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- Herausgeber: John Wiley & Sons
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- Sprache: Englisch
The new edition of the highly regarded laboratory manual for courses in food microbiology
Analytical Food Microbiology: A Laboratory Manual develops the practical skills and knowledge required by students and trainees to assess the microbiological quality and safety of food. This user-friendly textbook covers laboratory safety, basic microbiological techniques, evaluation of food for various microbiological groups, detection and enumeration of foodborne pathogens, and control of undesirable foodborne microorganisms. Each well-defined experiment includes clear learning objectives and detailed explanations to help learners understand essential techniques and approaches in applied microbiology.
The fully revised second edition presents improved conventional techniques, advanced analytical methodologies, updated content reflecting emerging food safety concerns, and new laboratory experiments incorporating commercially available microbiological media. Throughout the book, clear and concise chapters explain culture- and molecular-based approaches for assessing microbial quality and safety of diverse foods. This expanded and updated resource:
- Reviews aseptic techniques, dilution, plating, streaking, isolation, and other basic microbiological procedures
- Introduces exercises and relevant microorganisms with pertinent background information and reference material
- Describes each technique using accessible explanatory text, detailed illustrations, and easy-to-follow flowcharts
- Employs a proven “building block” approach throughout, with each new chapter building upon skills from the previous chapter
- Provides useful appendices of microbiological media, recommended control organisms, available supplies and equipment, and laboratory exercise reports
With methods drawn from the authors’ extensive experience in academic, regulatory, and industry laboratories, Analytical Food Microbiology: A Laboratory Manual, Second Edition, is ideal for undergraduate and graduate students in food microbiology courses, as well as food processors and quality control personnel in laboratory training programs.
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Table of Contents
COVER
TITLE PAGE
COPYRIGHT PAGE
PREFACE
PART I: BASICS OF FOOD MICROBIOLOGY LABORATORY
CHAPTER 1: LABORATORY SAFETY
BACKGROUND
PERSONAL SAFETY IN THE LABORATORY
MATERIALS IN MICROBIOLOGY LABORATORY
PRACTICAL ASPECTS
REFERENCE
CHAPTER 2: SAMPLING FOR MICROBIOLOGICAL ANALYSIS OF FOOD AND PROCESSING ENVIRONMENT
THEORETICAL ASPECTS
PRACTICING SAMPLING AND SAMPLE PREPARATION
CHAPTER 3: ENUMERATION OF MICROORGANISMS IN FOOD
INTRODUCTION
PLATE COUNT METHOD
MOST PROBABLE NUMBER METHOD
SELECTED REFERENCES
QUESTIONS
CHAPTER 4: PRACTICING BASIC TECHNIQUES
INTRODUCTION
OBJECTIVES
MEDIA
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: DILUTION AND PLATING
SESSION 2: POPULATION COUNTING AND COLONY STREAKING
SESSION 3: MORPHOLOGICAL EXAMINATION
PART II: FOOD MICROBIOTA
CHAPTER 5: AEROBIC MESOPHILIC PLATE COUNT
INTRODUCTION
OBJECTIVES
MEDIA
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: SAMPLING, HOMOGENIZATION, DILUTION, PLATING, AND INCUBATION
SESSION 2: COLONY COUNTING AND ISOLATION
SESSION 3: EXAMINING COLONY AND CELL MORPHOLOGIES
CHAPTER 6: MESOPHILIC SPOREFORMING BACTERIA
INTRODUCTION
OBJECTIVES
MEDIA AND INCUBATION CONDITIONS
MICROSCOPY METHODS
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: SAMPLE PREPARATION, PLATING, AND INCUBATION
MATERIALS AND EQUIPMENT
PROCEDURE
SESSION 2: SPORE ENUMERATION AND EXAMINING SPORULATION
MATERIALS AND EQUIPMENT
PROCEDURE
SESSION 3: MICROSCOPIC EXAMINATION OF SPORES
CHAPTER 7:
Pseudomonas
SPECIES AND OTHER SPOILAGE PSYCHROTROPHS
INTRODUCTION
OBJECTIVES
MEDIA AND TESTS
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: SAMPLING AND PLATING
PROCEDURE
SESSION 2: ENUMERATION OF
Pseudomonas
SPECIES AND TESTING FOR ENZYMATIC ACTIVITY
PROCEDURE
SESSION 3: DETECTING ENZYMATIC ACTIVITY
SESSION 4: ENUMERATION OF AEROBIC PSYCHROTROPHS
CHAPTER 8: DETECTION AND ENUMERATION OF
Enterobacteriaceae
IN FOOD
INTRODUCTION
OBJECTIVES
MEDIA AND TESTS
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: PREPARATION OF MOST PROBABLE NUMBER TUBES
SESSION 2: PRESUMPTIVE MPN DETERMINATION
SESSION 3: PRESUMPTIVE MPN DETERMINATION AND CONFIRMATION
SESSION 4: CONFIRMATION
CHAPTER 9: EXAMINATION AND ENUMERATION OF FOODBORNE FUNGI
INTRODUCTION
OBJECTIVES
MEDIA
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: EXAMINATION OF FOOD FOR MOLDINESS AND PREPARING SAMPLES FOR FUNGI ENUMERATION
SESSION 2: EXAMINATION OF PRE‐MOUNTED FUNGAL SPECIMENS
SESSION 3: DETERMINING FUNGI POPULATION AND ISOLATE MORPHOLOGY
PART III: FOODBORNE PATHOGENS
CHAPTER 10:
Staphylococcus aureus
INTRODUCTION
SELECTED REFERENCES
EXERCISE I: ENUMERATION, ISOLATION, AND IDENTIFICATION OF
Staphylococcus aureus
IN FOOD
SESSION 1: SAMPLING, HOMOGENIZATION, DILUTION, AND INOCULATION OF MPN TUBES
SESSION 2: SCREENING MPN TUBES USING
Staphylococcus
SELECTIVE‐DIFFERENTIAL MEDIUM
SESSION 3: INSPECTING AND ISOLATING PRESUMPTIVE
Staphylococcus aureus
COLONIES
SESSION 4: CONFIRMING
Staphylococcus aureus
IDENTITY AND CALCULATING
S. AUREUS
MPN
EXERCISE 2: EXPRESSION OF ENTEROTOXIN GENES BY
Staphylococcus aureus
FOOD ISOLATES
SESSION 1: DETECTION OF SEA GENE: DNA EXTRACTION AND PREPARING FOR PCR
SESSION 2: DETECTION OF SEA GENE: GEL ELECTROPHORESIS
SESSION 3: DETECTION OF SEA GENE: ELECTROPHORESIS RESULTS OBSERVATION
SESSION 4: DETECTING THE EXPRESSION OF STAPHYLOCOCCAL ENTEROTOXINS USING ELISA
CHAPTER 11:
Listeria monocytogenes
INTRODUCTION
PROCEDURE OVERVIEW
SELECTED REFERENCES
EXERCISE I: DETECTION OF
Listeria
SPECIES USING CULTURE‐BASED METHOD
SESSION 1: SAMPLE PREPARATION AND ENRICHMENT
SESSION 2: ISOLATION
SESSION 3: IDENTIFICATION
SESSION 4: MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION
EXERCISE II: DETECTION OF
Listeria monocytogenes
USING PCR‐BASED METHOD
SESSION 1: SAMPLE PREPARATION AND ENRICHMENT
SESSION 2: DNA EXTRACTION AND PCR
SESSION 3: GEL ELECTROPHORESIS
SESSION 4: ELECTROPHORESIS RESULTS
CHAPTER 12:
Salmonella enterica
INTRODUCTION
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: PRE‐ENRICHMENT
SESSION 2: SELECTIVE ENRICHMENT
SESSION 3: SAMPLE SCREENING AND
Salmonella
ISOLATION
MATERIALS AND EQUIPMENT
PROCEDURE
SESSION 4: ISOLATION
SESSION 5: IDENTIFICATION
SESSION 6: SCORING API STRIPS
CHAPTER 13: SHIGA TOXIN‐PRODUCING
Escherichia coli
INTRODUCTION
OBJECTIVES
DETECTION OF STEC IN FOOD
MEDIA
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: ENRICHMENT
SESSION 2: RAPID SCREENING BY MULTIPLEX PCR
SESSION 3: GEL ELECTROPHORESIS
SESSION 4: ISOLATION
SESSION 5: ISOLATION (CONTINUED)
SESSION 6: SCREENING ISOLATES FOR VIRULENCE GENES: PCR
SESSION 7: SCREENING OF ISOLATES FOR VIRULENCE GENES: GEL ELECTROPHORESIS AND ISOLATE SELECTION
SESSION 8: ISOLATE IDENTITY CONFIRMATION: BIOCHEMICAL TESTING
PART IV: CONTROL OF FOODBORNE MICROORGANISMS
CHAPTER 14: THERMAL RESISTANCE OF MICROORGANISMS IN FOOD
INTRODUCTION
OBJECTIVES
MEDIA
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: INOCULATION AND THERMAL TREATMENTS OF TWO MICROORGANISMS
SESSION 2: HEAT TREATMENTS OF
Enterococcus
sp. AT 60 AND 65°C
SESSION 3: COUNTING
ENTEROCOCCUS
SP. SURVIVORS
SESSION 4: DETERMINATION OF D‐VALUES AND Z‐VALUE
CHAPTER 15: PRODUCTION OF BACTERIOCINS IN MILK
INTRODUCTION
OBJECTIVES
MEDIA
PROCEDURE OVERVIEW
SELECTED REFERENCES
SESSION 1: FERMENTATION
SESSION 2: ANALYSIS OF FERMENTED MILK
SESSION 3: LACTIC ACID BACTERIA COUNT (CONTINUED) AND BACTERIOCIN BIOASSAY
SESSION 4: BACTERIOCIN BIOASSAY (CONTINUED)
APPENDIX I LABORATORY EXERCISE REPORT
OUTLINE
DATA PRESENTATION
MISCELLANOUS WRITING ISSUES
APPENDIX II BACTERIAL AND FUNGAL STRAINS RECOMMENDED FOR USE AS CONTROL ORGANISMS
APPENDIX III MICROBIOLOGICAL MEDIA
MEDIA PREPARATION GUIDELINES
MEDIA RECIPES
APPENDIX IV SUPPLIES AND EQUIPMENT AVAILABILITY
EQUIPMENT
SUPPLIES
INDEX
END USER LICENSE AGREEMENT
List of Tables
Chapter 3
TABLE 3.1 Microbial population count in cooked meat.
TABLE 3.2 Most Probable Number (MPN) estimatesa based on the number of positive t...
Chapter 4
TABLE 4.1 Counts of test culture population in the sample tube, based on serial d...
TABLE 4.2 Microscope examination of isolated bacteria from streaked PCA plates, a...
Chapter 5
TABLE 5.1 Approximate representation of different categories of foodborne microor...
TABLE 5.2 {Add a descriptive title for this data set, including food sample used,...
TABLE 5.3 {Add a descriptive title for this data, including food sample used, med...
Chapter 6
TABLE 6.1 Sporeforming bacteria of importance in food.
TABLE 6.2 {add a descriptive title and footnotes for this data, including food sa...
TABLE 6.3 {add a descriptive title and footnotes for this data, including food sa...
Chapter 7
TABLE 7.1 {Add descriptive title}
TABLE 7.2 Proteolytic and lipolytic activity of Pseudomonas spp. isolated from r...
TABLE 7.3 {Add descriptive title}
Chapter 8
TABLE 8.1 {add a descriptive title and footnotes for this data, including food sa...
TABLE 8.2 {Add a descriptive title and footnotes for this data, including food sa...
Chapter 9
TABLE 9.1 Characteristics of foodborne molds. The anamorph‐teleomorph relationshi...
TABLE 9.2 Characteristics of foodborne yeasts. The anamorph‐telemorph relationshi...
TABLE 9.3 {Provide a descriptive title}
TABLE 9.4 {add a descriptive title for this data. Add also appropriate footnotes}
TABLE 9.5 Fungi colony and population counts in foods analyzed by ______ and ____...
TABLE 9.6 Colony and cell morphology of fungi isolated from ________ {food analyz...
Chapter 10
TABLE 10.1 Characteristics of selected
Staphylococcus
spp.
TABLE 10.2 Results of tests needed to determine Staphylococcus aureus most‐probab...
TABLE 10.3 (Propose a meaningful title)
TABLE 10.4 Forward and reverse primers for Staphylococcus aureus enterotoxin A ge...
TABLE 10.5 {Use appropriate title}
TABLE 10.6 {add a descriptive title and footnotes for this data, including food s...
Chapter 11
TABLE 11.1 Differentiation of Listeria spp. by biochemical testing and blood hemo...
TABLE 11.2 Detection of
Listeria monocytogenes
in food and environmental samples.
TABLE 11.3 {add a descriptive title for this data, including food sample used, so...
TABLE 11.4 {add a descriptive title for this data, including food sample used, so...
Chapter 12
TABLE 12.1 Description of observed enrichment media and result of immunoassay tes...
TABLE 12.2 Characteristics of Salmonella on isolation and biochemical identificat...
TABLE 12.3 (
Add a descriptive title, including food and how results were obt
...
TABLE 12.4 Biochemical tests included in the API‐20E test strip, test interpretat...
TABLE 12.5 McFarland standards and approximate cell density.
Chapter 13
TABLE 13.1 {Add a descriptive title, including food and how results were obtained...
TABLE 13.2 {
Add a descriptive title, including food and how results were obt
...
Chapter 14
TABLE 14.1 Surviving populations of Pseudomonas sp. and Enterococcus sp. when ino...
TABLE 14.2 {Add a descriptive title, describing the results of heating Enterococc...
TABLE 14.3 {Add a descriptive title including food and how results were obtained}
Chapter 15
TABLE 15.1 Population count and pH of milk fermented (or unfermented) with a lact...
TABLE 15.2 Diameter of inhibition areas resulting from spotting 5 μl of nisin st...
TABLE 15.3 Bacteriocin concentrations (IU/ml)
a
in samples of fermented milk.
Part 3
TABLE III.1 Modes of transmission of microbial foodborne diseases.
Part 4
TABLE IV.1 Antimicrobial factors and associated technologies used in control of f...
List of Illustrations
Chapter 2
Figure 2.1 Food sampling.
Figure 2.2 Food blender (left) and stomacher (right).
Figure 2.3 Decimal dilution of food homogenate.
Chapter 3
Figure 3.1 Example of a dilution scheme, showing the dilutions (prepared fro...
Figure 3.2 Darkfield Quebec colony counter with a Petri plate mounted for co...
Figure 3.3 Procedure to estimate microbial population count in samples using...
Figure 3.4 Decision tree for applying microbial colony and population count ...
Figure 3.5 Applying colony and population counting rules.
Figure 3.6 Dilution and inoculation scheme for most probable number techniqu...
Chapter 4
Figure 4.1 Types of culture media that are commonly used in microbiological ...
Figure 4.2 Basic techniques as practiced in this exercise.
Figure 4.3 Dilution and plating scheme used in basic technique exercise.
Figure 4.4 Labeling the bottom of a Petri plate.
Figure 4.5 Three‐phase streaking.
Figure 4.6 Smear preparation and Gram staining.
Chapter 5
Figure 5.1 An overview of the aerobic mesophilic plate count method.
Figure 5.2 Suggested dilution scheme for aerobic mesophilic plate count exer...
Figure 5.3 A sample of results of market food analyzed using the mesophilic ...
Chapter 6
Figure 6.1 Life cycle of a spore‐forming bacterium. Dark shades (e.g., cell ...
Figure 6.2 Transmission electron micrograph of
Bacillus subtilis
spores (Cou...
Figure 6.3 Outline of procedure for counting aerobic and anaerobic mesophili...
Chapter 7
Figure 7.1 Scheme of testing food for
Pseudomonas
spp. and other psychrotrop...
Chapter 8
Figure 8.1 Detection and enumeration of
Enterobacteriaceae
in food.
Figure 8.2 Most‐probable number (MPN)
Enterobacteriaceae
enrichment broth (E...
Chapter 9
Figure 9.1 Classification of fungi associated with food. Some genera are gro...
Figure 9.2 Scheme of testing food for fungi.
Figure 9.3 Slide culture assembly
Figure 9.4 Hand‐held hard cheese shredder.
Chapter 10
Figure 10.1 Enumeration and identification of
Staphylococcus aureus
in food....
Figure 10.2 Colonies of
Staphylococcus aureus
(a) and
S. epidermidis
(b) on ...
Figure 10.3 Detection of staphylococcal enterotoxin gene (
sea
) in food isola...
Figure 10.4 Basics of gene expression in bacterial cell.
Figure 10.5 Enzyme‐linked immunoassay used in detection of staphylococcal en...
Figure 10.6 Microfiltration to prepare cell‐free culture supernatant.
Chapter 11
Figure 11.1 Detection of
Listeria
spp. in food and environmental sample usin...
Figure 11.2 Detection of
Listeria monocytogenes
in food and environmental sa...
Chapter 12
Figure 12.1 Method for detection of
Salmonella
in food.
Figure 12.2 Scoring the incubated API‐20E strip for food isolate and positiv...
Chapter 13
Figure 13.1 Complete procedure for the detection of Shiga toxin‐producing
Es
...
Figure 13.2 Detection of Shiga toxin‐producing
Escherichia coli
(STEC) in fo...
Figure 13.3 Detection of Shiga toxin‐producing
Escherichia coli
in food samp...
Figure 13.4 Gel showing multiplex PCR products resulting from
Escherichia co
...
Figure 13.5 Matrix of 24 inculcated microfuge tubes, prepared for PCR‐assist...
Chapter 14
Figure 14.1 Illustration of a survivor plot of an organism.
Figure 14.2 Illustration of a thermal resistance plot for an organism.
Figure 14.3 Procedure to determine thermotolerance of inoculated bacteria in...
Figure 14.4 Linear‐linear graphing paper.
Chapter 15
Figure 15.1 Bioassay for quantifying antimicrobial activity in a fermentate ...
Figure 15.2 Bioassay for quantifying antimicrobial activity in milk fermenta...
Figure 15.3 Inhibition areas resulting from spotting dilutions of nisin stoc...
Figure 15.4 Dose response plot depicting the linear relationship between log
Figure 15.5 Procedure overview of production of antimicrobial ingredients.
Figure 15.6 Microfiltration of bacteriocin‐containing fermentate.
Part 2
Figure II.1 Typical links in the food supply chain.
Figure II.2 Taxonomy of foodborne bacteria, drawn from Bergey’s manual of sy...
Part 3
Figure III.1 Enzyme‐linked immunosorbent bioassay (ELISA) technique for dete...
Figure III.2 Polymerase chain reaction (PCR) method for amplifying unique DN...
Guide
COVER PAGE
TITLE PAGE
COPYRIGHT PAGE
PREFACE
TABLE OF CONTENTS
BEGIN READING
APPENDIX I LABORATORY EXERCISE REPORT
APPENDIX II BACTERIAL AND FUNGAL STRAINS RECOMMENDED FOR USE AS CONTROL ORGANISMS
APPENDIX III MICROBIOLOGICAL MEDIA
APPENDIX IV SUPPLIES AND EQUIPMENT AVAILABILITY
INDEX
WILEY END USER LICENSE AGREEMENT
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ANALYTICAL FOOD MICROBIOLOGY
A Laboratory Manual
Second Edition
AHMED E. YOUSEF
The Ohio State University
Columbus, Ohio, USA
JOY G. WAITE‐CUSIC
Oregon State University
Corvallis, Oregon, USA
JENNIFER J. PERRY
University of Maine
Orono, Maine, USA
This edition first published 2022© 2022 John Wiley & Sons, Inc.
Edition HistoryWiley (1e, 2003)
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.
The right of Ahmed E. Yousef, Joy G. Waite‐Cusic, and Jennifer J. Perry to be identified as the author(s) of this work has been asserted in accordance with law.
Registered Office(s)John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA
Editorial Office111 River Street, Hoboken, NJ 07030, USA
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Limit of Liability/Disclaimer of WarrantyThe contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting scientific method, diagnosis, or treatment by physicians for any particular patient. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.
Library of Congress Cataloging‐in‐Publication Data
Names: Yousef, Ahmed Elmeleigy, author. | Waite‐Cusic, Joy G., author. | Perry, Jennifer J., author.Title: Analytical food microbiology : a laboratory manual / Ahmed E. Yousef, The Ohio State University, Columbus, OH, USA, Joy G. Waite‐Cusic, Oregon State University, Corvallis, OR, USA, Jennifer J. Perry, University of Maine, Orono, ME, USA.Other titles: Food microbiologyDescription: Second edition. | Hoboken, NJ : Wiley, [2022] | Revison of: Food microbiology / Ahmed E. Yousef, Carolyn Carlstrom. c2003. | Includes bibliographical references and index.Identifiers: LCCN 2021033026 (print) | LCCN 2021033027 (ebook) | ISBN 9780470425114 (paperback) | ISBN 9781119428039 (adobe pdf) | ISBN 9781119428015 (epub)Subjects: LCSH: Food–Microbiology–Laboratory manuals.Classification: LCC QR115 .Y686 2021 (print) | LCC QR115 (ebook) | DDC 664.001/579–dc23LC record available at https://lccn.loc.gov/2021033026LC ebook record available at https://lccn.loc.gov/2021033027
Cover Design: WileyCover Image: Courtesy of Ahmed Yousef
PREFACE
Microbiological testing of food is an important component of the global food system. Results from these tests support food safety and quality programs by helping researchers and processors monitor fermentations, validate and verify the efficacy of processing treatments, and demonstrate the efficacy of sanitation programs. Well‐trained food microbiologists are needed in the food industry, supporting industries, and regulatory agencies to conduct laboratory analyses that serve these functions.
There is no shortage of published methods for enumerating food microbiota or detecting foodborne pathogens. However, using these methods in teaching or training settings requires considerable adaptation and simplification. Professional analysts have an eight‐hour work shift, which provides ample and flexible time to complete a variety of microbiological analyses. On the contrary, most teaching laboratory or training sessions are short, often less than three hours; therefore, the use of official and standard methods in teaching and training environments is impractical.
Many of the methods presented in this book were designed from the authors’ combined experiences as microbiologists in academic, regulatory, and industry laboratories. Starting decades ago, with simple teaching exercises at the Ohio State University, we built full analytical protocols that echo recent advances in science yet are executable in diversely equipped laboratories, albeit to different degrees of completeness. The laboratory exercises have been structured so that each provides students or trainees an opportunity to learn a new technique or approach in microbiology. The learning objectives are listed explicitly below each exercise title and explained in detail within the body of the exercises. The book’s initial exercises meet the needs for basic training in food microbiology; these activities also prepare students and trainees for more advanced training in subsequent exercises.
The book has four main parts. The first part covers safety considerations and reviews basic microbiological techniques that may have been covered in previous introductory biology or microbiology courses. Included in Part I are simple exercises to help students with limited background in applied microbiology or to refresh experienced students with essential microbiological techniques. This part also emphasizes terminology and sets the stage for the approaches used in the remainder of the book. Part II includes exercises to evaluate various microbiological groups of significance to food quality, starting with mesophilic aerobic bacteria and ending with foodborne fungi. Pathogen detection is covered in Part III of the book. Both culture‐ and molecular‐based approaches are included in this section. Part IV covers the microbiological aspects of technologies used in the control of food microbiota. Included in this part are two exercises that familiarize students with microbiological control by thermal treatments and antimicrobial peptides.
This book evolved from simple exercises that have been offered as a food microbiology laboratory course at the Ohio State University (OSU), Columbus, Ohio, USA, since the early 1990s. As the course became popular, interest in documenting these exercises into a published book was expressed by OSU alumni as well as colleagues from other institutions, including those from developing countries. In response to this interest, the teaching methods were compiled in the Food Microbiology Laboratory Manual, which was authored by A. E. Yousef and C. Carlstrom and published in 2003 by John Wiley. The book became a popular textbook in several universities and its Spanish translation was published in 2006 in Spain. Considering the continuous advances in the food microbiology field, it took great effort to evolve course methods and test them repeatedly before offering them to the students as teaching exercises. Although many teaching assistants over the years helped with this effort, the contributions of Joy Waite‐Cusic and Jennifer Perry were the most significant. They continued to develop and test these exercises post‐graduation from OSU, and while they serve as faculty members at Oregon State University and University of Maine, respectively. Both J. Waite‐Cusic and J. Perry are co‐authors of the current book.
Methods included in this book were customized to fit two‐hour laboratory periods and for a class that meets two or three times per week for at least 12 weeks per semester. Like any methodology publication, users may find errors or points of confusion despite our best efforts. These will be reported and corrected when future versions of the book are developed.
We recognize and appreciate the help we received from many teaching assistants, staff members, and visiting scholars while preparing and refining the exercises in this book. Many teaching assistants have contributed to the annual offerings of the Food Microbiology Laboratory course at OSU. They alerted us to exercise shortcomings, suggested solutions, and even tested corrective measures. We particularly appreciate the help we received from C. Carlstrom, A. Abdelhamid, E. Huang, Y‐K. Chung, and B. Lado‐Diono. Matthew Mezydlo, Department of Microbiology, OSU, shared his excellent microbiology expertise and provided valuable advice that was integral to the success of our effort. We also thank the hundreds of students at OSU who have practiced these methods over the past three decades and pushed us to provide a better compilation of exercises. A special thank you goes to Dr. Patrick Dunne, who was a great supporter of our effort in putting this book together.
Ahmed E. Yousef Professor, The Ohio State University, Columbus, Ohio, USA
Joy G. Waite‐Cusic Associate Professor, Oregon State University, Corvallis, Oregon, USA
Jennifer J. Perry Associate Professor, University of Maine, Orono, Maine, USA
PART IBASICS OF FOOD MICROBIOLOGY LABORATORY
The first part of this book introduces the laboratory aspects of microbiology as applied to food. It includes four chapters, covering laboratory safety, food sampling, microbial enumeration, and an exercise in practicing the knowledge gained in the previous three chapters. This part also serves as a review of techniques and applications covered in introductory microbiology courses. Students will execute basic exercises that will help them recall and sharpen the skills gained in previous microbiology courses.
CHAPTER 1LABORATORY SAFETY
This chapter is intended to help students and other analysts maintain safety for themselves and coworkers while receiving microbiological training or executing laboratory exercises. Although the chapter does not cover every aspect of laboratory safety, it familiarizes laboratory workers with essential components of that safety. Successful and safe execution of a laboratory exercise by a student or a professional analyst requires the compliance with safety guidelines presented here, as well as those found in other resources. Additionally, it is important to emphasize that observance of common‐sense safety precautions is important in many situations.
BACKGROUND
Live microorganisms that are handled in microbiology laboratories may cause laboratory‐acquired infections (LAI). Safety of students in these laboratories requires observing and obeying a set of laboratory safety rules. It is essential that one becomes familiar with the food microbiology laboratory setup and safety guidelines before conducting any exercises. This knowledge not only minimizes the risk of LAI, but also leads to efficient use of time and laboratory resources. Once these rules are read and understood, students should sign a form indicating that they have read, understood, and are willing to comply with these rules. Completion of this step is needed before students can practice basic techniques and the instructor can ensure their compliance with the safety guidelines. Additionally, online or in‐person training may be required before students use certain microbiology laboratories.
Laboratory Environment and Personal Safety
There are many facets to the laboratory environment, ranging from tangible items such as fixtures, equipment, supplies, and waste disposal containers to more conceptual aspects such as safety. Laboratory instructors and students should be familiar enough with the laboratory environment to respond appropriately to safety issues and emergencies.
Microbiology laboratory equipment includes basic items such as incubators, refrigerators, water baths, autoclaves, centrifuges, and microscopes, to more contemporary equipment such as gel electrophoresis systems, multiple‐well (or microtiter) plate readers, and polymerase chain reaction (PCR) thermocyclers. Microbiology laboratories often contain biological safety cabinets and chemical fume hoods. A large array of small laboratory tools that are used routinely include pipettes, Bunsen burners, cell spreaders, streaking loops, and thermometers. Supplies and disposable items used in various exercises are also part of the laboratory environment.
Students will be able to complete various laboratory exercises successfully when all required equipment is available and in working order. To this end, a list of required equipment is included with each laboratory exercise in this manual. The students must understand the function of each piece of equipment and how to use it safely and correctly.
Food microbiology involves the study of numerous organisms, including those known to cause human diseases. Therefore, careful work habits are important to prevent the spread of disease to analysts or other workers who may use the laboratory space. Familiarity with the laboratory environment itself and with the procedures required to keep that environment safe and clean is a key component in good microbiology laboratory work.
Biosafety Levels
The health authorities in the US have created a publication regarding biosafety in microbiological and biomedical laboratories (see the reference listed at the end of this chapter). This manual sets forth guidelines for best practices for four biosafety levels. Each biosafety level has a particular set of protocols that laboratory users must follow to minimize risks to the laboratory workers and the public. There are protocols for primary containment (protection of individual workers) and secondary containment (protection of the public). The protocols provide guidance on laboratory security, laboratory practice and technique, required safety precautions, facility design and construction, and required training of supervisors and workers, as well as specific information regarding animal facilities, clinical facilities, and transportation of materials. The publication also contains listings of specific organisms and their assignment to risk groups (RG) with their recommended biosafety level. The following is a brief description of the four biosafety levels.
Biosafety level 1 (BSL‐1)
Appropriate for handling agents (RG‐1) that are not known to cause disease and are well characterized; examples include
Bacillus subtilis
and non‐pathogenic
Escherichia coli
.
Required protective devices include doors, sinks for handwashing, easily cleaned work surfaces, screened windows, and bench tops that are impervious to water.
No special construction or ventilation is required.
Primary barriers (lab coat and gloves) are required.
Biosafety level 2 (BSL‐2)
Appropriate for handling moderate‐risk agents (RG‐2). These include agents associated with human disease, but for which immunization or antibiotic treatment is available; examples include
Salmonella
and Measles virus.
All precautions for BSL‐1 are observed, plus doors must be lockable and marked with biohazard signs, eyewash station is available, air does not recirculate to non‐laboratory areas, and autoclave is available.
Special work areas (e.g., biosafety cabinets) should be assigned for activities that might generate aerosols or splashing and for handling large volume or high concentration of organisms.
Biosafety level 3 (BSL‐3)
Appropriate for handling agents that may cause serious and potentially lethal infections (RG‐3). These include agents that are transmittable by aerosols. Examples of these agents are
Mycobacterium tuberculosis
and St. Louis encephalitis virus.
All precautions for BSL‐2 are observed plus separate facility or zone with double door entry; inward only airflow with 10–12 air changes/hour; water‐resistant walls, floors, and ceilings; filtered vacuum lines; and respiratory protection may be required.
Biosafety level 4 (BSL‐4)
Appropriate for handling exotic agents (RG‐4) that (a) pose high risk of life‐threatening diseases, (b) are transmittable by infectious aerosols, and (c) for which no treatment is available. An example of these agents is Ebola virus.
All precautions for BSL‐3 are observed plus single‐pass dedicated air system; walls, ceilings, and floors create an internal seal; all liquid effluent and solid waste are decontaminated; entry doors cannot be opened simultaneously; communication system; emergency generator, positive pressure personnel suit, and showering upon exit are required.
Note: Food microbiology laboratories that accommodate pathogen work are classified as BSL‐2.
Decontamination and Waste Disposal
Contaminated but reusable laboratory utensils and glassware should be decontaminated before cleaning. Chemical disinfection or autoclaving are often used in this case. Contaminated disposable items (e.g., disposable gloves, pipettes, and agar plates) should be placed in designated biohazard boxes with liners. Disposal of these biohazard boxes should be managed by a professional service, which may subject these items to incineration or other validated decontamination method.
PERSONAL SAFETY IN THE LABORATORY
It is important that students become familiar with the personal protective equipment required for working in a microbiology laboratory as well as safety‐associated procedures and etiquette.
Personal Protective Equipment
Personal protective equipment (PPE) is needed to protect against physical, chemical, and biological laboratory hazards. Availability of this equipment is important but adequate training on how to use it is equally important. The following is a partial list of PPE, but others may be needed:
Safety glasses:
These should be cleaned and sanitized before and after use. Alcohol wipes may be adequate for the sanitization.
Laboratory gowns or coats:
These should be used and kept in the laboratory.
Face masks and face shields:
Masks and shields protect against splashes and aerosolized droplets and particles. These are also essential to prevent the spread of infectious airborne agents.
Gloves:
Use of disposable gloves is essential for most laboratory activities.
Observing Personal Safety
Appropriate attire.
Wear appropriate clothing for laboratory work. Closed‐toe shoes must be worn during each laboratory period. No sandals, open‐toe shoes, or bare feet are permitted. Shorts may be disallowed.
Winter coats and backpacks are stored away from the bench.
Keep these items at a safe distance from the laboratory bench and preferably outside of the laboratory. Coat hangers, cupboards, or preferably lockers should be used to store these items temporarily while students are working in the laboratory.
Items brought to the bench are subject to contamination; these should be kept to a minimum.
Keep away all books, notebooks, calculators, laptop computers, and similar items. Technically, two sheets of paper and a pencil are all that need to be brought to the bench. One of these papers contains the laboratory exercise summary, outline, or flow chart, and other is used for recording data. It is advisable that these two sheets are kept in a transparent plastic sleeve that can be sanitized at the end of the exercise.
Mobile phones should not be handled during the laboratory period.
Laboratory coat.
It is mandatory that a laboratory coat or similar protective covering is worn during each laboratory period. The coat must have either buttons or a zipper. The laboratory coat should be labeled with the student’s name and be kept completely buttoned or zipped for the duration of the laboratory period. After completing the work, coats should be kept in the laboratory. The instructor will point out appropriate coat storage, if available. Before coats are removed from the laboratory, they should be properly decontaminated; this can be accomplished by autoclaving.
Eye protection.
Safety goggles should be worn all the time in the laboratory.
Washing hands.
Washing hands minimizes or prevents the transfer of organisms between the analyst and the food to be analyzed and vice versa. Hands should be washed before starting any exercise (to avoid contaminating items being analyzed) and after the exercise (to prevent spreading contaminants outside the laboratory).
Use disposable gloves.
Even though disposable gloves may not be required for some experiments, it is advisable to wear these gloves for all activities in the laboratory. Analysts who have allergies to latex should wear gloves made of alternative materials (e.g., nitrile rubber). Once the work is completed, the gloves should be disposed of properly in the appropriate biohazard containers. Analysts should never leave the laboratory with gloves on. It is a sign of great carelessness when analysts are seen in hallways or elevators wearing disposable gloves. This can also be the cause of serious cross‐contamination on non‐laboratory surfaces, such as doorknobs.
Clean and sanitize the laboratory bench.
Use disinfectant and paper towels to wipe the laboratory bench both before and after any exercise. These paper towels should be disposed of in the regular trash, unless directed otherwise by the instructor.
Never begin laboratory work without the prior permission of the laboratory instructor or supervisor.
Generally, students are not allowed to work until
after
the instructor’s presentation on the day’s activities. If arriving early, the student may use this time to change into appropriate dress, review the exercises to be completed, inspect the laboratory for locations of needed equipment, and similar activities.
Eating or drinking in the laboratory is forbidden.
The laboratory environment is not an appropriate place for eating or drinking. In fact, any activity that might involve putting something into the mouth, (e.g., chewing gum, chewing tobacco, using a throat lozenge, smoking, habitually chewing on a pencil) may provide an opportunity for a pathogen to infect the analyst.
Applying cosmetics in the laboratory, including lip balm or lotion, is not allowed.
Anything that is applied may trap contaminants on the skin or introduce contaminants into the laboratory environment. Insertion of contact lenses is not permitted in the laboratory.
Avoid touching eyes, skin, or hair, particularly with worn gloves.
These activities can lead to body contamination with harmful microorganisms.
Miscellaneous.
Sitting on the laboratory bench is not permitted. Keep the laboratory as neat as possible at all times. At the end of each laboratory period, check and arrange all materials neatly. Return all materials to their proper places or dispose of them appropriately when your work is finished.
Never remove equipment, media, or microbial cultures from the laboratory.
Label all materials properly so that they can be identified easily.
Tubes should be labeled using label tape and a marker. Petri dishes should be labeled on the bottom (the side with the agar) with student name, the organism, type of medium, incubation temperature, and date.
Use pipettes carefully.
Pipettes can be hazardous if not used properly. Mouth pipetting is both a poor technique and a safety hazard; therefore, it is not permitted. Pipette bulbs, manual pipette aids, and semiautomatic pipetters (with pipette tips) are available for use. Forcing a pipette into either a bulb or a pipette aid may lead to breakage and should be avoided. It should be cautioned that improper use of pipettes can lead to dripping or generation of hazardous aerosols.
Be familiar with the available safety equipment and supplies.
Know the locations of the first‐aid kit, safety showers, eyewash stations, fire extinguishers, fire blankets, and fire alarms.
Avoid fire hazards.
Hair that is shoulder length or longer must be tied back or pinned up to minimize the risk of it catching on fire. Similarly, hats with brims should be avoided as the brim might come near the flame. Hats such as baseball caps may be worn facing backward to keep the brim away from flames. For safety, constantly be aware of any burners near you. Always use your own burner. Do NOT reach across the bench to use someone else’s burner. Some of the liquids present in the laboratory are flammable; keep these away from the Bunsen burner.
Handling fire emergencies.
Students should be aware of the location of available fire safety equipment (e.g., fire extinguishers, fire blankets) and the nearest exits in case of larger fires.
Alcohol fires are among the most common laboratory fires. Should a jar of alcohol catch fire, placing the lid over the jar quickly may suffocate the fire. Alternatively, cover the burning jar with a slightly bigger glass jar, such as a beaker. Keep flame away from staining bottles as these often contain alcohol.
If anyone’s hair or clothing should catch on fire, obtain a fire blanket, wrap the person in the blanket, and have them roll on the floor to extinguish the flames.
Any fire should be reported immediately to the laboratory supervisor.
If a major fire occurs, proceed to the nearest exit. DO NOT USE ELEVATORS!
Handling first‐aid emergencies.
Students should be aware of the location of the first‐aid kit in the laboratory. The kit should contain gauze bandages, adhesive bandages, bandage tape, sterile swabs, burn cream, antiseptic wipes, and hydrogen peroxide.
Get the instructor’s assistance before using the first‐aid kit.
Mercury spills.
While many laboratories have switched from mercury to alcohol thermometers, some laboratories may still be using mercury thermometers. Mercury is a hazardous material that requires special cleanup procedures. If a mercury thermometer is broken, the analyst should notify the instructor immediately. The students in the vicinity of the spill should not touch the mercury, should move away from the area, and should prevent others from entering the area.
No bicycles, skateboards, roller skates, or similar devices are permitted in the laboratory or the hallway.
These items can create a tripping hazard in the laboratory, to passersby in the hall, or during an emergency.
Report any personal injuries to the laboratory instructor/supervisor.
In case of accidents, mandatory accident reporting forms must be filled out as soon as possible.
MATERIALS IN MICROBIOLOGY LABORATORY
In many teaching laboratories, each student (or a group of students) is assigned a storage drawer (or a similar compartment) containing materials commonly used in the laboratory. Students should be sure that the drawer contains all the materials indicated in the course instructions and that all materials are returned and stored at the end of each session. Typical tools contained in this storage space may include inoculating loop, inoculating needle, microscope slides, cover slips, microscope lens cleaner, lens cleaning paper, lens oil, wax marking pencils or permanent markers, pipette bulbs, bibulous paper, and matches or a striker for lighting the Bunsen burner. Some of the consumable materials may be used up during the course of the term and students should learn where replacement materials are kept. At times, the items from a storage location may be misplaced. If this occurs, the student should not take supplies from someone else’s drawer.
Some laboratory communal supplies may also be used up during the course of the school term. These items may include paper towels, disinfectant solutions, Gram stain reagents, other staining agents or reagents, adhesive tape, and other frequently used materials. Students should determine where these items are stocked so that they can replenish supplies.
Students should know where to obtain distilled water. In most laboratories, special distilled water taps are used; these are often located near the regular hot‐ and cold‐water taps. The distilled water taps are frequently spring‐loaded to prevent anyone from leaving the tap open and wasting water. Often these taps have a tab on the handle labeled “DW.”
At the beginning of every laboratory session, the student should determine the location of all water baths, incubators, or other equipment that will be shared during that session. Students should collect all media and supplies required to perform the experiment. Many microbiological growth media look similar; therefore, caution should be taken to carefully and correctly label media. Students should not collect more media than will be used during the exercise. Careful reading of the laboratory exercise should allow students to determine the correct number of plates and tubes needed for each exercise.
The microbiology laboratory contains many materials that are potentially dangerous if used outside the laboratory environment. Students should never remove slides, plates, or tubes from the laboratory. After use in the laboratory, materials are either prepared for reuse or discarded. Each laboratory has a system for material disposal, protocols for which items are reused and which are discarded, locations where reusable materials should be placed at the end of the laboratory, expectations for what to clean manually by students, etc. Students must be familiar with proper disposal and proper clean up to ensure that materials are not wasted, biohazard containers do not contain excess materials, and everyone’s safety is preserved.
Used culture tubes should not be returned to the laboratory exercise set‐up area, unless the instructor specifically tells students to do so. Only unused media should be returned to the set‐up area.
Reusable materials may include some glassware, such as test tubes, bottles, and flasks. This reusable glassware should then be placed in the designated location for each type of item. Depending on their contents, tubes, bottles, and flasks may need to be autoclaved before washing. These items should be separated from items that do not require autoclaving. Some other items, such as blender jars, may not require autoclaving and may be manually washed by students. These items should be washed according to the designated protocol and placed in the designated drying area.
Non‐reusable materials are disposed of in either hazardous or non‐hazardous waste containers. Paper towels used with disinfectant to wipe off laboratory benches may be placed in the containers for non‐hazardous waste (i.e., regular trash). Gauze or lens paper used to clean microscope lenses before or after use is also safe to be placed in the regular trash. Items that have not been exposed to microorganisms do not require special disposal.
Biohazard containers
(e.g., special marked bags in cardboard boxes or cans with plastic liners) are used to discard contaminated materials. These materials include all disposable gloves, culture‐containing disposable Petri plates, and disposable test tubes. Contaminated materials (i.e., those exposed to laboratory microorganisms) are typically autoclaved or incinerated. Contaminated broken glassware should be disposed of in the sharps container. Broken, uncontaminated glassware should be placed in a receptacle designated for that purpose (e.g., the broken glass box). If syringes are used for a laboratory exercise, they should be disposed of in the sharps container designated by the instructor.
Spilling or splashing of cultures can happen. In case of small spills, the student needs to encircle and flood the area with excess disinfectant, allow disinfectant to sit for the proper amount of time, and wipe the area with paper towels or other provided absorbent towels, wiping toward the center to prevent spread of the contaminant. These towels are considered contaminated and should be disposed of in the biohazard container. In case of larger spills, the instructor should be notified immediately. Any broken glassware should be disposed of in a sharps container. Appropriate disposable gloves should be worn during the cleaning process and should be discarded after the spill has been addressed.
It is recommended that students tape their inoculated agar plates together at the end of each exercise or keep them in a designated group container (small plastic bin) to make retrieving the group’s plates easier at the beginning of the subsequent session and make them easier to handle and inspect by instructors. Plates should be placed in the correct orientation and in the designated location for incubation.
PRACTICAL ASPECTS
In addition to keeping yourself and other laboratory members safe, the proper exercise of safety protocols and etiquette allows for the timely completion of laboratory sessions. Lack of preparation before arriving to the laboratory may prevent students from finishing the exercise within the allotted time.
Before the Laboratory Session
Carefully read the laboratory exercise and understand why and how it is executed.
Summarize the practical steps to be carried out during the session on a single sheet of paper. This “exercise summary” should be one of only two papers allowed on the bench during the execution of the exercise. The second is a blank paper for writing observations and recording results. As indicated earlier, the exercise summary (plus the recording sheet) are ideally kept in a plastic sleeve and placed on the bench while executing the exercise. The plastic sleeve should be sanitized properly before leaving the laboratory.
Immediately Before Entering the Laboratory
Finish or dispose of any food or drink items/containers.
Turn off electric devices (e.g., mobile phones, laptop computers, tablets, etc.); these should be stowed appropriately for the duration of the laboratory session.
Immediately After Entering the Laboratory
Enter the teaching laboratory when the instructor/supervisor is available; more than one instructor should be available to supervise the session.
Keep your belongings (e.g., backpack, winter coat, etc.) in the designated area, which is preferably outside the laboratory; bring only the exercise summary and a blank sheet of paper to the bench.
Put the exercise summary (and the blank sheet) in the provided plastic sleeve; this is a sanitizable pocket for protection against spills. Present the exercise summary on the bench to be reviewed by the instructor.
Hair that is longer than shoulder length must be tied up.
Wash hands in the laboratory sink using the soap and disposable towels provided.
Put on a lab coat; when not in use, these should be stored in the laboratory throughout the course.
Put on disposable gloves and sanitize the bench; a quaternary ammonium solution or alcohol is often used for bench sanitization.
Listen carefully to the instructor’s short presentation; this presentation may include seating chart, assignment for the food to be analyzed, potential pitfalls, etc.
Start the exercise when instructed to do so.
While Executing the Exercise
Be aware of whether you are working individually or in groups of two or more. If working in groups, part of the work could be carried out individually and the other part is done cooperatively. If working in a group, make sure you communicate clearly with laboratory partner(s) before starting the exercise.
Start the laboratory exercise and observe the safety rules described earlier.
Do your best to complete the work efficiently and diligently.
Make sure you share the progress of the exercise or problems encountered with one of the instructors.
Record your observations or results. The exercise summary sheet or a separate sheet of paper may be used for recording. Alternatively, hand‐held electronic notepads may be provided by instructors for note taking and data collection.
Immediately After Completing the Laboratory Exercise
Show your work (mounted microscope slide, reaction results, colony counts, etc.) to the instructor.
If asked, transfer the data collected to the class computer or class data sheet.
Dispose of work items correctly.
Sanitize the bench using the sanitizer provided (often a quaternary ammonium sanitizer or alcohol).
Remove disposable gloves and place them in the biohazard container.
Store lab coat appropriately.
Wash hands.
Take your belongings and exit the laboratory.
REFERENCE
Centers for Disease Control and Prevention. (2020).
Biosafety in microbiological and biomedical laboratories
. 6
th
ed. U.S. Department of Health and Human Services, Washington, DC, USA.
CHAPTER 2SAMPLING FOR MICROBIOLOGICAL ANALYSIS OF FOOD AND PROCESSING ENVIRONMENT
It is a challenge to be able to assess the microbiological quality and safety of food accurately. The approach often used is a stepwise procedure that includes sampling, sample preparation, laboratory analysis, data collection, and result interpretation. Errors in each of these steps cumulatively determine the reliability of the overall procedure. Sampling can be an elaborate exercise (Figure 2.1), and analysts consider it the most error‐prone step. Poorly planned and executed sampling operations compromise the analyst’s ability to assess the quality or safety of food. This chapter includes two main sections: “Theoretical aspects,” which provides the knowledge needed for proper sampling, and “Practicing sampling and sample preparation,” which is a simplified practical exercise.
THEORETICAL ASPECTS
This section covers the theoretical principles of sampling and sample‐size calculations. Additionally, techniques that may be followed during sampling and sample preparation of food or processing environment are covered.
Sampling Principles
Introduction
In the simplest sense, a “sample” may be defined as a small and manageable quantity intended to represent the whole. The whole is commonly referred to as the “population,” and in relevance to the subject matter of this book, the population is the food lot. Foods vary considerably in physical, chemical, and microbiological characteristics. These variations dictate the way a food is sampled and analyzed. Physically, food could be in a solid, gel, or liquid state, at different degrees of hydrophobicity, with ingredients in homogeneous or heterogeneous distribution. Compositionally, foods vary in water content, pH, presence of antimicrobial ingredients, and many other attributes. Foods differ in microbial burden and profile; this depends on whether the food is raw or processed, and the type of processing it received. The goal of the analysis also varies. Some foods are analyzed for the enumeration of indicator microorganisms (e.g., coliforms), whereas others are tested for the presence of pathogens (e.g., Listeria monocytogenes). These factors must be taken into account to determine appropriate sampling, sample preparation, and microbial recovery methodology.
Figure 2.1 Food sampling.
Foods subject to analysis are usually found in sizable quantities located in a storage facility, ship container, tanker, retail display case, vendor stand in open market, etc. With consideration of the size of the food lot and expected variations among multiple samples from the same lot, a number of samples are collected for analysis on the hope that they accurately represent the entire lot. If samples taken are not a good representation of the whole, whether it is the fault of the sample collector or due to an error inherent in the sampling plan, the laboratory results will be misleading. Therefore, sampling operations should be planned well. Additionally, every effort should be made to avoid mishandling or contamination of the collected sample. Correctly withdrawn, handled, and analyzed samples may serve as evidence of the quality or the safety of the whole.
Although food is emphasized in this book, sampling and analysis of water and processing environment will also be addressed. A sample of water from a stream is described as a “specimen.” Similarly, samples from circulating cleaning or rinsing solution or swabs from a moving conveyer belt are also considered specimens. In these situations, the population sampled is not static and thus getting a representative portion is a challenging task.
Preparing A Sampling Plan
Sampling is an essential step in any procedure for assessing the microbiological quality or safety of food. Sampling is an integral part of food inspection, which is practiced for commercial or legal reasons. Researchers experimenting with food need sampling schemes that lead to statistically meaningful results. Regardless of the ultimate goal of the analysis, sampling should be planned and executed properly. The following are steps used in preparing a sound sampling plan.
Identify the Reasons for Sampling
Sampling is a key and critical step in microbiological analyses that are done for many reasons including: (i) assessing the general microbiological quality of a raw product or an ingredient; (ii) validating a food processing operation; (iii) assuring the safety of the processed food; and (iv) evaluating the sanitary condition of a food processing environment. Each of these cases require a carefully considered sampling plan.
Assess the Size, Nature, and Uniformity of the Lot to be Sampled
The population (i.e., the food lot) from which the samples are to be taken could be made of discrete units or bulk in a container. For example, a food lot of half‐and‐half coffee cream could be a stack of wholesale boxes, each containing multiple smaller retail boxes, and the latter containing multiple single‐serve (0.4 oz.) units. Alternatively, the lot could be bulk flour in a store bin or sack, milk in a tanker, or loose grains in a silo.
Sampling starts by taking a number of units from the stacks of the lot or a portion of the bulk; these are described as gross (or primary) samples (Figure 2.1). Subsets of gross samples constitute the laboratory samples. The analyst who receives laboratory samples should further reduce them to test samples. For example, a laboratory sample could be a 10 lb cheese block, from which a 25 g test sample is withdrawn. In this particular example, it is desirable to collect several test samples to overcome the lack of uniformity from the edge to the center of the cheese block. Once the test sample is subjected to laboratory analysis, it is no longer described as a “sample.” Instead, the analyst should use descriptive words such as food homogenate, test solution, cell suspension, cell pellet, culture supernatant, isolate, etc. Note that the number of samples to be withdrawn from the lot is determined as described later.
Determine the Acceptable Quality Level or the Tolerable Safety Risk
The acceptable quality or safety level should be identified before sample size is determined. A supermarket chain importing strawberries may accept only a truckload that produces less than 1% moldy samples among all the samples analyzed. Determination of “moldiness” may be done subjectively (e.g., visual inspection) or analytically (e.g., fungi count on microbiological media). Note that the latter approach is time consuming, and the product could suffer significant quality damage while waiting for results to be obtained.
Sampling for assessing food safety risks should be planned carefully. In addition to the factors discussed earlier, this sampling plan also should consider food status within the supply chain (e.g., raw or ready‐to‐eat), degree of processing (e.g., minimally processed or retorted), intended consumer population (e.g., infants or adults), and other factors. Analysts, for example, may be asked to determine the prevalence of Salmonella enterica on the surface of raw shell eggs produced in a cluster of farms that switched from a caged to a cage‐free or free‐range hen system. In a different scenario, analysts may be sampling for S. enterica in pasteurized shell egg from a company that introduced pasteurization as a new technology in egg processing and compare that with normal incidence of S. enterica in raw shell eggs. Although the food product and the targeted pathogen is the same in both examples, some pathogen‐positive samples are allowed in the first example (on‐farm), but none is allowed in the second example. Zero tolerance is common for infectious pathogens in ready‐to‐eat foods, whereas some degree of contamination is allowed in raw foods that are supposed to be cooked or processed before consumption.
Determine the Number of Samples to be Withdrawn
