Compendium. of Methods. for the Microbiological Examination of Foods APHA PRESS. Mary Lou Tortorello. Yvonne Salfinger.

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1 Compendium of Methods for the Microbiological Examination of Foods Editors Yvonne Salfinger Mary Lou Tortorello % A P H L ASSOCIATION OF PUBLIC HEAL1 HEALTH LABORATORIES* APHA PRESS AH IMPRINT OF AMERICAN PUBLIC HEALTH ASSOCIATION WASHINGTON,DC 2015

2 Contents Editorial Board Authors Reviewers Preface xvn xix xxiii xxv Section I General Methods Chapter 1 Laboratory Quality Management Systems Introduction The Role of Management in Laboratory QA Laboratory Operational Framework and QC Requirements Personnel Equipment/Instrumentation Laboratory Glassware and Plasticware Sample Management Analytical Methods Culture Media and Reagent Preparation or Test Kits Accreditation of Testing Laboratories 10 Chapter 2 Sampling Plans, Sample Collection, Shipment, and Preparation for Analysis Introduction General Considerations Equipment, Materials, and Reagents Precautions Procedures 14 Chapter 3 Microbiological Monitoring of the Food Processing Environment Introduction Environmental Sampling Strategies Sampling of Surfaces: Equipment and Physical Plant Principle of Monitoring the Microbiological Flora Precautions Limitations Rinse Solution Method for Sampling Containers and Processing Equipment Systems Surface Contact Methods Microbiological Air-Sampling Strategies Air-Sampling Methods Alternative Methods Aerosol Sampling and Measurement Guidelines 40 Chapter 4 Microscopic Methods Introduction General Concepts Types of Microscopy Useful in Food Microbiology Media, Reagents, and Stains Quantitative Applications Image Processing and Analysis Conclusions and Future Perspectives 58

3 Compendium of Methods for the Microbiological Examination of Foods h Chapter 5 Cultural Methods for the Enrichment and Isolation of Microorganisms Introduction Enrichment Methods Pre-enrichment Selective Enrichment Quality Assurance of Enrichment and Isolation Method 71 Chapter 6 Culture Methods for Enumeration of Microorganisms Introduction Principle General Description Precautions Procedures Alternative Methods Anaerobic or Other Atmospheres 85 Chapter 7 Cell Injury and Methods of Analysis Introduction Types of Stress Cellular Repair and Cross Protection Virulence Recovery and Detection of Sublethally Injured Bacteria Enrichment Methods Plating Methods Limitations and Conclusions 93 Chapter 8 Mesophilic Aerobic Plate Count Introduction General Considerations Principle General Description of Method Precautions Limitations Procedures Alternative Approved Methods Interpretation 100 Chapter 9 Enterobacteriaceae, Coliforms, and Escherichia Coli as Quality and Safety Indicators Introduction Definitions Precautions Equipment, Materials, and Reagents Sample Preparation The Enterobacteriaceae Coliforms Fecal Coliform Group Escherichia coli Interpretation of Data: The Value of Enterobacteriaceae, Coliforms, and. coli as Indicators of Quality and Indexes of Pathogens 116 Chapter 10 Enterococci Introduction General Considerations Equipment, Materials, and Reagents Precautions Enumeration of Enterococci Confirmation of Enterococci 124 Chapter 11 Rapid Methods for the Detection and Identification of Foodborne Pathogens Introduction Antibody-Based Methods 127

4 Contents 11.3 Nucleic Acid Amplification Methods Matrix-Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry 139 Chapter 12 Molecular Typing and Differentiation Introduction Serotyping PCR-RFLP and AFLP ARISA SSCP Phage Typing Sequencing of Individual Genes or Intergenic Regions Multilocus Sequence Typing Multiple-Locus Variable Number Tandem Repeats PCR-Based Genomic Fingerprinting Techniques (REP, ERIC, BOX) Ribotyping Pulsed-Field Gel Electrophoresis IPFGE) Optical Mapping Whole-Genome Sequencing Generalized Protocols 162 Section II Physiological Groups of Microorganisms Chapter 13 Psychrotrophic Microorganisms Introduction Review of Methods Used to Enumerate Psychrotrophs in Foods General Recommendations Sample Preparation Equipment, Media, Materials, and Reagents Procedures for Enumeration of Psychrotrophs Precautions Rapid Detection Interpretation 182 Chapter 14 Thermoduric Microorganisms and Heat-Resistance Measurements Introduction Heat-Resistance Measurements Methods for the Determination of Heat Resistance Equipment, Materials, and Procedures Interpretation 197 Chapter 15 Lipolytic Microorganisms Introduction General Considerations Equipment, Materials, and Reagents Procedures Precautions 205 Chapter 16 Proteolytic Microorganisms Introduction General Considerations Equipment, Material, and Reagents Procedures Precautions Interpretation 212 Chapter 17 Halophilic and Osmophilic Microorganisms Introduction Halophilic Microorganisms Osmophilic Microorganisms 218 Chapter 18 Pectinolytic Microorganisms and Pectinases Introduction Screening of Pectinolytic Microorganisms Visualization of Microbial Pectinases by Electrophoretic Techniques 224 vii

5 Compendium of Methods for the Microbiological Examination of Foods I Assays of Bacterial and Fungal Pectinases Identification of Novel Pectic Enzymes by Genome Mining 225 Chapter 19 Acid-Producing Microorganisms Introduction General Considerations Limitations Equipment, Materials, and Reagents Procedures 231 Chapter 20 Probiotics Introduction General Considerations Precautions Limitations Procedures Equipment, Materials, and Reagents/Media Interpretation Confirmation Alternative Viable Count Methods 264 Chapter 21 Yeasts and Molds Introduction General Considerations Precautions General Methods Procedures Interpretation New Methods Under Development 280 Chapter 22 Detection and Enumeration of Heat-Resistant Molds Introduction General Considerations Equipment, Materials, and Reagents Procedures Precautions Limitations Interpretation 22.8 Taxonomy of Important Heat-Resistant Molds 290 Chapter 23 Mesophilic Aerobic Endospore-Forming Bacilli Introduction Special Considerations Sources of Error Equipment, Materials, and Media Procedures Modifications Interpretation 303 Chapter 24 Mesophilic Anaerobic Sporeformers Introduction General Considerations Precautions Limitations Procedures Equipment, Materials, and Media Interpretation of Results 315 Chapter 25 Aciduric Flat Sour Sporeformers Introduction General Considerations Principles General Description of Methods

6 H Contents 25.5 Precautions Procedures Interpretation 325 Chapter 26 Thermophilic Flat Sour Sporeformers Introduction General Considerations Equipment, Materials, and Reagents Precautions Procedure Interpretation of Results 332 Chapter 27 Thermophilic Anaerobic Sporeformers Introduction General Considerations Equipment, Materials, and Solutions Precautions Procedure Interpretation 337 Chapter 28 Sulfide Spoilage Sporeformers Introduction General Considerations Sampling Requirements Precautions and Limitations Procedure Equipment, Materials, and Reagents Interpretation 343 Section III Microbial Genera Chapter 29 Aeromonas, Plesiomonas, and Arcobacter Aeromonas Plesiomonas Arcobacter 353 Chapter 30 Campylobacter Introduction General Considerations Sampling Requirements Precautions Limitations Procedures Equipment, Materials, and Reagents Injury Interpretation 370 Chapter 31 Bacillus cereus and Bacillus cereus Toxins Bacillus cereus Introduction Bacillus cereus General Considerations Bacillus cereus Precautions and Limitations Bacillus cereus Sample Requirements Bacillus cereus Equipment, Media, and Reagents Bacillus cereus Procedure Bacillus cereus Interpretation of Data Bacillus cereus New Technologies Bacillus Enterotoxins Introduction Bacillus Enterotoxins Enterotoxin Production and Extraction of Foods Bacillus Enterotoxins Screening for Enterotoxigenicity and Bacillus spp. Enterotoxins in Foods Using 3M Tecra BDEVIA ELISA Bacillus Enterotoxins Bacillus cereus Enterotoxin Reversed Passive Latex Agglutination (BCET-RPLA) Diarrheal Toxin Detection Kit Bacillus Enterotoxins Equipment Bacillus Enterotoxins Toxin Extraction or Production 386 IX

7 Compendium of Methods for the Microbiological Examination of Foods I Bacillus Enterotoxins Assay Method Bacillus Enterotoxins New Technologies 387 Chapter 32 Clostridium botulinum and Its Toxins Introduction General Considerations Sampling Requirements Precautions Limitations Procedures Equipment, Materials, and Reagents Interpreting Data 400 Chapter 33 Clostridium perfringens Introduction Sampling Equipment and Supplies Special Reagents and Media Recommended Controls Precautions and Limitations of Methods Procedure Interpretation of Data 408 Chapter 34 Pathogenic Escherichia coli Introduction Conventional Isolation Procedures Rapid Methods Serotyping Pathogenicity Testing Summary 421 Chapter 35 Listeria Introduction General Considerations Media, Reagents, and Equipment Precautions and Limitations of the Methods Procedures Interpretation of Data 438 Chapter 36 Salmonella Introduction Treatment of Sample Equipment and Supplies Precautions and Limitations of Reference and Rapid Methods Reference Method Interpretation of the Data-Reference Method Rapid Detection Assays 463 Chapter 37 Shigella Introduction General Considerations Sampling Requirements Equipment, Materials, and Reagents Procedures Injury Interpretation Precautions and Limitations 484 Chapter 38 Cronobacter Species Introduction Cronobacter spp. Description Cronobacter spp. in Foods Analysis of Cronobacter spp. in Foods Pathogenicity Testing 500

8 i Contents 38.6 Epidemiology Studies Precautions 504 Chapter 39 Staphylococcus aureus and Staphylococcal Enterotoxins Introduction General Considerations for Isolation of Enterotoxigenic Staphylococci Sampling Requirements and Handling of Samples Precautions and Limitations of Methods Equipment, Reagents, and Media Procedures Additional Tests S. aureus as an Agent of Foodborne Illness Examining Staphylococcal Isolates for Enterotoxin Production Extraction of Enterotoxins From Foods for ELISA Systems ELISA-Based Enterotoxin Testing VIDAS Staph Enterotoxin II Visual ELISA: Polyvalent (Types A-E) Screening for Determining Enterotoxigenicity and Identifying Staphylococcal Enterotoxins in Foods 520 Chapter 40 Vibrio Introduction General Considerations Equipment, Materials, and Reagents Precautions Limitations Procedures Interpretation 541 Chapter 41 Yersinia Introduction General Considerations Analysis of Food for Yersinia Equipment, Materials, and Media Procedures 554 Chapter 42 Waterborne and Foodborne Parasites Introduction Waterborne- and Vegetation-Associated Parasites: Prevalence, Disease, Transmission, and Methods of Identification Foodborne Parasites: Prevalence, Disease, Transmission, and Methods of Identification 42.4 Emerging Methods 578 Chapter 43 Toxigenic Fungi and Fungal Toxins Introduction Mycotoxins Mycotoxicoses Mycotoxin-Producing Molds Handling of Toxigenic Fungi and Mycotoxins Key Laboratory Facilities Requirements Cleaning, Decontamination, and Waste Disposal Media and Procedures 592 Chapter 44 Foodborne Viruses Introduction Enteric Virus Outbreaks Associated With Foods Classical Cell Culture-Based Assays Molecular Approaches: Extraction of Viral RNA Molecular Approaches: Assay of Viral RNA 604 Section IV Food Commodities Chapter 45 Meat and Poultry Products Introduction Raw Meat and Poultry Products Ready-To-Eat Meat and Poultry Products 623

9 Compendium of Methods for the Microbiological Examination of Foods 45.4 Pathogens Sampling Requirements Recommended Methods 627 Chapter 46 Eggs and Egg Products Introduction General Considerations Normal Flora Flora Changes in Spoilage Pathogens of Concern Indicators of a Lack of Sanitation Recommended Methods Interpretation of Data 639 Chapter 47 Milk and Milk Products Introduction Raw Milk Pasteurized Milk Dried Products Butter Frozen Dairy Products Concentrated Products Fermented Dairy Products Acid-Producing Bacteria in Dairy Foods Additional Reading 658 Chapter 48 Fish, Crustaceans, and Precooked Seafood Introduction General Considerations Fresh and Frozen Fish and Crustaceans Precooked Crustacean Products Breaded and Prepared Seafood Products Salted and Smoked Products Other Seafood Products Modified Atmosphere-Packaged Products Control Sampling Requirements Recommended Methods Interpretation of Results 671 Chapter 49 Molluscan Shellfish: Oysters, Mussels, and Clams Introduction Examination of Shellfish Preparation of Sample for Examining Shellfish in the Shell Microbiological Procedures Interpretation of Data Rapid Methods for Indicator Organisms International Microbiological Standards 683 Chapter 50 Fruits and Vegetables Introduction General Processes/Control of Organisms for Fruits and Vegetables Fresh Fruits and Vegetables Fresh-Cut Fruits and Vegetables Canned Fruits and Vegetables Frozen Fruits and Vegetables Dehydrated Fruits and Vegetables Recommended Methods Result Interpretation 693 Chapter 51 Fermented and Acidified Vegetables Introduction General Considerations Normal Flora 701

10 ^ Contents 51.4 Microbiota Changes in Spoilage Pathogenic Microorganisms Sampling Requirements Recommended Methods Interpretation of Data 712 Chapter 52 Gums and Spices Introduction General Considerations Sampling Requirements Spoilage Normal Flora Pathogens Recommended Methods Control Interpretation of Data 725 Chapter 53 Salad Dressings Introduction Composition Factors Affecting the Microbiological Stability and Safety of Dressings The Role of Acids New Product Trends Normal Microbiota Change in Spoilage Microbiota Microorganisms of Public Health Significance Microbial Indicators of Poor Sanitation Testing: Equipment, Materials, and Solutions Recommended Methods Challenge Study Design Advances in Rapid Microbiology Interpretation of Data 736 Chapter 54 Natural Sweeteners and Starches Introduction General Considerations Sampling Requirements Normal and Indicator Flora Spoilage Organisms Pathogens Recommended Methods Interpretation of Data 742 Chapter 55 Cereal and Cereal Products Introduction Factors Affecting Bioload Methods 749 Chapter 56 Confectionery Products Introduction General Considerations Spoilage of Confectionery Pathogens Microbial Control Procedures Recommended Methods Interpretation of Data 755 Chapter 57 Nut Meats Introduction General Considerations Normal Microbiota Pathogens: Bacteria Fungi and Mycotoxins Recommended Methods 762 H xiii

11 Compendium of Methods for the Microbiological Examination of Foods I Sampling Requirements Control Interpretation 764 Chapter 58 Juices and Juice-Containing Beverages Introduction Normal Microflora Pathogenic Microorganisms Spoilage Microflora Recommended Methods Interpretation of Data 774 Chapter 59 Ready-To-Drink Beverages Introduction General Considerations Sampling Requirements Spoilage Normal Microflora Pathogens Recommended Methods Equipment, Media, and Reagents Control Interpretation 788 Chapter 60 Bottled Water Introduction Types of Microorganisms Equipment, Media, and Reagents Recommended Methods Interpretation of Data 795 Chapter 61 Canned Foods Tests for Commercial Sterility Introduction General Considerations Sampling Requirements Recommended Methods Interpretation 802 Chapter 62 Canned Foods Tests for Cause of Spoilage Introduction Microbiology of Canned Foods Methods for Diagnosing Spoilage in Canned Foods Background Information Preliminary Steps Culture Procedures for Low-Acid Canned Foods Culture Procedures for Acid or Acidified Foods 817 Chapter 63 Pet Food Introduction General Considerations Normal Flora Spoilage Mycotoxins Pathogens Recommended Methods Microbial Control 834 Chapter 64 Beer and Wine Introduction Fermentation Spoilage Microorganisms Packaging and Shelf Life Quality Assurance Strategies Microbiological Methods of Analysis 843

12 i Contents 64.7 Microbiological Culture Media Microbiological Identification 846 Section V Reference Chapter 65 Investigation of Foodborne Illness Outbreaks Introduction Purpose of Investigation Theory of Investigation Equipment, Materials, and Solutions Investigative Procedures Analysis and Conclusion Molecular Subtyping Techniques Bacteria Molecular Subtyping Techniques Viruses Conclusion 861 Chapter 66 Microbial Food Safety Risk Assessment Introduction Risk Assessment: A Tool for Decision-Making Approaches to Microbial Food Safety-Risk Assessment Steps of Risk Assessment Mathematical Approaches to Risk Assessment Monte Carlo Analysis Uncertainty and Variability Data Considerations Applications of Risk Assessment Summary 880 Section VI Appendix Chapter 67 Microbiological Media, Reagents, and Stains Introduction General Considerations and Safety in the Preparation and Use of Media Equipment Alphabetical Listing of Culture Media Dyes and Reagents Stains Alphabetical Listing of Stains 947 Chapter 68 Measurement of Water Activity, Acidity, and Brix Water Activity Acidity Brix 961 Index 965 -I XV

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