Pharmaceutical microbiology

1. Introduction and Scope ... 1 1.1 Introduction ... 1 1.2 Historical Development of Microbiology ... 3 1.2.1. The Microscope ... 3 1.2.2. Spontaneous Generation Vs Biogenesis ... 4 1.2.3. Fermentation ... 6 1.2.4. Germ Theory ... 6 1.2.5. Classical Laboratory Methods and Pure Cultures ... 7 1.2.6. Immunity ... 8 1.2.7. Medical Microbiology ... 9 1.2.8. Pharmaceutical Microbiology ... 10 1.2.9. Industrial Microbiology ... 14 1.2.10. Emergence of Molecular Biology ... 15 1.2.11. Emergence of Virology ... 17 1.2.12. Microorganisms as Geochemical Agents ... 19 1.2.13. Microbiology in the New Millennium ... 19. 2. Structure and Function : Bacterial Cells ... 23 2.1 Introduction ... 23 2.2 Characteristic Features ... 23 2.2.1. Shape ... 23 2.2.2. Size ... 23 2.2.3. Reproduction ... 24 2.2.4. Formation of Colony ... 24 2.2.5. Mutation ... 24 2.2.6. Motility ... 24 2.2.7. Food and Oxygen Requirements ... 24 2.2.8. Temperature Requirements ... 24 2.3 Activities ... 25 2.4 Organization of Microbial Cells ... 25 2.4.1. Type of Cells ... 26 2.4.1.1. Eukaryotic Cells ... 27 2.4.1.2. Prokaryotic Cells ... 33 2.5 Archaeobacteria and Eubacteria ... 37 2.5.1. Methanogenic Bacteria [Methanogens] ... 38 2.5.2. Extreme Halophiles ... 40 (ix) 2.5.3. Thermoacidophiles ... 41 2.5.3.1. Thermoplasma ... 41 2.5.3.2. Sulfolobus ... 41 2.6 The Bacterial Cells ... 42 2.6.1. Typical Bacterial Cells ... 43 2.6.2. Capsules and Slimes ... 44 2.6.3. Flagella and Fimbria ... 46 2.6.3.1. Flagella ... 46 2.6.3.2. Fimbria [or Pili] ... 48 2.6.4. Cell Envelope ... 49 2.6.5. Gram-Positive and Gram-Negative Bacteria ... 51 2.6.6. Significance of Teichoic Acids ... 53 2.6.7. The Cell Membrane ... 54 2.6.8. Bacterial Cytoplasm ... 55 2.6.9. Ribosomes ... 57 2.6.10. Cellular Reserve Materials ... 58 3. Characterization, Classification and Taxonomy of Microbes ... 62 3.1 Introduction ... 62 3.2 Characterization ... 62 3.2.1. Morphological Characteristics ... 63 3.2.2. Chemical Characteristics ... 64 3.2.3. Cultural Characteristics ... 64 3.2.4. Metabolic Characteristics ... 66 3.2.5. Antigenic Characteristics ... 66 3.2.6. Genetic Characteristics ... 67 3.2.6.1. DNA Base Composition ... 67 3.2.6.2. Sequence of Nucleotide Bases in DNA ... 68 3.2.7. Pathogenecity ... 69 3.2.8. Ecological Characteristics ... 69 3.3 Classificiation ... 70 3.3.1. Difficulties Encountered in Classification of Microorganisms ... 70 3.3.2. Objectives of Classification ... 70 3.3.3. Genetic Methods of Classifying Microbes ... 71 3.3.3.1. Genetic Relatedness ... 71 3.3.3.2. The Intuitive Method ... 72 3.3.3.3. Numerical Taxonomy ... 72 3.3.4. Systemetized Classification ... 75 3.3.4.1. Natural Classification ... 75 3.3.4.2. Phyletic Calssification ... 75 (x) 3.3.4.3. Linnear Binomial Scheme ... 76 3.3.4.4. Phenotypic Classification ... 77 3.3.4.5. Microscopic Examination ... 79 3.3.4.6. Cataloguing rRNA ... 80 3.3.4.7. Computer Aided Classification ... 81 3.3.4.8. Bacterial Classification ... 82 3.4 Taxonomy ... 87 3.5 The Kingdom Prokaryotae ... 88 3.5.1. Actinomyctes ... 89 3.5.1.1. General Characteristics ... 89 3.5.1.2. Significance of Actinomycetes ... 90 3.5.1.3. Classification ... 91 3.5.1.3.1. Whole Cell Carbohydrate Patterns of Aerobic Actinomycetes ... 91 3.5.1.3.2. Major Constituents of Cell Wall Types of Actinomycetes ... 91 3.5.1.3.3. Groups of Actinomycetes Based on Whole Cell Carbohydrate Pattern and Cell Wall Type ... 92 3.5.1.3.4. Actinomycetes with Multiocular Sporangia ... 92 3.5.1.4. Actinomycetes and Related Organisms ... 93 3.5.1.4.1. Group ... 93 3.5.1.4.2. Genus ... 94 3.5.1.4.3. Order ... 97 3.5.1.4.4. Family ... 98 3.5.2. Bacteria ... 102 3.5.2.1. Salient Features ... 103 3.5.2.2. Structure and Form of the Bacterial Cell ... 104 3.5.2.2.1. Size and Shape ... 105 3.5.2.2.2. Structure ... 105 3.5.3. Rickettsia and Coxiella ... 107 3.5.4. Spirochaetes ... 108 4. Identification of Microorganisms ... 112 4.1 Introduction ... 112 4.2 Morphology ... 113 4.3 Selective and Diagnostic Media ... 113 4.3.1. Differential Media ... 116 4.3.1.1. Eosin Methylene Blue Agar [EMB-Agar] ... 116 (xi) 4.3.1.2. MacConkey Agar ... 116 4.3.1.3. Hektoen Enteric Agar [HE-Agar] ... 116 4.3.2. Enrichment Media ... 116 4.3.2.1. Blood Agar ... 116 4.3.2.2. Chocolate Agar ... 117 4.3.3. Characteristic Media ... 117 4.3.3.1. Triple Sugar Iron Agar [TSI-Agar] ... 117 4.4 Cultural Characteristics ... 119 4.5 Biochemical Tests (or Properties) ... 120 4.5.1. Carbohydrate (Sugar) Fermentation ... 120 4.5.2. Litmus Milk ... 120 4.5.3. Indole Production ... 120 4.5.4. Methyl Red Test [MR-Test] ... 121 4.5.5. Voges-Proskauer Test [VP-Test] ... 121 4.5.6. Citrate Utilization ... 121 4.5.7. Nitrate Reduction ... 122 4.5.8. Ammonia Production ... 122 4.5.9. Urease Test ... 122 4.5.10. Production of Hydrogen Sulphide ... 123 4.5.11. Reduction of Methylene Blue ... 123 4.5.12. Production of Catalase [Tube Catalase Test] ... 123 4.5.13. Oxidase Reaction ... 123 4.5.14. Egg-Yolk Reaction ... 124 4.5.15. Growth in Presence of Potassium Cyanide ... 124 4.5.16. Composite Media ... 124 4.6 Profile of Microbial Stains ... 127 4.6.1. Preparation of Bacterial Specimens for Light Microscopy ... 128 4.6.1.1. Standard Preparations ... 128 4.6.1.2. Preparation of Smears for Staining ... 128 4.6.1.3. Gram Staining ... 129 4.6.1.4. Differential Staining ... 131 4.6.1.4.1. Gram’s Stain ... 131 4.6.1.4.2. Acid-Fast Stain ... 131 4.6.1.5. Miscellaneous Staining ... 131 4.6.1.5.1. Capsule Staining ... 132 4.6.1.5.2. Endospore Staining ... 132 4.6.1.5.3. Flagella Staining ... 133 (xii) 4.6.2. Microscopy : The Differential Instruments ... 133 4.6.2.1. Concepts ... 133 4.6.2.2. Microscope Variants ... 134 4.6.2.2.1. Bright-Field Microscope ... 134 4.6.2.2.2. Dark-Field Microscope ... 136 4.6.2.2.3. Phase-Contrast Microscope ... 136 4.6.2.2.4. Differential Interference Contrast (DIC) Microscope ... 139 4.6.2.2.5. Fluorescence Microscope ... 139 4.6.2.2.6. Electron Microscope ... 141 4.6.2.2.6.1. Transmission Electron Microscope (TEM) ... 142 4.6.2.2.6.2. Scanning Electron Microscope (SEM) ... 143 5. Nutrition, Cultivation and Isolation : Bacteria-Actinomycetes-Fungi-Viruses ... 146 5.1 Introduction ... 146 5.2 Bacteria ... 146 5.2.1. Nutrition of Microorganisms ... 146 5.2.2. Cultivation of Bacteria ... 147 5.2.2.1. Binary Fission ... 148 5.2.2.2. Normal Growth Curve of Microorganisms ... 149 5.2.2.3. The Lag Phase of Microbial Growth ... 150 5.2.2.4. Translational Periods Between Various Growth Phases ... 150 5.2.2.5. Synchronous Growth ... 151 5.2.2.6. Effect of Nutritional Concentration Vs Growth Rate of Bacterial Culture ... 152 5.2.2.7. Growth Determining Techniques ... 152 5.2.3. Isolation of Bacteria ... 154 5.2.3.1. Selective and Diagnostic Media ... 154 5.2.3.2. Bismuth Sulphate Agar ... 154 5.2.3.3. Selective Media for Staphylococci ... 155 5.3 Actinomycetes ... 155 5.4 Fungi ... 156 5.4.1. Reproduction of Fungi ... 158 5.4.1.1. Asexual Reproduction ... 158 5.4.1.2. Sexual Reproduction ... 159 5.4.2. Industrial Importance of Fungi ... 159 (xiii) 5.4.2.1. Production of Wines and Beer ... 159 5.4.2.2. Production of Bakery Products ... 160 5.4.2.3. Production of Cheeses ... 160 5.5 Viruses ... 160 5.5.1. Bacteriophages ... 161 5.5.2. Growth of Bacteriophages in the Laboratory ... 164 5.5.3. Bacteriophage Lambda : The Lysogenic Cycle ... 164 6. Microbial Genetics and Variations ... 167 6.1 Introduction ... 167 6.2 Microbial Genetics ... 169 6.2.1. Structure and Function of Genetic Material ... 169 6.2.2. Genotype and Phenotype ... 170 6.2.3. Adaption and Mutation ... 170 6.2.4. DNA and Chromosomes ... 171 6.2.5. DNA Replication ... 172 6.2.6. Rate DNA Replication ... 174 6.2.7. Flow of Genetic Information ... 175 6.2.8. Bacterial Transformation ... 177 6.2.9. Bacterial Transcription ... 180 6.2.10. Bacterial Translation ... 182 6.2.11. Bacterial Conjugation ... 186 6.2.12. Bacterial Transduction ... 188 6.2.12.1. Generalized Transduction ... 189 6.2.12.2. Specialized Transduction ... 190 6.2.13. Bacterial Transfection ... 192 6.2.14. Phage Conversion ... 192 6.3 Microbial Variations [Genetic Manipulation in Microorganisms] ... 193 7. Microbial Control By Physical and Chemical Methods ... 198 7.1 Introduction ... 198 7.2 Physical Methods ... 198 7.2.1. Heat ... 199 7.2.2. Moist Heat ... 200 7.2.2.1. Boiling ... 201 7.2.2.2. Autoclaving ... 201 7.2.2.3. Pasteurization ... 204 7.2.2.4. Dry-Heat Sterilization ... 205 7.2.2.5. Filtration ... 205 (xiv) 7.2.2.6. Cold ... 207 7.2.2.7. Desiccation ... 208 7.2.2.8. Osomotic Pressure ... 208 7.2.2.9. Radiation ... 209 7.2.2.9.1. Ionizing Radiation ... 209 7.2.2.9.2. Nonionizing Radiation ... 210 7.3 Chemical Methods ... 214 7.3.1. Effective Disinfection—Fundamentals ... 214 7.3.2. Disinfectant—Critical Evaluation ... 215 7.3.2.1. Use-Dilution Tests ... 215 7.3.2.2. Filter Paper Method ... 216 7.3.3. Disinfectant Variants ... 216 7.3.3.1. Alcohols ... 216 7.3.3.2. Aldehydes ... 217 7.3.3.3. Chlorohexidine ... 218 7.3.3.4. Gaseous Chemosterilizers ... 218 7.3.3.5. Heavy Metals and Derivatives ... 219 7.3.3.6. Halogens ... 219 7.3.3.7. Organic Acids and Derivatives ... 221 7.3.3.8. Oxidizing Agents ... 222 7.3.3.9. Phenol and Phenolics ... 223 7.3.3.10. Quaternary Ammonium Compounds [QUATS] ... 224 7.3.3.11. Surface-Active Agents ... 226 7.4 Experimental Parameters Influencing the Antimicrobial Agent Activity ... 228 7.4.1. Population Size ... 228 7.4.2. Population Composition ... 228 7.4.3. Concentration of Antimicrobial Agent ... 229 7.4.4. Duration of Exposure ... 229 7.4.5. Temperature ... 229 7.4.6. Local Environment ... 229 8. Sterility Testing : Pharmaceutical Products ... 231 8.1 Introduction ... 231 8.2 Test for Sterility : Pharmaceutical Products ... 233 8.2.1. Membrane Filtration ... 233 8.2.2. Direct Inoculation ... 239 8.2.2.1. Nutrient Broth ... 239 8.2.2.2. Cooked Meat Medium and Thioglycollate Medium ... 240 8.2.2.3. Sabouraud Medium ... 240 (xv) 8.3 Sampling : Probability Profile ... 243 8.4 Overall Conclusions ... 245 9. Immune Systems ... 246 9.1 Introduction ... 246 9.1.1. Discrimination ... 247 9.1.2. Specificity ... 247 9.1.3. Anamnesis ... 247 9.1.4. Transferability by Living Cells ... 247 9.2 Types of Specific Immunity ... 248 9.2.1. Acquired Immunity ... 248 9.2.2. Active Immunity ... 249 9.2.3. Cell-Mediated Immunity ... 249 9.2.4. Congenital Immunity ... 249 9.2.5. Herd Immunity ... 249 9.2.6. Humoral Immuity [or B-Cell Mediated Immunity] ... 249 9.2.7. Local Immunity ... 250 9.2.8. Natural Immunity ... 250 9.2.9. Passive Immunity ... 250 9.3 Duality of Immune Systems ... 250 9.4 Immunological Memory ... 251 9.5 Natural Resistance and Nonspecific Defence Mechanisms ... 252 9.5.1. Natural Resistance ... 253 9.5.1.1. Species Resistance ... 253 9.5.1.2. Racial Resistance ... 253 9.5.1.3. Individual Resistance ... 254 9.5.1.4. External Defence Mechanisms ... 254 9.5.2. Internal Defense Mechanisms ... 256 9.5.3. Nonspecific Defense Mechanisms ... 256 9.5.3.1. Complement System ... 257 9.5.3.2. Phagocytosis ... 259 9.5.3.2.1. Functions of Phagocytes ... 260 9.5.3.2.2. Mechanism of Phagocytosis ... 260 9.5.3.3. Natural Killer Cells [NK Cells] ... 262 9.5.3.4. Interferons [IFNs] ... 263 9.5.3.4.1. Salient Features ... 263 9.5.3.4.2. Interferon : An Ideal Antiviral Substance ... 264 (xvi) (xvii) 9.5.3.4.3. Interferon Based on Recombinant DNA Technology ... 264 9.5.3.4.4. Classical Recombinant Interferons [r IFNs] ... 265 10. Microbiological (Microbial) Assays : Antibiotics–Vitamins–Amino Acids ... 268 10.1 Introduction ... 268 10.1.1. Importance and Usefulness ... 268 10.1.2. Principle ... 269 10.1.3. Methodologies ... 269 10.1.3.1. Cylinder-Plate Method ... 269 10.1.3.2. Turbidimetric (or Tube Assay) Method ... 269 10.1.4. Present Status of Assay Methods ... 270 10.2 Variants in Assay Profile ... 270 10.2.1. Calibration of Assay ... 270 10.2.2. Precision of Assay ... 271 10.2.3. Accuracy of Assay ... 272 10.2.4. Evaluation of Assay Performance ... 272 10.3 Types of Microbiological (Microbial) Assays ... 273 10.3.1. Agar-Plate Diffusion Assays (Method A) ... 273 10.3.1.1. One-Dimensional Assay ... 273 10.3.1.2. 2D-or 3D-Assay ... 274 10.3.1.3. Dynamics of Zone Formation ... 274 10.3.1.4. Management and Control of Reproducibility ... 275 10.3.1.5. Measurement of Zone of Inhibition ... 277 10.3.1.6. Calibration ... 277 10.3.1.6.1. Standard Curves ... 277 10.3.1.6.2. 2-By-2-Assay ... 278 10.3.2. Rapid-Reliable-Reproducible Microbial Assay Methods ... 279 10.3.2.1. Urease Activity ... 279 10.3.2.2. Luciferase Assay ... 280 10.4 Radioenzymatic [Transferase] Assays ... 281 10.4.1. Calibration ... 282 10.4.2. Non-Isotopic Modification ... 283 10.5 Analytical Methods for Microbial Assays ... 283 10.5.1. High Performance Liquid Chromatography [HPLC] ... 283 10.5.2. Reverse-Phase Chromatography [RPC] ... 286 10.5.3. Ion-Pair (or Paired-Ion) Chromatography [IPC] ... 286 10.6 Examples of Pharmaceutical Microbial Assays ... 287 10.6.1. Antibiotic Assays ... 287 10.6.1.2. Standard Preparation and Units of Activity ... 287 10.6.1.2. Preparation of Standard Solution ... 289 10.6.1.3. Preparation of Sample Solution ... 289 10.6.1.4. Test Organisms ... 291 10.6.1.5. Preparation of Inoculum ... 294 10.6.1.5.1. For Method A ... 294 10.6.1.5.2. For Method B ... 294 10.6.1.6. Temperature Control ... 294 10.6.1.7. Spectrophotometer ... 295 10.6.1.8. Cylinder-Plate Assay Receptacles ... 295 10.6.1.9. Turbidimetric Assay Receptacles ... 295 10.6.1.10. Assay Designs ... 295 10.6.1.10.1. Methods ... 296 [A] Cylinder-Plate or Cup-Plate Method ... 296 A-1. One Level Assay with Standard Curve ... 297 A-2. Two Level Factorial Assay ... 298 A-3. Other Designs ... 298 [B] Turbidimetric or Tube Assay Method ... 298 10.7 Assay of Antibiotics by Turbidimetric (or Nephelometric) Methods ... 300 10.7.1. Assay of Chlorotetracycline ... 300 10.7.2. Cognate Assays ... 301 10.7.3. Assay of Vitamins ... 301 10.7.3.1. Calcium Pantothenate ... 302 10.7.3.2. Niacin (or Niacinamide) ... 304 10.7.3.2. Vitamin B12 [or Cyanocobalamin] ... 306 10.7.4. Assay of Amino Acids ... 307 Glossary ... 308 Index ... 342