Microbiology: Laboratory Theory and Application

Cover......Page 1
Title page......Page 3
Copyright......Page 4
Preface......Page 5
Acknowledgments......Page 7
Contents......Page 9
Introduction: Safety and Laboratory Guidelines......Page 15
A Word About Experimental Design......Page 20
Data Presentation: Tables and Graphs......Page 21
Data Presentation: Be Creative, But Complete......Page 22
Section 1: Fundamental Skills for the Microbiology Laboratory......Page 25
Exercise 1-1: Glo-Germ™ Hand Wash Education System......Page 26
Exercise 1-2: Nutrient Broth and Nutrient Agar Preparation......Page 28
Aseptic Transfers and Inoculation Methods......Page 31
Exercise 1-3: Common Aseptic Transfers and Inoculation Methods......Page 32
Exercise 1-4: Streak Plate Methods of Isolation......Page 39
Exercise 1-5: Spread Plate Method of Isolation......Page 43
Section 2: Microbial Growth......Page 47
Exercise 2-1: Ubiquity of Microorganims......Page 48
Exercise 2-2: Colony Morphology......Page 50
Exercise 2-3: Growth Patterns on Slants......Page 58
Exercise 2-4: Growth Patterns in Broth......Page 59
Exercise 2-5: Evaluation of Media......Page 60
Exercise 2-6: Agar Deep Stabs......Page 62
Exercise 2-7: Fluid Thioglycollate Medium......Page 64
Exercise 2-8: Anaerobic Jar......Page 66
Exercise 2-9: The Effect of Temperature on Microbial Growth......Page 68
Exercise 2-10: The Effect of pH on Microbial Growth......Page 70
Exercise 2-11: The Effect of Osmotic Pressure on Microbial Growth......Page 72
Control of Pathogens: Physical and Chemical Methods......Page 74
Exercise 2-12: Steam Sterilization......Page 75
Exercise 2-13: The Lethal Effect of Ultraviolet Radiation on Microbial Growth......Page 78
Exercise 2-14: Chemical Germicides: Disinfectants and Antiseptics......Page 80
Section 3: Microscopy and Staining......Page 83
Exercise 3-1: Introduction to the Light Microscope......Page 84
Exercise 3-2: Calibration of the Ocular Micrometer......Page 90
Exercise 3-3: Microscopic Examination of Eukaryotic Microbes......Page 93
Exercise 3-4: Microscopic Examination of Pond Water......Page 98
Bacterial Structure and Simple Stains......Page 109
Exercise 3-5: Simple Stains......Page 114
Exercise 3-6: Negative Stains......Page 117
Exercise 3-7: Gram Stain......Page 119
Exercise 3-8: Acid-Fast Stains......Page 124
Exercise 3-9: Capsule Stain......Page 129
Exercise 3-10: Endospore Stain......Page 131
Exercise 3-11: Parasporal Crystal Stain......Page 135
Exercise 3-12: Wet Mount and Hanging Drop Preparations......Page 136
Exercise 3-13: Flagella Stain......Page 138
Exercise 3-14: Morphological Unknown......Page 140
Section 4: Selective Media......Page 143
A Word About Selective Media......Page 144
Exercise 4-1: Phenylethyl Alcohol Agar......Page 145
Exercise 4-2: Columbia CNA With 5% Sheep Blood Agar......Page 147
Exercise 4-3: Bile Esculin Test......Page 149
Exercise 4-4: Mannitol Salts Agar......Page 151
Exercise 4-5: MacConkey Agar......Page 153
Exercise 4-6: Eosin Methylene Blue Agar......Page 156
Exercise 4-7: Hektoen Enteric Agar......Page 159
Exercise 4-8: Xylose Lysine Desoxycholate Agar......Page 161
Section 5: Differential Tests......Page 163
A Word About Biochemical Tests and Acid-Base Reactions......Page 164
Introduction to Energy Metabolism Tests......Page 165
Exercise 5-1: Reduction Potential......Page 166
Exercise 5-2: Oxidation–Fermentation Test......Page 169
Exercise 5-3: Phenol Red Broth......Page 172
Exercise 5-4: Methyl Red and Voges-Proskauer Tests......Page 175
Exercise 5-5: Catalase Test......Page 179
Exercise 5-6: Oxidase Test......Page 182
Exercise 5-7: Nitrate Reduction Test......Page 185
Exercise 5-8: Citrate Test......Page 189
Exercise 5-9: Malonate Test......Page 191
Exercise 5-10: Decarboxylation Tests......Page 193
Exercise 5-11: Phenylalanine Deaminase Test......Page 196
Exercise 5-12: Starch Hydrolysis......Page 198
Exercise 5-13: Urea Hydrolysis......Page 201
Exercise 5-14: Casein Hydrolysis Test......Page 204
Exercise 5-15: Gelatin Hydrolysis Test......Page 206
Exercise 5-16: DNA Hydrolysis Test......Page 208
Exercise 5-17: Lipid Hydrolysis Test......Page 210
Exercise 5-18: ONPG Test......Page 212
Exercise 5-19: PYR Test......Page 214
Exercise 5-20: SIM Medium......Page 216
Exercise 5-21: Triple Sugar Iron Agar / Kligler Iron Agar......Page 220
Exercise 5-22: Lysine Iron Agar......Page 223
Exercise 5-23: Litmus Milk Medium......Page 225
Exercise 5-24: Bacitracin, Novobiocin, and Optochin Susceptibility Tests......Page 228
Exercise 5-25: Blood Agar......Page 231
Exercise 5-26: CAMP Test......Page 234
Exercise 5-27: Coagulase Tests......Page 236
Exercise 5-28: Motility Test......Page 238
Exercise 5-29: API 20 E Identification System for Enterobacteriaceae and Other Gram-negative Rods......Page 240
Exercise 5-30: Enterotube® II......Page 246
Exercise 5-31: Bacterial Unknowns Project......Page 251
Section 6: Quantitative Techniques......Page 257
Exercise 6-1: Standard Plate Count (Viable Count)......Page 258
Exercise 6-2: Urine Culture......Page 262
Exercise 6-3: Direct Count (Petroff-Hausser)......Page 264
Exercise 6-4: Closed-System Growth......Page 266
Exercise 6-5: Plaque Assay of Virus Titre......Page 269
Exercise 6-6: Thermal Death Time Versus Decimal Reduction Value......Page 273
Section 7: Medical Microbiology......Page 277
Exercise 7-1: Snyder Test......Page 278
Exercise 7-2: Lysozyme Assay......Page 280
Exercise 7-3: Antimicrobial Susceptibility Test (Kirby-Bauer Method)......Page 282
Exercise 7-4: Clinical Biofilms......Page 286
Exercise 7-5: Morbidity and Mortality Weekly Report (MMWR) Assignment......Page 288
Exercise 7-6: Epidemic Simulation......Page 290
Exercise 7-7: Identification of Enterobacteriaceae......Page 292
Exercise 7-8: Identification of Gram-positive Cocci......Page 298
Exercise 7-9: Identification of Gram-positive Rods......Page 303
Section 8: Environmental Microbiology......Page 309
Exercise 8-1: Winogradsky Column......Page 310
Introduction to the Nitrogen Cycle......Page 313
Exercise 8-2: Nitrogen Fixation......Page 314
Exercise 8-3: Nitrification: The Production of Nitrate......Page 319
Exercise 8-4: Ammonification......Page 322
Exercise 8-5: Denitrification: Nitrate Reduction......Page 324
A Word About Trophic Groups......Page 326
Sulfur Cycle—Introduction......Page 327
Exercise 8-6: Photosynthetic Sulfur Bacteria......Page 329
Exercise 8-7: Chemolithotrophic Sulfur-Oxidizing Bacteria......Page 332
Exercise 8-8: Sulfur-Reducing Bacteria......Page 335
Exercise 8-9: Bioluminescence......Page 338
Exercise 8-10: Soil Slide Culture......Page 340
Exercise 8-11: Soil Microbial Count......Page 343
Exercise 8-12: Membrane Filter Technique......Page 346
Exercise 8-13: Multiple Tube Fermentation Method for Total Coliform Determination......Page 350
Section 9: Food Microbiology......Page 355
Exercise 9-1: Methylene Blue Reductase Test......Page 356
Exercise 9-2: Making Yogurt......Page 358
Section 10: Microbial Genetics......Page 359
Exercise 10-1: Extraction of DNA from Bacterial Cells......Page 360
Exercise 10-2: Restriction Digest......Page 364
Exercise 10-3: Bacterial Transformation: The pGLO™ System......Page 368
Exercise 10-4: Polymerase Chain Reaction......Page 373
Exercise 10-5: Ultraviolet Radiation Damage and Repair......Page 378
Exercise 10-6: Ames Test......Page 381
Exercise 10-7: Phage Typing of E. coli Strains......Page 385
Section 11: Hematology and Serology......Page 387
Exercise 11-1: Differential Blood Cell Count......Page 388
Exercise 11-2: Precipitin Ring Test......Page 391
Exercise 11-3: Radial Immunodiffusion......Page 394
Exercise 11-4: Slide Agglutination......Page 396
Exercise 11-5: Blood Typing......Page 398
Exercise 11-6: Mononucleosis Hemagglutination Test......Page 401
Exercise 11-7: Quantitative Indirect ELISA......Page 403
Section 12: Eukaryotic Microbes......Page 411
Exercise 12-1: The Fungi—Common Yeasts and Molds......Page 412
Yeasts of Medical or Economic Importance......Page 413
Molds of Medical or Economic Importance......Page 414
Exercise 12-2: Fungal Slide Culture......Page 420
Amoeboid Protozoans Found in Clinical Specimens......Page 423
Ciliate Protozoan Found in Clinical Specimens......Page 424
Flagellate Protozoans Found in Clinical Specimens......Page 425
Sporozoan Protozoans Found in Clinical Specimens......Page 426
Trematode Parasites Found in Clinical Specimens......Page 430
Cestode Parasites Found in Clinical Specimens......Page 431
Nematode Parasites Found in Clinical Specimens......Page 435
Oxidation of Glucose: Glycolysis, Entner-Doudoroff and Pentose-Phosphate Pathways......Page 439
Oxidation of Pyruvate: The Krebs Cycle and Fermentation......Page 442
Transfers Using a Sterile Cotton Swab......Page 447
Stab Inoculation of Agar Tubes Using an Inoculating Needle......Page 448
Spot Inoculation of an Agar Plate......Page 449
Appendix C: Transfer from a Broth Culture Using a Glass Pipette......Page 451
Filling a Glass Pipette......Page 452
Inoculation of Broth Tubes with a Pipette......Page 453
Inoculation of Agar Plates with a Pipette......Page 454
Filling a Digital Pipettor......Page 455
Inoculation of Broth Tubes with a Digital Pipettor......Page 456
Inoculation of Agar Plates with a Pipettor......Page 457
Theory......Page 459
Instructions for Use of the Spectronic D20+......Page 460
Alternative Procedure for Exercise 6-1, Standard Plate Count (Viable Count)......Page 463
Alternative Procedure for Exercise 6-5, Plaque Assay of Virus Titre......Page 464
Alternative Procedure for Exercise 6-6, Thermal Death Time Versus Decimal Reduction Value......Page 466
Gel Preparation and Staining for Exercises 10-2 and 10-4......Page 469
Media......Page 473
Reagents......Page 485
Stains......Page 486
Gram Stain Reagents......Page 487
Simple Stains......Page 488
Data Sheets......Page 489
Section 1: Fundamental Skills for the Microbiology Laboratory......Page 491
Section 2: Microbial Growth......Page 501
Section 3: Microscopy and Staining......Page 539
Section 4: Selective Media......Page 569
Section 5: Differential Tests......Page 585
Section 6: Quantitative Techniques......Page 649
Section 7: Medical Microbiology......Page 669
Section 8: Environmental Microbiology......Page 693
Section 9: Food Microbiology......Page 721
Section 10: Microbial Genetics......Page 725
Section 11: Hematology and Serology......Page 745
Section 12: Eukaryotic Microbes......Page 759
Glossary......Page 771
Index......Page 779