Microbiology Laboratory Theory & Application

Microbiology Laboratory Theory & Application


Author:
Michael J. Leboffe & Burton E. Pierce
Published in: Morton Publishing Company
Release Year: 2010
ISBN: 978-0-89582-830-9
Pages: 786
Edition: Third edition
File Size: 34 MB
File Type: pdf
Language: English



Description of Microbiology Laboratory Theory & Application


This Microbiology Laboratory Theory & Application third edition in many ways is like another first edition. We have added 20 new exercises, incorporated four more exercises from MLTA Brief Edition, and have substantially rewritten several others. Every exercise has been screened and updated for better clarity, comprehensiveness, and appropriate placement in the manual. We have added three more of the boxed “A Word About” features to offer expanded introductory material in Sections 4, 5, and 8. Finally, we replaced many older photographs, and perhaps most important, employed a new artist to update the illustrations and enhance the overall beauty of the Microbiology Laboratory Theory & Application book.

Content of Microbiology Laboratory Theory & Application



Introduction Safety and Laboratory Guidelines  1

A Word About Experimental Design  6

Data Presentation: Tables and Graphs 7

Data Presentation: Be Creative, But Complete!8

SECTION 1 Fundamental Skills for the Microbiology Laboratory11

EXERCISE 1-1 Glo-GermTM Hand Wash Education System12

Basic Growth Media 14

EXERCISE 1-2 Nutrient Broth and Nutrient Agar Preparation 14

Aseptic Transfers and Inoculation Methods 17

EXERCISE 1-3 Common Aseptic Transfers and Inoculation Methods18

EXERCISE 1-4 Streak Plate Methods of Isolation  25

EXERCISE 1-5 Spread Plate Method of Isolation29

SECTION 2 Microbial Growth33

Diversity and Ubiquity of Microorganisms  34

EXERCISE 2-1 Ubiquity of Microorganims34

EXERCISE 2-2 Colony Morphology36

EXERCISE 2-3 Growth Patterns on Slants44

EXERCISE 2-4 Growth Patterns in Broth 45

Environmental Factors Affecting Microbial Growth 46

EXERCISE 2-5 Evaluation of Media46

Aerotolerance48

EXERCISE 2-6 Agar Deep Stabs  48

EXERCISE 2-7 Fluid Thioglycollate Medium 50

EXERCISE 2-8 Anaerobic Jar  52

EXERCISE 2-9 The Effect of Temperature on Microbial Growth  54

EXERCISE 2-10 The Effect of pH on Microbial Growth  56

EXERCISE 2-11 The Effect of Osmotic Pressure on Microbial Growth58

Control of Pathogens: Physical and Chemical Methods60

EXERCISE 2-12 Steam Sterilization61

EXERCISE 2-13 The Lethal Effect of Ultraviolet Radiation

on Microbial Growth 64

EXERCISE 2-14 Chemical Germicides: Disinfectants and Antiseptics66

SECTION 3 Microscopy and Staining69

Microscopy70

EXERCISE 3-1 Introduction to the Light Microscope70

EXERCISE 3-2 Calibration of the Ocular Micrometer  76

EXERCISE 3-3 Microscopic Examination of Eukaryotic Microbes  79

EXERCISE 3-4 Microscopic Examination of Pond Water 84

Bacterial Structure and Simple Stains 95

EXERCISE 3-5 Simple Stains 100

EXERCISE 3-6 Negative Stains  103

Differential and Structural Stains  105

EXERCISE 3-7 Gram Stain105

EXERCISE 3-8 Acid-Fast Stains  110

EXERCISE 3-9 Capsule Stain 115

EXERCISE 3-10 Endospore Stain 117

EXERCISE 3-11 Parasporal Crystal Stain121

EXERCISE 3-12 Wet Mount and Hanging Drop Preparations 122

EXERCISE 3-13 Flagella Stain 124

EXERCISE 3-14 Morphological Unknown 126

SECTION 4 Selective Media 129

A Word About Selective Media130

Selective Media for Isolation of Gram-Positive Cocci 131

EXERCISE 4-1 Phenylethyl Alcohol Agar131

EXERCISE 4-2 Columbia CNA With 5% Sheep Blood Agar  133

EXERCISE 4-3 Bile Esculin Test 135

EXERCISE 4-4 Mannitol Salts Agar 137

Selective Media for Isolation of Gram-Negative Rods139

EXERCISE 4-5 MacConkey Agar 139

EXERCISE 4-6 Eosin Methylene Blue Agar 142

EXERCISE 4-7 Hektoen Enteric Agar145

EXERCISE 4-8 Xylose Lysine Desoxycholate Agar 147

SECTION 5 Differential Tests  149

A Word About Biochemical Tests and Acid-Base Reactions  150

Introduction to Energy Metabolism Tests 151

EXERCISE 5-1 Reduction Potential  152

EXERCISE 5-2 Oxidation–Fermentation Test  155

Fermentation Tests 158

EXERCISE 5-3 Phenol Red Broth158

EXERCISE 5-4 Methyl Red and Voges-Proskauer Tests  161

Tests Identifying Microbial Ability to Respire 165

EXERCISE 5-5 Catalase Test 165

EXERCISE 5-6 Oxidase Test  168

EXERCISE 5-7 Nitrate Reduction Test  171

Nutrient Utilization Tests  175

EXERCISE 5-8 Citrate Test175

EXERCISE 5-9 Malonate Test177

Decarboxylation and Deamination Tests  179

EXERCISE 5-10 Decarboxylation Tests  179

EXERCISE 5-11 Phenylalanine Deaminase Test182

Tests Detecting Hydrolytic Enzymes  184

EXERCISE 5-12 Starch Hydrolysis184

EXERCISE 5-13 Urea Hydrolysis  187

EXERCISE 5-14 Casein Hydrolysis Test  190

EXERCISE 5-15 Gelatin Hydrolysis Test  192

EXERCISE 5-16 DNA Hydrolysis Test 194

EXERCISE 5-17 Lipid Hydrolysis Test 196

EXERCISE 5-18 ONPG Test198

EXERCISE 5-19 PYR Test  200

Combination Differential Media202

EXERCISE 5-20 SIM Medium  202

EXERCISE 5-21 Triple Sugar Iron Agar / Kligler Iron Agar 206

EXERCISE 5-22 Lysine Iron Agar  209

EXERCISE 5-23 Litmus Milk Medium 211

Antibacterial Susceptibility Testing214

EXERCISE 5-24 Bacitracin, Novobiocin,

and Optochin Susceptibility Tests 214

Other Differential Tests217

EXERCISE 5-25 Blood Agar217

EXERCISE 5-26 CAMP Test220

EXERCISE 5-27 Coagulase Tests 222

EXERCISE 5-28 Motility Test  224

Multiple Test Systems  226

EXERCISE 5-29 API 20 E Identification System for Enterobacteriaceae

and Other Gram-negative Rods226

EXERCISE 5-30 Enterotube® II 232

Bacterial Unknowns Project237

EXERCISE 5-31 Bacterial Unknowns Project237

SECTION 6 Quantitative Techniques 243

EXERCISE 6-1 Standard Plate Count (Viable Count) 244

EXERCISE 6-2 Urine Culture 248

EXERCISE 6-3 Direct Count (Petroff-Hausser) 250

EXERCISE 6-4 Closed-System Growth  252

EXERCISE 6-5 Plaque Assay of Virus Titre 255

EXERCISE 6-6 Thermal Death Time Versus Decimal Reduction Value 259

SECTION 7 Medical Microbiology 263

EXERCISE 7-1 Snyder Test264

EXERCISE 7-2 Lysozyme Assay  266

EXERCISE 7-3 Antimicrobial Susceptibility Test (Kirby-Bauer Method) 268

EXERCISE 7-4 Clinical Biofilms  272

EXERCISE 7-5 Morbidity and Mortality Weekly Report

(MMWR) Assignment274

EXERCISE 7-6 Epidemic Simulation 276

EXERCISE 7-7 Identification of Enterobacteriaceae  278

EXERCISE 7-8 Identification of Gram-positive Cocci  284

EXERCISE 7-9 Identification of Gram-positive Rods  289

SECTION 8 Environmental Microbiology 295

EXERCISE 8-1 Winogradsky Column296

Introduction to the Nitrogen Cycle299

EXERCISE 8-2 Nitrogen Fixation 300

EXERCISE 8-3 Nitrification: The Production of Nitrate305

EXERCISE 8-4 Ammonification  308

EXERCISE 8-5 Denitrification: Nitrate Reduction  310

A Word About Trophic Groups 312

Sulfur Cycle—Introduction 313

EXERCISE 8-6 Photosynthetic Sulfur Bacteria 315

EXERCISE 8-7 Chemolithotrophic Sulfur-Oxidizing Bacteria 318

EXERCISE 8-8 Sulfur-Reducing Bacteria321

EXERCISE 8-9 Bioluminescence 324

EXERCISE 8-10 Soil Slide Culture326

EXERCISE 8-11 Soil Microbial Count 329

EXERCISE 8-12 Membrane Filter Technique 332

EXERCISE 8-13 Multiple Tube Fermentation Method

for Total Coliform Determination  336

SECTION 9 Food Microbiology 341

EXERCISE 9-1 Methylene Blue Reductase Test  342

EXERCISE 9-2 Making Yogurt344

SECTION 10 Microbial Genetics 345

EXERCISE 10-1 Extraction of DNA from Bacterial Cells346

EXERCISE 10-2 Restriction Digest350

EXERCISE 10-3 Bacterial Transformation: The pGLOTM System  354

EXERCISE 10-4 Polymerase Chain Reaction359

EXERCISE 10-5 Ultraviolet Radiation Damage and Repair364

EXERCISE 10-6 Ames Test 367

EXERCISE 10-7 Phage Typing of E Coli Strains 371

SECTION 11 Hematology and Serology373

EXERCISE 11-1 Differential Blood Cell Count  374

Simple Serological Reactions  377

EXERCISE 11-2 Precipitin Ring Test  377

EXERCISE 11-3 Radial Immunodiffusion 380

EXERCISE 11-4 Slide Agglutination  382

EXERCISE 11-5 Blood Typing  384

EXERCISE 11-6 Mononucleosis Hemagglutination Test387

EXERCISE 11-7 Quantitative Indirect ELISA 389

SECTION 12 Eukaryotic Microbes  397

EXERCISE 12-1 The Fungi—Common Yeasts and Molds  398

Yeasts of Medical or Economic Importance 399

Molds of Medical or Economic Importance  400

EXERCISE 12-2 Fungal Slide Culture 406

EXERCISE 12-3 Examination of Common Protozoans

of Clinical Importance  409

Amoeboid Protozoans Found in Clinical Specimens 409

Ciliate Protozoan Found in Clinical Specimens  410

Flagellate Protozoans Found in Clinical Specimens 411

Sporozoan Protozoans Found in Clinical Specimens 412

EXERCISE 12-4 Parasitic Helminths  416

Trematode Parasites Found in Clinical Specimens  416

Cestode Parasites Found in Clinical Specimens 417

Nematode Parasites Found in Clinical Specimens  421

APPENDIX A Biochemical Pathways425

Oxidation of Glucose: Glycolysis, Entner-Doudoroff

and Pentose-Phosphate Pathways 425

Oxidation of Pyruvate: The Krebs Cycle and Fermentation428

APPENDIX B Miscellaneous Transfer Methods  433

Transfers Using a Sterile Cotton Swab  433

Stab Inoculation of Agar Tubes Using an Inoculating Needle 434

Spot Inoculation of an Agar Plate 435

APPENDIX C Transfer from a Broth Culture Using a Glass Pipette 437

Filling a Glass Pipette  438

Inoculation of Broth Tubes with a Pipette439

Inoculation of Agar Plates with a Pipette 440

APPENDIX D Transfer from a Broth Culture Using a Digital Pipette441

Filling a Digital Pipettor 441

Inoculation of Broth Tubes with a Digital Pipettor  442

Inoculation of Agar Plates with a Pipettor443

APPENDIX E The Spectrophotometer  445

Theory 445

Instructions for Use of the Spectronic D20+446

APPENDIX F Alternative Procedures for Section 6 449

Alternative Procedure for Exercise 6-1, Standard Plate Count

(Viable Count)  449

Alternative Procedure for Exercise 6-5,

Plaque Assay of Virus Titre450

Alternative Procedure for Exercise 6-6, Thermal

Death Time Versus Decimal Reduction Value  452

APPENDIX G Agarose Gel Electrophoresis455

Gel Preparation and Staining for Exercises 10-2 and 10-4455

APPENDIX H Medium, Reagent, and Stain Recipes 459

Media 459

Reagents 471

Solutions 472

Stains 472

Gram Stain Reagents473

Simple Stains474

Data Sheets  475

SECTION 1 Fundamental Skills for the Microbiology Laboratory477

SECTION 2 Microbial Growth 487

SECTION 3 Microscopy and Staining525

SECTION 4 Selective Media  555

SECTION 5 Differential Tests 571

SECTION 6 Quantitative Techniques635

SECTION 7 Medical Microbiology655

SECTION 8 Environmental Microbiology679

SECTION 9 Food Microbiology707

SECTION 10 Microbial Genetics  711

SECTION 11 Hematology and Serology  731

SECTION 12 Eukaryotic Microbes 745

Glossary757

Index765
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