Preface iii

Introduction: Laboratory Operations and Safety xiii

Basic Rules for Microbiology Laboratory Safety xv

PART 1 General Microscopy and Aseptic Technique

1 Laboratory Safety: Introduction to the Microscope 3

Laboratory Safety and Procedures, 4

Laboratory Procedures, 4

Simple Stain Technique, 5

The Microscope, 6

Microscope Components (Plate 1), 7

Eukaryotic Versus Prokaryotic Cells (Plates 2A, 2B), 10

Laboratory Cleanup, 10

Microscope Cleanup, 10

Discards, 11

General Cleanup, 11

Speaking of Safety, 11

2 Transfer and Isolation Techniques, Microbes in the Environment 17

Transfer Technique, 17

Tube-to-Tube Transfers, 18

Basic Aseptic Technique Procedures, 18

Isolation Techniques: Streak Plate and Pour Plate (Optional), 22

Streak Plate (Plate 3), 22

Prepare the Following Streak Plates Using the Cultures Provided, 25

To Lap or Not to Lap— That is the Question, 25

Pour Plate (Plate 4) Optional, May be Done as a Demonstration, 25

Microbes in the Environment, 26

Human Environment: Procedure, 27

Classroom/School Environment: Procedure, 27

Incubation, 27

Results, 27

Laboratory Cleanup, 28

Discards, 28

General Cleanup, 28

PART 2 Microbial Morphology, Differential Stains

3 Cultural and Cellular Morphology 33

Cultural Characteristics of Bacteria, 33

Procedure: Plates (Plate 5), 34

Procedure: Agar Slants, 35

Procedure: Broth (Plate 6), 35

Microbial Cellular Morphology, 35

Smear Preparation, 36

Broth Preparation, 37

Agar Slant and Plate Preparation, 37

Simple Stain Procedure, 37

Results from the Simple Stain Procedure (Plates 7–16), 38

Results from Prepared Slide, 38

Laboratory Cleanup, 38

4 Bacterial Growth 43

Factors Needed for Bacterial Growth, 44

Measuring Bacterial Growth, 45

Procedure for Preparation of Spread Plate, 45

Procedure for Demonstration of a Bacterial Growth Curve, 47

5 Gram Stain and Acid-Fast Stain 53

The Gram Stain, 53

The Gram Stain Technique (Traditional Method) (Plate 17A, B, C), 54

Gram Stain: (Alternative Method), 55

The Acid-Fast Stain Technique (Plate 18), 56

Acid-Fast Technique, 56

Laboratory Cleanup, 58

Incubation, 58

Discards, 58

General Cleanup, 58

Slides and Microscopes, 58

6 Endospore Stain, Capsule Stain, and the Hanging Drop Technique 61

The Endospore Stain, 61

The Spore Stain Technique (Plates 19, 20), 62

Results, 63

The Capsule Stain, 63

The Capsule Stain Technique (Plate 21), 63

The Hanging Drop Technique, 64

Results, 64

7 Fungi 69

The Fungi, 69

Results, 72

8 Viruses—Visualization and Enumeration 77

Viruses, 77

Bacteriophage Enumeration, 78

Results, 80

9 Parasitology 85

PART 3 Microbial Control and Biochemistry

10 Microbial Sensitivity Testing 97

Part 1: Physical Methods Ultraviolet Light Sensitivity, 98

Sterilization by the Use of UV Light, 98

The Penetrating Power of UV Light (Plate 34), 99

Heat Sensitivity (may be done as an alternative to the UV light procedure), 101

Effect of Cold Temperature and Slow Freezing (Optional), 102

Part 2: Chemical Methods Chemical Sensitivity, 102

Chemotherapeutic Agent Testing: The Kirby–Bauer Plate, 103

The Kirby–Bauer Technique, 104

Results, 107

11 Bacterial Biochemistry 113

Carbohydrate Metabolizing Enzymes, 114

Results, 115

Carbohydrate Fermentation (Phenol Red Broth) Tubes (Plate 37), 115

Amino Acid and Nitrogen Metabolism, 116

Results, 117

Decarboxylase Tubes (Plate 42), 117

12 Gas Requirements of Microorganisms 123

Growth of Anaerobes (may be done as a demonstration), 124

Demonstration of Catalase, 125

13 Specialized Media 129

Examples, 130

Inoculation of Blood Agar Plates for Demonstration of Alpha Hemolysis and Transport Medium, 131

Inoculation of Blood, Phenylethyl Alcohol, Mannitol Salt, Macconkey, and Eosin–Methylene Blue Agar Plates, 132

Results, 133

Inoculation of Triple Sugar Iron Agar and Sulfide–Indole Motility Medium, 133

Results, 134

Results of Transport Medium, 134

PART 4 Medical Microbiology

14 Genetics 143

Ames Test, 144

UV Light Procedure, 145

Kirby–Bauer Procedure, 145

Results: Ames Test (Plate 55), 145

Results: UV Light Procedure (Plate 35), 146

Results: Kirby–Bauer Test (Plates 36, 55), 146

15 Epidemiology 149

Handwashing Procedure (Done with Three Students), 150

Results: Handwashing (Plate 56), 151

Fomite and Direct Transmission of Microbes, 151

Results: Fomite and Direct Transmission, 152

Airborne Infections: Cough and Sneeze Plates, 152

Results: Airborne and Cough/Sneeze Plates, 153

Microbes in Makeup (Optional), 153

Results: Microbes in Makeup, 153

16 Specimen-Handling Protocols 159

Quantitative Urinalysis, 160

Results: Urine Samples, 162

Specimens from the Gastrointestinal Tract, 163

Results: GI Tract Isolation Technique, 163

17 Specific Laboratory Tests 169

Catalase Test, 171

Alternative Method (to be done at the end of this exercise), 171

Results: Catalase Test (Plate 46), 171

Bacitracin Sensitivity—Demonstration, 171

Results: Bacitracin Sensitivity (Plate 57), 172

Oxidase Test, 172

Results: Oxidase Test (Plate 59), 172

Coagulase Test, 172

Rapid Slide Test, 172

Tube Test, 172

Results: Coagulase Tube Test (Plate 60), 172

Novobiocin Sensitivity, 172

Results: Novobiocin Sensitivity, 173

Camp Test (Plate 58), 173

Results: Camp, 173

18 Serology 179

Elisa Test: Test for Toxins A and B from Clostridium Difficile, 180

Results, 182

Rapid Identification of Group A Antigen, 182

Results, 184

Differentiation of Streptococci Using Latex Agglutination, 184

Slidex Strepto-Kit^®, 184

Results, 186

Procedure Controls, 186

PART 5 Identification of a Bacterial Unknown

19 Identification of Enteric Pathogens: Traditional Methods 191

Results, 194

IMViC Test (See Plate 61), 194

Results of Gelatin Hydrolysis, 194

Results of the Oxidase Test (See Plate 59), 194

20 Identification of Enteric Pathogens: Rapid Identification Methods 201

EnteroPluri Test (Plate 62), 201

Compartment 1: Glucose Fermentation and Gas Production, 202

Compartments 2 and 3: Lysine and Ornithine Decarboxylation, 202

Compartment 4: Sulfide and Indole Production, 202

Compartments 5–8: Adonitol, Lactose, Arabinose, and Sorbitol Fermentation, 202

Compartment 9: Voges–Proskauer, 202

Compartment 10: Phenylalanine Deaminase and Dulcitol, 202

Compartment 11: Urea, 202

Compartment 12: Citrate, 202

Api® 20 E System (Plate 63), 203

Microtube 1: Ortho-Nitrophenyl-Beta-D-Ortho-Nitrophenyl-Beta-D-Galactopyranoside (Onpg), 203

Microtubes 2–4: Arginine Dihydrolase (Decarboxylase), Lysine Decarboxylase, and Ornithine Decarboxylase (Adh, Ldc, Odc), 203

Microtube 5: Citrate (Cit), 203

Microtube 6: Sulfide (H2s), 203

Microtube 7: Urea (Ure), 203

Microtube 8: Tryptophan Deaminase (Tda), 203

Microtube 9: Indole (Ind), 203

Microtube 10: Voges–Proskauer (Vp), 203

Microtube 11: Gelatin Hydrolysis (Gel), 203

Microtubes 12–20: Utilization of the Carbohydrates Glucose, Mannitol, Inositol, Sorbitol, Rhamnose, Saccharose (Sucrose), Melibiose, Amygdalin, and Arabinose (Glu, Man, Ino, Sor, Rha, Sac, Mel, Amy, And Ara), 203

Inoculation of Rapid Identification Media, 204

Inoculation of EnteroPluri Test, 204

Inoculation of the Api 20 E System, 204

Results, 205

21 Identification of a Bacterial Unknown: The Gram-Negative Unknown 209

Review, 209

Comparison Chart, 210

Flowchart, 210

Computer Identification, 210

ID Value Worksheet for EnteroPluri Test II 212

Results, 213

22 Identification of a Bacterial Unknown: The Gram-Positive Cocci 217

PART 6 Food and Environmental Microbiology

23 Identification and Quantitation of Microbial Numbers in a Water Sample 225

Presumptive Test: Analysis of a Contaminated Water Sample for the Presence of Coliforms, 226

Results, 227

Quantitation of Microbial Number in a Water Sample, 227

Results (Plate 64), 228

24 Identification of Microbes in Beef and Poultry and the Quantitation of Microbial Numbers 231

Microbial Presence in a Food Product and Colony Counts, 232

Results, 233

Recognition of Organism Genera, 233

25 Soil Microbiology 237

Part 1: The Recovery of Microorganisms from Soil, 238

Results, 239

Isolation Of Soil Bacteria, 239

Results, 239

Part 2: Isolation of Soil Bacteriophages of Bacillus Subtilis, 239

Results, 240

26 Microbial Ecology 245

Competition Between Bacteria, 246

Results, 246

Bacteriocin Production, 246

Results, 247

Bacterial–Fungal Interaction (Plate 65), 247

Results, 247

27 Biofilms 251

Growth of Dental Plaque in situ, 252

Results, 253

Soil Biofilm, 253

Results, 253

Growth of Bacterial Biofilms in vitro, 253

Results, 254

Bibliography 261

Answer Key for the Exercise Pre-Tests 263

Appendix 1 Flow Charts and Comparison Charts for the Identification of Enteric Gram Negative Rods Using Traditional Testing Procedures 265

Appendix 2 Flow Chart for the Identification of Enterobacteriaceae based on the API 20 E Rapid Identification System 269

Appendix 3 Gram-Positive Flow Chart 275

Photo Credit List 277

Index 279

Photographic Atlas PA-1

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