Microbiology/ a laboratory manual Cappuccino, James G.

Part I Basic Laboratory Techniques for Isolation, Cultivation, and Cultural
Characterization of Microorganisms 1
Introduction 1
Experiment 1: Culture Transfer techniques 7
Experiment 2: Techniques for Isolation of Pure Cultures 13
Part A: Isolation of Discrete Colonies frbm a Mixed Culture 13
Part B: Isolation of Pure Cultures from a Spread-Plate or Streak-Plate
Preparation 14
Experiment 3: Cultural Characteristics of Microorganisms 21
Part II Microscopy 27
Introduction 27
Experiment 4: Microscopic Examination of Stained Cell Preparations 29
Experiment 5: Microscopic Examination of Living Bacterial Preparations 37
Experiment 6: The Microscopic Measurement of Microorganisms 41
Part III Bacterial Staining 45
Introduction 45
Experiment 7: Simple Staining 51
Experiment 8: Negative Staining 55
Experiment 9: Gram Stain 59
Experiment 10: Acid-Fast Stain (Ziehl-Neelsen Method) 65
Experiment 11: Spore Stain (Schaeffer-Fulton-Method) 71
Experiment 12: Capsule Stain 75
Part IV Cultivation of Microorganisms: Nutritional and Physical
Requirements, and Enumeration of Microbial Populations 79
Introduction 79
Experiment 13: Nutritional Requirements: Media for the Routine Cultivation of
Bacteria 83 . *
Experiment 14: Use of Differential and Selective Media 89
Experiment 15: Physical Factors: Temperature 95 '
Experiment 16: Physical Factors: pH of the Extracellular Environment 99
Xiv CONTENTS
Experiment 17: Physical Factors: Atmospheric Oxygen Requirements 103
Experiment 18: Techniques for the Cultivation of Anaerobic Microorganisms 109
Experiment 19: Serial Dilution-Agar Plate Procedure to'Quantitate Viable Cells 115
Experiment 20: The Bacterial Growth Curve 121
Part V Biochemical Activities of Microorganisms 129
Introduction 129
Experiment 21: Extracellular Enzymatic Activities of Microorganisms 131
Experiment 22: Carbohydrate Fermentation 137
Experiment 23: Triple Sugar-Iron Agar Test 1.43
Experiment 24: IMViC Test 147
Part A: Indole Production Te^ 147
Part B: Methyl Red Test 148
Part C: Voges-Proskauer Test 149
Part D: Citrate Utilization Test 150
Experiment 25: Hydrogen Sulfide Test 157
Experiment 26: Urease Test 161
Experiment 27: Litmus Milk Reactions 165
Experiment 28: Nitrate Reduction Test 171
Experiment 29: Catalase Test 175
Experiment 30: Oxidase Test 179
Experiment 31: Genus Identification of Unknown Bacterial Cultures 183
Part VI The Protozoa 187
Introduction 187
Experiment 32: Free-Living Protozoa 189 •
Experiment 33: Parasitic Protozoa 193
Part VII The Fungi 199
Introduction 199
Experiment 34: Cultivation and Morphology of Molds 201
Experiment 35: Yeast Morphology 205
Experiment 36: Identification of Unknown Fungi 209
Part VIII The Viruses 215
Introduction 215
Experiment 37: Cultivation and Enumeration of Bacteriophages 219
Experiment 38: Isolation of Coliphages from Raw Sewage 225
Experiment 39: Staphylococcal Phage Typing 231
Part IX Physical and Chemical Agents for the Control of
CONTENTS
Microbial Growth 235
Introduction 235
Experiment 40: Physical Agents of Control: Moist Heat 237
Experiment 41: Physical Agents of Control: Environmental Osmotic Pressure 245
XV
Experiment 42: Physical Agents of Control: Electromagnetic Radiations 249
Experiment 43: Chemical Agents of Control: Chemotherapeutic Agents 253
Part A: The Kirby-Bauer Antimicrobial Susceptibility Test Procedure 254
Part B: Synergistic Effect of Drug Combinations 257
Experiment 44: Determination of Penicillin Activity in the Presence and Absence of
Penicillinase 263
Experiment 45: Chemical Agents of Control: Disinfectants and Antiseptics 267
■ i Part A: Phenol Coefficient 271
Part B: Agar Plate-Sensitivity Method 272
Part X Microbiology of Food 277
Introduction 277
Experiment 46: Methylene Blue Reductase Test 279
Experiment 47: Microbiological Analysis of Food Products: Bacterial Count 283
Experiment 48: Wine Production 287
Experiment 49: Sauerkraut Production 291
Part XI Microbiology of Water 295
Introduction 295
Experiment 50: Standard Qualitative Analysis of Water 297
Part A: Presumptive Test: Determination of the Most Probable Number of Coliform
Bacteria 297
Part B: Confirmed Test 299
Part C: Completed Test 300
Experiment 51: Quantitative Analysis of Water: Membrane Filter Method 303
Part XII Microbiology of Soil 309
Introduction 309
Experiment 52: Nitrogen Cycle 311
Part A: Ammonification 311
Part B: Nitrification 312
Part C: Denitrification 313
Part D: Nitrogen Fixation 314
Experiment 53: Microbial Populations in Soil: Enumeration 323
Experiment 54: Isolation of Antibiotic-Producing Microorganisms and Determination of
Antimicrobial Spectrum of Isolates 329
Part A: Isolation of Antibiotic-Producing Microorganisms 329
Part B: Determination of Antimicrobial Spectrum of Isolates 331 .
Experiment 55: Isolation of Pseudomonas Species by Means of the Enrichment Culture
Technique 335
Part XIII Bacterial Genetics 341
introduction 341
Experiment 56: Enzyme Induction 343
Experiment 57: Bacterial Conjugation 347
Vi CONTENTS
Experiment 58: Isolation of a Streptomycin-Resistant Mutant 353
Experiment 59: The Ames Test: A Bacterial Test System for Chemical
Carcinogenicity 357
Part XIV Medical Microbiology 363
Introduction 363
Experiment 60: Microbial Flora of the Mouth: Determination of Susceptibility to Dental
Experiment 61: Normal Microbial Flora of the Throat and Skin 369
Experiment 62: Identification of Human Staphylococcal Pathogens 373
Experiment 63: Identification of Human Streptococcal Pathogens 381
Experiment 64: Identification of Streptococcus pneumoniae 387
Experiment 65: Identification of Enteric Microorganisms Using Computer-Assisted
Multitest Microsystems 391
Enterotube Multitest System and Encise II 392
API (Analytical Profile Index) System 393
Experiment 66: Isolation and Presumptive Identification of Campylobacter 401
Experiment 67: Microbiological Analysis of Urine Specimens 405
Experiment 68: Microbiological Analysis of Blood Specimens 411
Experiment 69: Species Identification of Unknown Bacterial Cultures 417
Part XV Immunology 425
CONTENTS
Introduction 425
Experiment 70: Precipitin Reaction: The Ring Test 427
Experiment 71: Precipitin Reaction: Immunodiffusion 431
Experiment 72: Agglutination Reaction: The Febrile Antibody Test 437
Experiment 73: Immunofluorescence 441
Experiment 74: Latex Agglutination Test 445
Experiment 75: Enzyme-Linked Immunosorbent Assay 449
Experiment 76. Agglutination Reaction: Mono-Test for Infectious Mononucleosis 453
Experiment 77: Sexually Transmitted Diseases: Rapid Immunodiagnostic
Procedures 457
Part A: Rapid Plasma Reagin Test for Syphilis 457
Part B: Genital Herpes: Isolation and Identification of Herpes Simplex Virus 458
Part C: Detection of Sexually Transmitted Chlamydial Diseases 459