Detection and Enumeration of Bacteria, Yeast, Viruses, and Protozoan in Foods and Freshwater (Methods and Protocols in Food Science)

This volume details methods and procedures used to detect and enumerate bacteria in food. Chapters guide readers through food and beverage matrices, techniques used to enumerate bacteria, mixed bacterial strains (naturally present or inoculated), yeast, viruses, protozoan in distinct food matrices, and freshwater. Authoritative and cutting-edge, Detection and Enumeration of Bacteria, Yeast, Viruses, and Protozoan in Foods and Freshwater aims to provide a basic understanding on detection and enumeration of microorganisms in foods. Dedication Preface Contents Contributors Chapter 1: Survival of Pathogens on Surfaces and the Influence of Inoculating Matrix on Survival Capabilities 1 Introduction 2 Materials 2.1 Preparation of Surfaces 2.2 Preparation of Culture Media 2.3 Preparation of Bacterial Strains 2.4 Preparation of Controlled Environment 3 Methods 3.1 Survival Based on Surface and Temperatures 3.2 Survival Based on Diluent Type 3.3 Survival at Different Starting Concentrations at a High Humidity 4 Conclusions References Chapter 2: Enumeration of Viable Cells of Bacteria in Food and Water with Flow Cytometry 1 Introduction 2 Materials 2.1 Inoculum Preparation 2.2 Dye Concentrated Solutions 3 Methods 3.1 Staining Procedure 3.2 Acquisition Settings and Data Collection 3.3 Correcting Fluorescence Spillover 3.4 Data Analysis 4 Notes References Chapter 3: Evaluation of Physiological Characteristics of Bacterial Cells in Foods and Water with Flow Cytometry 1 Introduction 2 Materials 2.1 Inoculum Preparation 2.2 Dye Concentrated Solutions 3 Methods 3.1 Staining Procedure 3.2 Acquisition Settings and Data Collection 3.3 Correcting Fluorescence Spillover 3.4 Data Analysis 4 Notes References Chapter 4: Detection of Sublethally Injured Cells by the Selective Medium Plating Technique 1 Introduction 2 Materials 2.1 Culturing Method 3 Methods 3.1 Determination of the Maximum Noninhibitory Concentration (MNIC) of the Selective Agent 3.2 Selective Medium Plating Technique 3.3 Interpretation of Results: Quantification of Sublethally Injured Cells 4 Notes References Chapter 5: Combining Culturing Technique and Metabarcoding to Study Microbiota in the Meat Industry 1 Introduction 2 Materials 2.1 Sample Collection 2.2 Culturing Method 2.3 Metabarcoding 3 Methods 3.1 Experimental Design 3.2 Sample Collection 3.3 Culturing Method 3.4 Metabarcoding 4 Notes References Chapter 6: Assessment of In Vitro Biofilms by Plate Count and Crystal Violet Staining: Is One Technique Enough? 1 Introduction 2 Materials 2.1 Quantification of Biofilms by Plate Count Technique 2.2 Quantification of Biofilms by Crystal Violet Staining Assay 3 Methods 3.1 Quantification of the Biofilms by Plate Count Technique 3.2 Quantification of Biofilm Mass by Crystal Violet Staining Assay 4 Notes References Chapter 7: Evolution Assays for the Isolation of Mutant Bacteria Resistant to Natural Antimicrobials 1 Introduction 2 Materials 2.1 Evolution Assay by Cyclic Exposure to Prolonged Sub-inhibitory Doses 2.2 Evolution Assay by Cyclic Exposure to Short Lethal Treatments 3 Methods 3.1 Evolution Assay by Cyclic Exposure to Prolonged Sub-inhibitory Doses 3.2 Evolution Assay by Cyclic Exposure to Short Lethal Treatments 4 Notes References Chapter 8: Preparing Yeast Suspension Through Serial Dilution for Enumeration 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 9: Standardizing Suspension of Yeast for Inoculation in Food Fermentations 1 Introduction 2 Materials 2.1 Growth Curve 2.1.1 Culture Media 2.1.2 Spectrophotometer 2.1.3 Dry Weight 2.2 McFarland Equivalence Turbidity 2.3 Preservation of Inocula 2.3.1 Freezer Freezing at -70 C 2.3.2 Lyophilization 2.3.3 Refrigerated Storage 3 Methods 3.1 Growth Curve 3.1.1 Spectrophotometer 3.1.2 Dry Weight 3.2 McFarland Equivalence Turbidity 3.3 Preservation of Inoculum 3.3.1 Freezer Freezing at -70 C 3.3.2 Lyophilization 3.3.3 Refrigerated Storage 4 Notes References Chapter 10: Enumerating Yeast in Foods and Water Using the Spread Plating Technique 1 Introduction 2 Materials 2.1 Basic Equipment for Preparing Culture Media and Enumerating Yeasts 2.2 Diluents (See Notes 1 and 2) 2.3 Culture Media (See Note 3) 2.3.1 Basal Media 2.3.2 Acidified Media 2.3.3 Biostatic Agents and Antibiotics 2.3.4 Selective Media 2.3.5 Differential Media 3 Method 3.1 Diluents (See Note 13) 3.1.1 Peptone Water (PW) (0.1% m/v) 3.1.2 Butterfield ́s Phosphate Buffer (PB) (0.1 M, pH 7.0) 3.1.3 Saline Solution (NaCl 0.85%) 3.2 Culture Media Preparation 3.2.1 Basal Media 3.2.2 Acidified Media 3.2.3 Biostatic Agents and Antibiotics Antibiotics Biostatic Agents DRBC Agar OGYE Agar 3.2.4 Selective Media DG18 Agar MEA Agar with 40% Glucose MEA or TGY Supplemented with 0.5% Acetic Acid MYGP Copper Agar 3.2.5 Differential Media DBDM Agar 3.3 Samples Preparation (See Note 15) 3.4 Serial Dilutions 3.5 Enumeration 3.6 Incubation (See Notes 20-22) 3.7 Counting the Colonies and Calculating the Results 3.8 Morphological Characterization 3.9 Purification and Maintenance of Yeast Culture 4 Notes References Chapter 11: Enumerating Distinct Yeast in the Same Food Sample 1 Introduction 2 Materials 2.1 Selective and Differential Culture Media 2.1.1 Medium (See Note 1) 2.2 Direct Microscopy 3 Methods 3.1 Selective and Differential Culture Media 3.1.1 Lysine Agar 3.1.2 Molybdate Agar 3.1.3 Wallerstein Laboratory Nutrient Agar (WL) 3.1.4 CHROMagar Candida 3.2 Direct Microscopy 3.2.1 Wet Mount Slides 3.2.2 Neubauer Chamber 4 Notes References Chapter 12: Detection and Quantification of Yeast Species in Food Samples for Quality Control 1 Introduction 2 Materials 2.1 Yeast Culture 2.2 Identification by Physiological Characterization (See Note 2) 2.3 Identification by Specific Primer Pairs by PCR 2.4 Identification by MALDI-TOF 2.5 Independent Culture Method: Identification and Quantification by qPCR 3 Methods 3.1 Yeast Culture 3.2 Identification by Physiological Characterization 3.3 Identification by Specific Primer Pairs by PCR (See Note 10) 3.4 Identification by MALDI-TOF (See Note 13) 3.5 Independent Culture Method: Identification and Quantification by qPCR 4 Notes References Chapter 13: Evaluation of Yeast Inoculated in Parallel to the Autochthonous Microbiota in Food Samples 1 Introduction 2 Materials 2.1 Cell Viability 2.2 3M Petrifilm Rapid Yeast and Mold Count 2.3 Slide Culture Technique 2.4 Pulse Field Gel Electrophoresis (PFGE) 2.5 Quantitative Polymerase Chain Reaction (qPCR) 2.6 Matrix-Assisted Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF) 3 Methods 3.1 3M Petrifilm Rapid Yeast and Mold Count (RYM) 3.2 Slide Culture Technique 3.3 PFGE Analysis 3.4 qPCR Analysis 3.5 MALDI-TOF Analysis 4 Notes References Chapter 14: Double-Layer Plaque Assay Technique for Enumeration of Virus Surrogates 1 Introduction 2 Materials 2.1 Single-Layer Plaque or Bottom Agar 2.2 Double-Layer Plaque or Top Agar 2.3 Trypticase Soy Broth 2.4 Overnight Host Bacteria Stock Cultures 3 Methods 3.1 Double Agar Overlay Plaque Assay (Fig. 1) 4 Data Analysis and Calculations 5 Notes References Chapter 15: Detection of Protozoan Parasites on Leafy Greens Using Multiplex PCR 1 Introduction 2 Materials 2.1 Leafy Green Processing (Washing) 2.2 Nucleic Acid Extraction 2.3 Polymerase Chain Reaction 3 Methods 3.1 Recovery of Protozoan Parasites from Leafy Greens 3.2 Nucleic Acid Extraction 3.3 Multiplex Polymerase Chain Reaction (mPCR) 4 Notes References Chapter 16: Viability of Trypanosoma cruzi in Food and Beverages 1 Introduction 2 Materials 2.1 Trypanosoma cruzi 2.2 Food Matrix 2.3 Sieving System 2.4 Eluent 2.5 Microscope 2.6 Experimental Host 2.7 Antibiotic 3 Methods 3.1 Trypanosoma cruzi 3.2 Food Matrix 3.3 Experimental Contamination of the Food Matrix 3.4 Experimental Groups 3.5 Processing of the Food Matrix 3.6 Sieving 3.7 Elution 3.8 Microscopy 3.9 Inoculum and Route of Inoculation 3.10 Experimental Infection 3.11 Antibiotic Therapy 3.12 Parasitemia 3.13 Animal Care 3.14 Statistical Analysis 3.15 Biosafety, Ethics, and Bioethics 3.16 Quality of Analytical Results 4 Notes References Chapter 17: Detection of Giardia Cysts and Cryptosporidium Oocysts in Edible Shellfish: Choosing a Target 1 Introduction 1.1 Overview of Strategies for the Detection of Cryptosporidium Oocysts and Giardia Cysts in Shellfish 2 Materials 2.1 Pre-Sampling Harvesting 2.2 Reagents 2.3 Materials 2.4 Sample Collection 3 Methods 3.1 Protocol 1: Detection of Protozoa through Liquid Materials from Mollusks 3.1.1 Bivalve Opening 3.1.2 Sample Processing Internal Content Gill Collection and Processing 3.2 Protocol 2: Detection of Protozoa through Homogenized Tissue Materials from Mollusks 3.2.1 Bivalves Opening 3.2.2 Sample Processing Gill and Gastrointestinal Tract Removal 3.3 Purification Using Immunomagnetic Separation (IMS) 3.4 Detection of Protozoa by Direct Immunofluorescence Assay 3.5 Detection of Protozoa by Molecular Methods 4 Notes References Chapter 18: Protocol for the Detection of Toxoplasma gondii Oocysts in Water Samples 1 Introduction 2 Materials 3 Methods 3.1 Indication 3.2 Sampling 3.3 Concentration 3.4 Filtration 3.5 Membrane Elution and Centrifuge Concentration 3.6 Purification 3.7 Detection 3.7.1 Microscopic Detection (See Note 2) 3.7.2 PCR Detection DNA Extraction PCR Targeting the rep529 4 Notes References Chapter 19: Detection of Toxoplasma gondii in Milk and Cheese 1 Introduction 2 Materials 2.1 For Detection of T. gondii in Milk 2.2 For Detection of T. gondii in Cheese 3 Methods 3.1 Milk Processing 3.2 Cheese Processing 4 Notes References Chapter 20: Detection of Toxoplasma Gondii in Meat 1 Introduction 2 Materials 2.1 Obtaining and Transporting Samples 2.2 Peptic Digestion 2.3 Bioassay in Swiss Mice 2.4 Molecular Analysis 3 Methods 3.1 Meat Obtention 3.2 Concentration 3.2.1 Peptic Digestion: Adapted from Dubey 3.3 Detection 3.3.1 Bioassay in Swiss Mice (see Note 8): Adapted from Dubey (see Note 9) 3.3.2 Molecular Analysis DNA Extraction and Purification Polymerase Chain Reaction (PCR) 4 Notes References Chapter 21: Detection of Toxoplasma Gondii and Cyclospora Cayetanensis in Oysters 1 Introduction 2 Materials 2.1 Aspiration of Hemolymph 2.2 Pepsin-HCl Digestion of Whole Tissue 2.3 Nucleic Acid Extraction 2.4 Polymerase Chain Reaction 3 Methods 3.1 Aspiration of Hemolymph 3.2 Pepsin-HCl Digestion of Whole Tissue 3.3 Nucleic Acid Extraction 3.4 Polymerase Chain Reaction (PCR) 4 Notes References Index