Food Microbiology Protocols (Methods in Biotechnology, 14) 9780896038677, 089603867X

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Food Microbiology Protocols

M E T H O D S

I N

B I O T E C H N O L O G Y™

Food Microbiology Protocols Edited by

John F. T. Spencer and

Alicia L. Ragout de Spencer Planta Piloto de Procesos Industriales Microbiologicos, San Miguel Tucumán, Argentina

Humana Press

Totowa, New Jersey

© 2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Biotechnology™ is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ' ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Patricia F. Cleary. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected], or visit our Website at www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-867-X/01 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Main entry under title: Food microbiology protocols. Methods in molecular biology™. Food microbiology protocols./edited by John F. T. Spencer and Alicia L. Ragout de Spencer. p. cm.—(Methods in biotechnology; 14) Includes bibliographical references and index. ISBN 0-89603-867-X (alk. paper) 1. Food—Microbiology. I. Spencer, John F. T. II. Ragout de Spencer, Alicia L. III. Series. QR115.F658 2001 664'.001'579—dc21 00-021718

Preface Two of the recent books in the Methods in Molecular Biology series, Yeast Protocols and Pichia Protocols, have been narrowly focused on yeasts and, in the latter case, particular species of yeasts. Food Microbiology Protocols, of necessity, covers a very wide range of microorganisms. Our book treats four categories of microorganisms affecting foods: (1) Spoilage organisms; (2) pathogens; (3) microorganisms in fermented foods; and (4) microorganisms producing metabolites that affect the flavor or nutritive value of foods. Detailed information is given on each of these categories. There are several chapters devoted to the microorganisms associated with fermented foods: these are of increasing importance in food microbiology, and include one bacteriophage that kills the lactic acid bacteria involved in the manufacture of different foods—cottage cheese, yogurt, sauerkraut, and many others. The other nine chapters give procedures for the maintenance of lactic acid bacteria, the isolation of plasmid and genomic DNA from species of Lactobacillus, determination of the proteolytic activity of lactic acid bacteria, determination of bacteriocins, and other important topics. A substantial number of the chapters deal with yeasts, microorganisms which, after all, have also been associated with human foods and beverages for many thousands of years. The emphasis in Food Microbiology Protocols is on techniques for the improvement of methods for yeast hybridization and isolation, and for improvement of strains of industrially important yeasts, to be used in food and beverage production. For instance, the chapters by Katsuragi describe techniques for isolation of hybrids obtained by protoplast fusion and conventional mating, by the use of fluorescent staining, and by separation using flow cytometry. Other chapters discuss the identification of strains by analysis of mitochondrial DNA and other techniques. There are chapters on the isolation of strains of starches used in the production of human foods, and an important chapter on obtaining and isolating thermotolerant strains for the high temperature production of beverage and industrial alcohol. Finally, there are methods for the production of polyhydroxy alcohols for low-calorie sweeteners. The material on yeasts overlaps only slightly with that in the excellent book, Yeast Protocols, edited by Ivor H. Evans, so investigators interested in industrial yeasts should avail themselves of both volumes.

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The chapters on spoilage organisms and pathogens include valuable information on the isolation and identification of most important species in these areas. Several of these are concerned with bacteria, yeasts, and molds, causing spoilage of poultry products, as well as causing disease in humans. Methods for identification by molecular biology techniques and by conventional plate counts are given. There are two reviews on topics of immediate interest. Finally, the editors and the publishers would like to thank all those authors who gave so freely of their time and energy in preparing these chapters. The editors wish especially to thank Dr. Faustino Siñeriz, Director of PROIMI, for allowing us to use the facilities at PROIMI in the preparation of this book, and for his kind encouragement in the work at all times. We also thank Dr. María E. Lucca for her able assistance in correcting the final version. John F. T. Spencer Alicia L. Ragout de Spencer

Contents Preface ............................................................................................................. v Contributors ..................................................................................................... ix

PART I SPOILAGE ORGANISMS 1 Psychrotrophic Microorganisms: Agar Plate Methods, Homogenization, and Dilutions Anavella Gaitán Herrera ........................................................................ 3 2 Biochemical Identification of Most Frequently Encountered Bacteria That Cause Food Spoilage Maria Luisa Genta and Humberto Heluane ...................................... 11 3 Mesophilic Aerobic Microorganisms Anavella Gaitán Herrera ...................................................................... 25 4 Yeasts and Molds Anavella Gaitán Herrera ...................................................................... 27 5 Coliforms Anavella Gaitán Herrera ...................................................................... 29 6 Genetic Analysis of Food Spoilage Yeasts Stephen A. James, Matthew D. Collins, and Ian N. Roberts .......... 37

PART II PATHOGENS 7

Conductimetric Method for Evaluating Inhibition of Listeria monocytogenes Graciela Font de Valdez, Graciela Lorca, and María Pía de Taranto ....................................................................... 55

8 Molecular Detection of Enterohemorrhagic Escherichia coli O157:H7 and Its Toxins in Beef Kasthuri J. Venkateswaran ................................................................. 61 9 Detection of Listeria monocytogenes by the Nucleic Acid SequenceBased AmplificationTechnique Burton W. Blais and Geoff Turner ..................................................... 67 10 Detection of Escherichia coli O157:H7 by Immunomagnetic Separation and Multiplex Polymerase Chain Reaction Ian G. Wilson ........................................................................................ 85

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11 Detection of Campylobacter jejuni and Thermophilic Campylobacter spp. from Foods by Polymerase Chain Reaction Haiyan Wang, Lai-King Ng, and Jeff M. Farber ................................ 95 12 Magnetic Capture Hybridization Polymerase Chain Reaction Jinru Chen and Mansel W. Griffiths ................................................ 107 13 Enterococci Anavella Gaitán Herrera .................................................................... 111 14 Salmonella Anavella Gaitán Herrera .................................................................... 113 15 Campylobacter Anavella Gaitán Herrera .................................................................... 119 16

Listeria monocytogenes Anavella Gaitán Herrera .................................................................... 125

PART III FERMENTED FOODS 17 Methods for Plasmid and Genomic DNA Isolation from Lactobacilli M. Andrea Azcárate-Peril and Raúl R. Raya ................................... 135 18 Methods for the Detection and Concentration of Bacteriocins Produced by Lactic Acid Bacteria Sergio A. Cuozzo, Fernando J. M. Sesma, Aída A. Pesce de R. Holgado, and Raúl R. Raya ....................... 141 19 Meat Protein Degradation by Tissue and Lactic Acid Bacteria Enzymes Silvina Fadda, Graciela Vignolo, and Guillermo Oliver ................ 147 20 Maintenance of Lactic Acid Bacteria Graciela Font de Valdez .................................................................... 163 21 Probiotic Properties of Lactobacilli: Cholesterol Reduction and Bile Salt Hydrolase Activity Graciela Font de Valdez and María Pía de Taranto ....................... 173 22 Identification of Exopolysaccharide-Producing Lactic Acid Bacteria: A Method for the Isolation of Polysaccharides in Milk Cultures Fernanda Mozzi, María Inés Torino, and Graciela Font de Valdez ................................................................ 183 23 Differentiation of Lactobacilli Strains by Electrophoretic Protein Profiles Graciela Savoy de Giori, Elvira María Hébert, and Raúl R. Raya ................................................................................... 191

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24 Methods to Determine Proteolytic Activity of Lactic Acid Bacteria Graciela Savoy de Giori and Elvira María Hébert .......................... 197 25 Methods for Isolation and Titration of Bacteriophages from Lactobacillus Lucía Auad and Raúl R. Raya .......................................................... 203 26 Identification of Yeasts Present in Sour Fermented Foods and Fodders Wouter J. Middelhoven ..................................................................... 209

PART IV ORGANISMS

IN THE

M ANUFACTURE

OF

OTHER FOODS AND BEVERAGES

27 Protein Hydrolysis: Isolation and Characterization of Microbial Proteases Marcela A. Ferrero ............................................................................. 227 28 Production of Polyols by Osmotolerant Yeasts Lucía I. C. de Figueroa and María E. Lucca .................................... 233 29 Identification of Yeasts from the Grape/Must/Wine System Peter Raspor, Sonja Smole Mozina, and Neza Cadez ................... 243 30 Carotenogenic Microorganisms: A Product-Based Biochemical Characterization José Domingos Fontana ................................................................... 259 31 Genetic and Chromosomal Stability of Wine Yeasts Matthias Sipiczki, Ida Miklos, Leonora Leveleki, and Zsuzsa Antunovics ........................................................................ 273 32 Prediction of Prefermentation Nutritional Status of Grape Juice: The Formol Method Barry H. Gump, Bruce W. Zoecklein, and Kenneth C. Fugelsang .................................................................. 283 33 Enological Characteristics of Yeasts Fabio Vasquez, Lucía I. C. de Figueroa, and Maria Eugenia Toro ....................................................................... 297 34 Utilization of Native Cassava Starch by Yeasts Lucía I. C. de Figueroa, Laura Rubenstein, and Claudio González ........................................................................... 307

PART V METHODS

AND

EQUIPMENT

35 Reactor Configuration for Continuous Fermentation in Immobilized Systems: Application to Lactate Production José Manuel Bruno-Bárcena, Alicia L. Ragout de Spencer, Pedro R. Córdoba, and Faustino Siñeriz .................................... 321

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36 Molecular Characterization of Yeast Strains by Mitochondrial DNA Restriction Analysis Maria Teresa Fernández-Espinar, Amparo Querol, and Daniel Ramón ................................................................................. 329 37 Selection of Yeast Hybrids Obtained by Protoplast Fusion and Mating, by Differential Staining, and by Flow Cytometry Tohoru Katsuragi ............................................................................... 335 38 Selection of Hybrids by Differential Staining and Micromanipulation Tohoru Katsuragi ............................................................................... 341 39 Flotation Assay in Small Volumes of Yeast Cultures Sandro Rogério de Sousa, Maristela Freitas Sanches Peres, and Cecilia Laluce ......................................................................... 349 40 Obtaining Strains of Saccharomyces Tolerant to High Temperatures and Ethanol Maristela Freitas Sanches Peres, Sandro Rogério de Sousa, and Cecilia Laluce ......................................................................... 355 41 Multilocus Enzyme Electrophoresis Timothy Stanley and Ian G. Wilson ................................................. 369 42 Bacteriocin Production Process by a Mixed Culture System Suteaki Shioya and Hiroshi Shimizu .............................................. 395

PART VI R EVIEWS 43 Nutritional Status of Grape Juice Bruce W. Zoecklein, Barry H. Gump, and Kenneth C. Fugelsang .................................................................. 415 44 Problems with the Polymerase Chain Reaction: Inhibition, Facilitation, and Potential Errors in Molecular Methods Ian G. Wilson ...................................................................................... 427 45 Problems with Genetically Modified Foods José Manuel Bruno-Bárcena, M. Andrea Azcarate-Peril, and Faustino Siñeriz ............................................................................. 481 Index

......................................................................................................... 485

Contributors Z SUZSA A NTUNOVICS • Department of Genetics, University of Debrecen, Debrecen, Hungary LUCÍA AUAD • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina M. A NDREA A ZCÁRATE -P ERIL • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán , Argentina BURTON W. BLAIS • Laboratory Services Division, Canadian Food Inspection Agency, Ottawa, Ontario, Canada JOSÉ MANUEL BRUNO-BÁRCENA • Departamento de Biotecnologia, Instituto Agrochimica y Tecnologia de Alimentos, Valencia, Spain N EZA C ADEZ • Food Science and Technology Department, University of Ljubljana, Ljubljana, Slovenia JINRU CHEN • Department of Food Science and Technology, University of Guelph, Guelph, Ontario, Canada MATTHEW D. COLLINS • Institute of Food Research, Norwich, UK PEDRO R. CÓRDOBA • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina SERGIO A. CUOZZO • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina SILVINA FADDA • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina JEFF M. FARBER • Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, Ontario, Canada MARIA TERESA FERNÁNDEZ-ESPINAR • Departamento de Biotecnologia, Instituto Agroquimia y Tecnologia de Alimentos, Valencia, Spain MARCELA A. FERRERO • Microbiology Laboratory, Universität de Lles Illes Balears, Illes Balears, Spain L UCÍA I. C. DE F IGUEROA • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina JOSÉ DOMINGOS FONTANA • Biomass Chemo/Biotechnology Laboratory, Curitiba, Pr., Brazil KENNTH C. FUGELSANG • Department of Viticulture and Enology, California State University-Fresno, Fresno, CA MARIA LUISA GENTA • PROIMI-MIRCEN, San Miguel Tucumán, Argentina

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GRACIELA SAVOY DE GIORI • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina CLAUDIO GONZÁLEZ • PROIMI-MIRCEN, San Miguel de Tucumán, Argentina BARRY F. GUMP • Department of Viticulture and Enology, California State University-Fresno, Fresno, CA ELVIRA M. HÉBERT • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina HUMBERTO HELUANE • PROIMI-MIRCEN, San Miguel Tucumán, Argentina ANAVELLA GAITÁN HERRERA • Pontificia Universidad Javeriana, Bogotá, Colombia AIDA A. DE R. PESCE HOLGADO • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina STEPHEN A. JAMES • Institute of Food Research, Norwich, UK TOHORU KATSURAGI • Nara Institute of Science and Technology, Graduate School of Biological Sciences, Ikoma, Nara, Japan CECILIA LALUCE • Instituto Quimica de Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP), Araquara, SP, Brazil L EONORA L EVELEKI • Department of Genetics, University of Debrecen, Debrecen, Hungary G RACIELA L ORCA • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina MARÍA E. LUCCA • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina WOUTER J. MIDDELHOVEN • Laboratorium voor Microbiologie, Department Biomoleculaire Wetenschappen, Universiteit Wageningen, Wageningen, The Netherlands IDA MIKLOS • Department of Genetics, University of Debrecen, Debrecen, Hungary SONJA SMOLE MOZINA • Food Science and Technology Department, University of Ljubljana, Ljubljana, Slovenia F ERNANDA M OZZI • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina LAI -KING N G • Bureau of Microbiology, Laboratory Centre for Disease Control, Health Canada, Winnipeg, Manitoba, Canada GUILLERMO OLIVER • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina MARISTELA FREITAS SANCHES PERES • Programa de Pós Graduação da Faculdade de Ciências e Letras de Ribeirão Preto USP, SP, Brazil AMPARO QUEROL • Departamento de Biotecnologia, Instituto Agrochimica y Tecnologia de Alimentos, Valencia, Spain

Contributors

xiii

DANIEL RAMÓN • Departamento de Biotecnologia, Instituto Agrochimica y Tecnologia de Alimentos, Valencia, Spain PETER RASPOR • Food Science and Technology Department, University of Ljubljana, Ljubljana, Slovenia RAÚL R. RAYA • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina IAN N. ROBERTS • Institute of Food Research, Norwich, UK LAURA RUBENSTEIN • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina FERNANDO J. M. SESMA • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina H IROSHI S HIMIZU • Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan S UTEAKI S HIOYA • Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan FAUSTINO SIÑERIZ • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina M ATTHIAS SIPICZKI • Department of Genetics, University of Debrecen, Debrecen, Hungary SANDRO ROGÉRIO DE SOUSA • Instituto Quimica de UNES, Araquara, SP, Brazil ALICIA L. RAGOUT DE SPENCER • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina JOHN F. T. SPENCER • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina T IMOTHY S TANLEY • Bacteriology Department, Belfast City Hospital, Belfast, Northern Ireland MARÍA PÍA DE TARANTO • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina MARÍA INÉS TORINO • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina MARIA EUGENIA TORO • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI),San Miguel Tucumán, Argentina GEOFF TURNER • Laboratory Services Division, Canadian Food Inspection Agency, Ottawa, Ontario, Canada GRACIELA FONT DE VALDEZ • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán , Argentina FABIO VAZQUEZ • Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), San Miguel Tucumán, Argentina

xiv

Contributors

KASTHURI J. VENKATESWARAN • Planetary Protection Sciences, Jet Propulsion Laboratory, NASA, Pasadena, CA GRACIELA VIGNOLO • Centro de Referencia para Lactobacilos (CERELA), San Miguel Tucumán, Argentina H AIYAN WANG • Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, Ontario, Canada I AN G. W ILSON • Bacteriology Department, Belfast City Hospital, Belfast, Northern Ireland BRUCE W. ZOECKLEIN • Department of Food Science and Technology, Virginia Tech, Blacksburg, VA

Psychrotrophic Microorganisms

I SPOILAGE ORGANISMS

1

Psychrotrophic Microorganisms

3

1 Psychrotrophic Microorganisms Agar Plate Methods, Homogenization, and Dilutions Anavella Gaitán Herrera 1. Introduction Isolation and enumeration of the microorganisms present in foods usually demands preliminary treatment of samples to release into a liquid medium those microorganisms present that may be included within the food. In a mixing procedure known as “stomaching,” the food sample (see Note 1) and diluent are put into a sterile plastic bag that is vigorously struck on its outer surfaces by paddles inside a stomacher, the compression and shearing forces break up solid pieces of food. After samples are removed for analysis, the bag and its remaining contents can be discarded and the equipment is ready for use. If a stomacher is not available, an electric blender with cutting blades revolving at high speed can be used (see Note 2). The nature of the diluent used is another highly important factor. Diluents such as tap or distilled water, saline solutions, phosphate buffers, and Ringer’s solution are toxic to microorganisms, especially if the time of contact is unduly prolonged. For that reason, we suggest that using 0.1% peptone solution, physiological saline solution with 0.1% peptone added, is the most reliable source. Peptone 0.1% in saline solution in 0.85% NaCl is recommended by the International Standards Organization (see Notes 3 and 4). The number of microorganisms by plate count has been one of the more commonly used microbiological methods for determination food quality. This method indicates the adequacy of sanitation and formation of an opinion on incipient spoilage (see Note 5). The plate count has a special application to imported foods for control of the standard of sanitation practiced in the manufacturing establishments (1). From: Methods in Biotechnology, Vol. 14: Food Microbiology Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

3

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Herrera

The three methods in common use for enumerating microorganisms are the standard-plate count, also called the aerobic plate count or pour plate, the “surface plate,” or “spread-drop” method and the drop-plate method (see Note 6). None of these procedures can be depended on to list all types of organisms present within the test specimens. Many cells may not grow because of specifically unfavorable conditions of nutrition, aeration, temperature, or duration of incubation (1). The temperature chosen depends on the purpose of the examination. The incubation temperature of microorganisms have different growth temperature ranges such as 0–7oC for psychrotrophs, 30–35oC for mesophiles, and 55oC for thermophiles. No single incubation temperature will absolutely exclude all organisms from another group (see Notes 7 and 8). Incubators are required with minimal fluctuation or variation of temperature throughout the incubation chamber. All incubators should be checked and calibrated frequently (2). The psychrotrophic or psychrophilic microorganisms are able to grow for 7–10 d (see Note 9) at commercial refrigeration temperatures (0–7oC). Species of Achromobacter, Acinetobacter, Alcaligenes, Bacillus, Flavobacterium, Streptococcus, and Pseudomonas are included among the psychrotrophic bacteria. Furthermore, some yeasts and molds, including Penicillium, Aspergillus, Geotrichum, and Botrytis, are able to grow well in refrigerated foods (see Note 10) in large numbers. These microorganisms can cause off flavors amd physical defects in foods. Their growth rates are highly temperature dependent (see Note 11). Their presence indicates a high potential for spoilage during extended storage. Most of these microorganisms are destroyed by mild heat treatment, but some heat-resistant types such as some species of Bacillus and Clostridium may survive. The presence of psychrotrophic microorganisms in heat-processed foods implies post-processing contamination. They are sources of heat-resistant proteolytic and lipolytic enzymes (3) that affect adversely the quality of the food during storage after heat treatment (see Note 12). The presence of psychrotropic microorganisms is also important in such foods as frozen turkey or chicken when they are thawed. 2. Homogenization and Dilution: Method 1 2.1. Materials 1. Mechanical blender, two-speed model or single speed with rheostat control. 2. Glass or metal blending jars of 1 L capacity, with covers, resistant to autoclave temperatures. One sterile jar (autoclaved at 121oC) for 15 min) is required for each sample to be analyzed. 3. Balance with weights. Capacity at least 2500 g, sensitivity 0.1 g. 4. Instruments for preparing samples: knives, forks, forceps, scissors, spoons, spatulas, and tongue depressors, all sterilized for use by autoclaving or by hot air. 5. Pipets: 1, 5, and 10 mL.

Psychrotrophic Microorganisms

5

6. Refrigerator cooled to 2–5oC. 7. Peptone dilution fluid or peptone salt dilution fluid, sterilized in the autoclave for each sample 450 mL in flask or bottle, 90 or 99 mL blanks in dilution bottles or similar containers. 8. Mechanical mincer, mechanical blender, operating at not less than 8000 rpm and not more than 45,000 rpm. 9. For Method 1, items 2–4, and 6: a. Pipets, bacteriological b. Sterile culture tubes for dilution fluid, 15–20 mL capacity c. Peptone salt dilution fluid

3. Microogranisms: Agar Plate Method Petri dishes, glass (100 × 15 mm) or plastic (90 × 15 mm) (see Note 7). Pipets, bacteriological, 1, 5, and 10 mL sterile. Water bath or incubator for tempering agar, 44–46oC. Incubator, 29–31oC. Colony counter. Tally register. Plate count agar (standard-methods agar) (see Notes 5 and 6). Drying cabinet or incubator for drying the surface of agar plates, preferably at 50oC. 9. Glass spreaders (Drigalski spatulas; hockey-stick–shaped glass rods). 10. Pipets, bacteriological, sterile, with divisions of 0.1 mL or less. 1. 2. 3. 4. 5. 6. 7. 8.

3.1. Materials Low-temperature incubator capable of maintaining a temperature of 1–7oC. Nonselective agar media such as standard or trypticase soy broth. Selective agar, crystal violet, or tetrazolium agar. Pipets, bacteriological, 0.1 mL sterile Materials for preparation and dilution of the food homogenate, as listed in Subheading 2.1. 6. Petri dishes, as described previously. 7. Colony counter. 1. 2. 3. 4. 5.

4. Methods 4.1. Homogenization and Dilutions: Method 1 1. Begin the examination as soon as possible after the sample is taken. Refrigerate the sample at 0–5oC if the examination cannot be started immediately after it reaches the laboratory. If the sample is frozen, thaw it in its original container (or in the container in which it was received at the laboratory) for a maximum of 18 h in a refrigerator at 2–5oC. If the sample can be easily comminuted (as in ice cream), proceed without thawing. 2. Tare the empty sterile blender jar, then weigh into it, 50 g representative of the food sample. If the contents of the package are obviously not homogenous

6

Herrera (frozen dinner), take a 50-g sample from a macerate of the whole dinner, or analyze each different food portion separately, depending on the purpose of the test. 3. Add to the blender jar, 450 mL of the peptone dilution fluid or peptone salt dilution fluid. This provides a dilution of 10 (+/–) 1. 4. Blend the food and dilute promptly. Start at low speed and then switch to high speed within a few seconds. Time the blending carefully to permit 2 min at high speed. Wait 2 or 3 min for foam to disperse. 5. Measure 1 mL of the 10 –1 dilution of the blended material, avoiding foam, into a 99-mL dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a 30-cm arc. Repeat this process using the progressively increasing dilutions 10–2, 10 –3, 10 –4, and 10 –5, or dilutions that experience indicates are desirable for the food being tested.

4.2. Homogenization and Dilutions: Method 2 1. Begin the examination as soon as possible after the sample is taken. Preferably, start analysis of unfrozen samples within 1 h after receipt. If the sample is frozen, thaw in the original container (or in the container in which it was received in the laboratory) in the refrigerator at 2.5oC and examine as soon as possible after thawing is complete or at least sufficient to permit suitable subsamples to be taken (maximum thawing time 18 h). 2. Proceed to step 3 if the sample is difficult to blend, grind, and mix twice in the mechanical mincer. 3. Weigh into a tared blended jar at least 10 g of sample, representative of the food. Add nine times as much dilution fluid as sample. This provides a dilution of 10:1. 4. Time of grinding must not exceed 2.5 min. 5. Mix the contents of the jar by shaking and pipet duplicate portions of 1 mL each into separate tubes containing 9 mL of dilution fluid. Carry out steps 7 and 8 on each of the diluted portions. 6. Mix the liquids carefully by aspirating 10 times with a sterile pipet. 7. Transfer with the same pipet, 1.0 mL to another dilution tube containing 9 mL of dilution fluid and mix with a fresh pipet. 8. Repeat steps 7 and 8 until the required number of dilutions are made. Each successive dilution will decrease the concentration 10-fold.

4.2.1. Example 1 Dilution 10–1, 350, and 330: (350 + 330)/2 × 10 = 3400 Dilution 10–2, 26, and 28: (26 + 28) 2 × 100 = 2700 X = (3400 + 2700)2 = 3050 30 × 102 CFU/g

Psychrotrophic Microorganisms

7

4.2.2. Example 2 90 colonies on dilution 10–1 = 2900 40 colonies on dilution 10–2 = 4000 4000/2900 = 2 Report the lower count 17 × 10-2

4.2.3. Computing the Estimated Standard Plate Count (ESPC) 1. If counts on individual plates do not fall within the range 30–300 colonies, report the calculated count as ESPC. Calculate the count as directed in steps 2–4. 2. If plates of all dilutions show more than 300 colonies, divide each of the duplicate plates for the highest dilution into convenient radial sections (e.g., 2, 4, 8) and count all of the colonies in one or more sections. Multiply the total in each case by the appropriate factor to obtain an estimate of the total number of colonies for the entire plate. Average the estimates for the two plates, multiply the dilution, and report the resulting count as the ESPC. 3. If the three are more than 200 colonies per one-eighth section of the plates made from the most dilute suspension, multiply 1600 (i.e., 200 X 8) by that dilution, and express ESPC as more than (>) the resulting number. In all such cases, it is advisable to report the dilution used in parentheses.

The Standard Plate Count 5.1. Methods 1. Prepare the sample. 2. To duplicate sets of Petri dishes, pipet 1 mL aliquots from 10–1, 10–2, 10–3, 10–4, and 10–5 dilutions, and a 0.1-mL aliquot from the 10–5 dilution to give 10–1 to 10–6 g of food per Petri dish. 3. Promptly pour into Petri dishes 10–15 mL of melted and tempered agar. Immediately mix the aliquots with the agar medium by tilting and rotating the Petri dishes. The sequence of steps is: a. Tilt dish to and fro five times, in one direction. b. Rotate it clockwise five times. c. Tilt it to and fro again five times in a direction at right angles to that used the first time. d. Rotate it counterclockwise five times. 4. When the agar is solidified, invert the Petri dishes and incubate at 29–31°C for 48 +/- 3 h.

5.2. The Surface-Spread Plate 1. Add 15 mL of melted, cooled (45–60°C Plate count agar to each Petri dish used and allow to solidify.

8

Herrera 2. Transfer 0.1 mL of each of the dilutions to the agar surface. Using the same pipet for each dilution. Test at least three dilutions, even if the approximate range of numbers of microorganisms in the food is known. Start with the highest dilutions and proceed to the lowest, filling and emptying the pipet three times before transferring the 0.1-mL portion to the plate. 3. Promptly spread the 0.1-mL portions on the surface of the agar plates using glass spreaders (Drigalsky spatulas). Use a separate spreader for each plate. Allow the surfaces of the plates to dry for 15 min. 4. Incubate the plates in an inverted position, for 3 d.

5.2.1. Computing the Standard Plate Count and the Estimated Plate Count 5.2.1.1. THE STANDARD PLATE COUNT (SPC) 1. Select two plates corresponding to one dilution and showing between 30 and 300 colonies per plate. Count all colonies on each plate, using the colony counter and tally register. Take the average of the two counts and multiply by the dilution factor. Report the resulting number as the SPC. 2. Two plates should be counted even if one of them should give a count of fewer than 30 or more than 300 colonies. Again, take the average of the two counts and multiply by the dilution factor and report the resulting number as the SPC. 3. If the plates from two consecutive decimal dilutions fall into the countable range of 30–300 colonies compute the SPC for each of the dilutions as directed above, and report the average of the two values obtained, unless the higher computed count is more than twice the lower one, in which case report the lower computed count as the SPC. 4. Report only two significant digits as the colony-forming unit per gram or milliliter.

5.2.1.2. EXAMPLE 1 1600 × 103 = >1600 000 > 16 × 103 Dilution 10–1: No colonies: in cases when there are no colonies in the plates made from the most concentrated suspension, report the ESPC as less than (