&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/4-84/013(R)
June 1988
Revision
USEPA Manual of
Methods for Virology
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June 1988
Foreword
Environmental measurements are required to determine the quality of ambient water,
the character of effluents, and the effects of pollutants on aquatic life. The Environmental
Monitoring Systems Laboratory-Cincinnati conducts research to develop, evaluate,
standardize and promulgate methods to:
• Measure the presence and concentration of physical and chemical pollutants in
water, wastewater, bottom sediments, and solid waste.
• Concentrate, recover, and identify enteric viruses, bacteria, and other microorga-
nisms in water, waste, soil and air.
• Determine the health and ecological effects of viruses, bacteria and parasites in
the environment.
• Measure the effects of pollution on freshwater, estuarine, and marine organisms,
including the phytoplankton, zooplankton, periphyton, macrophyton. macroinverte-
brates, and fish.
• Automate the measurement of the physical, chemical, and biological quality of water.
• Conduct an Agencywide quality assurance program to assure standardization and
quality control of systems for monitoring water and wastewater.
This manual was prepared and updated in order to meet mandates of the Congress
of the United States of America as directed in the Water Quality Act of 1987 (PL
100-4), the Safe Drinking Water Act (PL 93-523) as amended by the Safe Drinking
Water Act Amendments of 1986 (PL 99-339), the Marine Protection, Research, and
Sanctuaries Act (PL 92-532), and the Resource Conservation and Recovery Act (PL
94-580). The manual presents a standardized, step-by-step procedure for recovering
viruses from most environmental samples other than air.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory—Cincinnati
Printed on Recycled Paper
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June 1988
Purpose
This manual provides procedures for collecting scientifically valid and legally
defensible information on human enteric viruses in water, wastewater and treated
effluents and in sludge, sediments and other solids as related to water quality problems,
pollution sources and control requirements. It focuses on practical and economical
virus monitoring technology and makes it possible for any competent water bacteriology
laboratory that can arrange for viral assays (and identifications) to evaluate and quantify
enteric viruses in environmental samples.
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June 1988
Table of Contents
Foreword i
Purpose ii
Figures xi
Tables xiii
Chapter 1 Introduction .1-1
1. Perspectives in Environmental Virology 1-1
2. The Viruses in Environmental Waters 1-2
3. Conclusions and Recommendations of the World Health
Organization (WHO) Scientific Group on Human Viruses
in Water, Wastewater and Soil 1-4
3.1 Conclusions of the Group 1-4
3.2 Recommendations of the Group 1-6
3.3 Summary 1-7
4. History of Methods Selection 1-7
4.1 Recommendations of the WHO Working Group and
the WHO Scientific Group 1-9
4.2 Recommendations in Standard Methods for Detecting
Viruses in Various Waters 1-10
4.3 Recommendations of the American Society for
Testing and Materials (ASTM) 1-11
5. The USEPA Manual 1-11
6. Bibliography 1-13
Chapter 2 Cleansing Laboratory Ware and Equipment 2-1
1. Precautions 2-2
2. Alternate Procedures 2-3
3. Preparation of Cleansing Compounds and Reagents 2-4
4. Procedure for Cleansing Laboratory Ware and Equipment 2-5
4.1 Cleansing with Detergent 2-5
4.1.1 General Laboratory Ware and
Washable Equipment 2-5
(a) Washing machine procedure 2-5
(b) Manual washing procedure 2-6
4.1.2 Test Tubes 2-7
4.1.3 Pipettes 2-8
4.1.4 Automatic Pipettor 2-9
4.1.5 Automatic Syringe 2-14
4.1.6 Disc Filter Holder 2-17
4.1.7 Dispensing Pressure Vessel 2-18
4.1.8 Plastic Screw Caps 2-19
in
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4.2 Cleansing with Acid 2-20
4.2.1 General Acid-Resistant Laboratory Ware 2-21
(a) Chromic acid procedure... T 2-21
(b) Nitric acid procedure , 2-22
4.2.2 Test Tubes 2-23
4.2.3 Pipettes . 2-25
4.3 Cleansing with Alkalies 2-27
5. Bibliography 2-28
Chapter 3 Sterilization and Disinfection 3-1
1. General Procedures 3-1
2. Sterilization Techniques 3-1
2.1 Solutions 3-1
2.2 Glassware, Autoclavable Plasticware, and Equipment ... 3-1
2.3 Contaminated Materials 3-7
3. Disinfection Techniques 3-8
4. Bibliography 3-9
Chapter 4. Quality Assurance 4-1
1. Introduction 4-1
1.1 Role in Research 4-1
1.2 Scope of Program 4-2
2. Sample Collection 4-2
2.1 Water and Sewage Samples 4-2
2.2 Chain of Custody 4-3
2.3 Sample Handling Procedures 4-3
2.4 Transport of Samples 4-3
3. Laboratory Facilities 4-4
3.1 Air Handling Systems 4-4
3.2 Disinfection of Laboratory 4-4
3.3 Space Allocation 4-4
3.4 Traffic 4-4
3.5 Bench Space Allocation 4-5
3.6 Lighting 4-5
3.7 Walls and Floors 4-5
3.8 Monitoring for Cleanliness in Work Areas 4-6
4. Laboratory Maintenance , 4-6
4.1 Cleaning 4-6
4.2 Storage 4-7
5. Laboratory Personnel 4-7
5.1 Professional Level 4-7
5.2 Supervisory and Senior Grade Level 4-8
5.3 Technical Level 4-8
5.4 Supervision of Personnel in Laboratory . ., 4-8
6. Laboratory Equipment and Instruments 4-9
6.1 Balances 4-9
iv
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June 1988
6.2 pH Meters 4-9
6.3 Distilled Water 4-9
6.4 Deionized Distilled Water 4-10
6.5 Ultraviolet Lights 4-10
6.6 Centrifuges 4-10
6.7 Downward Flow Laminar Hoods 4-11
6.8 Thermometers 4-11
6.9 Refrigerators 4-11
6.10 Dispensing Apparatus 4-11
6.11 Steam Autoclaves 4-11
6.12 Gas Sterilizers 4-12
6.13 Hot-Air Ovens 4-12
6.14 Roller Drum Apparatus 4-12
6.15 Freezers 4-12
6.16 Incubators 4-12
6.17 Security -. 4-13
7. Laboratory Supplies 4-13
7.1 Laboratory Ware 4-13
7.2 Media and Chemicals 4-13
7.3 Membrane Filters -. 4-14
7.4 Sintered-Glass Filters 4-14
8. Laboratory Procedures 4-14
8.1 Cell Cultures 4-14
8.1.1 Test for Sterility - 4-14
8.1.2 Preparation of Cell Lines 4-14
8.1.3 Preparation of Cell Cultures 4-1B
8.1.4 Record Keeping 4-15
8.2 Virus Assays 4-15
8.2.1 Preparation for Assays 4-15
8.2.2 Volume Assayed 4-16
8.2.3 Time of Assay- 4-16
8.2.4 Controls 4-16
8.2.5 Counting Plaques 4-16
8.2.6 Disposition of Data . . . „ 4-16
9. Bibliography 4-25
Chapter 5. Virus Adsorption-Elution (Viradel) Disc Filter Procedures
for Recovering Viruses from Sewages, Effluents, and Waters 5-1
1. Adsorption—Method One 5-1
1.1 Preparation 5-1
1.1.1 Apparatus and Materials 5-1
1.1.2 Media and Reagents 5-3
1.2 Procedure 5-3
1.2.1 Assembly of Apparatus 5-3
1.2.2 Salt Supplementation 5-7
1.2.3 Adjustment of pH 5-7
1.2.4 Filtration of Salted, pH-Adjusted Sample 5-8
2. Adsorption—Method Two 5-9
2.1 Preparation 5-9
2.1.1 Apparatus and Materials 5-9
2.1.2 Media and Reagents 5-11
2.2 Procedure 5-12
2.2.1 Preparation and Implementation 5-12
(a) Assembly of apparatus 5-14
(b) Treatment of prefilters 5-17
(c) Salt supplementation 5-23
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(d) Adjustment of pH 5-23
(e) Dechlorination 5-24
(f) Fluid proportioner 5-24
2.2.2 Filtration of Sample 5-27
t
3. Elution and Reconcentration 5-30
3.1 Procedure for Eluting Viruses from Filter 5-30
3.1.1 Apparatus and Materials 5-30
3.1.2 Media and Reagents 5-30
3.1.3 Procedure 5-31
3.2 Procedure for Processing Solids 5-32
3.2.1 Apparatus and Materials 5-32
3.2.2 Media and Reagents 5-33
3.2.3 Procedure 5-33
3.3 Organic Flocculation Concentration Procedure
of Katzenelson 5-36
3.3.1 Apparatus and Materials 5-36
3.3.2 Media and Reagents 5-36
3.3.3 Procedure 5-36
4. Bibliography 5-40
Chapter 6 Virus Adsorption-Elution (Viradel) Cartridge Filter
Procedures for Recovering Viruses from Sewage, Effluents,
and Waters • 6-1
1. Adsorption—Method One , 6-1
1.1 Preparation 6-1
1.1.1 Apparatus and Materials 6-2
1.1.2 Media and Reagents 6-4
1.2 Procedure v 6-5
1.2.1 Preparation and Implementation 6-5
(a) Assembly of apparatus 6-5
(b) Salt supplementation 6-9
(c) Adjustment of pH 6-10
(d) Dechlorination 6-10
(e) Fluid proportioner 6-11
1.2.2 Filtration of Sample 6-13
2. Adsorption—Method Two 6-15
2.1 Preparation 6-15
2.1.1 Apparatus and Materials 6-15
2.1.2 Media and Reagents 6-20
2.2 Procedure 6-21
2.2.1 Preparation and Implementation 6-23
(a) Assembly of apparatus 6-23
(b) Salt supplementation 6-24
(c) Adjustment of pH 6-25
(d) Dechlorination 6-25
(e) Fluid proportioner 6-26
2.2.2 Filtration of Sample 6-29
3. Elution and Concentration—Method One 6-31
3.1 Procedure for Eluting Viruses from Filters 6-31
3.1.1 Apparatus and Materials 6-31
3.1.2 Media and Reagents 6-34
3.1.3 Rearrangement of Apparatus 6-34
(a) Rearrangement for Method One 6-34
(b) Rearrangement for Method Two 6-35
f: »"
vi
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June 1988
3.1.4 Elution Procedure 6-37
3.2 Reconcentration—Method A. Membrane Disc Procedure 6-38
3.2.1 Apparatus and Materials 6-38
3.2.2 Media and Reagents 6-39
3.2.3 Procedure 6-40
(a) Assembly of apparatus 6-40
(b) Adjustment of pH of eluate 6-40
(c) Filtration of eluate 6-43
(d) Elution of viruses from filter 6-43
3.3 Reconcentration—Method B. Aluminum
Hydroxide-Hydroextraction Procedure 6-45
3.3.1 Apparatus and Materials 6-45
3.3.2 Media and Reagents 6-46
3.3.3 Procedure 6-47
(a) Preparation of dialysis bag 6-47
(b) Flocculation and hydroextraction 6-48
4. Elution and Concentration—Method Two 6-53
4.1 Procedure for Eluting Viruses from Filters 6-53
4.1.1 Apparatus and Materials 6-53
4.1.2 Media and Reagents 6-55
4.1.3 Rearrangement of apparatus 6-55
(a) Rearrangement for Method One 6-55
(b) Rearrangement for Method Two 6-56
4.1.4 Elution Procedure 6-57
4.2 Organic Flocculation Concentration Procedure of
Katzenelson 6-58
4.2.1 Apparatus and Materials 6-58
4.2.2 Media and Reagents 6-59
4.2.3 Procedure 6-59
5. Bibliography 6-62
Chapter 7 Method for Recovering Viruses from Sludges (and Other Solids) ... 7-1
1. Extraction of Viruses from Sludges 7-1
1.1 Preparation 7-1
1.1.1 Apparatus and Materials 7-1
1.1.2 Media and Reagents 7-2
1.2 Procedure 7-3
1.2.1 Conditioning of Sludge 7-3
1.2.2 Elution of Viruses from Sludge Solids 7-6
2. Concentration of Viruses from Sludge Eluates 7-9
2.1 Organic Flocculation Concentration Procedure of
Katzenelson 7-9
2.1.1 Apparatus and Materials 7-9
2.1.2 Media and Reagents 7-10
2.1.3 Procedure 7-10
3. Bibliography 7-14
Chapter 8 Method for Reduction of Cytotoxicity of Sample Concentrates 8-1
Revised
April 1986
1. Virus Recovery from Samples 8-1
2. Storage of Sample Concentrates 8-1
3. Predetermine Cytotoxicity of Sample Concentrate 8-1
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June 1988
4. Processing of Deeply Colored Sample Concentrates 8-1
5. Reduction of Toxicity of Sample Concentrate 8-1
5.1 Apparatus and Materials 8-1
5.2 Media and Reagents 8-1
5.3 Procedure 8-2
6. Plaque Procedure for Titrating Viruses 8-2
6.1 Preparation 8-2
6.1.1 Apparatus and Materials 8-2
6.1.2 Media and Reagents 8-2
6.2 Procedure 8-3
6.3 Counting Viral Plaques 8-3
7. Bibliography 8-3
Chapter 9 Cell Culture Preparation and Maintenance 9-1
Revised
Jan. 1987
1. Introduction 9-1
2. Medium Preparation 9-1
2.1 Apparatus and Materials 91
2.2 Media and Reagents 9-2
3. Preparation of Cell Culture Media 9-2
3.1 Technique 9-2
3.2 General Procedures 9-2
3.3 Medium Preparation 9-2
3.3.1 Sources of Cell Culture Media 9-2
3.3.2 Procedure for Preparation of EDTA-Trypsin 9-2
3.3.3 Procedure for Preparation of Growth Medium 9-3
3.3.4 Procedure for Preparation of Trypan Blue Solution . .9-3
3.3.5 Procedure for Preparation of Stock Antibiotic
Solutions 9-3
4. Procedure for Verifying Sterility of Liquids 9-4
4.1 Procedure for Verifying Sterility of Small Volumes
of Liquids 9-4
4.2 Procedure for Verifying Sterility of Large Volumes
of Liquids 9-4
4.3 Visual Evaluation of Media for Microbial Contaminants . . . .9-4
5. Procedures for Preparation of Stock BGM Cell Cultures 9-4
5.1 General Procedures 9-4
6. Procedure for Passage of BGM Cells 9-5
6.1 General Procedure 9-5
6.2 Procedure for Performing Viable Cell Counts 9-5
6.3 Procedure for Changing Medium on Cultured Cells 9-6
7. Procedure for Preparation of BGM Cell Cultures for
Virus Assay 9-6
7.1 Preparation of Cell Culture Bottles or Flasks 9-6
7.2 Preparation of Cell Culture Tubes 9-6
viii
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June 1988
8. Procedure for Preservation of BGM Cell Line 9-6
8.1 Preparation of Cells for Storage 9-6
8.2 Procedure for Freezing Cells 9-7
8.3 Procedure for Thawing Cells 9-7
9. Bibliography 9-7
Chapter 10 Cell Culture Procedures for Assaying Plaque-Forming Viruses .... 10-1
Revised
Dec. 1987
1. Introduction 10-1
2. Cell Monolayer Procedure 10-1
2.1 Sample Inoculation of Cell Monolayer for Virus Assay ....10-1
2.1.1 Apparatus and Materials 10-1
2.1.2 Media and Reagents 10-2
2.1.3 Procedure for Preparation of Stock
Antibiotic Solutions 10-2
2.1.4 Preparation of Maintenance Media 10-2
2.1.5 Procedure for Inoculating Test Sample 10-3
2.2 Agar Overlay Procedure for Plaque Assay 10-3
2.2.1 Apparatus and Materials 10-3
2.2.2 Media and Reagents 10-4
2.2.3 Preparation of Medium 199 10-4
2.2.4 Preparation of Pancreatin Solution for Use
in Detecting Reovirus 10-5
2.2.5 Preparation of Overlay Medium for Plaque
Assay 10-5
2.2.6 Preparation of Overlay Agar for Plaque Assay 10-5
2.2.7 Preparation of Agar Overlay Medium 10-5
2.2.8 Addition of Overlay Agar to Cell Culture
Test Vessels 10-6
2.3 Counting Viral Plaques 10-6
2.3.1 Counting Technique 10-6
2.3.2 Calculation of Virus Titer 10-6
2.4 Procedure for Verifying Sterility of Liquids 10-6
2.4.1 Procedure for Verifying Sterility of Small
Volumes of Liquids 10-6
2.4.2 Procedure for Verifying Sterility of Large
Volumes of Liquids 10-6
2.4.3 Visual Evaluation of Media for
Microbial Contaminants 10-6
3. Suspended Cell Procedure 10-7
3.1 Sample Inoculation of Suspended Cells for Virus Assay . . . 10-7
3.1.1 Apparatus and Materials 10-7
3.1.2 Procedure for Inoculating Test Sample 10-7
3.2 Agar Overlay Procedure for Plaque Assay 10-7
3.2.1 Apparatus and Materials 10-7
3.2.2 Media and Reagents 10-8
3.2.3 Procedure for Preparation of Stock
Antibiotic Solutions 10-8
3.2.4 Preparation of Medium 199 10-9
3.2.5 Preparation of Overlay Medium for Plaque
Assay 10-9
3.2.6 Preparation of Overlay Agar for Plaque Assay 10-9
3.2.7 Preparation of Agar Overlay Medium 10-9
3.2.8 Procedure 10-10
3.3 Counting Viral Plaques 10-10
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June 1988
3.3.1 Technique 10-10
3.3.2 Virus Enumeration 10-10
3.4 Procedure for Verifying Sterility of Liquids 10-10
3.4.1 Procedure for Verifying Sterility of Small
Volumes of Liquids 10-10
3.4.2 Procedure for Verifying Sterility of Large
Volumes of Liquids 10-10
3.4.3 Visual Evaluation of Media for
Microbial Contaminants 10-10
4. Bibliography 10-11
Chapter 11 Virus Plaque Confirmation Procedure 11-1
Revised
March 1988
1. Introduction 11-1
2. Recovery of Virus from Plaque 11-1
2.1 Apparatus, Materials and Reagents 11-1
2.2 Procedure 11-1
2.2.1 Procedure for Obtaining Viruses from Plaque 11-1
2.2.2 Procedure for Inoculating Viruses Obtained from
Plaques onto Cell Cultures 11-1
(a) Cell culture processing 11-2
(b) Procedure for Samples Tested Immediately .... 11 -2
(c) Procedure for Stored Samples 11-2
3. Bibliography 11-3
Chapter 1 2 Identification of Enteroviruses 12-1
Revised
May 1988
1. Introduction 12-1
2. Procedure for Typing Viruses 12-1
2.1 Apparatus and Materials 12-1
2.2 Media and Reagents 12-1
2.3 Procedure 12-2
2.3.1 Preparation of Microtiter Plates 12-2
2.3.2 Preparation of Virus for Identification 12-2
2.3.3 Addition of Antiserum Pools to Microtiter Plate .... 12-2
2.3.4 Addition of Virus to Microtiter Plates 12-2
2.3.5 Preparation of Cell Suspension and Completion
of Microtiter Test 12-3
3. Bibliography 12-4
Appendix
List of Vendors A-1
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June 1988
List of Figures
Figure No. Page
5-1 Flow Diagram of Method for Recovering Viruses from
Small Volumes (100 ml to 20 Liters) of Water, Sewage,
or Effluent 5-4
5-2 Schematic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL) Disc
Filter Procedure for Small Volume Filtrations 5-5
5-3 Photographic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL) Disc
Filter Procedure for Small Volume Filtrations 5-6
5-4 Flow Diagram of Method for Recovering Virusesfrom Large
Volumes (More than 20 Liters) of Water, Sewage, or
Effluents 5-13
5-5 Schematic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL) Disc
Filter Procedure for Large Volume Filtrations 5-15
5-6 Photographic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL) Disc
Filter Procedure for Large Volume Filtrations 5-16
5-7 Schematic Representation of Apparatus for Treatment of
Prefilters with Tween 80 to Prevent Adsorption of Viruses
to the Prefilters in the Virus Adsorption-Elution (VIRADEL)
Disc Filter Procedure for Large Volume Filtrations 5-18
5-8 Photographic Representation of Apparatus for Treatment
of Prefilters with Tween 80 to Prevent Adsorption of
Viruses to the Prefilters in the Virus Adsorption-Elution
(VIRADEL) Disc Filter Procedure for Large Volume
Filtrations 5-19
5-9 Flow Diagram of Reconcentration Procedure (Organic
Flocculation Procedure of Katzenelson) 5-35
6-1 Flow Diagram of Method One for Concentrating Viruses
from Large Volumes (More than 200 Liters) of Clean
Waters 6-6
6-2 Schematic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL)
Cartridge Filter Procedure for Large Volume Filtrations of
Clean (Non-Turbid) Waters 6-7
6-3 Photographic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL)
Cartridge Filter Procedure for Large Volume Filtrations of
Clean (Non-Turbid) Waters 6-8
6-4 Schematic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL)
Cartridge Filter Procedure for Large Volume Filtrations of
Turbid Waters 6-18
XI
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June 1988
6-5 Photographic Representation of Apparatus for Recovering
Viruses by the Virus Adsorption-Elution (VIRADEL)
Cartridge Filter Procedure for Large Volume Filtrations of
Turbid Waters 6-19
6-6 Flow Diagram of Method Two for Concentrating Viruses
from Large Volumes (More than 200 Liters) of Turbid
Waters 6-22
6-7 Flow Diagram of High pH Procedure (Basic Glycine, pH
10.5) for Eluting Viruses from Cartridge Filters and for
Reconcentrating Viruses from Clear Eluates by the
Membrane Filter Procedure 6-32
6-8 Flow Diagram of High pH Procedure (Basic Glycine, pH
10.5) for Eluting Viruses from Cartridge Filters and for
Reconcentrating Viruses from Turbid Eluates by the
AI(OH)3-Hydroextraction Procedure 6-33
6-9 Schematic Representation of Apparatus for Reconcen-
tration—Method A, a Membrane Disc Procedure for
Reconcentrating Viruses from Glycine Eluates 6-41
6-10 Photographic Representation of Apparatus for Reconcen-
tration—Method A, a Membrane Disc Procedure for
Reconcentrating Viruses from Glycine Eluates 6-42
6-11 Flow Diagram of Beef Extract Method for Eluting Viruses
from Cartridge Filters with Buffered 3% Beef Extract and
for Concentrating Eluted Viruses by the Katzenelson
Organic Flocculation Procedure 6-54
7-1 Flow Diagram of Method for Recovering and Concentrating
Viruses in Sludges 7-4
12-1 Representation of Microtiter Plate Preparation 12-2
12-2 Photographic Representation of Microtiter Plate
Preparation 12-3
XII
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June 1988
List of Tables
Table No. Page
3-1 Quantities of Deionized Distilled Water to be Added to
Vessels to Facilitate Sterilization During Autoclaving 3-3
3-2 Time-Temperature Couplings for Dry Sterilization 3-4
4-1 Monitoring Laboratory Equipment 4-17
4-2 Standards for Deionized Distilled Water 4-23
4-3 Laboratory Ware Maintenance 4-24
9-1 Guide for Preparation of BGM Stock Cultures 9-5
9-2 Guide for Preparation of Virus Assay Cell Cultures 9-7
10-1 Guide for Virus Inoculation, Suspended Cell Concentration
and Overlay Volume of Agar Medium 10-3
XIII
•&-U.S. GOVCtNMENT HUNTING OFFICE: M*2 •
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