Food microbiology and laboratory practice

Foo d Microbiolog y

a n d Laborator y Practic e

Chris Bell, Paul Neaves

& Anthon y P. William s

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Foo d Microbiolog y an d

Laborator y Practic e

Chris Bell

Consultant Food Microbiologist, UK

Paul Neaves

Consultant Food Microbiologist, UK

Anthony P. Williams

Consultant Food Microbiologist, UK

DAI HOC THA. NlAJrEN

TRUNGTAM KOCLIfU

Blackwell

Science

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f: Chris Bell, Paul Neaves and Anthony Williams. 2005

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Contents

Foreword ix

A cknowledgements x

Preface xi

1 The Structure and Habit of Microorganisms I

1.1 Introduction 1

1.2 Microorganisms associated with foods 2

1.2.1 Bacteria 2

1.2.2 Fungi 8

1.2.3 Viruses 13

1.2.4 Protozoa 13

1.2.5 Toxic algae 14

1.3 The origin of names 15

1.3.1 Bacterial classification 15

1.4 Microbial grouping in practice 15

1.4.1 Total colony counts 16

1.4.2 Indicator organisms 16

1.4.3 Enterobacteriaceae and coliforms 17

1.4.4 Gram-negative psychrotrophs 18

1.4.5 Lactic acid bacteria 18

1.4.6 Yeasts and moulds 19

1.5 Further reading 19

2 Factors Affecting the Growth, Survival and Death of Microorganisms 21

2.1 Introduction 21

2.2 Some important characteristics of food contaminant microorganisms 21

2.2.1 Characteristics that can be studied in the laboratory 21

2.2.2 Characteristics that inhibit study in a (normal routinel

laboratory 22

2.3 The characteristics of microbial growth 23

2.3.1 The factors that affect microbial growth 24

2.4 Further reading 39

3 Fundamentals of the Microbial Ecology of Foods I: Food Spoilage and

Food-borne Illness 40

3.1 Introduction 40

3.2Số hóa bởi Trung tâm Học liệu – ĐHTN http://www.lrc-tnu.edu.vn Microbial contamination - sources, routes and control 40

iv Contents

3.3 The fate of microorganisms in food 42

3.4 The consequences of microbial growth in foods 43

3.4.1 Food spoilage 43

3.4.2 Food-borne illness 50

3.5 Further reading 56

4 Fundamentals of the Microbial Ecology of Foods II: Food Preservation and

Fermentation 58

4.1 Introduction 58

4.2 Controlling shelf life by preservation systems 59

4.2.1 Temperature of processing and storage 60

4.3 Microbial fermentations and biotechnology 62

4.4 Further reading 67

5 Applieations of Microbiology in the Food Industry 68

5.1 Introduction 68

5.2 Hazard Analysis Critical Control Point (HACCP)-based Systems

and Microbiology 68

5.3 Risk assessment and microbiology 75

5.4 Raw food materials/ingredients and microbiology 77

5.5 Hygiene monitoring and microbiology 79

5.5.1 Swabs 80

5.5.2 Solid or liquid samples 82

5.5.3 Personnel 83

5.5.4 Air 83

5.5.5 Test results and their interpretation 84

5.6 Process monitoring and microbiology 84

5.7 Finished products and microbiology 86

5.7.1 Conformance to microbiological criteria 86

5.7.2 Product shelf life evaluations 87

5.7.3 Microbiological challenge testing 90

5.8 Trouble-shooting, crisis management and microbiology 91

5.9 Further reading 92

6 Laboratory Design and Equipment 93

6.1 Introduction 93

6.1.1 Standards required for the design and construction of a

microbiology laboratory 93

6.2 The building 94

6.3 Internal structure, fittings and services 96

6.4 Work flow 99

6.5 Equipment 100

6.6 Further reading ]]3

7 Laboratory Operation and Practice 115

7.1 Introduction 115

7.2 Standar Số hóa bởi Trung tâm Học liệu – ĐHTN http://www.lrc-tnu.edu.vn d operating procedures 115

Contents v

7.3 Laboratory staff and personal practices 116

7.4 Laboratory housekeeping 121

7.4.1 General 121

7.4.2 Laboratory waste disposal 121

7.4.3 Environmental monitoring 122

7.5 Laboratory quality standards and procedures 122

7.5.1 General 122

7.5.2 Laboratory assessment / accreditation and external quality

assessment (proficiency testing) 123

7.5.3 Practices 124

7.5.4 General procedures 125

7.5.5 Reference cultures 139

7.5.6 'Uncertainty of Measurement' of microbiological test

results 145

7.6 Further reading 147

8 Laboratory Standards of Operation: Accreditation and Documentation 148

8.1 Introduction 148

8.1.1 Laboratory accreditation: assessment criteria 148

8.2 Standard operating procedures 155

8.3 Sample processing documentation 155

8.4 Summary 171

8.5 Further reading 171

9 Conventional Microbiological Methods I: Equipment, Basic Techniques

and Obtaining Samples 172

9.1 Introduction 172

9.2 Basic tools of the food microbiologist 173

9.2.1 Sampling equipment and terminology 173

9.2.2 Pipettes 175

9.2.3 Loops, wires and spreaders 175

9.2.4 Other laboratory equipment 176

9.3 Microbiological media 179

9.3.1 Diluents 180

9.3.2 Liquid growth media 180

9.3.3 Gel (solid) growth media 183

9.3.4 General purpose growth media 183

9.3.5 Enrichment media 184

9.3.6 Pre-enrichment media 184

9.3.7 Diagnostic media 185

9.3.8 Preparation of microbiological media 186

9.4 Basic techniques of food microbiology 186

9.4.1 Aseptic technique 186

9.4.2 Pouring an agar plate 188

9.4.3 Streaking an agar plate 193

9.4.4 Slopes and stab technique 194

9.4.5 Detection of gas production in broth cultures 196

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vi Contents

9.4.6 Sterilisation and disposal 197

9.5 Incubation conditions 201

9.5.1 Atmosphere composition 201

9.5.2 Incubation temperature 204

9.5.3 Incubation time 205

9.6 Microbiological techniques 205

9.7 Obtaining and handling samples 207

9.7.1 Laboratory handling of factory samples 209

9.7.2 Statistical sampling 209

9.8 Safety in the food microbiology laboratory 210

9.9 Further reading 211

10 Conventional Microbiological Methods II: Microbiological Examination

of Samples 212

10.1 Introduction 212

10.1.1 The need for confirmation 212

10.2 Enumeration techniques 213

10.2.1 Colony counts 213

10.2.2 Most probable number (MPN) techniques 224

10.3 Detection tests 227

10.3.1 Sensitivity of detection tests 227

10.3.2 How to do a detection test for Salmonella 229

10.4 Environmental monitoring 231

10.4.1 Swabs 231

10.4.2 Contact plates, dip slides and exposure (settle) plates 232

10.5 Recognition of microbial growth after incubation 233

10.5.1 Assessment of microbial growth and reactions 233

10.5.2 Growth on non-selective media 234

10.5.3 Reactions on selective-diagnostic media 235

10.6 Automation and proprietary tests 237

10.6.1 Automation of repetitive procedures 237

10.6.2 Proprietary tests 237

10.7 Further reading 238

11 Confirmation Tests 240

11.1 Introduction 240

11.2 Preliminary confirmation tests 241

11.2.1 General 241

11.2.2 The microscope and microscopy 244

11.2.3 Staining techniques 248

11.2.4 Microscopy using live cultures 251

11.3 Basic biochemical tests 253

11.3.1 The catalase test 253

11.3.2 The oxidase test 254

11.3.3 Tests for carbohy drate utilisation 255

11.4 Further biochemical tests 257

11.4.1 Oxidation-Fermentation (O-F) test 257

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Contents vii

11.4.2 Indole test 258

11.4.3 Methyl red test 258

11.4.4 Voges-Proskauer test 259

11.4.5 Citrate utilisation 259

1 i .4.6 ONPG test 260

11.4.7 Decarboxylation of lysine and ornithine 260

11.4.8 Hydrogen sulphide production 261

11.4.9 Urease test 262

11.4.10 Gelatin liquefaction 262

11.4.11 Nitrate reduction 263

11.5 Some confirmation tests for specific organisms or groups

of organisms 264

11.5.1 Growth in different atmospheres 264

11.5.2 Heat-resistance 265

11.5.3 Coagulase test 265

11.5.4 Eijkman test 265

11.5.5 Slide agglutination test 266

11.5.6 CAMP test 267

11.6 Some examples of confirmation test profiles 268

11.7 Proprietary kits and reagents 268

11.7.1 Miniaturised, conventional tests 272

11.7.2 Other proprietary tests 273

11.8 Control cultures and other performance checks 274

11.8.1 Control cultures for diagnostic reactions on isolation

media 274

11.8.2 Control cultures for basic confirmation tests 274

11.8.3 Control cultures for biochemical confirmation tests 275

11.8.4 Other performance/quality monitoring procedures 275

11.9 Further reading 276

12 Introduction to'Alternative'Microbiological Methods 278

12.1 Introduction 278

12.2 The evolution of'alternative'microbiological methods 280

12.3 The principles and applications of some 'alternative'

microbiological methods 283

12.3.1 Direct counts 283

12.3.2 Detection of metabolic activity during sample incubation 285

12.3.3 Detection of metabolic activity without sample incubation 287

12.3.4 Detection of cell components 289

12.3.5 Fingerprinting methods 292

12.4 Microbial toxins 295

12.5 Further reading 296

Glossary of terms 297

References 307

Index 311

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Forewor d

It gives me great pleasure to write the foreword of this excellent training aid for those

working in food industry laboratories and those meeting the training needs of such

laboratory personnel. The authors collectively have a vast amount of experience both

in the laboratory practice of food microbiology and in the teaching of the subject and

are also much-valued consultants in the fields of food hygiene and food safety. Their

aim in producing this book has been to provide basic information and sources for

further information in order to help in the development of high standards for food

microbiology technicians for the next generation and the future generations to come.

They have succeeded admirably.

Many texts tend to concentrate on a specific area of laboratory practice, for

example food microbiology methods or laboratory design and operation. Here the

authors have discussed in a single volume all the practical aspects relevant to the

operation of a food microbiology laboratory as would be required for the testing of

factory samples of food and their ingredients. In twelve, well illustrated, chapters they

describe basic food microbiology including behaviour of organisms, food spoilage,

food-borne illness, food preservation and food industry applications, HACCP.

laboratory design, equipment, operation and accreditation and methods both con￾ventional and 'alternative'. Each topic is described in terms that are clear and easy to

understand and is supported by many figures and tables and also suggestions for

further reading. The structure of the book is logical and will take those with a minimal

knowledge of the subject forward to a fuller understanding of not only of what to do

and how to do it but also that ever important 'why it is done' and finally what the

results mean. As a food microbiologist of some 35 years experience I learned many

new facts from this book as well as having my memory jogged about the information I

had all but forgotten.

My overall impression of this book is that it covers the what, how and why of the

basic microbiology of food and its examination in an easy to read and understand

manner yet it also stimulates the reader to look further to expand on the knowledge

gained. It has a very 'common sense' approach and it will be an invaluable tool to

laboratory technicians in the food industry and to those teaching the subject.

Dr Diane Roberts BSc, PhD, CBiol, FIBiol, FIFST.

Former Deputy Director

PHLS Food Safety Microbiology Laboratory

Central Public Health Laboratory

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Acknowledgements

The authors thank the following companies for their kind help and support in the

supply of information for use in this book:

Astec Microflow Ltd

bioMericux SA

BioTek Instruments Ltd

Celsis Ltd

Don Whitley Scientific Ltd

Hygiena International Ltd

Millipore (UK) Ltd

Norpath Laboratories Ltd

Novation Ltd

PriorClavc Ltd

Pyser-SGI Ltd

Scientific Laboratory Supplies Ltd

Seward Ltd

Sterilin Ltd

Tecra Diagnostics UK

Westward Laboratories Ltd

Woodside Consulting

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Prefac e

The ever-increasing public interest and concern over food safety, as well as com￾mercial pressure to improve food quality and extend product shelf life, has imposed

more responsibility and pressure on all those involved in the microbiological exam￾ination of foods and related samples. The examinations, whether in public health

laboratories, food industry or contract testing service laboratories, serve a number of

purposes.

Microbiological examination of foods forms an essential part of product shelf life

assessment, food product challenge testing in relation to microbiological safety and

new product development. Microbiological examination of foods in which problems

have occurred or are suspected to have occurred may help to discover the causes of

outbreaks of illness, reasons for, or failure to achieve shelf life or non-conformance to

product specifications and the causes of customer complaints. Although in recent

years there has rightly been a shift in emphasis away from end-product micro￾biological testing to process control through the use of HACCP and similar systems,

there remains a place for microbiological examination for verifying that the pro￾duction processes are functioning as planned. This includes supplier performance

monitoring, environmental and personal hygiene monitoring and process perfor￾mance verification to provide data for trend analysis.

Whatever the reason for the examination, there is an absolute need to provide

reliable microbiological test results. To do this requires laboratories to develop and

maintain high standards in the design and construction of the laboratory facility and

the testing environment, and also of equipment, tools and materials, methods, staff

practices and documentation.

The customers of any microbiology laboratory, whether the smallest of factory

laboratories or the largest of contract testing service laboratories, rely upon the

laboratory to provide a 'true' result. As a consequence of microbiological test results

that are 'out of specification', particularly where human pathogens are involved.

actions may be taken by customers, ranging from increasing the levels of process

checking or equipment cleaning to more commercially costly product rejection, ces￾sation of production on a particular manufacturing line or even, factory closure. All

actions, though, carry some cost.

It is essential, therefore, that the laboratory staff and management are confident

that their facilities, methods, procedures and, importantly, staff competence and

practices will, and can be demonstrated to, deliver consistently 'true' information to

their customers so that relevant decisions can be taken in relation both to food safety

and to wholesomeness.

In order to maintain the high standards required, staff must be suitably trained to

understand what they are to do, how they are to do it and why they must do it in a

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xii Preface

prescribed way. A properly trained microbiology technician provided with the right

tools, equipment and environment is a valuable asset and makes a positive con￾tribution to the reliability of test results and ongoing confidence of the laboratory's

customers.

Too many incorrect or inaccurate results have been, and still are. attributable to

poor laboratory practices, and a lack of knowledge and understanding about

microorganisms and related microbiological quality and safety consequences. Such

mistakes have in the past damaged the food industry and food microbiology

laboratories severely, both commercially, as well as in reputation and lost jobs.

This book is written as an aid for teachers, trainers and trainees in food micro￾biology laboratories and on practical food microbiology training courses. It aims to

provide the basic information and further information sources that will help in the

development of high standards for the next and future generations of food micro￾biology technicians.

Plate section

Illustrations referred to in the text as plates arc to be found in one of the two colour

plate sections. A number of photomicrographs are taken from real laboratory

investigations and reflect the range of shapes and distribution of cells that may be

encountered in routine food microbiology work.

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1 Th e Structur e an d Habit o f Microorganism s

1.1 Introduction

Free-living creatures can be divided into five Kingdoms comprising animals, plants,

algae and protozoa, fungi and bacteria. Various classifications have been proposed,

but the simple grouping as follows is useful:

• Animalia (animals)

• Plantae (plants)

• Protista (algae and protozoa)

• Fungi (yeasts, moulds, mushrooms and toadstools, rusts and smuts, wilts)

• Monera (bacteria and related organisms).

The term 'microorganism' applies to members of three of these Kingdoms, namely the

Protista, the Monera and the Fungi, together with other biological forms not included

in the free-living Kingdoms, such as viruses and prions. All microorganisms have one

essential characteristic in common: all or part of their free-living structure is very

small. A precise definition of what constitutes 'small' probably does not exist but

most microbiologists would agree that a maximum value of around 100 microns (um)

might be appropriate (I pm [I micron] = l / 1000mm), although many micro￾organisms are between I and 10 pm in size and some (viruses, that are not free-living)

can be around one fiftieth (l / 50) pm or even smaller.

In descending order of size, the major groups of microorganisms comprise the

protozoa and algae, the yeasts and the spores of moulds, the bacteria and, finally, the

viruses. Single cells of microorganisms cannot be seen with the naked eye, but the

protozoa, algae, moulds and yeasts can be seen easily with a relatively low power of

magnification, e.g. x 100—400, and indeed when moulds and yeasts are fully grown

into a cluster known as a colony, they become clearly visible to the naked eye (mould

on bread or cheese is a familiar example). All of these organisms are capable of self￾replication through complex mechanisms of sexual and asexual reproduction. They

possess a true nucleus enclosed in a nuclear membrane that contains their genetic

material within complex chromosomes, i.e. they are eukaryotic.

The bacteria are also capable of self-replication but possess simple chromosomes

and no nuclear membrane, i.e. they arc prokaryotic; their cell division does not

normally involve sexual reproduction, although many are. in fact, capable of some

form of sexual activity. Bacteria are smaller than most eukaryotes and can only be

seen clearly as individual cells with the aid of the higher powers of a light microscope.

Plates 1-6 are located in the colour plate section at p. 100.

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2 Chapter 1

e.g. x 1000 magnification. However, like moulds and yeasts, when bacteria are grown

into a cluster known as a colony, they also become clearly visible to the naked eye.

Viruses can only replicate within the cells of a host organism, i.e. they are parasitic.

They arc parasites of members of the free-living Kingdoms and are even smaller and

simpler than bacteria, and it requires the high magnification of an electron micro￾scope ( x 10000 and higher) in order for them to be seen.

In recent years, prions have been described as the causal agent of bovine spongi￾form encephalopathy (BSE - 'mad cow disease') and related illnesses. These contain

no genetic material in the conventional sense, i.e. DNA (deoxyribonucleic acid) or

RNA (ribonucleic acid); they appear to be distorted protein molecules and are so

small that, to date, even the most powerful electron microscopes have been unable to

allow them to be seen.

Plate 1 (see colour plate sections for all plates) shows some representatives of the

Microbial Kingdoms. A comprehensive description of the features of the various

microorganisms is outside the scope of this book and the reader is referred to Adams

and Moss (2000) for more detailed information.

1.2 Microorganisms associated with foods

Food microbiology is the study of the microorganisms associated with foods. Bac￾teria, yeasts and moulds are mostly capable of growth in food whilst, although viruses

and protozoa cannot grow in food, their physical transmission via food has long been

recognised. However, in recent years, our ability to investigate the agents responsible

for food-borne illness has developed considerably, and different types of protozoa.

the toxins produced by algae and histamine produced in foods through bacterial

growth now also need to be understood in relation to food safety by the food

microbiologist, as well as prions, even though the latter may not be microorganisms in

the true sense of the word.

In practical food microbiology, therefore, it is important to understand the nature

and ecology (relationship of organisms to their surroundings) of different microbial

groups as well as the basic details of microbial structure and function. Where growth

of an organism occurs in a food material, the food microbiologist needs to know

whether the outcome could be food spoilage, food-borne illness (from infections or

from toxins) or food enhancement, e.g. in the form of a desirable fermentation. It is

also important to understand that some organisms can be studied easily in a routine

microbiology laboratory, while for others, special facilities such as controlled con￾tainment may be needed for safe handling of the organism. Access to databases for

help in identifying an organism or for obtaining information relating to outbreaks of

illness may also be required.

1.2.1 Bacteria

Morphology and cell staining

Bacteria are the single most significant group of microorganisms in food micro￾biology; they are prokaryotes. with a rather simple structure and relatively little

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