Method CMMEF

American Public Health Association
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2015 by the American Public Health Association
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of this volume.
Georges C. Benjamin, MD, FACP, FACEP(E), FNAPA, FRSPH(Hon), FFPH(Hon), Executive Director
Printed and bound in the United States of America

Typesetting: The Charlesworth Group
Cover Design: Alan Giarcanella
Printing and Binding: Sheridan Books, Inc.
Library of Congress Cataloging-in-Publication Data
Compendium of methods for the microbiological examination of foods / editors, Yvonne Salfinger and
Mary Lou Tortorello. - - Fifth edition.
p. ; cm.
Microbiological examination of foods
Includes bibliographical references and index.
ISBN 978-0-87553-273-8 (hardcover)
I. Salfinger, Yvonne, editor. II. Tortorello, Mary Lou, 1951-, editor. III. American Public Health
Association, issuing body. IV. Title: Microbiological examination of foods.
[DNLM: 1. Food Microbiology- -Laboratory Manuals. 2. Food Analysis- -methods- -Laboratory Manuals.
3. Food Contamination- -analysis- -Laboratory Manuals. 4. Food Inspection- -methods- -Laboratory
Manuals. QW 25]
QR115
664.0019579- -dc23
2015016836
Contents
Editorial Board .............................................................................................................................. xvii

Authors ........................................................................................................................................... xix
Reviewers ...................................................................................................................................... xxiii
Preface ........................................................................................................................................... xxv
Section I General Methods
Chapter 1 Laboratory Quality Management Systems .............................................................. 3
1.1 Introduction ............................................................................................................................... 3
1.2 The Role of Management in Laboratory QA ............................................................................ 3
1.3 Laboratory Operational Framework and QC Requirements ...................................................... 5
1.4 Personnel ................................................................................................................................. 6
1.5 Equipment/Instrumentation ...................................................................................................... 8
1.6 Laboratory Glassware and Plasticware .................................................................................... 8
1.7 Sample Management ............................................................................................................... 8
1.8 Analytical Methods .................................................................................................................. 9
1.9 Culture Media and Reagent Preparation or Test Kits ............................................................... 9
1.10 Accreditation of Testing Laboratories ..................................................................................... 10
Chapter 2 Sampling Plans, Sample Collection, Shipment, and Preparation
for Analysis ................................................................................................................ 13
2.1 Introduction ............................................................................................................................ 13
2.2 General Considerations .......................................................................................................... 13
2.3 Equipment, Materials, and Reagents ..................................................................................... 13
2.4 Precautions ............................................................................................................................ 14
2.5 Procedures ............................................................................................................................. 14
Chapter 3 Microbiological Monitoring of the Food Processing Environment ..................... 27
3.1 Introduction ............................................................................................................................ 27
3.2 Environmental Sampling Strategies ........................................................................................ 28
3.3 Sampling of Surfaces: Equipment and Physical Plant ............................................................ 29
3.4 Principle of Monitoring the Microbiological Flora ................................................................... 30
3.5 Precautions ............................................................................................................................ 30
3.6 Limitations ............................................................................................................................. 30
3.7 Rinse Solution Method for Sampling Containers and Processing Equipment Systems ......... 30
3.8 Surface Contact Methods ...................................................................................................... 31
3.9 Microbiological Air-Sampling Strategies ................................................................................. 35
3.10 Air-Sampling Methods ............................................................................................................ 36
3.11 Alternative Methods .............................................................................................................. 39
3.12 Aerosol Sampling and Measurement Guidelines .................................................................... 40
Chapter 4 Microscopic Methods ............................................................................................... 45
4.1 Introduction ............................................................................................................................ 45
4.2 General Concepts .................................................................................................................. 45
4.3 Types of Microscopy Useful in Food Microbiology ................................................................ 46
4.4 Media, Reagents, and Stains ................................................................................................. 50
4.5 Quantitative Applications ....................................................................................................... 54
4.6 Image Processing and Analysis .............................................................................................. 57
4.7 Conclusions and Future Perspectives .................................................................................... 58
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Compendium of Methods for the Microbiological Examination of Foods |
Chapter 5 Cultural Methods for the Enrichment and Isolation

of Microorganisms .................................................................................................... 67
5.1 Introduction ............................................................................................................................ 67
5.2 Enrichment Methods ............................................................................................................. 67
5.3 Pre-enrichment ...................................................................................................................... 67
5.4 Selective Enrichment ............................................................................................................. 69
5.5 Quality Assurance of Enrichment and Isolation Method ........................................................ 71
Chapter 6 Culture Methods for Enumeration of Microorganisms ........................................ 75
6.1 Introduction ............................................................................................................................ 75
6.2 Principle ................................................................................................................................. 75
6.3 General Description ............................................................................................................... 75
6.4 Precautions ............................................................................................................................ 75
6.5 Procedures ............................................................................................................................. 75
6.6 Alternative Methods .............................................................................................................. 85
6.7 Anaerobic or Other Atmospheres .......................................................................................... 85
Chapter 7 Cell Injury and Methods of Analysis ...................................................................... 89
7.1 Introduction ............................................................................................................................ 89
7.2 Types of Stress ...................................................................................................................... 89
7.3 Cellular Repair and Cross Protection ...................................................................................... 90
7.4 Virulence ................................................................................................................................ 90
7.5 Recovery and Detection of Sublethally Injured Bacteria ......................................................... 90
7.6 Enrichment Methods ............................................................................................................. 91
7.7 Plating Methods ..................................................................................................................... 92
7.8 Limitations and Conclusions .................................................................................................. 93
Chapter 8 Mesophilic Aerobic Plate Count .............................................................................. 95
8.1 Introduction ............................................................................................................................ 95
8.2 General Considerations .......................................................................................................... 95
8.3 Principle ................................................................................................................................. 96
8.4 General Description of Method .............................................................................................. 96
8.5 Precautions ............................................................................................................................ 97
8.6 Limitations ............................................................................................................................. 97
8.7 Procedures ............................................................................................................................. 98
8.8 Alternative Approved Methods .............................................................................................. 98
8.9 Interpretation ....................................................................................................................... 100
Chapter 9 Enterobacteriaceae, Coliforms, and Escherichia Coli as Quality
and Safety Indicators ............................................................................................. 103
9.1 Introduction .......................................................................................................................... 103
9.2 Definitions ............................................................................................................................ 104
9.3 Precautions .......................................................................................................................... 105
9.4 Equipment, Materials, and Reagents ................................................................................... 108
9.5 Sample Preparation .............................................................................................................. 109
9.6 The Enterobacteriaceae ....................................................................................................... 109
9.7 Coliforms ............................................................................................................................. 110
9.8 Fecal Coliform Group ........................................................................................................... 112
9.9 Escherichia coli .................................................................................................................... 112
9.10 Interpretation of Data: The Value of Enterobacteriaceae, Coliforms, and E. coli as
Indicators of Quality and Indexes of Pathogens ................................................................... 116
Chapter 10 Enterococci .............................................................................................................. 121
10.1 Introduction .......................................................................................................................... 121
10.2 General Considerations ........................................................................................................ 121
10.4 Precautions .......................................................................................................................... 123
10.5 Enumeration of Enterococci ................................................................................................. 124
10.6 Confirmation of Enterococci ................................................................................................. 124
Chapter 11 Rapid Methods for the Detection and Identification of
Foodborne Pathogens ............................................................................................ 127
11.1 Introduction .......................................................................................................................... 127
11.2 Antibody-Based Methods ..................................................................................................... 127
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11.3 Nucleic Acid Amplification Methods .................................................................................... 130

11.4 Matrix-Assisted Laser Desorption/IonizationTime-of-Flight Mass Spectrometry ............... 139
Chapter 12 Molecular Typing and Differentiation .................................................................. 153
12.1 Introduction .......................................................................................................................... 153
12.2 Serotyping ............................................................................................................................ 153
12.3 PCR-RFLP and AFLP ............................................................................................................ 154
12.4 ARISA .................................................................................................................................. 154
12.5 SSCP .................................................................................................................................... 154
12.6 Phage Typing ....................................................................................................................... 155
12.7 Sequencing of Individual Genes or Intergenic Regions ........................................................ 155
12.8 Multilocus Sequence Typing ................................................................................................ 155
12.9 Multiple-Locus Variable Number Tandem Repeats .............................................................. 156
12.10 PCR-Based Genomic Fingerprinting Techniques (REP, ERIC, BOX) ..................................... 159
12.11 Ribotyping ............................................................................................................................ 160
12.12 Pulsed-Field Gel Electrophoresis (PFGE) .............................................................................. 160
12.13 Optical Mapping ................................................................................................................... 160
12.14 Whole-Genome Sequencing ................................................................................................ 161
12.15 Generalized Protocols .......................................................................................................... 162
Section II Physiological Groups of Microorganisms
Chapter 13 Psychrotrophic Microorganisms ........................................................................... 175
13.1 Introduction .......................................................................................................................... 175
13.2 Review of Methods Used to Enumerate Psychrotrophs in Foods ....................................... 178
13.3 General Recommendations .................................................................................................. 180
13.4 Sample Preparation .............................................................................................................. 181
13.5 Equipment, Media, Materials, and Reagents ....................................................................... 181
13.6 Procedures for Enumeration of Psychrotrophs .................................................................... 181
13.7 Precautions .......................................................................................................................... 181
13.8 Rapid Detection ................................................................................................................... 181
13.9 Interpretation ....................................................................................................................... 182
Chapter 14 Thermoduric Microorganisms and Heat-Resistance
Measurements ........................................................................................................ 191
14.1 Introduction .......................................................................................................................... 191
14.2 Heat-Resistance Measurements .......................................................................................... 192
14.3 Methods for the Determination of Heat Resistance ............................................................ 194
14.4 Equipment, Materials, and Procedures ................................................................................ 194
14.5 Interpretation ....................................................................................................................... 197
Chapter 15 Lipolytic Microorganisms ...................................................................................... 201
15.1 Introduction .......................................................................................................................... 201
15.4 Procedures ........................................................................................................................... 205
15.5 Precautions .......................................................................................................................... 205
Chapter 16 Proteolytic Microorganisms .................................................................................. 209
16.1 Introduction .......................................................................................................................... 209
16.3 Equipment, Material, and Reagents ..................................................................................... 210
16.4 Procedures ........................................................................................................................... 211
16.5 Precautions .......................................................................................................................... 211
16.6 Interpretation ....................................................................................................................... 212
Chapter 17 Halophilic and Osmophilic Microorganisms ........................................................ 213
17.1 Introduction .......................................................................................................................... 213
17.2 Halophilic Microorganisms ................................................................................................... 214
17.3 Osmophilic Microorganisms ................................................................................................. 218
Chapter 18 Pectinolytic Microorganisms and Pectinases ...................................................... 223
18.1 Introduction .......................................................................................................................... 223
18.2 Screening of Pectinolytic Microorganisms ........................................................................... 224
18.3 Visualization of Microbial Pectinases by Electrophoretic Techniques ................................... 224
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18.4 Assays of Bacterial and Fungal Pectinases .......................................................................... 225

18.5 Identification of Novel Pectic Enzymes by Genome Mining ................................................. 225
Chapter 19 Acid-Producing Microorganisms ........................................................................... 229
19.1 Introduction .......................................................................................................................... 229
19.3 Limitations ........................................................................................................................... 230
19.5 Procedures ........................................................................................................................... 231
Chapter 20 Probiotics ................................................................................................................. 237
20.1 Introduction .......................................................................................................................... 237
20.3 Precautions .......................................................................................................................... 257
20.4 Limitations ........................................................................................................................... 257
20.5 Procedures ........................................................................................................................... 257
20.6 Equipment, Materials, and Reagents/Media ........................................................................ 259
20.7 Interpretation ....................................................................................................................... 259
20.8 Confirmation ........................................................................................................................ 259
20.9 Alternative Viable Count Methods ....................................................................................... 264
Chapter 21 Yeasts and Molds ................................................................................................... 277
21.1 Introduction .......................................................................................................................... 277
21.3 Precautions .......................................................................................................................... 278
21.4 General Methods ................................................................................................................. 278
21.5 Procedures ........................................................................................................................... 278
21.6 Interpretation ....................................................................................................................... 279
21.7 New Methods Under Development ..................................................................................... 280
Chapter 22 Detection and Enumeration of Heat-Resistant Molds ....................................... 287
22.1 Introduction .......................................................................................................................... 287
22.4 Procedures ........................................................................................................................... 288
22.5 Precautions .......................................................................................................................... 290
22.6 Limitations ........................................................................................................................... 290
22.7 Interpretation ....................................................................................................................... 290
22.8 Taxonomy of Important Heat-Resistant Molds ..................................................................... 290
Chapter 23 Mesophilic Aerobic Endospore-Forming Bacilli .................................................. 299
23.1 Introduction .......................................................................................................................... 299
23.2 Special Considerations ......................................................................................................... 300
23.3 Sources of Error ................................................................................................................... 301
23.4 Equipment, Materials, and Media ........................................................................................ 301
23.5 Procedures ........................................................................................................................... 301
23.6 Modifications ....................................................................................................................... 302
23.7 Interpretation ....................................................................................................................... 303
Chapter 24 Mesophilic Anaerobic Sporeformers .................................................................... 305
24.1 Introduction .......................................................................................................................... 305
24.3 Precautions .......................................................................................................................... 308
24.4 Limitations ........................................................................................................................... 308
24.5 Procedures ........................................................................................................................... 309
24.7 Interpretation of Results ...................................................................................................... 315
Chapter 25 Aciduric Flat Sour Sporeformers .......................................................................... 319
25.1 Introduction .......................................................................................................................... 319
25.3 Principles ............................................................................................................................. 321
25.4 General Description of Methods .......................................................................................... 321
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25.5 Precautions .......................................................................................................................... 321

25.6 Procedures ........................................................................................................................... 322
25.7 Interpretation ....................................................................................................................... 325
Chapter 26 Thermophilic Flat Sour Sporeformers .................................................................. 329
26.1 Introduction .......................................................................................................................... 329
26.4 Precautions .......................................................................................................................... 331
26.5 Procedure ............................................................................................................................ 331
Chapter 27 Thermophilic Anaerobic Sporeformers ................................................................ 335
27.1 Introduction .......................................................................................................................... 335
27.3 Equipment, Materials, and Solutions .................................................................................... 336
27.4 Precautions .......................................................................................................................... 336
27.5 Procedure ............................................................................................................................ 336
27.6 Interpretation ....................................................................................................................... 337
Chapter 28 Sulfide Spoilage Sporeformers ............................................................................. 341
28.1 Introduction .......................................................................................................................... 341
28.3 Sampling Requirements ....................................................................................................... 342
28.4 Precautions and Limitations ................................................................................................. 342
28.5 Procedure ............................................................................................................................ 342
28.7 Interpretation ....................................................................................................................... 343
Section III Microbial Genera
Chapter 29 Aeromonas, Plesiomonas, and Arcobacter ......................................................... 347
29.1 Aeromonas .......................................................................................................................... 347
29.2 Plesiomonas ......................................................................................................................... 351
29.3 Arcobacter ........................................................................................................................... 353
Chapter 30 Campylobacter ........................................................................................................ 365
30.1 Introduction .......................................................................................................................... 365
30.4 Precautions .......................................................................................................................... 366
30.5 Limitations ........................................................................................................................... 366
30.6 Procedures ........................................................................................................................... 366
30.8 Injury .................................................................................................................................... 370
30.9 Interpretation ....................................................................................................................... 370
Chapter 31 Bacillus cereus and Bacillus cereus Toxins ......................................................... 375
31.1 Bacillus cereusIntroduction ............................................................................................... 375
31.2 Bacillus cereusGeneral Considerations ............................................................................. 376
31.3 Bacillus cereusPrecautions and Limitations ...................................................................... 377
31.4 Bacillus cereusSample Requirements ............................................................................... 378
31.5 Bacillus cereusEquipment, Media, and Reagents ............................................................. 378
31.6 Bacillus cereusProcedure ................................................................................................. 378
31.7 Bacillus cereusInterpretation of Data ................................................................................ 380
31.8 Bacillus cereusNew Technologies .................................................................................... 381
31.9 Bacillus EnterotoxinsIntroduction ...................................................................................... 381
31.10 Bacillus EnterotoxinsEnterotoxin Production and Extraction of Foods .............................. 382
31.11 Bacillus EnterotoxinsScreening for Enterotoxigenicity and Bacillus spp.
Enterotoxins in Foods Using 3M Tecra BDEVIA ELISA ....................................................... 383
31.12 Bacillus EnterotoxinsBacillus cereus Enterotoxin Reversed Passive
Latex Agglutination (BCET-RPLA) Diarrheal Toxin Detection Kit .......................................... 385
31.13 Bacillus EnterotoxinsEquipment ....................................................................................... 386
31.14 Bacillus EnterotoxinsToxin Extraction or Production ......................................................... 386
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31.15 Bacillus EnterotoxinsAssay Method ................................................................................. 387

31.16 Bacillus EnterotoxinsNew Technologies ........................................................................... 387
Chapter 32 Clostridium botulinum and Its Toxins ................................................................. 391
32.1 Introduction .......................................................................................................................... 391
32.4 Precautions .......................................................................................................................... 394
32.5 Limitations ........................................................................................................................... 395
32.6 Procedures ........................................................................................................................... 395
32.8 Interpreting Data .................................................................................................................. 400
Chapter 33 Clostridium perfringens ......................................................................................... 403
33.1 Introduction .......................................................................................................................... 403
33.2 Sampling .............................................................................................................................. 404
33.3 Equipment and Supplies ...................................................................................................... 404
33.4 Special Reagents and Media ................................................................................................ 405
33.5 Recommended Controls ...................................................................................................... 405
33.6 Precautions and Limitations of Methods .............................................................................. 405
33.7 Procedure ............................................................................................................................ 406
33.8 Interpretation of Data ........................................................................................................... 408
Chapter 34 Pathogenic Escherichia coli ................................................................................... 411
34.1 Introduction .......................................................................................................................... 411
34.2 Conventional Isolation Procedures ....................................................................................... 413
34.3 Rapid Methods .................................................................................................................... 416
34.4 Serotyping ............................................................................................................................ 418
34.5 Pathogenicity Testing ........................................................................................................... 420
34.6 Summary ............................................................................................................................. 421
Chapter 35 Listeria ...................................................................................................................... 425
35.1 Introduction .......................................................................................................................... 425
35.3 Media, Reagents, and Equipment ........................................................................................ 431
35.4 Precautions and Limitations of the Methods ....................................................................... 432
35.5 Procedures ........................................................................................................................... 432
Chapter 36 Salmonella ............................................................................................................... 445
36.1 Introduction .......................................................................................................................... 445
36.2 Treatment of Sample ........................................................................................................... 453
36.3 Equipment and Supplies ...................................................................................................... 453
36.4 Precautions and Limitations of Reference and Rapid Methods ........................................... 454
36.5 Reference Method ............................................................................................................... 455
36.6 Interpretation of the Data-Reference Method ...................................................................... 463
36.7 Rapid Detection Assays ....................................................................................................... 463
Chapter 37 Shigella .................................................................................................................... 477
37.1 Introduction .......................................................................................................................... 477
37.5 Procedures ........................................................................................................................... 479
37.6 Injury .................................................................................................................................... 483
37.7 Interpretation ....................................................................................................................... 483
37.8 Precautions and Limitations ................................................................................................. 484
Chapter 38 Cronobacter Species .............................................................................................. 487
38.1 Introduction .......................................................................................................................... 487
38.2 Cronobacter spp. Description ............................................................................................... 487
38.3 Cronobacter spp. in Foods ................................................................................................... 493
38.4 Analysis of Cronobacter spp. in Foods ................................................................................. 493
38.5 Pathogenicity Testing ........................................................................................................... 500
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38.6 Epidemiology Studies ........................................................................................................... 503

38.7 Precautions .......................................................................................................................... 504
Chapter 39 Staphylococcus aureus and Staphylococcal Enterotoxins ................................ 509
39.1 Introduction .......................................................................................................................... 509
39.2 General Considerations for Isolation of Enterotoxigenic Staphylococci ................................ 511
39.3 Sampling Requirements and Handling of Samples .............................................................. 512
39.4 Precautions and Limitations of Methods .............................................................................. 513
39.5 Equipment, Reagents, and Media ........................................................................................ 513
39.6 Procedures ........................................................................................................................... 514
39.7 Additional Tests ................................................................................................................... 516
39.8 S. aureus as an Agent of Foodborne Illness ........................................................................ 516
39.9 Examining Staphylococcal Isolates for Enterotoxin Production ............................................ 518
39.10 Extraction of Enterotoxins From Foods for ELISA Systems ................................................. 518
39.11 ELISA-Based Enterotoxin Testing ........................................................................................ 519
39.12 VIDAS Staph Enterotoxin II .................................................................................................. 519
39.13 Visual ELISA: Polyvalent (Types AE) Screening for Determining Enterotoxigenicity
and Identifying Staphylococcal Enterotoxins in Foods .......................................................... 520
Chapter 40 Vibrio ........................................................................................................................ 527
40.1 Introduction .......................................................................................................................... 527
40.4 Precautions .......................................................................................................................... 535
40.5 Limitations ........................................................................................................................... 535
40.6 Procedures ........................................................................................................................... 536
40.7 Interpretation ....................................................................................................................... 541
Chapter 41 Yersinia .................................................................................................................... 549
41.1 Introduction .......................................................................................................................... 549
41.3 Analysis of Food for Yersinia ............................................................................................... 551
41.5 Procedures ........................................................................................................................... 554
Chapter 42 Waterborne and Foodborne Parasites ................................................................. 565
42.1 Introduction .......................................................................................................................... 565
42.2 Waterborne- and Vegetation-Associated Parasites: Prevalence, Disease,
Transmission, and Methods of Identification ....................................................................... 568
42.3 Foodborne Parasites: Prevalence, Disease, Transmission, and Methods of Identification ... 572
42.4 Emerging Methods .............................................................................................................. 578
Chapter 43 Toxigenic Fungi and Fungal Toxins ...................................................................... 583
43.1 Introduction .......................................................................................................................... 583
43.2 Mycotoxins .......................................................................................................................... 583
43.3 Mycotoxicoses ..................................................................................................................... 589
43.4 Mycotoxin-Producing Molds ................................................................................................. 590
43.5 Handling of Toxigenic Fungi and Mycotoxins ....................................................................... 592
43.6 Key Laboratory Facilities Requirements ............................................................................... 592
43.7 Cleaning, Decontamination, and Waste Disposal ................................................................. 592
43.8 Media and Procedures ......................................................................................................... 592
Chapter 44 Foodborne Viruses .................................................................................................. 595
44.1 Introduction .......................................................................................................................... 595
44.2 Enteric Virus Outbreaks Associated With Foods .................................................................. 596
44.3 Classical Cell CultureBased Assays .................................................................................... 597
44.4 Molecular Approaches: Extraction of Viral RNA ................................................................... 598
44.5 Molecular Approaches: Assay of Viral RNA ......................................................................... 604
Section IV Food Commodities
Chapter 45 Meat and Poultry Products .................................................................................... 619
45.1 Introduction .......................................................................................................................... 619
45.2 Raw Meat and Poultry Products .......................................................................................... 620
45.3 Ready-To-Eat Meat and Poultry Products ............................................................................ 623
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45.4 Pathogens ............................................................................................................................ 627

45.6 Recommended Methods ..................................................................................................... 627
Chapter 46 Eggs and Egg Products .......................................................................................... 633
46.1 Introduction .......................................................................................................................... 633
46.3 Normal Flora ........................................................................................................................ 634
46.4 Flora Changes in Spoilage .................................................................................................... 635
46.5 Pathogens of Concern ......................................................................................................... 636
46.6 Indicators of a Lack of Sanitation ......................................................................................... 637
Chapter 47 Milk and Milk Products .......................................................................................... 645
47.1 Introduction .......................................................................................................................... 645
47.2 Raw Milk .............................................................................................................................. 645
47.3 Pasteurized Milk .................................................................................................................. 648
47.4 Dried Products ..................................................................................................................... 649
47.5 Butter ................................................................................................................................... 650
47.6 Frozen Dairy Products .......................................................................................................... 650
47.7 Concentrated Products ........................................................................................................ 651
47.8 Fermented Dairy Products ................................................................................................... 652
47.9 Acid-Producing Bacteria in Dairy Foods ............................................................................... 654
47.10 Additional Reading ............................................................................................................... 658
Chapter 48 Fish, Crustaceans, and Precooked Seafood ........................................................ 661
48.1 Introduction .......................................................................................................................... 661
48.3 Fresh and Frozen Fish and Crustaceans .............................................................................. 661
48.4 Precooked Crustacean Products .......................................................................................... 665
48.5 Breaded and Prepared Seafood Products ............................................................................ 665
48.6 Salted and Smoked Products ............................................................................................... 666
48.7 Other Seafood Products ...................................................................................................... 666
48.8 Modified AtmospherePackaged Products .......................................................................... 668
48.9 Control ................................................................................................................................. 669
Chapter 49 Molluscan Shellfish: Oysters, Mussels, and Clams ............................................ 677
49.1 Introduction .......................................................................................................................... 677
49.2 Examination of Shellfish ....................................................................................................... 679
49.3 Preparation of Sample for Examining Shellfish in the Shell .................................................. 679
49.4 Microbiological Procedures .................................................................................................. 682
49.6 Rapid Methods for Indicator Organisms .............................................................................. 683
49.7 International Microbiological Standards ................................................................................ 683
Chapter 50 Fruits and Vegetables ............................................................................................ 687
50.1 Introduction .......................................................................................................................... 687
50.2 General Processes/Control of Organisms for Fruits and Vegetables .................................... 687
50.3 Fresh Fruits and Vegetables ................................................................................................ 688
50.4 Fresh-Cut Fruits and Vegetables .......................................................................................... 690
50.5 Canned Fruits and Vegetables .............................................................................................. 691
50.6 Frozen Fruits and Vegetables................................................................................................ 692
50.7 Dehydrated Fruits and Vegetables ........................................................................................ 692
50.8 Recommended Methods ...................................................................................................... 693
50.9 Result Interpretation ............................................................................................................. 693
Chapter 51 Fermented and Acidified Vegetables .................................................................... 697
51.1 Introduction ........................................................................................................................... 697
51.2 General Considerations ......................................................................................................... 697
51.3 Normal Flora.......................................................................................................................... 701
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51.4 Microbiota Changes in Spoilage ............................................................................................ 703

51.5 Pathogenic Microorganisms .................................................................................................. 705
51.6 Sampling Requirements ........................................................................................................ 705
51.8 Interpretation of Data ............................................................................................................ 712
Chapter 52 Gums and Spices ..................................................................................................... 719
52.1 Introduction ........................................................................................................................... 719
52.4 Spoilage ................................................................................................................................ 721
52.5 Normal Flora.......................................................................................................................... 721
52.6 Pathogens ............................................................................................................................. 722
52.8 Control .................................................................................................................................. 725
Chapter 53 Salad Dressings........................................................................................................ 731
53.1 Introduction ........................................................................................................................... 731
53.2 Composition .......................................................................................................................... 731
53.3 Factors Affecting the Microbiological Stability and Safety of Dressings................................ 731
53.4 The Role of Acids.................................................................................................................. 732
53.5 New Product Trends ............................................................................................................. 732
53.6 Normal Microbiota................................................................................................................. 733
53.7 Change in Spoilage Microbiota.............................................................................................. 733
53.8 Microorganisms of Public Health Significance....................................................................... 733
53.9 Microbial Indicators of Poor Sanitation.................................................................................. 734
53.10 Testing: Equipment, Materials, and Solutions ....................................................................... 735
53.12 Challenge Study Design ........................................................................................................ 736
53.13 Advances in Rapid Microbiology ........................................................................................... 736
Chapter 54 Natural Sweeteners and Starches ......................................................................... 739
54.1 Introduction ........................................................................................................................... 739
54.4 Normal and Indicator Flora .................................................................................................... 740
54.5 Spoilage Organisms .............................................................................................................. 741
54.6 Pathogens ............................................................................................................................. 741
Chapter 55 Cereal and Cereal Products .................................................................................... 745
55.1 Introduction ........................................................................................................................... 745
55.2 Factors Affecting Bioload ...................................................................................................... 745
55.3 Methods................................................................................................................................ 749
Chapter 56 Confectionery Products ........................................................................................... 751
56.1 Introduction ........................................................................................................................... 751
56.3 Spoilage of Confectionery ..................................................................................................... 751
56.4 Pathogens ............................................................................................................................. 753
56.5 Microbial Control Procedures ................................................................................................ 753
Chapter 57 Nut Meats ................................................................................................................. 759
57.1 Introduction ........................................................................................................................... 759
57.3 Normal Microbiota................................................................................................................. 760
57.4 Pathogens: Bacteria .............................................................................................................. 761
57.5 Fungi and Mycotoxins ........................................................................................................... 761
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57.8 Control .................................................................................................................................. 763
57.9 Interpretation......................................................................................................................... 764
Chapter 58 Juices and Juice-Containing Beverages................................................................ 769
58.1 Introduction ........................................................................................................................... 769
58.2 Normal Microflora ................................................................................................................. 769
58.3 Pathogenic Microorganisms .................................................................................................. 771
58.4 Spoilage Microflora ............................................................................................................... 772
Chapter 59 Ready-To-Drink Beverages...................................................................................... 779
59.1 Introduction ........................................................................................................................... 779
59.4 Spoilage ................................................................................................................................ 781
59.5 Normal Microflora ................................................................................................................. 783
59.6 Pathogens ............................................................................................................................. 784
59.8 Equipment, Media, and Reagents ......................................................................................... 786
59.9 Control .................................................................................................................................. 786
59.10 Interpretation......................................................................................................................... 788
Chapter 60 Bottled Water ........................................................................................................... 791
60.1 Introduction ........................................................................................................................... 791
60.2 Types of Microorganisms...................................................................................................... 792
60.3 Equipment, Media, and Reagents ......................................................................................... 793
Chapter 61 Canned FoodsTests for Commercial Sterility ................................................... 797
61.1 Introduction ........................................................................................................................... 797
61.5 Interpretation......................................................................................................................... 802
Chapter 62 Canned FoodsTests for Cause of Spoilage ....................................................... 805
62.1 Introduction ........................................................................................................................... 805
62.2 Microbiology of Canned Foods.............................................................................................. 805
62.3 Methods for Diagnosing Spoilage in Canned Foods.............................................................. 808
62.4 Background Information ........................................................................................................ 808
62.5 Preliminary Steps .................................................................................................................. 808
62.6 Culture Procedures for Low-Acid Canned Foods .................................................................. 814
62.7 Culture Procedures for Acid or Acidified Foods .................................................................... 817
Chapter 63 Pet Food .................................................................................................................... 823
63.1 Introduction ........................................................................................................................... 823
63.3 Normal Flora.......................................................................................................................... 824
63.4 Spoilage ................................................................................................................................ 827
63.5 Mycotoxins ........................................................................................................................... 829
63.6 Pathogens ............................................................................................................................. 830
63.8 Microbial Control ................................................................................................................... 834
Chapter 64 Beer and Wine .......................................................................................................... 841
64.1 Introduction ........................................................................................................................... 841
64.2 Fermentation......................................................................................................................... 841
64.3 Spoilage Microorganisms ...................................................................................................... 842
64.4 Packaging and Shelf Life....................................................................................................... 843
64.5 Quality Assurance Strategies ................................................................................................ 843
64.6 Microbiological Methods of Analysis..................................................................................... 843
xiv |
| Contents
64.7 Microbiological Culture Media ............................................................................................... 845

64.8 Microbiological Identification ................................................................................................. 846
Section V Reference
Chapter 65 Investigation of Foodborne Illness Outbreaks ..................................................... 851
65.1 Introduction ........................................................................................................................... 851
65.2 Purpose of Investigation ....................................................................................................... 851
65.3 Theory of Investigation.......................................................................................................... 852
65.4 Equipment, Materials, and Solutions..................................................................................... 852
65.5 Investigative Procedures ....................................................................................................... 856
65.6 Analysis and Conclusion........................................................................................................ 858
65.7 Molecular Subtyping TechniquesBacteria .......................................................................... 859
65.8 Molecular Subtyping TechniquesViruses ........................................................................... 860
65.9 Conclusion ............................................................................................................................ 861
Chapter 66 Microbial Food Safety Risk Assessment............................................................... 863
66.1 Introduction ........................................................................................................................... 863
66.2 Risk Assessment: A Tool for Decision-Making...................................................................... 863
66.3 Approaches to Microbial Food Safety-Risk Assessment ....................................................... 865
66.4 Steps of Risk Assessment .................................................................................................... 866
66.5 Mathematical Approaches to Risk Assessment .................................................................... 870
66.6 Monte Carlo Analysis ............................................................................................................ 873
66.7 Uncertainty and Variability ..................................................................................................... 874
66.8 Data Considerations .............................................................................................................. 876
66.9 Applications of Risk Assessment .......................................................................................... 876
66.10 Summary............................................................................................................................... 880
Section VI Appendix
Chapter 67 Microbiological Media, Reagents, and Stains ...................................................... 885
67.1 Introduction ........................................................................................................................... 885
67.2 General Considerations and Safety in the Preparation and Use of Media ............................. 885
67.3 Equipment............................................................................................................................. 891
67.4 Alphabetical Listing of Culture Media.................................................................................... 892
67.5 Dyes and Reagents............................................................................................................... 933
67.6 Stains .................................................................................................................................... 947
67.7 Alphabetical Listing of Stains ................................................................................................ 947
Chapter 68 Measurement of Water Activity, Acidity, and Brix.............................................. 951
68.1 Water Activity ....................................................................................................................... 951
68.2 Acidity ................................................................................................................................... 957
68.3 Brix........................................................................................................................................ 961
Index ................................................................................................................................................965
| xv
Editorial Board
Y M
vonne Salfinger joined the Compendium editorial ary Lou Tortorello attended Northern Illinois
board, representing the Association of Public Health University (BS) and Loyola University of Chicago
Laboratories (APHL). She graduated from Florida (MS, biological sciences). She received a PhD in
State University (BS) and earned an MS in community health microbiology from Cornell University where she researched
from the University of North Florida. She is pursuing a DrPH the structure and biosynthesis of lipopolysaccharide endotoxin
from the University of South Florida. After working at the Florida for her dissertation. At Cornell, she pursued post-graduate
Department of Health for 21 years, she moved to the Florida work, focusing on bacterial gene transfer mechanisms,
Department of Agriculture and Consumer Services, initially as improved dairy starter cultures, and rapid assays for detection
the quality manager, to pursue ISO/IEC 17025 accreditation for of pathogens; there, she also taught an undergraduate course,
the Division of Food Safety laboratories; in 2007, accreditation General Microbiology. Since 1991, she has worked as a
was accomplished. In 2003, she became the chief for the Bureau of research microbiologist with the Division of Food Processing
Food Laboratories, Division of Food Safety, Florida Department Science and Technology, U.S. Food and Drug Administration,
of Agriculture and Consumer Services. She was also involved in Bedford Park, Illinois. She currently serves as the chief of the
with the Food Emergency Response Network in several activities Food Technology Branch. Her professional experience also
relating to the analysis of foods during emergency events, includes work as the product manager of a serum diagnostic
including the establishment of Standard Operating Procedures. test for the HIV/AIDS virus at the Abbott Laboratories
After retiring from the Department of Agriculture and Consumer Diagnostics Division in Abbott Park, Illinois. She maintains
Services in 2012, she relocated to Denver, Colorado, where she research interests in improved microbiological methods for
now works as a consultant for APHL and the Association of Food foodborne pathogens, especially rapid methods, sample
and Drug Officials to assist with the implementation of ISO/IEC preparation, and the behavior and control of microbial
17025 across the regulatory food testing laboratories within the pathogens in foods and food processing environments.
United States. Over the years, she has contributed to public health Furthermore, she is co-editor of the Encyclopedia of Food
internationally: With the Centers for Disease Control and Microbiology, serves on the Editorial Board of Journal of Food
Prevention (CDC) and the Global Health program of APHL, Protection, and is chief editor of Food Microbiology.
she worked on the post-Hurricanes Mitch and Georges project in
El Salvador. With CDC, she provided technical quality assurance
to the tuberculosis laboratory in Ivanovo, Russia.
Gary R. Acuff (board), Texas A&M University, College Station Keith Ito (board), University of California, Davis, Dublin
Mark Carter (board), MC Squared Enterprises, Flossmoor, Ruth L. Petran (board), Ecolab, St. Paul, Minnesota
Illinois Yvonne Salfinger (co-editor), retired from Florida Department
Victor Cook (board), retired from U.S. Department of of Agriculture and Consumer Services, Tallahassee
Agriculture, Washington, D.C. Mary Lou Tortorello (co-editor), U.S. Food and Drug Admin-
Stephanie Doores (associate editor), Penn State University, istration, Bedford Park, Illinois
University Park Burton Wilcke (associate editor), The University of Vermont,
David Goldman (board), U.S. Department of Agriculture, Burlington
Washington, D.C.
| xvii

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American Public Health Association

2015 by the American Public Health Association

Printed and bound in the United States of America

Library of Congress Cataloging-in-Publication Data

Editorial Board .............................................................................................................................. xvii

Chapter 5 Cultural Methods for the Enrichment and Isolation

11.3 Nucleic Acid Amplification Methods .................................................................................... 130

18.4 Assays of Bacterial and Fungal Pectinases .......................................................................... 225

25.5 Precautions .......................................................................................................................... 321

31.15 Bacillus EnterotoxinsAssay Method ................................................................................. 387

38.6 Epidemiology Studies ........................................................................................................... 503

45.4 Pathogens ............................................................................................................................ 627

51.4 Microbiota Changes in Spoilage ............................................................................................ 703

57.7 Sampling Requirements ........................................................................................................ 762

64.7 Microbiological Culture Media ............................................................................................... 845

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