&EPA
United States
Environmental Protection
Agency
Environmental Monitoring arid
Support Laboratory
Cincinnati OH 45268
EPA-600/4-84-013
February 1984
Research and Development
USEPA Manual of
Methods for Virology
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USEPA MANUAL OF METHODS FOR VIROLOGY
by
Gerald Berg, Ph.D., Robert S. Safferman, Ph.D.,
Daniel R. Dahling, Donald Herman, and Christen J. Hurst, Ph.D.
Environmental Monitoring and Support Laboratory-Cincinnati
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
11
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring and Support Laboratory - Cincinnati conducts research to:
o Develop and evaluate methods to measure the presence and
concentration of physical, chemical, and radiological pollutants
in water, wastewater, bottom sediments, and solid wastes.
o Investigate methods for the concentration, recovery, and
identification of viruses, bacteria, and other microbiological
organisms in water and to determine the responses of aquatic
organisms to water quality.
o Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for
monitoring water and wastewater.
o Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
This manual was prepared in order to meet mandates of the Congress of
the United States of America as directed in the Clean Water Act (PL 95-217),
the Safe Drinking Water Act (PL 93-523), 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.
Robert L. Booth, Acting Director
Environmental Monitoring and Support
Laboratory - Cincinnati
iii
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PURPOSE
"This manual makes it possible for any competent water bacteriology
laboratory that can arrange for viral assays (and identifications) by a
competent virology laboratory to concentrate and recover viruses from
waters and from sludges and other solids." (See Chapter 1, Section 5.)
iv
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TABLE OF CONTENTS
Page
Foreword ill
Purpose iv
Figures xiv
Tables xvii
Acknowledgements xviii
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 Materials (ASTM) 1-11
5. THE USEPA MANUAL 1-11
6. BIBLIOGRAPHY 1-12
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
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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 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 Alkalais 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 3-6
3. DISINFECTION TECHNIQUES 3-6
4. BIBLIOGRAPHY 3-7
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 4-2
2.3 Sample Handling Procedures 4-3
2.4 Transport of Samples 4-3
3. LABORATORY FACILITIES 4-3
3.1 Air Handling Systems 4-3
3.2 Disinfection of Laboratory 4-4
3.3 Space Allocation 4-4
3.4 Traffic 4-4
3.5 Bench Space Allocation 4-4
3.6 Lighting 4-5
3.7 Walls and Floors 4-5
3.8 Monitoring for Cleanliness in Work Areas 4-5
4. LABORATORY MAINTENANCE 4-6
4.1 Cleaning 4-6
4.2 Storage 4-6
vi
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Page
5. LABORATORY PERSONNEL 4-7
5.1 Professional Level 4-7
5.2 Supervisory and Senior Grade Level 4-7
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
6.2 pH Meters 4-9
6.3 Deionized Distilled Water 4-9
6.4 Distilled Water 4-9
6.5 Ultraviolet Lights 4-10
6.6 Centrifuges 4-10
6.7 Laminar Flow Hoods 4-10
6.8 Thermometers 4-10
6.9 Refrigerators 4-10
6.10 Dispensing Apparatus ' 4-10
6.11 Steam Autoclaves 4-11
6.12 Gas Sterilizers 4-11
6.13 Hot-Air Ovens 4-11
6.14 Roller Drum Apparatus 4-11
6.15 Freezers - 4-11
6.16 Incubators 4-12
6.17 Security 4-12
7. LABORATORY SUPPLIES 4-12
7.1 Laboratory Ware 4-12
7.2 Media and chemicals 4-12
7.3 Membrane Filters 4-13
7.4 Sintered-Glass Filters 4-13
8. LABORATORY PROCEDURES 4-13
8.1 Cell Cultures 4-13
8.1.1 Test for Sterility 4-13
8.1.2 Preparation of Cell Lines 4-13
8.1.3 Preparation of Cell Cultures 4-13
8.1.4 Record Keeping 4-14
8.2 Virus Assays 4-14
8.2.1 Preparation for Assays 4-14
8.2.2 Volume Assayed 4-14
8.2.3 Time of Assay 4-14
8.2.4 Controls 4-15
8.2.5 Counting Plaques 4-15
8.2.6 Disposition of Data 4-15
9. BIBLIOGRAPHY 4-24
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
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Page
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 5-1T
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 5-22
(d) Adjustment of pH 5-23
(e) Dechlorination 5-23
(f) Fluid proportioner 5-24
2.2.2 Filtration of Sample 5-26
3. ELUTION AND RECONCENTRATION 5-29
3.1 Procedure for Eluting Viruses From Filter 5-29
3.1.1 Apparatus and Materials 5-29
3.1.2 Media and Reagents 5-30
3.1.3 Procedure 5-30
3.2 Procedure for Processing Solids 5-32
3.2.1 Apparatus and Materials 5-32
3.2.2 Media and Reagents 5-32
3.2.3 Procedure 5-33
3.3 Organic Flocculation Procedure
of Katzenelson 5-34
3.3.1 Apparatus and Materials 5-34
3.3.2 Media and Reagents 5-36
3.3.3 Procedure 5-36
4. BIBLIOGRAPHY 5-39
Chapter 6 VIRUS ADSORPTIOH-ELUTION (VIRADEL) CARTRIDGE FILTER
PROCEDURES FOR RECOVERING VIRUSES FROM SEWAGES,
EFFLUENTS, AND WATERS 6-1
1. ADSORPTION — METHOD ONE 6-1
1.1 Preparation 6-1
1.1.1 Apparatus and Materials 6-1
1.1.2 Media and Reagents 6-4
1.2 Procedure , 6-4
1.2.1 Preparation and Implementation 6-6
viii
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Page
(a) Assembly of apparatus 6-6
(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 6-28
3. ELUTION AMD 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
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
IX
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Page
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
(AMD 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 7-8
2.1 Organic Flocculation Concentration
Procedure of Katzenelson 7-8
2.1.1 Apparatus and Materials 7-9
2.1.2 Media and Reagents 7-9
2.1.3 Procedure 7-10
3. BIBLIOGRAPHY 7-14
Chapter 8 METHOD FOR RECOVERING VIRUSES FROM TOXIC SLUDGES
AND SOLIDS 8-1
1. EXTRACTION OF VIRUSES FROM SLUDGES 8-1
1.1 Preparation 8-1
1.1.1 Apparatus and Materials 8-1
1.1.2 Media and Reagents 8-3
1.2 Procedure 8-3
1.2.1 Conditioning of Sludge 8-3
1.2.2 Elution of Viruses from
Sludge Solids 8-7
2. CONCENTRATION OF VIRUSES FROM SLUDGE ELUATES 8-8
2.1 organic Flocculation Concentration
Procedure of Katzenelson 8-8
2.1.1 Apparatus and Materials 8-9
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2.1.2 Media and Reagents 8-10
2.1.3 Procedure 8-10
3. BIBLIOGRAPHY 8-14
Chapter 9 PREPARATION AMD USE OF CELL CULTURES 9-1
1. INTRODUCTION 9-1
2. PREPARATION 9-1
2.1 Apparatus and Materials 9-1
2.2 Media and Reagents 9-3
3. PROCEDURE FOR PREPARATION OF BGM CELL CULTURES 9-5
3.1 General Procedures 9-5
3.2 Procedure for Passage of BGM Cells 9-7
3.3 Procedure for performing Viable
Cell Counts 9-10
3.4 Procedure for changing Medium on
Cultured Cells 9-11
4. PLAQUE PROCEDURE FOR RECOVERING'OR TITRATING
VIRUSES 9-12
4.1 Inoculating Virus-containing Sample
onto Cell Cultures 9-12
4.2 Counting Viral Plaques 9-14
4.3 Reduction of Sample-associated Toxicity 9-14
5. PROCEDURE FOR VERIFYING STERILITY OF LIQUIDS 9-15
5.1 Procedure for Verifying Sterility of
Small Volumes of Liquids 9-15
5.2 Procedure for Verifying Sterility of
Large Volumes of Liquids 9-16
6. PREPARATION OF CELL CULTURE MEDIA 9-16
6.1 Techni que 9-16
6.1.1 Equipment Care 9-16
6.1.2 Disinfection of Work Area 9-16
6.1.3 Aseptic Technique 9-17
6.1.4 Dispensing Filter-Sterilized Media 9-17
6.2 Sterility Testing 9-17
6.2.1 Coding Media 9-17
6.2.2 Sterility Test 9-17
6.2.3 Storage of Media and Media
Components 9-17
6.2.4 Sterilization of NaHC03-containing
Solutions 9-17
6.3 MEDIA FORMULATIONS 9-18
6.3.1 Sources of Cell Culture Media 9-18
6.3.2 Constraints, Modifications, and
Conditions in Media Formulations 9-18
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Page
7. PREPARATION OF MEDIA AND STAINS FOR CELL CULTURES 9-19
7.1 Growth Medium 9-19
7.1.1 Formula 9-19
7.1.2 Procedure 9-19
7.2 Maintenance Medium 9-20
7.2.1 Formula 9-20
7.2.2 Procedure 9-20
7.3 Agar Overlay Medium 9-21
7.3.1 Formula 9-21
7.3.2 Procedure 9-21
7.4 Eagle's Minimum Essential Medium
with Hanks' Balanced Salt Solution 9-23
7.4.1 Formula 9-23
7.4.2 Procedure 9-24
7.5 Eagle's Minimum Essential Medium For use
In Preparing Growth Medium 9-26
7.5.1 Formula 9-26
7.5.2 Procedure 9-26
7.6 2X Eagle's Minimum Essential Medium Without
Phenol Red For use In overlay Medium 9-28
7.6.1 Formula 9-28
7.6.2 Procedure 9-28
7.7 Hanks' Balanced Salt Solution, 10X Stock 9-30
7.7.1 Formula 9-30
7.7.2 Procedure 9-30
7.8 100X Amino Acids Stock for Eagle's Minimum
Essential Medium (Without Cysteine and
Tyrosine) 9-31
7.8.1 Formula 9-31
7.8.2 Procedure 9-31
7.9 100X Vitamins Stock for Eagle's Minimum
Essential Medium 9-33
7.9.1 Formula 9-33
7.9.2 Procedure 9-33
7.10 Leibovitz's L-15 Medium 9-35
7.10.1 Formula 9-35
7.10,2 Procedure 9-36
7.11 Earle's Balanced Salt Solution, 10X Stock 9-37
7.11.1 Formula 9-37
7.11.2 Procedure 9-37
7.12 Sodium Bicarbonate, 7.5% 9-38
7.12.1 Formula 9-38
7.12.2 Procedure 9-38
7.13 Magnesium Chloride, 1% 9-38
7.13.1 Formula 9-38
7.13.2 Procedure 9-39
7.14 Trypsin-EDTA Solution 9-39
7.14.1 Formula 9-39
7.14.2 Procedure 9-40
xii
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Page
7.15 Neutral Red, 0.If 9-41
7.15.1 Formula 9-41
7.15.2 Procedure 9-41
7.16 Phenol Red, 0.5% 9-41
7.16.1 Formula 9-41
7.16.2 Procedure 9-41
7,17 Trypan Blue Solution for Cell Counting
Procedure 9-42
7.17.1 Formula 9-42
7.17.2 Procedure 9-42
7.18 Stock Solutions of Antibiotics for Cell
•Culture and overlay Media 9-43
7.18.1 Formula 9-43
7.18.2 Procedure 9-43
7.18,3 Use Levels for stock Solutions
of Antibiotics 9-43
8. BIBLIOGRAPHY 9-45
Chapter 10 VIRUS PLAQUE CONFIRMATION PROCEDURE 10-1
1. RECOVERY OF VIRUSES FROM PLAQUE 10-1
1.1 Apparatus and Materials 10-1
1.2 Procedure 10-2
1.2.1 Procedure for obtaining Viruses
from Plaque 10-2
1.2.2 Procedure for inoculating
Viruses Obtained from Plaques
onto Cell Cultures 10-3
(a) Procedure for Samples Tested
Immediately 10-3
(b) Procedure for Samples Stored
at -70° C Before Testing 10-4
CHAPTER 11 IDENTIFICATION OF ENTEROVIRUSES 11-1
1. PROCEDURE FOR TYPING VIRUSES 11-1
1.1 Apparatus and Materials 11-1
1.2 Media and Reagents 11-2
1.3 Procedure 11-2
1.3.1 Preparation of Microtiter Plates 11-2
1.3.2 Preparation of Virus for
Identification 11-5
1.3.3 Addition of Antiserum Pools to
Microtiter Plate 11-5
1.3.4 Addition of Virus to Microtiter
Plates 11-6
1,3.5 Preparation of Cell Suspension and
Completion of Hicrotiter Test 11-7
2. BIBLIOGRAPHY 11-10
APPENDIX
LIST OF VENDORS A-I
xili
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FIGURES
Figure Title 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 Viruses from
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
Prefliters 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
xi v
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Figure Title Page
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-
El uti on (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 6-5
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
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
xv
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Figure Title Page
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 Al(OH)3-Hydroextraction
Procedure 6-33
6-9 Schematic Representation of Apparatus for
Reconcentration ~ Method A, a Membrane Disc
Procedure for Reconcentrating Viruses from
Glycine Eluates 6-41
6-10 Photographic Representation of Apparatus for
Reconcentration — 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
Flow Diagram of Method for Recovering and
Concentrating Viruses in Sludges
7-1
7-4
8-1 Flow Diagram of Method for Recovering and
Concentrating Viruses in Toxic Sludges 8-4
11-1 Schematic Representation of Microtiter Plate
Preparation 11-3
11-2 Photographic Representation of Microtiter Plate
Preparation 11-4
xvi
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TABLES
Table Title Page
3-1 Quantities of Deionized Distilled Water to be Added
to Vessels to Facilitate Sterilization During
Autoclaving 3-3
4-1 Monitoring Laboratory Equipment 4-16
4-2 Standards for Laboratory Pure Distilled Water 4-22
4-3 Laboratory Ware Maintenance 4-23
9-1 Guide for Determining Volume of Cell Culture Medium,
Virus Sample Inoculum, and Overlay Medium to be
Used with Various Sized Cell Culture Vessels 9-8
xvn
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ACKNOWLEDGEMENTS
The authors gratefully acknowledge Mary Ellen Rohr, who drafted the
chapter on Quality Assurance and,proofread many parts of the manual; Kim
HcClellan, Tamara Goyke, Betty A. Wright, Cynthia L. Barnes, and Alice B.
Lyles for their laboratory simulations of the methods in Chapters 5-8;
F. Dlanne White and Joan G. Lobitz for their valuable secretarial skills;
and William E. Faulkner for many of the procedures in Chapter 2. Special
acknowledgement goes to Dwight G. Ballinger who pioneered in methods
development and quality assurance techniques, and promoted the detailed
approach which has become the cornerstone of the methods in this manual.
xviii
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CHAPTER 1
INTRODUCTION
PERSPECTIVES IN ENVIRONMENTAL VIROLOGY
The ability to multiply, to direct processes in the cells they
infect, and the ability to mutate are the only characteristics of
life that the virus is capable of manifesting, in essence, the virus
is alive only when it infects. Outside of living cells, the virus is
inert. Its essential viability in the hostile environment outside
the cell is time-marked. Yet, among those viruses excreted by
infected people into sev/age discharged into rivers, streams, and
lakes, many often survive to reach the water intakes and recreational
areas of downstream communities. If that sewage or its treated
effluents or sludges are discharged to the land instead, sufficient
numbers of viruses may survive to contaminate crops or ground waters
in the aquifers below. If the discharge is to the oceans, viruses
may contaminate recreational beach waters or approved shellfish-
harvest waters. Over the years, cases of such contamination have
been documented repeatedly even in the apparent absence of indicator
bacteria.
The smallest numbers of viruses detectable in cell cultures, the
most sensitive hosts for many viruses, may be sufficient to infect
susceptible individuals who consume them. Thus, any number of viruses
that reaches a water intake or that is consumed by a recreationalist
is a potential hazard. To detect such small numbers of viruses in
water requires concentrating viruses1from large volumes of water.
- 1-1 -
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In the past several years, a growing awareness of the waterborne
virus problem has developed within the scientific community. This
awareness has resulted in the development of a number of techniques
for recovering viruses from waters of various qualities. These
waters range from sewage to tap water. The techniques that have been
developed include filter adsorption-elution, glass powder adsorption-
elution, ultrafiltration, polyelectrolyte adsorption, aluminum
hydroxide adsorption, protamine precipitation, hydroextraction,
two-phase separation, organic flocculation, and alginate membrane
filtration. Some of these methods are modestly efficient in limited
circumstances. None of them has universal potential at present.
There is endless change in the chemical quality of waste and
receiving waters, and the unpredictable effects of such change on the
efficiencies of the methods for quantitatively concentrating viruses
from waters is a problem that may long be with us. Thus, methods may
always require selection and flexibility to meet the needs of
changing situations. Guidance for such selection and flexibility is
given herein.
2. THE VIRUSES IN ENVIRONMENTAL WATERS
Enteroviruses (polioviruses, coxsackieviruses [groups A and B],
echoviruses, and hepatitis A virus), rotaviruses and other reoviruses
(Reoviridae), adenoviruses, and Norwalk-type agents — a total of
more than 100 different serological types -- constitute the major
enteric virus complement of human origin. Most of these viruses have
been detected in sewage and in receiving waters over the years.
Members of other virus groups have been recovered from human feces
and urine, but none has been reported with great frequency or in
large numbers in sewage or in receiving waters.
- 1-2 -
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Viruses of non-human sources abound in environmental waters. Some of
these viruses, such as reoviruses, may infect man; the significance
of certain other viruses from non-human sources is as yet
undetermined.
The numbers of viruses detected per liter of sewage range from
less than 100 infective units to more than 100,000 infective units.
In temperate climates, the numbers generally increase in the warmer
months and decrease in the colder months, reflecting overall
infection and excretion patterns in the community. In the tropics,
the numbers of viruses in sewage are highest during the rainy
season. Since viruses do not multiply outside of susceptible living
cells, dilution in hostile receiving waters and the toll of time
eventually reduce the numbers of viruses to levels often barely
detectable by the best techniques available, even when 1,000-L
quantities of water are tested. In receiving streams, however, such
numbers of viruses, in terms of the daily water intake requirements
of even small communities, are not small.
When one considers the low efficiencies of the methods that we
have for concentrating these viruses, that the cell culture systems
used for detecting viruses are usually sensitive to less than half of
the virus types excreted by man, that the plaque procedure usually
used for detecting and quantifying viruses is itself relatively
inefficient, and that there are undoubtedly viruses in sewage that
have not yet been detected and identified, it seems reasonable to
surmise that the numbers of viruses we now detect in environmental
waters are probably an order of magnitude or more below the
quantities actually present there. The numbers of viruses that reach
recreational waters and intakes downstream of outfalls may thus be
very large indeed.
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3. CONCLUSIONS AND RECOMMENDATIONS OF THE WORLD HEALTH ORGANIZATION
(WHO) SCIENTIFIC GROUP ON HUMAN VIRUSES IN WATER, WASTEWATER AND
SOIL*
In 1979, the World Health Organization (WHO) published the
report of a WHO Scientific Group on Human Viruses in Water,
Wastewater and Soil. The Group included USEPA participation. The
Conclusions and Recommendations of the Group follow and are quoted
directly:
3.1 [Conclusions]
While bacterial contamination of water and soils and
the associated health risks have been thoroughly studied,
attention is now increasingly being focused on the hazards
associated with virus contamination of water. The
Scientific Group reviewed the current state of knowledge on
the subject and concluded that the contamination of water
and soil by wastev/ater and human faeces containing enteric
viruses may pose real public health problems. This is also
applicable to areas of the world in which the major
waterborne bacterial diseases have been brought under
control.
There are over 100 different types of enteric
viruses, all considered pathogenic to man. Their
concentration in wastewater may reach 10 000-100 000/1, and
they have the ability to survive for months in water and in
soil, in some instances, the ingestion of a single
infectious unit can lead to infection in a certain
proportion of susceptible humans.
On numerous occasions viral hepatitis A epidemics
have been waterborne. Many outbreaks of viral hepatitis A
have resulted from eating shellfish grown in sewage-
contaminated estuarine and coastal waters. It is also
probable that a significant proportion of the reported
waterborne gastroenteritis outbreaks of nonbacterial
etiology have been associated with waterborne viruses
(e.g., rotaviruses).
While the Scientific Group recognized that massive
water-borne outbreaks of virus-associated diseases have
been detected only on limited occasions, it concluded that
*Human Viruses in Water, Wastewater and Soil, Report of a WHO
Scientific Group, Technical Report Series 639. World Health
Organization, Geneva, Switzerland, 1979. 50 pp.
- 1-4 -
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the constant exposure of large population groups to even
relatively small numbers of enteric viruses in large
volumes of water can lead to an endemic state of virus
dissemination in the community, which can and should be
prevented.
Bacteria used as conventional indicators to evaluate
the safety of potable water supplies have been shown to be
significantly less resistant than viruses to environmental
factors and to water and wastewater treatment processes.
As a result, enteric viruses may be present in water that
manifests little or no sign of bacterial pollution.
Where surveys have been carried out, viruses have
been detected in the drinking-water supply system of a
number of cities, despite the fact that these supplies have
received conventional water treatment, including filtration
and disinfection, which are considered adequate for
protection against bacterial pathogens. Plans for the
recycling of .wastewater for domestic consumption are being
considered in some cities, while many others are drawing
their water supply from contaminated surface sources
carrying a significant proportion of wastewater. In both
situations the risk of viruses penetrating the supply
system must be carefully evaluated so that adequate
monitoring and treatment can be provided.
Methods for the concentration and enumeration of
viruses in large volumes of water have been developed but
are not yet standardized. Through the use of such methods
large water samples can be monitored for viruses on a
routine basis.
Water treatment methods capable of accomplishing
effective virus removal and inactivation are now available,
so that conventional water treatment plants can be suitably
modified to deal with this problem. The formation of
carcinogenic compounds when water containing organic
material is chlorinated may give rise to a potential health
problem. However, in situations in which there is a risk
of waterborne communicable disease there should be no
hesitation in continuing current water disinfection with
chlorine until alternate techniques for effective virus
inactivation are developed.
Viruses present in wastewater and sludge applied to
land for irrigation, fertilization or disposal purposes can
survive in soil for periods of weeks or even months.
Edible crops, contaminated either by contact with virus-
laden soil or by wastewater sprinkler-irrigation, can
harbour viruses for sufficient periods of time to survive
harvesting and marketing, and thus their eventual
consumption constitutes a potential health risk.
Only limited data are available on the health risks
resulting from the dispersion of viruses in aerosols
created by sewage treatment and land disposal systems.
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However, a potential hazard does exist and steps to reduce it
may be warranted. Disinfection of effluent prior to land
disposal, particularly in the case of sprinkler-irrigation in
the vicinity of inhabited areas, could be an effective
preventive measure.
3.2 [Recommendations]
(1) Wherever possible, drinking-water should be free
from human enteric viruses. To ensure that this goal is
being achieved, a 100-1 to 1000-1 sample should be tested by
the most sensitive method available. In all cases of
intentional direct wastewater reuse for domestic consumption,
this procedure should be considered essential and should be
applied at least in large urban areas in which potable
supplies are derived from virus-polluted sources, such as
surface water containing a significant proportion of
wastewater either untreated or insufficiently treated to
inactivate viruses. Further consideration should be given to
the establishment of recommended virus concentration limits
for water for recreational purposes, and wastewater effluent
and sludge for agricultural use.
(2) Where virological facilities can be provided, it
is desirable to monitor wastewater effluents, raw-water
sources and drinking-water for the presence of viruses. This
will provide baseline data to evaluate the health risk faced
by the population.
(3) In the light of the greater resistance of many
enteric viruses to disinfection and other treatment processes
compared to that of bacteria utilized as pollution indicators,
drinking-water derived from virus-contaminated sources should
be treated by methods of proved high efficiency for removing
or inactivating viruses and not only bacteria. Particular
emphasis should be given in such cases to ensure the effective
disinfection of drinking-water with, for example, free
available chlorine residuals of 0.5 mg/1 maintained for a
contajct time of 30-60 minutes or an ozone residual of 0.2-0.4
mg/1 maintained for 4 minutes.
(4) Because of the ability of viruses to survive for
long periods in seawater, it is recommended that coastal
bathing and shellfish growing areas should be protected from
contamination by wastewater and sludge. Virus monitoring of
these areas is a desirable measure.
(5) Control procedures should be instituted in all
situations in which wastewater or sludge is used for
irrigation or fertilization, to prevent the contamination of
vegetables and fruits which are to be eaten raw. (Moreovei—
even though they may eventually be cooked—contaminated raw
vegetables are liable to pollute other food in the kitchen.)
Where it is nevertheless planned to irrigate such crops or
where sprinkler-irrigation is to be used near populated
areas, the effluent should be treated so that it reaches a
high microbiological quality approaching that of drinking-
water.
- 1-6 -
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(6) Since the factors that influence the movement
of viruses in soil are still not fully understood, and
since effluent and soil conditions vary so greatly, caution
should be exercised if wastewater irrigation or land
disposal takes place in.the vicinity of wells supplying
drinking-water. Careful study of local conditions is
required and the cautious siting of such wells and routine
virological monitoring of the water are advised as safety
measures.
(7) Further research is necessary into the health
risks associated with viruses in water and soil. These
studies should include the development and evaluation of
methods of detecting viruses and alternative indicators of
virus pollution (e.g., phages) and the improvement of
treatment methods for the inactivation and removal of
viruses from water and Wastewater. The dissemination and
survival of viruses in the natural environment should also
be investigated.
(8) A standard method should be developed for the
concentration and detection of viruses-in large volumes of
drinking-water (e.g., 100-1000 1) based on a full
evaluation in different laboratories of present
techniques. Such an attempt would facilitate the
development of virus-monitoring programmes and would ensure
a maximum degree of comparability of results. A laboratory
quality-control system should be developed to enable
participating laboratories to standardize their
procedures.
3.3 Summary
Although not a direct response to the efforts of the WHO
Scientific Group, this manual should make possible the
monitoring operations envisioned by that group.
4. HISTORY OF METHODS SELECTION
In 1965, a symposium on "Transmission of Viruses by the
Water Route" included a major segment on methods for recovering
viruses from the water environment. The focus on methods,
within the context of the water transmission problem, resulted
in a growing interest in methods research over the years that
followed.
- 1-7 -
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In 1975, a WHO Working Group on Bacteriological and
*
Virological Examination of Water met in Germany to recommend
the promulgation of methods for recovering bacteria and viruses
from various environmental waters and sludges. The USEPA
participated. Although methods for recovering bacteria are
well-advanced, methods for recovering viruses are not.
Nonetheless, the Sub-group on Virological Examination, with some
reservations, selected several methods for promulgation which it
**
believed were the best methods currently available. The
American Public Health Association, The American Water Works
Association and the Water Pollution Control Federation, through
their jointly published Standard Methods, and the American
Society for Testing Materials have also recommended methods for
recovering viruses from the water environment. The methods
described in this USEPA manual have seen the benefit of the
research and experience of the years that have passed since
1965. Nonetheless, the current state-of-the-art requires that
the following caveats are considered:
*Report of a Working Group on Bacteriological and Virological Examination
of Water (World Health Organization in collaboration with the Federal
Republic of Germany, Mainz, Germany, April 21-25, 1975). Water
Research, 10:177-178, 1976.
Lund, E. 1982. Virological Examination, _3:462-509. Jji Suess, M. J., ed.,
Examination of Water for Pollution Control, Pergamon Press, New York.
**The mandate of the sub-group did not include tap and ocean waters, but
some of the methods described herein are directly applicable to such
waters.
- 1-8 -
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- Changes in the quality of waters sampled may
affect markedly the efficiency of each method
described. Few studies are available that compare
the efficiency of one method with another under the
same conditions.
- None of the methods described has been studied
with more than a few virus types. Most studies
have been laboratory and not field studies. None
of the methods is equally efficient for the
recovery of all of the types of viruses frequently
found in environmental waters.
- Some of the techniques described are labor-
intensive. Some require expensive equipment. In a
methodology so rapidly evolving, there is a risk of
obsolescence and obvious economic consequences.
4.1 Recommendations of the WHO Working Group and the WHO Scientific
Group
Both the aforementioned WHO Working Group on
Bacteriological and Virological Examination of Water and the WHO
Scientific Group on Human Viruses in Water, Wastewater and Soil
suggested tentatively for concentrating viruses from 0.2- to 5-L
volumes of wastewater and other waters a microporous filter
adsorption-elution technique, adsorption-precipitation with
Various salts, polyethylene glycol hydroextraction, aqueous
polymer two-phase separation, and soluble alginate filtration.
These Groups tentatively suggested tangential flow ultra-
filtration and flow-through adsorption-elution systems for
concentrating viruses from 5- to 400-L volumes of relatively
clean waters.
- 1-9 -
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The WHO Groups also recommended tentative methods for
recovering viruses from solids in waters and from sludges.
These methods were based on elution, with beef extract, serum,
or other proteinaceous materials, of viruses from the solids.
The tentative methods recommended by the two WHO Groups
have not been presented yet as operational procedures that can
be followed readily in the laboratory. Several of those methods
(but not the subsequent viral assays) are intended for use in
bacteriological laboratories that are minimally equipped and
staffed. Both Groups recommended that the tentative methods
undergo round-robin* testing.
**
4.2 Recommendations in Standard Methods for Detecting Viruses in
Various Waters
The 15th edition of Standard Methods presents a
microporous filter adsorption-elution technique, an aluminum
hydroxide adsorption-precipitation technique, and a polyethylene
glycol hydroextraction technique, all as tentative standard
methods for recovering viruses from waters and wastewaters. The
filter adsorption-elution technique is recommended for
concentrating viruses from only a few liters of any water
(single-stage filter adsorption-elution technique) and from
large volumes of purer waters (two-stage filter adsorption-
*Tests done under identical conditions by several participating
laboratories to determine the effectiveness, precision, and accuracy
of a method.
**Standard Methods for the Examination of Water and Wastewater, 15th Edition.
American Public Health Association, American Water Works Association,
Water Pollution Control Federation, Washington, D.C., 1981.
- 1-10 -
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elution technique). The latter technique may be used to
concentrate viruses from volumes of 1,000 L and more of finished
waters, standard Methods recommends the aluminum
hydroxideadsorption-precipitation technique and the polyethylene
glycol hydroextraction technique only for small volumes of waste
and other relatively highly contaminated waters.
The Standard Methods procedures have not been round-robin
tested.
The 15th edition of Standard Methods does not recommend
methods for recovering viruses from solids in water or from
sludges, but it does describe virus assay procedures.
Although the methods in Standard Methods have been
written in a manner intended as procedural, Standard Methods
recommends that testing with these methods "should be done only
by competent and specially trained water virologists having
adequate facilities."
4.3 Recommendations of the American Society for Testing Materials
(ASTM) ,
Most of the methods described in this USEPA manual have
been round-robin tested by the ASTM. A formal acceptance of
these methods as ASTM methods is pending.
5. THE USEPA MANUAL
The USEPA manual contained herein is state-of-the-art. The
manual comprises the best methodology available today, and it will be
revised frequently so that it remains state-of-the-art.
Each method in this manual has been presented as a step-by-step
procedure that should be easily followed by technicians trained in
bacteriology and familiar with aseptic techniques and safety
- 1-11 -
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procedures. Each method has been subjected to numerous successful
laboratory simulations by both experienced and inexperienced
technical personnel. Only the assays for viruses, which must be done
in cell cultures or in animals, require the skills of trained
virologists.
This manual makes it possible for any competent water
bacteriology laboratory that can arrange for viral assays (and
identifications) by a competent virology laboratory to concentrate
and recover viruses from waters and from sludges and other solids.
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6. BIBLIOGRAPHY
Human Viruses in Water, Wastewater and Soil, Report of a WHO
Scientific Group, Technical Report Series 639. World Health
Organization, Geneva, Switzerland, 1979. 50 pp.
Lund, E. 1982. Virological Examination, .3:462-509. In Suess, M. J.,
ed., Examination of Water for Pollution Control, Pergamon Press,
New York.
Report of a Working Group on Bacteriological and Virological
Examination of Water (World Health Organization in collaboration
with the Federal Republic of Germany, Mainz, Germany,- April
21-25, 1975). Water Research, 10:177-178, 1976.
Standard Methods for the Examination of Water and Wastewater, 15th
Edition. American Public Health Association, American Water
Works Association, Water Pollution Control Federation,
Washington, D.C., 1981.
- 1-13 -
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CHAPTER 2
CLEANSING LABORATORY WARE AND EQUIPMENT*
Laboratory ware and equipment that are not chemically clean are
responsible for considerable losses in personnel time and supplies in
many laboratories. These losses may occur as down time when experiments
clearly have been adversely affected and as invalid data that are often
attributed to experimental error. Chemical contaminants that adversely
affect experimental results are not always easily detected. The problem
of improper washing is usually worst in large laboratories with common
preparation facilities that are staffed with personnel of limited
training who often believe that if it's clean enough to eat from it's
clean enough to use in the laboratory.
The key to an effective preparation facility lies in the careful
training of hands-on personnel who must be made to understand that a
preparation facility is not really a kitchen (as the preparation facility
is so often referred to, perhaps aggravating the problem). It is, of
course, imperative that the supervisor of the preparation facility
understands and appreciates the need for chemically clean laboratory
ware. Competent supervisors who understand the need, even with personnel
who do not understand, can achieve the quality of cleanliness that is
necessary.
laboratory ware comprises laboratory glassware and plasticware.
- 2-1 -
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1. PRECAUTIONS
1.1 Sterilize contaminated laboratory ware and equipment before
cleansing them (see Chapter 3).
1.2 During the washing process, do not allow laboratory ware or
equipment to dry until after the final rinse in deionized
distilled water. Detergent that has dried on laboratory ware or
equipment is difficult to remove.
1.3 Transport strong acids only in appropriate safety carriers.
1.4 Once detergent solution or acid used to clean a vessel has been
rinsed away, do not touch lip or inside of vessel with hands.
Detergent or acid on hands or gloves and oil even from clean
skin are sources of contamination.
1.5 Check cleansed laboratory ware and equipment for residual
detergent and acid in accordance with recommendations in
Chapter 4.
1.6 Use non-toxic stainless steel, non-toxic glass, non-toxic
non-breakable plastic, or other non-toxic materials for plumbing
that carries deionized distilled water. Do not use copper
plumbing. Do not use plumbing tnat contains any ions that may
be toxic.
1.7 If a washing machine is used, ensure that jets are strong enough
to reach all parts of deep vessels. Ensure also that jets are
not so powerful they fill narrow-necked vessels and prevent
draining during the time that water is being delivered. Ensure
that jets and drains are not clogged. Ensure that washing
machine operates properly. Check timing of wash and rinse
cycles, nescale lime deposits with descaler when necessary.
1.8 Use only cold water for tap water rinsing. Hot water may
contain grease or oil removed from plumbing.
- 2-2 -
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1.9 Use only cold water to wash laboratory ware heavily contaminated
with proteinaceous material. Hot water may coagulate such
material. Laboratory ware contaminated with infectious
material, however, must be sterilized before it is cleansed (see
Chapter 3, Section 1-.3).
1,. 10 Inspect washed laboratory ware and equipment for cleanliness.
Recleanse unclean laboratory ware by appropriate procedures.
Check laboratory ware and equipment for damage. Repair or
replace damaged laboratory ware and equipment as appropriate.
1.11 In a multi-purpose laboratory in which different levels of
cleanliness are required, code all laboratory ware and
equipment, cleanse to specifications, and return to owners.
Always report cleansing problems, breakage, and damage to owners.
2. ALTERNATE PROCEDURES
2.1 Disposable laboratory ware may be used when available.
2.2 Cleansing procedures described herein are adequate for most
laboratory situations. Less rigorous procedures may be used
when quality control tests show they are adequate for
laboratory's needs.
2.3 Distilled water (see Chapter 4) may be used in place of
deionized distilled water for rinsing whenever quality control
tests show that distilled water is adequate.
2.4 When contaminants refractory to chromic acid and HN03
procedures are encountered on acid-resistant laboratory ware or
equipment, aqua regia may be used to cleanse the ware or
equipment in the manner described for concentrated chromic acid.
- 2-3 -
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3. PREPARATION OF CLEANSING COMPOUNDS AMD REAGENTS :
3.1 Liquid detergent compound for machine-washing glassware and
equipment (MIR-A-KOL, Du Bois Chemical Co., or equivalent). ,
Use according to manufacturer's instructions.
3.2 Detergent powder for hand-washing glassware and equipment (Buell
Cleaner, Mo. 222, Polychem Corp., or equivalent).
Use according to manufacturer's instructions.
3.3 Nitric acid (HMOg), 10%.
To prepare 10% HMOo, pour 100 mL of concentrated HMOo slowly
into 900 ml of cold deionized distilled water. TO AVOID
DANGEROUS SPLATTERS, MEVER POUR HATER INTO CONCENTRATED ACID
(see also CAUTIOM, Section 4.2).
3.4 Chromic acid (dichromate solution).
To prepare chromic acid, dissolve 40 g of sodium dichromate
rQ) or potassium dichromate (KCr) in 1
liter of concentrated sulfuric acid. Dissolve KgCr^ in
the acid on a magnetic stirrer. Na?Cr?07 is more soluble
but more expensive than K^Cr.^. TAKE CARE TO AVOID
EXPOSURE TO ACID (see CAUTIOM, Section 4.2). Potassium and
sodium dichromate are strong oxidizing agents and must be
handled cautiously.
3.5 Aqua regia
Prepare aqua regia in laboratory fume hood only.
To prepare aqua regia, pour 250 ml. of fuming (technical grade)
HNOg into 750 -ml. of fuming (technical grade) HCl , and mix
carefully. Take care to avoid dangerous splatters and exposure
to fumes (see CAUTION, Section 4.2).
-------
4. PROCEDURES FOR CLEANSING LABORATORY WARE AND EQUIPMENT
Laboratory ware and equipment may be cleansed in several ways. Those
used for cell cultures may require special care.
4.1 Cleansing with Detergent
4.1.1 General laboratory ware and washable equipment.
(a) Washing machine procedure.
Equip washing machine with capability for
delivering four deionized distilled water rinses.
The water jets in some washing machines are not
strong enough to reach all walls in tall vessels.
This results in poor washing and rinsing. The
water jets in other washing machines are too strong
for test tubes and similar vessels and for many
other narrow-necked vessels. Jets that are too
powerful hold detergent and rinse water in place
and do not allow them to drain properly. If
washing machine is unable to wash or rinse
adequately, use procedure described in Section
4.1.1. Step (b).
(a.l) Immerse washable vessels in detergent
solution, and soak them overnight.
If vessels are too large to immerse, fill
them to brim with detergent solution, and
soak them overnight.
(a.2) Brush-wash vessels with hot (50-60° C)
detergent solution.
Hot tap water that exceeds 50° C is
adequate for preparing detergent solution.
- 2-5 -
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(a.3) Machine-wash vessels.
Follow manufacturer's instructions
carefully. Add four deionized distilled
water rinses if not included in
manufacturer's instructions.
(a.4) Drain and air dry vessels, or dry vessels in
drying chamber.
(a.5) Sterilize vessels by appropriate method (see
Chapter 3).
(b) Manual washing procedure.
Use fresh detergent solution daily.
Solutions that are saved may become heavily
contaminated with bacteria.
(b.l) Immerse vessels in detergent solution, and
soak vessels overnight.
(b.2) Brush-wash vessels with hot (50-60° C)
detergent solution.
Hot tap water that exceeds 50° C is
adequate for preparing detergent solution.
(b.3) Swish-rinse vessels 10 times with cold tap
water .
To swish-rinse, pour into the vessel a
volume of tap water equal to about 10% of
the volume of the vessel, and swish water
around entire surface with each rinse.
(b.4) Swish-rinse vessels five times with
deionized distilled water.
(b.5) Drain and air dry vessels, or dry vessels in
drying.chamber.
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(b.6) Sterilize by appropriate method (see
Chapter 3).
4.1.2 Test Tubes
Test tubes may be. washed by procedure described In
Section 4.1.1, Step (a) unless a washing machine is
unavailable or washing machine jets are so powerful they
do not allow adequate evacuation of tubes and thus
Interfere with washing and rinsing. In either event, the
procedure that follows may be used instead of the washing
machine procedure.
(a) Place test tubes open end up into covered wire
basket, place basket into stainless steel or
plastic vessel sufficient in size to allow complete
immersion of tubes, and fill vessel with hot
detergent solution.
(b) Steam autoclave (100° C) immersed tubes for 30
minutes.
(c) Empty vessel and tubes, and run cold tap water in
to flush out detergent solution.
Introduce tap water into bottom of vessel with a
hose connnected to tap. Wax pencil and other scum
will wash over rim of vessel.
(d) Fill and empty tubes in vessel 10 times with cold
tap water.
(e) Fill and empty tubes in vessel five times with
deionized distilled water.
(f) Drain and air dry tubes, or dry tubes in drying
chamber.
(g) Sterilize screw-cap tubes.
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(g.l) Place-screw cap tubes in test tube racks,
and cover them with a sheet of aluminum foil.
(g.2) Sterilize tubes in dry heat oven (maintain
170° C [340° F] for one hour).
(h) Sterilize other tubes.
(h.l) Plug tubes with cotton, or protect mouths of
tubes with caps or with semi-permeable
plastic inserts.
(h.2) Sterilize tubes with cotton plugs in dry
heat oven (maintain 170° C [340° F] for
one hour).
(h.3) Autoclave tubes with caps or plastic inserts
at 121° C for 30 minutes.
4.1.3 Pipettes
(a) Remove cotton plugs from pipettes.
If necessary, remove cotton plugs by forcing a jet
of air or water through delivery tips of pipettes.
(b) Place pipettes, with tips up, into pipette holder.
(c) Place pipette holder into a pipette jar, and fill
jar with hot (50-60° C) detergent solution.
Hot tap water that exceeds 50° C is adequate for
preparing detergent solution. Pipettes must be
completely immersed. If air bubbles are present in
pipettes, raise and lower pipette holder several
times to remove bubbles.
(d) Soak pipettes in detergent solution for 24 hours.
Raise and lower pipette holder five or six times
during the 24 hour period to agitate detergent
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solution and thus help remove sofl and debrfs from
pipettes.
(e) Place pipette holder into automatic pipette washer,
and rinse pipettes through 10 cycles of cold tap
water.
(f) Rinse pipettes through five cycles of deionized
distilled water.
(g) Remove pipettes from automatic pipette washer, and
allow pipettes to drain and air dry.
(h) Plug pipettes with cotton.
(i) Sterilize pipettes in dry heat oven (maintain
170° C [340° F] for one hour).
4.1.4 Automatic Pipettor (Brewer-type)
Immediately after pipettor has been used, fill reservoir
with tap water and carefully pump sufficient water
through the system to remove cellular debris and other
materials that might adhere to apparatus. Determine
whether syringe delivers properly without cannula
connected.
(a) Remove tubing from reservoir, and remove syringe
from pipettor; autoclave valve, tubing, reservoir,
and syringe at 121° C for 60 minutes.
(b) Disassemble syringe, and remove cannula.
(c) Cleanse syringe, tubing, reservoir, valve, and
cannula.
(c.l) Syringe
(c.1.1) Rinse plunger and barrel of syringe
with copious quantities of cold tap
water.
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If plunger and barrel require
further cleansing, soak them
overnight in 10% HNO^ or In 10%
chromic acid (See Section 4.2.1,
Step [b] and Step la]), and repeat
Step (c.1.1). CAUTION; Take care
when using acid (see CAUTION,
Section 4.2). Do not expose metal
to acid.
(c.1.2) Rinse plunger and barrel with
copious quantities of deionized
distilled water.
(c.1.3) Soak plunger and barrel of syringe
overnight in deionized distilled
water.
(c.1.4) Allow plunger and barrel of syringe
to drain and air dry.
(c.1.5) Proceed to Step (d).
(c.2) Tubing
(c.2.1) Rinse tubing copiously with cold
tap water.
If tubing does not come clean,
place it in hot (50-60° C)
detergent solution, remove air
bubbles, and allow tubing to soak
for 24 hours. Then, repeat Step
(c.2.1).
(c.2.2) Rinse tubing copiously with
deionized distilled water.
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(c.2.3) Soak tubing overnight in deionized
distilled water.
(c»2.4) Allow tubing to drain and air dry.
(c.2.5) Proceed to Step (d).
(c.3) Reservoir
(c.3.1) Fill reservoir with hot
(50-60° C) detergent solution,
and soak reservoir overnight.
Hot tap water that exceeds 50° C
is adequate for preparing detergent
solution.
(c.3.2) Brush-wash reservoir with hot
(50-60° C) detergent solution.
If reservoir does not come clean,
rinse it with tap water, and soak
it overnight in 10% HMO, or in
10% chromic acid. See Section
4.2.1, Step (b) and Step (a).
CAUTION; Take care when using acid
(see CAUTION. Section 4.2).
(c.3.3) Rinse reservoir 10 times with cold
tap water.
(c.3.4) Swish-rinse reservoir five times
with deionized distilled water.
(c.3.5) Allow reservoir to drain and air
dry.
(c.3.6) Proceed to Step (d).
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(c.4) Valve
If syringe has been delivering properly with
thecannula removed (see Section 4.1.4), no
further attention to valve Is needed. If
syringe has not been delivering properly
with the cannula removed, go to Step (c.4.1).
(c.4.1) Remove valve from apparatus.
(c.4.2) Soak valve overnight in 10% HMO,
or in 10% chromic acid.
CAUTION: Take care when using acid
(see CAUTION. Section 4.2).
(c.4.3) Rinse valve copiously with cold tap
water.
(c.4.4) Rinse valve copiously with
deionized distilled water.
(c.4.5) Allow valve to drain and air dry.
(c.4.6) Return valve to apparatus.
(c.4.7) Proceed to Step (d).
(c.5) Cannula
(c.5.1) Connect cannula to a clean
syringe.
(c.5.2) Force 50 ml of deionized distilled
water through cannula.
If cannula is unobstructed, go to
Step (c.5.3). If cannula is
completely or partially obstructed
go to Step (c.5.5).
(c.5.3) Allow cannula to drain and air
dry.
- 2-12 -
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(c.5.4) Proceed to Step (d).
(c.5.5) If cannula is obstructed, remove
cannula from syringe, and soak
cannula for 24 hours in 10% HN03
or in 10% chromic acid.
Move cannula up and down in acid to
remove air bubbles that may have
formed.
CAUTION: Take care when using acid
(see CAUTION, Section 4.2).
(c.5.6) Rinse cannula copiously in cold tap
water.
(c.5.7) Connect cannula to syringe.
(c.5.8) Force 50 ml of cold tap water
through cannula.
(c.5.9) Force 50 ml of deionized distilled
water through cannula.
(c.5.10) Remove cannula from syringe.
(c.5.11) Allow cannula to drain and air dry.
(c.5.12) Proceed to Step (d).
(d) Reassemble syringe.
(e) Reconnect tubing to reservoir and to syringe.
(f) Connect cannula to appropriate tubing.
(g) Pour 100 mL of deionized distilled water into
reservoir, cover opening of reservoir with aluminum
foil, protect cannula with glass tube cover, and
wrap syringe, interconnecting tubing, and protected
cannula in cloth.
- 2-13 -
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(h) Autoclave assembled apparatus at 121° C for 30
minutes.
Use slow exhaust.
4.1.5 Automatic Syringe (Cornwall-type)
Immediately after syringe has been used, fill reservoir
with tap water and pump sufficient water through the
system to remove cellular debris and other materials that
might adhere to apparatus. Determine whether syringe is
delivering properly without cannula connected.
(a) Remove tubing from reservoir, and autoclave entire
apparatus at 121° C for 60 minutes.
(b) Disassemble syringe.
(c) Cleanse syringe, tubing, and cannula, and replace
valves, if necessary.
(c.l) Syringe
(c.1.1) Rinse plunger and barrel of syringe
with copious quantities of cold tap
water.
If plunger and barrel require
further cleansing, soak them
overnight in 10% HN03 or in 10%
chromic acid (see Section 4.2.1,
Step [b] and Step [a]), and repeat
Step (c.1.1).
CAUTION: Take care when using acid
(see CAUTION, Section 4.2). Do not
expose metal to acid.
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(c.1.2) Rinse plunger and barrel of syringe
with copious quantities of
deionized distilled water.
(c.1.3) Soak plunger and barrel of syringe
overnight in deionized distilled
water.
(c.1.4) Allow plunger and barrel of syringe
to drain and air dry.
(c.1.5) Proceed to Step (d).
(c.2) Tubing
(c.2.1) Rinse tubing with copious
quantities of cold tap water.
If tubing does not come clean,
place it in hot (50-60° C)
detergent solution, remove air
bubbles, and allow tubing to soak
for 24 hours. Then, repeat Step
(c.2.1).
(c.2.2) Rinse tubing with copious
quantities of deionized distilled
water.
(c.2.3) Soak tubing overnight in deionized
distilled water.
(c.2.4) Allow tubing to drain and air dry.
(c.2.5) Proceed to Step (d).
(c.3) Valves
If syringe has been delivering properly with
the cannula removed (see Section 4.1.5), no
further attention to valves is needed. If
, ^
- 2-15 -
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syringe has not been delivering properly
with the cannula removed, check inlet and
outlet valves. Replace either valve, or
both valves, if damaged or hard.
(c.4) Cannula
(c.4.1) Connect cannula to a clean syringe.
(c.4.2) Force 50 ml of deionized distilled
water through cannula.
If cannula is completely or
partially obstructed, go to Step
(c.4.5).
(c.4.3) Allow cannula to drain and air dry.
(c.4.4) Proceed to Step (d).
(c.4.5) If cannula is obstructed, remove
cannula from syringe, and soak
cannula for 24 hours in 10% HN03
or in 10% chromic acid.
Move cannula up and down in acid to
remove air bubbles that have formed.
CAUTION: Take care when using acid
(see CAUTION. Section 4.2).
(c.4.6) Rinse cannula copiously in cold tap
water.
(c.4.7) Connect cannula to syringe.
(c.4.8) Force 50 ml of cold tap water
through cannula.
(c.4.9) Force 50 ml of deionized distilled
water through cannula.
- 2-16 -
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(c.4.10) Remove cannula from syringe.
(c.4.11) Allow cannula to drain and air
dry.
(c.4.12) Proceed to Step (d).
(d) Reassemble syringe.
(e) Connect cannula to syringe.
(f) Protect cannula with glass tube cover, and wrap
syringe and protected cannula in cloth.
(g) Autoclave apparatus at 121° C for 30 minutes.
Use slow exhaust.
4.1.6 Disc Filter Holder.
(a) Disassemble disc filter holder, and discard
membrane.
(b) Rinse filter holder components with copious
quantities of cold tap water.
If debris remains after tap water rinse, brush-wash
filter holder with hot (50-60° C) detergent
solution. Remove refractory debris with non-
abrasive scrubber. Use fine grade steel wool only
if absolutely necessary. Rinse again with copious
quantities of cold tap water.
(c) Rinse filter holder components with copious
quantities of deionized distilled water.
(d) Allow filter holder components to drain and air
dry.
Check gaskets for distortion (flattened areas), and
replace gaskets if necessary.
(e) Attach tubes to inlet and outlet ports of filter
holder.
- 2-17 -
-------
(f) Clamp shut outlet port.
(g) Place filter support on base of filter holder.
(h) Fill base of holder with deionized distilled
water.
(i) Place membrane filter on filter support.
(j) Cover filter with deionized distilled water.
(k) Reassemble disc filter holder.
Do not tighten down top of filter holder.
(1) Autoclave filter and filter holder at 121° C for
20 minutes.
Use slow exhaust.
(m) Allow filter holder to cool, and tighten down top
of holder.
4.1.7 Dispensing pressure vessel.
(a) Remove lid from dispensing pressure vessel.
(b) Rinse pressure vessel and lid with copious
quantities of cold tap water.
If debris remains after tap water rinse, brush-wash
vessel and lid with hot (50-60° C) detergent
solution. Remove refractory debris with non-
abrasive scrubber. Use fine grade steel wool only
if absolutely necessary. Rinse again with copious
quantities of cold tap water.
(c) Swish-rinse pressure vessel and lid five times with
deionized distilled water.
To swish-rinse, pour into the vessel a volume of
water equal to about 10% of the volume of the
vessel, and swish water around entire surface with
each rinse.
- 2-18 -
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(d) Allow vessel and lid to drain and air dry.
(e) Pour TOO. ml of deionized distilled water into
vessel.
(f) Cover vessel opening with aluminum foil, and wrap
lid with aluminum foil.
Be certain vent/relief valve on lid is open.
(g) Autoclave vessel and lid at 121° C for 30
minutes, and dry both in autoclave for 10 minutes.
4.1.8 Plastic Screw-caps.
(a) Place caps in stainless steel or plastic vessel
containing detergent solution.
(b) Steam autoclave (100° C) caps in detergent
solution for 15 minutes.
(c) Pour water from vessel, and rinse caps with copious
quantities of cold tap water.
Run hose< from tap to bottom of vessel to achieve
thorough rinsing.
(d) Rinse caps with copious quantities of deionized
distilled water.
Run hose from deionized distilled water line to
bottom of vessel to achieve thorough rinsing.
(e) Place caps in upright position on towel, and allow
caps to drain and air dry.
(f) Place caps in upright position in glass petri
plates. . '•
(g) Place petri plates in petri plate cannister.
(h) Autoclave caps at 121° C for 30 minutes.
Leave top off cannister during autoclaving to allow
penetration of steam.
- 2-19 -
-------
(i) Allow plates and caps to cool, and secure cover on
cannister.
4.2 Cleansing with Acid
Either chromic acid or 10% HNO^ may be used to cleanse
glassware. Ten percent HNOg requires longer contact (24
hours) with tubes than chromic acid requires, but residual
o is not as likely to be toxic to cell cultures as residual
chromic acid is.
Do not expose metals or other materials to acids unless certain
that those substances are acid-resistant. CAUTION: Chromic
acid and other acids may react violently with organ ics or other
oxidizable substances. Take care to avoid such reactions.
Cleanse laboratory ware with detergent solutions before
cleansing them with acids. Chronic acid and UNCL are strong
acids capable of producing severe burns even when used in
relatively dilute solutions. When working with these or with
other strong acids, avoid inhalation of fumes. Protect eyes
with safety goggles or with full face mask. Protect clothing
with acid-resistant laboratory coat or apron. If eyes are
accidentally exposed to acid, immediately wash them with copious
quantities of water for at least 15 minutes. Consult a
physician immediately thereafter. If other parts of the body
are exposed to acid, immediately remove clothing over exposed
areas and wash exposed areas with copious quantities of water.
Consult a physician immmediately thereafter if affected area is
large or if exposure has been lengthy. Subsequently, wash
exposed areas of clothing with copious quantities of water.
- 2-20 -
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4.2.1 General Acid-resistant Laboratory Ware.
(a) Chromic acid procedure.
Glassware arid other acid-resistant laboratory ware
cleansed with chromic acid may retain some chromium
ions even after extensive rinsing. For some work,
these ions may be undesirable. Chromic acid may be
toxic to cells. Glassware and other laboratory
ware used for cell culture work, if washed with
chromic acid, may subsequently need to be washed
with detergent solution to remove chromium ions
(see Section 4.1).
(a.l) Thoroughly rinse loose debris and residues
from vessel with tap water.
(a.2) Pour into vessel from an acid reservoir a
volume of chromic acid equal to about 10% of
the capacity of the vessel.
Take care to avoid splatter. Small vessels
may be immersed in a vat of acid. Do not
allow acid to contact skin (see CAUTION,
Section 4.2). When necessary, wear acid-
resistant gloves. Gloves must possess good
gripping qualities, because acid makes
yessels sii ppery.
(a.3) Rotate vessel so that acid covers entire
inside area of vessel.
Allow chromic acid to remain in contact with
vessel for about five minutes.
(a.4) Pour acid from vessel into acid reservoir.
- 2-21 -
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Chromic acid is reusable until oxidized
(green). If chromic acid is oxidized,
dispose of it safely as with other toxic
wastes.
, (a.5) Fill and empty vessel with cold tap water 10
times.
Be certain that all acid is removed from
outside of vessel.
(a.6) Swish-rinse vessel five times with deionized
• • distilled water.
To swish-rinse, pour into the vessel a
volume of water equal to about 10% of the
volume of the vessel, and swish water around
entire surface with each rinse.
(a.7) Drain and air dry vessel, or dry vessel in
drying chamber.
(a.8) Sterilize vessel by appropriate method (see
Chapter 3).
(b) Nitric acid procedure.
(b.l) Rinse loose debris from vessel with tap
water.
(b.2) Fill vessel to brim with 10% HN03.
Small vessels may be immersed in a vat of
acid. Dp not allow acid to contact skin
(see CAUTION. Section 4.2). When necessary,
wear acid-resistant gloves. Gloves must
possess good gripping qualities, because
acid makes vessels slippery.
- 2-22 -
-------
(b.3) Allow 10% HN03 to remain in contact with
vessel surface for 24 hours.
(b.4) Carefully.pour acid down acid-resistant
sewer drain, and flush acid away with
copious quantities of tap water.
(b.5) Fill and empty vessel 10 times with cold tap
water.
Be certain that all acid is removed from
outside of vessel.
(b.6) Swish-rinse vessel five times with deionized
distil led water.
To swish-rinse, pour into the vessel a
volume of water equal to about 10% of the
volume of the vessel, and swish water around
entire surface with each rinse.
(b.7) Drain and air dry vessel, or dry vessel in
drying chamber.
(b.8) Sterilize vessel by appropriate method (see
Chapter 3).
4.2.2 Test Tubes
CAUTION: Take care to avoid splatter. Do not allow acid
to contact skin (see CAUTION, Section 4.2), When
necessary, wear acid-resistant gloves. Gloves must
possess good gripping qualities because acid makes tubes
slippery.
(a) Rinse loose debris from tubes with tap water.
(b) Place tubes open end up into covered acid-
resistant wire basket, and place basket into
acid-resistant vessel.
- 2-23 -
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(c) Fill vessel with,chromic acid or with 10% HNO-j.
If chromic acid is used, allow acid to remain in
contact with tubes for about five minutes. If 10%
HMO- is used, allow acid to remain in contact
with tubes for 24 hours.
(d) Pour acid from tubes.
Chromic acid is reusable until oxidized (green) and
may be poured back into reservoir. If chromic acid
is oxidized, dispose of it safely as with other
toxic wastes. Wash waste HNO^ down acid-
resistant drain with copious quantities of tap
water.
(e) Run cold tap water into vessel to flush acid from
tubes.
Run tap water through a hose into bottom of
vessel. Wax pencil and other scum will wash over
rim of vessel.
(f) Fill and empty tubes in vessel 10 times with cold
tap water.
(g) Fill and empty tubes in vessel five times with
deionized distilled water.
Tubes for cell culture work that have been cleansed
with chromic acid must be cleansed with detergent
solution. For such tubes, proceed to Section 4.1.2.
(h) Drain and air dry tubes, or dry tubes in drying
oven.
(i) Sterilize screw-cap tubes.
- 2-24 -
-------
(i.l) Place screw-cap tubes in test tube racks,
.. and cover them,with a sheet of alumimum
;•-. '... - ... foil.
...'.'. (i«2) Sterilize tubes ,in dry heat oven (maintain
170° C [340°.F] for one hour).
(j) Sterilize other tubes.
(j.l) Plug tubes with cotton, or protect mouths of
tubes with semi-permeable plastic inserts.
(j.2) Sterilize tubes with cotton plugs in dry
heat oven (maintain 170° C [340° F] for
one hour).
(j.3) Autoclave tubes with plastic inserts at
121° C for 30 minutes.
4.2.3 Pipettes
(a) Remove cotton plugs from pipettes.
If necessary, remove, cotton plugs by forcing a jet
of air or water through delivery tips of the
pipettes.
(b) Place pipettes, with tips up, into an
acid-resistant plastic pipette holder.
(c) Carefully place pipette holder into an
acid-resistant pipette jar filled with 10% HNO^.
CAUTION: Take care to avoid acid splatter. Do not
, allow acid to contact eyes or skin (see CAUTION,
Section 4.2). Wear eye protection and an
acid-resistant laboratory coat or an acid-
resistant apron. When necessary, wear
- 2-25 -
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acid-resistant gloves with good gripping
qualities. Acid makes pipettes and pipette holders
slippery.
(d) Carefully raise and lower pipette holder several
times to force air bubbles from pipettes.
(e) Soak pipettes in acid for 24 hours.
Carefully raise and lower pipette holder five or
six times during the 24-hour period to agitate acid
and thus help remove contaminants and debris from
pipettes.
(f) Carefully remove pipette holder from pipette jar,
and place holder and pipettes in automatic pipette
washer.
Take care to avoid acid splatter.
(g) Immediately rinse pipettes through 10 cycles of
cold tap water.
Do not allow acid dripping from pipettes to remain
in contact with metal parts of automatic pipette
washer. Acid may damage metal.
(h) Rinse pipettes through seven cycles of deionized
distilled water.
(i) Remove pipettes from automatic washer, and allow
pipettes to drain and air dry.
(j) Plug pipettes with cotton.
(k) Place pipettes in pipette canisters, and sterilize
in dry heat oven (maintain 170° C [340° F] for
one hour).
- 2-26 -
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4.3 Cleansing with Alkalies
Alkalies such as sodium metasilicate, trisodium phosphate,
sodium carbonate, and soft soaps can be used to cleanse
laboratory ware and equipment. Alkalies, however, tend much
more than acids to etch at least the glassware.
- 2-27 -
-------
5. BIBLIOGRAPHY
Paul, John. Cell and Tissue Culture, 5th Edition, Churchill
Livingstone, New York, 1975.
Standard Methods for the Examination of Water and Wastewater, 15th
Edition, American Public Health Association, Washington, D.C.,
1981.
- 2-28 -
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CHAPTER 3
STERILIZATION AND DISINFECTION
1. GENERAL PROCEDURES
1.1 Use aseptic techniques for handling test waters, sewages,
sludges, and cell cultures.
1.2 Sterilize apparatus and containers that will come into contact
with test waters, sludges, or elutants, all solutions added to
test waters unless otherwise indicated, and all elutants.
1.3 Sterilize all contaminated materials (including all blood and
blood products) before discarding.
1.4 Disinfect all spills and splatters.
2. STERILIZATION TECHNIQUES
2.1 Solutions
Unless otherwise indicated, sterilize all solutions except those
used for cleansing, standard buffers, hydrochloric acid (HCl),
sodium hydroxide (MaOH), Freon and other organic materials, and
disinfectants by autoclaving them at 121° C for 15 minutes.
HCl, MaQH, Freon, and disinfectants as used herein are self-
sterilizing (bactericidal and fungicidal)_. When autoclaving
buffered beef extract, use a vessel large enough to accommodate
foaming.
2.2 Autoclavable Glassware, Autoclavable Plasticware, Dialysis
Tubing, and Equipment
- 3-1 -
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Water may speed heat transfer in larger vessels during
autoclaving and thereby speed the sterilization process.
Add deionized distilled water to vessels in quantities indicated
in Table 3-1. Lay large vessels on sides in autoclave, if
possible, to facilitate displacement of air in vessels by
flowing steam.
2.2.1 Cover openings into glassware, autoclavable plasticware,
and equipment with aluminum foil before autociaving.
2.2.2 Sterilize glassware, unless otherwise noted, in a dry
heat oven at a temperature of 170 C for one hour (see
Table 3-2 for acceptable alternative time-temperature
couplings).
2.2.3 Autoclave at 121° C for one hour plasticware that can
withstand autoclaving.
Plasticware requires more time to sterilize than glass-
ware because plastic transfers heat more slowly than
glass.
2.2.4 Sterilize stainless steel vessels in an autoclave at
121° C for 30 minutes.
Vent-relief valves on vessels so equipped must be open
during autoclaving and closed immediately when vessels
are removed from autoclave.
2.2.5 Sterilize with ethylene oxide tubing and plasticware that
cannot withstand autoclaving.
CAUTION: Avoid exposure to ethylene oxide fumes.
Ethylene oxide is toxic.
Sterilize materials in 12% ethylene oxide (20-50%
relative humidity). Expose dry materials to ethylene
oxide for two hours. Expose materials that are not dry
for four hours.
- 3-2 -
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TABLE 3-1
Quantities of deionized distilled water to be added to vessels to
facilitate sterilization during autoclaving.
Quantity of
Vessel Size (Liter) Deioniz.ed Distilled Water (mL)
2 and 3 25
4 50
8 100
24 500
54 1000
*Add to vessel the volume of deionized distilled water indicated, cover
mouth of vessel with aluminum foil, lay vessel on its side if
possible, and autoclave.
- 3-3 -
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TABLE 3-2
Time-temperature couplings for dry sterilization.
Temperature
o C op Hours
140 285 3
150 300 2.5
160 320 2
170 340 1
- 3.4 .
-------
2.2.6 Autoclave dialysis tubing at 121° C for 15 minutes.
Fill dialysis bag two-thirds full with deiom'zed
distilled water before autoclaving.
2.2.7 Autoclave membrane filters in situ in filter holders at
121° C for 20 minutes.
To speed sterilization and to prevent filters from
cracking, seat (and cover) filters in a small volume of
deionized disti11ed water.
Open vent/relief valves on filter holders before
autoclaving, and close vent/relief valves immediately
after autoclaving.
2.2.8 Sterilize cartridge filters according to manufacturer's
instructions.
2.2.9 Autoclave apparatus except pumps, cartridge filter
holders, and combination-type pH electrodes at 121° C
for 30 minutes.
2.2.10 Sterilize drums, other vessels, and other apparatus too
large for autoclaves by chlorination.
Fill vessels or apparatus with deiom'zed distilled water
containing 10-15 mg of chlorine (NaQCl) per liter,
adjusted to pH 6-7 with HCl. Dechlorinate chlorinated
water in vessels or apparatus after 30 minutes by adding
sufficient sodium thiosulfate (Na2$2p3) solution to
yield 50 mg per liter. Allow 15 minutes for
dechlorination, and drain water from vessels or apparatus.
2.2.11 Sterilize pumps and cartridge filter holders with
chlorine or with ethylene oxide.
(a) Chlorine procedure.
- 3-5 -
-------
(a.l) For 30 minutes, recirculate through .pumps
and cartridge filter holders 4 liters of
deionized distilled water containing 10-15
mg of chlorine (NaQCl) per liter, adjusted
to pH 6-7 with HC1.
(a.2) Dechlorinate pumps and cartridge filter
holders by passing through them 1 liter of a
solution containing 50 mg of sodium
thiosulfate (Na2$203) per liter of
deionized distilled water.
(b) Ethylene oxide procedure.
CAUTION: Avoid exposure to ethylene oxide fumes.
Ethylene oxide is toxic.
(b.l) Sterilize pumps and cartridge filter holders
in a gas sterilizer by exposing them to 12%
ethylene oxide (30-50% relative humidity)
for four hours at 55-60° C.
(b.2) Aerate pumps and cartridge filter holders in
a gas aerator to remove residual ethylene
oxide (as recommended by the sterilizer
manufacturer), or maintain pumps and
cartridge filter holders at 37° C for a
minimum of three days before using them.
2.2.12 Sterilize pH electrodes with chlorine or with HCl.
Sterilize electrodes before and after each use.
(a) Chlorine procedure.
- 3-6 -
-------
(a.l) Immerse tip of electrode for one minute into
deionized distilled water that contains
10-15 mg of chlorine (NaOCl) per liter,
adjusted to pH 6-7 with HCl.
(a.2) Dechlorinate electrode by immersing tip into
sterile deionized distilled water that
contains 50 mg of Na9S9Oo per liter
C, L. O
and then rinsing tip with sterile deionized
distilled water.
(b) HCl procedure.
(b.l) Immerse tip of pH electrode into 1 M HCl for
one minute.
Use only fresh 1 M HCl prepared daily from
concentrated HCl.
(b.2) Rinse electrode tip with sterile deionized
distilled water.
2.2.12 Sterilize working instruments such as scissors and
forceps by autoclaving them at 121° C for 30 minutes.
Working instruments such as scissors and forceps may be
sterilizedbetweenuses by^jmmersing them in 70% ethanol
and flaming them.
2.3 Contaminated materials.
Autoclave contaminated materials for one hour at 121° C. Be
sure that steam can enter contaminated materials freely, if
volume of contaminated materials is unusually large, exposure
time at 121° C must be increased appropriately.
- 3-7 -
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3. DISINFECTION TECHNIQUES
3.1 Disinfect spills and other contamination on surfaces that do not
stain with a solution of 0.5% I2 in 70% ethanol.
3.2 Disinfect spills and other contamination on surfaces that stain
with a solution of 0.1% HOC1.
Q.1% HOC! may be prepared by appropriately diluting an NaQCl
solution (Clorox, The Clorox Co., or equivalent) and adjusting
its pH to 6-7 with dilute HC1.
- 3-8 -
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4. BIBLIOGRAPHY
Block, S. S. ed., Disinfection, Sterilization, and Preservation.
Philadelphia, 2nd edition, Lee and Febiger, 1977.
Davis, B. D., Dulbecco, R., Eisen, H.N., and Ginsberg, H.S.,
Microbiology, 3rd edition, Harper and Row, Publishers, New York,
1980.
- 3-9 -
-------
-------
CHAPTER 4
QUALITY ASSURANCE*
1. INTRODUCTION
1.1 Role in Research
1.1.1 In research for any purpose, the quality of data must be
protected. Quality assurance plays a key role in the
production and protection of scientifically valid data through
a variety of planned and systematic activities and
procedures. A laboratory quality control program is the
orderly application of practices necessary to remove or reduce
errors in any laboratory operation that are attributable to
personnel, equipment, supplies, sampling procedures, and
analytical methods.
1.1.2 A quality control program must be practical, integrated, and
require only a reasonable amount of time or it is likely to
be bypassed. When properly administered, a balanced,
conscientiously applied quality control program assures the
production of uniformly high quality data without interfering
with the primary analytical functions of the laboratory.
*Many of the sections of this chapter were adapted from Microbiological Methods
for Monitoring the Environment. I, Hater and Hastes, EPA-600/8-78-071, 1978.
- 4-1 -
-------
When possible, this laboratory program should be supplemented
by participation of the laboratory in an interlaboratory
quality control program.
1.2 Scope of Program
This chapter on Quality Assurance deals with sample collection,
facilities, maintenance, personnel, equipment and instruments,
supplies, and procedures. See Microbiological Methods for Monitoring
the Environment. I. Water and Wastes, EPA-600/8-78-071, U. S.
Environmental Protection Agency, Cincinnati, Ohio, 1978, for
discussions of statistics applicable to microbiology (p. 225), and for
the development of a quality control program (p. 244). For the
latter, also see Interim Guide!ines and Specifications for Preparing
Quality Assurance Project Plans, QAMS-005/80, Office of Monitoring
Systems and Quality Assurance, Office of Research and Development, U.
S. Environmental Protection Agency, Washington, D. C., 1980. For
discussions of safety, see Biosafety in Microbiological and Biomedical
Laboratories (Draft), Centers for Disease Control, Atlanta, Georgia,
and National institutes of Health, Bethesda, Maryland, 1983.
2. SAMPLE COLLECTION
2.1 Water and Sewage Samples
2.1.1 Water and sewage samples collected must be representative of
the particular environment sampled.
2.1.2 Sample sites and sampling frequency must provide representative
characteristics and variabilities.
2.1.3 The number of samples collected must rest within the processing
capability of the laboratory.
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2.2 Chain of Custody
A strict chain of custody procedure is required for all samples in
legal enforcement actions. (For chain of custody procedures, see
Handbook for Sampling and Sample Preservation of Water and Wastewater,
NTIS, PB83-124503, 1982, pp. 345-355.)
2.3 Sample Handling Procedures
2.3.1 Aseptic technique must be maintained during sampling.
2.3.2 Sterile containers and equipment must be used in all sampling
procedures.
2.3.3 When a sample is received in the laboratory, the integrity of
the sample container and the condition of the sample must be
checked and recorded.
2.3.4 It must be ascertained that the sample has been properly
labelled.
2.3.5 In enforcement cases, a check must be made to assure that the
chain of custody procedure has been followed.
2.4 Transport of Samples
Proper conditions must be met to maintain viability of viruses during
transport.
2.4.1 Samples must be refrigerated or iced immediately upon
collection.
2.4.2 All samples that cannot be processed within 24 hours must be
frozen and stored at -70° C immediately.
Freezing and thawing of virus samples must be kept to a minimum.
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3. LABORATORY FACILITIES
3.1 Air Handling Systems
3.1.1 All laboratories should be maintained under negative air
pressure.
3.1.2 Biological safety cabinets should be available for work
requiring sterile conditions and protection for personnel and
samples.
3.2 Disinfection of Laboratory
Laboratories should be equipped with ultraviolet lights (see
Table 4-1) for general decontamination of rooms during periods when
personnel are absent.
Take precautions to prevent entry of personnel into laboratories when
UV lights are on.
3.3 Space Allocation
3.3.1 Laboratories should provide separate rooms for processing in
each of the following categories: potable, surface, and ground
v/aters; sewage and wastewater effluents; sludges, silts, and
other solids; cell cultures; and virus identification.
3.3.2 Freezers, incubators, and instruments should be housed in rooms
where they can be accessed without disturbing ongoing
laboratory effort.
3.3.3 The areas provided for preparation and sterilization of media,
glassware, and equipment should be separate from other
laboratory work areas but close enough for convenience.
3.4 Traffic
3.4.1 Visitors and through-traffic must be minimized in work areas.
3.4.2 Signs must be posted on doors to limit access to work areas.
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3.5 Bench Space Allocation
3.5.1 Sufficient clean bench space must be available for work to be
performed efficiently.
For routine studies the minimum area recommended for each
worker is six linear feet. Research or other analyses that
demand specialized equipment may require significantly more
space per worker. These estimates of bench space are exclusive
of areas used for preparatory and supportive activities.
3.5.2 Bench tops should be set at heights of 36-38 inches.
This height is usually comfortable for work in a standing or
sitting position.
3.5.3 Depth of bench tops should be 28-30 inches.
3.5.4 Desk tops of sit-down benches should be set at a height of
30-31 inches.
This height is required to accommodate microscopy, plaque
counting, calculating, and writing.
3.5.5 Bench tops should be stainless steel, epoxy plastic, or other
smooth impervious material which is inert and
corrosion-resistant.
3.5.6 Bench tops should be seamless or have seams sealed with
impervious material.
3.6 Lighting
Laboratory lighting must be even, screened to reduce glare, and
provide about 100 footcandles of light intensity on working surfaces.
3.7 Walls and Floors
t
3.7.1 Walls should be covered with waterproof paint, enamel, or other
surface material that provides a smooth finish which is easily
cleaned and disinfected.
' - 4-5 -
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3.7.2 Floors should be covered with good quality tiles or other heavy
duty material which can be maintained with skid-proof wax.
3.8 Monitoring for Cleanliness in Work Areas
3.8.1 High standards of microbiological cleanliness must be
maintained in work areas.
3.8.2 Laboratory surfaces and laboratory air should be monitored for
microorganisms by one or more procedures.
Tests should be run on a weekly or on some other time basis
based on experience to monitor counts in the same work areas
over time and to allow comparisons between different work
areas. Microbial densities in the air should not exceed 15
colony-forming units per 930 square centimeters (~1 square
foot) of agar medium exposed per 15 minutes of exposure.
For a detailed description of these monitoring procedures, see
Microbiological Methods for Monitoring the Environment.
I. Water and Wastes. EPA-600/8-78-017, 1978, pp. 195-197.
LABORATORY MAINTENANCE
4.1 Cleaning
4.1.1 Laboratory benches must be cleaned after each use and at the
end of each working day; shelves, floors, and windows must be
cleaned on a scheduled basis.
4.1.2 Work benches must be wiped down with disinfectant before and
after each use.
Dry-dusting is not permissible in a virology laboratory.
4.1.3 Floors must be wet-mopped and treated with a disinfectant
solution to reduce contamination of air in the laboratory.
Sweeping or dry-mopping is not permissible in a virology
laboratory.
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4.1.4 Spills and leaks must be cleaned up immediately and disinfected
when necessary.
4.2 Storage
4.2.1 Laboratory areas must be kept free of clutter.
Clutter can be controlled, by cleaning up work areas immediately
after each use and by conducting a weekly clean-up of the
laboratory.
4.2.2 Equipment and supplies should be stored when not in use.
5. LABORATORY PERSONNEL
Virologists, other microbiologists, technicians, and support personnel in
the environmental virology laboratory must have training and experience
appropriate for the laboratory's program. The variety and complexity of
the tasks and tests performed determine the professional bench and
on-the-job-training required.
B.I Professional Level
5.1.1 Professional staff perform scientific work in connection with
identification, culture, study, and control of viruses and
other organisms.
Most of the work is performed in a laboratory environment and
is generally concerned with research and development,
monitoring, regulations, and public health.
5.1.2 Professional responsibilities require the ability to apply and
adapt scientific theories and principles of microbiology at a
level that allows making limited independent decisions.
5.1.3 The basic educational requirement is a BS/BA degree in virology
or microbiology or a BS/BA degree in biology with a minor in
virology or microbiology.
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5.2 Supervisory and Senior Grade Level
5.2.1 The Supervisory and Senior Grade level staff retain the key
positions of responsibility for planning and directing the
systematic research on a virus problem area and for organizing,
evaluating, and documenting the results pertinent to these
activities.
5.2.2 Professional responsibilities include direct leadership in
assigned subject matter areas, exercising full and independent
responsibility for the development of criteria, methods, and
virus data of general applicability for use by others,
interpreting the scope and scientific quality of the data, and
serving as a specialist in virology.
5.3 Technician Level
5.3.1 The technician level staff typically assist professionals by
doing routine tests, performing tasks involving a series of
steps, and maintaining records of experiments.
5.3.2 Technician responsibilities include performing repetitive
tasks, some understanding of the work done in the laboratory
and the relationships of various tasks, recognizing readily
observable events or reactions, and making precise measurements.
5.4 Supervision of Personnel in Laboratory
5.4.1 The laboratory should be directed by a Professional Virologist.
In a small laboratory where the staff consists of a single
non-professional technician, an approved consultant virologist
must be available for guidance and counselling.
5.4.2 Work assignments in the laboratory must have readily definable
objectives.
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5.4.3 The supervisor or consultant must review staff performance at
least annually and laboratory procedures used at least
quarterly.
Sample collecting and handling, media and glassware
preparation, sterilization, routine testing procedures,
counting, data handling, quality control techniques, and
laboratory safety are areas requiring examination.
6. LABORATORY EQUIPMENT AMD. INSTRUMENTS
Quality control of laboratory apparatus includes servicing and monitoring
the operation of incubators,, water baths, hot-air sterilizing ovens,
autoclaves, water stills, refrigerators, freezers, and other laboratory
equipment. Each item of equipment must be tested to^ verify that it meets
the manufacturer's specifications and the user's needs for accuracy and
precision. (See Table 4-1 for check list.)
6.1 Balances
Balances must be kept clean and protected from corrosion, checked
monthly with weights meeting class S standards, and serviced
annually.
6.2 pH Meters
Before each use, pH meters must be standardized with two standard
buffers (pH 4.0, 7.0, 10.0) bracketing pH of sample.
Buffer solutions must not be reused.
6.3 Distilled Water
6.3.1 Conductivity of distilled water must be monitored at least
daily, and preferably continuously, with a conductivity meter.
For continuous monitoring, an in-line meter should be used.
6.3.2 The water still must be drained and cleaned at least monthly.
6.3.3 The water reservoir must be cleaned at least quarterly.
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6.4 Deionized Distilled Water (see Table 4-2)
6.4.1 Conductivity of deionized distilled water must be monitored at
least daily and preferably continuously with a conductivity
meter. For continuous monitoring, an in-line meter should be
used.
6.4.2 Deionized distilled water must be monitored for bacteria
monthly.
6.4.3 Chemical analysis of deionized distilled water may be done when
necessary by chemists trained in such procedures.
6.4.4 Cartridges of deionizing resins must be replaced as indicated
by manufacturer or on the basis of analytical tests.
6.5 Ultraviolet Lights
Ultraviolet lights must be checked quarterly.
When less than 80% of the rated initial output is emitted, the lights
must be replaced. Also perform spread plate irradiation test
quarterly. For appropriate procedures, see Microbiological Methods
for Monitoring the Environment. I. Water and Wastes,
EPA-600/8-78-017, 1978, pp. 198-199.
6.6 Centrifuges
6.6.1 Centrifuges must contain a safety interlock.
6.6.2 Centrifuges must be disinfected and cleaned frequently.
6.6.3 On centrifuges not equipped with a built-in tachometer,
rheostat controls must be checked against a tachometer at
various loadings every six months to insure proper
gravitational forces. Rheostats should never be used as final
indicators of centrifuge speed when centrifuges are equipped
with built-in tachometers.
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6.7 Downward Flow Laminar Hoods
6.7.1 Downward flow laminar hoods must be free of clutter, and all
surfaces must be cleaned and swabbed with a disinfectant before
and after each use.
6.7.2 Hoods must be tested at least once annually to ensure proper
operation.
CAUTION: The integrity of the air curtain may be compromised
by air currents produced in hoods with clutter.
6.8 Thermometers
Thermometers must be calibrated at least once annually against
National Bureau of Standards (MBS) certified thermometers or
equivalents.
6.9 Refrigerators
Refrigerator temperatures must be monitored and recorded daily.
6.10 Dispensing Apparatus
Dispensing apparatus must be checked for accuracy of delivery volume
at each volume change and periodically throughout extended runs.
Apparatus must be recalibrated when necessary.
6.11 Steam Autoclaves
Steam autoclaves must be equipped with steam filters.
Steam autoclaves must be monitored at each use with temperature
recording charts and indicator tapes. Autoclave operation must also
be checked weekly with maximum-minimum thermometers and spore strips
or suspensions. Autoclaves should be checked weekly with a
thermocouple inserted into simulated worst case material.
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6.12 Gas Sterilizers
Gas sterilizers must be monitored at each use with recording charts
and indicator tapes. Such sterilizers must also be checked weekly
with spore strips or suspensions.
CAUTION: Ethylene oxide is toxic. Proper precautions must be taken
to protect personnel against exposure to ethylene oxide.
6.13 Hot-Air Ovens
Hot-air ovens must be monitored at each use with temperature indicator
tapes and thermometers or recording charts calibrated in the
160-180° C range. Hot air ovens must also be checked weekly with
spore strips.
6.14 Roller Drum Apparatus
Each roller drum apparatus for cell cultures must have an alarm that
signals power failure.
6.15 Freezers
6.15.1 All -70° C freezers must be equipped with temperature-
recording charts and alarms to signal excessive temperature
changes.
6.15.2 All -20° C freezers must be equipped with temperature-
recording charts or with thermometers.
6.15.3 Freezers should be cleaned and defrosted at least every six
months.
6.16 Incubators
6.16.1 Walk-in incubators must be equipped with temperature-recording
charts and alarms.
6.16.2 Reach-in incubators must contain automatic high temperature
cut-offs and must be checked daily with thermometers immersed
in water.
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6.17 Security
All incubators, freezers, and refrigerators should be secured with
locks.
7. LABORATORY SUPPLIES
7.1 Laboratory Ware
7.1.1 Laboratory ware must be thoroughly cleansed (see Chapter 2) and
rinsed in deionized distilled water (see Table 4-3).
7.1.2 Unless otherwise indicated, glassware must be sterilized in a
dry heat oven at 170° C for one hour; autoclavable
plasticware must be sterilized in an autoclave at 121° C for
one hour.
Use indicator tape to assure that sterilization temperature has
at least been reached; record date of sterilization on tape.
7.1.3 Whenever feasible, cell culture vessels should be discarded
after one use.
7.1.4 Laboratory ware should be tested for acid and alkaline
residuals and detergents by the procedures described in
Microbiological Methods for Monitoring the Environment. I.
Water and Wastes, EPA-600/8-78-017, 1978, pp. 199-200.
Laboratory ware that has not come clean and laboratory ware
with acid, alkaline, or detergent residuals must be recleansed.
7.2 Media and Chemicals
7.2.1 All chemicals and media must be dated upon receipt.
Unless otherwise indicated, only the purest grade of
commercially available chemicals, usually reagent-grade, may be
used. Caked media and media in opened containers for more than
six months must be discarded.
7.2.2 When appropriate, media must be pretested for sterility and
nutritional quality,and lots ordered from approved batches.
- 4-13 - '
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7.3 Membrane Filters
Membrane filters must be checked by a bubble test for air leaks and
must meet federal government specifications (for bubble test
procedure, see Microbiological Methods for Monitoring the
Environment. I. Water and Wastes, EPA-600/8-78-017, 19789 p. 205).
7.4 Sintered-Glass Filters
Sintered-glass filters must be checked periodically for retention of
bacteria (for testing retention characteristics see 1983 Annual Book
of ASTM Standards, Vol. 11.02, p. 856).
8. LABORATORY PROCEDURES
8.1 Cell Cultures
8.1.1 Test for Sterility
Test all cell culture media for sterility before use.
To test cell culture media for sterility, incubate all media at
37° C for one week prior to use. If gross contamination is
not visible after incubation, media must be tested in
thioglycollate broth.
8.1.2 Preparation of Cell Lines
(a) To reduce risk of contaminating one cell line with
another, prepare only one cell line in a given room at
any one time, and cleanse and disinfect work area
thoroughly before introducing another cell line.
(b) To reduce risk of massive microbiological contamination,
prepare separately media and reagents for each cell line.
(c) Use only heat-inactivated serum (56° C for 30 minutes)
in preparation of media.
(d) Personnel must wear protective clothing, changing after
each use.
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8.1.3 Preparation of Cell Cultures
(a) Trypsinize and dispense cells into fresh stock media in a
downward flow laminar hood.
(b) Check cell density by packed cell volume, or if feasible,
by direct cell count before and after distributing
cells.
(c) Test all distributed media for sterility in
thioglycollate broth to determine whether contamination
of media occurred during preparation of cell cultures.
(d) Test cell cultures at least once a month for Mycoplasma
contamination (flycotrim Mycoplasma Detection System, Hana
Media, Inc., or equivalent, may be used to test for
Mycoplasma).
8.1.4 Record Keeping
A continuous record must be kept of cell line passages.
8.2 Virus Plaque Assays
8.2.1 Preparation for Assay
(a) Cell cultures must be washed by replacement of culture
medium in cell culture vessels with serum-free medium
four hours or less before cultures are used to assay for
viruses.
(b) Cell cultures must be checked microscopically and
macroscopically periodically after seeding for growth of
cultured cells and for contamination.
(c) The same cell culture batch, and thereby cell cultures of
the same age, must be used for any given assay.
(d) uninoculated overlay controls must be included in each
assay to detect endogenous viruses.
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(e) Cell line sensitivity must be tested routinely against
reference viruses.
8.2.2 Volume Assayed
A minimum of 10% of each processed sample eluate must be
assayed; however, the total volume of each processed drinking
water sample eluate must be assayed.
8.2.3 Time of Assay
Processed samples must be refrigerated immediately at 4° C
and should be assayed as quickly as possible.
If a sample cannot be assayed within 8 hours after processing,
it must be frozen quickly and stored at -70° C until
assayed.
8.2.4 Controls
Diluents and/or elutants used in processing samples should be
assayed as controls.
8.2.5 Counting Plaques
Viral plaques must be counted from first day of appearance;
viral plaques must be marked as they are counted.
8.2.6 Disposition of Data
All data must be reviewed for consistency and adequacy and
properly documented and stored.
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TABLE 4-1
Monitoring Laboratory Equipment
Item
Monitoring Procedure
1. Balance
c.
d.
Use an analytical balance with a sensitivity
of 1 mg or less at a 10 g load for weighing 2 g
or less. For weighing larger quantities, use a
balance with a sensitivity of 50 mg at a 150 g load,
Check balance monthly with a set of certified
class S weights.
Wipe balance and weights clean after each use.
Protect weights from laboratory humidity and
corrosion.
Contract, on an annual basis, with a qualified
expert for balance maintenance.
2. pH Meter
a. Compensate pH meter for temperature with each use.
b. Date standard buffer solution when first opened,
and check monthly with another pH meter. Discard
buffer solution if the pH is more than +_ 0.1
pH unit from the manufacturer's stated value or
if it is contaminated with microorganisms.
c. Standardize pH meter with two standard buffers
(pH 4.0, 7.0, 10.0) bracketing pH of sample,
before each use.
d. Do not re-use buffer solutions.
e. Contract, on an annual basis, with a qualified
expert for pH meter maintenance.
3. Water Still a.
Drain and clean still at least monthly, according
to instructions from the manufacturer.
Drain and clean distilled water reservoir at least
quarterly.
Monitor distilled water daily for conductance.
Conductivity should not exceed 2 mho/cm at 25° C.
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TABLE 4-1
(Continued)
Monitoring Laboratory Equipment
Item
Monitoring Procedure
4. Water Deionizer
a. Monitor deionized distilled water for conductance
at least daily and continuously when possible.
Conductivity may not exceed 0.1 ymho/cm at
25° C. Monitor for trace metals and other toxic
compounds when necessary (see Table 4-2).
b. Replace cartridges of deionizing resins as
indicated by manufacturer or as indicated by
analytical results.
c. Monitor bacterial counts at exit point of deionizer
unit. Replace cartridges when standard plate count
exceeds 1,000 CFU/mL.
5. Ultraviolet Lamps a. Clean ultraviolet lamps monthly by wiping them with
a soft cloth moistened with ethanol.
b. Test ultraviolet lamps with a light meter
quarterly; if lamps emit less than 80% of their
rated initial output, replace them.
c. Perform spread plate irradiation test quarterly.
For procedure, see Microbiological Methods for
Monitoring the Environment. I. Water and Wastes,
EPA-600/8-78-017, pp. 198-199.
6. Centrifuges
a. Disinfect and clean centrifuges frequently.
b. Check brushes and bearings for wear every six
months.
c. For centrifuges not equipped with built-in
tachometers, check rheostat control against a
tachometer at various loadings every six months to
ensure proper gravitational fields.
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TABLE 4-1
(Continued)
Monitoring Laboratory Equipment
Item
Monitoring Procedure
7. Microscope
a. Allow only trained technicians to use microscopes.
b. Appoint one laboratory worker to be responsible for
the care of the microscopes.
c. Clean optics and stage of microscope after every
use. Use only lens paper for cleaning.
d. Keep microscopes covered when not in use.
e. Establish annual maintenance on contract.
8. Downward Flow
Laminar Hood
a. With an appropriate instrument, check filters in
hood monthly for plugging or obvious dirt
accumulation. Clean or replace filters as needed.
b. Check hood for leaks and for appropriate rate of air
flow every three months.
c. Expose blood agar plates to air flow in hood for
one hour once per month to measure contamination.
d. Every two weeks, remove plug from outlet of hood,
and clean ultra-violet lamps with a soft cloth
moistened with ethanol.
e. Test ultraviolet lamps quarterly with a light
meter. If lamp emits less than 80% of its rated
output, replace lamp.
f. Perform maintenance as directed by the manufacturer.
g. Once a week, measure efficiency of air flow at hood
face with a pressure monitor control device.
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TABLE 4-1
(Continued)
Monitoring Laboratory Equipment
Item
Monitoring Procedure
9. Thermometers and a.
Recording Devices
Check the accuracy of thermometers and temperature
recording instruments, in the monitoring range, at
least annually against an NBS certified thermometer
or equivalent. Thermometer graduations should not
exceed the deviation permitted in the analytical
method. Check mercury columns for breaks.
Record calibration checks in a quality control
record. Mark NBS calibration correction on each
thermometer or on the outside of the incubator,
refrigerator, or freezer containing the thermometer.
Record daily temperature checks on charts, and
retain records for at least six months.
10. Refrigerator
a. Check and record refrigerator temperature daily.
b. clean refrigerator monthly.
c. identify and date all material in refrigerator.
d. Defrost unit, and discard outdated materials in
refrigerator and freezer compartments every six
months.
11. Dispensing Apparatus a.
b.
c.
d.
e.
Check accuracy of delivery from dispensing apparatus
with an NBS class A, graduated cylinder at the start
of each volume change and periodically throughout
extended runs.
Lubricate moving parts of apparatus according to
manufacturer's instructions and at least once per
month.
Correct immediately any leaks, loose connections, or
malfunctions in apparatus.
After dispensing agar or medium, pass a large volume
of hot deionized distilled water through dispenser
to remove traces of agar o»* medium.
At the end of the work day, disassemble parts that
have come in contact with disposed fluid, wash well,
rinse with deionized distilled water, and dry.
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TABLE 4-1
(Continued)
Monitoring Laboratory Equipment
Item Monitoring Procedure
12. Steam Autoclave a. Equip autoclave with steam filter.
b. Record temperature in autoclave continuously with
recording thermometer.
c. Verify that autoclave maintains uniform operating
temperature.
d. Test performance of autoclave at each use with
indicator tape and weekly with maximum-minimum
thermometer and with spore strips or suspensions.
If evidence of contamination occurs, identify and
eliminate cause.
e. Test performance of autoclave weekly with a
thermocouple inserted into simulated worst case
material.
f. Procure semi-annual preventive maintenance
inspections.
13. Hot Air Oven a. Equip oven with a thermometer accurate in
160-180° C range.
b. Each time a hot air oven is used, monitor
performance of oven with temperature indicator tape
and thermometer or temperature recording chart.
c. Monitor sterilization weekly with,spore strips.
14. Freezers a. Check temperatures in freezers continuously with a
recording thermometer.
b. Equip each freezer with a temperature/power alarm
system.
c. Identify and date all materials in freezers.
d. Clean and defrost freezers every six months.
Discard outdated materials.
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TABLE 4-1
(Continued)
Monitoring Laboratory Equipment
Item Monitoring Procedure
15. Incubators a. If partially-submersible glass thermometer is used
(Air/Water-Jacket) to monitor incubator temperature, bulb and stem must
be immersed in water to the mark on stem.
b. Monitor temperatures in incubators continuously with
recording thermometers. Measure temperatures daily
on top and bottom shelves of incubators.
Periodically measure temperatures on all shelves in
use. (For walk-in incubators, expand test points
proportionately.)
c. Equip each incubator with a temperature/power alarm
system.
d. Whenever possible, locate incubators where room
temperature is in the 16-27° C range.
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TABLE 4-2
Standards for Deionized Distilled Water
Parameter
Ideal
Monitoring
Frequency
Limit
Chemical Tests
Conductivity
PH
Total Organic
Carbon
Trace Metal,, Single
Trace Metals, Total
(Cd, Cr, Cu, Ni, Pb, Zn)
Ammonia/Amines
Free chlorine
With each use
Optional
Optional
Optional
Optional
Optional
Optional
0.1 mho/cm
at 25° C
5.5-7.5
1.0 mg/liter
0.05 mg/liter
1.0 mg/liter
0.1 mg/liter
None detectable
by amperometric
titration
Bacteriological Test
Standard Plate Count for
Freshly Dispensed Water
Monthly
1,000 CFU/mL
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TABLE 4-3
Laboratory Ware Maintenance
1. Utensils and Vessels
Use utensils and vessels of non-corrodible and
non-contaminating materials such as Pyrex glass,
stainless steel, and appropriate plastics.
2. Laboratory Ware (Reusable) a.
With each use, examine laboratory ware especially
screw-capped dilution vessels and flasks, for
chipped or broken edges and etched surfaces.
Discard chipped or badly-etched laboratory ware.
b. Inspect laboratory ware after cleansing. Water
should sheet without beading significantly. If
water beads excessively on the cleansed surfaces,
recleanse the laboratory ware.
c. Test laboratory ware for acid or alkaline residues
by adding bromthymol blue indicator to
representative laboratory ware items (see Section
7.1.4).
d. Test laboratory ware for residual detergent (see
Section 7.1.4).
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9. BIBLIOGRAPHY
Biosafety in Microbiological and Biomedical Laboratories (Draft), Centers
for Disease Control, Atlanta, Georgia, and National Institutes of
Health, Bethesda, Maryland, 1983. 90 pp.
Handbook for Sampling and Sample Preservation of Water and Wastewater,
NTIS, PB83-124503, U. S. Environmental Protection Agency, Cincinnati,
Ohio, 1982, pp. 345-355.
Interim Guidelines and Specifications for Preparing Quality Assurance
Project Plans, QAMS-005/80, Office of Monitoring Systems and Quality
Assurance, Office of Research and Development, U. S. Environmental
Protection Agency, Washington, D. C., 1980, 40 pp.
Water and Environmental Technology, 1983 Annual Book of ASTM Standards,
Vol. 11.02, American Society for Testing and Materials, Philadelphia,
Pennsylvania.
Winter, J. A., R. H. Bordner, and P. V. Scarpino. Microbiological
Methods for Monitoring the Environment. I. Water and Wastes,
EPA-600/8-78-071, U. S. Environmental Protection Agency, Cincinnati,
Ohio, 1978.
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CHAPTER 5
VIRUS ADSORPTION-ELUTION (VIRADEL) DISC FILTER PROCEDURES
FOR 'RECOVERING VIRUSES FROM SEWAGES, EFFLUENTS, AND WATERS
Waters that contain chlorine and cannot be processed immediately must be
dechlorlnated immediately upon collection. Immediate dechl on' nation may be
accomplished by placing into the collection vessel 0.8 ml of a 10% solution
of sodium thi'osulfate (NaSO) for each liter of water to be
collected. That quantity of NdgSgOg is sufficient for neutralizing 15
mg of chlorine per liter.
Use aseptic techniques and sterile materials and apparatus only. Sterilize
all contaminated materials before discarding them (see Chapters 2 and 3).
1. ADSORPTION — METHOD ONE
This procedure may be used for volumes of 100 mL to 20 liters for all
sewages and for all heavily polluted waters.
1.1 Preparation
1.1.1 Apparatus and Materials
Unless thumb-screw-drive-clamps are to be used to connect
tubing to equipment, install quick-disconnect connectors on
the ports of all apparatus.
(a) Disc filter holders ~ 47, 90, 142, or 293 mm diameters
(Millipore Corp., or equivalent).
Use only pressure type filter holders. The diameter of
the holder used depends upon the volume and turbidity
of the water tested. Experience with the clogging
- 5-1 -
-------
potential of the volumes of sewage, effluents, or other
waters under study dictates the diameter of the filter
holders used. See Sections 1.2 and 2 for further
guidance.
(b) Virus-adsorbing disc filters -- 0.45-jjm pore size
(Millipore Corp. HA series, or equivalent).
Select diameter of filter appropriate for the disc
filter holder that is used.
(c) Fiberglass prefilters (Millipore Corp., AP15 and AP20,
or equivalents).
(d) Dispensing pressure vessel — 20-liter capacity
(Millipore Corp., or equivalent).
(e) Positive air or nitrogen pressure source equipped with
pressure gauge.
Pressure source, if laboratory air line or pump, must
be equipped with oil filter, if source is capable of
producing high pressure, deliver to pressure vessel and
filter holder no more pressure than recommended by the
filter manufacturer.
(f) Carboy, autoclavable plastic, or flask of a size
sufficient to collect total volume of sample.
(g) pH meter, measuring to an accuracy of at least 0.1 pH
unit, equipped with combination-type electrode (Van
London Co., or equivalent, for electrode only).
(h) Autoclavable inner-braided tubing with metal
quick-disconnect connectors or with thumb-screw-drive-
el amps for connecting tubing to equipment to be used
under pressure.
- 5-2 -
-------
Quick-disconnects can be used only after equipment has
been properly adapted.
(i) Magnetic stirrer and stir bars.
(j) Filling bell attached to inner-braided tubing.
1.1.2 Media and Reagents
(a) Hydrochloric acid (HCl) — 1 M.
Prepare 1 liter of 1 M hydrochloric acid solution.
This solution may be stored at room temperature for
several months.
(b) Sodium hydroxide (NaOH) — 1 M.
Prepare 100 mL of 1 M NaOH. This solution may be
stored at room temperature for several months.
(c) Magnesium chloride (MgCl '61^0) — 1 M.
Prepare 50 mL of 1 M MgCI,, for each liter of sample.
1.2 Procedure (see Figure 5-1 for flow diagram of procedure)
Usually virus-adsorbing filters with diameters of 47 or 90 mm,
coupled with prefilters of appropriate size, are adequate for raw
sewage and primary effluents where volumes of 200 mL or less need to
be filtered. Filters of larger diameter are required for the larger
volumes of secondary and tertiary effluents that must be processed.
1.2.1 Assembly of Apparatus (see Figures 5-2 and 5-3)
Use inner-braided tubing to make all connections between
apparatus to be used under pressure.
(a) Remove top of filter holder C.
(b) With two sets of forceps, place 0.45-jjm virus-adsorbing
filter onto support screen of holder.
- 5-3 -
-------
WATER, SEWAGE, OR EFFLUENT
Water, sewage, or effluent that
contains chlorine must be
collected in vessels that
contain 0.8 ml of a 10%
solution of Na2S203 for each
liter of sample.
On magnetic stirrer, add 1 M
(to final concentration of 0.05 M).
SALTED WATER, SEWAGE, OR EFFLUENT
On magnetic stirrer, adjust pH of
salted water, sewage, or effluent to
3.5 _+ 0.1 with 1 M HC1.
SALTED, pH-ADJUSTED WATER, SEWAGE, OR EFFLUENT
Filter salted, pH-adjusted water,
sewage, or effluent through a filter
stack consisting of AP20 and APIS
fiberglass prefilters and a 0.45-um
virus-adsorbing filter, in that order.
Viruses adsorb to virus "-adsorbing filter,
to prefilters, and to solids trapped on
prefilters.
VIRUS-BEARING FILTER, PREFILTERS, AND SOLIDS
Place buffered 3% beef extract (BE)
(pH 9) onto filter stack, allow 30
minutes contact, and force BE through
filters with positive pressure.
ELUATE
Assay for viruses
(see Chapter 9).
Concentrate viruses by organic
flocculation procedure of
Katzenelson (see Figure 5-9).
•*
Figure 5-1. Flow Diagram of Method for Recovering Viruses from Small Volumes
(100 mL to 20 Liters) of Water, Sewage, or Effluent.
- 5-4 -
-------
A - Pressure Source
(Compressedl air or l\h)
A, - Pressure Regulator
AA - Laboratory Pressure System
B - Pressure Vessel
B, - Inlet Port
en
i
en
B2
B3
C
C,
C2
C3
D
E
- Vent/Relief Valve
- Outlet Port
- Filter Holder
- Inlet Port
- Vent/Relief Valve
- Outlet Port
- Filling Bell
- Receiving Vessel
Figure 5-2. Schematic Representation of Apparatus for Recovering Viruses
by the Virus Adsorption-Elution (VIRADEL) Disc Filter
Procedure for Small Volume Filtrations (see Figure 5-3
for Photographic Representation of Apparatus).
-------
I
01
Figure 5-3. Photographic Representation of Apparatus for Recovering Viruses
by the Virus-Adsorption-Elution (VIRADEL) Disc Filter
Procedure for Small Volume Filtrations (see Figure 5-2
for Annotated Schematic Representation of Apparatus).
-------
(c) With two pairs of forceps, place AP 15 prefliter on top
of 0.45-um filter.
(d) With two pairs of forceps, place AP 20 prefilter on top
of AP 15 prefilter.
(e) Replace and tighten down top of filter holder C.
(f) Connect positive pressure source A or AA to inlet port
B, of 20-liter pressure vessel B.
(g) Connect outlet port BO of pressure vessel B to inlet
port C-, of filter holder C.
(h) Place filling bell D, with inner-braided tubing
attached, over opening of flask or carboy (E) of a size
sufficient to.collect the total volume of sample.
(i) Connect free end of tube on the filling bell to outlet
port Co of filter holder C.
1.2.2 Salt Supplementation of Sample.
(a) Place stir bar into container holding sample.
(b) Place sample container on magnetic stirrer, and stir at
speed sufficient to develop vortex.
(c) Add sufficient quantity of 1 M MgCl2 to bring the
concentration of MgClp in the sample to 0.05 M.
1.2.3 Adjustment of pH of Sample.
Optimal conditions of pH vary for concentrating different
viruses, especially viruses from different taxonomic groups.
Conditions that favor recovery of enteroviruses are described
below.
(a) Place pH electrode into salted water sample.
(b) Add sufficient 1 M HCl to bring pH of salted sample to
3.5 + 0.1.
- 5-7 -
-------
Rapid mixing of acid into sample is important because
slow mixing may result in pH levels sufficiently low in
parts of the sample to inactivate viruses.
(c) Turn off magnetic stirrer.
(d) Remove pH electrode from sample.
1.2.4 Filtration of Salted, pH-adjusted Sample (from Section 1.2.3,
Step [b]).
(a) Remove top from pressure vessel B.
(b) Pour salted pH-adjusted sample into pressure vessel B.
To prevent transfer of stir bar into pressure vessel,
hold another stir bar or magnet underneath flask when
decanting sample.
(c) Replace top on pressure vessel B and tighten down.
(d) Wrap vent/relief valve C« on top of filter holder C
with disinfectant-soaked gauze, and open valve about
one-half turn.
(e) Apply pressure sufficient to purge trapped air from
filter holder C.
(f) Close vent/relief valve C2 as soon as sample begins
to flow from valve.
(g) Wipe up spilled sample with laboratory disinfectant.
(h) Increase pressure sufficiently to force sample through
the filter (usually 0.4-1.5 kg/cm2).
(i) When all of sample has passed through filters, turn off
pressure source A or AA.
(j) Wrap vent/relief valve B? with disinfectant-soaked
gauze, and open valve to relieve pressure in pressure
vessel B.
- 5-8 -
-------
(k) When pressure is relieved, close vent/relief valve B,,.
(1) Discard filtrate.
(m) Elute viruses from filters immediately as described in
Section 3.1.
2. ADSORPTION — METHOD TWO
This method is recommended for volumes larger than 20 liters but not
larger than 400 liters (e.g., tertiary effluents, surface waters, ground
waters, and tap waters). The usefulness of this method is limited by the
clarity of the water that is filtered. Prefilters must be replaced as
they clog. More than ten changes of prefilters are generally impractical.
Usually, 20 liters or less of river or ocean water clog a prefilter with
a diameter of 293 mm. For chlorinated waters that contain sufficient
solids to require elution, do not use this method. Instead, use the
Viradel Cartridge Filter Procedure Method Two in Chapter 6.
2.1 Preparation
2.1.1 Apparatus and Materials
Unless thumb-screw-drive-clamps are to be used to connect
tubing to equipment, install quick-disconnect connectors on
the ports of all apparatus except on the additive pumps.
Provide physical support as necessary for equipment that is
not free-standing.
(a) Disc filter holders — 142 and 293 mm diameter
(Millipore Corp., or equivalent).
(b) Virus-adsorbing disc filters for 142 mm filter holder
— 0.45-)jm pore size (Millipore Corp., HA series, or
equivalent).
(c) Fiberglass prefilters for 293 mm filter holder
(Millipore APIS and AP20, or equivalents).
- 5-9 -
-------
(d) Dispensing pressure vessel -- 20-liter capacity
(Millipore Corp., or equivalent).
(e) Positive pressure source equipped with pressure gauge.
Pressure source, if laboratory air line or pump, must
be equipped with oil filter. If source is capable of
producing high pressure, deliver to pressure vessel and
filter holder no more pressure than recommended by the
filter manufacturer.
(f) Plastic-coated drum(s) — 200-liter capacity, or other
container of size suitable to hold sample if sample is
not pumped directly from source.
(g) Sterilizable self-priming water pump that delivers
approximately 25-50 liters per minute.
Pump is not needed if sampled water is under pressure,
e.g., tap water.
(h) Carboy, autoclavable plastic with nipple on bottom
fitted with tube clamped to a dispensing Y (clamp tube
closed between nipple and Y) -- 20-liter capacity.
If the water at the sampling site is to be drawn
directly from a pressurized source and is to be
dechlorinated, then two similarly fitted carboys are
needed, otherwise only one carboy is needed.
(1) Fluid proportioner consisting of fluid-driven motor
with four additive pumps (Johanson and Son Machine
Corp., Model M 14 Q with one P-562 and one P-750
additive pump affixed to each side of the fluid-driven
motor, or equivalent).
- 5-10 -
-------
Assemble fluid proporti'oner, and connect tubing In
accordance with manufacturer's instructions.
(j) Mixing chamber (Johanson and Son Machine Corp., C-SS, or
equivalent).
(k) pH meter, measuring to an accuracy of at least 0.1 pH
unit, equipped with a combination-type electrode (Van
London Co., or equivalent, for electrode only).
(1) Tee, stainless steel, with three female NPT* ports.
Equip center port with pH electrode in-line adapter (Van
London Co., or equivalent, for adapter only).
(m) Autoclavable inner-braided tubing with metal
quick-disconnect connectors or with thumb-screw-drive-
clamps for connecting tubing to equipment to be used
under pressure.
Quick-disconnect connectors can be used only after
equipment has been properly adapted<
(n) Filling bell attached to inner-braided tubing.
(o) Magnetic stirrer and stir bars.
(p) Sterile aluminum foil.
(q) Water meter (Badger Meter Inc., or equivalent).
2J.2 Media and Reagents
(a) Hydrochloric acid (HC1) — 0.12 M and 12 M
(concentrated) solutions.
Prepare 100 mL of 0.12 M HC1.
* National Pipe Thread
- 5-11 -
-------
(b) Sodium thiosulfate (Na2S203*5H20) — 40%
stock solution (with respect to Na^Og'ShLO).
Prepare 50 mL of 40% (w/v) stock solution for each 100
liters of water to be processed. Prepare one liter of
Na^SpOg solution by dissolving 400 g of Na^SpOp' 5H.2Ł
in 500 mL of deionized distilled water and bringing
final volume of solution to one liter with deionized
distilled water. If lesser quantities ofNa^O-
are needed, lesser quantities may be prepared. Sodium
thiosulfate is used for dechlorinating waters that
cannot be dechlorinated except immediately prior to test
procedure (e.g., tap water tested directly at source).
For dechlorinating all other waters, see page 5-1.
(c) Magnesium chloride (Mgd2*6H20) — 5 M stock
solution.
Prepare 1 liter of solution for each 100 liters of water
to be processed.
(d) Tween 80 — 0.1% (v/v) prepared in deionized distilled
water.
Prepare 6 liters of 0.1 % Tween 80.
2.2 Procedure (see Figure 5-4 for flow diagram of procedure)
Usually, prefliters with diameters of 293 mm and virus-adsorbing
filters with diameters of 142 mm are appropriate for volumes greater
than 20 liters.
2.2.1 Preparation and Implementation
It is usually convenient to sterilize each piece of apparatus
and equipment one or more days before it is used (see Chapter
3). It is convenient to sterilize apparatus in small units
- 5-12 -
-------
WATER, SEWAGE, OR EFFLUENT
Filter sample through AP20 and APIS
prefllters. Replace and save clogged
pre filters.
PREFI1.TERED VIATFR, SEWAGE, OR EFFLUEHT
nechlorinate sample, if
necessary. To dechlorinate,
flow 0.03 H Ha2S203
(to final concentration of
0.0003 M) continuously Into
sample.
By continuous flow,
add to sample sufficient
acidified (with 12 H HCl)
1 H MgCl2 to bring
pH of sample to 3.5 ^ 0.1
and concentration of MgClj
1n sample to 0.05 II.
SOLIDS-BEARING AP20 PREFILTER
Collect solids from
AP20 prefliter.
VIRUS-BEARING SOLIDS
H1x IDS buffered
beef extract (BE)
(pH 7.0 + 0.1) with
solids for 30 minutes,
centrifuge, and
discard solids.
Viruses elate
from solids.
BEEF EXTRACT ELUATE
Assay for viruses
(See Chapter 9).
PPEFILTERED, SALTED, pH-ADJUSTED WATER, SEWAGE,
OR FFFLUEHT '
Filter sample through
0.45-)im virus-adsorbing
filter. Maintain pH 3.5
+_ 0.1 by readjusting
additive feed pumps
appropriately.
VIRUS-BEARIMG FILTER
Place buffered 3S beef extract (BE)
(pH 9+_0.1) on virus-bearing
filter, allow contact between BE
and filter for 30 ninutes, and force
BE through filter.
Viruses elute. from filter.
BEFF EXTRACT F.LUATE
Concentrate viruses by organic
flocculation procedure of Katzenelson
(See Figure 5-9).
Concentrate viruses
by organic floccula-
tion technique of
Katzenelson (see
Figure 5-9).
Assay for viruses
(See Chapter 9).
Figure 5-4. Flow Diagram of Method for Recovering Viruses from Large Volumes
(More than 20 Liters) of Hater, Sewage, or Effluents.
- 5-13 -
-------
when sterilization Is accomplished by steam or ethylene
oxide. However, it is advisable to assemble and connect
units of apparatus that are to be sterilized by
chlorination. The interconnected apparatus can be
disassembled after the ch1orination procedure is completed,
the ports covered with aluminum foil, and the units stored
until used.
(a) Assembly of apparatus (see Figures 5-5 and 5-6)
Use inner-braided tubing to make all connections for
apparatus to be used under pressure. To simplify
procedures and maintain sterility, the apparatus is
totally assembled at this time although sections of the
apparatus will need to be disassembled and reassembled
later.
(a.l) If sample is under pressure (e.g., tap water),
connect water source H to inlet port I, of
filter holder I (293 mm). If sample is not
under pressure, connect sample source to inlet
port HH, of self-priming water pump HH, and
connect outlet port HhL of pump HH to inlet
port I, of filter holder I.
(a.2) Connect outlet port I3 of filter holder I to
inlet port J_ of fluid proportioner J.
(a.3) Connect outlet port J, of fluid proportioner J
to inlet port M-, of mixing chamber M.
Mixing chamber must be supported to prevent it
from falling.
- 5-14 -
-------
H - Pressurized Water Source (Tap)
HH - Self-Priming Water Pump
(Connected to water source)
HH,- - Inlet Port
HH2 - Outlet Port
i
h
ii
Is
J
- Filter Holder (Prefilter)
- Inlet Port
- Vent/Relief Valve
- Outlet Port
- Fluid Proportioner
A
J,a
J,b
J4
K
L
M
M,
Ma
N
0
0,
en
i
en
i
. Chemical Feed (P750)
Additive Pumps (Larger)
-"Chlorine Neutralizer (P562)
.Additive Pumps (Smaller).
- Inlet Port
- Hose Adapter Body
- Outlet Port
- Carboy (Acid-salt solution)
- Carboy (Chlorine neutralizer)
- Mixing Chamber
- Inlet Port
- Outlet Port
- Pipe Tee
- pH Meter
- pH Electrode
- pH Electrode In-Line Adapter
- Filter Holder
(Virus-adsorbing filter)
- Inlet Port
- Vent/Relief Valve
- Outlet Port
- Water Meter
- Inlet Port
- Outlet Port
Figure 5-5. Schematic Representation of Apparatus for Recovering. Viruses
by the Virus Adsorption-Elution (VIRADEL) Disc Filter Procedure.
for Large Volume Filtrations (See Figure 5-6 for Photographic
Representation of Apparatus).
-------
Figure 5-6. Photographic Representation of Apparatus for Recovering Viruses
by the Virus Adsorption-Elution (VIRADEL) Disc Filter Procedure
for Large Volume Filtrations (See Figure 5-5 for Annotated
Schematic Representation of Apparatus).
-------
(a.4) Connect outlet port M2 of mixing chamber M to
one arm of pipe tee N.
Support pipe tee N to protect electrode, if
:. '.-'^'^C'-'- "''"'•" "-"'-.-^f-"'-'>.r^.-..... •-.'•
necessary. ' . ' t . .
(a.5) Lock pH electrode 0-, into pH electrode in-line
adapter 0? in center post of pipe tee N.
Same pH electrode (after sterilization) that is
used to adjust pH in Step (d.4) may be used.
(a.6) Connect other arm of pipe tee N to inlet port
P, of filter holder P.
(a.7) Connect outlet port P., of filter holder P to
. - ^
inlet port' Q, of water meter Q.
(a.8) Connect outlet port Q~ of water meter Q to
discard.
(b) Treatment of prefilters with Tween 80 to prevent
adsorption of viruses (see Figures 5-7 and 5-8; also
see Figures 5-5 and, 5-6).
Treat APIS and AP20 prefilters separately. AP15 and
AP20 prefilters cannot be readily distinguished one
from the other.
(b.l) Remove top of filter holder I.
(b.2) With two sets of forceps, place AP15 prefilters
onto support screen of filter holder I.
Up to 10 prefilters may be stacked in filter
holder I for treatment. The number of
prefilters stacked is the number that experience
suggests will be needed to filter the waters to
( be tested. In the absence of experience, treat
- 5-17 -
-------
OR
T - Pressure Source (Compressed air or
T, - Pressure Regulator
TT - Laboratory .Pressure System
U - Pressure Vessel
U, - Inlet Port
U2 - Vent/Relief Valve
U3 - Outlet Port
- Filter Holder
- Inlet Port
- Vent/Relief Valve
- Outlet Port
oo
i
Figure 5-7. Schematic Representation of Apparatus for Treatment of Prefliters
with Tween 80 to Prevent Adsorption of Viruses to the PrefiHers
in the Virus Adsorption-Elution (VIRADEL) Disc Filter Procedure
for Large Volume Filtrations (See Figure 5-8 for Photographic
Representation of Apparatus).
-------
I
tn
ID
Figure 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
(See Figure 5-7 for Annotated Schematic Representation
-------
five prefliters of each type for relatively
clear waters and 10 prefliters of each type for
more turbid waters. Unused Tween-treated
prefliters may be stored aseptically at 4° C
for up to two weeks.
(b.3) Replace and tighten down top of filter holder I.
(b.4) Open vent/relief valve I?.
(b.5) Disconnect tube from inlet port I, of filter
holder I.
Protect sterility of exposed tube.
(b,6) With a new length of tubing, connect inlet port
I, of filter holder I to outlet port IL of
20-liter pressure vessel u.
(b.7) Connect pressure source T or TT to inlet port
U, of pressure vessel U.
(b.8) Remove top of pressure vessel U.
(b.9) Pour 2 liters of 0.1% Tween 80 into pressure
vessel U.
(b.10) Replace top on pressure vessel U and tighten
down.
Check vent/relief valve ug on pressure vessel
U to be certain it is closed.
(b.ll) Disconnect tube at inlet port J,, of fluid
proportioner J, and place end of tube into
6-liter flask V.
(b.12) Cover inlet port J2 of fluid proportioner J
with sterile aluminum foil.
- 5-20 -
-------
p
(b.13) Apply pressure (T or TT) (about 0.4 kg/cm)
sufficient to force Tween 80 through prefilters.
(b.14) Close vent/relief valve !„ on filter holder I
as soon as Tween 80 flows through vent, and
allow all of the Tween 80 to flow through the
prefilters.
(b.15) Turn off pressure source (T or TT)
(b.16) Relieve pressure in pressure vessel U by opening
vent/relief valve IL.
(b.17) Remove tube from flask V, discard Tween 80', and
return tube to flask V.
(b.18) Remove top of pressure vessel U.
(b.19) Pour 4 liters of deionized distilled water into
pressure vessel U.
(b.20) Replace and tighten down top of pressure vessel
U.
(b.21) Close vent/relief valve IL.
(b.22) Open vent/relief valve I~ on filter holder I.
2
(b.23) Apply pressure (about 0.4 kg/cm ) sufficient
to force water through prefilters (prefilter
rinse).
(b.24) Close vent/relief valve !„ on filter holder I
as soon as water flows through vent, and allow
all of the deionized distilled water to flow
through the prefilters.
(b.25) Turn off pressure source (T or TT).
(b.26) Relieve pressure in pressure vessel U by opening
vent/relief valve IL.
- 5-21 -
-------
(b.27) Discard rinse water, and replace tube from
outlet port I_ of filter holder I into same
flask.
(b.28) Remove top of filter holder I.
(b.29) With two sets of forceps, remove the AP15
prefilters from filter holder I, and place the
prefilters on aluminum foil.
(b.30) Cover the stack of prefilters with another piece
of fo i1.
(b.31) Repeat steps (b.2) through (b.10) and (b.13)
through (b.30) with AP20 prefilters.
(b.32) Remove aluminum foil from inlet port J? of
fluid proportioner J.
(b.33) Remove tube from 6-liter flask, and connect to
inlet port J~ of fluid proportioner J.
(b.34) With two sets of forceps, remove top AP15
prefilter from stack.
(b.35) Place the AP15 prefilter onto support screen of
filter holder I.
(b.36) With two sets of forceps, remove top AP20
prefilter from stack, and lay the AP20 prefilter
on top of AP15 prefilter.
(b.37) Replace and tighten down top of filter holder I.
(b.38) Disconnect tube from outlet port Uo of
pressure vessel U, and cover tube end with
aluminum foil.
(b.39) Disconnect tube from inlet port I, on filter
holder I.
- 5-22 -
-------
(b.40) Reconnect tube from pressure source H or HH^
to inlet port I, on filter holder I.
(c) Preparation of salt supplement
Preparation of sufficient salt supplement for 400
liters of processed water is described below. If less
water is to be processed, proportionately less salt
supplement needs to be prepared.
(c.l) Remove cover from 20-liter carboy K.
(c.2) Pour 8 liters of deionized distilled water into
carboy K.
(c.3) Add 4 liters of 5 M MgCl2 solution to the
deionized distilled water in carboy K.
(c.4) Replace cover loosely on carboy K.
(d) Preparation of acid for adjustment of pH
(d.l) Pour 380 ml of test water into a 600-mL beaker.
(d.2) Place stir bar into test water.
(d.3) Place beaker on magnetic stirrer, and stir at
speed sufficient to develop vortex in test water.
(d.4) Place pH electrode into test water.
pH meter must be standardized before it is used.
(d.5) Add sufficient 0.12 M HC1 to test water to
obtain pH 3.5 +0.1.
(d.6) Record volume of 0.12 M HCl used.
(d.7) Add to salt solution from Step (c.3) above a
volume of 12 M HCl equal to 11 times the
quantity of 0.12 M HCl needed to produce the
required pH in the 380-mL volume of test water.
- 5-23 -
-------
(d.8) Bring acid-salt solution to 20 liters with
deionized distilled water, and mix solution well.
(e) Preparation of Na?S?0^ solution for dechlorination
Step (e) applies only to chlorinated waters processed
directly from a source (e.g., tap water). All
chlorinated test waters obtained from sources outside
of the processing facility must be dechlorinated
immediately when the samples are obtained (see page
5-1). Preparation of sufficient NagSgOp for
dechlorinating 400 liters of processed water is
described below. If less water is to be processed,
proportionately less Na^SgO^ needs to be prepared.
(e.l) Remove cover from 20-liter carboy L.
(e.2) Pour 10 liters of deionized distilled water into
carboy L.
(e.3) Add 186 mL of 40% Na2$203 solution to the
deionized distilled water in carboy L to give a
final molarity of 0.03, and mix solution well.
(e.4) Replace cover loosely on carboy L.
(f) Fluid proportioner
(f.l) Connect a long length of tubing to each end of
dispensing Y on 20-liter carboy K that contains
the acid-salt solution prepared in Step (d.7)
above.
Tubing is already in place if additive pumps are
sterilized with chlorine (see Section 2.2.1).
In this instance, disconnect tubing from bottom
of additive pump J, and continue with Step
(f.2).
- 5-24 -
-------
(f.2) Remove cover from top of carboy K.
(f.3) Place free end of each tube into mouth of carboy
K.
(f.4) Release pinch clamp, and allow acid-salt
solution to flow into tubes.
(f.5) Remove tubes from mouth of carboy K, and insert
tubes into the inlet (bottom) ports of larger
additive pumps J, .
i a
Allow acid-salt solution to flow freely into
tubing, but manipulate tubes to prevent
overflow.
(f.6) Replace cover loosely on carboy K.
(f.7) Adjust the calibration on the metering rod for
each pump J-, to a setting of 3.2.
This calibration equals delivery rate of 1 part
of acid-salt solution to each 19 parts of test
water. If dechlorination is not necessary,
leave the ports of the two remaining additive
pumps J-., covered (see Section 2.2.1), and go
to Step (f.15).
If pressurized source is used, water should
first be run for a length of time sufficient to
cleanse spigot.
(f.8) Connect a long length of tubing to each end of
dispensing Y on 20-liter carboy L that contains
. the 0.03 M Na^SpO- solution prepared in
Step (e) above.
- 5-25 -
-------
Tubing may already be In place if pumps are
sterilized with chlorine (see Section 2.2.1).
In this Instance, disconnect tubing from bottom
of additive pumps, and continue with Step (f.9).
(f.9) Remove cover from top of carboy L.
(f.10) Place free end of each tube into mouth of
carboy L.
(f.ll) Release pinch clamp, and allow NapSpCU
solution to flow into tubes.
(f.12) Remove the tubes from mouth of carboy L, and
insert tubes into the inlet (bottom) ports of
smaller additive pumps J,, .
Allow Na?S_gO, solution to flow freely into
tubes, but manipulate tubes to prevent overflow.
(f.13) Replace cover loosely on carboy L.
(f.14) Adjust the calibration on the metering rod for
each additive pump J-,, to a 1.3 setting.
This calibration equals delivery rate of 1 part
of 0.03 M NapSpOg solution to each 99
parts of test water.
(f.15) Disconnect tube from inlet port M, of mixing
chamber M, and connect tube to discard.
(f.16) To remove air from tubes, prime pumps by
hand-operating metering rods in a reciprocating
motion.
(f.17) Reconnect tube from outlet port J. of fluid
proportioner J to inlet port M-, of mixing
chamber M.
- 5-26 -
-------
2.2.2 Filtration of Sample
(a) Make initial reading on water meter Q, and record
reading.
(b) Remove top of filter holder P.
(c) With two sets of forceps, place 0.45 pm virus-adsorbing
filter onto support screen of holder.
(d) Replace and tighten down top of filter holder P.
(e) Open vent/relief valves l? and P? on filter holders
I and P.
(f) Open pressurized water source H or start water pump HH
and purge trapped air from filter holders I and P.
(g) Close vent/relief valves l? and P? on filter
holders I and P as soon as sample begins to flow from
valves.
(h) Wipe up spilled sample with laboratory disinfectant.
(i) Read pH meter 0 to ascertain that proper pH is achieved.
Check meter periodically to be certain that proper pH
is maintained. If pH readjustment is necessary,
appropriately alter settings on metering rods for
additive pumps P-750.
(j) When appropriate volume has been filtered, or if flow
rate becomes significantly reduced, turn off pressure
either at pressurized source H or at water pump HH.
(k) Open vent/relief valves I? and-P_ on filter holders
I and P.
(1) Disconnect tube from pressurized source H or water pump
HH, and connect free end of tube to positive air or
nitrogen pressure source.
- 5-27 -
-------
(m) Close vent/relief valve I2 on filter holder I.
(n) Apply pressure sufficient to force remaining sample
water from filter holder I.
(o) Turn off pressure at positive air or nitrogen pressure
source.
(p) Open vent/relief valve !„ on filter holder I.
(q) Disconnect hose from positive pressure source, and
reconnect to pressurized source H or water pump HH.
(r) Remove top of filter holder I.
(s) Replace clogged prefilters with new prefilters as
described in Steps (b.34) through (b.37).
If appropriate volume of sample has been filtered, do
not insert new filter into filter holder.
(t) Place each set of clogged prefilters on aluminum foil,
and cover.
See Section 3.2 for processing solids on clogged
prefilters.
If appropriate volume of sample has been filtered,
proceed to Step (cc).
(u) Close vent/relief valve !„ on filter holder I.
(v) Continue filtration procedure.
Bleed air from both filter holders I and P at
vent/relief valves Ig and P^ each time apparatus is
opened to replace prefilters. As many changes of
prefilters should be made as are necessary to process
entire sample.
Steps (q) through (t) may be completed for each set of
prefilters as filtration procedure continues.
- 5-28 -
-------
(w) 'Uncover one set .of';pne|jTters.
(x) With spatula, ;.ser'a;jj| .so ji dsif rom top pref liter (AP20).
(y) Place solids in a''tared,beaker, and cover mouth of
beaker with aluminum.foil. : -, "
(z) Maintain, beaker at 4° C.,,,. •'•:•'•'..
.See Section 3.2 for .process i hg- solids.
(aa) After requ i red ;vol ume; of water-1
has been filtered, turn
off pressure either at pressurized source H or at water
pump HH. • • • '
(bb) Open vent/relief .valves, !„ and .-P- on filter holders
(cc) Disconnect at pipe- tee ,N the tube leading to inlet port
P, of filter holder -P, , and connect free end of tube
to positive pressure. sp.urce.
(dd) Close vent/re,lief valve P? on filter holder P.
(ee). Apply •. pressure sufficient to force remaining sample
-water from filter holder P. '
(ff) Make final reading on- water;meter.
Subtract initial reading from final reading to
determine total. voj ume filtered. Subtract volume of
acid-salt solution and, if used, volume of
Na0S^Oo solution from 'total volume filtered to
--••' -^"7"t—-O ' - . t -",-,"/-' •'",-" •• •l,~:-~:.--~ . • • "- ' ...-•_ . ._._
determine vol ume .bf^water sampled .
(gg) .turn off pressure at .positive pressure source.
(hh) Open vent/relief valve ,pp on filter holder P.
(ii) .. Disconnect tube from outlet port P_ of filter holder
' - . • ' '-",-.'• ~ - .'-•-• *5
P, and replace with 'tube connected to filling bell.
(jj) Elute viruses from viru.$radsbrb;tng filter as described
in Section 3. •, ; ;
' , .'.' • - 5-29 -
-------
3. ELUTIOM AMD RECOHCENTRATION
3.1 Procedure for Eluting Viruses from Filters (see Figures 5-2 and 5-3,
and Figures 5-5 and 5-6)
3.1.1 Apparatus and Materials
(a) Positive pressure source equipped with pressure gauge.
Gauge necessary only if pressure source is capable of
producing pressures exceeding tolerances of equipment.
Pressure source, if laboratory air line or pump, must
be equipped with an oil filter. If source is capable
of producing high pressure, deliver to pressure vessel
and filter holder no more pressure than recommended by
manufacturer.
(b) pH meter, measuring to an accuracy of at least 0.1 pH
unit, equipped with a combination-type electrode.
(c) Autoclavable inner-braided tubing fitted with metal
quick-disconnect connectors or with thumb-screw-drive-
clamps for connecting tubing to equipment.
(d) Magnetic stirrer and stir bars.
3.1.2 Media and Reagents
(a) Sodium hydroxide (NaOH) — 1 M.
Prepare 500 ml of 1 H NaOH. This solution may be
stored for several months at room temperature.
(b) Glycine.
(c) Beef extract powder (Grand Island Biological Co., or
equivalent).
Prepare buffered 3% beef extract by dissolving 60 gm of
beef extract powder and 7.5 g of glycine (final glycine
concentration = 0.05 M) in 2 liters of deionized
- 5-30 -
-------
distilled water. Autoclave beef extract solution, and
adjust pH to 9 with 1 M NaOH.
3.1.3 Procedure
(a) Place filling bell attached to outlet port of filter
holder C (Method 1, Figures 5-2 and 5-3) or P
(Method 2, Figures 5-5 and 5-6) on receiving flask.
To prevent toppling, it may be necessary to support
flask.
(b) Disconnect tube from inlet port of filter holder C
(Method 1, Figures 5-2 and 5-3) or P (Method 2, Figures
5-5 and 5-6).
(c) Open vent/relief valve on filter holder.
(d) Pour into inlet port of filter holder 0.45 ml of beef
extract (pH 9) for each square cm of effective filter
area.
Determine total effective filter area from
manufacturer's specifications. Volume of beef extract
thus needed for 142 mm filter is 44 mL.
(e) Close vent/relief valve on top of filter holder.
(f) Connect tube to inlet port of filter holder.
(g) Allow beef extract to remain in contact with filter(s)
for 30 minutes.
(h) Apply pressure sufficient to force beef extract through
filter(s).
Lower receiving flask and tilt filter holder to permit
complete evacuation of buffered 3% beef extract from
filter(s).
- 5-31 -
-------
(i) Turn off pressure at source.
(j) Open vent/relief valve on filter holder.
(k) Unless beef extract eluate is reconcentrated or assayed
for viruses immediately, refrigerate eluate immediately
at 4° C, and maintain at that temperature until
eluate is reconcentrated or is assayed for viruses.
If reconcentration or assay for viruses cannot be
undertaken within eight hours, store eluate immediately
at -70° C. The number of cell cultures necessary for
the viral assay may be reduced by reconcentrating the
viruses in the beef extract by the organic flpeculation
procedure of Katzenelson (see section 3.3).
3.2 Procedure for Processing Solids
Often more viruses are recovered from the solids in waters than from
the waters from which the solids are obtained.
3.2.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) Membrane filter apparatus for sterilization — 47 mm
diameter filter holder with 30-mL slip tip syringe
(Millipore Corp., Swinnex filter No. SX0004700, or
equivalent for filter holder only).
(c) Membrane filters, 47 mm diameter — 5-, 1.2-, 0.65-,
and 0.45-nm pore sizes (Millipore Corp., HA series, or
equivalent).
Place filter with 0.45-^m pore size on support screen
of Swinnex filter holder, and stack the remaining
filters on top in order of increasing pore size.
- 5-32 -
-------
Filters stacked in tandem as described tend to clog
more slowly when turbid material is filtered through
them.
(d) Refrigerated centrifuge capable of attaining
2,500 x _g.
3.2.2 Media and Reagents
(a) Disodium hydrogen phosphate (Na^HPCLVhLO).
(b) Citric acid.
(c) Beef extract powder (Gibco, or equivalent).
Prepare buffered (pH 7.0) 10% beef extract by
dissolving 10 g beef extract powder, 1.34 g
Na^HPO.VHpO, and 0.12 g citric acid in 100 ml
of deionized distilled water.
3.2.3 Procedure
(a) Weigh beaker that contains solids scraped from
prefilters (from Section 2.2.2, Step [z]).
Calculate weight of solids by subtracting tare weight
of beaker from weight of beaker with solids.
(b) Place stir bar into beaker.
(c) Measure into beaker 3 ml of 10% buffered beef extract
for every gram of solids.
(d) Place beaker on magnetic stirrer, and stir for 30
minutes.
Viruses elute from solids.
(e) Pour suspension of solids and buffered beef extract
eluate into 250-mL centrifuge bottle.
- 5-33 -
-------
Glass centrifuge bottles may not be able to withstand g
force that will be applied. To prevent transfer of
stir bar into centrifuge bottle, hold another stir bar
or magnet underneath beaker when decanting solids.
(f) Centrifuge suspension for 30 minutes at approximately
2,500 x Ł.
(g) Decant buffered beef extract eluate into beaker of
appropriate size, and discard solids.
The number of cell cultures necessary for the viral
assay may be reduced by reconcentrating the viruses in
the beef extract by the organic flocculation procedure
of Katzenelson. If viruses in eluate are to be
reconcentrated, proceed to Step (h). If
reconcentration is not required, proceed to Step (i).
(h) Add 7 mL of deionized distilled water to each 3 mL of
eluate if reconcentration is required, and proceed
according to Section 3.3.
(i) Load eluate into 30-mL syringe.
(j) Place tip of syringe into filter holder, and place
filter holder on a 125-mL receiving flask.
(k) Force eluate through filters into 125-mL receiving
flask.
Take care not to put pressure on receiving flask. If
filter clogs, invert filter, draw remaining fluid from
top _gf c|ogged jj Her jmto_syri nge, and rep 1 ace f i 1 ter
holder and filters.
- 5-34 -
-------
BEEF EXTRACT (BE) ELUATE (3% OR 10% BE)
If concentration of BE is 10%, reduce
it to 3% with deionized distilled
water. If volume of BE is less
than 100 ml, add sufficient 3% BE
to bring total volume to 100 ml.
3% BEEF EXTRACT ELUATE (100 ml)
On magnetic stirrer, add 1 M HCl until
pH of 3% BE reaches 3.5 j^ 0.1.
Precipitate forms.
Stir for 30 minutes longer.
Maintain pH 3.5 +_ 0.1 with 1 M HCl
and 1 M NaOH.
SUSPENDED BEEF EXTRACT PRECIPITATE
Centrifuge precipitated BE suspension
at 4° C for 15 minutes at 2,500 x _g_.
Record volume of supernate. Discard
supernate.
VIRUS-BEARING PRECIPITATE
Add to precipitate 5 mL of 0.15 M
Na2HP04'7H20 for each
100 mL of supernate discarded.
Stir on magnetic stirrer until
precipitate dissolves.
Adjust pH of dissolved precipitate
to 7.0-7.5 with 1 M HCl or 1 M NaOH.
DISSOLVED PRECIPITATE
Assay for viruses
(See Chapter 9.)
Figure 5-9. Flow Diagram of Reconcentration Procedure (Organic
Flocculation Procedure of Katzenelson).
- 5-35 -
-------
Steps (i) thru (k) may be repeated as often as
necessary to filter entire volume of eluate.
(1) Refrigerate eluate immediately at 4° C and maintain
at that temperature until eluate is assayed for
viruses.
If assay for viruses cannot be undertaken within eight
hours, store eluate immediately at -70° C.
3.3 Organic Flocculation Concentration Procedure of Katzenelson (see
Figure 5-9 for flow diagram of procedure).
It is preferable to assay eluted viruses in the beef extract eluate
without further concentrating them, because some loss of viruses
may occur in concentration. However, the numbers of cell cultures
needed for assays may be reduced by further concentrating the
viruses.
3.3.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) pH meter, measuring to an accuracy of at least 0.1 pH
unit, equipped with a combination-type electrode.
(c) Refrigerated centrifuge capable of attaining 2,500 x Ł,
3.3.2 Media and Reagents
(a) Disodium hydrogen phosphate (Na2HP04*7H20) --
0.15 M.
(b) Hydrochloric acid (HC1) — 1 M.
(c) Sodium hydroxide (NaOH) — 1 M.
3.3.3 Procedure
(a) If concentration of beef extract in eluate is 10%,
reduce it to 3% with deionized distilled water; if
volume of beef extract eluate is less than 100 mL, add
- 5-36 -
-------
sufficient 3% beef extract to bring total volume to
TOO ml.
(b) Place stir bar in flask that contains beef extract
eluate.
(c) Place flask that contains beef extract eluate on
magnetic stirrer, and stir at a speed sufficient to
develop vortex.
To minimize foaming (which may inactivate viruses), do
not mix faster than necessary to develop vortex.
(d) Insert pH electrode into beef extract eluate.
(e) Add 1 M HC1 to flask slowly until pH of beef extract
reaches 3.5 _+ 0.1.
A precipitate will form. If pH is accidentally
reduced below 3.4, add 1 M NaQH until pH is 3.5 -
0.1. Avoid, if possible, reducing pH below 3.4
because some inactivat ion of viruses may pccur.
(f) Continue to stir for 30 minutes more, and maintain pH
at 3.5 +_ 0.1.
(g) Remove pH electrode from beef extract.
(h) Remove cap from 250-mL screw-capped centrifuge
bottle.
Glass centrifuge bottles may not be able to withstand
g force that will be applied.
(i) Pour contents of flask into 250-mL screw-capped
centrifuge bottle.
To prevent transfer of stir bar into centrifuge
bottle, hold another stir bar or magnet against bottom
of flask when decantihg contents.
- 5-37 -
-------
(j) Replace and tighten down cap on screw-capped
centrifuge bottle.
(k) Centrifuge precipitated beef extract suspension in
refrigerated centrifuge (4° C) for 15 minutes at
2,500 x _Ł.
(1) Remove cap from screw-capped centrifuge bottle.
(m) Pour supernate into graduated cylinder, and record
volume.
(n) Discard supernate.
(o) Place a stir bar into centrifuge bottle containing the
precipitate.
(p) Add to the precipitate 5 ml of 0.15 M Na2HP04 for
each 100 ml of supernate decanted.
(q) Replace and tighten down cap on screw-capped
centrifuge bottle.
(r) Place the centrifuge bottle on a magnetic stirrer, and
stir slowly until precipitate has dissolved
completely.
Support bottles as necessary to prevent toppling.
Avoid foaming which may inactivate or aerosolize
viruses. Precipitate may be partially dissipated with
spatula before or during stirring procedure.
(s) Remove cap from screw-capped centrifuge bottle.
(t) Measure pH of concentrate (dissolved precipitate). If
pH is above or below 7.0-7.5, adjust to that range
with either 1 M HCl or 1 M NaOH.
- 5-38 -
-------
(u) Replace and tighten down cap on screw-i-capped
centrifuge bottle.
(v) Refrigerate concentrate immediately at 4° C, and
maintain at that temperature until assay for viruses
is undertaken.
If assay for viruses cannot be undertaken within eight
hours, store concentrate immediately at -70° C.
(w) Assay for viruses in accordance with instructions
given in Chapter 9.
- 5-39 -
-------
4. BIBLIOGRAPHY
Berg, G., D. R. Dahling, and D. Berman. 1971. Recovery of Small
Quantities of Viruses from Clean Waters on Cellulose Nitrate
Membrane Filters. Appl. Microbiol. 22:608-614.
Cliver, D. 0. Enterovirus Detection by Membrane Chromatography. Jji
"Transmission of Viruses by the Water Route," edited by G. Berg.
John Wiley and Sons, New York, 1967, pp. 139-149.
Cliver, D. 0. 1968. Virus Interactions with Membrane Filters.
Biotechnol. Bioeng. 10:877-889.
Dahling, D. R., and R. S. Safferman. 1979. Survival of Enteric
Viruses Under Natural Conditions in a Subarctic River.
Appl. Environ. Microbiol. 38:1103-1110.-
Farrah, S. R., and G. Bitton. 1978. Elution of Poliovirus Adsorbed
to Membrane Filters. Appl. Environ. Microbiol. 36:982-984.
Farrah, S. R., S. M. Goyal, C. P. Gerba, C. Wallis, and J. L.
Melnick. 1978. Concentration of Poliovirus from Tap Water onto
Membrane Filters with Aluminum Chloride at Ambient pH Levels.
Appl. Environ. Microbiol. 35:624-626.
Katzenelson, E., B. Fattal, and T. Hostovesky. 1976. Organic
Flocculation: an Efficient Second-Step Concentration Method for the
Detection of Viruses in Tap Water. Appl. Environ. Microbiol.
32:638-639.
Rao, N. U., and N. A. Labzoffsky. 1969. A Simple Method for the
Detection of Low Concentration of Viruses in Large Volumes of Water
by the Membrane Filter Technique. Can. J. Microbiol. 15:399-403.
Wallis, C., and J. L. Melnick. 1967. Concentration of Viruses from
Sewage by Adsorption on Millipore Membranes. Bull. W.H.O.
36:219-225.
Wall is, C., and J. L. Melnick. 1967. Concentration of Enteroviruses
on Membrane Filters. J. Virol. 1:472-477.
- 5-40 -
-------
CHAPTER 6
VIRUS ADSORPTION-ELUTION (VIRADEL) CARTRIDGE FILTER PROCEDURES
FOR RECOVERING VIRUSES FROM SEWAGES, EFFLUENTS, AND WATERS
In principle, the Virus Adsorption-Elution (VIRADEL) Cartridge Filter
Procedures described in this chapter are the same as Method 2 described in
Chapter 5. The VIRADEL cartridge filter procedures require much greater
volumes of elutant than Method 2 in Chapter 5 requires, but the cartridge
filter procedures may be used for sample volumes greater than 200 liters
and perhaps for volumes greater than 2000 liters.
Waters that contain chlorine and cannot be processed immediately must be
dechlorinated immediately upon collection. Immediate dechlorination may
be accomplished by placing into the collection vessel 0.8 mL of a 10%
solution of sodium thiosulfate (Na^SgOg) for each liter of water to
be collected. That quantity of ^5203 is sufficient for
neutralizing 15 mg of chlorine per liter.
Use aseptic techniques and sterile materials and apparatus only.
Sterilize all contaminated materials before discarding them (see Chapters
2 and 3).
Provide physical support as necessary for equipment that is not
free-standing.
1. ADSORPTION — METHOD ONE
This procedure may be used for all waters that do not require
prefi1trati on.
1.1 Preparation
- 6-1 -
-------
1.1.1 Apparatus and Materials
Install quick-disconnect connectors on ports of all
apparatus except on additive pumps.
(a) Holder for 10-inch* cartridge filter (Fulflo, Model
No. F15-10, Commercial Filter Division, Carborundum
Co., or equivalent).
(b) Cartridge filter, pleated epoxy-fiberglass --
10-inch, 0.45-pi pore size (DUO-FN 10-E-0.45 A ECIS,
Filterite Corp., or equivalent).
(c) Plastic-coated drums — 200-liter capacity, or other
containers of size suitable to hold sample, if
sample is not pumped directly from source.
(d) Sterilizable self-priming water pump that delivers
approximately 25-50 liters per minute.
Pump is not needed if sampled water is under
pressure, e.g., tap water.
(e) Carboy, autoclavable plastic with nipple on bottom
fitted with tubing clamped to a dispensing Y (clamp
tubing closed between nipple and Y) — 20-liter
capacity.
If the water at the sampling site is to be drawn
directly from a pressurized source and is to be
dechlorinated, then two similarly fitted carboys are
needed. Otherwise, only one carboy is needed.
Twice the number of carboys is needed under these
conditions if water volumes greater than 400 liters
are to be processed.
*Size is given in inches when commercially designated only in that unit.
• - 6-2 -
-------
Fluid proportioner consisting of fluid-driven motor
with four additive pumps (Johanson and Son Machine
Corp., Model M14Q with one P-562 and one P-750
additive pump affixed to each side of the
fluid-driven motor, or equivalents).
Assemble fluid proportioner, and connect tubing in
accordance with manufacturer's instructions.
(g) Mixing chamber (Johanson and Son Machine Corp.,
C-SS, or equivalent).
(h) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode
(Van London Co., or-equivalent, for electrode only).
(i) Tee, stainless steel, with three female NPT* ports.
Center port equipped with pH electrode in-line
adapter (Van London Co., or equivalent, for adapter
only).
(j) Autoclavable inner-braided tubing fitted with metal
quick-disconnect connectors for connecting tubing to
equipment to be used under pressure.
Quick-disconnect connectors can be used only after
equipment has been properly adapted.
(k) Magnetic stirrer and stir bars.
(1) Water meter (Badger Meter Inc., or equivalent).
(m) Positive pressure source equipped with pressure
gauge.
*National Pipe Thread
- 6-3 -
-------
Pressure source, if laboratory air line or pump,
must be equipped with oil filter. If source is
capable of producing high pressure, deliver to
filter holder no more pressure than recommended by
manufacturer.
1.1.2 Media and Reagents
(a) Hydrochloric acid (HCl) — 0.12 and 12 M
(concentrated) solutions.
Prepare 100 to 500 mL of 0.12 M HCl. This solution
may be stored for several months at room temperature.
(b) Sodium thiosulfate (Na2$203*5H20) — 40%
(w/v) stock solution (with respect to Na?S?03
•5H20).
Prepare one liter of NagSgCU solution by
dissolving 400 g of Na.,So03'5H,0 in 500
mL of deionized distilled water and bringing final
volume of solution to one liter with deionized
distilled water. This solution may be stored in
dark, rubber-stoppered bottle for up to one month at
room temperature. This solution is to be used to
dechlorinate water that cannot be dechlorinated
except immediately prior to test procedure (e.g.,
tap water tested directly at source). For
dechlorinating all other waters, see Page 6-1.
(c) Aluminum chloride (A1C13'6H20) -- 3 M stock
solution.
Prepare 100 mL of 3 M AlCK for each 400 liters of
water to be processed.
- 6-4 -
-------
1.2 Procedure (see Figure 6-1 for flow diagram of procedure)
In this procedure an apparatus is described that can be used with
clean waters, such as tap waters, where only a 0.45 jjm pleated
epoxy-fiberglass cartridge filter is needed. For waters that are
sufficiently turbid so that the volume filtered will clog this
filter, prefliters are required and Method 1 cannot be used. For
turbid waters, use Method 2 described in Section 2. Experience
usually dictates the method of choice. (CAUTION: Turbid water
may clog the fluid proportioner and, if abrasive, may damage it).
1.2.1 Preparation and Implementation
It is usually convenient to sterilize each piece of
apparatus and equipment one or more days before it is used
(see Chapter 3). It is convenient to sterilize apparatus
in small units when sterilization is accomplished by steam
or ethylene oxide. However, it is advisable to assemble
and connect units of apparatus that are to besterilizedby
chlorination. The interconnected apparatus can be
disassembled after the chlorination procedure is completed,
the ports covered with aluminum foil,and the units stored
until used.
(a) Assembly of apparatus (see Figures 6-2 and 6-3).
Use inner-braided tubing fitted with quick-
disconnect connectors to make all connections for
apparatus to be used under pressure.
(a.l) If sample is under pressure, connect water
source A (e.g., tap water), to inlet port B2
of fluid proportioner B. If sample is not
under pressure, connect water source to inlet
port AA-, of self-priming water pump AA.
- 6-5 -
-------
WATER
Dechlorinate water, if necessary.
To dechlorinate, flow 0.03 M
Na?S?Oo to final concentra-
tion of 0.0003 M continuously
into water.
By continuous flow, add to water
sufficient acidified (with 12 M
HC1) 0.01 M A1C13 to bring pH of
water to 3.5 _+ 0.1 and concentration
of A1C13 to 0.0005 M.
SALTED, pH-ADJUSTED WATER
Filter water through virus-adsorbing
0.45 jUm pleated epoxy-fiberglass
cartridge filter. Maintain pH 3.5 +_
0.1 by readjusting additive feed
pumps appropriately.
VIRUS-BEARING PLEATED FILTER
V
Go to elution and concentration procedure, Sections 3 and 4.
Figure 6-1. Flow Diagram of Method One for Concentrating Viruses
from Large Volumes (More than 200 Liters) of Clean Waters.
- 6-6 -
-------
A - Pressurized Water Source (Tap)
AA - Self Priming Water Pump
(Connected to water source)
AA, - Inlet Port
AA2 - Outlet Port
B - Fluid Proportioner
B,a - Chemical Feed (P750)
Additive Pumps (Larger)
- Chlorine Neutralizer (P562)
Additive Pumps (Smaller)
- Inlet Port
- Hose Adapter Body
- Outlet Adapter
- Carboy (Acid-salt solution)
- Carboy (Chlorine neutralizer)
- Mixing Chamber
B,b
B2
B3
B4
C
D
E
en
i
AA,
AA
E,
E2
F
G
G,
G2
H
H,
H2
H3
- Inlet Port
- Outlet Port
- Pipe Tee
- pH Meter
- pH Electrode
- pH Electrode In-Line Adapter
- Cartridge Holder (Pleated filter)
- Inlet Port
- Vent/Relief Valve
- Outlet Port
- Water Meter
- Inlet Port
- Outlet Port
"1
I
c
H,
H
, i
"3 "1
\>- I /
' v
Discard
Figure 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
(See Figure 6-3 for Photographic Representation of Apparatus).
-------
en
i
00
Figure 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) Haters
(See Figure 6-2 for Annotated Schematic Representation of Apparatus).
-------
Lonnect outlet port AAo or water pump AA to
inlet port Bp of fluid proportioner B.
(a.2) Connect outlet port B4 of fluid
prop-ortioner 'B to inlet port E, of mixing
chamber E.
(a.3) Connect outlet port E2 of mixing chamber E
to one arm of pipe tee F.
(a.4) Lock pH electrode G-, into pH electrode
in-line adapter Gp.
The same pH electrode (after sterilization)
that is used to adjust pH in Step (c.4) may
be used.
(a.5) Connect other arm of pipe tee F to inlet port
H-, of cartridge filter holder H.
(a.6) Connect outlet port H3 of cartridge filter
holder H to inlet port I, of water meter I.
(a.7) Connect outlet port I~ of water meter I to
discard.
(b) Preparation of salt supplement
Preparation of sufficient salt supplement for 400
liters of processed water is described below. If
more or less water is to be processed,
proportionately more or less salt supplement needs
to be prepared. When more salt supplement is
needed, prepare it in another carboy.
(b.l) Remove cover from 20-liter carboy C.
(b.2) Pour 10 liters of deionized distilled water
into carboy C, and add 67 ml of 3 M A1C1-
solution to the deionized distilled water.
- 6-9 -
-------
(b.3) Replace cover loosely on carboy C.
(c) Preparation of acid for adjustment of pH
(c.l) Pour 380 ml of test water into a 600-mL
beaker.
(c.2) Place stir bar into test water.
(c.3) Place beaker on magnetic stirrer, and stir at
speed sufficient to develop vortex in test
water.
(c.4) Place pH electrode into test water.
pH meter must be standardized before it is
used.
(c.5) Adjust pH of test water to 3.5 +_ 0.1 with
0.12 M HC1.
(c.6) Record volume of 0.12 M HCl used.
(c.7) Add to carboy C a volume of 12 M HCl equal to
11 times the quantity of 0.12 M HCl needed to
reduce the pH in the 380 ml volume of test
water to 3.5 +^0.1.
(c.8) Bring the volume of acid-salt solution to 20
-liters with deionized distilled water, and
mix solution well.
(d) Preparation of Na^S^ solution for
dechlorination
Step (d) applies only to chlorinated waters
processed directly from a source (e.g., tap water).
All chlorinated test waters obtained from sources
outside of the processing facility must be
dechlorinated immediately when the samples are
- 6-10 -
-------
obtained (see Page 6-1). Preparation of sufficient
Na0S00, for dechlorinating 400 liters of
1 " ' -X- " * • -U__L.__—. - .. , ....... . I — I-
(e.2) Remove cover from top of carboy C.
(e.3) Place free end of each tube into mouth of
carboy C.
- 6-11 -
-------
(e.4) Release pinch clamp, and allow acid-salt
solution to flow into tubes.
(e.5) Remove tubes from mouth of carboy C, and
insert tubes into the inlet (bottom) ports of
(larger) additive pumps B, .
Allow acid-salt solution to flow freely into
tubing, but manipulate tubes to prevent
overflow.
(e.6) Replace cover loosely on carboy C.
(e.7) Adjust the calibration on the metering rod
for each pump B, to a 3.2 setting.
This calibration equals delivery rate of 1
part of acid-salt solution to each 19 parts
of test water. If dechlorination is not
necessary, leave the ports of the two
remaining (smaller) additive pumps B,b
covered (see Section 1.2.1), and go to Step
(e.15).
(e.8) Connect a long length of tubing to each end
of dispensing Y on 20-liter carboy D that
contains the 0.03 M ^2S2°3 solution
prepared in Steps (d.l-d.4) above.
Tubing is already in place if pumps are
sterilized with chlorine (see Section
1.2.1). In this instance, disconnect tubing
from bottoms of additive pumps, and continue
with Step (e.9).
(e.9) Remove cover from top of carboy D.
- 6-12 -
-------
(e.10) Place free end of each tube into mouth of
carboy D.
(e.ll) Release pinch clamp, and allow Na^O^
solution to flow into tubes.
(e.12) Remove tubes from mouth of carboy D, and
insert tubes into the inlet (bottom) ports of
(smaller) additive pumps B,, .
Allow Na?S0po solution to flow freely
into tubes, but manipulate tubes to prevent
overflow.
(e.13) Replace cover loosely on carboy D.
(e.14) Adjust the calibration on the metering rod
for each additive pump B,b to a 1.3 setting.
This calibration equals delivery rate of one
part of 0.03 M Na^SpOo solution to each
99 parts of test water.
(e.15) Disconnect tube from inlet port E-. of
mixing chamber E, and connect tube to discard.
(e.16) To remove air from tubes, prime all additive
pumps by hand-operating metering rods in a
reciprocating motion.
(e.17) Reconnect tube from outlet port B. of fluid
propertioner B to inlet port E-, of mixing
chamber E.
1.2.2 Filtration of Sample
(a) Unscrew base of cartridge filter holder H.
(b) Center 0.45-jjm pleated epoxy-fiberglass cartridge
filter into base of filter holder H.
- 6-13 -
-------
(c) Screw base of cartridge filter holder H into its top
section, and wrench-tighten to seal.
(d) Make initial reading on water meter I, and record
reading.
(e) Open vent/relief valve H? on top of cartridge
filter holder H.
(f) Open source valve A or start water pump AA to
provide maximum flow through system.
(g) Close vent/relief valve H- on cartridge filter
holder H as soon as water flows through valve.
(h) Wipe up spilled water with laboratory disinfectant.
(i) Read pH meter G to ascertain that proper pH is
achieved.
Read meter periodically to be certain that proper pH
is maintained. If pH readjustment is necessary,
appropriately alter settings on metering rods for
(larger) additive pumps B-. .
(j) After required volume of water has been filtered,
close source valve A or turn off water pump AA.
(k) Open vent/relief valve H? on top of cartridge
filter holder H to relieve pressure in system.
(J) Close vent/relief valve H?.
Wipe up spills with disinfectant, as necessary.
(m) Disconnect tubing from inlet port H-] of cartridge
filter holder H.
Disinfect spills at disconnect.
(n) Connect free end of tubing to discard.
- 6-14 -
-------
(o) Elevate cartridge filter holder H, and invert to
drain.
(p) Make final reading on water meter I, and record
reading.
Subtract initial reading from final reading to
determine total volume filtered. Subtract volume of
acid-salt solution used and volume of Na^O-?
solution, if used, from total volume filtered to
determine volume of water sample filtered.
(q) Elute viruses from filter as described in Sections 3
and 4.
2. ADSORPTION — METHOD TWO
This procedure may be used for waters that require prefiltration.
2.1 Preparation
2.1.1 Apparatus and Materials
Install quick-disconnectconnectors onports of all
apparatus except on additive pumps.
(a) Cartridge filter, pleated epoxy-fiberglass —
10-inch*, 0.45-jum pore size (DUO-FN 10-E-0.45
N-ECIS, Filterite Corp., or equivalent).
(b) Cartridge filter, honeycomb-wound fiberglass yarn —
10-inch, l-/jm and 5-|im pore sizes (K 27, 1-jjm and K
19, 5-jun, Commercial Filter Division, Carborundum
Co., or equivalent), as needed.
*Size is given in inches when commercially designated only in that unit.
- 6-15 -
-------
One or more fiberglass-wound filters needs to be
used only when it Is anticipated that the pleated
filter will clog before the filtration procedure is
complete. In the absence of experience,
honeycomb-wound filters should be used for all
waters except tap waters, but may be used for tap
waters, if necessary.
(c) Holders for 10-inch cartridge filters (Fulflo, Model
No. F15-10, Commercial Filter Division, Carborundum
Co., or equivalent).
One ho'lder is needed for pleated filter. An
additional holder is needed for each honeycomb-wound
cartridge that is to be used.
(d) Plastic-coated drums -- 200-liter capacity, or other
containers of size suitable to hold sample, if
sample is not pumped directly from source.
(e) Sterilizable self-priming water pump that delivers
approximately 25-50 liters per minute.
Pump Is not neededif sample water is 'under
pressure, e.g., tap water.
(f) Carboy, autoclavable plastic with nipple on bottom
fitted with tubing clamped to a dispensing Y (clamp
tubing closed between nipple and Y) — 20-liter
capacity.
If the water at the sampling site is to be drawn
directly from a pressurized source and is to be
dechlorinated, then two similarly fitted carboys are
needed. Otherwise, only one carboy is needed.
- 6-16 -
-------
Twice the number of carboys is needed, under these
conditions, if water volumes greater than 400 liters
are to be processed.
(g) Four stainless steel pipe plugs (Johanson and Son
Machine Corp., A40, or equivalent).
(h) Hose adapter for fluid proportioner equipped with
four hose fittings (quad system) (Johanson and Son
Machine Corp., A34-Q and A33 or equivalent).
(i) Fluid proportioner consisting of fluid-driven motor
with four additive pumps (Johanson and Son Machine
Corp., Model M 14Q with one P-562 and one P-750
additive pump affixed to each side of the
fluid-driven motor, or equivalents).
Assemble fluid proportioner in accordance with the
manufacturer's instructions except when otherwise
indicated. If four tube fittings with attached
tubing are connected to hose adapter H^, remove
the tubing from the fittings, and replace the
fittings with four stainless steel pipe plugs (see
Figures 6-4 and 6-5 for location of hose adapters).
Then, screw the four tube fittings into hose adapter
B. Connect a 1.8 meter (6-foot) length of tubing to
the top port on each additive pump. Connect the
free end of each tube leading from the outlet (top)
port of each of the four additive pumps to a tube
fitting on hose adapter B.
(j) Mixing chamber (Johanson and Son Machine Corp.,
C-SS, or equivalent).
- 6-17 -
-------
CO
AA,
AA
A - Pressurized Water Source (Tap)
AA - Self Priming Water Pump
(Connected to test water source)
AA, - Inlet Port
AA2 - Outlet Port
B - Hose Adapter Body
C - Mixing Chamber
Ci - Inlet Port
C2 - Outlet Port
D - Pipe Tee
E - pH Meter
Et - pH Electrode
E2 - pH Electrode In-Line Adapter
F - Cartridge Holder (Honeycomb filter)
F, - Inlet Port
F2 - Vent/Relief Valve
F3 - Outlet Port
G - Cartridge Holder (Honeycomb filter)-
G, - Inlet Port
G2 - Vent/Relief Valve
G3 - Outlet Port
H - Fluid Proportioner
H,a - Chemical Feed (P750) Additive
Pumps (Larger)
H,b - Chlorine Neutralizer (P562)
H2
H3
H4
I
J
K
K,
K2
K3
L
L,
La
- Inlet Port
- Hose Adapter Body
- Outlet Port
- Carboy (Acid-salt solution)
- Carboy (Chlorine neutralizer)
- Cartridge Holder (Pleated filter)
- Inlet Port
- Vent/Relief Valve
- Outlet Port
- Water Meter
- Inlet Port
- Outlet Port
Additive Pumps (Smaller)
Figure 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
(See Figure 6-5 for Photographic Representation of Apparatus).
-------
Figure 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
(See Figure 6-4 for Annotated Schematic Representation of Apparatus)
-------
(k) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode
(Van London Co., or equivalent, for electrode only).
(1) Tee, stainless steel, with three female NPT ports.
Center port equipped with pH electrode in-line
adapter (Van London Co., or equivalent, for
electrode and adapter only).
(m) Autoclavable inner-braided tubing with metal
quick-disconnect connectors for connecting tubing to
equipment to be used under pressure.
Quick-disconnect connectors can be used only after
equipment has been properly adapted.
(n) Magnetic stirrer and stir bars.
(o) Water meter (Badger Meter Inc., or equivalent).
(p) Positive pressure source equipped with pressure
gauge.
Pressure source, if laboratory air line or pump,
must be equipped with oil filter. If source is
capable of producing high pressure, deliver to
filter holder no more pressure than recommended by
manufacturer.
2.1.2 Media and Reagents
(a) Hydrochloric acid (HCl) — 0.12 and 12 M
(concentrated) solutions.
Prepare 100-500 mL of 0.12 M HCl. This solution may
be stored for several months at room temperature.
- 6-20 -
-------
(b) Sodium thiosulfate (Na2S203'5H20) - 40$
(w/v) stock solution (with respect to Na^SpOo
•5H20).
Prepare one liter of NagSpOp solution by
dissolving 400 g of Na,,So03'SHpO in 500
mL of deionized distilled water and bringing final
volume of solution to one liter with deionized
oti
distilled water. Solution may be stored in dark,
rubber-stoppered bottle for up to one month at room
temperature.
Solution is to be used for water that cannot be
dechlorinated except immediately prior to test
procedure (e.g., water tested directly at source).
For dechlorinating all other waters, see Page 6-1.
(c) Aluminum chloride (AlCl^GhLO) — 3 M stock
solution.
Prepare 100 mL of 3M A1C1., for each 400 liters of
water to be processed.
2.2 Procedure (see Figure 6-6 for flow diagram of procedure)
In this procedure, an apparatus is described that can be used for
waters so turbid that the volume filtered will clog a 0.45-um
pleated epoxy-fiberglass cartridge filter. This apparatus is
similar to that described in Method One of this chapter except
that honeycomb-wound fiberglass filters are installed in advance
of the pleated filter to remove particulate matter in the water,
and in-line placement of the equipment is modified to allow
adjustment of the pH and salt concentration of the test waters
before those waters are prefiltered. Experience usually
- 6-21 -
-------
TURBID WATER
(Water or effluent)
Dechlorinate sample, if necessary.
To dechlorinate, flow 0.03 M
NapSgO., to final concentra-
tion of 0.0003 M continuously
into sample.
By continuous flow, add to sample
sufficient acidified (with 12 M HCl)
0.01 M AlCl- to bring pH of sample
to 3.5 _+ 0.1 and concentration of
A1C13 to 0.0005 M.
SALTED, pH-ADJUSTED SAMPLE
Filter water through cartridge pre-
filters (Use 1-jjm honeycomb-wound
fiberglass yarn filter for river
waters and waters of similar turbid-
ity, and use 5-^im honeycomb-wound
fiberglass yarn filter preceding
l-jum filter for secondary and
tertiary effluents) and then through
virus-adsorbing 0.45-/jm pleated
cartridge filter.
Maintain pH 3.5 _+ 0.1 by readjusting
additive feed pumps appropriately.
Viruses adsorb to virus-adsorbing
filter. Viruses adsorbed to
particulates trapped on prefilters.
VIRUS-BEARING PLEATED FILTER—PARTICULATE-BEARING PREFILTER(S)
Go to elution and concentration procedure, Sections 3 and 4.
Figure 6-6. Flow Diagram of Method Two for Concentrating Viruses
from Large Volumes (More than 200 Liters) of Turbid Waters.
- 6-22 -
-------
dictates whether prefiltration is needed. In the absence of
experience, use procedure in Section 1. ADSORPTION — METHOD ONE
for tap waters and for other waters of similar clarity. Use a 1
jum honeycomb-wound fiberglass yarn cartridge filter preceding the
0.45-jum pleated filter for surface waters and for other waters of
similar clarity, and add a 5-jjm honeycomb-wound fiberglass yarn
cartridge filter preceding the 1-jjm filter for secondary and
tertiary effluents and for other waters of similar clarity.
2.2.1 Preparation and Implementation
It is usually convenient to sterilize each piece of
apparatus and equipment one or more days before it is used
(see Chapter 3). It is convenient to sterilize apparatus
in small units when sterilization is accomplished by steam
or ethylene oxide. It is convenient to assemble and
connect all units of apparatus that are to be sterilized
by chlorination. The interconnected apparatus can be
disassembled after chlorination, the ports covered with
aluminum foil and the units stored until used.
(a) Assembly of apparatus (see Figures 6-4 and 6-5)
Use inner-braided tubing fitted with quick-
disconnect connectors to make all connections for
equipment under pressure.
(a.l) If sample is under pressure, connect water
source A to either port of hose adapter B.
If sample is not under pressure, connect
water source to inlet port AA, of self-
priming water pump AA. Connect outlet port
of water pump AAp to either port of hose
adapter B.
- 6-23 -
-------
(a.2) Connect remaining port of hose adapter B to
inlet port C-| of mixing chamber C.
(a.3) Connect outlet port C2 of mixing chamber C
to one arm of pipe tee D.
(a.4) Lock pH electrode E-| into pH electrode
in-line adapter E2-
Same pH electrode (after sterilization) that
is used to adjust pH in Step (c.4) may be
used.
(a.5) Connect other arm of pipe tee D to inlet port
F-i of cartridge holder F.
(a.6) Connect outlet port F-, of cartridge holder
F to inlet port G-, of cartridge holder 6.
(a.7) Connect outlet port 63 of cartridge holder
G to inlet port H2 of fluid proportioner H.
(a.8) Connect outlet port H4 of fluid
proportioner H to inlet port K-| of
cartridge holder K.
(a.9) Connect outlet port K3 of cartridge holder
K to inlet port L-i of v/ater meter L.
(a.10) Connect outlet port L2 of v/ater meter I, to
discard.
(b) Preparation of salt supplement
Preparation of sufficient salt supplement for 400
liters of processed water is described below. If
more or less water is to be processed,
proportionately more or less salt supplement needs
to be prepared. When more salt supplement is
needed, prepare it in another carboy.
- 6-24 -
-------
(b.l) Remove cover from 20-liter carboy I.
(b.2) Pour 10 liters of deiorrized distilled water
into carboy I, and add 67 mL of 3 M AlCl,
solution to the deionized distilled water.
(b.3) Replace cover loosely on carboy I.
(c) Preparation of acid for adjustment of pH
(c.l) Pour 380 mL of test water into a 600-mL
beaker.
(c.2) Place stir bar into test water.
(c.3) Place beaker on magnetic stirrer, and stir at
speed sufficient to develop vortex in test
water.
(c.4) Place pH electrode into test water.
pHmeter must be standardized before it is
used.
(c.5) Adjust pH of test water to 3.5 +_ 0.1 with
0.12 M HC1.
(c.6) Record volume of 0.12 M HCl used.
(c.7) Add to carboy I a volume of 12 M HCl equal to
11 times the quantity of 0.12 M HCl needed to
produce the required pH in the 380-mL volume
of test water.
(c.8) Bring acid-salt solution to 20-liters with
deionized distilled water, and mix solution
well.
(d) Preparation of Na2$203 solution for
dechlorination
- 6-25 -
-------
Step (d) applies only to chlorinated waters
processed directly from a source. All chlorinated
test waters obtained from sources outside of the
processing facility must be dechlorinated
Immediately when the samples are obtained (see Page
6-1). Preparation of sufficient Ma^Og for
dechl on'nating 400 liters of processed water Is
described below.
If more or less water Is to be processed,
proportionately more or less Na?SJDo needs to
be prepared. When more NagSgOg is needed,
prepare it in another carboy.
(d.l) Remove cover from 20-liter carboy J.
(d.2) Pour 10 liters of deionized distilled water
into carboy J.
(d.3) Add 186 nt of 40% Na2$203 solution to
the deionized distilled water in carboy J to
give a final molarity of 0.03, and mix
solution well.
(d.4) Replace cover loosely on carboy J.
(e) Fluid proportioner
(e.l) Connect a long length of tubing to each end
of dispensing Y on 20-liter carboy I that
contains the acid-salt solution prepared in
Step (c.8) above.
Tubing is already in place if additive pumps
are sterilized with chlorine (see Section
2.2.1).
- 6-26 -
-------
in this instance, disconnect tubing from
bottom of additive pumps H, , and continue
with Step (e.2).
(e.2) Remove cover from top of carboy I.
(e.3) Place free end of each tube into mouth of
carboy I.
(e.4) Release pinch clamp, and allow acid-salt
solution to flow into tubes.
(e.5) Remove tubes from mouth of carboy I, and
insert tubes into inlet (bottom) ports of
(larger) additive pumps H-, .
Allow acid-salt solution to flow freely into
tubing, but manipulate tubes to prevent
overf1ow.
(e.6) Replace cover loosely on carboy I.
(e.7) Adjust the calibration on the metering rod
for each pump H-, to a 3.2 setting.
This calibration equals delivery rate of one
part of acid-salt solution to each 19 parts
of test water. If dechlprination is not
necessary, leave the ports of the two
remaining additive pumps H,fa covered (see
Section 2.2.1), and go to Step (e.15).
(e.8) Connect a long length of tubing to each end
of dispensing Y on 20-liter carboy J that
contains the 0.03 M Ma^S^Do solution
prepared in Steps (d.l-d.4) above.
- 6-27 -
-------
Tubing is already In place If pumps are
sterilized with chlorine (see Section 2.2.1).
In this instance, disconnect tubing from
bottoms of additive pumps, and continue with
Step (e.9).
(e.9) Remove cover from top of carboy J.
(e.10) Place free end of each tube into mouth of
carboy J.
(e.ll) Release pinch clamp, and allow Na^O.,
solution to flow into tubes.
(e.12) Remove tubes from mouth of carboy J, and insert
tubes into inlet (bottom) ports of (smaller)
additive pumps Hlb.
Allow MaSO solution to flow freely
into tubes, but manipulate tubes to prevent
overflow.
(e.13) Replace cover loosely on carboy J.
(e.14) Adjust the calibration on the metering rod for
each additive pump Hlb to a 1.3 setting.
This calibration equals delivery rate of one
part of 0.03 M Na2Sg03 solution to each
99 parts of test water.
(e.15) Disconnect tube from inlet port C-, of mixing
chamber C, and connect tube to discard.
(e.16) TO remove air from tubes, prime additive pumps
by hand-operating metering rods in a
reciprocating motion.
(e.17) Reconnect tube from outlet port of hose adapter
B to inlet port C-| of mixing chamber C.
- 6-28 -
-------
2.2.2 Filtration of Sample
(a) Unscrew base of cartridge filter holder F.
(b) Center 5-jim honeycomb-wound fiberglass yarn
cartridge filter into base of filter holder F.
(c) Screw base of cartridge filter holder F back into
its top section, and wrench-tighten to seal.
(d) Unscrew base of cartridge filter holder G.
(e) Center 1-jim honeycomb-wound fiberglass yarn
cartridge filter into base of filter holder 6.
(f) Screw base of cartridge filter holder 6 back into
its top section, and wrench-tighten to seal.
(g) Unscrew base of cartridge filter holder K.
(h) Center 0.45-jim pleated epoxy-fiberglass cartridge
filter into base of filter holder K.
(i) Screw base of cartridge filter holder K back into
its top section, and wrench-tighten to seal.
(j) Make initial reading on water meter L, and record
reading.
(k) Open vent/relief valves F2, G2, and K2 on top
of cartridge filter holders F, G, and K.
(1) Open source valve A or start water pump AA to
provide maximum flow through system.
(m) Close vent/relief valves F?, G?, and K2 on top
of cartridge filter holders F, G, and K as soon as
water,flows through valves.
(n) Wipe up spilled water with laboratory disinfectant.
(o) Read pH meter E to ascertain that proper pH is
achieved.
- 6-29 -
-------
Read meter periodically to be certain that proper
pH is maintained. If pH readjustment is necessary,
appropriately alter settings on metering rods for
(larger) additive pumps H-. .
(p) After required volume of water has been filtered,
close source valve A or turn off water pump AA.
(q) Open vent/relief valves F9, G9, and K9 on top
Cri t. t. Ł•
of cartridge filter holders F, G, and K to relieve
pressure in system.
(r) Close vent/relief valves F2, G2» and K2*
Wipe up spills with disinfectant as necessary.
(s) Disconnect tubing from source A or from water pump
outlet AA2.
Disinfect spills at disconnect.
(t) Connect free end of tubing to discard.
(u) Elevate cartridge filter holders F, G, and K, and
invert to drain.
(v) Take final reading on water meter, and record
reading.
Subtract initial reading from final reading to
determine total volume filtered. Subtract volume
of acid-salt solution used and, if used, volume of
solution from total volume filtered
to determine volume of water sampled.
(w) Elute viruses from filters as described in Sections
3 and 4.
- 6-30 -
-------
3. ELUTION AND RECONCENTRATION -- METHOD ONE
This method may be used for eluting viruses not significantly
inactivated at pH levels of about 10.5 in 15 minutes at ambient
temperatures. To elute viruses that cannot be safely recovered by
this procedure, see Section 4.
3.1 Procedure for Eluting Viruses from Cartridge Filters (see Figures
6-7.1 and 6-8.1 for flow diagrams of procedure)
3.1.1 Apparatus and Materials
(a) Positive pressure source equipped with a pressure
gauge.
Gauge necessary only if pressure source is capable
of producing pressures exceeding tolerances of
equipment. Pressure source, if laboratory air line
or pump, must be equipped with an oil filter. If
source is capable of producing high pressure,
deliver to pressure vessel and filter holder no more
pressure than recommended by manufacturer.
(b) Dispensing pressure vessel -- 4 liters (Millipore
Corp., or equivalent).
(c) Beakers, graduated — 2 liters.
One beaker is needed for each filter that is eluted.
(d) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode.
(e) Autoclavable inner-braided tubing fitted with metal
quick-disconnect connectors on one end and glass
elbow on the other.
Make glass elbow from a 13-cm length (approximate
P.P. 6 mm) of glass tubing by making a 40 degree
bend about 5 cm from one end. Connect tubing onto
- 6-31 -
-------
Figure 6-7.1
Virus Elution
Procedure
Figure 6-7.2 Virus Reconcentration
Procedure
VIRUS-BEARING CARTRIDGE FILTER
Force 1600 mL of basic glycine solution (0.05 M glycine,
pH 10.5+^0.1) through cartridge filter slowly (Apply
pressure no greater than 0.4 kg/cm ).
Viruses elute from filter.
Check pH of eluate. If pH is less than 9.5, repeat^
elution procedure with fresh basic glycine solution,
and combine eluates.
If precipitate forms in eluate, reconcentrate viruses
by Al(OH),-hydroextraction procedure.
CAUTION: Reconcentration by Al(OH),-hydroextraction
procedure must begin immediately, because pH of
eluate must be reduced immediately to prevent
inactivation of viruses.
BASIC ELUATE
Mix rapidly into eluate sufficient acid glycine solution
(0.05 M glycine, pH 2) to bring pH of eluate to 3.5 + 0.1.
If eluate becomes turbid during or after acidification,
terminate procedure and reconcentrate viruses by
Al(OH),-hydroextraction procedure.
CAUTION: Begin Al(OH), procedure immediately, because pH
of eluate must be reduced immediately to prevent
inactivation of viruses.
ACIDIFIED ELUATE
Filter acidified eluate through 0.45-jim virus-adsorbing
disc filter.
Viruses adsorb to filter.
VIRUS-BEARING DISC FILTER
Elute viruses by forcing successively two 5-mL volumes
of basic glycine solution (0.05 M glycine, pH 10.5 _+ 0.1)
through disc filter.
Check pH of eluate. If pH is less than 9.5, repeat
elution procedure with fresh basic glycine solution, and
combine eluates.
\ls
BASIC ELUATE
Adjust pH of combined eluates to 7.0-7.5 with acid glycine
solution (0.05 M glycine, pH 2).
NEUTRALIZED ELUATE
Add sufficient fetal calf serum to neutralized eluate to
yield final serum concentration of 2%.
STABILIZED NEUTRALIZED ELUATE
I
Assay for viruses (See Chapter 9).
Figure 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 -
-------
rfgure 6-8.1 Virus FTutfon
Procedure
VIRUS-BF.ARING CARTRIWF FILTER
Force 1600 nL of basic glyclne solution (0.05 H glycine,
pH 10.5^0.1) through cartridge filter slowly (Apply
pressure no greater than 0.4 kg/en2).
Viruses elute fron filter.
Check pH of eluate. If pH 1s less than 9.5. repeat
elution procedure with fresh basic glycine solution,
and combine eluates.
Figure 6-8.2 Virus Reconcentration
Procedure
BASIC ELUATF.
Stir sufficient 0.3 M A1C13 Into eluate to obtain final
concentration of 0.003 H.
1
SALTED aUATE
Adjust pH of salted eluate to 7.0 + 0.1 with 1 M Na2C03.
An Al(OH), floe forns as Ha-CO, is added.
Stir for five minutes more. Allow floe to settle for 30 ninutes.
SETTLED VIRUS-BEARING Al(OH)3 FLOC
Aspirate all but several nL of liquid above settled floe,
discard aspirated fluid, and centrifuge floe at 1,000 x Ł
for 30 ninutes. Save floe, discard supernate.
CEMTPIFUGED VIRUS-BEARIMG Al(OH)3 FLOC
Measure floe volune, and nix three volumes of buffered fetal
calf serun (BFCS)-glycine, pH 11.5 + 0.1 into Al(OH)3 floe.
Viruses elute from floe.
FLOC-BFCS ELUATE MIXTURE
i Centrifuge floc-eluate mixture at 1,000 x Ł for three ninutes.
Discard floe.
BFCS ELUATE
Adjust pH of BFCS eluate to 7.0 .+_ 0.1 with acid glycine
solution (1 M glycine, pH 2).
I
NEUTRALIZED BFCS ELUATE
Pour neutralized BFCS eluate into a dialysis bag, and dialyze
BFCS against polyethylene glycol at 4° C until 10-20 nt of
fluid remains in bag.
DIALYSATE
Suspend bag containing dialysate in chilled phosphate-buffered
saline. Maintain at 4°-10° C for one hour.
ISOTOHIC DIALYSATE
Add antibiotics.
Assay for viruses (See Chapter 9).
Figure 6-8. Flow Diagrar.i of High pH Procedure (Basic Glycine, pH 10.5) for
Fluting Viruses fron Cartridge Filters and for Reconcentrating
Viruses from Turbid Eluates by the Al(OH)3-Hydroextraction
Procedure.
- 6-33 -
-------
longer end of elbow. One elbow witn attacnea timing
Is needed for each filter that is eluted.
(f) Magnetic stirrer and stir bars.
3.1.2 Media and Reagents
(a) Sodium hydroxide (NaOH) -- 10 M.
Prepare 500 ml of 10 M NaOH.
(b) Basic glycine solution — 0.05 M glycine, adjusted
to pH 10.5 +; 0.1 with 10 M NaOH.
Autoclave glycine solution before adjusting pH.
Prepare 3 liters of basic 0.05 M glycine solution.
(c) Hydrochloric acid (HC1) — 12 M (concentrated) HC1
solution.
(d) Acid glycine solution — 0.05 M glycine, adjusted to
pH 2 with 12 M HC1.
Autoclave glycine solution before adjusting pH.
Prepare 3 liters of acid 0.05 Mglycine solution.
3.1.3 Rearrangement of Apparatus
(a) Rearrangement for Method One (see Figures 6-2 and
6-3).
(a.l) Disconnect at pipe tee F, the tubing leading
to inlet port H, of filter holder H.
(a.2) Connect free end of tubing from inlet port
H-, of filter holder H to outlet port of
pressure vessel.
Pressure vessel is not shown in Figures 6-2
and 6-3.
(a.3) Connect inlet port of pressure vessel to
positive air pressure source.
(a.4) Disconnect tubing from outlet port H3 of
filter holder H.
- 6-34 -
-------
(a.5) Hook glass elbow with 40 degree bend onto
pouring spout of a 2-liter glass beaker.
Raise aluminum foil covering beaker enough to
expose only the pouring spout.
(a.6) Connect free end of tubing from glass elbow
to outlet port H3 of filter holder H.
(a.7) Crimp aluminum foil cover over glass elbow.
(a.8) Elute viruses from filter as described in
Section 3.1.4 below.
(b) Rearrangement for Method Two (see Figures 6-4 and
6-5).
(b.l) Disconnect at pipe tee D, the tubing leading
to the inlet port F, of filter holder F.
(b.2) Connect free end of tubing from inlet port
p., of filter holder F to outlet port of
pressure vessel.
Pressure vessel is not shown in Figures 6-4
and 6-5.
(b.3) Connect inlet port of pressure vessel to
positive pressure source.
(b.4) Disconnect tubing from outlet port F3 of
filter holder F.
(b.5) Hook glass elbow with 40 degree bend onto
pouring spout of a 2-liter glass beaker.
Raise aluminum foil covering beaker enough to
expose only the pouring spout.
(b.6) Connect free end of tubing from glass elbow
to outlet port F^ of filter holder F.
(b.7) Crimp aluminum foil cover over glass elbow.
- 6-35 -
-------
(b.8) Elute viruses from filter as described in
Section 3.1.4 below.
(b.9) Disconnect tubing from outlet port of
pressure vessel.
(b.10) Connect free end of tubing from inlet port
G-] of filter holder G to outlet port of
pressure vessel.
(b.ll) Disconnect tubing from outlet port 63 of
filter holder G.
(b.12) Hook glass elbow with 40 degree bend onto
pouring spout of a 2-liter glass beaker.
Raise aluminum foil covering beaker enough to
expose only the pouring spout.
(b.13) Connect free end of tubing from glass elbow
to outlet port GO of filter holder G.
(b.14) Crimp aluminum foil cover over glass elbow.
(b.15) Elute viruses from filter as described in
Section 3.1.4 below.
(b.16) Disconnect tubing from outlet port of
pressure vessel.
(b.17) Disconnect at outlet port f-L of fluid
proportioner H, the tubing leading to the
inlet port K-, of filter holder K.
(b.18) Connect free end of tubing from inlet port
K, of filter holder K to outlet port of
pressure vessel.
(b.19) Disconnect tubing from outlet port K, of
filter holder K.
- 6-36 -
-------
(b.20) Hook glass elbow with 40 degree bend onto
pouring spout of a 2-liter graduated glass
beaker.
Raise aluminum foil covering beaker enough to
expose only the pouring spout.
(b.21) Connect free end of tubing from glass elbow
to outlet port K3 of filter holder K.
(b.22) Crimp aluminum foil cover over glass elbow.
(b.23) Elute viruses from filter as described in
Section 3.1.4 below.
3.1.4 Elution Procedure
(a) Remove top of pressure vessel.
(b) Pour into pressure vessel 1600 ml of basic glycine
solution (pH 10.5 +_ 0.1).
(c) Replace top of pressure vessel.
(d) Close vent/relief valve on pressure vessel.
(e) Open vent/relief valve on cartridge filter holder.
(f) Apply pressure sufficient to purge trapped air from
filter apparatus.
(g) Close vent/relief valve on cartridge filter holder
as soon as basic glycine solution begins to flow
from valve.
(h) Wipe up spilled liquid with laboratory disinfectant.
(i) Increase pressure to that sufficient to force basic
glycine solution through the filter.
2
Do not exceed a pressure of 0.4 kg/cm so that
basic glycine solution passes through cartridge
filter slowly thereby maximizing elution contact
period.
- 6-37 -
-------
(j) Turn off pressure at source.
(k) Open vent/relief valve on pressure vessel.
(1) Check pH of eluate.
If pH of eluate is below 9.5, repeat elution
procedure with fresh elutant, combine eluates in
graduated beaker, and reconcentrate. Instructions
for reconcentrating viruses begin in Section 3.2.
Reconcentration must begin immediately, because pH
of eluate must be reduced immediately to prevent
inactivation of viruses.
3.2 Reconcentration — Method A. Membrane Disc Procedure (see Figure
6-7.2 for flow diagram of procedure)
Where it canbe used, the membranedisc procedure is the
preferred method for reconcentrating viruses from the eluates
resulting from the procedures described in the preceding section
(Section 3.1.4). However, in some eluates, a precipitate is
present that impedes filtration of the eluate through a membrane
filter. Reconcentrate such eluates by the aluminumhydroxide-
hydroextraction procedure described in Section 3.3. If, during
acidification in the membrane procedure, turbidity occurs in
previously clear eluates, discontinue acidification and
reconcentrate these eluates by the aluminum hydroxide-
hydroextraction procedure. Optionally, for any given sample, all
clear eluates may be pooled and all turbid eluate's may be pooled
for reconcentration.
3.2.1 Apparatus and Materials
(a) High pressure disc filter holders — 47mm diameter
(Millipore Corp., XX4504700, or equivalent).
- 6-38 -
-------
(b) Virus-adsorbing disc filter, mixed esters of
cellulose -- 0.45-pi pore size (Millipore HA, or
equivalent),, .,- .
(c) Dispensing pressure vessel -- 20-liter capacity
(Millipore Corp., XX6700L20, or equivalent).
(d) Positive pressure source equipped with pressure
gauge.
Gauge necessary only if pressure source is capable
of producing pressures exceeding tolerances of
equipment. Pressure source, if laboratory air line
or pump, must be equipped with oil filter. If
source is capable of producing high pressure,
deliver to pressure vessel and filter holder no more
pressure than recommended by manufacturer.
(e) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode.
(f) Autoclavable inner-braided tubing with metal
quick-disconnect connectors or with thumb-
screw-drive-clamps for connecting tubing to
equipment.
(g) Magnetic stirrer and stir bar.
(h) Filling bell connected to inner-braided tubing.
3.2.2 Media and Reagents
(a) Hydrochloric acid (HCl) -- 12 M (concentrated) HCl
solution.
(b) Acid glycine solution, 0.05 M, adjusted to pH 2 with
12 M HCl.
Autoclave glycine solution before adjusting pH.
- 6-39 -
-------
(c) Sodium hydroxide (NaOH) -- 10 M.
Prepare 500 mL of 10 M NaQH.
(d) Basic glycine solution, 0.05 M, adjusted to pH 10.5 +_
0.1 with 10 M NaOH.
Prepare 3 liters of basic glycine solution. Autoclave
glycine solution before adjusting pH.
(e) Fetal calf serum.
3.2.3 Procedure
(a) Assembly of Apparatus (See Figures 6-9 and 6-10)
(a.l) Remove top of filter holder C.
(a.2) With forceps, lay 0.45-um virus-adsorbing
filter onto support screen of holder.
(a.3) Replace and tighten down top of filter holder C.
(a.4) Connect positive pressure source A or AA to
inlet port B of pressure vessel B.
(a.5) Connect outlet port B3 of pressure vessel B
to inlet port C-, of filter holder C.
(a.6) Place filling bell D, with inner-braided tubing
attached, over opening of flask E, and connect
free end of the tubing to the outlet port C?
of filter holder C.
(b) Adjustment of pH of eluates from Section 3.1.4, Step
0)
Add with rapid, continuous stirring sufficient acid
glycine solution to bring pH of eluate to 3.5 jf 0.1.
It is important to mix acid glycine solution into
sample rapidly, because slow mixing may result in pH
levels sufficiently low in parts of the sample to
- 6-40 -
-------
A - Pressure Source
(Compressed air or N2>
A, - Pressure Regulator
AA - Laboratory Pressure System
B - Pressure Vessel
B, - Inlet Port
B2 - Vent/Relief Valve
en
B3 - Outlet Port
C - Filter Holder
C, - Inlet Port
C2 - Outlet Port
D - Filling Bell
E - Receiving Vessel
Figure 6-9. Schematic Representation of Apparatus for Reconcentration —
Method A, a Membrane Disc Procedure for Reconcentrating
Viruses from Glycine Eluates
(See Figure 6-10 for Photographic Representation of Apparatus).
-------
Figure 6-10. Photographic Representation of Apparatus for Reconcentration —
Method A, a Membrane Disc Procedure for Reconcentrating
Viruses from Glycine Eluates
(See Figure 6-9 for Annotated Schematic Representation of Apparatus).
-------
inactivate viruses. If eluate becomes turbid during
or after acidification, terminate procedure and
reconcentrate viruses.by the aluminum hydroxide-
hydroextraction procedure described in Section 3.3.
Reconcentration with the aluminum hydroxide-
hydroextraction procedure must begin immediately,
because pH of eluate must be reduced immediately to
prevent inactivation of viruses.
(c) Filtration of eluate.
(c.l) Remove top of pressure vessel B.
(c.2) Pour eluate into pressure vessel B.
(c.3) Replace and tighten down top of pressure
vessel B.
(c.4) Apply pressure from source A or AA sufficient
to force sample through the filter (usually
0.4-1.5 kg/cm2).
(c.5) Turn off pressure at source A or AA.
(c.6) Open vent/relief valve B2 of pressure
vessel B.
(c.7) When pressure is relieved, close vent/relief
valve B?.
(d) Elution of viruses from filter.
(d.l) Disconnect tubing from outlet port C? of
filter holder C.
(d.2) Place 100-mL beaker under outlet port Cp of
filter holder C.
(d.3) Disconnect tubing from inlet port C-, of
filter holder C.
- 6-43 -
-------
(d.4) Pour 5 ml of basic glycine solution into
inlet port C-, of filter holder C.
(d.5) Reconnect tubing to inlet port C1 of filter
holder C.
(d.6) Apply sufficient pressure from source A or AA
to force basic glycine solution through
filter.
(d.7) Turn off pressure at source A or AA.
(d.8) Open vent/relief valve B? on pressure
vessel B.
(d.9) When pressure is relieved, close vent/relief
valve B?.
(d.10) Disconnect tubing from inlet port C, of
filter holder C.
(d.ll) Pour another 5 mL of basic glycine solution
into inlet port C1 of filter holder C.
(d.12) Repeat steps (d.5-d.8), collecting total 10
mL of eluates in the same beaker.
Check pH of combined eluates. If pH isbelow
9.5, repeat steps (d.9-d. 12) with fresh basic
glycine solution.
(d.13) Adjust pH of combined eluates to 7.0-7.5 with
acid glycine solution.
(d.14) Measure total volume of neutralized eluate.
(d.15) Add sufficient fetal calf serum to
neutralized eluate to yield a final serum
concentration of 2%.
- 6-44 -
-------
(d.16) Refrigerate neutralized eluate at 4 C
immediately, and maintain at that temperature
until eluate is assayed for viruses.
If assay for viruses cannot be undertaken
within eight hours, store eluate immediately
at -70° C.
3.3 Reconcentration — Method B. Aluminum Hydroxide-Hydroextraction
Procedure (see Figure 6-8.2 for flow diagram of procedure)
Use the aluminum hydroxide-hydroextraction procedure to
reconcentrate viruses from turbid eluates that result from the
procedures described in Section 3.1.4 and from eluates that
become turbid upon acidification during the membrane disc
procedure described in Section 3.2.3, Step (b).
3.3.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with combination-type electrode.
(c) Instrument tray, stainless steel — overall
dimensions 43 cm x 10 cm x 5 cm (Vollrath Co., No.
83170, or equivalent).
(d) Oialyzer tubing, molecular weight cutoff 12,000,
3-cm diameter (Arthur H. Thomas Co., No. 3787-D42,
or equivalent).
(e) Clamps, dialyzer tubing (Arthur H. Thomas Co., No.
3787-N30, or equivalent).
(f) Centrifuge tubes, screw-capped, round-bottom ~
50-100 m|_.
- 6-45 -
-------
3.3.2 Media and Reagents
(a) Hydrochloric acid (HC1) — 12 M (concentrated) HC1
solution.
(b) Sodium hydroxide (NaOH) -- 10 M.
Prepare 500 ml of 10 M NaOH.
(c) Aluminum chloride (AlClg) -- 0.3 M.
Prepare 500 ml of 0.3 M A1CU.
(d) Sodium carbonate (Na2C03) — 1 M.
Prepare 100 ml of 1 M Na^CO^
(e) Glycine.
(f) Acid glycine solution — 1 M glycine adjusted to pH
2 with 12 M HC1.
Prepare 2 liters of acid glycine solution.
(g) Basic fetal calf serum (BFCS) with glycine -- 1 M
glycine in fetal calf serum (PCS) adjusted to pH
11.5 + 0.1 with 10 M NaOH.
Prepare 100 ml of BFCS with glycine. To prepare
BFCS with glycine, autoclave glycine powder in a
covered vessel, add PCS, and adjust pH.
(h) Phosphate-buffered saline -- Solution A: Sodium
chloride (NaCl), 40 g; potassium chloride (KC1),
1 g; calcium chloride (CaClp), 0.5 g; magnesium
chloride (MgClp'SHpO), 0.5 g; deionized
distilled water to 4 liters. Solution B; Sodium
phosphate, dibasic (Na2HP04), 1 g; potassium
phosphate, monobasic, (KH^O^), 1 g; deionized
distilled water to 1 liter. Prepare solutions A and
B separately, then mix them together in a ratio of
1:1.
- 6-46 -
-------
(i) Polyethylene glycol -- 20,000 MW.
Three kg of polyethylene glycol are required.
(j) Antibiotics
Prepare as indicated for medium in Chapter 9,
Section 7.18.
3.3.3 Procedure
(a) Preparation of dialysis bag.
(a.l) Soak a 40-cm length of dialyzer tubing in
deionized distilled water for five minutes.
(a.2) Fold one end of the tubing over itself to
form a 2-cm overlap.
(a.3) Center dialyzer tubing clamp over overlap,
and lock clamp in place to form dialysis bag.
(a.4) Grasp undamped end of dialysis bag between
thumb and forefinger, and rub the facing
surfaces against each other.
This procedure separates the facing surfaces
and opens the dialysis bag.
(a.5) Fill dialysis bag two-thirds full with
deionized distilled water.
(a.6) Fold free end of dialysis bag over itself to
form a 2-cm overlap.
(a.7) Center dialyzer tubing clamp over overlap,
and lock clamp in place.
(a.8) Squeeze dialysis bag gently to force water
against clamped ends.
If water leaks through either clamped end,
remove clamp from faulty seal and repeat
Steps (a.6-a.8).
- 6-47 -
-------
(a.9) Squeeze dialysis bag near clamps, exerting
sufficient pressure to test the integrity of
the tubing.
If water leaks through tubing, discard tubing
and repeat Steps (a.1-a.9).
(a.10) Sterilize dialysis bag by the procedure
described in Chapter 3.
(a.ll) After sterilization, recheck dialysis bag for
leaks by repeating Steps (a.8-a.9).
(a.12) Store dialysis bag in deionized distilled
water at 4° C.
(b) Flocculation and hydroextraction.
(b.l) Measure volume of eluate against graduations
on beaker (from Section 3.1.4, Step [1]).
(b.2) Place stir bar into eluate.
(b.3) Place beaker on magnetic stirrer, and stir at
speed sufficient to develop vortex in eluate.
(b.4) Add sufficient volume of 0.3 M A1C1- to
eluate to obtain a final A1C13
concentration of 0.003 M.
(b.5) Place pH electrode into eluate.
(b.6) Adjust pH of eluate to 7 + 0.1 with 1 M
Na2C03.
An A1(OH)0 floe forms as Na0CO0 is
11 — • • ' J Ł o
added.
(b.7) After pH 7 +_ 0.1 is obtained, stir for five
minutes.
(b.8) Remove pH electrode.
(b.9) Turn off magnetic stirrer.
- 6-48 -
-------
(b.10) Allow floe to settle for 30 minutes.
(b.ll) Aspirate liquid to 2-3 cm above level of floe.
(b.12) Discard aspirated liquid.
(b.13) Resuspend settled floe in remaining liquid.
Resuspend floe by swirling beaker or by
placing beaker on magnetic stirrer and
activating stirrer.
(b.14) Pour suspended floe into round bottom
centrifuge tube(s).
Screw-capped centrifuge tubes with a capacity
of 50-100 ml are usually adequate. To
prevent transfer of stir bar into centrifuge
tube") hold another stir bar or magnet
underneath beaker when decanting contents.
(b.15) Centrifuge tube(s) at 1000 x Ł for three
minutes.
(b.16) Decant or aspirate supernate.
(b.17) Discard supernate.
(b.18) Measure approximate volume of Al(OH)3
residue.
To a centrifuge tube similar to that
containing the A1(OH)- residue, add water
to a level equal to the height of the
residue, and estimate volume of residue by
measuring the volume of water in a graduated
cylinder or pipette.
(b.19) Add to each volume of Al(OH)3 residue,
three volumes of BFCS-glycine, pH 11.5 + 0.1.
- 6-49 -
-------
(b.20) Shake centrifuge tube(s) for five seconds to
mix contents.
(b.21) Centrifuge each Al(OH)3 -- BFCS-glycine
suspension for three minutes at 1000 x Ł.
(b.22) Pour BFCS-glycine supernate(s) into beaker.
(b.23) Place stir bar into beaker.
(b.24) Place beaker on magnetic stirrer, and stir
contents of beaker at a speed sufficient to
develop vortex.
(b.25) Place pH electrode into BFCS-glycine.
(b.26) Adjust pH of BFCS-glycine to 7+0.1 with 1 M
acid glycine solution.
(b.27) Turn off magnetic stirrer.
(b.28) Remove pH electrode.
(b.29) Discard residue from Step (b.21).
(b.30) Remove dialysis bag from storage, and wipe
exterior of bag with towel.
(b.31) Remove clamp from one end of dialysis bag.
(b.32) With sterile scissors, remove end of dialysis
bag by cutting across the bag through center
of clamp impression.
This procedure removes inside edge of bag
that had been exposed to contamination.
(b.33) Discard water from dialysis bag.
(b.34) Pour neutralized BFCS-glycine eluate into
dialysis bag.
(b.35) Fold open end of the bag over itself to form
a 2-cm overlap.
- 6-50 -
-------
(b.36) Center dialyzer tubing clamp over overlap,
and lock clamp in place.
(b.37) Place a 1-cm layer of polyethylene glycol
into instrument tray.
(b.38) Place dialysis bag on polyethylene glycol.
(b.39) Add sufficient polyethylene glycol to cover
dialysis bag.
(b.40) Place cover on instrument tray.
(b.41) Maintain instrument tray overnight at 4 C.
Hydroextract until.approximately 10 to 20 ml
of concentrated neutralized BFCS-glycine
remain in bag.
(b.42) Rinse polyethylene glycol from outside
surface of dialysis bag with deionized
distilled water.
(b.43) Pour 900 ml of chilled (4-10° C) phosphate-
buffered saline into a 1-liter beaker.
Maintain phosphate-buffered saline at
4-10° C; carry out Steps (b.44-b.47) in
coJhd^^njorMrijco'td by other means available.
(b.44) Place beaker of phosphate-buffered saline on
magnetic stirrer.
(b.45) Place stir bar into phosphate-buffered
saline, and stir at a speed sufficient to
develop vortex.
(b.46) Immerse dialysis bag into phosphate-buffered
saline.
Care must be taken not to puncture bag with
stir bar.
- 6-51 -
-------
(b.47) Stir for one hour.
(b.48) Remove dialysis bag from phosphate-buffered
saline and wipe exterior of bag with towel.
jo; , . • ,• , ; • . '' •
(b.49)~Remove clamp from one end of dialysis bag.
.,0: •
(b.50) With sterile scissors, remove end of dialysis
bag by cutting across the bag through center
of clamp impression.
This procedure removes inside edge of bag
that had been exposed to contamination.
(b.51) Pour concentrate into 100-mL graduated beaker.
(b.52)~Hold dialysis bag in fully inverted position
over beaker.
(b.53) Place upper end of bag between forefinger and
middle finger in a scissors grip.
(b.54) Squeeze bag between fingers in scissors grip.
(b.55) Pull fingers down over length of bag to
remove remaining concentrate.
that fingers donot contaminate
concentrate.
(b.56) Determine volume of concentrate in beaker.
(b.57) Add antibiotics to concentrate in accordance
with instructions given for medium in
Chapter 9, Section 7.18.
(b.58) Refrigerate concentrate immediately at
4° C, and maintain at that temperature
until concentrate is assayed for viruses.
If assay for viruses cannot be undertaken
within eight hours, store concentrate
immediately at -70° C.
- 6-52 -
-------
4. ELUTION AND RECONCENTRATION — METHOD TWO
This method may be used for eluting viruses from filters that cannot
be safely eluted with Method One (this method should be as effective as
: {•
Method One for eluting viruses sensitive to pH 10.5).
4.1 Procedure for Eluting Viruses from Filters (see Figure 6-11.1 for
flow diagram of procedure)
4.1.1 Apparatus and Materials
(a) Positive pressure source equipped with a pressure
gauge.
Gauge necessary only if pressure source is capable
of producing pressures exceeding tolerances of
equipment. Pressure source, if laboratory air line
or pump, must be equipped with an oil filter. If
source is capable of producing high pressure,
deliver to pressure vessel and filter holder no more
pressure than recommended by manufacturer.
(b) Dispensing pressure vessel — 4 liters (Millipore
Corp., or equivalent).
(c) Beaker, graduated — 2 liters.
(d) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode.
(e) Autoclavable inner-braided tubing fitted with metal
quick-disconnect connectors on one end and glass
elbow on the other.
Make glass elbow from a 13-cm length (approximate
P.P., 6 mm) of glass tubing by making a 40 degree
bend about 5 cm from one end. Connect tubing onto
longer end of elbow.
(f) Magnetic stirrer and stir bars.
- 6-53 -
-------
Figure 6-11.1 Virus Elution
Procedure
VIRUS-BEARIIIG CARTRIDGE FILTER
Force 1600 nL of buffered
3% beef extract (BE), pH 9
through cartridge fllter(s)
slowly (Apply pressure no
greater than 0.4 kg/cm ).
FILTERED ELUATE (3% BE)
If concentration of
viruses Is not necessary
Assay eluate (3% BE) for
viruses (See Chapter 9).
If concentration of viruses
Is necessary proceed below.
Figure 6-11.2 Virus Concentration
Procedure (Katzenelson
Organic Ftocculatlon
Procedure)
FILTERED ELUATE (3% BE)
On magnetic stlrrer, adjust pH
of filtered eluate (3% BE)
to 3.5 ^0.1 with 1 (I HC1.
Floe begins to form.
Mix filtered eluate (3S BE)
and forming floe on magnetic
stlrrer for 30 minutes.
FLOCCEP ELUATE
Centrifuge flocced eluate at
2,500 x Ł for 15 minutes
at 4° C. Discard supernate,
retain floe.
FLOC
Add 0.15 II Ma2HP04 (l/20th
volume of 3% BE) to floe,
and nix.
Floe dissolves on mixing.
DISSOLVED
Adjust pH to 7.0-7.5 with
1 II HC1 or 1 H HaOH
v Assay dissolved floe for
viruses (See Chapter 9).
Figure 6-11. Flow Diagram of Beef Extract Method for Elutlng Viruses from Cartridge
Filters with Buffered 3% Beef Extract and for Concentrating Eluted
Viruses by the Katzenelson Organic Flocculatlon Procedure.
-------
4.1.2 Media and Reagents
(a) Sodium hydroxide (NaOH) — 1 M.
Prepare 500 ml of 1 M NaQH.[ This solution may be
i
stored for several months at room temperature.
(b) Glycine.
(c) Beef extract powder (Grand Island Biological Co., or
equivalent).
Prepare buffered 3% beef extract by dissolving 60 g
of beef extract powder and 7.5 g of glycine (final
concentration = 0.05 M) in 2 liters of distilled
water. Autoclave beef extract solution, and adjust
pH to 9 + 0.1 with 1 M NaOH.
4.1.3 Rearrangement of Apparatus
(a) Rearrangement for Method One (see Figures 6-2 and
6-3).
(a.l) Disconnect at pipe tee F, the tubing leading
to inlet port H-, of filter holder H.
(a.2) Connect free end of tubing from inlet port
H-| of filter holder H to outlet port of
pressure vessel.
Pressure vessel is not shown in Figures 6-2
and 6-3.
(a.3) Connect inlet port of pressure vessel to
positive air pressure source.
(a.4) Disconnect tubing from outlet port HO of
filter holder H.
(a.5) Hook glass elbow with 40 degree bend onto
pouring spout of a 2-liter glass beaker.
- 6-55 -
-------
Raise aluminum foil covering beaker enough to
expose only the pouring spout.
(a.6) Connect free end of tubing from glass elbow
to outlet port H3 of filter holder H.
(a.7) Crimp aluminum foil cover over glass elbow.
(a.8) Elute viruses from filter as described in
Section 4.1.4 below.
(b) Rearrangement for Method Two (see Figures 6-4 and
6-5).
(b.l) Disconnect at pipe tee D, the tubing leading
to the inlet port F-i of filter holder F.
(b.2) Connect free end of tubing from inlet port
F-j of filter holder F to outlet port of
pressure vessel.
Pressure vessel is not shown in Figures 6-4
and 6-5.
(b.3) Connect inlet port of pressure vessel to
positive pressure source.
(b.4) Disconnect tubing from outlet port 63 of
filter holder 6.
(b.5) Disconnect at outlet port H, of fluid
proportioner H, the tubing leading to the
inlet port K-, of filter holder K.
(b.6) Connect free end of tubing from inlet port
K-j of filter holder K to outlet port 63
of filter holder G.
(b.7) Disconnect tubing from outlet port K3 of
filter holder K.
- 6-56 -
-------
(b.8) Hook glass elbow with 40 degree bend onto
pouring spout of a 2-liter graduated glass
beaker.
Raise aluminum foil covering beaker enough to
expose only the pouring spout.
(b.9) Connect free end of tubing from glass elbow
to outlet port K3 of filter holder K.
(b.10) Crimp aluminum foil cover over glass elbow.
(b.ll) Elute viruses from filter as described in
Section 4.1.4 below.
4.1.4 Elution Procedure
(a) Remove top of pressure vessel.
(b) Pour into pressure vessel 1600 ml of buffered 3%
beef extract (pH 9).
(c) Replace top of pressure vessel.
(d) Close vent/relief valve on pressure vessel.
(e) Open vent/relief valve on cartridge filter holder.
If more than one cartridge filter is used, open
valves on all holders.
(f) Apply pressure sufficient to purge trapped air from
filter apparatus.
(g) Close vent/relief valve on (each) cartridge filter
holder as soon as buffered 3% beef extract solution
begins to flow from valve.
(h) Wipe up spilled liquid with laboratory disinfectant.
(i) Increase pressure to that sufficient to force
buffered 3% beef extract solution through the
filter(s).
o
Do not exceed a pressure of 0.4 kg/cm so that
- 6-57 -
-------
buffered 3% beef extract solution passes through
cartridge filter(s) slowly thereby maximizing
elation contact period. When air enters line from
pressure vessel, elevate and invert filter holder(s)
to permit complete evacuation of buffered 3% beef
extract from filters.
(j) Turn off pressure at source.
(k) Open vent/relief valve on pressure vessel.
(1) Proceed to Section 4.2 immediately.
If concentration of viruses cannot be undertaken
immediately, eluate may be stored for up to eight
hours at 4° C before reconcentration.
If reconcentration cannot be undertaken within eight
hours, store eluate immediately at -70° C.
Instructions for reconcentrating viruses begin in
Section 4.2.
4.2 Organic Flocculation Concentration Procedure of Katzenelson (see
Figure 6-11.2 for flow diagram of procedure)
It is preferable to assay eluted viruses in the beef extract
eluate without further concentrating them because some loss of
viruses may occur in concentration. However, the numbers of cell
cultures needed for assays may be reduced by further
concentrating the viruses.
4.2.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode.
- 6-58 -
-------
(c) Refrigerated centrifuge capable of attaining
2500 x jj.
Each sample centrifuged at 2500 x g will consist of
about 1600 ml.
4.2.2 Media and Reagents
(a) Disodium hydrogen phosphate (Ma2HP04*7H20)
— 0.15 M.
(b) Hydrochloric acid (HC1) — 1 M.
(c) Sodium hydroxide (NaOH) ~ 1 M.
4.2.3 Procedure
(a) Place stir bar into graduated beaker containing
buffered 3% beef extract eluate from 4.1.4 (1).
(b) Place beaker that contains the beef extract on
magnetic stirrer, and stir at a speed sufficient to
develop vortex.
To minimize foaming (which may inactivate yijruses),
do not mix faster thannecessary to develop vertex.
(c) Insert pH electrode into beef extract eluate.
(d) Add 1 M HC1 to flask slowly until pH of beef extract
reaches 3.5 +_ 0.1.
A precipitate will form. If pH is accidentally
reduced below 3.4, add 1 M MaOH until pH is 3.5 +
0.1. Avoid reducing pH below 3.4 because some
inactivation of viruses may occur.
(e) Remove pH electrode from beaker, and continue to
stir for 30 minutes more.
(f) Remove caps from screw-capped centrifuge bottles.
- 6-59 -
-------
Glass centrifuge bottles may not be able to
withstand g force that will be applied.
(g) Pour contents of beaker into centrifuge bottles.
To prevent transfer of stir bar into centrifuge
bottle, hold another stir bar or magnet against
bottom of beaker when decanting contents.
(h) Replace and tighten down caps on centrifuge
bottles.
(i) Centrifuge precipitated beef extract suspension in
refrigerated centrifuge (4° C) for 15 minutes at
2500 ?l 9-
(j) Remove caps from screw-capped centrifuge bottles.
(k) Pour supernates into graduate cylinder, and record
volumes.
(1) Discard supernates.
(m) Place a stir bar into each centrifuge bottle that
contains precipitate.
(n) To each precipitate, add 5 mL of 0,15 M Na2HP04
'7H20 for each 100 mL of supernate decanted.
(o) Replace and tighten down caps on centrifuge bottles,
(p) Place each centrifuge bottle on a magnetic stirrer,
and stir each precipitate slowly until it has
dissolved completely.
Support bottles as necessary to prevent toppling.
Avoid foaming which may inactivate or aerosolize
viruses. Precipitate may be partially dissipated
with spatula before or during stirring procedure.
(q) Remove caps from screw-capped centrifuge bottles.
- 6-60 -
-------
(r) Remove foil cover from 250-mL beaker.
(s) Combine the dissolved precipitates in beaker.
To prevent transfer of stir bar into beaker, hold
another stir bar or magnet:against the bottom of the
centrifuge bottle when decanting concentrate.
(t) Measure pH of concentrate (dissolved precipitate).
If pH is above or below 7.0-7.5, adjust to that
range with either 1 M HCl or 1 M NaOH.
(u) Replace foil cover securely on beaker.
(v) Refrigerate concentrate immediately at 4° C, and
maintain at that temperature until assay for viruses
is undertaken.
If assay for viruses cannot be undertaken within
eight hours, store concentrate immediately at
-70°_C.
(w) Assay for viruses in accordance with instructions
given in Chapter 9.
- 6-61 -
-------
5. BIBLIOGRAPHY
Block, J.-C., Joret, J.-C., Morlot, M., Foliguet, J.-M. (1978).
Recovery of Enteroviruses in Surface Waters by Adsorption-Elution
on Glass Microfibers. TSM-L'Eau, 73:181-4. French.
Dahling, D. R., Safferman, R. S. (1979). Survival of Enteric Viruses
under Natural Conditions in a Subarctic River. AppH. Environ.
Microbiol. 38:1103-10.
Farrah, S. R., Gerba, C. P., Goyal, S. M., Wallis, C., Melnick, J. L.
(1977). Regeneration of Pleated Filters Used to Concentrate
Enteroviruses from Large Volumes of Tap Water. Appl. Environ.
Microbiol. 33:308-11.
Farrah, S. R., Gerba, C. P. Wallis, C., Melnick, J. L. (1976).
Concentration of Viruses from Large Volumes of Tap Water Using
Pleated Membrane Filters. Appl. Environ. Microbiol. 31:221-6.
Farrah, S. R., Goyal, S. M., Gerba, C. P., Wallis, C., Melnick, J. L.
(1977). Concentration of Enteroviruses from Estuarine Water.
Appl. Environ. Microbiol. 33:1192-6.
Fattal, B., Katzenelson, E., Hostovesky, T., Shuval, H. I. (1977).
Comparison of Adsorption-Elution Methods for Concentration and
Detection of Viruses in Water. Water Res. 11:955-8.
Gerba, C. P., Farrah, S. R., Goyal, S. M., Wallis, C., Melnick, J. L.
(1978). Concentration of Enteroviruses from Large Volumes of Tap
Water, Treated Sewage, and Seawater. Appl. Environ. Microbiol.
35:540-8.
Hill, W. F., Jr., Jakubowski, W., Akin, E. W., Clarke, N. A. (1976).
Detection of Virus in Water: Sensitivity of the Tentative
Standard Method for Drinking Water. Appl. Environ. Microbiol.
31:254-61.
- 6-62 -
-------
Jakubowski, W., Chang, S.-L., Ericksen, T. H., Lippy, E. C., Akin, E. W.
(1978). urge-Volume Sampling of Water Supplies for
Microorganisms. J. Amer. Water Works Assn. 70:702-6.
Katzenelson, E., Fattal, B., Hostovesky, T. (1976). Organic
Flocculation: an Efficient Second-Step Concentration Method for
the Detection of Viruses in Tap Water. Appl. Environ. Microbiol.
32:638-9.
Landry, E. F., Vaughn, J. M., Thomas, M. Z>, Vicale, T. J. (1978).
Efficiency of Beef Extract for the Recovery of Poliovirus from
Wastewater Effluents. Appl. Environ. Microbiol. 36:544-8.
Payment, P., Gerba, C. P., Wallis, C., Melnick, J. L. (1976). Methods
for Concentrating Viruses from Large Volumes of Estuarine Water on
Pleated Membranes. Water Res. 10:893-6.
Payment, P., Trudelet, M., Pavilanis, V. (1978). Evaluation of the
Efficiency and the Technique of Adsorption-Elution of Poliovirus 1
on Fiberglass Filters: Application to the Virologic Analysis of
100 ml to 1,000 Liters of Water. Can. J. Microbiol. 24:1413-16.
French.
Sobsey, M. D., Gerba, C. P., Wallis, C., Melnick, J. L. (1977).
Concentration of Enteroviruses from Large Volumes of Turbid
Estuary Water. Can. J. Microbiol. 23:770-8.
Wellings, F. M., Lewis, A. L., Mountain, C. W. Viral Concentration
Techniques for Field Sample Analysis. J^TI "Virus Aspects of
Applying Municipal Waste to Land," Symposium Proceedings, edited
by L. B. Baldwin, J. M. Davidson, and J. F. Gerber. Center for
Environmental Programs, university of Florida, Gainesville (1976),
45-51.
- 6-63 -
-------
-------
CHAPTER 7
METHOD FOR RECOVERING VIRUSES FROM SLUDGES (AND OTHER SOLIDS)
The method described below may be used for raw primary and raw activated
sludges, and for such sludges after they have been digested mesophilically
or thermophilically. Although limited supporting experimental data are
available, the method is probably also useful for other sludges, soils, and
dredge spoils. See Figure 7-1 for flow diagram of the method. If sludges,
soils, or dredge spoils are toxic to cell cultures used for assay of
viruses, obtain fresh sample material, and use method described in Chapter
8.
Use aseptic techniques and sterile materials and apparatus only. Sterilize
all contaminated materials before discarding them (see Chapters 2 and 3).
1. EXTRACTION OF VIRUSES FROM SLUDGES
1.1 Preparation
1.1.1 Apparatus and Materials
(a) Refrigerated centrifuge capable of attaining 10,000 x
Ł and screw-capped centrifuge bottles that can
withstand 10,000 x Ł.
Each sample centrifuged at 10,000 x g will consist of
about 100 mL.
(b) pH meter, measuring to an accuracy of at least 0.1 pH
unit, equipped with a combination-type electrode.
- 7-1 r
-------
(c) Magnetic stirrer and stir bars.
(d) Membrane fi,l,ter apparatus for sterilization — 47-mm
diameter filter holder and 50-mL slip tip syringe
(Millipore Corp., Swinnex filter, No. SX0047000, or
equivalent for filter holder only).
If final eluate must be concentrated by the organic
flocculation procedure of Katzenelson (see Section
2), membrane filter apparatus is not required.
(e) Disc filters, 47 mm diameter — 3.0-, 0.45-, and
0.25-jun pore size filters (Filterite Corp., Duo-Fine
series, or equivalent). Filters must be cut to
proper size from sheet filters.
Disassemble Swinnex filter holder. Place filter
with 0.25-;jm pore size on support screen of filter
holder, and stack the remaining filters on top in
order of Increasing pore size. Reassemble and
tighten filter holder. Filters stacked in tandem as
described tend to clog more slowly when turbid
material is filtered thr'dugH..them. Prepare several
filter stacks.
If final eluate must be.concentrated by the organic
flocculation procedure of Katzenelson (see
Section 2), disc filters are not required.
1.1.2 Media and Reagents
(a) Disodium hydrogen phosphate (Na_HP04*7H20).
(b) Citric acid.
(c) Beef extract powder (Grand Island Biological Co., or
equivalent).
- 7-2 -
-------
Prepare buffered 10% beef extract by dissolving 10 gm
beef extract powder, 1.34 g Na0HPO/i'7H00 and
'' I _ ... -- -I - r. .1. ._ I • II *lŁ\l I I f| ' Cm* ' ™ " '
0.12 g citric acid in 100 ml of deionized distilled
water. Dissolve by stirring for about two hours on a
magnetic stirrer. Autoclave at 121° C for 15
minutes.
(d) Hydrochloric acid (HCl) — 5 M.
(e) Aluminum chloride (A1C13*6H20) — 0.05 M.
Autoclave AlC13solution at 121° G for 15 minutes.
(f) Sodium hydroxide (NaOH) — 5 M.
1.2 Procedure (See Figure 7-1.1)
1.2.1 Conditioning of Sludge
In the absence of experience that dictates otherwise, use
1QO-m|_ volumes for sludges, 100-mL volumes of a 5%
suspension in deionized distilled water for dredge spoils
(v/v) or soils (w/v), and lOO-g quantities for digested
dewatered sludges and for other samples difficult to
measure volumetrically.
(a) Measure 100 ml of we11-mixed sludge in a graduated
100-mL cylinder.
Sludge must be mixed vigorously immediately before it
is poured into cylinder because sludge solids, which
contain most of the viruses, begin to settle out
immediately after mixing stops.
(b) Place stir bar into a 250-mL beaker.
(c) Pour the 100 ml of measured sludge from the cylinder
into the 250-mL beaker.
It may be necessary to pour sludge several times
- 7-3 -
-------
Figure 7-1.1
Virus Recovery
procedure
SLUDGE (100 ml)
On magnetic stirrer, add AlCl-j (to final
concentration 0.0005 H), adjust pH of salted
sludge to 3.5 +^ 0.1 with 5 M HC1, and nix
vigorously for 30 nlnutes.
SALTED, pH-ADJUSTED SLUDGE
Centrifuge salted, pH-adjusted sludge at
2,500 x Ł for 15 nlnutes. Discard supernate,
retain sludge sol Ids.
SLUDGE SOLIDS
Add 100 mL of buffered 10X beef extract (BE)
(pH 7.0 ^0.1) to sludge solids. Mix
resuspended sludge solids on magnetic
stirrer for 30 minutes to elute viruses.
RESUSPEHDED SLUDGE SOLIDS
Centrifuge resuspended sludge solids at
10,000 x Ł for 30 minutes. Discard
sol Ids, retain eluate (supernate).
ELUATE
If concentration of viruses 1s not necessary,
•filter eluate through membrane stack.
If concentration of viruses jiŁ necessary, proceed
to Virus Concentration Procedure (Figure 7-1.2)
FILTERED ELUATE (1 OS BE)
Assay eluate (10% BE) for
viruses (See Chapter 9).
Figure 7-1.2 Virus Concentration
Procedure
ELUATE
Filter eluate through membrane stack.
FILTERED ELUATE (1 OS BF)
Add sufficient volume of deionlzed distilled
water to filtered eluate to reduce BE
concentration from 10% to 3%.
Begin Katzenelson organic flocculatlon procedure.
DILUTED FILTERED ELUATE (3% BE)
On magnetic stirrer, adjust pH of filtered
eluate (3% BE) to 3.5 + 0.1 with 1 H HC1.
Floe begins to form.
Mix filtered eluate (3% BE) and forming floe
on magnetic stirrer for 30 minutes.
FLOCCED ELUATE
Centrifuge flocced eluate at 2,500 x g for
15 minutes at 4° C. Discard supernate,
retain floe.
FLOC FROM ELUATE
Add 0.15 tl tla2HP04 (l/20th volume of
diluted beef extract) to floe, and nix.
Floe dissolves on nixing.
DISSOLVED FLOC
Adjust pH to 7.0-7.5 with
1 II HCl or 1 H NaOH
Assay dissolved floe for
viruses (See Chapter 9).
Figure 7-1. Flow Diagram of Method for Recovering and
Concentrating Viruses In Sludges
- 7-h -
-------
from beaker to cylinder and back In order to remove
all sludge solids to beaker.
CAUTION: Take care to avoid formation of aerosols.
(d) Place beaker on magnetic stirrer, and stir at speed
sufficient to develop vortex.
(e) Add 1 ml of 0.05 M AlClg to mixing sludge.
Final concentration of AlCK in sludge is
approximately 0.0005 M.
(f) Place combination-type pH electrode into mixing
sludge.
pH meter must be standardized at pH 4.
(g) Adjust pH of sludge to 3.5 _+ 0.1 with 5 M HC1.
If pH falls below 3.4, readjust it with 5 M NaOH.
When sludge adheres to electrodes, clean electrodes
by moving them up and down gently in mixing sludge.
(h) Continue mixing for 30 minutes more.
The pH of the sludge should be checked at frequent
intervals. If the pH drifts up, readjust it to 3.5
+ 0.1 with 5 M HC1. If the pH drifts down, readjust
it with 5 M NaOH.
(i) Turn off stirrer, and remove pH electrode from
sludge.
(j) Remove cap from a screw-capped centrifuge bottle.
Glass centrifuge bottles may not withstand
10,000 x g force that will be applied.
(k) Pour conditioned sludge into centrifuge bottle.
To prevent transfer of stir bar into centrifuge
bottle when decanting sludge, hold another stir bar
- 7-5 -
-------
or magnet against bottom of beaker. Sludge that
adheres to stir bar in the beaker may be removed by
manipulation with a pipette. It may be necessary to
pour sludge several times from centrifuge bottle to
beaker and back In order to remove all sludge solids
to bottle.
CAUTION: Take care to avoid formation of aerosols.
(1) Replace and tighten down cap on centrifuge bottle.
(m) Centrifuge conditioned sludge at 2,500 x Ł for 15
minutes at 4° C.
(n) Remove cap from centrifuge bottle.
(o) Decant supernate into beaker.
(p) Replace cap on centrifuge bottle.
(q) Discard supernate.
1.2.2 Elution of Viruses from Sludge Solids
(a) Remove cap from centrifuge bottle that contains
sedimented, conditioned sludge [from Section 1.2.1,
Step (p)].
(b) Place stir bar into centrifuge bottle.
(c) Add 100 ml of buffered 10% beef extract to the
sedimented, conditioned sludge.
The volume of buffered 10% beef extract used to
elute viruses from the conditioned sludge is equal
to the original volume of the sludge sample [Section
1.2.1, Step (a)].
(d) Replace and tighten down cap on centrifuge bottle.
(e) Place centrifuge bottle on magnetic stirrer, and
- 7-6 -
-------
stir at speed sufficient to develop vortex.
To minimize foaming (which may inactivate viruses),
do not mix faster than necessary to develop vortex.
Care must be taken to prevent bottle from toppling.
Stabilize bottle as necessary.
(f) Continue mixing for 30 minutes.
(g) Turn off stirrer.
(h) Remove cap from centrifuge bottle.
(i) With long forceps or magnet retriever, remove stir
bar from centrifuge bottle.
(j) Replace and tighten down cap on centrifuge bottle.
(k) Centrifuge conditioned sludge-eluate mixture at
10,000 x Ł for 30 minutes at 4° C.
(1) Remove cap from centrifuge bottle.
(m) Decant eluate into beaker, discard sludge sediment.
The number of cell cultures necessary for the viral
assay may be reduced by concentrating the viruses in
the beef extract by the organic flocculation
procedure of Katzenelson. Some loss of viruses may
occur with this procedure. If viruses in eluates
are to be concentrated, proceed immediately to
Section 2. If concentration is not required,
proceed to Step (n).
(n) Place a filter holder that contains a filter stack
on a 250-ml_ Erlenmeyer receiving flask.
(o) Load 50-mL syringe with eluate.
(p) Place tip of syringe into filter holder.
- 7-7 -
-------
(q) Force eluate through filter stack into 250-mL
receiving flask.
Take care not to break off tip of syringe and to
minimize pressure on receiving flask because such
pressure may splinter or topple the flask. If
filter stack begins to clog badly, empty loaded
syringe into beaker containing unfiltered eluate,
fill syringe with air, and inject air into filter
stack to force residual eluate from filters.
Continue filtration procedure with another filter
holder and filter stack. Discard contaminated
filter holders and filter stacks. Steps (n) thru
(q) may be repeated as often as necessary to filter
entire volume of eluate. Disassemble each filter
holder and examine bottom filters to be certain they
have not ruptured. If a bottom filter has ruptured,
repeat Steps (n) through (q) with new filter holders
and filter stacks.
(r) Refrigerate eluate immediately at 4 C, and
maintain it at that temperature until it is assayed
for viruses.
If assay for viruses cannot be undertaken within
eight hours, store eluate immediately at -70° C.
- 7-8 -
-------
2. CONCENTRATION OF VIRUSES FROM SLUDGE ELUATES
2.1 Organic Flocculation Concentration Procedure of Katzenelson
It is preferable to assay eluted viruses in the beef extract
eluate without concentrating them because some loss of viruses
may occur in concentration. However, the numbers of cell
cultures needed for assays may be reduced by concentrating the
viruses in the eluate.
2.1.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode.
(c) Refrigerated centrifuge capable of attaining 2,500 x
Ł and screw-capped centrifuge bottles.
Each sample centrifuged at 2,500 x g will consist of
> :• about 330 ml.
(d) Membrane filter apparatus for sterilization — 47-mm
diameter filter holder and 50-mL slip tip syringe
(Millipore Corp., Swinnex filter, No. SX0004700, or
equivalent, for filter holder only).
(e) Disc filters, 47 mm diameter — 3.0-, 0.45-, and
0.25-pm pore size filters (Filterite Corp., Duo-Fine
series, or equivalent). Filterite must be cut to
proper size from sheet filters.
*Katzenelson, E., B. Fattal, and T. Hostovesky. 1976. Organic
flocculation: an efficient second^step concentration method for the
detection of viruses in tap water. Appl. Environ. Microbiol. 32:638-639.
- 7-9 -
-------
Disassemble Swinnex filter holder. Place filter with
0.25 Jim pore size on support screen of filter holder,
and stack the remaining filters on top in order of
increasing pore size. Reassemble and tighten filter
holder. Filters stacked in tandem as described tend
to clog more slowly when turbid material is filtered
through them. Prepare several filter stacks.
2.1.2 Media and Reagents
(a) Disodium hydrogen phosphate (Na2HP04*7H20)
— 0.15 M.
(b) Hydrochloric acid (HCl) -- 1 M.
(c) Sodium hydroxide (NaOH) — 1 M.
2.1.3 Procedure (See Figure 7-1.2)
(a) Place a filter holder that contains a filter stack on
a 250-mL Erlenmeyer receiving flask.
(b) Load 50-mL syringe with eluate from Section 1.2.2,
Step (m).
(c) Place tip of syringe into filter holder, and force
eluate through filter stack.
Take care not to break off tip of syringe and to
minimize pressure on receiving flask, because such
pressure may splinter or topple the flask. If filter
stack begins to clog badly, empty loaded syringe into
beaker containing unfiltered eluate, fill syringe
with air, and inject air into filter stack to force
residual eluate from filters. Continue filtration
procedure with another filter holder and filter
stack. Discard contaminated filter holders and
- 7-10 -
-------
filter stacks. Steps (a) thru (c) may be repeated as
often as necessary to filter entire volume of
eluate. Disassemble each filter holder and examine
bottom filters to be certain they have not ruptured.
If a bottom filter has ruptured, repeat Steps (a)
through (c) with new filter holders and filter stacks.
(d) Remove filter holder from top of Erlenmeyer flask,
pour eluate into graduated cylinder, and record
volume.
(e) Pour eluate into 600-mL beaker.
(f) For every 3 ml of beef extract eluate, add 7 ml of
deionized distilled water to the 600-mL beaker.
The concentrationof beef extract is now 3%.This
dilution is necessary because10% beef extract often
does not processwell by the organic flpeculation
conce ntrati on procedure.
(g) Record the total volume of the diluted filtered beef
extract.
(h) Place stir bar in beaker that contains diluted
filtered beef extract.
(i) Place beaker that contains the diluted filtered beef
extract on magnetic stirrer, and stir at a speed
sufficient to develop vortex.
To minimize foaming (which may inactivate viruses),
do not mix faster than necessary to develop vortex.
(j) Insert combination-type pH electrode into diluted,
filtered beef extract.
pH meter must be standardized at pH 4.
- 7-11 -
-------
(k) Add 1 M HC1 to flask slowly until pH of beef extract
reaches 3.5 JH 0.1.
A precipitate will form. If pH is accidentally
reduced below 3.4, add 1 H NaOH until pH is 3.5 +
0.1. Avoid reducing pH below 3.4 because some
inactivation of viruses may occur.
(1) Continue to stir for 30 minutes more.
(m) Turn off stirrer.
(n) Remove caps from screw-capped centrifuge bottles.
Use one or more bottles, as needed. Glass centrifuge
bottles may not withstand 2,500 x g force that will
be applied.
(o) Remove electrode from beaker, and distribute contents
of beaker evenly among centrifuge bottles.
To prevent transfer ofstir bar into a centrifuge
bottle, hold another stir bar or magnet against
bottom of beaker when decanting contents.
(p) Replace and tighten down caps on centrifuge bottles.
(q) Centrifuge precipitated beef extract suspensions at
2,500 x Ł for 15 minutes at 4° C.
(r) Remove caps from centrifuge bottles.
(s) Pour off, and discard supernates.
(t) Place a small stir bar into each centrifuge bottle
that contains precipitate.
(u) Replace covers loosely on centrifuge bottles.
(v) Raise caps from tops of centrifuge bottles and divide
a volume of 0.15 M Na2HP04*7H20 equal to
1/20 of the volume recorded in Step (g) equally among
- 7-12 -
-------
the precipitates in the centrifuge bottles.
The volume of 0.15 M NaHPO'yHO in which
the precipitate will be dissolved is equal to 5 ml
for each 100 ml of diluted beef extract.
(w) Replace and tighten down caps on centrifuge bottles.
(x) Place each bottle on a magnetic stirrer, and stir
each precipitate slowly until precipitate has
dissolved completely.
Support bottles as necessary to prevent toppling.
Avoid foaming which may inactivate or aerosolize
viruses. Precipitate may be partially dissipated
with spatula before or during stirring procedure.
(y) Remove caps from centrifuge bottles.
(2) Combine the dissolved precipitates in a small beaker.
To prevent transfer of stir bars into beaker, hold
another stir bar or magnet against the bottom of each
centrifuge bottle when decanting dissolved
precipitates.
(aa) Measure pH of dissolved precipitates.
If pH is above or below 7.0-7.5, adjust to that range
with either 1 M HC1 or 1 M NaOH.
(bb) Refrigerate dissolved precipitates immediately at
4° C, and maintain at that temperature until assay
for viruses is undertaken.
If assay for viruses cannot be undertaken within
eight hours, store dissolved precipitates immediately
at -70° C.
(cc) Assay for viruses in accordance with instructions
given in Chapter 9.
- 7-13 -
-------
3. BIBLIOGRAPHY
Berg, G., D. Berman, and R. S. Safferman. 1982. A method for
concentrating viruses recovered from sewage sludges. Can. J.
Microbiol. 28:553-556.
Berg, G., and D. R. Dahling. 1980. Method for recovering viruses from
river water solids. Appl. Environ. Microbiol. 39:850-853.
Berman, D., G. Berg, and R. S. Safferman. 1981. A method for
recovering viruses from sludges. J. Virol. Methods. 3:283-291.
Brashear, D. A., and R. L. Ward. 1982. Comparison of methods for
recovering indigenous viruses from raw wastewater sludge. Appl.
Environ. Microbiol. 43:1413-1418.
Farrah, S. R., P. R. Scheuerman, and G. Bitton. 1981. Urea-lysine
method for recovery of enteroviruses from sludge. Appl. Environ.
Microbiol. 41:455-458.
Glass, J. S., R. J. Van Sluis, and W. A. Yanko. 1978. Practical
method for detecting poliovirus in anaerobic digester sludge.
Appl. Environ. Microbiol. 35:983-985.
Goddard, M. R., J. Bates, and M. Butler. 1981. Recovery of
indigenous enteroviruses from raw and digested sewage sludges.
Appl. Environ. Microbiol. 42:1023-1028.
Hurst, C. J., S. R. Farrah, C. P. Gerba, and J. L. Melm'ck. 1978.
Development of quantitative methods for the detection of
enteroviruses in sewage sludges during activation and following
land disposal. Appl. Environ. Microbiol. 36:81-89.
Katzenelson, E., B. Fattal, and T. Hostovesky. 1976. Organic
flocculation: an efficient second-step concentration method for
the detection of viruses in tap water. Appl. Environ. Microbiol.
32:638-639.
- 7-14 -
-------
Lund, E., and C.-E. Hedstrom. 1966. The use of an aqueous polymer
phase system for enterovirus isolations from sewage. Am J.
Epidemic!. 84:287-291.
Lund, E., and V. Ronne. 1973. On the isolation of virus from sewage
treatment plant sludges. Water Res. 7:863-871.
Malina, J. F., Jr., K. R. Ranganathan, B. P. Sagik, and B. E. Moore.
1975. Poliovirus inactivation by activated sludge. J. Water
Pollut. Control Fed. 47:2178-2183.
Nielsen, A., and B. Lydholm. 1980. Methods for the isolation of
virus from raw and digested wastewater sludge. Water Res.
14:175-178.
Pancorbo, 0. C., P. R. Scheuerman, S. R. Farrah, and G. Bitton. 1981.
Effect of sludge type on poliovirus association with and recovery
from sludge solids. Can. J. Microbiol. 27:279-287.
Sattar, S. A., and J. C. N. Westwood. 1979. Recovery of viruses from
field samples of raw, digested, and lagoon-dried sludges. Bull.
World Health Org. 57:105-108.
Subrahmanyan, T. P. 1977. Persistence of enteroviruses in sewage
sludge. Bull. World Health Org. 55:431-434.
Turk, C. A., B. E. Moore, B. P. Sagik, and C. A. Sorber. 1980.
Recovery of indigenous viruses from wastewater sludges, using a
bentonite concentration procedure. Appl. Environ. Microbiol.
40:423-425.
Ward, R. L., and C. S. Ashley. 1976. Inactivation of poliovirus in
digested sludge. Appl. Environ. Microbiol. 31:921-930.
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1976.
Demonstration of solids-associated virus in wastewater and
sludge. Appl. Environ. Microbiol. 31:354-358.
- 7-15 -
-------
Wolf, H. W., R. S. Safferman, A. R. Mixon, and C. E. Stringer. 1974.
Virus inactivation during tertiary treatment. J. Amer. Water
Works Assn. 66:526-531.
- 7-16 -
-------
CHAPTER 8
METHOD FOR RECOVERING VIRUSES FROM TOXIC SLUDGES AND SOLIDS
The method described below may be used for raw primary sludges and for
other sludges toxic to cells used for assaying viruses. Although limited
experimental support is available, the method is probably also useful for
toxic soils and toxic dredge spoils. See Figure 8-1 for flow diagram of
the method.
Use aseptic techniques and sterile materials and apparatus only.
Sterilize all contaminated materials before discarding them (see*Chapters
2 and 3).
1. EXTRACTION OF VIRUSES FROM SLUDGES
This procedure, which requires Freon, must be done in a hood that is
vented to the outdoors.
1.1 Preparation
1.1.1 Apparatus and Materials
(a) pH meter, measuring to an accuracy of 0.1 pH unit,
equipped with a combination-type electrode.
(b) Magnetic stirrer and stir bars.
(c) Funnel, Buchner, porcelain, Coors, plate diameter
126 mm (American Scientific Co., F7300-8, or 7
equivalent).
- 8-1 -
-------
Equip funnel with rubber stopper and insert into
2-liter filtering flask. CAUTION: Use only flask
that can withstand vacuum applied. Connect rubber
tube from sidearm of filtering flask through
disinfectant trap to laboratory vacuum line.
(d) Disc filter, AP25, 127 mm diameter (Mi Hi pore Corp.,
AP series, or equivalent).
Place AP25 filter onto plate of Buchner funnel.
(e) Membrane filter apparatus for removing bacteria and
fungi — 47-mm diameter filter holder and 50-mL slip
tip syringe (Mi Hi pore Corp., Swinnex filter, No.
SX0047000, or equivalent for filter holder only).
If final eluate must be concentrated by the organic
flpeculation procedure of Katzenelson (see Section
2), membrane filter apparatus is not required.
(f) Disc filters, 47-mm diameter — 3.0-, 0.45-, and
0.25-|im pore size filters (Filterite Corp., Duo-Fine
series, or equivalent). Filters must be cut to
proper size from sheet filters.
Disassemble Swinnex filter holder. Place filter
with 0.25-fm pore size on support screen of filter
holder, and stack the remaining filters on top in
order of increasing pore size. Reassemble and
tighten filter holder. Filters stacked in tandem as
described tend to clog more slowly when turbid
material is filtered through them. Prepare several
filter stacks.
- 8-2 -
-------
If final eluate must be concentrated by the organic
flocculatign procedure of Katzenelson (see
Section 2), disc filters are not required.
1.1.2 Media and Reagents
(a) Disodium hydrogen phosphate (Na2HP04"7H20).
(b) Citric acid.
(c) Beef extract powder (Grand Island Biological Co., or
equivalent).
Prepare buffered 10% beef extract by dissolving 10 g
beef extract powder, 1.34 g NagHPO^'/HgO
and 0.12 g citric acid in 100 mL of deionized
distilled water. Dissolve by stirring for about two
hours on a magnetic stirrer.
(d) Hydrochloric acid (HC1) — 5 M and 1 M.
(e) Aluminum chloride (A1C13'6H2Q) — 0.05 M.
(f) Aluminum chloride (A1C13*6H20) ~ 0.0005 M,
pH 3.5 + 0.1.
Prepare 500 mL of 0.0005 M A1C13> and autoclave it
at 121° C for 15 minutes. Adjust to pH 3.5 + 0.1
with 1 H HC1.
(g) Sodium hydroxide (MaOH) — 5 M.
(h) Freon (DuPont Freon TF, or equivalent).
Freon does not require sterilization.
1.2 Procedure (See Figure 8-1.1)
1.2.1 Conditioning of Sludge
In the absence of experience that dictates otherwise, use
100-mL volumes for sludges. 100-mL volumes of a 5%
suspension in deiom'zed distilled water for dredge spoils
- 8-3 -
-------
Figure 8-1.1
Virus Recovery
Procedure
SLUDGE (100 UL)
On magnetic stlrrer, add AlCl, (to final
concentration 0.0005 M), adjust pH of salted
sludge to 3.5 +_ 0.1 with 5 H HC1, and mix
vigorously for 30 nlnutes.
SALTED, pH-ADJUSTED SLUDGE
Add 75 nL of Freon to mixing sludge, and
continue vigorous mixing for 5 nlnutes.
I
FREOH-TREATED, SALTED, pH-ADJUSTED SLUDGE
Pour sludge-Freon mixture Into Buchner
funnel that contains an AP25 filter,
and apply vacuum. Safely dispose of
chemicalIy-n1crob1olog1cally contaminated
flask and contents.
SLUDGE SOLIDS
When sol Ids on filter appear dry, wash solIds
five tines with 100-nL amounts of 0.0005 H
AlClj, pH 3.5 +^0.1.
HASHED SLUDGE SOLIDS
Pour 100-nL of buffered lot beef extract (BE)
(pH 7.0 Ł 0.1) onto washed sludge solids,
wait 10 minutes, and apply suction.
ELUATE
If concentration of viruses 1s not necessary,
filter eluate through membrane stack.
If concentration of viruses jŁ necessary, proceed
to Virus Concentration Procedure (Figure 8-1.2)
FILTERED ELUATE (lOi BE)
^Assay eluate (101 BE) for
viruses (See Chapter 9).
Figure 8-1.2
Virus Concentration
Procedure
aUATE
Filter eluate through membrane stack.
FILTERED ELUATE (101 BE)
Add sufficient volume of delonlzed distilled
water to filtered eluate to reduce BE
concentration from 101 to 31.
Begin Katienelson organic flocculatlon procedure.
DILUTED FILTERED ELUATE (31 BE )
On magnetic stlrrer, adjust pH of filtered
eluate (31 BE) to 3.5 + 0.1 with 1 H HCl.
Floe begins to form.
H1x filtered eluate (31 BE) and forming floe
on magnetic stlrrer for 30 minutes.
FLOCCED aUATE
Centrifuge flocced eluate at 2,500 x
for
15 minutes at 4
retain floe.
C. Discard supernate,
FLOC FROM ELUATE
Add 0.15 H Ha2HP04 (l/20th volume of
diluted beef extract) to Hoc, and mix.
Floe dissolves on mixing.
DISSOLVED ROC
Adjust pH to 7.0-7.5 with
1 H HCl or 1 H NaOH
.Assay dissolved floe for
viruses (See Chapter 9).
Figure 8-1.
Flow Dligrw of Method for Recovering and
Concentrating Viruses 1n Toxic Sludges
- 8-4 -
-------
(v/v) or soils (w/v), and 100-g quantities for digested
dewatered sludges and for other samples difficult to
measure volumetrically.
(a) Measure TOO ml of well-mixed sludge in a graduated
cylinder.
Sludge must be mixed vigorously immediately before
it is poured into cylinder because sludge solids,
which contain most of the viruses, begin to settle
out immediately after mixing stops.
(b) Place stir bar into a 600-mL beaker.
(c) Pour the 100 ml of measured sludge from the 100-mL
cylinder into the 600-mL beaker.
It may be necessary to pour sludge several times
from beaker to cylinder and back in order to remove
all sludge solids to beaker.
CAUTION: Take care to avoid formation of aerosols.
(d) Place beaker on magnetic stirrer, and stir at speed
sufficient to develop vortex.
(e) Add 1 ml of 0.05 M A1C13 to mixing sludge.
Final concentration of AlCl- in sludge is
approximately 0.0005 M.
(f) Place combination-type pH electrode into mixing
sludge.
pH meter must be standardized at pH 4.
(g) Adjust pH of sludge to 3.5 +_ 0.1 with 5 M HC1.
If pH falls below 3.4, readjust it with 5 M NaOH.
When sludge adheres to electrodes, clean electrodes
by moving them up and down gently in mixing sludge.
- 8-5 -
-------
(h) Continue mixing for 30 minutes more.
The pH of the sludge should be checked at frequent
intervals. If the pH drifts up, readjust it to 3.5
+ 0.1 with 5 M HC1. If the pH drifts down,
readjust it with 5 M NaOH.
(i) Remove pH electrode from sludge.
(j) Add 75 mL of Freon to mixing sludge.
All procedures involving Freon must be done in hood
vented to outdoors.
(k) Readjust magnetic stirrer to speed sufficient for
thorough mixing, and mix for five minutes.
Because Freon is heavier than water and settles to
bottom, care must be taken that aqueous and Freon
layers intermix thoroughly.
(1) Turn off stirrer.
(m) Turn on vacuum connected to sidearm flask that
holds Buchner funnel containing AP25 filter.
(n) Pour sludge-Freon mixture into Buchner funnel.
To prevent transfer of stir bar into Buchner
funnel, hold another stir bar or magnet against
bottom of beaker when decanting contents.
(o) As soon as sludge solids on AP25 filter begin to
appear dry, pour 100 mL of 0.0005 M AlClg (pH 3.5
_+ 0.1) onto solids.
To avoid possible inactivation of viruses, do not
allow filtered solids to dry. Cover all solids on
filter in order to ensure thorough wash and maximal
removal of toxic substances.
- 8-6 -
-------
(p) When all wash liquid has passed through solids on
filter, repeat wash procedure in Step (o) four more
times.
(q) Turn off vacuum.
(r) Replace filtering flask.
Eluted viruses will be collected in second
filtering flask. Safely dispose of
chemically-microbiologically contaminated flask and
contents.
1.2.2 Elution of Viruses from Sludge Solids
(a) Pour 100 mL of buffered 10% beef extract onto
solids on AP25 filter.
Cover all solids on filter in order to ensure
proper elution. Allow beef extract to remain on
solids for ten minutes before going to Step (b).
(b) Turn on vacuum.
(c) When all beef extract has passed through solids on
filter, turn off vacuum.
(d) Disconnect tube from sidearm of filtering flask.
(e) Remove Buchner funnel from filtering flask, and
discard funnel and contents.
(f) Decant eluate from filtering flask into 250-mL
beaker.
The number of cell cultures necessary for the viral
assay may be reduced by concentrating the viruses
in the beef extract by the organic flpeculation
procedure of Katzenelson. some loss of viruses may
occur with this procedure. If viruses in eluate
- 8-7 -
-------
are to be concentrated, proceed Immediately to
Section 2. If concentration is not required,
proceed to Step (g).
(g) Place a filter holder that contains a filter stack
on a 250-mL receiving flask.
(h) Load 50-mL syringe with eluate.
(i) Place tip of syringe into filter holder.
(j) Force eluate through filter stack into 250-mL
receiving flask.
Take care not to break off tip of syringe and to
minimize pressure on receiving flask because such
pressure may splinter or topple the flask. If
filter stack begins to clog badly, empty loaded
syringe into beaker containing unfiltered eluate,
fill syringe with air, and inject air into filter
stack to force residual eluate from filters.
Continue filtration procedure with another filter
holder and filter stack. Discard contaminated
filter holders and filter stacks. Steps (g)
through (j) may be repeated as often as necessary
to filter entire volume of eluate. Disassemble
filter holder and examine bottom filter to be
certain it has not ruptured. If bottom filter has
ruptured, repeat Steps (g) through (j) with another
filter holder and filter stack.
(k) Refrigerate eluate immediately at 4° c, and
maintain it at that temperature until it is assayed
for viruses.
- 8-8 -
-------
If assay for viruses cannot be undertaken within
eight hours, store eluate immediately at -70° C.
2. CONCENTRATION OF VIRUSES FROM SLUDGE ELUATES
2.1 Organic Flocculation Concentration Procedure of Katzenelson
It is preferable to assay eluted viruses in the beef extract
eluate without concentrating them because some loss of viruses
may occur in concentration. However, the numbers of cell
cultures needed for assays may be reduced by concentrating the
viruses in the eluate.
2.1.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) pH meter, measuring to an accuracy of at least 0.1
pH unit, equipped with a combination-type electrode.
(c) Refrigerated centrifuge capable of attaining 2,500
x Ł and screw-cap centrifuge bottles.
Each sample centrifuged at 2,500 x g will consist
of about 330 ml.
(d) Membrane filter apparatus for sterilization —
47-mm diameter filter holder and 50-mL slip tip
syringe (Millipore Corp., Swinnex filter, No.
SX0004700, or equivalent for filter holder only).
(e) Disc filters, 47-mm diameter ~ 3.0-, 0.45-, and
0.25-^m pore size filters (Filterite Corp., Duo-Fine
*Katzenelson, E., B. Fattal, and T. Hostovesky. 1976. Organic
floccupation: an efficient second-step concentration method for the
detection of viruses in tap water. Appl. Environ. Microbiol.
32:638-639.
- 8-9 -
-------
series, or equivalent). Filters must be cut to
proper size from sheet filters.
Disassemble Swinnex filter holder. Place filter
with 0.25-^m pore size on support screen of filter
holder, and stack the remaining filters on top in
order of increasing pore size. Reassemble and
tighten filter holder. Filters stacked in tandem
as described tend to clog more slowly when turbid
material is filtered through them. Prepare several
filter stacks.
2.1.2 Media and Reagents
(a) Disodium hydrogen phosphate (Ma2HP04*7H20)
-- 0.15 M.
(b) Hydrochloric acid (HC1) — 1 M.
(c) Sodium hydroxide (NaOH) — 1 M.
2.1.3 Procedure (See Figure 8-1.2)
(a) Place filter holder on 250-mL Erlenmeyer receiving
flask.
(b) Load 50-mL syringe with eluate from Section 1.2.2,
Step (f).
(c) Place tip of syringe into filter holder, and force
eluate through filter stack.
Take care not to break off tip of syringe and to
minimize pressure on receiving flask because such
pressure may splinter or topple the flask. If
filter stack begins to clog badly, empty loaded
syringe into beaker containing unfiltered eluate,
fill syringe with air, and inject air into filter
- 8-10 -
-------
stack to force residual eluate from filters.
Continue filtration procedure with another filter
holder and filter stack. Discard contaminated
filter holders and filter stacks. Steps (a)
through (c) may be repeated as often as necessary
to filter entire volume of eluate. Disassemble
filter holder and examine bottom filter to be
certain it has not ruptured. If bottom filter has
ruptured, repeat Steps (a) through (c) with another
filter stack.
(d) Remove filter holder from top of Erlenmeyer flask,
pour eluate into graduated cylinder, and record
volume.
(e) Pour eluate into 600-mL beaker.
(f) For every 3 mL of beef extract eluate, add 7 mL of
deionized distilled water to the 600-mL beaker.
The concentration of beef extract is now 3%. This
dilution is necessary because 10% beef extract does
not always process well by the organic flocculation
conee n t r a t i on procedure.
(g) Record the total volume of the diluted, filtered
beef extract.
(h) Place stir bar in beaker that contains diluted,
filtered beef extract.
(i) Place beaker that contains the diluted filtered
beef extract on magnetic stirrer, and stir at a
speed sufficient to develop vortex.
-8-11 -
-------
To minimize foaming (which may Inactivate viruses),
do not mix faster than necessary to develop
vortex.
(j) Insert combination-type pH electrode into diluted,
filtered beef extract.
pH meter must be standardized at pH 4.
(k) Add 1 M HCl to flask slowly until pH of beef
extract reaches 3.5 +_ 0.1.
A precipitate will form. If pH is accidentally
reduced below 3.4, add 1 M NaOH until pH fs 3.5 +
0.1. Avoid reducing pH below 3.4 because some
i nac tivati on__of_ _v1 ruses may occur.
(1) Continue to stir for 30 minutes more.
(m) Turn off stirrer.
(n) Remove caps from screw-capped centrifuge bottles.
Use one or more bottles, as needed. Glass
centrifuge bottles may not be able to withstand
2,500 x g force that will be applied.
(o) Remove electrode from beaker, and distribute
contents of beaker evenly among centrifuge bottles.
To prevent transfer of stir bar into centrifuge
bottles, hold another stir bar or magnet against
bottom of each beaker when decanting contents.
(p) Replace and tighten down caps on centrifuge bottles.
(q) Centrifuge precipitated beef extract suspensions at
2,500 x Ł for 15 minutes at 4° C.
(r) Remove caps from centrifuge bottles.
- 8-12 -
-------
(s) Pour off and discard supernates.
(t) Place a small stir bar into each of the centrifuge
bottles that contains precipitate.
(u) Replace covers loosely on centrifuge bottles.
(v) Raise caps from tops of centrifuge bottles, and
divide a volume of 0.15 M Na2HP04 equal to 1/20
of the volume recorded in Step (g) equally among
the precipitates in the centrifuge bottles.
The volume of 0.15 M Na2HP04 in which the
precipitate will be dissolved is equal to 5 ml for
each 100 mL of diluted beef extract.
(w) Replace and tighten down caps on centrifuge bottles.
(x) Place each bottle on a magnetic stirrer, and stir
each precipitate slowly until precipitate has
dissolved completely.
Support bottles as necessary to prevent toppling.
Avoid foaming which may inactivate or aerosolize
viruses. Precipitate may be partially dissipated
with spatula before or during stirring procedure.
(y) Remove caps from centrifuge bottles.
(z) Combine the dissolved precipitates in a small
beaker.
To prevent transfer of stir bars into beaker, hold
another stir bar or magnet against the bottom of
each centrifuge bottle when decanting dissolved
precipitates.
(aa) Measure pH of dissolved precipitates.
- 8-13 -
-------
If pH is above or below 7.0-7.5, adjust to that
range with either 1 M HCl or 1 M NaOH.
(bb) Refrigerate dissolved precipitates immediately at
4° C, and maintain at that temperature until
assay for viruses is undertaken.
If assay for viruses cannot be undertaken within
eight hours, store dissolved precipitates
immediately at -70° C.
(cc) Assay for viruses 1n accordance with instructions
given in Chapter 9.
- 8-14 -
-------
3. BIBLIOGRAPHY
Berg, G., D. Berman, and R.. S. Safferman. 1982. A method for
concentrating viruses recovered from sewage sludges. Ccm._J.
Mlcroblol. 28:553-556.
Berg, G., and D. R. Dahllng. 1980. Method for recovering
viruses from river water solids. Appl. Environ. Mlcroblol.
39:850-853.
Berman, D., G. Berg, and R. S. Safferman. 1981. A method for
recovering viruses from sludges. J. Vlrol. Methods. 3:283-291.
Brashear, D. A.» and R. L. Ward. 1982. A comparison of methods for
recovering Indigenous viruses from raw wastewater sludge. Appl.
Environ. Hlcroblol. 43:1413-1418.
Farrah, S. R., P. R. Scheuerman, and G. Bitton. 1981. Urea-lyslne
method for recovery of enteroviruses from sludge. Appl.
Environ. Hlcroblol. 41:455-458.
Goddard, M. R.j J. Bates, and M. Butler. 1981. Recovery of
Indigenous enteroviruses from raw and digested sewage sludges.
Appl. Environ. Hlcroblol. 42:1023-1028.
Katzenelson, E., B. Fattal, and T. Hostovesky. 1976. organic
flocculatlon: an efficient second-step concentration method for
the detection of viruses In tap water. Appl.Environ.
Mlcroblol. 32:638-639.
Lund, E., and C.-E. Hedstrom. 1966. The use of an aqueous polymer
phase system for enterovlrus Isolations from sewage. Am. J.
Ep1dem1ol. 84:287-291.
Nielsen, A. L.» and B. Lydholm. 1980. Methods for the Isolation of
virus from raw and digested wastewater sludge. Water Res.
14:175-178.
- 8-15 -
-------
Sattar, S. A., and J. C. N. Westwood. 1976. Comparison of four
eluents in the recovery of indigenous viruses from raw sludge.
Can. J. Microbiol. 22:1586-1589.
Sattar, S. A., and J. C. N. Westwood. 1979. Recovery of viruses
from field samples of raw, digested, and lagoon-dried sludges.
Bull. World Health Qrg. 57:105-108.
Turk, C. A., B. E. Moore, B. P. Sagik, and C. A. Sorber. 1980.
Recovery of indigenous viruses from wastewater sludges, using a
bentonite concentration procedure. Appl. Environ. Microbiol.
40:423-425.
Ward, R. L., and C. S. Ashley. 1976. Inactivation of poliovirus in
digested sludge. Appl. Environ. Microbiol. 31:921-930.
- 8-16 -
-------
CHAPTER 9
PREPARATION AND USE OF CELL CULTURES
1. INTRODUCTION
This chapter outlines procedures and media for culturing Buffalo
green monkey (BGM) kidney cells* and for assaying viruses recovered in
these cells. BGM cells are a continuous line derived from African Green
monkey kidney cells. The characteristics of this line were described by
A. L. Barren, C. Olshevsky, and M. M. Cohen in 1970.** Use of BGM cells
for recovering viruses from environmental samples was described by
D. R. Dahling, G. Berg, and D. Berman in 1974.***
This chapter is intended as guidance for the competent virologist
who is preparing to recover, assay, and identify viruses in environmental
samples. Cells other than BGM may be used when deemed preferable by a
competent virologist. Although BGM cells are very sensitive to many
enteroviruses, these cells are not sensitive
*BGM cells are available to qualified laboratories and may be obtained
from [)r. R. S. Safferman, Chief of Virology, EMSL, U. S. EPA,
Cincinnati, Ohio 45268.
**Barron, A. L., C. Olshevsky, and M. M. Cohen. 1970. Characteristics
of the BGM Line of Cells from African Green Monkey Kidney. Archiv.
for pie Gesamte Virusforschung. 32:389-392.
***Dahling, D. R., G. Berg, and D. Berman. 1974. BGM, A Continuous Cell
Line More Sensitive Than Primary Rhesus and African Green Kidney
Cells for the Recovery of Viruses from Water, Health Laboratory
Sciences. 11:275-282.
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to other enteroviruses or to certain other viruses that may occur in
environmental samples. Thus, to maximize the numbers of viruses
recovered from environmental samples, several cell lines may need to
be used.
In this chapter, only the plaque technique for assaying viruses
is described. Liquid culture procedures may also be used. Plaque
procedures allow greater counting accuracy. Liquid cultures often
yield greater sensitivity.
2. PREPARATION
2.1 Apparatus and Materials
2.1.1 Glassware, Pyrex glass, clear (Corning Glass Works, or
equivalent).
Storage vessels must be equipped with air-tight
closures.
2.1.2 Magnetic stirrer and stir bars.
2.1.3 Autoclavable inner-braided tubing with metal
quick-disconnect connectors for tubing to be connected to
equipment under pressure.
2.1.4 Positive pressure air or nitrogen source equipped with
pressure gauge.
Pressure source, if laboratory air line or pump, must be
equipped with oil filter. Deliver to filter holder no
more pressure than recommended by manufacturer.
2.1.5 Dispensing pressure vessel — 20-liter capacity
(Hillipore Corp., or equivalent).
2.1.6 Disc filter holders — 142 or 293 mm diameter (Mi Hi pore
Corp., or equivalent).
Use only pressure type filter holders.
- 9-2 -
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2.1.7 Virus-adsorbing disc filters -- 0.22-jum pore size
(Millipore Corp., GS series, or equivalent).
2.1.8 Fiberglass prefilters (Mi Hi pore Corp., API 5 and AP20, or
equivalents).
Stack APIS and AP20 prefilters and 0.22-jum membrane
filter into disc filter holder with AP20 prefilter on top
and 0.22-^im membrane filter on bottom.
2.1.9 Cell culture vessels, Pyrex borosilicate glass (Corning
Glass Works, or equivalent), soda or flint glass
prescription (Rx) bottles (Brockway Glass Co., Inc., or
equivalent), plastic (Falcon Tissue Culture Labware,
Becton, Dickinson and Co., or equivalent), disposable
glass roller bottles (Bellco Biological Glassware, or
equivalent), or disposable plastic roller bottles
(Corning Glass Works, or equivalent).
Vessels (tubes, flasks, bottles) for growth of cell
cultures must be clear glass or plastic to allow
observation of the cultures. Plastic vessels must be
treated by the manufacturer to allow cells to adhere
properly. Vessels for cell cultures must be equipped
with air-tight closures.
2.1.10 Screw caps, black with rubber liners (24-414 for 6 02.*
prescription (Rx) bottles, Brockway Glass Co., Inc., or
equivalent).
*Size is given in oz. when commercially designated only in that unit.
- 9-3 -
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Caps for larger culture bottles usually supplied with
bottles.
2.1.11 Roller apparatus (7730-Series, Bellco Biological
Glassware, or equivalent).
2.1.12 pH meter measuring to an accuracy of at least 0.1 pH
unit.
2.1.13 Incubator capable of maintaining the temperatures of cell
cultures at 36.5° +_ 1° C.
2.1.14 Waterbath, equipped with circulating device to assure
even heating at 36.5° +< 1° C, 56° +• 1° C, and
60°±1° C.
2.1.15 Light microscope, with conventional light source,
equipped with lenses to provide 40, 100, and 400X total
magnifications.
2.1.16 Inverted light microscope equipped with lenses to provide
40, 100, and 400X total magnifications.
2.2 Media and Reagents
To avoid exposure of cells to toxic chemical contaminants,
chemicals applied to cell cultures must be reagent grade or
equivalent in purity. Compounds such as neutral red, trypan
blue, and phenol red that are not usually sold at the reagent
grade level must be obtained in the purest form available.
2.2.1 Fetal calf serum, filter-sterilized, heat-inactivated at
56° C for 30 minutes, certified free of viruses and
mycoplasma (Grand island Biological Co., or equivalent).
Test toxicity of sample of serum, on cells before
purchasing serum lot.
2.2.2 Agar (Bacto-Agar, Difco Laboratories, or equivalent).
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2.2.3 Milk, sterile, homogenized, whole, fluid (Real-Fresh
Brand, or equivalent).
2.2.4 Trypsin, 1:300 powder (BBL, Becton, Dickinson and Co., or
equivalent) or Trypsin, 1:250 powder (Difco Laboratories,
or equivalent).
2.2.5 Sodium (Tetra) Ethylenediamine Tetraacetate Powder
(Versene), technical grade, (Fisher Scientific Company, or
equivalent).
2.2.6 Thioglycollate medium (Bacto Dehydrated Fluid
Thioglycollate Medium, Difco Laboratories, or
equivalent).
2.2.7 Water, sterile, distilled, deionized.
See Chapter 4.
2.2.8 Fungizone, Mycostatin, and Neomycin (E. R. Squibb and
Sons, or equivalent), Gentamicin (Schering-Plough Corp.,
or equivalent), Penicillin G and Dihydrostreptomycin
sulfate (Eli Lilly and Co., or equivalent), Tetracycline
(Pfizer, inc., or equivalent).
Use antibiotics of tissue culture or injection grade
only.
2.2.9 Vitamins, amino acids, salts, acids, dyes, research grade
or best grade available (Sigma Chemical Co., or
equivalent).
3. PROCEDURE FOR PREPARATION OF BGM CELL CULTURES
3.1 General Procedures
The BGM cell line grows readily on the inside surfaces of plastic
flat-sided vessels, glass bottles, and glass test tubes.
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B6M cell cultures can be purchased from several companies for
plaque assay or plaque confirmation procedures. Although it is
labor Intensive for a laboratory to maintain the B6M cell line
and to prepare cultures, it is much less expensive to prepare
cultures than to purchase them. To reduce the risk of
contamination, cell cultures should be prepared in controlled
facilities used for no other purpose.
3.1.1 Pass and maintain BGM stock cultures in 16 to 32 oz.
(or equivalent in growth area), flat-sided, glass bottles
or in plastic cell culture flasks.
If available, roller bottles and roller apparatus units
are preferable to flat-sided bottles or flasks for
growing cells because roller cultures require less medium
than flat-sided bottles per unit of cell monolayer
surface. For growing cells in roller bottles, adjust
roller apparatus rotation speed to one-half revolution
per minute.
3.1.2 Prepare cell cultures for plaque assays in vessels with
growth areas of 45 sq. cm (6 oz.) or larger.
Vessels with relatively large surface areas are used to
accommodate the large sample inoculums in environmental
virology studies. Vessels with smaller surface areas may
be used, if necessary. Only flat-sided cell culture
vessels can be used for plaque assays.
3.1.3 Prepare cultures for plaque confirmation in 16 x 150 mm
glass or plastic cell culture tubes.
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3.1.4 Except during handling operations, maintain BGM cells at
36.5° +_ 1° C in air-tight cell culture vessels.
(a) Maintain in constant motion roller bottles that
contain cells.
(b) Maintain flat-sided cell culture bottles or flasks
that contain cells in a stationary position with the
flat side (cell monolayer side) down.
(c) Maintain cell culture tubes in either stationary
racks or in rotating drums slanted upwards at an
angle of approximately 15° (almost horizontal) so
that the fluid inside extends upward one-third to
two-thirds the length of the tube.
Cell culture vessels must be stored in a way such
that their liquid contents do not touch the Inner
surface of the vessels' caps.
3.1.5 To reduce shock to cells, warm growth media, maintenance
media, washing solutions used for removing toxic materials
from inoculated cell cultures, and all other solutions to
36.5° +_ 1° C before placing them on cell monolayers.
3.1.6 Test all media and solutions to be used in cell culture
operations to assure their microbiological sterility (see
Chapter 4).
3.1.7 Introduce only trypsin-EDTA solution and sample inoculums
directly onto cell monolayer surface.
Inoculums are introduced gently onto monolayer surfaces
directly to minimize loss of viruses through adherence of
inoculums onto other surfaces inside the cell culture
vessels.
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3.1.8 introduce all materials other than trypsin-EDTA solution or
inoculum onto a part of the inside container wall that is
not covered by cell monolayer surface.
This precaution is taken to dissipate force used to
introduce fluids.
3.1.9 Pass stock BGM cell cultures at approximately seven-day
intervals.
3.1.10 Prepare cultures for plaque assay and plaque confirmation
three to seven days before cultures are to be inoculated
with virus-containing material.
3.1.11 Before discarding, autoclave all media and washing solutions
that have been in contact with cells or that contain serum.
3.2 Procedure for Passage of BGM Cells
3.2.1 Pour spent medium from cell culture vessels, and discard the
medium.
To prevent splatter, a gauze-covered beaker may be used to
collect spent medium.
3.2.2 Add to the cell cultures a volume of warm (36.5° +_ 1° C)
trypsin-EDTA solution equal to 40% of the volume of medium
replaced (see Table 9-1).
Pour the trypsin-EDTA solution directly onto the cells.
3.2.3 Allow trypsin-EDTA solution to remain in contact with the
cells at 36.5° +^1° C until cell monolayer can be shaken
loose from inner surface of cell culture vessel (about 10
minutes).
If necessary, a sterile rubber policeman (or scraper) may be
used to physically remove the cell sheet from the
- 9-8 -
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TABLE 9-1
Guide for Determining Volume of Cell Suspension, Cell Culture Medium,
Virus Sample Inoculum, and Overlay Medium to be Used with
Various Sized Cell Culture Vessels
Vessel Type
arid sTze"~
(Flat-Sided Glass
or Plastic Bottle)
1 oz.
2 oz. or 25 cm
4 oz.
6 oz.
p
8 oz. or 75 cm
f
16 oz. or 150 cm*
32 oz.
Volume of Fluid in ml
Cell Suspension,
Growth, or
Maintenance Medium
4
8
12
15
20
40
50 not
Virus Sample
Inoculum
0.1
0.2
0.3-0.4
0.5-1.0
0.5-1.5
1.0-3.0
commonly used
Agar Overlay
Medi urn
5
10
15
20
25
50
not commonly used
Roller Apparatus
(Glassor plastic Bottle)
64 oz.
100
cannot be used cannot be used
Cell Culture Tube
(Glass or Plastic)
16 x 150 mm
O.lrl.O
cannot be used
*Size is given in oz. when commercially designated only in that unit.
- 9-9 -
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bottle. However, this procedure should be used only as a
last resort because of the risk of cell culture
contamination inherent in such manipulations.
3.2.4 Pour the suspended cells into centrifuge tubes or bottles.
To facilitate collection and resuspension of cell
pellets, use tubes or bottles with conical bottoms.
Centrifuge tubes and bottles used for this purpose must
be able to withstand the g-force applied.
3.2.5 Centrifuge cell suspension at 1,000 x _g for 10 minutes to
pellet cells.
3.2.6 Pour off and discard the supernate.
3.2.7 Suspend the pelleted cells in growth medium.
The quantity of medium used for resuspending pelleted
cells varies from 10 ml to more than 1 liter, depending
upon the volume of the individual laboratory's need for
cell cultures. Resuspend passaged cells in large volumes
of medium to allow thorough mixing of cell pellets (to
•reduce sampling error) and to minimize the significance
of the loss of the 1 ml of cell suspension required for
the cell counting procedure. Do not dilute cells to a
concentration of less than 6x10 per ml. because
viable cell counts (see Section 3.3) cannot be done with
lesser concentrations of cells.
3.2.8 Perform a viable cell count on this concentrated
suspension (see Section 3.3).
3.2.9 Dilute the cell suspension in growth medium to a
concentration of approximately 2.5 x 10 viable cells
per ml of medium.
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3.2.10 Dispense the cell suspension into cell culture vessels.
The quantities of suspension that must be used for
culture vessels of different sizes are shown in Table
9-1.
3.3 Procedure for Performing Viable Cell Counts
3.3.1 Add 0.5 ml of cell suspension (or diluted cell
suspension) to 1.0 ml of 0.5% trypan blue solution.
Dilution compensated for in Section 3.3.6, Step (b).
3.3.2 Disperse cells by repeated pipetting.
Avoid introducing air bubbles into the suspension,
because air bubbles may interfere with subsequent filling
of hemocytometer chambers.
3.3.3 With a capillary pipette, carefully fill hemocytometer
chambers on both sides of a slip-covered hemocytometer
slide.
Do not under or over fill the chambers.
3.3.4 Rest slide on flat surface for about 10 minutes to allow
trypan blue to penetrate cell membranes of nonviable
cells.
3.3.5 Under 100X total magnification, count and total the cells
in the four large corner sections of both hemocytometer
chambers.
Include in the count cells lying on the lines marking the
top and left margins of the sections, and ignore cells on
the lines marking the bottom and right margins.
Trypan blue is excluded by living cells. Therefore, to
quantify only viable cells, count only cells that are
clear in color. DO not count cells that are blue.
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3.3.6 Determine the concentration of viable cells in the cell
suspension (or diluted cell suspension) with the
following equations:
(a) total number of average number
viable cells in f 8 of viable cells
the 8 sections ' per section
(b) average number average number of
of viable cells viable cells per mL
per section x 3 x 10,000 = of cell suspension
(or per mL of diluted
cell suspension)
To obtain an accurate cell count, the optimal total
number of cells per hemocytometer section should be
between 20 and 50. This range is equivalent to between
6.0 x IP5 and 1.5 x IP6 cells per mL of cell
suspension. If the cell suspension contains a larger
concentration of cells, that portion of the cell
suspension to be used for the counting procedure may
first require dilution with growth medium, if such a
dilution is made, be certain to factor this dilution into
the cell count.
3.4 Procedure for Changing Medium on Cultured Cells
Three to four days after seeding with an appropriate number of
cells, monolayers normally become 95 to 100% confluent, and
growth medium becomes acidic. Growth medium on confluent stock
cultures should then be replaced with maintenance medium.
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If stock culture cell monolayers have not reached 95 to 100%
con-Fluency by this time and the medium on these cultures has not
become acidic, then the medium should not be changed until the
monolayers do reach 95 to 100% confluency. If three to four
days after passage, monolayers are not yet 95 to 100% confluent
and the medium in which they are Immersed has become acidic,
then the medium must be replaced with fresh growth medium
instead of with maintenance medium.
It should not be necessary to replace growth medium on cultures
for plaque assay or plaque confirmation until four hours or less
before cultures are to be inoculated with viruses. If these
cultures are not needed for plaque assay or plaque confirmation
at the time they become confluent and the growth medium acidic,
replace the growth medium with maintenance medium.
3.4.1 Pour spent medium from cell culture vessels, and discard
the spent medium.
3.4.2 Add to the cell culture vessels a volume of fresh
maintenance medium equal to the volume of spent medium
discarded.
4. PLAQUE PROCEDURE FOR RECOVERING OR TITRATING VIRUSES
To titrate viruses, inoculate multiple dilutions in appropriate
numbers of replicate cell cultures.
4.1 Inoculating Sample onto Cell Cultures
4.1.1 From one to four hours before cultures are to be
inoculated, replace medium in culture vessels with an
equal volume of maintenance medium.
4.1.2 Maintain cultures at 36.5° -f 1° c until they are to
be inoculated.
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4.1.3 Before culture vessels are inoculated, identify them with
an indelible marker.
4.1.4 Pour medium from cell culture vessels, and discard
medium.
4.1.5 Carefully inoculate into each culture vessel the volume
of sample which is correct for vessels of that size.
See Table 9-1 for inoculum sizes appropriate for commonly
used cell culture vessels.
If inoculum may be toxic, see Section 4.3.
4.1.6 Immediately rock inoculated culture vessel gently to
achieve uniform distribution of sample on surface of cell
monolayer.
Uniform distribution of sample inoculum results in
uniform distribution of plaques and thereby facilitates
accurate plaque counting.
4.1.7 Incubate inoculated cell cultures for two hours at room
temperature (22-25° C) to permit viruses to adsorb onto
and to infect cells.
4.1.8 Apply warm (46° C) agar overlay medium to each culture
vessel.
See Table 9-1 for the amount of overlay medium that
should be added.
Pour medium onto side of cell culture vessel opposite the
cell monolayer, allow medium to cool momentarily, and
then place the culture vessel, monolayer side down, on a
stationary table or bench at room temperature (22-25°
C) so that the agar will distribute evenly as it
solidifies.
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4.1.9 Cover cell culture vessels with a sheet of aluminum foil,
a tightly woven cloth, or some other suitable cover to
prevent photoinactivation of virions.
Agar begins to harden almost immediately and fully
solidifies in 30 to 60 minutes.
4.1.10 Invert culture vessels, and incubate in the dark at
36.5°±1°C.
4.2 Counting Viral Plaques
4.2.1 Begin counting and marking plaques in cultures two days
after overlaying, and repeat procedure every two days for
a total of 10 days (for enteroviruses) after overlaying.
4.2.2 Record plaque counts at each reading.
Virus titers are calculated from total count.
4.2.3 Calculate virus titers (plaque-forming units [PFU]) for
each inoculated virus-containing sample.
Total number of plaques per culture yesjej (or average
number of plaques per culturevesselIfseveral vessels
have been inoculated with the same sample) to obtaini the
virus tjrter of sample in terms of PFU per inoculum
volume, jo obtain PFU per ml, multiply the number of PFU
by the reciprocal of the inoculum volume (and by the
dilution, if a dilution was made).
4.3 Reduction of Sample-Associated Toxicity
This procedure may result in the loss of virions and is
to be used only if there is a likelihood that inoculum
will be toxic to cell cultures.
4.3.1 Inoculate cell cultures with samples that contain
viruses.
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4.3.2 Incubate inoculated cultures for two hours at room
temperature (22-25° C) to allow virions to adsorb onto
and to infect cells.
4.3.3 Pour inoculum from cell culture vessels, discard
inoculum, and add to each culture 1 mL of washing
solution [0.85% (w/v) NaCl containing 2% (v/v) fetal calf
2
serum] for each 5-cm of cell surface area.
4.3.4 Gently rock the washing solution twice across the cell
monolayer, and pour off arid discard the washing
solution.
4.3.5 Overlay washed cultures with agar overlay medium.
See Section 4.1.8.
See Table 9-1 for quantity of overlay required.
5. PROCEDURE FOR VERIFYING STERILITY OF LIQUIDS
There are many techniques available for verifying the sterility of
1j qu1ds such as c?l1 culture^ medi a: and thei Two
techniques, described be!ow, arfe standard in mar^ 1abpratories. The
capabi1jties of these techniques, however, are 1imited to detecting
microorganisms that grow unaided on the test medium utilized.
Viruses, mycoplasma, and microorganisms that possess fastidious
growth requirements or that require living host systems will not be
detected. Nonetheless, with the exception of a few special
contamination problems, the test procedures and microbiological media
listed below should prove adequate. Do not add antibiotics to media
or media components until after sterility of the media and components
has been demonstrated.
5.1 Procedure for Verifying Sterility of Small Volumes of Liquids
5.1.1 Inoculate 5-20 mL, as appropriate, of the material to be
- 9-16 -
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tested for sterility into sterile ttriogly coll ate broth
(20-100 ml, as appropriate).
5.1.2 Shake the mixture, and incubate it at 36.5° +_ 1° C.
5.1.3 Examine the inoculated broth daily for seven days to
determine whether growth of contaminating organisms has
occurred.
Vessels that contain thioglycollate medium must be
tightly sealed before and after medium is inoculated. In
some instances, growth medium that contains MaHCOo but
no antibiotics may be used as detection medium.
5.2 Procedure for Verifying Sterility of Large Volumes of Liquids
5.2.1 Filter 50-100 mL of the liquid tested for sterility
through a 47 mm diameter, 0.22-pn pore size membrane
filter.
5.2.2 Remove filter from its holder, and place filter on
surface of solidified nutrient agar in a Petri dish.
Place filter face up on agar.
5.2.3 Incubate Petri dish at 36.5° +_ 1° C, and examine
filter surface daily for seven days to determine whether
growth of contaminating organisms has occurred.
6. PREPARATION OF CELL CULTURE MEDIA
This section is a guide for preparation of media for growing BGM
cells and for performing agar overlay plaque assays for viruses that
multiply in BGM cells.
6.1 Technique
6.1.1 Equipment Care
Carefully wash and sterilize equipment used for preparing
media before each use.
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6.1.2 Disinfection of Work Area
Thoroughly disinfect surfaces on which media preparation
equipment is to be placed.
6.1.3 Aseptic Technique
Use aseptic technique when preparing and handling media
or media components.
6.1.4 Dispensing Filter-Sterilized Media
To avoid post-filtration contamination, dispense
filter-sterilized media into storage vessels in a
microbiological laminar flow hood.
Dispense sterile media into storage containers through
clear glass filling bells.
6.2 Sterility Testing
6.2.1 Coding Media
Assign a lot number to each batch of media or media
component prepared.
6.2.2 Sterility Test
Test each lot of medium and medium components to confirm
sterility before the lot is used for cell culture or
plaque assay (See Section 5).
6.2.3 Storage of Media and Media Components
Store media and media components in clear air-tight
containers.
6.2.4 Sterilization of NaHCO^-containing Solutions
Sterilize media and other solutions that contain NaHCOo
by positive pressure filtration.
Negative pressure filtration of such solutions increases
their pHs and reduces their buffering capacities.
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6.3 Media Formulations
6.3.1 Sources of Cell Culture Media
Commercially-prepared liquid cell culture media and media
components are available from several sources. Cell
culture media can also be purchased in powder form that
requires only dissolution in deionized distilled water
and sterilization. Media from commercial sources are
usually quality controlled carefully and quite adequate.
However, media can also be prepared in the laboratory
from chemicals. Such preparations are labor intensive
and may be expensive but allow quality control of the
process at the level of the preparing laboratory.
6.3.2 Constraints, Modifications, and Conditions in Media
Formulations
(a) Do not attempt to prepare Leibovitz's L-15 medium
in a form more concentrated than that normally used
for growing cells (IX concentration).
(b) Prepare Eagle's minimum essential medium (MEM) in
concentrations indicated below (up to 10X greater
than that normally used for growing cells).
Certain components for Eagle's MEM may be prepared
in concentrations up to 100X greater than those
normally used for growing cells (See below).
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7. PREPARATION OF MEDIA AND STAINS FOR CELL CULTURES
7.1 Growth Medium
7.1.1 Formula (Preparation of 1 liter)
Eagle's MEM with Hanks' BSS 450.0 mL
Leibovitz's L-15 medium 450.0 mL
NaHCOg, 7.5% solution 7.0 mL
Fetal calf serum 100.0 mL
Penicillin-streptomycin, stock solution 1.0 mL
Tetracycline, stock solution 0.5 mL
Fungizone, stock solution 0.2 mL
7.1.2 Procedure
(a) Filter through a 0.22-jum membrane any ingredient in the
formula that is not sterile.
(b) Place 450 mL of Eagle's MEM with Hanks' BSS into a clear
glass vessel.
(c) Maintain continuous stirring.
(d) Add Leibovitz's L-15 medium and 7.5% NaHCOg to the
Eagle's MEM with Hanks' BSS.
(e) Store medium at 4° C until needed.
(f) Add fetal calf serum and antibiotics to medium
immediately before medium is used.
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7.2 Maintenance Medium
7.2.1 Formula (Preparation of 1 liter)
If medium is to be used for washing cells before cell
cultures are to be inoculated with viruses, replace the
50 ml of fetal calf serum with 50 ml of deionized
distilled water.
Deionized distilled water ....... 850.0 ml
Earle's BSS 10X stock 100.0 ml
Phenol red, 0.5% 1.0 ml
MaHCO,, 7.5% 7.0 mL
O
Fetal calf serum . . 50.0 ml
Penicillin-streptomycin, stock solution . 1.0 ml
Tetracycline, stock solution 0.5 ml
Fungi zone, stock solution 0.2 ml
7.2.2 Procedure
(a) Filter through a 0.22-/im membrane any ingredient in
the formula that is not sterile.
(b) Place 850 mL of deionized distilled water into a
clear glass vessel.
(c) Maintain continuous stirring.
(d) Add Earle's BSS 10X stock, phenol red, and 7.5%
NaHC03 to the deionized distilled water.
(e) Store medium at 4° C until needed.
(f) Add fetal calf serum and antibiotics to medium
immediately before medium is used.
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7.3 Agar Overlay Medium
7.3.1 Formula (Preparation of 1 liter)
Mixture A
2X Eagle's MEM without phenol red for use in
overlay medium 415.0 mL
Fetal calf serum 20.0 ml
NaHC03, 7.5% . . . 30.0 ml
MgCl2, 1% 10.0 ml
Neutral red, 0.1% 15.0 ml
Penicillin-streptomycin, stock solution 1.0 ml
Tetracycline, stock solution . 0.5 ml
Fungizone, stock solution 0.2 ml
Mixture B
Agar 15.0 g
Deionized distilled water 500.0 ml
7.3.2 Procedure
Do not prepare Mixtures A and B in advance of the day on
which they are to be used.
*
(a) Mixture A
(a.l) Filter through a 0.22-fim membrane any
ingredient in the formula that is not
sterile.
(a.2) Place 415 ml of 2X Eagle's MEM without
phenol red into a clear glass vessel.
(a.3) Maintain constant stirring.
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(a.4) Add the fetal calf serum, NaHC03, Mgd2,
neutral red, and antibiotics to the 2X
Eagle's MEM (Mixture A).
(a.5) Warm Mixture A to 36.5° + 1° C in a
water bath.
If Mixture A is prepared more than one hour
before it is to be used, store it at 4° C,
and warm it to 36.5° +_ 1° C about 30
minutes before it is needed.
(b) Mixture B
(b.l) Place 500 ml of deionized distilled water
into a glass vessel that can withstand
autoclaving.
(b.2) Add the agar to the deionized distilled
water.
(b.3) Autoclave agar and water at 121° C for 15
minutes.
(b.4) Cool dissolved agar to 56° + 1° C in a
water bath.
(c) Immediately before overlay medium is to be placed
on cell cultures, combine Mixtures A and B and add
10 ml of sterile whole milk.
(d) Mix quickly by swirling.
If a large number of cell cultures is to be
overlayed, maintain medium in a 36.5° ± 1° C
water bath during overlay procedure.
(e) Overlay cells immediately.
See Table 9-1 for quantity of overlay required.
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7.4 Eagle's Minimum Essential Medium (MEM) with Hanks' Balanced
Salt Solution
7.4.1 Formula (Preparation of 1 liter)
This medium may also be prepared as a ten-fold (IPX)
concentrate, and components of the formula may be prepared
in even higher concentrations (See below). Formulations for
preparing this medium in IX and 2X concentrations (the
latter for use in preparing overlay medium for plaque
assays) from more concentrated sub-components are presented
in later sections.
Deionized distilled water 750.0 ml
Inorganic salts
CaClg HO.O mg
KC1 400.0 mg
KH2P04 60.0 mg
MgCl2'6H20 100.0 mg
MgS04*7H20 100.0 mg
Nad 8000.0 mg
Na2HP04 60.0 mg
Ami no Acids
L-Arginine HC1 84.0 mg
L-Cystine 48.0 mg
L-Glutamine 300.0 mg
Glycine 30.0 mg
L-Histidine HCl-H20 42.0 mg
L-Isoleucine 105.0 mg
L-Leucine 105.0 mg
L-Lysine HC1 146.2 mg
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L-Methiom'ne 30.0 mg
L-Phenylalam'ne 66.0 mg
L-Serine 42.0 mg
L-Threonine 95.0 mg
L-Tryptophan . 16.0 mg
L-Tyrosine 73.0 mg
L-Valine 93.6 mg
Vitamins
Choline chloride 4.0 mg
Folic acid . 4.0 mg
-i-lnositol 7.0 mg
Micotinamide 4.0 mg
Pantothenic acid 4.0 mg
Pyridoxal HC1 4.0 mg
Riboflavin 0.4 mg
Thiamine HC1 . 4.0 mg
Other components
Glucose . 1000.0 mg
Phenol red 5.0 mg
7.4.2 Procedure
(a) Place 750 mL of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add the ingredients listed to the deionized
distilled water.
Allow each ingredient to go into solution before
adding the next ingredient.
- 9-25 -
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(d) Adjust the volume of the solution to 1 liter with
deionized distilled v/ater.
(e) Filter-sterilize medium through a Q.22-jim
membrane.•
(f) Store medium at 4° C.
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7.5 Eagle's Minimum Essential Medium for Use in Preparing Growth
Medi urn
7.5.1 Formula (Preparation of 1 liter)
Solution A
Deiom"zed distilled water 750 mL
Hanks' BSS 10X stock 100 mL
Phenol red, 0.5% 1 ml
Vitamins 100X stock 10 mL
Ami no acids 100X stock 10 mL
Solution B
neionized distilled water 25 mL
L-Tyrosine 73 mg
Solution C
Deionized distilled water 25 mL
L-Cysteine 48 mg
MaOH, IN AS needed
7.5.2 Procedure
(a) Place 750 mL of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add to the deionized distilled water the
ingredients for Solution A.
Allow each ingredient to go into solution before
adding the next ingredient.
(d) Dissolve 73 mg of L-tyrosine in 25 mL of deionized
distilled water (Solution B).
- 9-27 -
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Boil over bunsen burner or hot plate until the
L-tyrosine goes into solution.
(e) Cool Solution B, and add Solution B to Solution A.
(f) Dissolve 48 mg of L-cysteine into 25 mL of
deionized distilled water, and add IN MaOH until
L-cysteine is neutralized (Solution C).
(g) Add Solution C to Solutions A and B.
(h) Adjust volume of medium (Solution A, B, C) to 1
liter with deionized distilled water.
(i) Filter-sterilize medium through a 0.22-^m
membrane.
(j) Store medium at 4° C.
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7.6 2X Eagle's Minimum Essential Medium Without Phenol Red for Use
in Overlay Medium
7.6.1 Formula (Preparation of 1 liter)
Solution A
Deionized distilled water 625 ml
Hanks' BSS 10X stock 200 ml
Vitamins 100X stock 20 ml
Ami no acids 100X stock 20 ml
Solution B
Deionized distilled water 50 ml
L-Tyrosine . 146 mg
Solution C
Deionized distilled water 50 ml
L-Cysteine 96 mg
MaOH, IN As needed
7.6.2 Procedure
(a) Place 625 ml of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add to the deionized distilled water the
ingredients for Solution A.
Allow each ingredient to go into solution before
adding the next ingredient.
(d) Dissolve 146 mg of L-tyrosine into 50 ml of
deionized distilled water (Solution B).
- 9-29 -
-------
Boll over bunsen burner or hot plate until the
L-tyrosine goes Into solution.
(e) Cool Solution B, and add Solution B to Solution A.
(f) Dissolve 96 mg of L-cysteine into 50 ml of
deionized distilled water, and add IN NaOH until
L-cysteine is neutralized (Solution C).
(g) Add Solution C to Solutions A and B.
(h) Adjust volume of medium (Solutions A, B, C) to 1
liter with deionized distilled water.
(i) Filter-sterilize medium through a 0.22-jum
membrane.
(j) Store medium at 4° C.
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7.7 Hanks' Balanced Salt Solution (Hanks' BSS) 10X Stock
7.7.1 Formula (Preparation of 1 liter)
Deiom"zed distilled water 750 ml
NaCl . . 80000 mg
KCl 4000 mg
MgS04'7H20 1000 mg
Na2HP04 600 mg
KH2P04 600 mg
MgCl2*6H20 1000 mg
Cad2 1400 mg
Glucose 10000 mg
7.7.2 Procedure
(a) Place 750 ml of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add ingredients to the deionized distilled water in
the order listed.
Allow each ingredient to go into solution before
adding the next one.
(d) Adjust volume of solution to 1 liter with deionized
distilled water.
(e) Filter-sterilize medium through a 0.22-^m membrane.
(f) Store medium at 4° C.
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7.8 100X Amino Acids Stock for Eagle's Minimum Essential Medium
(Without Cysteine and Tyrosine)
7.8.1 Formula (Preparation of 1 liter)
Deionized distilled water ... 750 ml
L-Arginine HCl 8400 mg
L-Histidine HCl*H20 4200 mg
L-Isoleucine 10500 mg
L-Leucine . 10500 mg
L-Lysine HCl 14620 mg
L-Methionine 3000 mg
L-Phenylalanine 6600 mg
L-Threonine 9500 mg
L-Tryptophan 1600 mg
L-Valine 9360 mg
Glycine . 3000 mg
L-Serine 4200 mg
L-Glutamine 30000 mg
7.8.2 Procedure
(a) Place 750 ml of deionized distilled water into a
clear glass vessel, and bring water to 60° +^1°
C in a water bath.
(b) Add an ami no acid (other than L-Glutamine) to the
water.
(c) Remove flask from waterbath, and stir over magnetic
stirrer until amino acid dissolves completely.
- 9-32 -
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(d) Return vessel to water bath, warm solution in
vessel to 60° +_ 1° C, weigh out another amino
acid, and repeat procedure until all amino acids
(except L-glutamine) have been dissolved.
(e) Cool solution of amino acids to 4° C.
(f) Add L-glutamine, and stir solution until the
L-glutamine has dissolved.
(g) Adjust volume of solution (amino acid stock) to
1 liter with deionized distilled water.
(h) Filter-sterilize solution through a 0.22-jm
membrane.
If amino acid stock is to be used for preparing a
medium that subsequently will be filter-sterilized,
f i1ter- s te r i 1i zatio n o f ami no ac ids stock is
unnecessary.
(i) Store stock solution at 4° C.
If amino acid stock solution is to be stored
without filter sterilization, store at -20° C.
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7.9 100X Vitamins Stock for Eagle's Minimum Essential Medium
7.9.1 Formula (Preparation of 1 liter)
Solution A
Deionized distilled water 750 ml
Choline chloride 400 mg
Nicotinamide 400 mg
Pantothenic acid 400 mg
Pyridoxal HC1 400 mg
Thiamine HCL 400 mg
Riboflavin 40 mg
i-Inositol 700 mg
Solution B
Deionized distilled water 125 ml
Folic acid 400 mg
NaOH, IN As needed
7.9.2 Procedure
(a) Place 750 ml of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add to the deionized distilled water the
ingredients listed under Solution A.
Allow each ingredient to go into solution before
adding the next one.
(d) Dissolve the folic acid in 125 ml of deionized
distilled water by continuous stirring.
- 9-34 -
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(e) Add IN MaOH slowly to the folic acid solution until
that solution becomes clear (Solution B).
(f) Add Solution B to Solution A.
(g) Adjust volume of combined solution (vitamin stock)
to 1 liter with deionized distilled water.
If vitamin stock solution appears turbid, add IN
MaOH until solution becomes clear.
(h) Filter-sterilize stock solution through a 0.22-jJ«n
membrane.
If vitamin stock is to be used for preparing a
medium that subsequently will be filter-
sterilized, filter-sterilization of vitamin stock
is unnecessary.
(i) Store vitamin stock solution at 4° C.
If vitamin stock solution is to be stored without
filter-sterilization, store at -20° C.
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7.10 Leibovitz's L-15 Medium
7.10.1 Formula (Preparation of 1 liter)
This medium can be prepared only in a IX concentration;
it cannot be prepared in more concentrated form.
Deionized distilled water 750 ml
INORGANIC SALTS
CaCl2 140.0 mg
KC1 400.0 mg
KH2P04 60.0 mg
MgCl2'6H20 200.0 mg
MgS04'7H20 200.0 mg
Nad 8000.0 mg
Ha2HP04 190.0 mg
AMIMO ACIDS
L-Alanine 225.0 mg
L-Arginine 500.0 mg
L-Asparagine 250.0 mg
L-Cysteine 120.0 mg
L-Glutamine 300.0 mg
Glycine 200.0 mg
L-Histidine 250.0 mg
L-Isoleucine . . . ., 125.0 mg
L-Leucine 125.0 mg
L-Lysine 75.0 mg
L-Methionine 75.0 mg
L-Phenylalanine 125.0 mg
L-Serine 200.0 mg
L-Threonine 300.0 mg
L-Tryptophan 20.0 mg
L-Tyrosine 300.0 mg
L-Valine 100.0 mg
- 9-36 -
-------
VITAMINS
DL-Ca Pantothenate 1.0 mg
Choline chloride 1.0 mg
Folic acid 1.0 mg
i-lnositol 2.0 mg
Nicotinamide 1.0 mg
Pyridoxine HC1 1.0 mg
Riboflavin-5'-phosphate, sodium 0.1 mg
Thiamine monophosphate 1.0 mg
OTHER COM>ONENTS
D (+) Galactose 900.0 mg
Phenol red 10.0 mg
Sodium pyruvate 550.0 mg
7.10.'2 Procedure
(a) Place 750 ml of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add the ingredients listed to the deionized
distilled water.
Allow each ingredient to go into solution before
adding the next one. Add the phenol red last so
that complete dissolution of each component can be
ascertained.
(d) Adjust volume of medium to 1 liter with deionized
distilled water.
(e) Filter-sterilize medium through a 0.22-/jm
membrane.
(f) Store medium at 4° C.
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7.11 Earle's Balanced Salt Solution (Earle's BSS) 10X Stock
7.11.1 Formula (Preparation of 1 liter)
Deionized distilled water 625 mL
NaCl 68000 mg
KCl 4000 mg
HgS04*7H20 2000 mg
NaH2P04*H20 1250 mg
CaClg 2000 mg
Glucose 10000 mg
7.11.2 Procedure
(a) Place 625 ml of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add ingredients, in the order listed, to the
deionized distilled water.
Allow each ingredient to go into solution before
adding the next one.
(d) Adjust volume of solution to 1 liter with deionized
distilled water.
(e) Filter-sterilize stock solution through a 0.22-^m
membrane.
(f) Store stock solution at 4° C.
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7.12 Sodium Bicarbonate, 7.5%
7.12.1 Formula (Preparation of 1 liter)
Deionized distilled water 750 ml
NaHC03 75 g
7.12.2 Procedure
(a) Place 750 ml of COLD deionized distilled water into
a clear glass vessel.
(b) Maintain constant stirring.
(c) Add NaHCOo to the deionized distilled water, and
stir until the NaHCOo is completely dissolved.
(d) Adjust volume to 1 liter with deionized distilled
water.
(e) Filter-sterilize solution through a 0.22-jjm
membrane.
Use positive pressure filtration only.
(f) Dispense solution into glass vessels immediately
after filtration.
Use only vessels with air-tight rubber stoppers, or
with air-tight screw caps.
(g) Store solution at 4° C.
7.13 Magnesium Chloride, 1%
7.13.1 Formula (Preparation of 1 liter)
Deionized distilled water 1 liter
MgCl2*6H20 10 g
- 9-39 -
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7.13.2 Procedure
(a) Place 1 liter of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add the MgCl2'6H20 to the deionized distilled
water.
Stir until the MgClg is completely dissolved.
(d) Autoclave solution at 121° C for 30 minutes.
(e) store solution at 4° C.
7.14 Trypsin-EDTA (Na2EDTA*2H20)* Solution
7.14.1 Formula (Preparation of 1 liter)
(a) Solution A
Deionized distilled water 220 ml
Trypsin, 1:250 3000 mg
Trypsin 1;300 may be substituted for trypsin 1:250.
If trypsin 1:300 is used, use 2.5 g of trypsin
instead of 3.0 g.
(b) Solution B
Deionized distilled water 778 ml
Nad 8000 mg
KC1 200 mg
KH2P04 200 mg
Na2HP04*7H20 1150 mg
Glucose 5000 mg
EDTA* (Versene) 1250 mg
*Disodium EDTA dihydrate
- 9-40 -
-------
(c) Additional components
HC1, IN . As needed
NaOH, IN As needed
7.14.2 Procedure
(a) Solution A
(a.l) Place 220 ml of deionized distilled water
into a clear glass vessel.
(a.2) Maintain constant stirring.
(a.3) Add the trypsin to the deionized distilled
water.
(a.4) Stir trypsin in water on a magnetic stirrer
until all of the trypsin is dissolved.
Expect to stir for at least two hours.
(b) Solution B
(b.l) Place 778 ml of deionized distilled water
into a clear glass vessel.
(b.2) Maintain constant stirring.
(b.3) Add NaCl, KCl, KH2P04, Na2HP04
*7H20, glucose, and EDTA to the deionized
distilled water.
(b.4) Stir until all ingredients are dissolved.
Expect to stir for at least two hours.
(c) Add Solution A to Solution B.
(d) Stir for two hours.
(e) Adjust pH of trypsin solution (Solution A and
Solution B combined) to 7.5-7.7 with HCl or NaOH.
(f) Filter-sterilize trypsin solution through a 0.22-/un
membrane.
(g) Store trypsin solution at 4° C or at -20° C.
- 9-41 -
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7.15 Neutral Red, 0.1%
7.15.1 Formula (Preparation of 1 liter)
Deionized distilled water 1 liter
Neutral red 1 g
7.15.2 Procedure
(a) Place 1 liter of deionized distilled v/ater into a
clear glass vessel.
(b) Maintain constant mixing.
(c) Add neutral red to the deionized distilled water.
Stir until the neutral red is completely
dissolved.
(d) Filter-sterilize neutral red solution through a
0.22 jura membrane.
(e) Store neutral red solution in the dark at ambient
temperatures.
7.16 Phenol Red, 0.5%
7.16.1 Formula (Preparation of 1 liter)
Deionized distilled water 1 liter
Phenol red 5 g
NaOH, IN As needed
7.16.2 Procedure
(a) Place 750 mL of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add phenol red to the deionized distilled water.
(d) Place pH electrodes into the mixture, and adjust pH
to 7 with IN MaOH.
Stir until phenol red has dissolved completely.
- 9-42 -
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(e) Adjust volume of phenol red solution to 1 liter
with deionized distilled water.
(f) Autoclave phenol red solution at 121° C for 15
minutes.
(g) Store phenol red solution at 4° C.
7.17 Trypan Blue Solution (0.5%) for Cell Counting Procedure
7.17.1 Formula (Preparation of 1 liter)
Deionized distilled water . ... 1 liter
Trypan blue 5.0 g
NaCl 8.5 g
7.17.2 Procedure
(a) Place 1 liter of deionized distilled water into a
clear glass vessel.
(b) Maintain constant stirring.
(c) Add the trypan blue and NaCl to the deionized
distilled water.
Sti r untij jtryjpjm bl ue and HaCj have di ssol ved
completely.
(d) Autoclave trypan blue stain at 121° C for 15
mi nutes.
(e) Store trypan blue stain at ambient room
temperature.
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7.18 Stock Solutions of Antibiotics for Cell Culture and Overlay Media
7.18.1 Formula (Stock Solutions)
Amphotericin B (Fungizone) 5 mg/mL
Gentamicin sulfate 50 mg/mL
Mystatin (Mycostatin) 50,000 units/ml
Neomycin sulfate 10 mg/mL
Penici11i n-streptomyci n
Penicillin 6 100,000 units/mL
Dihydrostreptomycin sulfate 125 mg/mL
(80% active)
Tetracycline hydrochloride 25 mg/mL
7.18.2 Procedure
(a) Prepare stock antibiotic solutions and suspensions
according to manufacturer's instructions.
If stock antibiotic solutions are not purchased in
a stej^le^ ^ be filter-.sterilized
through a 0.22-jum membrane before they are used.
(b) store antibiotic solutions at 4° C except
amphotericin B.
Store amphotericin B at -20°^.
7.18.3 Use Levels for Stock Solutions of Antibiotics/100 mL of
Medium
(a) Amphotericin B 0.02 mL
(b) Gentamicin sulfate 0.10 mL
(c) Nystatin* 0.20 mL
*Nystat1n may be used in place of or in addition to amphotericin B to
control fungi.
- 9-44 -
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(d) Penicillin G-dihydrostreptomycin sulfate . .0.15 ml
(e) Tetracycline hydrochloride 0.05 ml
(f) Neomycin sulfate** 0.10 ml
**Neomycin may be used in agar overlays when microorganisms resistant to
the antibiotics normally used in the overlays are encountered.
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8. BIBLIOGRAPHY
Barren, A. L., C. Olshevsky, and M. M. Cohen. 1970.
Characteristics of the BGM Line of Cells from African
Green Honkey Kidney. Archiv. for Die Gesamte
Virusforschung. 32:389-392.
Dahling, D. R., G. Berg, and D. Berman. 1974. BGM, A
Continuous Cell Line More Sensitive than Primary Rhesus
and African Green Kidney Cells for the Recovery of Viruses
from Water, Health Laboratory Sciences. 11:275-282.
Eagle, H. 1959. Ami no Acid Metabolism in Mammalian Cell
Cultures. Science. 130:432-437.
Leibovitz, A. 1963. The Growth and Maintenance of
Tissue-Cell Cultures in Free Gas Exchange with the
Atmosphere. American Jour. Hygiene. 78:173-180.
Laboratory Manual in Virology, Edition Two. Ontario Ministry
of Health, Toronto, Ontario, Canada, 1974. 375 pp.
Paul, J. 1975. Cell and Tissue Culture, Fifth Edition.
Churchill Livingstone, Medical Division of Longman Group
Limited, London, Great Britain, 484 pp.
Rovozzo, G. C., and C. N. Burke. 1973. A Manual of
Basic Virological Techniques. Prentice-Hall, Inc.,
Englewood Cliffs, Hew Jersey, 287 pp.
- 9-46 -
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CHAPTER 10
VIRUS PLAQUE CONFIRMATION PROCEDURE
The technique described In this chapter may be used for confirming viral
plaques in cell cultures adhering to glass or plastic surfaces or in cells
suspended in overlay agar.
Use aseptic techniques and sterile materials and apparatus only. Sterilize
all contaminated materials before discarding them (see Chapters 2 and 3).
1. RECOVERY OF VIRUS FROM PLAQUE
1.1 Apparatus and Materials
1.1.1 Disposable Pasteur pipettes -- 22.9 cm (long tip).
Flame pipette gently about 2 cm from end of tip until tip
bends to approximate angle of 45°.
1.1.2 Rubber bulb — 1 mL capacity.
1.1.3 Cell culture in roller tube.
Use culture appropriate for virus likely to be recovered.
1.1.4 Test tube rack for roller tube cultures.
1.1.5 Storage medium, Earle's balanced salt solution containing 2%
heat-inactivated fetal calf serum (see Chapter 9).
Storage medium is necessary only if plaque sample material
is to be stored before confirmation procedure is completed.
Whenever possible, plaque sample material should be
inoculated onto a cell culture immediately, because storage
of such sample material even at -70° C may result in some
reduction in confirmation counts.
- 10-1 -
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1.1.6 Five-mL screw-capped (with rubber insert) vial.
Place 2 mL of storage medium in 5-mL screw-capped vial.
1.2 Procedure
1.2.1 Procedure for obtaining viruses from plaque.
Decision to test plaque material for viruses immediately or
to store material at -70° C for later testing must be made
before proceeding further.
(a) Place rubber bulb onto top of Pasteur pipette.
(b) Remove screw cap or stopper from plaque bottle (If
plaque is in petri dish, raise cover from dish
sufficiently to allow entry into dish).
(c) Squeeze rubber bulb on top of Pasteur pipette to expel
air.
(d) Penetrate agar directly over edge of plaque with tip
of Pasteur pipette.
(e) Gently force tip of pipette through agar to surface of
vessel, and scrape cells from edge of plaque.
If cells are present as a monolayer on the surface of
the vessel, surface must be repeatedly scratched and
gentle suction applied to insure that virus-cell-agar
plug enters pipette. If cells are suspended in the
agar, scraping of vessel surface with pipette is
unnecessary.
(f) Aspirate plug from plaque into pipette.
(g) Remove pipette from plaque bottle (or petri dish).
(h) Replace and tighten down screw cap or stopper on
plaque bottle (If plaque is in petri dish, replace
cover on dish).
- 10-2 -
-------
If sample Is to be tested in cell culture immediately,
proceed to Section 1.2.2, Step (a). If sample must be
stored, proceed to Section 1.2.2, Step (b).
1.2.2 Procedure for inoculating viruses obtained from plaques onto
cell cultures.
(a) Procedure for samples tested immediately.
(a.l) Remove cap from cell culture tube.
(a.2) Place tip of Pasteur pipette containing
virus-eel1-agar plug into medium in cell
culture tube.
Tilt cell culture tube as necessary to
faci1i tate procedure.
(a.3) Force agar from Pasteur pipette into cell
culture medium by gently squeezing rubber
bulb.
Squeeze bulb repeatedly to wash contents of
pipette into cell culture medium.
(a.4) Withdraw pipette from cell culture tube,
replace and tighten down screw-cap on tube, and
discard pipette.
(a.5) Place cell culture tube in rack for roller tube
cultures.
(a.6) incubate cell culture at 36.5° C +_ 1° C,
and examine cells daily for cytopathic effects
(CPE).
- 10-3 -
-------
Incubate under conditions and for a period of
time appropriate for the virus likely to be
recovered. See Chapter 9 for cell culture and
cell culture examination techniques.
If confirmation is to be completed by
identifying viruses (enteroviruses) recovered,
proceed to Chapter 11.
(b) Procedure for samples stored at -70° C before
testing.
(b.l) Thaw vial containing storage medium in a
36° C water bath, and remove cap from vial.
(b.2) Place tip of Pasteur pipette containing virus
cell-agar plug into storage medium.
(b.3) Force agar from Pasteur pipette into storage
medium by gently squeezing rubber bulb.
Squeeze bulb repeatedly to wash contents of
pipette into storage medium.
(b.4) Withdraw pipette from vial, replace and tighten
down screw-cap onto vial, and discard pipette.
(b.5) Store vial at -70° C.
When confirmation is to be completed, thaw
sample quickly in warm water, and proceed to
Step (b.6).
(b.6) Remove cap from cell culture tube.
(b.7) Remove cap from storage vial containing thawed
sample.
- 10-4 -
-------
(b.8) With a 2-mL pipette, inoculate complete
contents of vial containing sample into cell
culture tube.
Take care to wash total contents of pipette
cell culture medium.
(b.9) Withdraw pipette from cell culture tube,
replace and tighten down screw-cap on tube,
discard pipette and sample vial.
(b.10) Place cell culture tube in rack for roller tube
cultures.
(b.ll) incubate cell culture at 36.5° C +_ 1° C,
and examine cells daily for cytopathic effects
(CPE).
incubate under conditions and for a period of
time appropriate for the virus likely to be
recovered. See Chapter 9 for cell culture and
cell culture examination techniques.
If confirmation is to be completed by
identifying viruses (enterovi ruses) recovered,
proceed to Chapter 1 1 .
- 10-5 -
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CHAPTER 11
IDENTIFICATION OF ENTEROVIRUSES
1. PROCEDURE FOR TYPING VIRUSES
A neutralization test for enterovi'ruses is described in this chapter. The
test procedure utilizes Lim Benyesh-Melm'ck (LB-M) antiserum pools A-H for
the identification of 37 enteroviruses, a single antiserum preparation for
the identification of coxsackievirus B3 and LB-M pools J-P for the
identification of 19 type A coxsackieviruses not identified by pools A-H.
The antiserum pools, with instructions for rehydration and storage and
with virus identification tables, were available until recently from the
National Institutes of Health (NIH). These antiserum pools have now been
depleted. New pools are being produced by the World Health Organization
(WHO) and should be available by the time this manual Is published. The
method described herein, with some modification to accomodate differences
in pool design, should be appropriate for the new pools.
The mlcrotlter method described herein is a modification of the method
described in the literature accompanying the NIH pools. The two methods
work equally well, but the mlcrotlter method requires much less antiserum.
1.1 Apparatus and Materials
1.1.1 Microtiter plates, 96 well, flat bottom.
1.1.2 Sealing tapes for mlcrotlter plates If plates are to be
Incubated in a non-C02 incubator (recommended method), or
- 11-1 -
-------
plastic lids for nricrotiter plates if plates are to be
incubated in a C02 incubator.
1.1.3 Micro-pipettors or pipettes capable of dispensing volumes of
0.025 and 0.05 ml.
1.1.4 Cornwall syringe, or equivalent, capable of delivering 0.2 ml
quantities.
1.1.5 Cotton-tipped applicators.
1.1.6 Magnetic stirrer and stir bars.
1.1.7 Narrow-tip felt marking pen.
1.2 Media and Reagents
1.2.1 Earle's Balanced Salt Solution (EBSS) (for dilution).
Prepare 40 ml for each virus to be identified.
1.2.2 Antiserum pools A-H and coxsackievirus B3 antiserum diluted
and prepared as described in NIH instruction sheets.
Store at -20° C until used.
1.2.3 Growth medium containing 5% gamma globulin-free or normal
fetal calf serum.
Prepare 30 ml of medium for each microtiter plate to be used.
Prepare antiserum pools J-P only when needed to type viruses
not identified by pools A-H or coxsackievirus B3 antiserum.
1.3 Procedure
1.3.1 Preparation of Microtiter Plates
Arrange each plate as indicated in Figures 11-1 and 11-2.
(a) With a narrow-tip felt marking pen, draw lines between
every two columns along the length of the plate.
(b) On one end of each plate, mark identification code of
samples tested.
Four viruses can be identified simultaneously on one
- 11-2 -
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E2 XXXXXXXX
El XXXXXXXX
E XXXXXXXX
B3 XXXXXXXX
H XXXXXXXX
Row G XXXXXXXX
F XXXXXXXX
E XXXXXXXX
D XXXXXXXX
C XXXXXXXX
B, XXXXXXXX
A XXXXXXXX
1234
Column
Figure 11-1. Schematic Representation of Microtiter Plate Preparation
(See Figure 11-2 for Photographic Representation of Microtiter
Plate Preparation).
- 11-3 -
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Figure 11-2.
Photographic Representation of Microtiter Plate Preparation
(See Figure 11-1 for Schematic Representation of Microtiter
Plate Preparation).
- 11-4 -
-------
piate. inns, numoer trie columns i, z, s, ana q_ to
designate duplicate wells for each virus.
(c) Mark identity of each antiserum on left side of plate
next to each row of wells.
(See Figure 11-1). Designate the first eight rows as A-H
to indicate LB-M pools A-H, designate row 9 as B3 to
indicate coxsackievlrus B3 antiserum, designate row 10 as
E to indicate virus control dilution made in Earle's
balanced salt solution (EBSS) (see Chapter 9), and
designate rows 11 and 12 as El and E2, respectively, to
indicate serial 10-fold dilutions of virus control in row
]IO.
1.3.2 Preparation of Virus for Identification
(a) Remove virus isolate from storage in -70° C freezer,
thaw, and mix well. Designate the virus isolate as No. 1.
(b) Dilute thawed virus to 10"5 in EBSS.
5 *?
Prepare 2 ml of 10" dilution of virus. The 10"
dilution is the working dilution of virus that will be
mixed with the antiserum pools in the microtiter plate
wells.
(c) From the 10 dilution, prepare a 1:2 dilution in EBSS.
This dilution will be transferred to row E of the
microtiter plate later.
(d) From the 1:2 dilution of virus prepare two serial 10-fold
dilutions (1:20 and 1:200).
These dilutions will be transferred to rows El and E2 of
microtiter plate later.
(e) Repeat Steps (a)-(d) with each virus isolate to be
- 11-5 -
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identified, designate these isolates 2-4, and proceed to
Section 1.3.3.
1.3.3 Addition of Antiserum Pools to Microtiter Plate
(a) Thaw the antiserum pools, and mix each antiserum pool
well.
(b) With a micro-dilutor tip or pipette, dispense 0.025 ml of
antiserum from pool A into each well in row A.
It is important to place tip of diTutor or pipette into
the bottom of the well and to expel all of the antiserum
in the pipette into the well.
(c) Repeat Section 1.3.3, Steps (a) and (b) with antiserum
pools B-H and with the antiserum for coxsackievirus B3,
placing antiserums into designated wells, and proceed to
Section 1.3.4.
1.3.4 Addition of Virus to Microtiter Plates
5
(a) Add 0.025 ml of the 10 dilution of virus No. 1 [from
Section 1.3.2, Step (b)] to each well in rows A-B3 of
column 1.
Take care to introduce the virus at the top of the
wells. Do not allow tip of diTutor or pipette to
touch an antiserum and thereby possibly cross-
contaminate other antiserums.
(b) Into the two wells marked E in column 1, add 0.05 ml of
the 1:2 dilution of virus No. 1 from Section 1.3.2,
Step (c).
(c) Into the two wells marked El in column 1, add 0.05 ml of
the 1:20 dilution of virus No. 1 from Section 1.3.2, Step
(d).
- 11-6 -
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(d) into the two wells marked E2 in column 1, add 0.05 mL or
the 1:200 dilution of virus No. 1 from Section 1.3.2,
Step (d).
(e) Repeat steps (a)-(d) with viruses No. 2-4, adding the
appropriate dilutions of the viruses to the appropriate
wells (See Figure 11-1).
(f)' Gently tap the sides of the microtiter plate with index
finger to mix the contents of the wells.
(g) Cover microtiter plates with lids or with a loose sterile
cover, and incubate plates at 36.5° C +^ 1° C for two
hours.
1.3.5 Preparation of Cell Suspension and Completion of Microtiter Test
Many host cell types, primary and continuous, are available for
propagating viruses. Usually, the host cell type in which a
virus is recovered from the environment is suitable for
identifying that virus by the microtiter neutralization test.
See Chapter 9 for methods for preparation of BGM cell
cultures. See Lennette, E. H. and Schmidt, N. J.,
Pi agnostic P rocedures for V^al, Rl cjcetts i alandChi amydial
Infections, American Public Health Association, Washington, D.
C., 1979, for methods for preparation of primary and other
continuous cell types, for suckling mouse procedures necessary
for identifying most Group A coxsackieviruses, and for methods
for identifying viruses other than enteroviruses.
(a) Trypsinize sufficient cells to yield a final cell count
appropriate for the cells used in the test.
For BGM cells a count of 30,000-50,000 cells per 0.2 ml
of cell culture medium is appropriate. The number of
- 11-7 -
-------
cells required for this test differs with different cell
types.
(b) Mix cells in medium for at least 15 minutes.
A longer period of mixing will generally not injure cells.
(c) After virus-antiserum mixtures have incubated for two
hours (Section 1.3.4, Step G), with a Cornwall syringe,
dispense 0.2 ml of cell suspension into each well.
Do not allow tip of syringe to touch contents of a well
and thereby possibly cross-contaminate the contents of
other wells. With cotton-tipped applicators, wipe up
spilled cells on the top of plates between and around
wells.
(d) Remove backing from sealing tape, seal each plate, and
incubate plates at 36.5° C +_ 1° C.
If plates are to be incubated in a C0g incubator, do
not seal plates.
(e) After three days of incubation, examine cells in wells
daily for five more days for cytopathic effects (CPE).
Use an inverted microscope to examine cells.
(f) When CPE develops, use identification tables provided
with antiserum pools to identify viruses.
If all wells evidence CPE and identification cannot be
made with virus identification tables, titrate virus and
repeat entire test with a virus dilution calculated to
add 200 infective doses to each well in Row E. Follow
this same procedure if all virus control wells in Rows 1
and 2 are negative and the pattern of results does not
allow identification with identification tables.
- 11-8 -
-------
If virus control wells show that an appropriate quantity
of virus infective doses has been used in test and cells
in at least one well containing antiserum show no CPE and
identification cannot be made with identification tables,
repeat tests with antiserum pools A-H and B3.
If, under this circumstance, CPE appears in all wells
containing virus and antiserum, repeat test but with
antiserum pools J-P instead of A-H and B3.
- 11-9 -
-------
2. BIBLIOGRAPHY
Laboratory Manual in Virology, Edition Two. Ontario Ministry of Health,
Toronto, Ontario, Canada, 1974. 375 pp.
Lennette, E. H. and Schmidt, N. J. 1979. Diagnostic Procedures for Viral,
Rickettsial and Chlatnydial Infections, American Public Health Association,
Washington, D.C. 1138 pp.
- 11-10 -
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APPENDIX
VENDORS*
American Scientific Products
2410 McGaw Road
Obetz, Ohio 43207
614-491-0050
Badger Meter inc.
Flow Products Division
4545 West Brown Deer Road
Milwaukee, Wisconsin 53223
414-355-0400
Becton, Dickinson and Company
Rutherford, New Jersey 07070
201-460-2232
Bellco Biological Glassware
Vineland, New Jersey 08360
609-691-1075
Bristol Laboratories
Division of Bristol-Myers Company
P. 0. Box 657
Syracuse, New York 13201
315-432-2000
Brockway Glass Company, Inc.
Parkersburg, West Virginia 26101
304-295-9311
Carborundum Company
Commercial Filters Division
Lebanon, Indiana 46052
317-482-3900
The Clorox Company
P. 0. Box 24305
Oakland, California 94623
415-271-7000
*List of vendors only indicates one possible source for products used
in this Manual, in most instances, many other vendors can supply
the same materials listed or acceptable alternatives.
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Corning Glass Works
P. 0. Box 5000
Corning, New York 14831
607-974-9000
Cos tar Division
Data Packaging Corporation
205 Broadway
Cambridge, Massachusetts 02139
617-492-1110
Department of Health and Human Services
United States Public Health Service
National Institutes of Health
Building WW, Room 7A03
Bethesda, Maryland 20205
202-496-2131
Difco Laboratories
P. 0. Box 1058A
Detroit, Michigan 48232
313-961-0800
Du Bois Chemical Company
1300 Du Bois Tower
Cincinnati, Ohio 45202
513-762-6000
. Eli Lilly and Company
307 E. McCarty Street
Indianapolis, Indiana 46285
317-261-2000
Falcon
Division of Becton, Dickinson and Company
Oxnard, California 93030
800-235-5953
Filterite Corporation
2033 Greenspring Drive
Timonium, Maryland 21093
301-252-0800
Fisher Scientific Company
585 Alpha Drive
Pittsburgh, Pennsylvania 15238
412-784-2600
Flow Laboratories, Inc.
7655 Old Springhouse Road
McLean, Virginia 22102
301-881-2900
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Grand Island Biological
3175 Staley Road
Grand Island, New York 14072
716-773-7616
Hana Media, Inc.
Berkeley, California 94710
415-549-0874
Johanson and Son Machine Corporation
259 Allwood Road
Clifton, Mew Jersey 07012
201-773-6160
Kansas City Biological, Inc.
P. 0. Box 5441
Lenexa, Kansas 66215
800-255-6032
Kimble
Division of Owens-Illinois
P. 0. Box 1035
Toledo, Ohio 43666
419-247-0727
Lederle Laboratories Division
American Cyanamid Company
P. 0. Box 149
Pearl River, New York 10965
914-735-5000
M. A. Bioproducts
Unit of Whittaker Corporation
Building 100, Biggs Ford Road
Walkersville, Maryland 21793
800-638-8174
Millipore Corporation
Bedford, Massachusetts 01730
617-275-9200
Norton Company
Plastics and Synthetics Division
P. 0. Box 350
Akron, Ohio 44309
216-630-9230
Pfizer Laboratories Division
Pfizer, inc.
235 East 42nd Street
New York, New York 10017
212-573-2323
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Polychem Corporation
12 Lyman St.
New Haven, Connecticut 06511
203-777-7363
Real Fresh, Inc.
1211 E. Noble Avenue
Visalia, California 93277
209-732-8005
Schering-Plough Corporation
Galloping Hill Road
Kenilworth, New Jersey 07033
201-931-2000
Sigma Chemical Company
P. 0. Box 14508
St. Louis, Missouri 63178
800-325-3010
E. R. Squibb and Sons, Inc.
P. 0. Box 4000
Princeton, New Jersey 08540
Arthur H. Thomas Company
Third and Vine St.
Philadelphia, Pennsylvania 19106
215-574-4500
609-921-4000
Van London Company
6103 Glenmont
Houston, Texas 77036
713-772-6641
The Vollrath Company
1236 North 18th Street
Sheboygan, Wisconsin 53081
4H_457_4851
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* U.S. GOVERNMENT PRINTING OFFICE: 1984-759-102/0857
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