MICROBIOLOGICALEXAMINATION ofWATER andWASTEWATER

Maria CsurosCsaba Csuros

With Contributions ByKlara Ver

LEWIS PUBLISHERSBoca Raton London New York Washington, D.C.

Contents

Chapter 1Scope and History of Microbiology1.1 Microbes and Microbiology

1.1.1 General Concepts of Microbiology1.1.2 Size of Microorganisms1.1.3 Unicellular and Noncellular Organisms1.1.4 Basic Characteristics of Living Systems 21.1.5 Investigation of Microorganisms 2

1.2 Brief History of Microbiology 21.2.1 The Microscope and Microbes 21.2.2 Spontaneous Generation vs. Biogenesis Theory 21.2.3 Aseptic Techniques 5

1.3 The Golden Age of Microbiology 51.3.1 Fermentation and Pasteurization 61.3.2 The Germ Theory of Disease 61.3.3 Vaccination 7

1.4 Chemotherapy 81.4.1 The First Synthetic Drugs 91.4.2 Antibiotics 9

1.5 Modern Developments in Microbiology 101.6 Naming and Classifying of Microorganisms 10

1.6.1 Naming of Microorganisms 101.7 The Diversity of Microorganisms 11

1.7.1 Bacteria 111.7.2 Fungi 111.7.3 Protozoa 121.7.4 Algae 121.7.5 Viruses 121.7.6 Multicellular Parasites 12

Chapter 2Procaryotic Cells 152.1 Introduction of Procaryotic and Eucaryotic Cells 152.2 The Procaryotic Cell 15

2.2.1 Morphology of the Bacterial Cell 152.2.1.1 Spiral Bacteria 162.2.1.2 Bacilli 172.2.1.3 Cocci 17

2.3 Structures External to the Cell Walls 182.3.1 Glycocalyx 182.3.2 Flagella 192.3.3 Pili 19

2.4 The Cell Wall 192.4.1 Gram Positive Bacteria 202.4.2 Gram Negative Bacteria 20

2.4.3 Atypical Cell Wall 202.4.4 Damage to the Cell Wall 21

2.5 Structures Internal to the Cell Wall 212.5.1 Plasma or Inner Membrane 212.5.2 Movement of Material Across Membranes 22

2.5.2.1 Diffusion 222.5.2.2 Transport by Carriers 232.5.2.3 Endocytosis and Exocytosis 23

2.5.3 Cytoplasm 232.5.4 Nuclear Area 242.5.5 Ribosomes 242.5.6 Inclusion 25

2.5.6.1 Metachromatic Granules 252.5.6.2 Polysaccharide Granules 262.5.6.3 Lipid Inclusions 262.5.6.4 Sulfur Granules 262.5.6.5 Gas Vacuoles 26

2.5.7 Endospores 262.5.7.1 Sporulation 262.5.7.2 Germination 26

Chapter 3Microbial Metabolism 293.1 Meaning of Metabolism 29

3.1.1 The First and Second Law of Thermodynamics 293.1.1.1 The First Law of Thermodynamics 303.1.1.2 The Second Law of Thermodynamics 30

3.1.2 Energy Transport through ADP and ATP 303.2 Metabolic Pathways 303.3 Enzymes 31

3.3.1 Mechanism of Enzyme Action 323.3.2 Naming the Enzymes 323.3.3 Temperature and pH Effect on Enzymes 323.3.4 Inhibition of Enzymes 33

3.3.4.1 Competitive Inhibition of Enzymes 333.3.4.2 Noncompetitive Inhibition of Enzymes 33

3.4 Nutritional Classification of Organisms 343.4.1 Energy Production 34

3.4.1.1 Carbon and Energy Sources 353.4.1.2 Nitrogen Sources 353.4.1.3 Other Chemical Requirements 35

3.5 Energy Production of Organisms 353.5.1 Oxidation-Reduction 363.5.2 Phosphorylation as ATP Formation in a Biological System 36

3.5.2.1 Substrate Level Phosphorylation 363.5.2.2 Oxidative Phosphorylation 363.5.2.3 Photophosphorylation 36

3.5.3 Formation of ATP during Respiration 363.5.3.1 Aerobic Respiration 373.5.3.2 Anaerobic Respiration 373.5.3.3 Fermentation 37

3.5.4 Formation of ATP during Fermentation 37

3.6 Carbohydrate Catabolism 373.6.1 Glycolysis 383.6.2 Alternative Pathway of Glycolysis by Bacteria 383.6.3 Transition Reaction 383.6.4 Krebs Cycle or Citric Acid Cycle 393.6.5 Respiratory Chain or Electron Transport System or Cytochrome System 413.6.6 Aerobic Respiration 423.6.7 Fermentation 43

Chapter 4Microbial Growth and Its Control 454.1 Microbial Growth 45

4.1.1 The Lag Phase 454.1.2 The Log Phase 454.1.3 The Stationary Phase 454.1.4 The Death Phase 45

4.2 Physical Requirements 464.2.1 Temperature 46

4.2.1.1 Cold Loving Microbes: Psychrophiles 464.2.1.2 Moderate Temperature Loving Microbes: Mesophiles 464.2.1.3 Heat Loving Microbes: Thermophiles 47

4.2.2 pH and Buffers 474.2.3 Osmotic Pressure 48

4.3 Chemical Requirements 484.3.1 Carbon 484.3.2 Nitrogen, Sulfur, and Phosphorus 484.3.3 Trace Elements 484.3.4 Oxygen 494.3.5 Organic Growth Factors 49

4.4 Control of Microbial Growth 504.4.1 Terms Related to Destruction of Organisms 51

4.4.1.1 Sterilization 514.4.1.2 Disinfection 514.4.1.3 Pasteurization 514.4.1.4 Germicide 514.4.1.5 Antiseptics 514.4.1.6 Asepsis 514.4.1.7 Degerming 514.4.1.8 Sanitization 51

4.5 Physical Methods of Microbial Control 524.5.1 High Heat 52

4.5.1.1 Moist Heat Sterilization 524.5.1.2 Dry Heat Sterilization 52

4.5.2 Filtration 524.5.3 Low Temperature 524.5.4 Desiccation 534.5.5 Radiation 53

4.5.5.1 Ionizing Radiation 534.5.5.2 Nonionizing Radiation 53

4.6 Chemical Methods of Microbial Control 534.6.1 Surfactants or Surface Acting Agents 53

4.6.1.1 Soap 53

4.6.1.2 Acid-Anionic Surface Acting Sterilizers 534.6.1.3 Quaternary Ammonium Compounds (Quats) 54

4.6.2 Organic Acids 544.6.3 Aldehydes 544.6.4 Ethylene Oxide 544.6.5 Oxidizing Agents 544.6.6 Phenol (Carbolic Acid) 554.6.7 Halogens 554.6.8 Alcohols 554.6.9 Heavy Metals 55

Chapter 5Microorganisms in the Environment 575.1 Biochemical Cycles 57

5.1.1 The Water Cycle or Hydrologic Cycle 575.1.2 The Carbon Cycle 58

5.1.2.1 The Greenhouse Effect 595.1.3 The Nitrogen Cycle 59

5.1.3.1 Nitrogen-Fixing Bacteria 605.1.3.2 Nitrifying Bacteria 615.1.3.3 Denitrifying Bacteria 62

5.1.4 The Sulfur Cycle 625.1.4.1 Sulfate Reducing Bacteria 625.1.4.2 Sulfur Reducing Bacteria 625.1.4.3 Sulfur Oxidizing Bacteria 63

5.1.5 The Phosphorus Cycle 635.2 Microorganisms Found in Air 63

5.2.1 Sources of Indoor Pollution 645.2.1.1 Outdoor Environment 645.2.1.2 ' Indoor Contamination 64

5.2.2 Factors Affecting Indoor Microbial Levels 655.2.3 Controlling Microorganisms in Air 65

5.2.3.1 Chemical Agents 655.2.3.2 Ultraviolet Radiation 655.2.3.3 Air Filtration 65

5.3 Microorganisms in Soil 665.3.1 Components of Soil 66

5.3.1.1 Inorganic Components 665.3.1.2 Organic Matter 665.3.1.3 Root Systems 675.3.1.4 Living Organisms 67

5.3.2 Microorganisms in Soil 675.3.2.1 Bacteria 675.3.2.2 Actinomycetes 685.3.2.3 Fungi 685.3.2.4 Cyanobacteria, Algae, Protista and Viruses 69

5.3.3 Abiotic Factors Influence Microorganisms in the Soil 695.3.3.1 Water and Oxygen 695.3.3.2 Soil pH 695.3.3.3 Temperature 69

5.3.4 Importance of Decomposers in the Soil 695.3.5 Soil Pathogens 70

5.4 Aquatic Microbiology 705.4.1 Freshwater Environment 70

5.4.1.1 Ground water 705.4.1.2 Surface Water 70

5.4.2 Factors Affecting Microorganisms in Aquatic Environments 715.4.2.1 Temperature 715.4.2.2 pH 715.4.2.3 Nutrients 715.4.2.4 Oxygen 715.4.2.5 Depth 71

5.4.3 Marine Environments 715.4.3.1 Temperature 715.4.3.2 pH 725.4.3.3 Salinity 725.4.3.4 Hydrostatic Pressure 725.4.3.5 Sunlight and Oxygen Concentration 725.4.3.6 Nutrient Concentrations 72

5.4.4 Water Pollution 725.4.4.1 Organic Wastes 735.4.4.2 Industrial Wastes 735.4.4.3 Synthetic Chemicals 735.4.4.4 Radioactive Substances 735.4.4.5 Agricultural, Mining, and Construction Activities 735.4.4.6 Heat 73

5.4.5 Pathogens in Water 735.5 Water Purification 74

5.5.1 Flocculation 745.5.2 Filtration 745.5.3 Chlorination 74

5.6 Sewage Treatment 755.6.1 Primary Treatment 755.6.2 Secondary Treatment 75

5.6.2.1 Trickling Filter Systems 765.6.2.2 Activated Sludge Systems 76

, 5.6.3 Tertiary Treatment 765.6.4 Septic Tanks 77

Chapter 6Bioremediation of Organic Contaminants 796.1 Bioremediation 79

6.1.1 The Objective of Bioremediation 796.1.2 Advantage of Bioremediation 796.1.3 Disadvantage of Bioremediation 806.1.4 Development of Bioremediation 806.1.5 Biotransformation by Subsurface Microorganisms 80

6.2 Application of Bioremediation 806.2.1 Wood Preservative Industries 816.2.2 Soil and Hazardous Waste Bioremediation 81

6.2.2.1 Bioreactors 826.2.2.2 Solid Phase Bioremediation 826.2.2.3 Soil Heaping 826.2.2.4 Composting 82

6.2.3 Degradation of Synthetic Chemicals in Soil 826.2.4 Biological Control of Groundwater Pollution 836.2.5 Modern Microbiological Concepts and Pollution Control 84

Chapter 7

Microbiological Quality of Environmental Samples 857.1 Monitoring Microbiological Quality 85

7.1.1 Waterborne Disease Outbreaks 857.1.1.1 Swimming Associated Outbreaks 86

7.1.2 Recovery of Pathogens from Environmental Samples 877.1.3 Indicator Bacteria 87

7.1.3.1 Coliform Group 877.1.3.2 Fecal Coliform Bacteria 87

7.1.4 Standards on Microbiological Quality 887.2 Introduction to Microbiological Parameters to the Sanitary Quality of

Environmental Samples 887.2.1 Heterotrophic Plate Count (HPC) 887.2.2 Coliform Bacteria Group 88

7.2.2.1 Total Coliform Group 887.2.2.2 Fecal Coliform, Escherichia coli (E. coli) 887.2.2.3 Fecal streptococcus 897.2.3.4 Klebsiella 89

7.3 Pathogenic Microorganisms 897.3.1 Pathogenic Bacteria 89

7.3.1.1 Salmonella 907.3.1.2 Salmonella typhi 907.3.1.3 Shigella 907.3.1.4 Vibrio cholera 907.3.1.5 Enteropathogenic Escherichia coli 907.3.1.6 Yersinia 907.3.1.7 Campylobacter 917.3.1.8 Legionella pneumophila 917.3.1.9 Clostridium 91

7.3.2 Viruses 927.3.2.1 Hepatitis A Virus (HAV) 937.3.2.2 NorwalkAgent 93

7.3.3 Pathogenic Protozoa 937.3.3.1 Giardia lamblia 937.3.3.2 Entamoeba histolytica 94

7.4 Iron and Sulfur Bacteria 947.5 Actinomycetes 967.6 Fungi 96

7.6.1 Aspergillus 967.6.2 Candida albicans 96

7.7 Regulations for Drinking Water Quality 967.7.1 Monitoring Agencies 97

7.7.1.1 World Health Organization (WHO) 97

7.7.1.2 European Economic Community (EEC) 977.7.1.3 Environmental Protection Agency (EPA) 97

7.7.2 Monitoring Requirements 987.7.3 Minimum Coliform Monitoring Requirements 987.7.4 Standards 997.7.5 Disinfection Requirements for All Public Water Supplies 997.7.6 Disinfection By-Product Regulations 100

7.8 Groundwater 1007.8.1 Classification of Groudwater 1007.8.2 Groundwater Standards 101

7.9 Surface Waters 1017.9.1 Surface Water Classification 1017.9.2 Surface Water Treatment Rule (SWTR) 1027.9.3 Surface Water Quality Standards 103

7.10 Wastewater 1037.10.1 Industrial Wastewater 1037.10.2 Permit for Industrial and Domestic Wastewater Effluents 1047.10.3 Minimum Treatment Standard of Domestic Wastewater Effluents 104

7.10.3.1 Effluents for Direct Surface Water Disposal or SurfaceWater Disposal via Ocean Outfall 104

7.10.3.2 Discharging to Open Ocean Water 1047.10.3.3 Effluent Disinfection 104

7.10.3.3.1 Basic Disinfection 1047.10.3.3.2 High-Level Disinfection 1057.10.3.3.3 Intermediate Disinfection 1057.10.3.3.4 Low-Level Disinfection 105

7.11 Soil and Sediment 1057.12 Air 106

Chapter 8Safety in Environmental Microbiology Laboratory 1078.1 Laboratory Facilities 1078.2 Laboratory Safety Considerations 108

8.2.1 General Laboratory Safety Rules 1088.2.2 Standard Safety Practices in a Microbiological Laboratory 1098.2.3 General Handling and Storage of Chemicals and Gases I l l8.2.4 Electrical Precautions I l l

8.3 Summarized Safety Check List for Environmental Microbiology Laboratories I l l8.3.1 Administrative Considerations I l l8.3.2 Personal Conduct 1148.3.3 Laboratory Equipment 1148.3.4 Disinfection/Sterilization 1158.3.5 Biohazard Control 1158.3.6 Handling and Storage of Chemicals and Gases 1158.3.7 Emergency Precautions 115

Chapter 9Laboratory Quality Assurance and Quality Control 1179.1 Introduction : 117

9.1.1 Quality Assurance (QA) 1179.1.2 Quality Control (QC) 117

9.1.3 Quality Assessment 1179.2 Requirements for Facilities and Personnel 117

9.2.1 Ventilation 1179.2.2 Laboratory Bench Areas 1179.2.3 Walls and Floors 1189.2.4 Laboratory Cleanliness 1189.2.5 Air Monitoring 118

9.2.5.1 RODAC Plates 1189.2.5.2 Air Density Plates 118

9.2.6 Personnel 1199.3 Quality Control for Laboratory Equipment and Instrumentation 119

9.3.1 Thermometers and Temperature-Recording Instruments 1199.3.2 Balances 1199.3.3 pH Meter 1209.3.4 Water Deionization Unit 1209.3.5 UV Sterilizer 1219.3.6 Membrane Filter Apparatus 1239.3.7 Centrifuge 1239.3.8 Hot-Air Oven 1259.3.9 Autoclave 1259.3.10 Refrigerators and Freezers 1269.3.11 Incubators 1269.3.12 Water Bath 1269.3.13 Safety Hood 1269.3.14 Microscope 1279.3.15 Spectrophotometer 127

9.4 Quality Control of Laboratory Supplies 1279.4.1 Glassware 1279.4.2 Glass, Plastic, and Metal Utensils for Media Preparation 1289.4.3 Culture Dishes 1289.4.4 Sterility Check on Glassware 1289.4.5 Chemicals and Reagents 1289.4.6 Dyes and Stains 1299.4.7 Membrane Filters and Pads 1299.4.8 Culture Media 129

9.4.8.1 Quality Control Criteria for Prepared Media 1309.5 Quality Control of Laboratory Pure Water 130

9.5.1 Checks and Monitoring Criteria 1309.5.2 Test for Bacterial Quality 130

9.5.2.1 Reagents 1319.5.2.2 Sample Preparation 1329.5.2.3 Preparation of Bacterial Suspension 1329.5.2.4 Test 1329.5.2.5 Calculation 1329.5.2.6 Interpretation of the Results 133

9.6 Analytical Quality Control Procedures 1339.6.1 Quality Control in Routine Analysis 133

9.6.1.1 Negative (Sterile) Control 1339.6.1.2 Positive Control 1349.6.1.3 Duplicate Analysis v 134

9.6.2 Measurement of Method Precision 134

9.6.3 Reference Sample 1349.6.4 Performance Sample 1349.6.5 Membrane Filter Method (MF) Verification 135

9.6.5.1 Total Coliform 1359.6.5.2 Fecal Coliforms 1369.6.5.3 Fecal Streptococci 136

9.7 Records and Data Reporting 1369.8 Interlaboratory Quality Control 136

Chapter 10Collecting and Handling Environmental Samples for Microbiological Examination 13910.1 Sampling 139

10.1.1 Sampling Program 13910.1.2 Type of Samples 13910.1.3 Sample Containers 13910.1.4 Dechlorinating Agent 14010.1.5 Chelating Agents 14010.1.6 Sampling Procedures 140

10.2 Sample Collection from Different Sources 14110.2.1 Collecting Potable Water Samples 141

10.2.1.1 Sampling from Distribution System 14110.2.1.2 Sampling from Wells 142

10.2.2 Collect Samples from River, Stream, Lake, Spring, or Shallow Well 14310.2.3 Sample Collection from Bathing Beaches 14310.2.4 Marine and Estuarine Sampling 14610.2.5 Sample Collection from Domestic and Industrial Discharges 14610.2.6 Collecting Samples from Sediments and Sludges 146

10.2.6.1 Bottom Sediments 14610.2.6.2 Sludges 147

10.2.7 Soil Sampling 14710.3 Sample Identification 14810.4 Sample Transportation, Preservation and Holding Time 149

10.4.1 Sample Transportation and Preservation 14910.4.2 Laboratory Custody Procedure 15010.4.3 Holding Time 15010.4.4 Discard Samples 151

Chapter 11

Laboratory Equipment and Supplies in the Environmental Microbiology Laboratory 15311.1 Laboratory Equipment 153

11.1.1 Incubators 15311.1.2 Hot-Air Sterilizing Oven 15311.1.3 Autoclaves 15311.1.4 pH Meter 15411.1.5 Balances 15511.1.6 Optical Counting Equipment 15511.1.7 Refrigerator 15611.1.8 Membrane Filtration Equipment 15611.1.9 Line Vacuum or Electric Vacuum Pump 15711.1.10 Inoculating Needles or Loops 157

11.1.11 Microscope 15711.1.11.1 Parts of the Microscope 15711.1.11.2 Light Microscope 15811.1.11.3 Focusing the Microscope 160

11.1.11.3.1 Low Power (4X or 10X) 16011.1.11.3.2 Higher Power 160

11.1.11.4 Rules for Using Microscopes 16111.1.11.5 Dark Field Microscopy 16111.1.11.6 Phase-Contrast Microscopy 16111.1.11.7 Fluorescent Microscopy 16211.1.11.8 Electron Microscopy 162

11.2 Laboratory Glassware 16211.2.1 Petri Dishes 162

11.2.1.1 Petri Dishes with Tight Fitting Lids 16211.2.1.2 Petri Dishes with Loose Fitting Lids 162

11.2.2 Pipets 16211.2.3 Graduated Cylinders 16611.2.4 Vacuum Filter Flask 16611.2.5 Safety Trap Flask 16711.2.6 Dilution (Milk Dilution) Bottles 16711.2.7 Fermentation Tubes and Vials 16811.2.8 Thermometers 16811.2.9 Cleaning Laboratory Glassware 16911.2.10 Glassware Sterilization 169

11.3 Chemicals and Reagents 16911.3.1 Chemicals and Reagents 16911.3.2 Laboratory Pure Water 169

Chapter 12Culture Media 17112.1 Culture Media and Culture 171

12.1.1 Chemically Defined Media 17112.1.2 Complex Media 17112.1.3 Selective and Differential Media 172

12.1.3.1 Selective Media 17212.1.3.2 Differential Media 172

12.1.4 Storage of Dehydrated Culture Media 17212.1.5 Preparation of Media 172

12.1.5.1 Preparation and Sterilization of Media 17312.1.6 Sterilization of the Media 17412.1.7 pH Check of the Media 17512.1.8 Storage of Culture Media 17512.1.9 Sterile Media from Commercial Sources 176

12.2 Bacterial Growth in Media 17612.2.1 Bacterial Growth in Liquid (Broth) Media 176

12.2.1.1 Observing Growth Patterns on Broth Media 17612.2.2 Bacterial Growth in Agar Slant Cultures 178

12.2.2.1 Preparation of Agar Slants 17812.2.3 Bacterial Selection by Sugar Fermentation 17812.2.4 Obtaining Pure Culture: Streak Plate Method 180

12.2.4.1 How to Prepare Agar Plates 181

12.2.4.2 How to Inoculate Agar Plates 18212.2.4.3 How to Incubate Agar Plates 182

Chapter 13Direct Measurement of Bacterial Growth 18313.1 Dilutions 183

13.1.1 Single Dilution 18313.1.2 Serial Dilution 18313.1.3 Prompt Use of Dilutions 18413.1.4 Dilution Water 184

13.1.4.1 Phosphate Buffered Dilution Water 18413.1.4.1.1 Stock Phosphate Buffer Solution 18413.1.4.1.2 Working Phosphate Buffer Solution 185

13.2 Plate Counts 18513.2.1 Pour Plate Method 18513.2.2 Spread Plate Method 18613.2.3 Streak Plate Method 186

13.3 Membrane Filter (MF) Technique 18613.3.1 Advantage of the Membrane Filter Technique 18713.3.2 Limitations of the Membrane Filter Technique 18713.3.3 Outline of the Membrane Filter Technique 187

13.4 Most Probable Number (MPN) Method 18813.4.1 Presumptive Test 18813.4.2 Confirmed Test 18813.4.3 Completed Test 18813.4.4 Calculation and Reporting of MPN Values 189

13.5 Staining Procedures 18913.5.1 Preparation of Bacterial Smears 18913.5.2 Gram Stain 189

13.6 Direct Microscopic Counts 191

Chapter 14Estimation of Bacterial Numbers by Indirect Methods 19314.1 Turbidity 19314.2 Biochemical Reactions and Enzymatic Tests 194

14.2.1 Catalase Test 19414.2.2 Clumping Factor Test (Slide Coagulase Test) 19414.2.3 Nitrate Reduction Test 19514.2.4 Oxidase Test (Kovacs Method) 19614.2.5 Indole Test 197

14.2.5.1 The Rapid Indole Test 19714.2.5.2 Regular Indole Test 197

14.2.6 Rapid Urease Test 19814.2.7 IMViC Tests 19814.2.8 Methyl Red and Voges-Proskauer Test 198

14.2.8.1 Methyl Red (MR) Test 19914.2.8.2 Voges-Proskauer (V-P) Reaction 199

14.2.9 Citrate Utilization Test 19914.2.10 Decarboxylase Test _. 200

14.2.11 Motility Test 20014.3 Rapid Identification Systems (Multitest Systems) 200

Chapter 15Methods for Analyzing Microbiological Quality of the Environment 20115.1 Methods and Techniques 201

15.1.1 Methods for the Total Coliform Group 20115.1.2 Methods for Fecal Coliforms 20115.1.3 Heterotrophic Plate Count 20215.1.4 Method for Fecal Streptococcus 20315.1.5 Detection of Stressed Organisms 203

15.1.5.1 Ambient Temperature Effect 20315.1.5.2 Chlorinated Effluents and Toxic Wastes 203

15.2 Rapid Detection Methods 20315.2.1 7 Hour Fecal Coliform Test 204

15.3 Methods for Microbiological Examination of Recreational Waters 20415.3.1 Swimming Pools 204

15.3.1.1 Disinfected Indoor Pools 20415.3.1.2 Disinfected Outdoor Pools 20415.3.1.3 Untreated Pools 204

15.3.2 Whirlpools 20515.3.3 Natural Bathing Beaches 205

15.4 Detection of Pathogenic Bacteria 20515.5 Detection of Soil Microorganisms 206

15.5.1 Sample Collection 20615.5.2 Sample Handling and Storage 20615.5.3 Moisture Determination 20615.5.4 Plate Count 20715.5.5 Most Probable Number (MPN) Method 207

Chapter 16Heterotrophic Plate Count 20916.1 Introduction 20916.2 Pour Plate Method 209

16.2.1 Equipment and Material 20916.2.2 Media Preparation 21016.2.3 Preparation of Agar 21016.2.4 Dilution Preparation 21016.2.5 Plate Preparation 21116.2.6 Counting 213

16.2.6.1 Select Plates Having 30 to 300 Colonies 21316.2.6.2 Plates Having More Than 300 Colonies 21316.2.6.3 Plates Have No Colonies 21416.2.6.4 Number of Colonies per Plate Greatly Exceeding 300 21416.2.6.5 Spreading Colonies Are on the Plate(s) 21416.2.6.6 Plates Are Uncountable 21416.2.6.7 Examples for Counting and Reporting 214

16.3 Spread Plate Method 21516.3.1 Equipment and Material 21516.3.2 Media and Agar Preparation, Sample Dilution 21516.3.3 Preparation of Agar Plates 215

16.3.4 Procedure 215.16.3.5 Counting and Reporting 216

16.4 Plate Count from Soils and Sediments 21616.4.1 Moisture Determination 21616.4.2 Preparation of Dilutions 21616.4.3 Calculation and Reporting 21716.4.4 Pour Plates 21716.4.5 Spread Count 217

Chapter 17Determination of Total Coliform 21917.1 Membrane Filter Technique 219

17.1.1 Introduction 21917.1.2 Application 21917.1.3 Equipment and Glassware 22017.1.4 Culture Media 221

17.1.4.1 Preparation of the M-Endo Broth Media 22117.1.4.2 Preparation of the M-Endo Agar Medium 22217.1.4.3 Preparation of Lauryl Tryptose Broth 22217.1.4.4 Preparation of Brilliant Green Bile Broth 22217.1.4.5 Preparation of Dilution Water 22217.1.4.6 Preparation of Magnesium Chloride Solution 222

17.1.5 Set Up Filtration Apparatus 22217.1.6 Suggested Sample Volumes for Membrane Filter Total Coliform 222

17.2 Membrane Filtration (MF) Procedure 22317.2.1 Procedure Using Broth Media 22317.2.2 Procedure Using Agar Media 22317.2.3 Counting and Recording Colonies 223

17.2.3.1 General Rules for Counting and Reporting 22417.2.3.2 Special Rules in Counting and Reporting for Potable Waters 225

17.2.4 Verification 22517.2.4.1 Verification Procedures 225

17.3 Delayed Incubation by the MF Method 22617.3.1 Application 22717.3.2 Equipment and Glassware 22717.3.3 Culture Media 227

17.3.3.1 Preparation of M-Endo Preservative Medium 22717.3.4 Procedure, Counting, and Recording 227

17.4 Most Probable Number (MPN) Method 22717.4.1 Introduction 22717.4.2 Application •., 22817.4.3 Equipment and Glassware 22817.4.4 Culture Media 228

17.4.4.1 Lauryl Tryptose Broth 22817.4.4.2 Brilliant Green Lactose Bile Broth (BGLB) 22817.4.4.3 Eosine Methylene Blue Agar (EMB Agar) 228

17.4.5 Dilution Water 22917.4.6 Procedure 230

17.4.6.1 Presumptive Test 23017.4.6.2 Confirmed Test 23017.4.6.3 Completion Test 230

17.4.7 Calculation 231

17.4.7.1 Calculation of Reported Value When 10, 1.0, and 0.1 mlPortions Are Used 231

17.4.7.2 Calculation of Reported Values When the Series of Decimal DilutionsIs Other Than 10, 1.0, and 0.1 ml 231

17.4.7.3 More Than Three Sample Volumes Are Inoculated 23117.5 Determination of Klebsiella 233

17.5.1 Introduction 23317.5.2 Membrane Filter Procedure 234

17.5.2.1 Apparatus 23417.5.2.2 Culture Medium 234

17.5.2.2.1 Modified M-FC Agar (M-FCIC) Agar 23417.5.2.2.2 M-KlebAgar 234

17.5.2.3 Procedure 23517.5.2.4 Counting Colonies 23517.5.2.5 Verification 235

17.6 Application for Soil, Sediment, and Sludge Samples 23517.7 Analytical Quality Control 236

Chapter 18Determination of Fecal Coliform 23718.1 Definition of the Fecal Coliform Group 23718.2 Membrane Filter Method 237

18.2.1 Application 23718.2.2 Equipment and Glassware 23718.2.3 Culture Media 237

18.2.3.1 Preparation of M-FC Broth 23818.2.3.1.1 Rosolic Acid Solution 23818.2.3.1.2 0.2JVNaOH 238

18.2.3.2 Preparation of M-FC Agar 23818.2.3.3 Preparation of Lauryl Tryptose Broth 23818.2.3.4 Preparation of EC Medium 23818.2.3.5 Preparation of Sterile Dilution Water 238

18.2.4 Procedure 23818.2.4.1 Set Up Filtration Apparatus 23818.2.4.2 Suggested Sample Volumes for the Membrane

Filter Fecal Coliform Test 23818.2.4.3 Procedure Using Broth Media 23918.2.4.4 Procedure Using Agar Media 239

18.2.5 Counting and Recording Colonies 23918.2.5.1 Countable Membranes with 20 to 60 Colonies 23918.2.5.2 Countable Membrane Filters with Less Than 20 Blue Colonies 23918.2.5.3 Membranes with No Colonies 23918.2.5.4 Countable Membranes with More Than 60 Colonies 24018.2.5.5 Uncountable Membranes with More Than 60 Colonies 240

18.2.6 Verification 24018.2.6.1 Procedure 240

18.3 Delayed Incubation MF Method 24018.3.1 Application 24018.3.2 Equipment and Glassware 24018.3.3 Culture Media 241

18.3.3.1 Preparation of the M-VFC Medium 24118.3.4 Counting and Recording Colonies 242

18.4 The Most Probable Number (MPN) Method 242

18.4.1 Application 24218.4.2 Equipment and Glassware 24218.4.3 Media 242

18.4.3.1 Lauryl Tryptose Broth 24218.4.3.2 EC Medium 24218.4.3.3 Dilution Water 242

18.4.4 Procedure 24218.4.5 Calculation and Reporting 244

18.5 The MUG Test 24418.6 Application for Soil, Sediment, and Sludge Samples 24418.7 Analytical Quality Control (QC) Procedure 244

Chapter 19Determination of Fecal Streptococcus 24519.1 Introduction 245

19.1.1 The Fecal Coliform and Fecal Streptococci, FC/FS Ratio 24519.2 Enterococci Portion of the Fecal Streptococcus Group 24619.3 Membrane Filter (MF) Technique 246

19.3.1 Application 24619.3.2 Equipment and Glassware 24619.3.3 Culture Media 247

19.3.3.1 Preparation of KF Streptococcus Agar 24719.3.3.1.1 1 Percent Solution of 2,3,5-triphenyl-

tetrazolium-chloride (TTC) 24719.3.3.1.2 10 Percent Sodium Carbonate, Na^Og 247

19.3.3.2 Preparation of Brain Heart Infusion Broth (BHI) 24719.3.3.3 Brain Heart Infusion Agar (BHI Agar) 24719.3.3.4 Brain Heart Infusion Broth with 40 Percent Bile 248

19.3.3.4.1 Preparation of 10 percent Oxgall Solution 24819.3.3.5 Dilution Water 248

19.3.4 Procedures 24819.3.4.1 Sample Volume 248

19.3.5 Counting and Recording Colonies 24819.3.6 Verification 248

19.4 Delayed MF Procedure 24919.5 Most Probable Number (MPN) Method 249

19.5.1 Culture Media 24919.5.1.1 Azide Dextrose Broth 24919.5.1.2 Pfizer Selective Enterococcus, (PSE) Agar 250

19.5.2 Procedure 25119.5.2.1 Presumptive Test 25119.5.2.2 Confirmed Test 251

19.5.3 Calculation 25119.6 Pour Plate Method 251

19.6.1 Media 25219.6.2 Procedure 25219.6.3 Counting and Reporting 252

19.6.3.1 Plates with 30 to 300 Colonies 25219.6.3.2 All Plates Greater than 300 Colonies 25319.6.3.3 All Plates with Fewer than 30 Colonies 25319.6.3.4 Plate with No Colonies 253

19.6.3.5 All Plates Are Crowded 25319.7 Procedures for Soils, Sediments, and Sludges 25419.8 Analytical Quality Control (QC) Procedures 254

Chapter 20Enterobacteriaceae 25520.1 Introduction 255

20.1.1 Coliform Organisms 25520.1.2 Differentiation of Enterobacteriaceae 255

20.2 ENTEROTUBEII 25620.2.1 Operation of ENTEROTUBE II 25620.2.2 To Read ENTEROTUBE II 25720.2.3 To Indicate Positive Reactions 25720.2.4 Ordering Information 257

20.3 BBL OXI/FERM Tube II 26020.4 API 20E System 260

20.4.1 Operation of API 20E 263

Chapter 21Iron and Sulfur Bacteria 26521.1 Introduction to Iron and Sulfur Bacteria 26521.2 Iron Bacteria 265

21.2.1 Identification of Iron Bacteria 26621.3 Sulfur Bacteria 267

21.3.1 Common Forms of Sulfur Bacteria 26721.3.1.1 Sulfate-Reducing Bacteria 26821.3.1.2 Photosynthetic Green and Purple Sulfur Bacteria 26821.3.1.3 Colorless Filamentous Sulfur Bacteria 26821.3.1.4 Aerobic Sulfur Oxidizers 268

21.3.2 Identification of Sulfur Bacteria 26921.3.2.1 Green and Purple Sulfur Bacteria 26921.3.2.2 Colorless Filamentous Sulfur Bacteria 26921.3.2.3 Colorless Nonfilamentous Sulfur Bacteria 27021.3.2.4 Colorless Small Sulfur Bacteria and Sulfate-Reducing Bacteria 270

21.4 Enumeration, Enrichment, and Isolation of Iron and Sulfur Bacteria 270

Chapter 22Detection of Actinomycetes 27122.1 General Introduction 271

22.1.1 Actinomycetes 27122.1.2 Streptomyces 271

22.2 Determination of Actinomycetes Density 27222.2.1 Plating Method 27222.2.2 Preparation and Dilution of Samples 27222.2.3 Preparation of Soil Samples 27222.2.4 Medium 27322.2.5 Procedure 27322.2.6 Counting 27322.2.7 Calculation and Reporting 274

22.2.7.1 Alternative Calculations 275

Appendix AExponential Notation 277A.I General Discussion 277

A. 1.1 Numbers Greater Than 10 in Exponential Notation 277A.1.2 Numbers Less Than 1 in Exponential Notation 278A. 1.3 Adding and Subtracting Numbers in Exponential Notation 278A. 1.4 Multiplying and Dividing Numbers in Exponential Notation 278

Appendix BInternational System of Units (Metric System) 279B.I Derived SI Units 279B.2 Derived SI Units with Special Names 279B.3 Prefixes 280B.4 Useful Conversion Factors 280

Appendix CUnits and Conversion Factors 281C.I Conversion to Metric Measures 281C.2 Conversion from Metric Measures 281

Appendix DBiochemical Oxygen Demand (BOD) 283D.I Scope and Application 283

D.I.I Carbonaceous vs. Nitrogenous BOD 283D.I.2 Dilution Requirements 283D.1.3 Summary of the Method 284D.1.4 Sampling and Storage 284D.1.5 Sample Collection for the Winkler Method 284D. 1.6 Interferences 285

D.2 Apparatus and Material 285D.3 Reagents 285D.4 Procedure 286

D.4.1 DO Meter Calibration 286D.4.2 Preanalysis Checking 287D.4.3 Analysis 287

D.5 Calculation 288D.5.1 BOD Concentration 288D.5.2 Correction Factor for Seed Control 288D.5.3 Dilution Factor 289D.5.4 Alternative Calculation 289

D.6 Quality Control 289D.6.1 Method Blank 289D.6.2 Seed Control 289D.6.3 Reference Standard (Glucose-Glutamic Acid Standard) 289D.6.4 DO Meter Performance Check 290D.6.5 Accuracy 290D.6.6 Precision 290

D.7 Safety 290

Appendix EDetermination of Solids 291E.I General Discussion 291

E.1.1 Sample Collection and Handling 291E.1.2 Sample Pretreatment 291

E.2 Determination of Total Solids (TS) Dried at 103 to 105°C 291E.2.1 Principle of the Method 291E.2.2 Interferences 291E.2.3 Apparatus and Material 292E.2.4 Procedure 292E.2.5 Calculation 292E.2.6 Quality Control 292

E.2.6.1 Method Blank 292E.3 Determination of Total Dissolved Solids (TDS) Dried at 180°C 292

E.3.1 Principle of the Method 292E.3.2 Interferences 293E.3.3 Apparatus and Material 293E.3.4 Reagents 293

E.3.4.1 Reference Standards, 500 mg per 1 293E.3.5 Procedure 293

E.3.5.1 Preparation of Evaporation Dishes 293E.3.5.2 Sample Analysis 294

E.3.6 Calculation 294E.3.7 Quality Control 295

E.3.7.1 Method Blank 295E.3.7.2 Reference Standard 295E.3.7.3 Duplicates 295

E.4 Determination of the Total Suspended Solids (TSS) Dried at 103 to 105°C 295E.4.1 Principle of the Method 295E.4.2 Interferences 295E.4.3 Apparatus and Material 295E.4.4 Procedure 295

E.4.4.1 Filter Preparation 295E.4.4.2 Sample Analysis 296

E.4.5 Calculation 296E.5 Fixed and Volatile Solids Ignited at 500°C 296

E.5.1 Principle of the Method 296E.5.2 Interferences 297E.5.3 Apparatus and Material 297E.5.4 Procedures 297E.5.5 Calculation 297

E.6 Total, Fixed, and Volatile Solids in Solid and Semisolid Samples 297E.6.1 Principle of the Method 297E.6.2 Interferences 297E.6.3 Apparatus and Material 298E.6.4 Procedure 298

E.6.4.1 Preparation of Evaporation Dishes 298E.6.4.2 Sample Analysis 298

E.6.5 Calculation 298E.6.6 Quality Control 298

E.7 Settleable Solids 298E.7.1 Principle of the Method 298E.7.2 Apparatus 298E.7.3 Procedure 299

Appendix FDetermination of pH 301F.I General Discussion 301F.2 Sample Collection and Holding Time 301F.3 Potentiometric Determination of pH 301

F.3.1 Principle of the Method 301F.3.2 Interferences 302F.3.3 Apparatus and Materials 302F.3.4 Reagents 302F.3.5 Meter Calibration 303F.3.6 Measurement of Aqueous Samples and Wastes

Containing Greater Than 20 Percent Water 303F.3.7 Measurement of Solid Samples 303F.3.8 General Rules 304

F.4 Quality Control (QC) 304F.4.1 Duplicate Analysis 304F.4.2 Initial Calibration Verification (ICV) 304F.4.3 Continuing Calibration Verification (CCV) 305F.4.4 Calculate Accuracy 305

Appendix GBacteriophages 307G.I Coliform Counts 307

G.I.I Total Coliforms 307G.1.2 Fecal Coliforms 307

Bibliography 309

Index 311