Table of Contents
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
