Microbiology: Laboratory Theory and Application Michael J. Leboffe, Burton E Pierce

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Cover title copyright Preface Acknowledgments INTRODUCTION: Safety and Laboratory Guidelines Student Conduct Basic Laboratory Safety Reducing Contamination of Self, Others, Cultures, and the Environment Guidelines Governing Handling of BSL-2 Organisms Disposing of Contaminated Materials Student Compliance with Laboratory Safety Regulations A Word About Experimental Design Data Presentation: Tables and Graphs Data Presentation: Be Creative, But Complete Student Safety Contract SECTION 1: Fundamental Skills for the Microbiology Laboratory Exercise 1-1: A Comparison of Hand-Cleansing Agents Basic Growth Media Exercise 1-2: Nutrient Broth and Nutrient Agar Preparation Aseptic Transfers and Inoculation Methods Exercise 1-3: Common Aseptic Transfers and Inoculation Methods Exercise 1-4: Streak Plate Methods of Isolation Exercise 1-5: Spread Plate Method of Isolation SECTION 2: Microbial Growth Diversity and Ubiquity of Microorganisms Exercise 2-1: Ubiquity of Microorganisms Exercise 2-2: Colony Morphology Exercise 2-3: Growth Patterns on Slants Exercise 2-4: Growth Patterns in Broth Environmental Factors Affecting Microbial Growth Exercise 2-5: Evaluation of Media Aerotolerance Exercise 2-6: Agar Deep Stabs Exercise 2-7: Fluid Thioglycollate Broth Exercise 2-8: Anaerobic Jar Effect of Environmental Factors on Bacterial Growth Rate Exercise 2-9: The Effect of Temperature on Microbial Growth Exercise 2-10: The Effect of pH on Microbial Growth Exercise 2-11: The Effect of Osmotic Pressure on Microbial Growth Control of Pathogens: Physical and Chemical Methods Exercise 2-12: Steam Sterilization Exercise 2-13: The Lethal Effect of UltravioletRadiation on Microbial Growth Exercise 2-14: Chemical Germicides:Disinfectants and Antiseptics SECTION 3: Microscopy and Staining Exercise 3-1: Introduction tothe Light Microscope Exercise 3-2: Calibration of the Ocular Micrometer Exercise 3-3: Microscopic Examination of Eukaryotic Microbes Exercise 3-4: Microscopic Examination of Freshwater Microbes Bacterial Structure and Simple Stains Exercise 3-5: Simple Stains Exercise 3-6: Negative Stains Differential and Structural Stains Exercise 3-7: Gram Stain Exercise 3-8: Acid-Fast Stains Exercise 3-9: Capsule Stain Exercise 3-10: Endospore Stain Exercise 3-11: Wet Mount and Hanging Drop Preparation Exercise 3-12: Flagella Stain Exercise 3-13: Morphological Unknown SECTION 4: Selective Media A Word about Selective Media Selective Media for Isolation of Gram-Positive Cocci Exercise 4-1: Phenylethyl Alcohol Agar Exercise 4-2: Columbia CNA with 5% Sheep Blood Agar Exercise 4-3: Bile Esculin Agar Exercise 4-4: Mannitol Salt Agar Selective Media for Isolation of Gram-negative Rods Exercise 4-5: MacConkey Agar Exercise 4-6: Eosin Methylene Blue Agar (Levine) Exercise 4-7: Hektoen Enteric Agar Exercise 4-8: Xylose Lysine Desoxycholate Agar SECTION 5: Differential Tests A Word about Biochemical Tests and Acid-Base Reactions Introduction to Energy Metabolism Tests Exercise 5-1: Reduction Potential Exercise 5-2: Oxidation–Fermentation Test Fermentation Tests Exercise 5-3: Phenol Red Broth Exercise 5-4: Methyl Red and Voges–Proskauer Tests Exercise 5-5: ONPG Test Exercise 5-6: Catalase Test Exercise 5-7: Oxidase Test Exercise 5-8: Nitrate Reduction Test Nutrient Utilization Tests Exercise 5-9: Citrate Utilization Test Exercise 5-10: Malonate Utilization Test Decarboxylation and Deamination Tests Exercise 5-11: Decarboxylation Tests Exercise 5-12: Phenylalanine Deaminase Test Tests Detecting Hydrolytic Enzymes Exercise 5-13: Starch Hydrolysis (Amylase Test) Exercise 5-14: DNA Hydrolysis (DNase Test) Exercise 5-15: Lipid Hydrolysis (Lipase Test) Exercise 5-16: Casein Hydrolysis (Casease Test) Exercise 5-17: Gelatin Hydrolysis (Gelatinase Test) Exercise 5-18: Urea Hydrolysis (Urease Test) Exercise 5-19: PYR Test Combination Differential Media Exercise 5-20: SIM Medium Sulfur Reduction, Indole Production, Motility Exercise 5-21: Triple Sugar Iron Agar / Kligler Iron Agar Exercise 5-22: Lysine Iron Agar Exercise 5-23: Litmus Milk Medium Antimicrobial Susceptibility Testing Exercise 5-24: Bacitracin, Novobiocin, and Optochin Susceptibility Tests Other Differential Tests Exercise 5-25: Blood Agar Exercise 5-26: CAMP Test Exercise 5-27: Coagulase and Clumping Factor Tests Exercise 5-28: Motility Test Multiple Test Systems Exercise 5-29: api 20 E Identification System for Enterobacteriaceae and Other Gram-negative Rods Exercise 5-30: EnteroPluri–Test (Enterotube® II) Bacterial Unknowns Project Exercise 5-31: Bacterial Unknowns Project SECTION 6: Quantitative Techniques Exercise 6-1: Standard Plate Count (Viable Count) Exercise 6-2: Urine Culture Exercise 6-3: Direct Count (Petroff–Hausser) Exercise 6-4: Closed-System Growth Exercise 6-5: Plaque Assay of Virus Titer Exercise 6-6: Thermal Death Time Versus Decimal Reduction Value SECTION 7: Medical Microbiology Exercise 7-1: Snyder Test Exercise 7-2: Lysozyme Assay Exercise 7-3: Antimicrobial Susceptibility Test: Disk Diffusion (Kirby-Bauer) Method Exercise 7-4: Clinical Biofilms Exercise 7-5: Morbidity and Mortality Weekly Report (MMWR) Assignment Exercise 7-6: Epidemic Simulation Exercise 7-7: Identification of Enterobacteriaceae Exercise 7-8: Identification of Gram-positive Cocci Exercise 7-9: Identification of Gram-positive Rods SECTION 8: Environmental Microbiology A Word about Trophic Groups Exercise 8-1: Winogradsky Column Introduction to the Nitrogen Cycle Exercise 8-2: Nitrogen Fixation Exercise 8-3: Nitrification: The Production of Nitrate Exercise 8-4: Ammonification Exercise 8-5: Denitrification: Nitrate Reduction Sulfur Cycle—Introduction Exercise 8-6: Photosynthetic Sulfur Bacteria Exercise 8-7: Chemolithotrophic Sulfur-Oxidizing Bacteria Exercise 8-8: Sulfur-Reducing Bacteria Exercise 8-9: Bioluminescence Exercise 8-10: Soil Slide Culture Exercise 8-11: Soil Microbial Count Water Quality Testing Exercise 8-12: Membrane Filter Technique Exercise 8-13: Multiple Tube Fermentation Methodfor Determining Water Quality SECTION 9: Food Microbiology Exercise 9-1: Methylene Blue Reductase Test Exercise 9-2: Making Yogurt Exercise 9-3: Yogurt CFU Density of an Active and Live Culture and Identification of a Yogurt Unknown SECTION 10: Microbial Genetics Exercise 10-1: Extraction of DNA from Bacterial Cells Exercise 10-2: Restriction Digest Exercise 10-3: Bacterial Transformation: the pGLO™ System Exercise 10-4: Polymerase Chain Reaction Exercise 10-5: Ultraviolet Radiation Damage and Repair Exercise 10-6: Ames Test Exercise 10-7: Phage Typing of E. Coli Strains SECTION 11: Hematology and Serology Exercise 11-1: Differential Blood Cell Count Introduction to Antigens and Antibodies Simple Serological Reactions Exercise 11-2: Precipitin Ring Test Exercise 11-3: Double Gel Immunodiffusion Exercise 11-4: Slide Agglutination Exercise 11-5: Blood Typing Exercise 11-6: ELISA for Detecting Antibodies in a Patient’s Sample (The Antibody Capture Method) SECTION 12: Eukaryotic Microbes Eukaryotic Supergroups—A Refresher Exercise 12-1: Representative Fungi of Medical and Economic Importance Exercise 12-2: Fungal Slide Culture Exercise 12-3: Examination of Simple Eukaryotic Pathogens Exercise 12-4: Parasitic Helminths APPENDICES APPENDIX A: Biochemical Pathways APPENDIX B: Miscellaneous Transfer Methods APPENDIX C: Transfer from a Broth Culture Using a Glass Pipette APPENDIX D: Transfer from a Broth Culture Using a Digital Pipette APPENDIX E: The Spectrophotometer APPENDIX F: Alternative Procedures for Section 6 APPENDIX G: Agarose Gel Electrophoresis APPENDIX H: Medium, Reagent, and Stain Recipes Glossary Index Copyright Title Page Dedication Contents Chapter 1: ‘I’m thinking’ – Oh, but are you? Chapter 2: Renegade perception Chapter 3: The Pushbacker sting Chapter 4: ‘Covid’: The calculated catastrophe Chapter 5: There is no ‘virus’ Chapter 6: Sequence of deceit Chapter 7: War on your mind Chapter 8: ‘Reframing’ insanity Chapter 9: We must have it? So what is it? Chapter 10: Human 2.0 Chapter 11: Who controls the Cult? Chapter 12: Escaping Wetiko Postscript Appendix: Cowan-Kaufman-Morell Statement on Virus Isolation Bibliography Index