement without overflow and risk of spillage when it is transferred. 10. Inappropriate articles such as floating material, paper, tissues etc. should not be placed in discard jars. 11. Jars should be properly disinfected frequently and no material should be left there for more than 24 h, failing which microbes may grow.
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2.6.3 Good Disposal Practices (GDP) Good Disposal Practices (GDP) are recommended to achieve this which are as follows: 2.6.3.1 Glassware Sterilize all glassware containing inoculated culture media by autoclaving at 121 °C for at least 60 min in an autoclave marked for disposal purposes. Scrap out or pour away any remaining culture media and thereafter clean the glassware using appropriate soap/detergent/chromic acid solution. Washing of Glassware 1. Pre-cleaning of glassware by rinsing thoroughly before placing in washing solution. 2. Remove any tape, label, residual parafilm etc. from the glassware. 3. Avoid letting soap dry onto glassware. 4. Keep large containers filled with water and small containers totally submerged in washing bucket until washed. 5. Rinse each item for at least 5–10 times thoroughly to remove soap firstly with tap water followed by distilled water/ deionized water. Then place the item on rack to drain. 6. Autoclaving of fluids in new glassware may lead to release of alkali and alteration of pH, therefore, new glassware requires neutralization. This is done by soaking the glassware in 2–3% hydrochloric acid for several hours followed by washing and rinsing with distilled water. 7. Washed glassware should be dried be leaving in the air or by placing them in hot-air oven at 160 °C for 20 min. 8. Before taking glassware to their appropriate storage locations, cool the items. 2.6.3.2 Petri-dishes Pack all Petri-dishes in autoclavable disposal bags after completion of work followed by their sterilization as mentioned above and final disposal in biohazard bins. 2.6.3.3 Loops and Microtips Discard into disinfectant solution in discard jars for overnight. These contents of discard jars should be poured in autoclavable disposal bags followed by its sterilization by autoclaving and finally disposed off in biohazard bin. Empty discard jars should also be autoclaved before further use to avoid undesirable contamination.
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2.6.3.4 Contaminated Samples Samples found contaminated with pathogens, are discarded only after sterilization at 121 °C for 60 min and disposed off in biohazard waste bin for incineration (as per biohazard waste management rules).
References 1. Balasubramaniam VM, Ting EY, Stewart CM, Robbins JA (2004) Recommended laboratory practices for conducting high-pressure microbial inactivation experiments. Innovative Food Sci Emerg Technol 5(3):299–306 2. Chosewood LC, Wilson DE (2009) Biosafety in microbiological and biomedical laboratories. US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institutes of Health, Washington, DC 3. Grainger J, Hurst J, Burdass D (2001) Basic practical microbiology: a manual. The Society for General Microbiology, Reading, pp 1–26 4. Kaushik P (2000) Introductory microbiology. Emkay Publications, New Delhi, pp VIII+1–VIII46 5. Kaushik P, Chauhan A (2009) Cyanobacteria: antibacterial activity. New India Publishing, New Delhi 6. Butson P, Hawitt K (2008) Microbiological control for non-sterile pharmaceuticals, monograph no. 2, pqg monograph no.12. Pharmig/The Chartered Quality Institute, Hertfordshire/London 7. Richardson JH (ed) (1994) Laboratory safety: principles and practices, 2nd edn. American Society for Microbiology, Washington, DC 8. Saha D, Jain VK, Jain B, Tandey R (2011) Good laboratory practice: design and utility. Asian J Pharm Technol 1(1):1–3 9. Sutton S, Singer D (2011) Microbiological best laboratory practices, USP(1117) value and recent changes to a guidance of quality laboratory practices. Am Pharm Rev 14:4–41
Chapter 3
Microbiological Culture Media: Types, Role and Composition
3.1 Introduction Culture media refers to the ‘mixture of nutrients in an appropriate ratio’ which provides the nutritional requirements of the microorganisms leading to their growth under in vitro conditions. Under natural conditions of the environment, microbes usually adapt to the habitats most suitable for their needs. However, in the laboratory, these requirements must be met by a culture medium. This is basically an aqueous solution to which all the necessary nutrients have been added. Culture media can be prepared by using raw materials depending upon the composition but now-a-days, dehydrated and ready-to-use media are available. Broad-spectrum culture media i.e. prepared using raw materials are economical and provide better opportunity for growth of novel microorganisms.
3.2 Types of Media Although depending on the type and combination of nutrients, different categories of media can be made but the general composition of a medium remains same and is as follows: (a) Solvent (usually distilled water) (b) Hydrogen donors and acceptors (c) Carbon source or energy source (e.g. Glucose) (d) Nitrogen source (e.g. Protein hydrolysates or peptones) (e) Inorganic nutrients (e.g. Carbon, Hydrogen, Oxygen, Nitrogen, Sulphur, Phosphorus, Potassium, Sodium, Calcium, Magnesium and Iron) (f) Trace elements (e.g. Mn, Mb, Cu, Co, Ni, Zn, V, B, Se, Si, W) (g) Growth factors (e.g. amino acids, purines, pyrimidines, vitamins, blood, serum) © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_3
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( h) Solidifying agent e.g. agar agar (i) Buffers (e.g. Sodium or Magnesium Phosphate, acetate or citrate salts are added to maintain pH during fermentation, but can chelate essential metals like Fe+2) (j) Selective Agents (e.g. toxic chemicals, antibiotics, inhibitory dyes) (k) Indicator Dyes (e.g. phenol red, neutral red, bromocresol purple to indicate the changes in pH of media, however, dyes can be toxic to sensitive or stressed cells).
3.3 Consistency-Based Classification of Media Solidifying or gelling agents such as agar agar (a polysaccharide fractionated from Red Algae i.e. Gelidium) have the tendency to become solidified below temperature 45 °C while it liquefies above this temperature. It is also the source for metals, minerals, sulphate and pyruvate. Depending upon the addition and quantity of this substance, media are of three types:
3.3.1 Liquid (Broth) Media Liquid (Broth) Media, such as nutrient broth, tryptic soy broth or glucose broth which are prepared without the use of agar agar. These media are specifically used in studies refers to microbial growth and metabolism in which it is necessary to have homogenous media conditions, to follow optical density, and to allow early sampling for analysis of substrates and metabolic products. Tubes and flasks with liquid cultures can be incubated with either static or shaken incubation.
3.3.2 Semisolid Media Semisolid Media are having the agar agar as solidifying agents, however, in concentration (usually ≤1%) which do not permit complete solidification of the media. These media are used in fermentation studies, in determining bacterial motility, and in promoting anaerobic growth.
3.3.3 Solid Media Solid Media, such as nutrient agar, do have the agar agar in concentration (≥1.5%) which provide complete solidification and are used for the surface growth of microorganisms in order to observe colony morphology, for pure culture isolation, often
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in the enumeration and isolation of bacteria from a mixed population by diluting the original bacteria suspension and spreading a small inoculum over the surface of the solidified medium and to observe specific biochemical reactions (extracellular enzymes diffusing away from the colony can be detected as a result of their action on insoluble substrates present in the agar medium) by spread plate and streak plate methodology. These media can be poured into test tube either in a slanting position, called as agar slant or in an upright position, called as agar deep tube or into a Petri dish, called as agar plate.
3.4 Composition-Based Classification of Media Different types of microbial culture media are composed depending upon the requirement of the concerned microorganisms to be grown and these media are as follows:
3.4.1 Chemically Defined Media (Synthetic Media) Chemically Defined Media (Synthetic Media) are media composed of known quantity and quality of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e., the exact chemical composition of the medium is known. Typically, they contain a simple sugar as the carbon and energy source, an inorganic nitrogen source, various mineral salts and if necessary, growth factors (purified amino acids, vitamins, purines and pyrimidines), example of such media are, BG-11 media for in vitro cultivation of blue green algae (Cyanobacteria).
3.4.2 Complex Media Complex Media are composed of complex materials rich in vitamins and nutrients. They contain water soluble extracts of plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and tryptone). Sugar, often glucose is added to serve as the main carbon and energy source. The combination of extracts and sugar creates a medium which is rich in minerals and organic nutrients, but since the exact composition is unknown, the medium is called complex.
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3.5 Function-Based Classification of Media Culture media are classified on the basis of their function, which are as follows:
3.5.1 All Purpose Media All Purpose Media for example, Tryptic Soy Agar, Nutrient Agar are among the culture media which do not have any special additives and support the growth of almost all types of bacterial species in the laboratory.
3.5.2 Selective/Differential Media Selective/Differential Media are media based on either of the two categories above supplemented with growth-promoting or growth-inhibiting additives. Selective medium is made selective for a particular genus/ species by adding the inhibitory agents to restrict the growth of undesired microorganisms. The additives may be species- or organism-selective (e.g., a specific substrate, or an inhibitor such as cyclohexamide (artidione) which inhibits all eucaryotic growth and is typically used to prevent fungal growth in mixed cultures). Most commonly used selective agents are azide, selenite, tellurite, bismuth, brilliant green, malachite green, crystal violet, bile, cetrimide, lauryl sulphate, tergitol, tween 80, dichloran etc. Addition of bile salts in culture media is found to be selective for Salmonella typhi. Addition of specific antibiotics is also shows significant selectivity for particular kind of microbe. EMB (Eosine Methylene Blue Agar) media is a selective media that inhibit the growth of Gram-positive bacteria while stimulate the growth of Gram-negative bacteria. McConkey Agar media is referred as differential media as it differentiates between lactose-fermenters and lactose-nonfermenters while allowing the growth of both types of organisms. Blood agar media is also a type of Selective/ Differential Media.
3.5.3 Enrichment Media Enrichment Media are special media having the specific growth factors which allow metabolically fastidious microorganisms to grow. They are similar to selective media but designed to increase the number of desired microbes to a detectable level without affecting the rest of the microbial population under incubation conditions to isolate the desired microorganisms from natural samples.
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3.5.3.1 Stressed Microbes and Their Recovery Several processing methods like drying, salting, heating, chilling etc. make the microbes stressed or damaged. These cells are not able to grow till the repair (recovery) through pre-enrichment methods which are as follows: (a) Addition of neutralizing substances to neutralize the effect of toxic, damaging chemicals such as wetting agents, surfactants, sulphydral compounds, β-lactamases etc. (b) Addition of lysozyme for germination of ‘dead’ spores. (c) Removal of peroxides by adding catalase or whole blood. (d) Recovery of membrane damage by adding pyruvate (e) Stabilization of membranes and RNA by adding magnesium. Stresses microbes usually recover and grow better between 33-37 °C.
3.5.4 Reducing Media Reducing Media are best suited for the growth of anaerobic microorganisms.
3.6 Culture Media: Check Points (a) Proper storage conditions i.e. temperature 25 ± 2 °C, relative humidity 50 ± 10%, protection from undesirable light. (b) Should carry labels identifying the contents, preparation date, shelf-life, storage conditions, batch number, expiry date, date of opening etc. (c) Discard outdated or expired media, or bottles with reduced clarity and darkening of color. (d) Don’t open the media bottles for too long time as media contents may be hygroscopic in nature. (e) Read instructions carefully before preparation of media. (f) Dissolve all ingredients in appropriate amount of distilled water prior to autoclaving using hot-plate with continuous stirring. Otherwise, they will set at the bottom of the vessel and will cause darkening during autoclaving. (g) While preparing the media, superheating should be avoided as it can cause complex formation of amino acids, phosphates and free metal ions, which in turn reduces the nutritive value and shelf-life of media. (h) Check pH of media before and after sterilization. If required, adjust with the help of diluted HCl or NaOH. (i) After sterilization, use the media as soon as possible and avoid keeping for longer as it can cause contamination.
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(j) New batch of culture media should be evaluated for their ‘Growth Promotion Ability’ both qualitatively and quantitatively prior to use for routine analysis and record should be maintained for the same.
3.7 Culture Media: Role of Ingredients (Table 3.1) Table 3.1 Name of ingredients and their role in media Name of ingredients Agar Ammonium dihydrogen phosphate Ammonium sulphate Asparagine Acicase
Role in media Solidifying agent Source of nitrogen
Good sources of ions that simulate metabolism Source of amino acid and nitrogen Source of nitrogenous compounds, carbon, sulphur and other essential nutrients Beef heart Source of amino acids and other nutrients Bile salt Inhibitor of gram-positive bacteria Brilliant green Inhibitor of gram-positive bacteria Bromothymol blue pH indicator Casein enzymic hydrolysate Source of nitrogenous and carbonaceous compounds, minerals, vitamins and trace ingredients for the growth of organisms Chloramphenicol Antibacterial agent Crystal violet Inhibitor of gram-positive bacteria Dextrose Energy and carbohydrate source 2,3,5 Triphenyl Tetrazolium Add to the media to enhance the visibility of bacterial growth, Chloride (TTC) produced red color colonies. Red colored compounds named formazan is formed Dipotassium and Buffering to the medium monopotassium phosphates Dipotassium phosphate pH controlling agent also provides the buffering system Disodium phosphate Used to maintain the buffering action of the medium D-sorbitol Energy source Eosin-Y Make medium selective, inhibit certain gram-positive bacteria Ethyl violet Added to selective medium, inhibits bacteria Other than Enterococci Glucose Carbon and energy source Glycerol Additional source of carbon Heart pancreatic digest Supports luxuriant growth of fastidious and non-fastidious organisms HM infusion B Source of nitrogenous compounds, carbon, sulphur and other essential nutrients Lactose Energy source (continued)
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Table 3.1 (continued) Name of ingredients L-cysteine hydrochloride L-cystine Lithium chloride L-malic acid Magnesium chloride Magnesium sulphate Malachite green Malt extract
Meat peptic digest Meat peptone Neutral red Pancreatic digest of casein Peptic digest of animal tissue Phenol red Polysorbate 80 Potassium tellurite Sodium azide Sodium caseinate Sodium chloride Sodium citrate Sodium deoxycholate Sodium propionate Sodium pyruvate Sodium thioglycolate Sodium thiosulphate Soya peptone Soyabean meal Starch Sucrose Sulphates Tryptone Yeast extract
Role in media Reducing agent Permits Clostridium to grow Inhibits contaminating microflora except target one Inhibitory to several bacterial species Enhance the osmotic pressure in the medium Good sources of ions that simulate metabolism Inhibitory agent Extra source of carbon, it also offers an acidic environment and nutrients favorable for growth and metabolism of yeasts and molds Supports luxuriant growth of fastidious and non-fastidious organisms Source of nitrogenous and carbonaceous compounds, minerals, vitamins and trace ingredients for the growth of organisms Indicator Supports luxuriant growth of fastidious and non-fastidious organisms Source of nitrogen, carbon, trace elements and other essential nutrients pH indicator Additional source of growth factor and fatty acid Inhibits contaminating microflora except target one Added to selective medium, inhibits bacteria Other than Enterococci Nitrogen source To maintain the osmotic balance/ equilibrium Source of carbon Inhibitor of Coliform bacteria and gram-positive bacteria Substrate in anaerobic fermentation Helps in recovery of injured cells Provide satisfactory anaerobiosis, permits Clostridium to grow Inactivator of halogen Additional growth factors Source of growth nutrients Use as a protective colloid against toxic substances present in the medium Energy source Source of sulphur and metallic ions Additional growth factors It supplies sulphur, trace elements, vitamin B as nutrients and additional growth factors
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3.8 Culture Media: Compositions 3.8.1 Actinomycetes Isolation Agar Actinomycetes isolation agar medium is used for the isolation and identification of actinomycetes from water and soil samples. This media is used for maintenance of actinomycetes group of microorganisms for shorter period (Table 3.2). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.2 Alkaline Peptone Water Peptic digest of animal tissue provides amino acids and other nitrogenous substances. Sodium chloride maintains osmotic equilibrium. This media is recommended as an enrichment medium having relatively high pH for the isolation and identification of Vibrio spp. from sea foods, infectious materials and other clinical specimens such as feces (Table 3.3). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.2 Composition of Actinomycetes isolation agar Sodium caseinate (2.0 g/L) Sodium propionate (4.0 g/L) Magnesium sulphate (0.1 g/L) Agar (15.0 g/L)
L-Asparagine (0.1 g/L) Dipotassium phosphate (0.5 g/L) Ferrous sulphate (0.001 g/L) pH after sterilization (8.1 ± 0.2)
Table 3.3 Composition of Alkaline Peptone Water Peptic digest of animal tissue (10.0 g/L)
Sodium chloride (10 g/L); pH after sterilization (8.4 ± 0.2)
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3.8.3 Ammonifying Bacteria Isolation Medium This is a highly selective medium recommended for the detection and isolation of ammonifying bacteria in environmental samples (Table 3.4). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.4 Anaerobic Agar Anaerobic Agar medium is used for the isolation of anaerobic bacteria such as Clostridium bacteria and other anaerobic microorganisms from food samples (Table 3.5). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.5 Antibiotic Assay Medium No-1 This medium is generally used for the microbiological assays of various antibiotics such as beta-lactam (e.g. Penicillin, Cephalosporins), Amikacin, Bacitracin, Cephalothin, Chloramphenicol, Chlortetracycline, Cloxacillin Cycloserine,
Table 3.4 Composition of Ammonifying Bacteria Isolation medium Potassium chloride (0.20 g/L) Ferrous sulfate (0.01 g/L) Magnesium sulfate heptahydrate (0.20 g/L) Peptone (10.0 g/L)
Table 3.5 Composition of Anaerobic Agar
Potassium di hydrogenphosphate (3.0 g/L) Sodium chloride (0.20 g/L) Calcium sulfate (0.10 g/L) pH after sterilization (5.6 ± 0.2)
Glucose (10.0 g/L) Sodium chloride (5.0 g/L) Sodium formaldehyde Sulfoxylate (1.0 g/L) Agar (20.0 g/L)
Tryptone (20 g/L) Sodium thioglycolate (2 g/L) Methylene blue (0.002 g/L) pH after sterilization (7.2 ± 0.2)
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Colistin, Demeclocycline, Erythromycin, Framycetin, Gentamicin, Kanamycin, Methacycline, Nafcillin, Neomycin, Novobiocin, Oxytetracycline, Paromomycin, Penicillin-G, Rifamycin sodium and many more in accordance with Indian and United State Pharmacopeia (Table 3.6). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.6 Antibiotic Assay Medium No-12 This medium is generally used for the microbiological assay of antifungal agents like Nystatin by using Saccharomyces cerevisiae as a test-organism (Table 3.7). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.7 Antibiotic Assay Medium No-19 This medium is generally used for the microbiological assay of Amphotericin B, Netamycin and Nystatin using Saccharomyces as a test organism (Table 3.8). Table 3.6 Composition of Antibiotic Assay Medium No-1
Peptone (6.0 g/L) Yeast extract (3.0 g/L) Dextrose (1.0 g/L) Casein enzymic hydrolysate (4.0 g/L) Beef extract (1.5 g/L) Agar (15.0 g/L), pH after sterilization (6.6. ± 0.2)
Table 3.7 Composition of Antibiotic assay medium No-12
Peptone (10.0 g/L) Sodium chloride (10.0 g/L) Dextrose (10.0 g/L) Yeast extract (5.0 g/L) Beef extract (2.5 g/L) Agar (15.0 g/L), pH after sterilization (6.1 ± 0.2)
Table 3.8 Composition of Antibiotic assay medium No-19
Peptone (9.4 g/L) Sodium chloride (10.0 g/L) Dextrose (10.0 g/L) Yeast extract (4.7 g/L) Beef extract (2.4 g/L) Agar (23.5 g/L), pH after sterilization (6.1 ± 0.2)
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Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.8 Antibiotic Assay Medium No-11 This medium is generally used for the microbiological assay of Neomycin, Erythromycin (Table 3.9). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.9 Baird–Parker Agar Medium This medium was developed by Baird Parker from the Tellurite-glycine formulation of Zebovitz et al. for isolation and enumeration of Staphylococci and it is recommended for isolation and enumeration of coagulase positive Staphylococci from water, food and other materials. Grey to black colonies with opaque zone due to coagulase activity appear onto this medium (Table 3.10). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.9 Composition of Antibiotic assay medium No-11
Peptone (6.0 g/L) Casein enzymic hydrolysate (4.0 g/L) Dextrose (1.0 g/L) Yeast extract (3.0 g/L) Beef extract (1.5 g/L) Agar (15.0 g/L), pH after sterilization (6.1 ± 0.2)
Table 3.10 Composition of Baird–Parker Agar medium Glycine (12.0 g/L) Sodium pyruvate (10.0 g/L) Lithium chloride (5.0 g/L) Agar (20.0 g/L)
Casein enzymic hydrolysate (10.0 g/L) Yeast extract (1.0 g/L) Beef extract (5.0 g/L) pH after sterilization (6.1 ± 0.2)
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3.8.10 BG-11 Medium This medium is used for the isolation and identification of cyanobacteria (Blue- Green algae) from water, soil and marine samples (Table 3.11). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.11 Biotin Assay Medium This media is recommended for the determination of biotin concentration by using microbiological assay of biotin using Lactobacillus plantarum ATCC 8014 as the test organism (Table 3.12). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.11 Composition of BG-11 Medium Dipotassium hydrogen phosphate (0.0314 g/L) Magnesium sulphate (0.036 g/L) Sodium carbonate (0.020 g/L) Citric acid (0.0056 g/L) pH after sterilization 7.1
Sodium nitrate (1.5 g/L) Calcium chloride dihydrate (0.0367 g/L) Disodium magnesium EDTA (0.001 g/L) Ferric ammonium citrate (0.006 g/L) Add sodium chloride (10 g/L) and vitamin B12 (1 g/L) for isolation of marine cyanobacteria
Table 3.12 Composition of Biotin Assay Medium Vitamin Assay Casamino Acids (12.0 g/L) Sodium acetate (20.0 g/L) DL-tryptophan (0.020 g/L) Guanine hydrochloride (0.02 g/L) Thiamine hydrochloride (0.02 g/L) Niacin (0.02 g/L) Pyridoxine Hydrochloridem (0.04 g/L) Dipotassium phosphate (1.0 g/L) Magnesium sulfate (0.4 g/L) Ferrous sulfate (0.020 g/L)
Dextrose (40.0 g/L) L-Cystine (0.2 g/L) Adenine sulfate (0.02 g/L) Uracil (0.02 g/L) Riboflavin (0.02 g/L) Calcium Pantothenate (0.02 g/L) p-Aminobenzoic acid (0.0002 g/L) Monopotassium phosphate (1.0 g/L) Sodium chloride (0.2 g/L) Manganese sulfate (0.020 g/L) pH (6.8 ± 0.2)
3.8 Culture Media: Compositions
35
3.8.12 Bismuth Sulphite Agar (BSA) Bismuth Sulphite Agar is a selective media which is recommended for the isolation and preliminary identification of Salmonella typhi and other Salmonellae from pathological materials, sewage, water supplies, food and other infected materials. Salmonella colonies grow black colonies with a surrounding metallic sheen resulting from hydrogen Sulphite production and reduction of Sulphite to black ferric sulphide (Table 3.13). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Do not autoclave this medium as overheating may abolish the selective nature of the media.
3.8.13 Brain–Heart Infusion Broth This media is recommended for the cultivation of fastidious microorganisms from variety of water, food and other infected material. It is also used to prepare the inoculum for antimicrobial susceptibility testing. Proteose peptone and infusions (calf brain and beef heart) serve as the sources of carbon, nitrogen, essential growth factors, amino acids and vitamins. Dextrose serves as a source of energy. Disodium phosphate helps in maintaining the buffering action of the medium (Table 3.14). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.13 Composition of Bismuth Sulphite Agar (BSA) Peptic digest of animal tissue (10.0 g/L) Dextrose (5.0 g/L) Ferrous sulphate (0.3 g/L) Brilliant green (0.025 g/L)
Beef extract (5.0 g/L) Disodium phosphate (4.0 g/L) Bismuth sulphite indicator (8.0 g/L) Agar (20.0 g/L) Final pH (7.7)
Table 3.14 Composition of Brain–heart Infusion broth Calf brain, infusion (200.0 g/L) Proteose peptone (10.0 g/L) Sodium chloride (5.0 g/L)
Beef heart, infusion (250.0 g/L) Dextrose (2.0 g/L) Disodium phosphate (2.5 g/L); Final pH (7.4 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
3.8.14 Blood Agar Medium Blood Agar Medium is a highly selective, used with blood or with antibiotic for the isolation and growth of fastidious bacteria such as Streptococci and Staphylococci species etc. This medium can be used with antibiotic (Table 3.15). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.15 Baird Parker Agar Medium Baird Parker Agar medium is used for the isolation, detection and enumeration of Staphylococcus species from food, water and drug samples (Table 3.16). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.16 Brilliant Green Agar (BGA) This media is recommended for the isolation and identification of Salmonella from water, food samples even it is used as a selective media for the isolation of Salmonella from drug and pharmaceuticals samples. This media is mainly recommended by APHA, FDA and by Indian Pharmacopoeia other scientific publications (Table 3.17).
Table 3.15 Composition of Blood Agar Medium
Tryptose (10.0 g/L) Peptone (10.0 g/L) Sodium chloride (5.0 g/L) Agar (15.0 g/L) Blood Final pH (7.3 ± 0.2)
Table 3.16 Composition of Baird Parker Agar medium Peptone (5.0 g/L) Yeast extract (1.0 g/L) Sodium puruvate (10.0 g/L) Agar (20.0 g/L) 50 ml concentrated egg yolk emulsion
Tryptone (10.0 g/L) Glycine (12.0 g/L) Lithium chloride (5.0 g/L) Final pH (7.0 ± 0.2) 3 ml sterile 3.5% potassium tellurite solution 50 ml egg yolk tellurite emulsion
3.8 Culture Media: Compositions
37
Table 3.17 Composition of Brilliant Green Agar (BGA) Proteose peptone (10.0 g/L) Lactose (10.0 g/L) Sodium chloride (5.0 g/L) Brilliant green (0.0125 g/L)
Yeast extract (3.0 g/L) Sucrose (10.0 g/L) Phenol red (0.08 g/L) Agar (12.0 g/L) Final pH (6.9 ± 0.2)
Table 3.18 Composition of Brilliant Green Bile Lactose (BGBL) broth Lactose monohydrate (10.0 g/L) Dehydrated oxbile (20.0 g/L)
Enzymatic digest of casein (10.0 g/L) Brilliant green (0.0133 g/L); pH after sterilization (7.2 ± 0.2)
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.17 Brilliant Green Bile Lactose (BGBL) Broth This is a selective medium which is used for the confirmation of coliform bacteria in water, soil and food samples (Table 3.18). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.18 Buffered Peptone Water (BPW) This media is used as a pre-enrichment medium for the recovery of injured Salmonella spp. from water, food and other contaminated material prior to selective enrichment and isolation of Salmonella. It is inhibitor free medium and is well buffered and provide the condition to the cell that have been injured during the p rocess of food preservations (Table 3.19). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
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3 Microbiological Culture Media: Types, Role and Composition
Table 3.19 Composition of Buffered peptone water (BPW)
Proteose peptone (10.0 g/L)
Disodium phosphate, anhydrous (3.5 g/L) Monopotassium phosphate (1.5 g/L) Sodium chloride (5.0 g/L); Final pH (7.2) ±0.2
Table 3.20 Composition of Buffered Sodium Chloride Peptone Solution Potassium dihydrogen phosphate (3.6 g/L) Sodium chloride (4.3 g/L)
Disodium hydrogen phosphate dihydrate (7.2 g/L) Peptone (1.0 g/L); Final pH (7.2 ± 0.2)
3.8.19 Buffered Sodium Chloride Peptone Solution This medium is generally recommended for carrying out the microbial limit test of drug and pharmaceuticals samples in accordance with the harmonized methodology of several Pharmacopoeias (Table 3.20). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.20 Cetrimide Agar It is used as a selective medium for the isolation and identification of Pseudomonas aeruginosa from food, water and drug and pharmaceuticals samples as per national as well as international methods. Cetrimide is a quaternary ammonium salt, which acts as a cationic detergent that reduces the surface tension in the point of contact and has precipitant, complexing and denaturing effects on bacterial membrane proteins (Table 3.21). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8 Culture Media: Compositions
39
Table 3.21 Composition of Cetrimide Agar Pancreatic digest of gelatin (20.0 g/L) Dipotassium sulphate (10.0 g/L) Agar (13.6 g/L)
Table 3.22 Composition of Chloramphenicol Yeast Extract Glucose Agar
Magnesium chloride (1.4 g/L) Cetrimide (0.3 g/L) pH after sterilization (7.2 ± 0.2)
Yeast extract (5.0 g/L) Dextrose (20 g/L) Chloramphenicol (0.1 g/L) Agar (14.9 g/L); pH after sterilization (6.6 ± 0.2)
Table 3.23 Composition of Columbia Agar Pancreatic digest of casein (10.0 g/L) Heart pancreatic digest (3.0 g/L) Sodium chloride (5.0 g/L) Agar (15.0 g/L)
Meat peptic digest (5.0 g/L) Maize starch (1.0 g/L) Yeast extract (5.0 g/L) pH after sterilization (6.6 ± 0.2)
3.8.21 Chloramphenicol Yeast Extract Glucose Agar (CYGA) This medium is used for the isolation and enumeration of yeast and mold from water, food and other contaminated samples. This medium contains antibiotic chloramphenicol (a thermostable antibiotic) which make it selective for Yeast and Mold by inhibiting the growth of bacteria. This medium is recommended by guidelines given by national and international body (Table 3.22). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.22 Columbia Agar Columbia Agar medium is used for the detection and isolation of fastidious bacteria from drug and pharmaceutical preparations in accordance with the microbial limit testing harmonized methodology of pharmacopoeia (Table 3.23). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
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3 Microbiological Culture Media: Types, Role and Composition
3.8.23 Cook Meat Medium (CMM) Cooked Meat Medium is used for cultivation of aerobes and anaerobes, especially pathogenic Clostridia and also for the maintenance of stock cultures. Cooked meat medium contains beef heart, the muscle protein which provide amino acids and other nutrients. Beef heart also contains glutathione, a reducing substance that permits the growth of obligate anaerobes (Table 3.24). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.24 Deoxycholate Citrate Agar (DCA) It is a selective medium generally used for the isolation and recovery of species belongs to Salmonella and Shigella from water, food and other contaminated samples. The use of this medium is written in national and international guidelines (Table 3.25). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Do not autoclave this medium as heat is detrimental to the medium.
3.8.25 Differential Reinforced Clostridia Broth Differential Reinforced Clostridia Broth is used for the isolation and identification of Clostridium spp. from water samples (Table 3.26).
Table 3.24 Composition of Cook Meat medium Beef heart, infusion (500.0 g/L) Sodium chloride (2.5 g/L)
Peptic digest of animal tissue (5.0 g/L) pH after sterilization (7.8 ± 0.2)
Table 3.25 Composition of Deoxycholate citrate agar Heart Infusion solids (10.0 g/L) Sodium deoxycholate (5.0 g/L) Sodium citrate (20.0 g/L) Lactose (10.0 g/L)
Proteose peptone (10.0 g/L) Neutral red (0.020 g/L) Ferric ammonium citrate (2.0 g/L) Agar (13.5 g/L), pH after sterilization (7.5 ± 0.2)
3.8 Culture Media: Compositions
41
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.26 Enterobacteria Enrichment Broth Enterobacteria Enrichment Broth, Mossel is used for selective enrichment of Enterobacteriaceae consists of Salmonella, Shigella and other enteric pathogens (Table 3.27). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.27 Eosin Methylene Blue (EMB) Agar It is selective medium used for the isolation and differentiation of Gram-negative enteric bacteria from water, food and other contaminated samples (Table 3.28).
Table 3.26 Composition of Differential Reinforced Clostridia Broth Peptic digest of animal tissue (10.0 g/L) Yeast extract (1.5 g/L) Sodium acetate, hydrated (5.0 g/L) L-cysteine hydrochloride (0.5 g/L)
Beef extract (10.0 g/L) Starch (1.0 g/L) Glucose (1.0 g/L) pH after sterilization (7.2 ± 0.2)
Table 3.27 Composition of Enterobacteria Enrichment Broth Pancreatic digest of gelatin (10.0 g/L) Dehydrated ox-bile (20.0 g/L) Potassium dihydrogen phosphate (2.0 g/L)
Glucose monohydrate (5.0 g/L) Disodium hydrogen phosphate, dihydrate (8.0 g/L) Brilliant green (0.015 g/L); pH after sterilization (7.2 ± 0.2)
Table 3.28 Composition of Eosin Methylene Blue (EMB) Agar Dipotassium phosphate (2.0 g/L) Lactose (5.0 g/L) Eosin – Y (0.4 g/L) Agar (13.5 g/L)
Peptic digest of animal tissue (10.0 g/L) Sucrose (5.0 g/L) Methylene blue (0.065 g/L) pH after sterilization (7.2 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.28 Ethyl Violet Azide Dextrose Agar Ethyl Violet Azide Dextrose Agar is recommended for the isolation and confirmation of Streptococci from water, food and other contaminated sample. Combination of 0.0083 g % of ethyl violet dye and 0.04 g % of azide provided the best selective action favoring growth of Streptococci (Table 3.29). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.29 Fluid Thioglycollate Medium (FTM) Fluid thioglycollate medium is recommended for sterility test of drug and pharmaceuticals (both injectable and noninjectable) as per guidelines given in Pharmacopoeia. In this medium aerobe, anaerobes and microaerophiles grow and produce turbidity. Sodium thioglycollate and L- cysteine act as reducing agents and expel oxygen from the medium to neutralize the toxic effects of mercurial preservatives and peroxides formed the medium, thereby promoting anaerobiosis, and making the medium suitable to test materials containing heavy metals. An increase in the oxygen content is indicated by the a color change of the redox indicator, resazurin, which is pink when oxidized and colorless when reduced (Table 3.30). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.29 Composition of Ethyl Violet Azide Dextrose Agar Dextrose (5.0 g/L) Dipotassium phosphate (2.7 g/L) Ethyl violet (0.00083 g/L) Sodium azide (0.065 g/L)
Casein enzymic hydrolysate (20.0 g/L) Monopotassium phosphate (2.7 g/L) Sodium chloride (5.0 g/L) Agar (15.0 g/L); pH after sterilization (7.0 ± 0.2)
3.8 Culture Media: Compositions
43
Table 3.30 Composition of Fluid thioglycollate medium Yeast extract (5.0 g/L) Glucose monohydrate (5.5 g/L) L-Cystine (0.5 g/L) Resazurin sodium (0.001 g/L)
Table 3.31 Composition of Glucose Agar
Pancreatic digest of casein (15.0 g/L) Sodium chloride (5.0 g/L) Sodium thioglycollate (0.5 g/L) Agar (0.750 g/L); pH after sterilization (7.1 ± 0.2)
Yeast extract (1.5 g/L) Tryptone (10.0 g/L) Glucose (10.0 g/L) Sodium chloride (5.0 g/L) Bromocresol purple (0.15 g/L) Agar (15.0 g/L); pH after sterilization (7.0 ± 0.2)
Table 3.32 Composition of Glucose-Salt-Teepol Broth Beef extract (3.0 g/L) Glucose (5.0 g/L) Methyl violet (0.002 g/L)
Peptic digest of animal tissue (10.0 g/L) Sodium chloride (30.0 g/L) Teepol (4.0 g/L); pH after sterilization (8.8 ± 0.2)
3.8.30 Glucose Agar Glucose Agar is used for determining the fermentation reactions of presumptive Enterobacteriaceae (Table 3.31). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.31 Glucose-Salt-Teepol Broth Glucose Salt Teepol Broth is recommended for enrichment of Vibrio parahaemolyticus and marine isolate from water, food and other contaminated samples. It is also used to enumerate the bacteria by MPN technique (Table 3.32). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
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3 Microbiological Culture Media: Types, Role and Composition
3.8.32 GN Broth GN Broth is recommended for selective enrichment of gram-negative enteric organisms (Table 3.33). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.33 Glucose Yeast Extract Agar This medium is recommended for enumeration and cultivation of Lactobacilli in drug and pharmaceuticals samples (Table 3.34). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.34 Hugh-Leifson Medium This medium is recommended to differentiate among anaerobic and aerobic breakdown of carbohydrate (Table 3.35).
Table 3.33 Composition of GN Broth Dextrose (1.0 g/L) Mannitol (2.0 g/L) Dipotassium phosphate (4.0 g/L) Sodium chloride (5.0 g/L)
Tryptose (20.0 g/L) Sodium citrate (5.0 g/L) Monopotassium phosphate (1.5 g/L) Sodium deoxycholate (0.5 g/L); pH after sterilization (7.0 ± 0.2)
Table 3.34 Composition of Glucose Yeast Extract Agar Yeast extract (5.0 g/L) Glucose (2.0 g/L) Dipotassium phosphate (0.5 g/L) Zinc sulphate (0.0016 g/L) Cobalt sulphate (0.0016 g/L) Agar (15.0 g/L)
Peptic digest of animal tissue (5.0 g/L) Monopotassium phosphate (0.5 g/L) Magnesium sulphate (0.010 g/L) Copper sulphate (0.0016 g/L) Sodium chloride (0.010 g/L) pH after sterilization (7.0 ± 0.2)
3.8 Culture Media: Compositions Table 3.35 Composition of Hugh-Leifson Medium
45
Peptone (2.0 g/L) Dipotassium phosphate (0.3 g/L) Bromothymol blue (0.030 g/L)
Table 3.36 Composition of-Agar Medium
Peptone (5.0 g/L) Dextrose (1.0 g/L) Agar (15.0 g/L)
Sodium chloride (5.0 g/L) Glucose (10.0 g/L) Agar (3.0 g/L); pH after sterilization (7.1 ± 0.2)
Yeast extract (2.5 g/L) Polysorbate 80 (1.0 g/L) pH after sterilization (3.7 ± 0.2)
Table 3.37 Composition of KG (Kim Goepfert) Agar base Peptic digest of animal tissue (1.0 g/L) Phenol red (0.025 g/L)
Yeast extract (0.5 g/L) Agar (18.0 g/L); pH after sterilization (6.8 ± 0.2)
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving and used as and when required.
3.8.35 K-Agar Medium This medium is used for the detection, isolation, cultivation of Alicyclobacillus in food and farm products as per standard (Table 3.36). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving and used as and when required.
3.8.36 KG (Kim Goepfert) Agar Base This medium with added supplements is recommended for helping fast and free spore formation which helps in distinctive Bacillus cereus from Bacillus thuringiensis (Table 3.37).
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3 Microbiological Culture Media: Types, Role and Composition
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving and used as and when required.
3.8.37 Lactose Broth This medium is recommended for the detection of coliform bacteria in water, food and other contaminated samples by using national and international guidelines (Table 3.38). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.38 MacConkey Broth MacConkey Broth medium is used for the isolation and enumeration of coliform from all kind of water like drinking water, package drinking water, mineral water, natural mineral water, swimming pool water, recreational water, sewage water etc. by using MPN Technique. This medium is also used for the selective enrichment of E. coli from drug and pharmaceuticals product in accordance with various pharmacopoeias (Table 3.39). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.38 Composition of Lactose broth Peptic digest of animal tissue (5.0 g/L) Lactose (2.0 g/L)
Beef extract (3.0 g/L) pH after sterilization (6.9 ± 0.2)
Table 3.39 Composition of MacConkey Broth Peptic digest of animal tissue (20.0 g/L) Sodium chloride (5.0 g/L) Lactose (10.0 g/L)
Bile salts (5.0 g/L) Bromocresol purple (0.010 g/L) pH after sterilization (7.4 ± 0.2)
47
3.8 Culture Media: Compositions
3.8.39 Mac Conkey Agar (MCA) This is a selective medium which is generally recommended for the selective isolation and identification of E. coli from water, food and drug and pharmaceuticals sample in accordance with the methods given by national and international organization. This medium is also recommended for the selective isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacteria (Table 3.40). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.40 Malt Extract Broth This medium is used for the isolation and enumeration of yeasts and molds in food and pharmaceutical samples. Malt Extract Broth is also used for the maintenance and mass cultivation of fungi (Table 3.41). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.40 Composition of Mac Conkey Agar Peptones (meat and casein) (3.0 g/L) Sodium chloride (5.0 g/L) Crystal violet (0.001 g/L) Bile salts (1.5 g/L)
Pancreatic digest of gelatin (17.0 g/L) Lactose monohydrate (10.0 g/L) Neutral red (0.030 g/L) Agar (13.5 g/L); pH after sterilization (7.1 ± 0.2)
Table 3.41 Composition of Malt Extract Broth Mycological peptone (3.0 g/L)
Malt extract (17.0 g/L); pH after sterilization (5.4 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
3.8.41 Mannitol Salt Agar Mannitol Salt Agar is a selective medium which is used for the isolation and identification of Staphylococcus aureus from food, water and other contaminated sample in accordance to national and international methodology. The additional property of lipase activity of S. aureus is detected by the addition of the egg yolk emulsion (Table 3.42). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.42 Mannitol Yeast Polymyxin Agar (MYP Agar) This medium is used for the isolation and identification of Bacillus spp. from food, water and other contaminated samples (Table 3.43). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.43 Minimal Agar This medium is used for the isolation and description of nutritional mutants of Escherichia coli (Table 3.44).
Table 3.42 Composition of Mannitol Salt Agar Peptones (10.0 g/L) Sodium chloride (75.0 g/L) Phenol red (0.025 g/L)
Beef extract (1.0 g/L) D-Mannitol (10.0 g/L) Agar (15.0 g/L); pH after sterilization (at 25 °C) 7.4 ± 0.2
Table 3.43 Composition of Mannitol Yeast Polymyxin Agar Peptic digest of animal tissue (10.0 g/L) Sodium chloride (10.0 g/L) Phenol red (0.025 g/L)
Meat extract (1.0 g/L) D-Mannitol (10.0 g/L) Agar (12.0 g/L); pH after sterilization (7.1 ± 0.2)
3.8 Culture Media: Compositions
49
Table 3.44 Composition of Minimal Agar Dextrose (1.0 g/L) Monopotassium phosphate (2.0 g/L) Ammonium sulphate (1.0 g/L) Agar (15.0 g/L)
Dipotassium phosphate (7.0 g/L) Sodium citrate (0.5 g/L) Magnesium sulphate (0.1 g/L) pH after sterilization (7.0 ± 0.2)
Table 3.45 Composition of Motility Test Medium Peptic digest of animal tissue (10.0 g/L) Agar (4.0 g/L)
Beef extract (3.0 g/L) pH after sterilization (7.4 ± 0.2)
Table 3.46 Composition of MR-VP Medium Buffered peptone (7.0 g/L) Dipotassium phosphate (5.0 g/L)
Dextrose (5.0 g/L) pH after sterilization (6.9 ± 0.2)
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.44 Motility Test Medium Motility Test Medium is used for detecting motility of microorganisms (Table 3.45). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.45 MR-VP Medium (Glucose Phosphate Broth) MR-VP Medium (Glucose Phosphate Broth) is recommended for the performance of the Methyl Red and Voges-Proskauer tests in differentiation of the coli-aerogenes group (Table 3.46).
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3 Microbiological Culture Media: Types, Role and Composition
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.46 Mueller Hinton Broth (MHB) This medium is highly recommended for the determination of minimum inhibitory concentration of extracts and other bioactive compounds. This is recommended to study antimicrobial susceptibility testing by CLSI guidelines (Table 3.47). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving. Calcium and magnesium ion concentrations are adjusted for MIC determination.
3.8.47 Muller Hinton Agar This medium is used for the determination of susceptibility of microorganism to antimicrobial agents including extracts and bioactive compounds by disc diffusion and agar well diffusion assay. This medium is highly recommended by CLSI and Kirby-Bauer (Table 3.48). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.47 Composition of Mueller Hinton Broth
HM infusion B (300.0 g/L) Acicase (17.5 g/L) Starch (1.5 g/L) pH after sterilization (7.3 ± 0.2)
Table 3.48 Composition of Muller Hinton Agar
HM infusion B (300.0 g/L) Acicase (17.5 g/L) Starch (1.5 g/L) Agar (17.0 g/L)pH after sterilization (7.3 ± 0.2)
3.8 Culture Media: Compositions
51
3.8.48 Nitrate Medium This medium is recommended for the nitrate reduction test of microorganisms (Table 3.49). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.49 Nutrient Agar Nutrient agar media is a general-purpose medium which is used for the cultivation, enumeration of microorganism from water, food and other contaminated materials. This medium is also used in preparing slants for the maintenance of both working and stock culture. It is also used for the biochemical and serological identification of microorganism (Table 3.50). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.50 Nutrient Broth It is general purpose medium used for the maintenance and cultivation of microorganisms (Table 3.51). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.49 Composition of Nitrate medium Peptic digest of animal tissue (5.0 g/L) Potassium nitrate (1.0 g/L)
Meat extract (3.0 g/L) pH after sterilization (7.0 ± 0.2)
Table 3.50 Composition of Nutrient Agar Peptic digest of animal tissue (5.0 g/L) Agar (15.0 g/L)
Beef extract (3.0 g/L) pH after sterilization (6.8 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
Table 3.51 Composition of Nutrient Broth Peptic digest of animal tissue (5.0 g/L) Yeast extract (1.5 g/L)
Table 3.52 Composition of Osmophilic Agar
Beef extract (1.5 g/L) pH after sterilization (7.4 ± 0.2)
Malt Extract (20 g/L) Sucrose (400 g/L)
Yeast Extract (5 g/L) Agar (20 g/L); pH after sterilization (7.4 ± 0.2)
3.8.51 Osmophilic Agar Osmophilic Agar medium is recommended for the detection, isolation and identification of osmophilic bacteria (Table 3.52). Instructions for use: Heat to dissolve, place in flasks and sterilize at 121 °C at 15 pounds of pressure for 15 minutes in a steam autoclave. For quantitation of osmophilic yeast and mold, aseptically add 2 mL of sterile 10% tartaric acid solution to each 100 mL of sterile and cooled (45 °C) medium prior to use. For quantitation of osmophilic bacteria, tartaric acid is not added to the osmophilic agar.
3.8.52 Osmophilic Dilution Blanks Dextrose, 400 g; purified water, 1000 mL. Dispense in dilution blanks in appropriate volumes. Cap and sterilize at 121 °C under 15 lbs. pressure for 15 minutes in a steam autoclave. Avoid overheating.
3.8.53 Pantothenate Assay Medium Pantothenate Assay Medium is recommended for the microbiological assay of Pantothenic acid or its salts using Lactobacillus plantarum ATCC 8014 as the test organism (Table 3.53). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
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53
Table 3.53 Composition of Pantothenate assay medium Casein acid hydrolysate (10.0 g/L) Sodium acetate (20.0 g/L) DL-Tryptophan (0.2 g/L) Guanine hydrochloride (0.020 g/L) Riboflavin (Vitamin B2) (0.0004 g/L) Niacin (0.001 g/L) p-Amino benzoic acid (PABA) (0.0002 g/L) Monopotassium phosphate (1.0 g/L) Magnesium sulphate (0.400 g/L) Ferrous sulphate (0.020 g/L)
Dextrose (40.0 g/L) L-Cystine (0.4 g/L) Adenine sulphate (0.020 g/L) Uracil (0.020 g/L) Thiamine hydrochloride (0.0002 g/L) Pyridoxine (0.0008 g/L) Biotin (0.0000008 g/L) Dipotassium phosphate (1.0 g/L) Sodium chloride (0.020 g/L) Manganese sulphate (0.020 g/L); pH after sterilization (6.8 ± 0.2)
Table 3.54 Composition of Pantothenate Inoculum broth Peptonized milk (15.0 g/L) Dextrose (10.0 g/L) Monopotassium phosphate (2.0 g/L)
Yeast extract (5.0 g/L) Polysorbate 80 (1.0 g/L) Tomato juice (100 ml) 5.0 g/L; pH after sterilization (6.8 ± 0.2)
3.8.54 Pantothenate Inoculum Broth Pantothenate Inoculum Broth is recommended for the preparation of inoculum used in microbiological assays of pantothenic acid or its salts (Table 3.54). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.55 Peptone Water Peptone Water is recommended as a growth medium to study the property of microorganism to ferment a specific carbohydrate and particularly suitable as a substrate in the study of indole production (Table 3.55). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
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Table 3.55 Composition of Peptone water Peptic digest of animal tissue (10.0 g/L)
Sodium chloride (5.0 g/L); pH after sterilization (6.8 ± 0.2)
Table 3.56 Composition of Plate count agar
Casein enzyme hydrolysate (5.0 g/L) Yeast extract (2.5 g/L) Dextrose (1.0 g/L) Agar (15.0 g/L); pH after sterilization (6.8 ± 0.2)
Table 3.57 Composition of Rappaport Vassiliadis medium Soya peptone (4.5 g/L) Dipotassium phosphate (0.4 g/L) Magnesium chloride, hexahydrate (29.0 g/L)
Sodium chloride (8.0 g/L) Potassium dihydrogen phosphate (0.6 g/L) Malachite green (0.036 g/L); pH after sterilization (5.2 ± 0.2)
3.8.56 Plate Count Agar Plate Count Agar is recommended for the determination of plate counts of microorganisms in food, water, wastewater and also from clinical samples (Table 3.56). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.57 Rappaport Vassiliadis Medium (RV) Rappaport Vassiliadis Salmonella Enrichment Broth is recommended for selective enrichment of Salmonella species from water, food and other contaminated samples. The medium contains soyabean meal which provides essential growth nutrients. Magnesium chloride raises the osmotic pressure in the medium. Malachite green is an inhibitory agent, other than Salmonella (Table 3.57). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8 Culture Media: Compositions
55
3.8.58 Sabouraud Dextrose Agar This medium is used for the enumeration of yeast and mold from food, water and drug and pharmaceutical samples (Table 3.58). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.59 Sabouraud Dextrose Broth This medium is recommended for cultivation aciduric microorganism, yeasts and moulds in variety of samples (Table 3.59). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.60 Selenite Cystine Broth (SCB) This medium is used as selective enrichment of Salmonella spp. from verity of food, water and other contaminated samples (Table 3.60).
Table 3.58 Composition of Sabouraud Dextrose Agar
Dextrose (40.0 g/L) Mycological, peptone (10.0 g/L) Agar (15.0 g/L) pH after sterilization (5.6 ± 0.2)
Table 3.59 Composition of Sabouraud Dextrose Broth Dextrose (20.0 g/L)
Peptone (10.0 g/L) pH after sterilization (5.6 ± 0.2)
Table 3.60 Composition of Selenite Cystine Broth Casein enzyme hydrolysate (5.0 g/L) Disodium phosphate (10.0 g/L)
Lactose (4.0 g/L) L-Cystine (.010 g/L); pH after sterilization (7.0 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Do not autoclave this medium as heating is detrimental.
3.8.61 Simmon’s Citrate Agar Simmon citrate agar medium is used for citrate utilization test to differentiate member of Enterobacteriaceae (Table 3.61). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.62 Skim Milk Agar It is used for cultivation and enumeration of microorganism from food and water sample. This is also used for the demonstration of coagulation and proteolysis of casein (Table 3.62). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.61 Composition of Simmon’s Citrate agar
Magnesium sulphate (0.2 g/L)
Ammonium dihydrogen phosphate (1.0 g/L) Sodium citrate (2.0 g/L) Sodium chloride (5.0 g/L) Di1potassium phosphate (1.0 g/L) L-Cystine (.010 g/L) Bromothymol blue (0.080 g/L) Agar (15.0 g/L)pH after sterilization (6.8 ± 0.2)
Table 3.62 Composition of Skim milk agar Skim milk powder (28.0 g/L) Yeast extract (2.5 g/L) Agar (15.0 g/L)
Casein enzyme hydrolysate (5.0 g/L) Dextrose (1.0 g/L) pH after sterilization (7.0 ± 0.2)
3.8 Culture Media: Compositions
57
3.8.63 Soyabean Casein Digest Broth Soyabean Casein Digest Medium is a general-purpose medium used for cultivation of a wide variety of microorganisms and recommended for sterility testing of molds and lower bacteria (Table 3.63). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.64 Soybean Casein Digest Agar This medium is used for the isolation and cultivation of all kinds of microorganisms from food, water, pharmaceutical samples and clinical samples. This medium is highly recommended for sterility testing by various pharmacopoeias (Table 3.64). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.65 Starch Agar This medium is used for the detection and isolation of starch hydrolyzing bacteria from food and farm samples (Table 3.65). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.63 Composition of Soyabean Casein Digest Broth Pancreatic digest of casein (17.0 g/L) Sodium chloride (5.0 g/L) Dibasic potassium phosphate (2.5 g/L)
Table 3.64 Composition of Soybean Casein Digest Agar
Peptic digest of soybean meal (3.0 g/L) Dextrose (2.5 g/L) pH after sterilization (7.3 ± 0.2)
Soya peptone (5.0 g/L) Tryptone (15.0 g/L) Sodium chloride (5.0 g/L) Agar (15.0 g/L); pH after sterilization (7.3 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
Table 3.65 Composition of Starch Agar Peptic digest of animal tissue (5.0 g/L) Starch, soluble (2.0 g/L)
Meat Extract (3.0 g/L) Agar (15.0 g/L); pH after sterilization (7.2 ± 0.2)
Table 3.66 Composition of TC-SMAC agar medium Meat peptone (3.0 g/L) Bile salts mixture (1.5 g/L) Neutral red (0.30 g/L) D-sorbitol (10.0 g/L)
Casein enzymic hydrolysate (17.0 g/L) Sodium chloride (5.0 g/L) Crystal violet (0.001 g/L) Agar (13.5 g/L); pH after sterilization (7.1 ± 0.2)
Table 3.67 Composition of Tergitol – 7 Agar Peptic digest of animal tissue (10.0 g/L) Meat extract (5.0 g/L) Bromo thymol blue (0.05 g/L) Agar (16.0 g/L)
Yeast extract (6.0 g/L) Lactose (20.0 g/L) Sodium heptadecyl sulphate-Tergitol- 7 (0.1 g/L) pH after sterilization (7.3 ± 0.2)
3.8.66 TC-SMAC Agar Medium This is a selective medium used for the detection, isolation and detection of Escherichia coli O157:H7 from food and farm products (Table 3.66). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.67 Tergitol – 7 Agar (T-7) This medium is used for the selective enumeration of coliform organisms (Table 3.67). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
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3.8 Culture Media: Compositions
3.8.68 Tetrathioanate Broth (TTB) This medium is used for the selective enrichment and isolation of Salmonella spp. from food, water and other contaminated samples (Table 3.68). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.69 Thiosulphate-Citrate-Bile Salts-Sucrose Agar (TCBS) TCBS Agar is recommended for the selective isolation and cultivation of Vibrio cholerae and other enteropathogenic Vibrio’s causing food poisoning (Table 3.69). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Do not atutoclave this medium as heating is detrimental.
3.8.70 Top Agar This medium is used for the determination of mutagenicity of chemicals by AMES test (Table 3.70). Table 3.68 Composition of Tetrathioanate broth Peptone, special (18.0 g/L) Sodium chloride (5.0 g/L) Dextrose (0.5 g/L) Sodium thiosulphate (38.0 g/L) Brilliant green (0.01 g/L)
Yeast extract (2.0 g/L) D-Mannitol (2.5 g/L) Sodium deoxycholate (0.5 g/L) Calcium carbonate (25.0 g/L) pH after sterilization (7.6 ± 0.2)
Table 3.69 Composition of Thiosulphate-Citrate-Bile Salts-Sucrose Agar Proteose peptone (10.0 g/L) Sodium thiosulphate (10.0 g/L) Oxgall (8.0 g/L) Sodium chloride (00.0 g/L) Bromo thymol blue (0.04 g/L) Agar (15.0 g/L)
Yeast extract (5.0 g/L) Sodium citrate (10.0 g/L) Sucrose (20.0 g/L) Ferric citrate (1.0 g/L) Thymol blue (0.04 g/L) pH after sterilization (8.6 ± 0.2)
Table 3.70 Composition of Top Agar Agar (6.0 g/L)
Sodium chloride (5.0 g/L)
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3 Microbiological Culture Media: Types, Role and Composition
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.71 Triple Sugar-Iron Agar Medium This Medium is used for identification of gram-negative bacteria (Table 3.71). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.72 Urease Broth Medium This medium is highly recommended for the detection of urease production by microorganisms (Table 3.72).
Table 3.71 Composition of Triple Sugar-Iron Agar Medium Peptone (20.0 g/L) Yeast extract (3.0 g/L) Sucrose (10.0 g/L) Ferrous sulphate (0.2 g/L) Sodium thiosulphate (0.3 g/L) Agar (12.0 g/L)
Beef extract (3.0 g/L) Lactose (10.0 g/L) Dextrose monohydrate (1.0 g/L) Sodium chloride (5.0 g/L) Phenol red (0.024 g/L) pH after sterilization (7.0 ± 0.2)
Table 3.72 Composition of Urease broth medium Peptic digest of animal tissue (1.5 g/L) Monopotassium phosphate (2.0 g/L) Agar (15.0 g/L)
Dextrose (1.0 g/L) Phenol red (0.012 g/L) 50 ml of sterile 40% urea solution, pH after sterilization (6.8 ± 0.2)
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61
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.73 Veal Infusion Agar Veal Infusion Agar is used for the cultivation of fastidious microorganisms (Table 3.73). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.74 Veal Infusion Broth Veal Infusion broth is used for the cultivation of fastidious microorganisms (Table 3.74). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.75 Violet Red Bile Agar Violet Red Bile Agar is selective medium used for the isolation, detection and enumeration of coliform bacteria in water, milk, other dairy food products (Table 3.75).
Table 3.73 Composition of Veal Infusion Agar
Veal infusion from (500.0 g/L) Proteose peptone (10.0 g/L) Sodium chloride (10.0 g/L) Agar (15.0 g/L); pH after sterilization (7.3 ± 0.2)
Table 3.74 Composition of Veal Infusion broth Veal infusion from (500.0 g/L) Sodium chloride (10.0 g/L)
Proteose peptone (10.0 g/L) pH after sterilization (7.3 ± 0.2)
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3 Microbiological Culture Media: Types, Role and Composition
Table 3.75 Composition of Violet Red Bile Agar Peptic digest of animal tissue (7.0 g/L) Bile salts mixture (1.5 g/L) Neutral red (0.030 g/L) Sodium chloride (5.0 g/L)
Yeast extract (3.0 g/L) Lactose (10.0 g/L) Crystal violet (0.002 g/L) Agar (15.0 g/L) pH after sterilization (7.3 ± 0.2)
Table 3.76 Composition of Vogel-Bonner (VB) Medium MgSO4.7H2 O (10.0 g/L) K2HPO4 (500.0 g/L)
Citric acid monohydrate (100.0 g/L) NaHNH4 PO4. 4H2 O (175.0 g/L)
Table 3.77 Composition of Willis and Hobb’s Medium with Neomycin Peptic digest of animal tissue (10.0 g/L) Sodium chloride (5.0 g/L) Neutral red (0.032 g/L)
Meat extract (10.0 g/L) Lactose (12.0 g/L) Agar (10.0 g/L) pH after sterilization (7.0 ± 0.2)
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Do not autoclave this medium as overheating is detrimental.
3.8.76 Vogel-Bonner (VB) Medium (Table 3.76) Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.77 W illis and Hobb’s Medium with Neomycin (Egg-Yolk Medium) Willis and Hobbs Medium Base is used for isolation and identification of Clostridium from food (Table 3.77).
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63
Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.78 Xylose Lysine Deoxycholate Agar Xylose-Lysine Deoxycholate Agar (XLD Agar) is a selective medium recommended for the isolation and enumeration of Salmonella typhi and other Salmonella species (Table 3.78). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
3.8.79 Yeast Extract Broth This medium is used for the detection, isolation, cultivation of yeasts including molds and other aciduric microorganisms (Table 3.79). Instructions for use: Add the ingredients in 1000 ml distilled water in a conical flask. Dissolve the ingredients completely by using hot plate or suitable instrument. Sterilize the medium by autoclaving.
Table 3.78 Composition of Xylose Lysine Deoxycholate Agar Yeast extract (3.0 g/L) Lactose (7.5 g/L) Sodium chloride (5.0 g/L) Sodium thiosulphate (6.8 g/L) Phenol red (0.08 g/L) Agar (15.0 g/L)
L-Lysine (5.0 g/L) Sucrose (7.5 g/L) Sodium deoxycholate (2.5.0 g/L) Ferric ammonium citrate (0.80 g/L) Xylose (3.5 g/L) pH after sterilization (7.4 ± 0.2)
Table 3.79 Composition of Yeast Extract Broth Yeast extract (5.0 g/L) Malt extract (3.0 g/L)
Peptic digest of animal tissue (5.0 g/L) Dextrose (10.0 g/L); pH after sterilization (6.2 ± 0.2)
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46. Krieg NR, Holt JG (1984) Bergey’s manual of systematic bacteriology, vol 1. Williams and Wilkins, Baltimore, p 1044 47. Likotrafiti E, Manderson KS, Fava F, Tuohy KM, Gibson GR, Rastall RA (2004) Molecular identification and anti-pathogenic activities of putative probiotic bacteria isolated from faeces of healthy elderly individuals. Microb Ecol Health Dis 16(2–3):105–112 48. MacFaddin JF (1985) Media for isolation-cultivation-identification-maintenance of medical Bacteria, vol 1. Williams and Wilkins, Baltimore 49. MacFaddin, JF (2000) Biochemical tests for identification of medical bacteria, 3rd edn. Lippincott, Williams & Wilkins, New York 50. Manual, HiMedia (1998) HiMedia Manual for microbiology laboratory practice:524 51. McFadden JF (1985) Biochemical test for identification of medical bacteria, 2nd edn. Williams and Wilkins, Baltimore, p 924 52. Microbiology, BD Bioscience, sparks Md (1998) 11th edn 53. Mueller JH, Hinton J (1941) Proc Soc Exp Biol Med 48:330 54. Murray PR, Baron JH, Pfaller MA, Jorgensen JH, Yolken RH (eds) (2003) Manual of clinical microbiology, vol 5, 8th edn, American Society for Microbiology, Washington, DC 55. Oberhofer TR (1985) Manual of non fermenting gram-negative bacteria. Chrchill Livingston, New York, p 154 56. Official Methods of Analysis of AOAC International (1995). Cuniff P (ed) 16th edn. AOAC International, Virginia 57. Pelczar MJ, Chan EC, Kreig MR (1986) Microbiology, 5th edn. McGraw Hill Book, New York 58. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Bacteriol Rev 35:171–205 59. Rappaport F, Henigh E (1952) J Clin Pathol 6:361 60. Scherrer R. Gram’s staining reaction, gram types and cell walls of bacteria 61. Simmons JS (1926) A culture medium for differentiating organism of typhoid-colon aerogenes group and for isolating certain fungi. J Infect Dis 39:209–2412 62. Singh D, Dimri AG, Goyal P, Chauhan A, Aggarwal ML, Chacko KM (2012) Microbiological evaluation of street-vended and restaurant’s food items. Curr Res Biol Pharm Sci 1(1):25–30 63. Vaughn RH, Osborne JT, Wedding GT, Tabachnick J, Beisel CG, Braxton T (1950) The utilization of citrate by Escherichia coli. J Bacteriol 60(2):119–127 64. Visintin S, Alessandria V, Valente A, Dolci P, Cocolin L (2016) Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa. Int J Food Microbiol 216:69–78 65. Voges O, Proskauer B (1898) Z Hyg Infekt 28:20 66. Wilson and Blair (1931) J Hyg 31:138
Chapter 4
Methods of Sterilization and Disinfection
4.1 Introduction Sterilization simply refers to eliminate all forms of life including viruses, bacteria, fungi and their spores from culture media or other equipments. Before inoculation of desired microbes, sterilization is done to ensure aseptic conditions as well as during subsequent handling of equipments or media; aseptic techniques are employed to prevent any undesired contamination. On the other hand, disinfection refers to the destruction of organisms that might cause disease or spoilage in food industries. Disinfection is usually done by chemicals and does not necessarily kill spores.
4.2 Methods for Sterilization There are several methods by which sterilization can be achieved and are as follows:
4.2.1 Heat Sterilization Use of heat denatures and coagulates vital proteins within the cell leading to disturbances in ‘Central-Dogma’ that results in cell death. Heat sterilization can be done by various means such as:
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_4
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4.2.2 Incineration Sterilization of inoculating wires or loops by holding them in a Bunsen flame (Red Heat sterilization) until they are red hot. This method is also applicable for the final disposal of laboratory wastes.
4.2.3 Moist Heat This process is also known as steam sterilization because steam with pressure is used to kill the microbes. This is highly efficient technique than that of other methods of sterilization such as dry heat. This method is applicable for the sterilization of culture media, aqueous solutions and even during the disposal of discarded cultures and contaminated materials. Autoclave (the technical version of a pressure cooker) is used for this purpose which follows automatic cycles of heating under pressure for the required time. During this process, air is removed in order to achieve the 121.1 °C temperature and 15 lb./in2 (psi) pressure of steam. The keeping time of material is about 15–20 minutes for successful sterilization; however, depending upon the load of material, this time can be increased to up to 30 minutes. Autoclaving for the purpose of disposal of contaminated materials, 60 minutes cycle is required. For successful autoclaving, the materials to be sterilized should be packed loosely. Under the conditions of autoclaving, all kind of microbes including spores will not survive.
4.2.4 Tyndalliztion This process is named after bacteriologist Tyndall and used to sterilize specific culture media that might be spoiled by exposure to higher temperature of autoclaving. Such media may contain carbohydrates or gelatin that can be easily hydrolysed. In this method, media are steamed at 100 °C for 30–45 minutes on each of 3 successive days with intermittent incubation. On the first day, vegetative bacteria are killed. Spores that survive will germinate during the subsequent incubation and produce vegetative cells that are killed during subsequent steaming. At the end of third day, media will be completely sterilized.
4.2.5 Dry Heat Steam heat sterilization has disadvantage for glassware due to its corrosive nature thus dry heat sterilization usually employs for materials which could either be corroded by steam or must remain dry before use, for example, metal instruments, glass
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petri dishes, flasks, pipettes and cotton wool. This process requires longer time intervals and higher temperatures than steam sterilization i.e. 160–170 °C for not more than 120 minutes.
4.2.6 Radiation Sterilization Heat-sensitive materials and environmental samples such as soil and sediment where structural changes caused by heat need to be avoided, radiations can be used for sterilization. This type of sterilization is also used in Bio-safety Cabinets, Laminar air flow chambers and clean rooms for providing aseptic conditions and to protect the operator from harmful microbes. Ultraviolet (UV) light cause the excitation of atoms and formation of thymine dimmers in nucleic acids that leads to fatal mutations. UV light cannot penetrate materials so is used mainly for surface treatments e.g. Laminar flow benches, and air and water. On the other hand, ionizing radiations have more penetration power causing ionization within cells. Gamma radiation generated through a Cobalt (Co60) source is used to sterilize complex matrices such as soil and foodstuff and even packaged materials like medical devices.
4.2.7 Filtration Sterilization Filtration sterilization operates through the exclusion rather than destruction of microorganisms. It is safe for the user and is employed for sensitive liquids and gases and some of media components which can’t withstand high temperature of autoclaving e.g. vitamin solutions. Different types of filters are used for this purpose. Depth filters which are made of columns packed with fibrous materials such as glass wool or cotton wool. The twisting and turning fibers entrap particles and so act as filters; they show little resistance to flow and are used mainly for gases or as pre-filters for membrane filters which are easily clogged. Membrane filters are most useful and act by screening out particles. Their effectiveness depends on the size of the membrane pores and the electrostatic attractions present. The most commonly used filters in microbiology are usually made of cellulose acetate or cellulose nitrate. Size of filter pores required to screen out the yeast cells are 0.45–1.2 μm, for bacteria 0.2 μm and for viruses & mycoplasmas 0.01–0.1 μm. Filtration is also employed for making air sterile in the working environment of clean rooms and bio-safety cabinets using pre-filters, fine filters and HEPA filters. HEPA are high efficiency particulate air filters which exclude the microorganism and let the sterile air comes in the clean room.
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4.3 Chemical Sterilization/Disinfection Several equipments such as optical instruments (Microscopes) and electrical devices are heat-sensitive and could be damaged by heat, therefore, chemical sterilization is recommended but due to the toxicity of the chemicals used, and this is not the most popular form of sterilization. Chemicals such as gaseous ethylene oxide, which alkylates amino, sulfhydryl, carboxyl and hydroxyl groups of microbial cell compounds; formaldehyde, used as a fumigant; and hydrogen peroxide vapour used in aseptic packaging. Some disinfectants cause health hazards thus it is advisable to wear protective coverings while using disinfectants, moreover, they should not be used to ‘sterilize’ materials when physical methods can be employed. Vegetative forms of microorganisms usually show the maximum susceptibility against the disinfectants while spores and some kind of viruses are resistant to these chemicals. The chemicals which are usually employs during the sterilization/disinfection in Microbiology laboratory are described as below:
4.3.1 Phenolics Highly effective chemicals against all kind of vegetative cells including Mycobacteria and fungi, however inactive against spores and viruses. Usually employs in discard jars and disinfection of surfaces but, not much effective in presence of rubber, wood and plastics and not compatible with cationic detergents. In the presence of large amounts of organic matter, this chemical should be used at higher concentrations i.e. 2–5% that of 1% which is usually employed in case of lesser organic matter. Fresh dilutions should be prepared daily and should not be stored for more than 24 h. Phenolic are harmful for eyes and skin, so proper precautions should be taken while using this chemical.
4.3.2 Hypochlorites This chemical releases chlorine which is highly effective against bacteria, fungi and spores but not useful in presence of proteins and plastics. They have corrosion properties; hence uses are limited to discard jars and surface disinfection only in approximately 2500 ppm concentration. This chemical decay fast and should be used within 24 h. It may cause irritation to eyes, lungs and skin.
4.3.3 Aldehydes Both gaseous (formaldehyde) and liquid (glutaraldehyde) are used as disinfectants and active against all kind of vegetative cells as well as spores. They remain active even in the presence of proteins, natural or synthetic materials or detergents.
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Formaldehyde along with potassium permanganate (KMnO4) is used for fumigation which effectively kills the microbes present in the air, however, this method is not much viable now-a-days as it adversely affects the eyes and respiratory system. 1:10 dilution of formalin (i.e. 4% formaldehyde) is used for surface cleaning in rooms and safety cabinets.
4.3.4 Alcohols During Microbiological practices, 70% ethanol or iso-propyl alcohol is prominently employed for cleaning and surface sterilization of equipments or working benches. They are most effective against vegetative bacteria than that of spores or fungi. They are relatively harmless to skin but may cause eye irritation. Special precautions should be taken at the time of their use in laminar air flow chamber or safety cabinets because of the simultaneous use of Bunsen flame inside these cabinets. Due to undesirable effects of disinfectants on the skin, eyes and respiratory system; proper PPEs such as disposable gloves and safety goggles, lab-coats, masks etc. should be worn by lab personnel while preparing the dilutions from stock solution of disinfectants.
4.3.5 Quaternary Ammonium Compounds (QAC) These cationic detergents are highly effective against most kind of microorganisms, however, can be inactivated in presence of plastic, proteins, anionic detergents or soaps. They have non-corrosive nature and have high stability upon dilution, thus, used at 1–2% dilution for cleaning. They are non-toxic and harmless to the skin and eyes hence more popular chemical disinfectants. Their preparation is used for fogging of air to make clean room contamination free during the microbiological practices.
4.3.6 Iodophores They are effective against all kind of microbes including spores and viruses but rapidly inactivated by proteins and not compatible with anionic detergents. These are diluted to 75–100 ppm iodine for surface disinfection and 1500 ppm as sporicide. Due to the presence to an indicator, they are brown or yellow in color which shows its activity i.e. they are active as long as remain color. They stain the skin which can be removed with sodium thiosulphate solution. They do not cause harm to skin but eye irritation can occur.
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References 1. Block SS (ed) (1991) Disinfection, sterilization and preservation, 4th edn. Lea and Febiger, Philadelphia 2. Block TD. Membrane filtration: a user’s guide and reference manual, Science 3. Grainger J, Hurst J, Burdass D (2001) Basic practical microbiology: a manual. The Society for General Microbiology. Marlborough House, Basingstoke road, Spencers wood, Reading RG 7, IAG, UK, pp 1–26 4. Kaushik P, Chauhan A (2009) Cyanobacteria: antibacterial activity. New India Publishing, New Delhi 5. McDonnell G, Russell AD (1999) Antiseptics and disinfectants: activity, action and resistance. Clin Microbial Rev 12(1):147–179 6. Microbiological control for non-sterile pharmaceuticals, monograph no. 2, pqg monograph no. 12 (2008) Pharmig and The Chartered Quality Institute, Pharmig, T5 The Maltings, Roydon road, Stanstead Abbotts, Hertfordshire SG12 8HG, UK 7. Russell AD (1990) Bacterial spores and chemical sporicidal agents. Clin Microbial Rev 3(2):99–119 8. Rutala WA, Weber DJ (1997) Uses of inorganic hypochlorite (bleach) in healthcare facilities. Clin Microbial Rev 10(4):597–610. Tech. Publishers, Madison, 1983
Chapter 5
Equipments and Instruments for Microbiological Laboratories
5.1 Introduction Equipments are essential part of any lab provided they are properly managed by Quality Assurance (QA) System. Equipment management, therefore, is of utmost importance to ensure that equipment is • • • • • •
properly installed, suitable for its intended purposes, user friendly, in a safe and serviceable condition, understood by users, meets safety and quality requirements. All these requirements are usually achieved by:
(a) proper selection, (b) justification of need, (c) acceptance upon delivery to ensure having all accessories and operating manuals and subsequent qualifications (IQ = Installation Qualification, OQ = Operational Qualification, PQ = Performance Qualification) revealing that it is undamaged and in proper working order. (d) proper training to users by trained personnel, (e) proper maintenance, repair, calibration and modification, (f) replacement and disposal, (g) Maintenance of a record having brief descriptions of acceptance, all inspections, maintenance, repair, calibration and any other modification.
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_5
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5.2 Major Equipments: Brief Description 5.2.1 Optical Microscope These optical instruments are required for visualizing the microorganisms like bacteria, yeast, molds as these can’t be seen by naked eyes. In lab, mostly bright-field microscopes are used in which dark image is seen against the brighter background. However, adjustment of diaphragm leads to dark-field observation i.e. bright image against dark background as in case of staining of curd for observation of Lactobacilli. Laboratory microscope often has eye piece of 10× magnification and objective lenses having 5×, 10×, 40× and 100× magnifications. For observation under 100× magnifications, a drop of immersion oil is usually applied over the microscopic slide. For better visualization, adequate illumination is required which is provided now-a-days in terms of artificial light source. For observation of microbial colonies on Petri-dishes, dissecting microscopes are also available. Special precautions should be taken while using the microscopes. Daily cleaning of lenses are also prerequisite for better imaging.
5.2.2 Water Bath Applicable for short-term incubation as it generates convection currents which keep the contents of the tube well mixed and accelerates reactions such as agglutination. These are also equipped with stirrer for homogenous heat transfer and thermostat to maintain the temperature. This instrument is also used to maintain the liquid state of molten culture media during the experiment to avoid the solidification.
5.2.3 Refrigerators These cabinets are specially designed to meet the low temperature requirements of the Microbiology lab such as storage of lyophilized vials (4–8 °C), mother cultures (−20 °C), stock cultures and working cultures of microorganisms. Various culture media, their ingredients and reagents are also required to be kept under refrigerated conditions as well as perishable samples such as water and food should also be refrigerated till their analysis and final disposal.
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5.2.4 Incubators Microorganisms need to be provided the proper and controlled conditions such as temperature, oxygen, humidity etc. in order to achieve optimal growth during their in vitro cultivation in culture media. Such controlled conditions are given in form of biological incubators. These equipments are equipped with thermostat which maintains the temperature of the cabinet. It is also having the circulation fan for homogenous and uniform circulation of air throughout the chamber. It is also available as BOD (Biological Oxygen Demand) incubator which provides the required amount of oxygen to the growing aerobic microbial cells for their optimal growth. For the growth of anaerobic bacteria, CO2 incubators are available that have an internal atmosphere of 5–8% CO2. Incubators should be kept separately in the laboratory where controlled temperature i.e. 25 ± 2 °C, relative humidity of 50 ± 10% and protection from sunlight should be provided. Incubators should never be overloaded with undesired materials such as Petri-dishes, flasks etc. and these items should be removed immediately after the completion of incubation period and observation of results. Since incubators are place where microorganisms are growing, there may be higher chances of their escape in the environment of incubators, therefore, regular cleaning of incubators is highly recommend using proper disinfectant.
5.2.5 Centrifuge These machines are based on the principle of centrifugal force to separate the components of any solution depending upon their sizes or density. They are also used to suspend the microbial cells growing in liquid culture media. General purpose centrifuge is capable of holding 15 ml and 50 ml tubes and usually operates at 4000 RPM (revolution per minute). Refrigerated centrifuges with changeable rotors are also available now-a-days. Swinging-bucket centrifuge is safer than the angle head because the former is less likely to distribute aerosols in uncapped tubes are used, however, for maximum safety, sealed buckets should be fitted. Lid of centrifuge should be closed during operation or spinning of rotors. Centrifuge tubes should be of identical lengths and thickness and should be used always in pairs, opposite to one another and to have approximately same quantity and weight to avoid ‘head wobble’ i.e. bursting during operation.
5.2.6 Biosafety Cabinets (BSC) These cabinets are specifically designed for carrying out the Microbiological analysis under complete aseptic conditions without the fear of contamination and at the same time, keep the analyst completely safe from undesired infections caused by
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microorganisms which may be present in the sample. These cabinets are equipped with HEPA (high efficiency particular air filters) having the pore size of 0.45 μm through which only clean air can pass and retain all organisms having the size greater than that of pore size of filter. Therefore, they are intended to capture and retain aerosols i.e. infectious organisms present in the air of cabinet. However, they are not meant to protect the worker from spillage and the poor techniques followed during the analysis. For better accuracy of these equipments, they should not be overloaded with unnecessary equipments and analysis should be done near the front and the analyst should avoid bringing hands out of the cabinet while working. The working surface and walls of the cabinet should be decontaminated on daily basis. Prior to start the analysis, the internal atmosphere of the cabinet should be sterilized by switch on the UV light for about 10–15 min. Precautions should be taken by switching off the UV before opening the cabinet and starting the analysis. Thereafter, proper cleaning and disinfection of the cabinet’s bench should be done by mopping it with the use of 70% isopropyl alcohol. Complete evaporation of alcohol takes place within 2–3 min, and then Bunsen burner should be flamed which will give additional advantage to maintain the sterility of internal environment of the cabinet. In between the analytical process, analyst should allow the settling of aerosols and intermittent cleaning by alcohol to avoid unnecessary contamination. After each set of analysis, analyst should also disinfect their hands and arms by using 70% isopropyl alcohol. In some of the modern Biosafety cabinets, Bunsen burners are not allowed as it may cause disturbance of airflow in the cabinet. Smoke Test is usually done to determine the presence and direction of air currents by burning a chemical such as Titanium tetrachloride (TTC) that produces a dense, visible vapour which responds to quite small air movements. Anemometer is used to measure airflow. Depending upon their application, they are of three types i.e. Class I, II and III. Class I cabinet retains aerosol by passing a current of air at the front of the cabinet i.e. vertical curtain and remove most of the organisms. The airflow is maintained between 0.7 to 1.0 m3/s ± 20% through the front of the cabinet. The clean air then passes through the fan, which maintains the airflow, and is exhausted to atmosphere, where any organisms that have not been retained on the filter are so diluted that they are no longer likely to cause infection if inhaled. Regular cleaning and replacement of filter, if required is also recommended. Class II cabinet is more complex in terms of its operation as 70% of the air is recirculated through filters and maintain the inside working area clean and sterile. Rest 30% of the air is exhausted to atmosphere and is replaced by a ‘curtain’ of room air, which enters at the working face. This prevents the escape of any particles or aerosols released in the cabinet. Class III cabinet is completely enclosed and prevent any leakage of particles into the room. These cabinets have designed in such a way so that air enters through filters and is exhausted to atmosphere through filters and have integral gloves with which analyst works.
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5.2.7 Hot-Air Oven Also known as dry-heat sterilizers, they are thermostatically controlled and fitted with circulating fans to ensure even temperatures in all parts of the equipment. It is usually required to sterilize the glass ware at 160–180 °C. Since air is not a good conductor of heat, therefore, ovens should not be overloaded and proper space should be there to allow the circulation of hot air in between the material to be sterilized. It usually works in three steps i.e. heating-up period which is the time, taken for the entire load to reach the sterilization temperature (approx. 1 hr); holding periods at different sterilization temperatures (160 °C or 180 °C) and cooling-down period carried out gradually to prevent glassware from cracking as a result of too rapid fall in temperature (approx. 2 hr). These equipments should be calibrated with thermocouples at regular intervals. Commercial chemical indicators should be used for its sterilization accuracy.
5.2.8 Autoclave These are the specific equipments utilized to carry out the steam heat sterilization using steam under pressure at approx. 121.1 °C temperature and 15 lb./in2 pressure for about 15–20 min. Laboratory bench autoclaves work like pressure cooker and have a metal chamber with a strong metal lid that can be fastened and sealed with a rubber gasket. It is equipped with an air and steam discharge tap, pressure gauge, safety valves and an electric immersion heater in the bottom of the chamber. During process, there must be sufficient water inside the chamber. The autoclave is then loaded, lid is closed, and safety valve is then adjusted to the required temperature and the heat is turned on. After boiling of water, steam will release inside the chamber which also lead to removal of air. After complete removal of air from the chamber, the steam pressure will rise inside the chamber until the desired pressure and temperature are reached. At this point, the process is held for 15 min to acquire complete sterilization following that heater is turned off and the autoclave is allowed to cool before opening the lid otherwise, glassware containing media or liquid may burst due to pressure. The efficiency of autoclaves is greatly affected by air and it works best if air is not present i.e. complete vacuum inside the chamber. However, in the presence of 50% air, the temperature of autoclave will be only 112 °C. Therefore, before commencing the sterilization process, air should be completely removed. Moreover, avoid overloading the autoclave and materials should be packed loosely for better accuracy of sterilization process and penetration of steam. Depending upon the load, sterilization time can be lengthened to ensure that this reaches the appropriate temperature. Proper precautions should be taken while operating autoclaves such as wearing full-face visors to avoid the harmful effects of steam and also wear thermal protective (asbestos) gloves.
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Autoclaved should be monitored on regular basis for their accuracy in terms of sterilization by either of three ways: (a) use of Bowie-Dick Autoclave Tape which is impregnated with a chemical. It is placed in the load. When adequate steam penetration is there, the color of tape will change. (b) use of Chemical Indicators which are in the form of sealed tubes or sachets and change colors in the presence of absolute time and temperature combination. (c) use of Biological Indicators i.e. spores of Bacillus sterothermophilus (ATCC 7935) and Clostridium sporogenes (ATCC 7955) are used as suspensions or adsorbed on carriers such as filter paper strips. These strips are placed at various locations in the load and after autoclaving, are inoculated to pre-sterilized broth media. Following incubation at appropriate temperature for given time period, any turbidity indicates unsuccessful processing.
5.2.9 Homogenizers, Blenders and Mixers These instruments are required for grinding, emulsifying and homogenizing the samples and require a fresh, sterile cup for each sample. They may release aerosols during operation. The metal cover should be decontaminated before and after use. However, in Stomacher, samples are homogenized in sterile plastic bags by the action of paddles. The bags are automatically sealed during the operation, hence, a little risk of aerosol dispersal. In this machine, large numbers of samples can be processed in a short time without pauses for washing and re-sterilizing.
5.2.10 Vortex Mixer It is useful device for mixing the contents of test tubes. These should be operated in Biosafety cabinets.
5.2.11 Weighing Balance In microbiology laboratory, weighing balance are mainly used for media preparation and weighing of standards and test samples used for the analysis. For most accurate measurement, weighing balances should be completely enclosed. For weighing test samples, the balances should be kept inside laminar and biosafety cabinets. For more accuracy clean the balances before and after use. Calibrate the balance as and when required. Always wear Gloves, Face mask and Apron while weighing the media taking care of the possible health hazards i.e. its carcinogenicity, disorders by inhalation of the media or allergic reactions etc.
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5.2.12 pH Meter A pH meter is an instrument used to measure the pH of media before and after sterilization. pH meter should be calibrated daily using buffer solution of different pH. Inspect the pH meter electrode for scratches, cracks, salt crystals build up or membrane/ junction deposits. Rinse off any salt build up with distilled water. Remove any membrane/ junction deposits. Drain the reference chamber, flush it with fresh filling solution and refill the chamber with fresh filling solution.
5.2.13 Colony Counting Device Colony counting device is used for counting of colonies on agar plates after incubation. Counting can be done manually. Normally, digital colony counting device are used in microbiology laboratory. Clean the surface of the instrument by dry cloth before and after every use.
5.2.14 Microwave Oven Microwave oven is used for melting solidified agar media for use (but not for sterilization).
5.3 Minor Equipments and Other Requirements 5.3.1 Bunsen Burners These are used for flame sterilization of inoculating loops, forceps, scissors, mouth of flask, test tubes etc. during the analysis. They are used in BSC Class I and II, and proper precautions should be taken care of while using burners such as no any flammable material should be present in the cabinet. While flaming the conical flasks or test tubes, cotton plugs should be kept at proper distance to avoid fire. Alcohol or other disinfectant should also be used while taking proper vigilance.
5.3.2 Inoculation Loops These loops are made up of Nicrome wire which at the end is in the form of a loop and used during the inoculation of microbial culture into the culture media by streak plate method. They should not be more than 5 cm long in order to minimize
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vibration and involuntary discharge of culture film. Loop size is usually kept at 3 mm diameter and should be completely closed so as to held fluid cultures in it. Aluminum holders are generally used to hold this wire and loop. Now-a-days, presterilized, ready-to-use, plastic disposable loops of 1 μl and 10 μl capacity, packed as single entity are also available.
5.3.3 Inoculation Needle Used for Inoculation from very small colonies or for transfer of small inocula from liquid media.
5.3.4 Forceps (Stainless Steel) Multipurpose uses such as transfer of sterile paper or antibiotic discs.
5.3.5 Spreaders They are used to spread the microbial inoculum by spreading during the spread plate method. They are usually made up of glass rods and L-shaped but pre- sterilized, ready-to-use, plastic spreaders are also available.
5.3.6 Microscopic Slides and Cover Slips Glass slides are generally used to prepare the smear of microbial culture to observe its microscopic structure under the microscope. Cover slips are used during preparation of permanent slides. The most convenient cover slips are of thickness grade No. 1 and size 16 mm2.
5.3.7 Pasteur Pipettes These pipettes are used to transfer liquid cultures and among the most dangerous lab item. Rubber teats should be used for better application of Pasteur pipettes. They are used once only and attempts to recycle them may result in cuts and pricks to fingers.
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5.3.8 Graduated Pipettes These straight-sided pipettes are made up of glass having 1-10 ml capacity. They must be cotton plugged at the suction end to prevent entry of microbes from teat or pipettor. Pipettes should be wrapped with aluminum foil before being sterilized in the hot air oven. Disposable pipettes are also available. Mouth Pipetting is Strongly Prohibited in All Microbiological Practices.
5.3.9 Glassware and Plasticware Soda-glass bottles and tubes are used for ordinary microbiological work, however, borosilicate glass is strongly recommended for most of the analytical work related to Microbiology so as to withstand the repeated sterilization process and longer use. Disposable and autoclavable plastic material is also available for routine analysis. Autoclavable plastics such as polypropylene, polycarbonate, nylon, polytetrafluororethylene (PTFE or Teflon), polyallomer, methylpentene polymer (TPX), vinyl tubing etc. withstand the autoclave conditions. Some plastics soften during autoclaving and may become distorted if not packed properly.
5.3.10 Petri-dishes They are used for in vitro cultivation of microbes by pour plate, spread plate or streak plate methods and are available in various sizes i.e. 50 mm, 90 mm, 150 mm etc. 90 mm Petri-dish is mostly used type for routine microbiological analysis. Borosilicate glass Petri-dishes are thin, easy to handle, with flat top & bottom, scratch-free during repeated use and stack safely without the fear of fall down but are fragile, expensive and must be washed with care. Thick pressed glass Petri- dishes are convex, scratched with use and washing, cannot stacked safely but are cheap and not fragile. Glass Petri-dishes are usually sterilized by hot air after wrapping in aluminum foil. Disposable plastic Petri-dishes which are pre-sterilized, packed individually or in batches in polyethylene bags, cheaper, can be stored for longer period of time without any deterioration and easy to handle, are now commonly used throughout the world.
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5.3.11 Test Tubes and Bottles Test tubes are very common glassware being used in most of the laboratories. They are easier to handle and require less space in storage containers and incubators. However, culture media may dry up during storage in the test tubes. Therefore, bottles are more convenient for longer storage of culture media. Test tubes are available in various sizes. Rimless test tubes are best suited for microbiological work. Cotton plugs are used.
5.3.12 Conical Flask Large volumes of liquid media for inoculation and liquid/media for short-term storage (non-absorbent cotton wool plug prevents contamination).
5.3.13 Marker Pen Labeling Petri dishes, test tubes, flasks, bottles and microscope slides.
5.3.14 Personal Protective Equipments (PPE) Lean laboratory coat/apron: protection of clothing, containment of dust on clothing; Safety spectacles: for dealing with chemicals, gloves, masks, head caps etc.
5.3.15 Autoclavable/Roasting Bag Holds contaminated items in autoclave to avoid spillages.
5.3.16 Thermometer Checking incubator/water bath temperatures.
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5.3.17 Spillage Kit Dealing with spilled cultures.
5.3.18 Disinfectants Treatment of work surface before and after use and spillages; disposal of used pipettes and microscope slides; in soap form for hand washing.
5.3.19 Ethanol (70% Industrial Methylated Spirit) Sterilization of metal forceps and glass spreaders by ignition, also Surface sterilization of bio-safety cabinets and laminar air flow benches.
5.3.20 Autoclave Indicator Tape Changes color in response to heat to distinguish those items that have received heat treatment.
5.3.21 Sterilizer Control Tube/Strip Changes color when correct temperature has been applied and held for the required length of time to effect sterilization.
5.3.22 Non-absorbent Cotton Wool Used to Plugs test tubes, flasks and pipettes to avoid contamination.
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5.4 Equipments: Quality Aspects Most of the laboratory exercises require complex automated and costly equipments and any error in these equipments may lead to false results, hence, regular monitoring and maintenance are of utmost importance to ensure the best quality of results. Proper monitoring of the equipments also saves costly repairs and possibility of equipment to be ‘out of order’. This can be accomplished by applying following: 1 . Regular cleaning of the equipments. 2. Proper and effective training to workers 3. Assigning the trained staff members for complete accountability for maintenance 4. Display of list of trained staff members for trouble-shooting 5. Daily monitoring and recording of the key aspects like temperature, humidity etc. 6. Calibration at proper interval by authorized external agency. 7. Intermediate check at proper interval 8. Preparation of Standard Operating Procedures (SOP) (a) Important information of equipment such as details, operation, maintenance, calibration frequency etc. (b) A well-structured manual, organized and indexed, will allow a microbiologist from one laboratory to perform any of the tests described any other. (c) It should be prepared in clear, concise and explicit descriptions of procedures by experienced senior member of the lab, and then verified and approved by other senior members of the organization. (d) Avoid unnecessarily complicated procedures and the possibility of staff commencing unauthorized short-cuts. (e) SOP should be place near to the relevant equipment and should be easily available to all relevant personnel. (f) SOP should have an ‘Issue Date’. This is the date on which SOP is issued to the concerned person of lab. (g) It is followed by ‘Effective Date’ i.e. the date on which implementing of SOP will start. Time interval between Issue and Effective dates is approximately 15 days during which the technical contents of SOP should read carefully by the authority and carry out modifications, if required. (h) Revision of the SOP is another important process and carries out either annually or once in two years. ‘Revision Date’ is also mentioned on SOP for such purpose. (i) Amendments can be done as and when required and reason for revision should also be mentioned. (j) Only designated personnel should be permitted to modify a SOP and should have approval from the quality manager. (k) It is the responsibility of senior staff to ensure that it is used and strictly adhered to.
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References 1. Grainger J, Hurst J, Burdass D (2001) Basic practical microbiology: a manual. The Society for General Microbiology, Reading, pp 1–26 2. Kaushik, P. 2000. Introductory microbiology. Emkay Publications, Post Box 9410, B-19 East Krishna Nagar, Swami Dayanand Marg, Delhi. pp VIII+1–46 3. Kaushik P, Chauhan A (2009) Cyanobacteria: antibacterial activity. New India Publishing, New Delhi 4. Butson P, Hawitt K (2008) Microbiological control for non-sterile pharmaceuticals, monograph no. 2, pqg monograph no. 12, Pharmig and The Chartered Quality Institute, Pharmig, T5 The Maltings, Roydon Road, Stanstead Abbotts, Hertfordshire SG12 8HG, United Kingdom
Chapter 6
Methods of Sampling for Environment, Food and Pharmaceutical Analysis
6.1 Introduction Sampling is an important aspect of microbiological experiments as it can influence the results significantly; therefore, sampling of any product which must be analyzed in the laboratory is done only by an expert of Microbiological practices and in a scientific manner. Collection of sample or sampling is a scientific method which needs to be practiced in efficient approach to avoid the possible contamination of undesired microorganisms which can, otherwise, interfere with the actual results. Following aspects should be taken into consideration while sampling: • Withdrawn samples should always have to be representative of the lot/batch. • Site characteristics and environmental factors should have to be recorded which will help during the interpretation of results later on. • Personnel protective equipments (PPEs) such as lab coat, face masks, sterile gloves, sterile caps etc. should be used while taking the samples. • Sample should be taken in pre-sterilized containers. • Sample format should be filled in triplicate with all relevant details like quantity of samples, location of sample, sampling date and signature of microbiologist and authorized persons under whose presence sample has been taken. The present section, therefore, dealt with the detailed procedures of sampling for air, water and drug & pharmaceutical products based on the author’s direct experiences of several years of industry.
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_6
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6.2 Method for Water Sampling ‘Water is life’ and this is also true for microbes. A diverse kind of microflora may be present in the water depending upon the type of water-bodies such as fresh water, saline water, potable water, sewage water, packaged water, mineral water etc. Since a number of infections are caused by water-borne microorganisms, the waters particularly the drinking water should be detected for the presence of such microbes. Absolute detection of microbes is, therefore, depends upon the scientific mode of sampling of water so as to avoid the undesirable contamination due to outside microbes. The following aspects should be taken care of while doing the sampling of drinking water: (a) Use sterilized polypropylene or glass bottle for sampling. (b) Sterilization of bottles can be done by using 70% isopropyl alcohol (IPA) or by exposing them with proper dose of gamma radiation. (c) Before sampling, wear personal protective equipment including gloves, mask, lab coat and cap. (d) Always keep ice box with ice pack or ice cubes and marker during sampling. (e) Firstly, wipe out the mouth of taps with 70% IPA and then let the water flow for a few seconds without any disturbance in the flow. (f) Take the bottle beneath the running tap in such a way so that water flow inside the bottle. While doing this, ensure that water does not flow outside the bottle. (g) The water should not touch the top edge of the bottle, there should be a gap of 2–3 cm. (h) Cap the bottle tightly and then wrap with aluminum foil. (i) Keep the bottle in ice box under cooling condition and bring the sample to the laboratory. Sample should reach to the laboratory within 3–4 hrs of sampling. (j) Record all the relevant information like location and quantity of sample, date of sampling etc.
6.3 Method for Soil Sampling There are various kind of microorganisms (Bacteria, actinomycetes and fungi etc.) present in the soil. Each organism has a different role i.e. beneficial as well as detrimental. Keeping in view the importance of sampling in qualitative and quantitative microbial analysis, the following aspects should be taken care of while doing the sampling of soil and surface sediment: (a) Use sterilized polypropylene or glass bottle for sampling. (b) Sterilization of bottles can be done by using 70% isopropyl alcohol (IPA) or by exposing them with proper dose of gamma radiation. (c) Before sampling, wear personal protective equipment including gloves, mask, lab coat and cap.
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( d) Always keep ice box with ice pack or ice cubes and marker during sampling. (e) Firstly, wipe out the mouth of taps with 70% IPA and then take the desired amount of soil using sterile spatula. (f) Cap the bottle tightly and then wrap with aluminum foil. (g) Keep the bottle in ice box under cooling condition and bring the sample to the laboratory. Sample should reach to the laboratory within 3–4 hrs of sampling. (h) Record all the relevant information like location and quantity of sample, date of sampling etc.
6.4 Method for Air Sampling Air is full of microorganisms including vegetative cells as well as spores of certain kind of bacterial and fungal species. Air sampling simply refers to the collection of air-borne microorganisms which upon contamination, may impact spoilage of products pertaining to the safety and quality issues. Several air-borne microbes are also responsible for severe human infections. Therefore, the absolute sampling is required to know the kind of microflora present in the air. Two methods pertaining to collection of microbes’ i.e. vegetative cells and spores are practices throughout.
6.4.1 Passive Method A settle plate method is a method in which sterile solidified agar plates are exposed in air at a certain time interval at different locations and allow the air carrying microbes to settle down on to the agar surface. The locations for settle plates can be one or more and are selected on the basis of the position of door, air inlet and air outlet. Upon incubation, these microbes grow in the form of colonies which can be counted using colony counter. In case of specified microorganisms, enrichment and selective media can be used and further biochemical tests can reveal the possible presence or absence of pathogens.
6.4.2 Active Method This method is based on the devices and the volume of the air passing through the device which further depends on the device being used. During active sampling, generally the instrument is kept at the center of room whereas if the area is too big, two or more location can be selected. In this case, the average count of these locations is taken at the time of reporting. Before starting the instrument for sampling wipe out the cover with 70% isopropyl alcohol and then place the plates for
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exposure. Care should be taken while put on and put off the plates. All the exposed plates must be transported in the laboratory within 3–4 hrs under cooled condition using ice-box so as to avoid undesirable growth of microorganisms.
6.5 Method for Food and Food Products Sampling Food is full of nutrients and a greatest source for microbial growth. Depending upon the nature of food like perishable or non-perishable, shelf life of food can be decided. Food sampling is highly critical in terms of nature of food as inaccurate sampling can lead to undesirable contamination and hence, the false positive results for the detection of specified pathogen. Therefore, it is recommended to withdraw food samples such as cooked food or packed food by an experienced technical person. The following procedure can be opted for best possible food sampling: 1. Take suitable polypropylene or PET (polyethylene terephthalate) sample container of appropriate volume and pack it in a poly bag and then seal it by using sealer machine. 2. Sterile the container either by 70% IPA or expose with proper dose of gamma radiation. 3. Before sampling, wear personal protective equipments including gloves, mask, lab coat and cap. 4. Always keep ice box with ice pack or ice cubes and marker during sampling. 5. At first, examine and note down the surrounding location where food container (from which you are going to withdraw the sample) are kept. 6. Using all the personnel protective equipments, remove the container from pack. 7. Mark the sample container with name of sample, date of lifting, location, initial of officer etc. before withdrawing the sample. 8. Sterilize the spoon (for sampling) with 70% IPA followed by evaporation of alcohol by keeping it standstill for a few seconds. 9. Open the food container and mix the food with pre-sterilized spoon. 10. Withdraw the defined quantity of sample in the sample container. Immediately close this and keep in ice box. 11. Bring the samples in the laboratory for analysis within 3–4 hrs of sampling.
6.6 Method for Pharmaceutical Products Sampling Pharmaceuticals preparations such as active pharmaceutical ingredients (API) are highly crucial samples received in the Microbiology laboratory for sampling and for further analysis like sterility assurance and detection of bacterial endotoxin. Sampling of APIs is vital in order to maintain the sterility of the entire bulk product; therefore, special attention should be paid off. Sampling of API should be
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carried out under strict aseptic conditions of clean room and biosafety cabinet. During sampling, pre-sterilized vial and lid should be used. Container of API should be wiped with 70% IPA prior to opening. With the help of rotator device take out the sample and then fill the vials. This is followed by sealing of vials with the help of sealer. After taking the sample, close the lid of container and seal it hermetically with sterile tape. Mark the vials for their identification information. Use these vials for experiments or store under ambient conditions until use.
References 1. Chauhan A, Goyal P, Verma A, Jindal T (2015) Microbiological evaluation of drinking water sold by roadside vendors of Delhi, India. Appl Water Sci. https://doi.org/10.1007/ s13201-015-0315-x 2. Eaton AD, Clesceri LS, Greenberg AW (eds) (2005) Standard methods for the examination of water and wastewater, 21st edn. APHA, Washington, DC 3. Indian Pharmacopoeia (2018) Govt. of India, Ministry of Health and Family Welfare, New Delhi, India 4. IS 1622 (1981) Methods of sampling and microbiological examination of water (first revision). Bureau of Indian Standards, Manak Bhavan, 9 Bhadur Shah Zafar Marg, New Delhi 110002 5. IS 5404 (1984) Methods for drawing and handling of food samples for microbiological analysis. Bureau of Indian Standards, Manak Bhavan, 9 Bhadur Shah Zafar Marg, New Delhi 110002 6. ISO 19458 (2006) Water quality – sampling for microbiological analysis 7. Kaushik P (2000) Introductory microbiology. Emkay Publications, Post Box 9410, B-19 East Krishna Nagar, Swami Dayanand Marg, Delhi, pp VIII+1–46 8. Kaushik P, Chauhan A (2009) Cyanobacteria: antibacterial activity. New India Publishing, New Delhi 9. STANDARD B, ISO B (2006) Water quality – sampling for microbiological analysis 10. United States Pharmacopoeia (2019) The United States Pharmacopoeial Convention Inc., Rockville
Chapter 7
Microbiological Methods for Water, Soil and Air Analysis
7.1 Introduction Microbiological examination of water is used to monitor and control the quality and safety of various types of water including potable water i.e. water intended for drinking or use in food preparation, treated recreational water such as swimming pools & spa pools and untreated water used for recreational purposes such as sea, river, and lake water. This can be achieved by proper protection and sanitation of water resources, and suitable treatment strategies and by inspecting and maintaining the water distribution system. Entry of pathogens into water sources is also one of key strategy in this regard. Fecal contamination of water is also a significant cause for various infections caused by pathogenic bacteria, fungi, viruses, protozoa, helminths etc. If indicator microorganisms are detected in a substance, it indicates the presence of fecal contamination and therefore possible presence of pathogenic microorganisms in the water. Indicator microorganisms are tested for because they are easier and cheaper to test for than all the possible pathogens that might be present. The most common indicators are total coliform bacteria, fecal coliforms, and Escherichia coli (E. coli). It is very important to note the presence of coliforms, fecal coliforms, or even Escherichia coli in water do not indicate the presence of pathogenic microorganisms but do suspect them. Presence of coliform or fecal coliform bacteria does not determine whether a sample will cause disease. Although, there are a number of potential pathogens which are associated with water; indicator organisms especially of fecal origin such as coliforms and E. coli, have been used as markers of risk. Other species of microorganisms such as enterococci, Clostridium perfringens, Klebsiella, Enterobacter, and Citrobacter are also used in water testing. The fecal coliform test was developed as a marker of fecal pollution when Salmonella typhi was the commonest known cause of waterborne diseases. The
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 A. Chauhan, T. Jindal, Microbiological Methods for Environment, Food and Pharmaceutical Analysis, https://doi.org/10.1007/978-3-030-52024-3_7
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marker most closely associated with illness was the enterococci count, although fecal coliforms were also independently associated with illness. Total coliforms and total counts were not independently associated with illness. This section pertains to the evaluation of water, soil and air for various microbiological parameters. The methods presented here have been written in a manner that they can be used for analysis of water, soil and air without any problems; hence, it may be noted that some of the methods may be used for other materials as well whereas there are certain methods meant to be used only for water. Thus, when water, soil and air is to be examined for microbiological parameters, all the methods here must be adopted as described.
7.2 M ethod for the Determination of Most Probable Number (MPN) of Coliforms in Water Sample 7.2.1 Method Overview Coliform bacteria are Gram-negative, lactose-fermenting, non-spore forming, indicator organisms belonging to family Enterobacteriaceae and denote the quality of water in terms of microbial contamination. These organisms might be pathogenic, thus proper precautions should be taken while handling the samples. This method describes the detailed procedure to determine the most probable number (MPN) of Coliforms in water which is calculated based on positive tubes containing acid and gas. When well-shaken water sample is inoculated in test tubes having MacConkey broth in both single and double strengths, coliforms grow and cause lactose fermentation that leads to production of lactic acid and Co2 gas, hence, color of media turns to yellow and gas appears in Durham’s tube, respectively. Based on these positive tubes, MPN is calculated by using MPN Table. Results are reported as ‘Probable Number of Coliforms per 100 ml’ by using MPN Table.
7.2.2 Scope of the Method This method is applicable for the analysis of water and wastewater samples such as drinking water, swimming pool water, packaged drinking water, reverse osmosis (RO) water, ground water, underground water, effluent and wastewater, water used for manufacturing of food and other industry products etc.
7.2 Method for the Determination of Most Probable Number (MPN) of Coliforms…
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7.2.3 Experimental Requirements (Table 7.1)
7.2.4 Experimental Procedure 7.2.4.1 Preparation of Media (a) Depending upon the number of samples to be tested, weigh accurately the appropriate quantity of media as per manual instructions and mix it properly in required volume of distilled water using conical flask of suitable size. (b) Usually the capacity of conical flask should be double than that of quantity of media to be prepared. (c) Dissolve all ingredients properly using the water bath or hot plate or heating mantle. (d) Check the pH of media and adjust using 0.1 N NaOH/ 0.1 N HCl, if required. (e) Plug the conical flask with non-absorbent cotton. (f) Wrap the cotton plug with aluminum foil followed by pasting of indicator tape to ensure sterilization. (g) Mark the glassware with name of media, date of preparation and initial of Microbiologist.
Table 7.1 List of materials, equipment and instruments Culture media, reagents and others MacConkey broth MacConkey agar Brilliant green bile lactose Dettol solution (1:40) (BGBL) broth Lactose broth Durham’s tube E. coli as ‘positive S. aureus as ‘negative control’ control’ Equipment’s & instruments Weighing balance pH meter Clean room Biosafety cabinets/ laminar air flow chamber Hot-air oven Test tubes Microscope Petri-dishes Quebec Colony counter Inoculation loop Vortex Bunsen burner/sprit lamp Face mask Lab coat
Nutrient agar Isopropyl alcohol (70%) Absorbent and non-absorbent cotton/ butter paper/tissue paper 0.1 N NaOH/ 0.1 N HCl
Autoclave Incubators Micropipettes Conical flasks Microscopic slides Gloves Autoclave chemical Indicator tape/ marker
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7.2.4.2 Sterilization of Media Place all the media in an autoclave and sterilize it at 121 °C for 15 minutes. After sterilization, take out the media by using asbestos gloves and keep at appropriate condition till the analysis. 7.2.4.3 Growth Promotion Test (Media Performance Test) New batch of culture media should be evaluated for their ‘growth promotion ability’ both qualitatively and quantitatively as applicable with specific bacterial culture prior to use for routine analysis. 7.2.4.4 Presumptive Test Initial test for possible presence of Coliforms. (a) Homogenize the water sample by shaking it thoroughly for twenty- five times. (b) Prepare three sets of MacConkey broth (MB), each set having five test tubes. All test tubes should have inverted Durham’s tubes. (c) First set of five test tubes will be having the double-strength MB which is prepared using the double quantity of dehydrated MB in appropriate volume of distilled water. On the other hand, the other two sets having single-strength MB is prepared by using the prescribed quantity. (d) Inoculate 10 ml homogenized sample in each of five tubes from first set having 10 ml of double-strength MB. (e) Inoculate 1.0 ml homogenized sample in each of five tubes from second set having 10 ml of single-strength MB. (f) Inoculate 0.1 ml homogenized sample in each of five tubes from third set having 10 ml of single-strength MB. (g) Incubate all above tubes at 37 ± 1 °C for 48 hrs. (h) Following incubation, observe all the tubes for acid formation (i.e. yellow in color) and gas production (i.e. bubble in Durham’s tube). (i) Consider all the tubes as ‘Positive’ in which acid and gas is observed. (j) Count the number of Positive tubes in all three sets of Double and Single strength media. (k) If no acid and gas is observed in any of the three sets of tubes, discontinue the test and record the results as less than 2 organisms/100 ml. 7.2.4.5 Confirmed Test This test is performed in continuation of presumptive test in order to confirm the observations of positive tubes.
7.2 Method for the Determination of Most Probable Number (MPN) of Coliforms…
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(a) Inoculate a loopful inoculum from each individual positive tube into 10 ml Brilliant Green Bile Lactose (BGBL) broth in three sets, respectively. (b) Incubate all BGBL tubes at 37 ± 1 °C for 48 hrs. (c) Observe the tubes for gas production i.e. bubble formation in Durham’s tube and refer these tubes as ‘Positive’. (d) Record the number of positive BGBL tubes in all three sets and proceed for the completed test. (e) In case gas production is not observed in any of the tube, discontinue the test and record the result as less than 2 organisms/100 ml. 7.2.4.6 Completed Test This is the final step in order to confirm the presence of Coliforms in given water sample. (a) Streak loopful inoculum from all positive BGBL tubes on MacConkey agar plates and Incubate all the plates at 37 ± 1 °C for 24 hrs. (b) Observe the plates for ‘pink colonies’. (c) Inoculate 10 ml Lactose broth having inverted Durham’s tube with typical or atypical colonies as observed above and incubate at 37 ± 1 °C for 24–48 hrs. (d) At the same time, also streak the above colonies on Nutrient agar (NA) slant followed by incubation at 37 ± 1 °C for 24 hrs. (e) Following incubation, observe the lactose broth tubes for gas formation. (f) Also perform Gram staining by taking the inoculum from NA slant. (g) Consider the test as completed for the presence of Coliforms, if organisms observed after staining are Gram negative, non-spore forming, rod shaped and if gas is produced in lactose broth (Figs. 7.5 and 7.6).
7.2.5 Interpretation and Expression of Results Based on number of positive tubes from all three sets of BGBL tubes as recorded in confirmed test, calculate the ‘Most Probable No. of Coliforms/100ml of Water Sample’ using the MPN table (Table 7.2) and record the results as ‘Number of MPN Coliforms/100 ml’ (Figs. 7.1, 7.2, 7.3 and 7.4).
7.2.6 Quality Control Check the laboratory records before conducting the experiments. While performing the experiment, quality control is achieved by running simultaneously, E. coli as ‘Positive Control’ and S. aureus as ‘Negative Control’. Three controls (i.e. media
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Table 7.2 MPN Table when five tubes are used per dilution and 95% confidence limit for various combinations
S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
Combination of positive tubes 0-0-0 0-0-1 0-1-0 0-2-0 1-0-0 1-0-1 1-1-0 1-1-1 1-2-0 2-0-0 2-0-1 2-1-0 2-1-1 2-2-0 2-3-0 3-0-0 3-0-1 3-1-0 3-1-1 3-2-0 3-2-1 4-0-0 4-0-1 4-1-0 4-1-1 4-1-2 4-2-0 4-2-1 4-3-0 4-3-1 4-4-0 5-0-0 5-0-1 5-0-2 5-1-0 5-1-1 5-1-2 5-2-0 5-2-1 5-2-2
MPN index per 100 ml