Cleanroom Microbiology forthe Non-Microbiologist

SECOND EDITION

David M. Carlben

CRC PRESSBoca Raton London New York Washington, D.C.

CONTENTS

1 The Scope of Microbiology 1I. Introduction 1II. Cells 3III. Binomial Nomenclature 3IV. Morphology 4V. Specific Microbial Groups 5

A. Bacteria 51. The Gram Stain 82. External Features of Bacterial Cells 103. The Bacterial Interior 144. Bacterial Genetics 16

B. Fungi 181. Molds 182. Yeasts 20

C. Algae 21D. Protozoa 22E. Viruses 23

1. The Viral Infection Cycle 252. Retroviruses 273. Oncoviruses 274. Viruses and Gene Therapy 27

F. Subviral Particles 281. Viroids 282. Satellite RNA 283. Prions 28

VI. Taxonomy of Microorganisms 29VII. Microscopy 30

A. Light Microscopy 311. Staining 312. Types of Light Microscopes 33

XV

XVI Cleanroom Microbiology for the Non-Microbiologist

B. Electron Microscopy 34VIII. Summary 37

Growth of Microorganisms 39I. Introduction 39

A. Scientific Notation 40II. Growth of Bacteria 40

A. Nutritional Requirements 40B. Environmental Requirements 43

1. Temperature 432. Atmosphere 443. pH 47

C. Binary Fission and the Bacterial Growth Curve 481. Microbe Math 482. Characteristics of Bacterial Populations 493. The Growth Curve 50

D. Continuous Growth 53E. Determining Bacterial Populations in Cultures 54

1. The Viable Plate Count 552. Filtration Methods 593- Total Cell Counts 594. Instrumental Methods 61

F. Microorganisms as Tools 65III. Growing Other Microorganisms: Fungi, Protozoa, Algae,

and Viruses 66A. Fungi 66B. Protozoa and Algae 67C. Viruses 67

1. Counting Viruses 68IV. Aseptic Technique 68V. Safety Issues 70VI. Summary 71

Controlling Growth and Activities of Microorganisms 73I. Introduction 73II. Definitions 73III. Physical Methods of Control 74

A. The Kinetics of Microbial Death 74B. Heat 77

1. Moist Heat 772. Dry Heat 813. Sterilizing Large Loads 82

C. Low Temperatures 84D. Radiation 84

1. Ionizing Radiation 852. Nonionizing Radiation 86

IV.

V.

VI.

VII.

E. Filtration.1. Dept2. Mem3. Meet4. Valid

Ensuring SterilitA. Overkill IsB. BioburderC. ValidationD. MaintainirChemical MethcA. DefinitionB. Factors InC. Disinfecta

1. Char2. Selec3. Valid4. Rotai

D. Sterilants.1. Char

E. Antibiotic:Control of MicrA. The NeedB. Water PuiC. Biofilms...Summary

4 Cleanroom FaciliiI.II.III.IV.V.VI.VII.VIII.

IX.

IntroductionCertified ClearuBioclean FacilitClean FacilitiesUnidirectional iHEPA and ULftAseptic Fill AreBarriers, CleanA. Biological

1. Clas;2. Clas;3. Clas;4. Prop5. BSC

B. Isolators.C. Clean BeiBiosafety Level;A. BSL4 FaciB. Positive-P

Contents XVII

E. Filtration 881. Depth Filters 892. Membrane Filters 893. Mechanisms of Filtration 894. Validation of Filtration Sterilization 91

IV. Ensuring Sterility 92A. Overkill Method 92B. Bioburden Method 93C. Validation of Sterilization Processes 94D. Maintaining Sterility 95

V. Chemical Methods of Control 96A. Definitions 96B. Factors Influencing Antimicrobial Chemical Agents 97C. Disinfectants 99

1. Characteristics of Specific Agents 992. Selection of Disinfectants 1013. Validation of Disinfectants 1024. Rotation of Disinfectants 103

D. Sterilants 1041. Characteristics of Specific Agents 104

E. Antibiotics 106VI. Control of Microorganisms in High-Purity Water Systems 107

A. The Need for Pure Water 107B. Water Purification Methods 108C. Biofilms 110

VII. Summary I l l

Cleanroom Facilities and Personnel Controls 113I. Introduction 113II. Certified Cleanrooms 114III. Bioclean Facilities — Viable vs. Nonviable Particles 115IV. Clean Facilities — General Considerations 116V. Unidirectional Air Flow 117VI. HEPA and ULPA Filters 120VII. Aseptic Fill Areas 123VIII. Barriers, Clean Zones, and Isolators 124

A. Biological Safety Cabinets 1241. Class I BSCs 1252. Class II BSCs 1253. Class III BSCs 1284. Proper BSC Operation 1295. BSC Certification 130

B. Isolators 131C. Clean Benches 134

IX. Biosafety Levels 134A. BSL4 Facilities 137B. Positive-Pressure Personnel Suits 137

xviii • Cleanroom Microbiology for the Non-Microbiologist

X. Cleanroom Garments 137A. Correct Gowning Procedures 139B. Sterile Gowning 142

XI. Personnel Practices and Training 142XII. Personal Hygiene 143XIII. General Personnel Practices 144XIV. Summary 145

5 Detection and Enumeration of Microorganisms in theCleanroom 147I. Introduction 147II. Overview of Microbiological Environmental Monitoring 147

A. Validation 147B. Monitoring Techniques 148

1. General Considerations 1482. Sampling Sites 1513. Frequency of Sampling 1524. Transporting Microbiological Samples 152

III. Monitoring of Air 153A. Air Samplers 153

1. The Andersen Sampler 1532. The Slit Sampler 1553. Portable Samplers 1564. Liquid Impingers 1595. Sampler Efficiencies 161

IV. Fallout Methods 162V. Surface Monitoring 163VI. Touch Plates and Other Personnel Monitoring 166VII. Microbiological Assessment of Liquids 167VIII. Microbiological Assessment of Solids and Semisolids 168IX. Monitoring for Specific Microorganisms 169

A. Bacteria 1691. Bacterial Nutrition and Choice of Growth Media 169

B. Fungi 1711. Fungal Nutrition and Choice of Growth Media 171

X. Rapid Methods of Identification 172XI. Testing for Bacteriostasis and Fungistasis 173XII. Detection of Biofilms 174XIII. Sterile Media Fill Tests 175XIV. Alert and Action Levels 175XV. The Sterility Suite 176XVI. Disposal of Cultures 176XVII. Summary 176

References 179

Index 183

THMIC

IMicrobiology is the brandthings, microorganisms. Tand protozoa. Viruses, wralso included in the studyparticles such as viroids ai

Microorganisms are unhforms of life are present, buconditions are too harsh fconly living things found at;hot springs of Yellowstoneand the extremely dry deseigrow in environments at tiwater have been isolated n

Natural microbial popul;garden soil is particularly(about 1/5 tsp.) may contaiof up to 7000 different sp<This mass represents 0.3%way, an acre of soil downbacteria. Scientists have estiithe living biological mass oi

The bodies of all animal:microorganisms. As many a;centimeter of our skin, andmilliliter. Nasal washings frc