Cleanroom Microbiology for the Non-Microbiologist

SECOND EDITION

David M. Carlben

CRC PRESS
Boca Raton London New York Washington, D.C.

CONTENTS
1 The Scope of Microbiology
Introduction Cells Binomial Nomenclature Morphology Specific Microbial Groups A. Bacteria 1. The Gram Stain 2. External Features of Bacterial Cells 3. The Bacterial Interior 4. Bacterial Genetics B. Fungi 1. Molds 2. Yeasts C. Algae D. Protozoa E. Viruses 1. The Viral Infection Cycle 2. Retroviruses 3. Oncoviruses 4. Viruses and Gene Therapy F. Subviral Particles 1. Viroids 2. Satellite RNA 3. Prions VI. Taxonomy of Microorganisms VII. Microscopy A. Light Microscopy 1. Staining 2. Types of Light Microscopes I. II. III. IV. V.

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Cleanroom Microbiology for the Non-Microbiologist

B. Electron Microscopy VIII. Summary Growth of Microorganisms I. Introduction A. Scientific Notation II. Growth of Bacteria A. Nutritional Requirements B. Environmental Requirements 1. Temperature 2. Atmosphere 3. pH C. Binary Fission and the Bacterial Growth Curve 1. Microbe Math 2. Characteristics of Bacterial Populations 3. The Growth Curve D. Continuous Growth E. Determining Bacterial Populations in Cultures 1. The Viable Plate Count 2. Filtration Methods 3- Total Cell Counts 4. Instrumental Methods F. Microorganisms as Tools III. Growing Other Microorganisms: Fungi, Protozoa, Algae, and Viruses A. Fungi B. Protozoa and Algae C. Viruses 1. Counting Viruses IV. Aseptic Technique V. Safety Issues VI. Summary Controlling Growth and Activities of Microorganisms I. Introduction II. Definitions III. Physical Methods of Control A. The Kinetics of Microbial Death B. Heat 1. Moist Heat 2. Dry Heat 3. Sterilizing Large Loads C. Low Temperatures D. Radiation 1. Ionizing Radiation 2. Nonionizing Radiation

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Contents

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Filtration 1. Depth Filters 2. Membrane Filters 3. Mechanisms of Filtration 4. Validation of Filtration Sterilization IV. Ensuring Sterility A. Overkill Method B. Bioburden Method C. Validation of Sterilization Processes D. Maintaining Sterility V. Chemical Methods of Control A. Definitions B. Factors Influencing Antimicrobial Chemical Agents C. Disinfectants 1. Characteristics of Specific Agents 2. Selection of Disinfectants 3. Validation of Disinfectants 4. Rotation of Disinfectants D. Sterilants 1. Characteristics of Specific Agents E. Antibiotics VI. Control of Microorganisms in High-Purity Water Systems A. The Need for Pure Water B. Water Purification Methods C. Biofilms VII. Summary

E.

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Cleanroom Facilities and Personnel Controls

I. II. III. IV. V. VI. VII. VIII. Introduction Certified Cleanrooms Bioclean Facilities Viable vs. Nonviable Particles Clean Facilities General Considerations Unidirectional Air Flow HEPA and ULPA Filters Aseptic Fill Areas Barriers, Clean Zones, and Isolators A. Biological Safety Cabinets 1. Class I BSCs 2. Class II BSCs 3. Class III BSCs 4. Proper BSC Operation 5. BSC Certification B. Isolators C. Clean Benches Biosafety Levels A. BSL4 Facilities B. Positive-Pressure Personnel Suits

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Cleanroom Microbiology for the Non-Microbiologist

Cleanroom Garments A. Correct Gowning Procedures B. Sterile Gowning XI. Personnel Practices and Training XII. Personal Hygiene XIII. General Personnel Practices XIV. Summary 5 Detection and Enumeration of Microorganisms in t h e Cleanroom I. Introduction II. Overview of Microbiological Environmental Monitoring A. Validation B. Monitoring Techniques 1. General Considerations 2. Sampling Sites 3. Frequency of Sampling 4. Transporting Microbiological Samples III. Monitoring of Air A. Air Samplers 1. The Andersen Sampler 2. The Slit Sampler 3. Portable Samplers 4. Liquid Impingers 5. Sampler Efficiencies IV. Fallout Methods V. Surface Monitoring VI. Touch Plates and Other Personnel Monitoring VII. Microbiological Assessment of Liquids VIII. Microbiological Assessment of Solids and Semisolids IX. Monitoring for Specific Microorganisms A. Bacteria 1. Bacterial Nutrition and Choice of Growth Media B. Fungi 1. Fungal Nutrition and Choice of Growth Media X. Rapid Methods of Identification XI. Testing for Bacteriostasis and Fungistasis XII. Detection of Biofilms XIII. Sterile Media Fill Tests XIV. Alert and Action Levels XV. The Sterility Suite XVI. Disposal of Cultures XVII. Summary

X.

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References Index