Microbiology. A Laboratory Manual

Laboratory Protocol xv
PART 1 Basic Laboratory Techniques
for Isolation, Cultivation, and
Cultural Characterization of
Microorganisms 1
Introduction 1
Experiment 1: Effectiveness of
Hand Washing 7
Experiment 2: Culture TransferTechniques 13
Experiment 3: Techniques for
Isolation of Pure Cultures 19
Part A: Isolation of Discrete
Colonies from a Mixed
Culture 19
Part B: Isolation of Pure
Cultures from a Spread-Plate or
Streak-Plate Preparation 22
Experiment 4: Cultural
Characteristics of
Microorganisms 29
PART 2 Microscopy 35
Introduction 35
Experiment 5: Microscopic 
Examination of Stained Cell
Preparations 37
Experiment 6: Microscopic
Examination of Living
Microorganisms Using a
Hanging-Drop Preparation or
a Wet Mount 45
Experiment 7: The
Microscopic Measurement of 
Microorganisms 51
PART 3 Bacterial Staining 55
Introduction 55
Experiment 8: Preparation of 
Bacterial Smears 59
Experiment 9: Simple 
Staining 65
Experiment 10: Negative 
Staining 71
Experiment 11: Gram Stain 75
Experiment 12: Acid-Fast 
Stain 81
Experiment 13: Differential 
Staining for Visualization of
Bacterial Cell Structures 87
Part A: Spore Stain (SchaefferFulton
Method)
87
Part B: Capsule Stain (Anthony
Method) 89
PART 4 Cultivation of Microorganisms:
Nutritional and Physical
Requirements, and
Enumeration of Microbial
Populations 97
Introduction 97
Experiment 14: Nutritional 
Requirements: Media for the 
Routine Cultivation of 
Bacteria 99
Experiment 15: Use of 
Differential, Selective, and
Enriched Media 105
Experiment 16: Physical Factors:
Temperature 115
Experiment 17: Physical
Factors: pH of the Extracellular
Environment 121
Experiment 18: Physical
Factors: Atmospheric Oxygen
Requirements 125
Experiment 19: Techniques for
the Cultivation of Anaerobic
Microorganisms 131
Experiment 20: Serial Dilution–
Agar Plate Procedure to Quantitate
Viable Cells 137
Experiment 21: The Bacterial
Growth Curve 145
PART 5 Biochemical Activities of
Microorganisms 153
Introduction 153
Experiment 22: Extracellular 
Enzymatic Activities of 
Microorganisms 155
Experiment 23: Carbohydrate
Fermentation 161
Experiment 24: Triple Sugar–Iron
Agar Test 167
Experiment 25: IMViC Test 171
Part A: Indole Production 
Test 172
Part B: Methyl Red Test 173
Part C: Voges-Proskauer 
Test 174
Part D: Citrate Utilization 
Test 175
Experiment 26: Hydrogen Sulfide 
Test 183
Experiment 27: Urease Test 187
Experiment 28: Litmus Milk 
Reactions 191
Experiment 29: Nitrate Reduction 
Test 197
Experiment 30: Catalase 
Test 201
Experiment 31: Oxidase 
Test 205
Experiment 32: Utilization of 
Amino Acids 209
Part A: Decarboxylase Test 209
Part B: Phenylalanine 
Deaminase Test 211
Experiment 33: Genus 
Identification of Unknown
Bacterial Cultures 215
PART 6 The Protozoa 221
Introduction 221
Experiment 34: Free-Living 
Protozoa 223
Experiment 35: Parasitic 
Protozoa 229
PART 7 The Fungi 237
Introduction 237
Experiment 36: Cultivation and 
Morphology of Molds 239
Part A: Slide Culture 
Technique 239
Part B: Mold Cultivation on 
Solid Surfaces 242
Experiment 37: Yeast Morphology,
Cultural Characteristics, and 
Reproduction 247
Experiment 38: Identification of 
Unknown Fungi 255
PART 8 The Viruses 261
Introduction 261
Experiment 39: Cultivation 
and Enumeration of
Bacteriophages 265
Experiment 40: Isolation of
Coliphages from Raw Sewage 271
PART 9 Physical and Chemical Agents
for the Control of Microbial
Growth 277
Introduction 277
Experiment 41: Physical Agents 
of Control: Moist Heat 279
Experiment 42: Physical Agents 
of Control: Environmental OsmoticPressure 285
Experiment 43: Physical Agents
of Control: Electromagnetic
Radiations 289
Experiment 44: Chemical Agents
of Control: Chemotherapeutic
Agents 293
Part A: The Kirby-Bauer
Antibiotic Sensitivity Test
Procedure 294
Part B: Synergistic Effect of
Drug Combinations 296
Experiment 45: Determination of
Penicillin Activity in the Presence
and Absence of Penicillinase 303
Experiment 46: Chemical Agents
of Control: Disinfectants and
Antiseptics 309
PART 10 Microbiology of Food 317
Introduction 317
Experiment 47: Microbiological 
Analysis of Food Products:
Bacterial Count 319
Experiment 48: Wine
Production 323
PART 11 Microbiology of Water 327
Introduction 327
Experiment 49: Standard 
Qualitative Analysis of Water 329
Experiment 50: Quantitative 
Analysis of Water: Membrane Filter
Method 337
PART 12 Microbiology of Soil 343
Introduction 343
Experiment 51: Microbial 
Populations in Soil:
Enumeration 345
Experiment 52: Isolation
of Antibiotic-Producing
Microorganisms and
Determination of Antimicrobial
Spectrum of Isolates 351
Part A: Isolation of Antibiotic-
Producing Microorganisms 352
Part B: Determination of
Antimicrobial Spectrum of
Isolates 353
Experiment 53: Isolation of
Pseudomonas Species by Means
of the Enrichment Culture
Technique 357
PART 13 Bacterial Genetics 363
Introduction 363
Experiment 54: Enzyme 
Induction 365
Experiment 55: Bacterial
Conjugation 371
Experiment 56: Isolation
of a Streptomycin-Resistant
Mutant 377
Experiment 57: The Ames Test: A
Bacterial Test System for Chemical
Carcinogenicity 381
PART 14 Biotechnology 387
Introduction 387
Experiment 58: Bacterial 
Transformation 389
Experiment 59: Isolation of 
Bacterial Plasmids 397
Experiment 60: Restriction 
Analysis and Electrophoretic 
Separation of Bacteriophage 
Lambda DNA 407
PART 15 Medical Microbiology 417
Introduction 417
Experiment 61: Microbial Flora 
of the Mouth: Determination of
Susceptibility to Dental Caries 419
Experiment 62: Normal Microbial
Flora of the Throat and Skin 423
Experiment 63: Identification
of Human Staphylococcal
Pathogens 431
Experiment 64: Identification
of Human Streptococcal
Pathogens 439
Experiment 65: Identification of
Streptococcus pneumoniae 447
Experiment 66: Identification 
of Enteric Microorganisms Using 
Computer-Assisted Multitest
Microsystems 453
Experiment 67: Isolation and
Presumptive Identification of
Campylobacter 463
Experiment 68: Microbiological
Analysis of Urine Specimens 467
Experiment 69: Microbiological
Analysis of Blood Specimens 473
Experiment 70: Species
Identification of Unknown
Bacterial Cultures 479
PART 16 Immunology 487
Introduction 487
Experiment 71: Precipitin 
Reaction: The Ring Test 489
Experiment 72: Agglutination 
Reaction: The Febrile Antibody
Test 493
Experiment 73: Immunofluorescence
499
Experiment 74: Enzyme-Linked
Immunosorbent Assay 503
Experiment 75: Sexually
Transmitted Diseases: Rapid
Immunodiagnostic
Procedures 507
Part A: Rapid Plasma Reagin
Test for Syphilis 507
Part B: Genital Herpes: Isolation
and Identification of Herpes
Simplex Virus 509
Part C: Detection of Sexually
Transmitted Chlamydial
Diseases 510
Appendices
Appendix 1: Scientific
Notation 515
Appendix 2: Methods for the
Preparation of Dilutions 517
Appendix 3: Microbiological
Media 519
Appendix 4: Biochemical Test
Reagents 525
Appendix 5: Staining
Reagents 528
Appendix 6: Experimental
Microorganisms 529
Art & Photo Credits 531