Clinical Microbiology & Advanced Microbiology Laboratory Manual

Preface 
Introduction 
Laboratory Safety 
General Information 
Microbiology Lab Safety Contract 
Laboratory Waste Disposal 

PART I CLINICAL MICROBIOLOGY LABORATORY

SECTION I.1 Laboratory Safety and Metric System 
I.1.1. Laboratory Safety 
I.1.2. Metric System Denominators, Units, Fraction, and Multiplication 
I.1.3. Exercise: Measurements in Clinical Microbiology 
I.1.4. Exercise: Hand Washing 
LABORATORY REPORT I.1. Diseases Caused by Bacteria, Measurements in Clinical Microbiology, Hand Washing 

SECTION I.2 Growing Bacteria 
I.2.1. Introduction to Microbes 
I.2.2. Exercise: Where are the Microbes? 
Reflection Questions 
Week 2: Making Your Observations 
LABORATORY REPORT I.2 Growing Bacteria 

SECTION I.3 Aseptic Transfer 
I.3.1. Aseptic Transfer Techniques 
I.3.2. Exercise: Agar Art 
I.3.3. Exercise: Aseptic Transfer 
LABORATORY REPORT I.3.1. Agar Art Observations 
LABORATORY REPORT I.3.2. Aseptic Transfer (Plates and Slants) 

SECTION I.4 Isolating Bacteria 
I.4.1. Streak Isolation 
I.4.2. Serial Dilutions Concept 
I.4.3. Counting Colony-Forming Units—Counting Bacteria 
I.4.4. Exercise: Four-Phase Streak 
I.4.5. Exercise: Serial Dilutions and Colony Forming Units Calculation 
LABORATORY REPORT I.4. Streak Isolation, Serial Dilutions, Plate Count, and CFUS Calculation 

SECTION I.5 Microscopy 
1.5.1. Using Your Microscope 
I.5.2. Exercise: Using Your Microscope 
LABORATORY REPORT I.5. Using Your Microscope 

SECTION I.6 Gram Staining 
I.6.1. Introduction to the Gram Staining 
I.6.2. Bacterial Smear Preparation 
I.6.3. Exercise: Gram Staining
LABORATORY REPORT #7 

SECTION I.7 Selective and Differential Media and Biochemical Differential Tests 
I.7.1. Basic Principles of Selective and Differential Media 
I.7.2. Mannitol Salt Agar 
I.7.3. Eosin–Methylene Blue Agar 
I.7.4. Sheep Blood Agar 
I.7.5. Urea Hydrolysis Test 
I.7.6. Citrate Utilization Test 
I.7.7. Catalase Test 
I.7.8. Sulfur Reduction, Indole Production, Motility Test 
I.7.9. Exercise: Selective and Differential Media and Biochemical Differential Tests 
LABORATORY REPORT I.7.1. Selective and Differential Media and Biochemical Differential Tests 
LABORATORY REPORT I.7.2. Differentiating Bacteria (Based on Case Study) 

SECTION I.8 Microbiology and Food 
I.8.1. Introduction 
I.8.2. Exercise: Microbiology of Food 
LABORATORY REPORT I.8. Microbiology of Food 

SECTION I.9 The Unknown Bacteria Identification Project 
I.9.1. Introduction to the Unknown Bacteria Identification Project 
I.9.2. Guidelines for Completing the Unknown Bacteria Identification Project 
I.9.3. Procedure for Completing the Unknown Bacteria Identification Project 
LABORATORY REPORT 1.9. Unknown Bacteria Identification 

PART II ADVANCED MICROBIOLOGY LABORATORY

SECTION II.1 Introduction to Microbiology Laboratory and Laboratory Safety and Metric System 
II.1.1. Introduction to Microbiology Laboratory and Laboratory Safety 
II.1.2. Metric System Denominators, Units, Fractions, and Multiplication 
II.1.3. Conversion of Metric Measurements to Imperial and Imperial to Metric 
II.1.4. Exercise: Hand Washing 
LABORATORY REPORT II.1. Safety and Waste Disposal, Metric System, and Hand Washing 

SECTION II.2 Aseptic Transfer 
II.2.1. Aseptic Transfer Techniques 
II.2.2. Streak Isolation 
II.2.3. Serial Dilutions Concept 
II.2.4. Counting Colony Forming Units
II.2.5. Exercise: Aseptic Transfer and Streak Isolation 
II.2.6. Exercise: Serial Dilutions and Colony Forming Units (CFUS)
Plate Count and Calculation 
LABORATORY REPORT II.2. Aseptic Transfer Techniques, Streak Isolation, Serial Dilutions, Plate Count, and CFUS Calculation 

SECTION II.3 Bacterial Colonies and Culture Characterization 
II.3.1. Bacterial Colonies Morphology 
II.3.2. Bacterial Culture Characteristics 
II.3.3. Exercise: Bacterial Colonies and Culture Characterization Using Agar Art 
LABORATORY REPORT II.3. Bacterial Colony Characterization Using Agar Art 

SECTION II.4 Microscopy 
II.4.1. Introduction to Light Microscope 
II.4.2. Working with Oil-Immersion Microscopy 
II.4.3. Field of View Calculation 
II.4.4. Preparation of Bacterial Smear for Microscopy 
II.4.5. Exercise: Observing Bacteria Through Oil-Immersion Microscopy 
II.4.6. Exercise: Preparing Bacterial Smear for Microscopy and Observing Using Oil-Immersion Microscopy 
LABORATORY REPORT II.4.1. Basics of Microscopy and FOV Calculation 
LABORATORY REPORT II.4.2. Bacterial Smear Preparation from Liquid and Solid Cultures and Observation Using Oil-Immersion Microscopy 

SECTION II.5 Bacterial Smear Staining Techniques 
II.5.1. Basic Principles of Staining 
II.5.2. Classification of Staining 
II.5.3. Simple Staining 
II.5.4. Gram Staining 
II.5.5. Acid-Fast Staining 
II.5.6. Capsule Staining 
II.5.7. Endospore Staining 
II.5.8. Exercise. Staining Bacterial Smears: Gram Staining, Capsule Staining, and Endospore Staining 
LABORATORY REPORT II.5. Staining Techniques: Gram Staining, Capsule Staining, and Endospore Staining 

SECTION II.6 Bacterial Culture Media 
II.6.1. Types of Bacterial Culture Media 
II.6.2. Media Preparation 
II.6.3. Growth Pattern in Broth 
II.6.4. Growth Pattern on Agar Slant 
II.6.5. Growth Pattern on Deep Stab Agar 
II.6.6. Growth Pattern on Agar Plate (Petri Dish) 
II.6.7. Exercise: Evaluation of Bacterial Respiratory Pattern Using Liquid Media and Thioglycolate Broth 
LABORATORY REPORT II.6. Bacterial Respiratory Pattern and Oxygen Requirement 

SECTION II.7 Selective and Differential Media 
II.7.1. Basic Principles of Selective and Differential Media 
II.7.2. Phenylethyl Alcohol Agar 
II.7.3. Mannitol Salt Agar 
II.7.4. Eosin–Methylene Blue Agar 
II.7.5. MacConkey Agar 
II.7.6. Sheep Blood Agar 
II.7.7. Exercise: Selective and Differential Media 
LABORATORY REPORT II.7. Selective and Differential Media 

SECTION II.8 Biochemical Differential Tests 
II.8.1. Phenol Red Test 
II.8.2. Methyl Red/Voges–Proskauer (MR-VP) Test 
II.8.3. Urea Hydrolysis Test 
II.8.4. Citrate Utilization Test 
II.8.5. Catalase Test 
II.8.6. Nitrate Reduction Test 
II.8.7. Sulfur Reduction, Indole Production, Motility Test 
II.8.8. Triple Sugar Iron Agar 
II.8.9. Exercise: Biochemical Differential Tests 
LABORATORY REPORT II.8. Biochemical Differential Tests 

SECTION II.9 Bacterial Gene Recombination 
II.9.1. Types of Bacterial Gene Recombination
II.9.2. Introduction to E. Coli pGLO Transformation 
II.9.3. Calculation of Transformation Efficiency 
II.9.4. Exercise: pGLO Transformation 
LABORATORY REPORT II.9. pGLO Transformation 

SECTION II.10 The Unknown Bacteria Identification Project 
II.10.1. Introduction to the Unknown Bacteria Identification Project
II.10.2. Guidelines for the Unknown Bacteria Identification Project
II.10.3. Exercise: Procedure for Completion of the Unknown Bacteria  Identification Project 
LABORATORY REPORT II.10. Unknown Bacteria Identification 

Appendix
Media Recipes 
Preparation of Broth-Based (Liquid) and Agar-Based (Solid) Media For General Use 
Selective and Differential Media 
Media for Differential Tests