EPA/814-B-95-002
June 1995
Virus Monitoring Protocol
for the Information
Collection Requirements Rule
United States Environmental Protection Agency
OGWDW/TSD/WSTB
26 West Martin Luther King Drive
Cincinnati, OH 45268
^<£) Printed on Recycled Paper
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The use of manufacturer trade names in this document
does not constitute endorsement by the United States
Environmental Protection Agency.
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Contents
About This Document v
Acknowledgments vi
Foreword: The Virus Monitoring Protocol vii
Chapter 1: Introduction 1
Laboratory Approval Requirements 1
Cell Culture Preparation .,„......, 1
Personnel Requirements 2
Principal Analyst/Supervisor ...2
Analyst 2
Technician 2
For More Information 2
Chapter 2: Sample Processing 3
Quality Control and Performance Evaluation Samples 3
QC Samples 4
PE Samples „ 5
Sample Arrival 5
Elution Procedure , 5
Apparatus and Materials , 5
Media and Reagents 6
Procedure 7
Organic Flocculation Concentration Procedure '...„ 10
Apparatus and Materials 10
Media and Reagents 11
Procedure 11
Chapter 3: Total Culturable Virus Assay 15
Quantal Assay 15
Apparatus and Materials 15
Media and Reagents 15
Sample Inoculation and CPE Development 16
Virus Quantisation 22
Reduction of Cytotoxicity in Sample Concentrates 24
Media and Reagents 24
Procedure for Cytotoxicity Reduction 24
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CONTENTS
Appendix A: Cell Culture Preparation and Maintenance A - 1
Preparation of Cell Culture Medium A -1
General Principles A -1
Apparatus and Materials A -1
Media and Reagents A - 2
Media Preparation Recipes A - 3
Preparation and Passage of BGM Cell Cultures A - 5
Vessels and Media for Cell Growth A - 5
General Procedure for Cell Passage A - 5
Procedure For Performing Viable Cell Counts A - 6
Procedure for Preservation of BGM Cell Line A - 8
Preparation of Cells for Storage A - 8
Procedure for Freezing Cells A - 9
Procedure for Thawing Cells A- 9
Appendix B: Sterilization and Disinfection B -1
General Guidelines B -1
Sterilization Techniques B -1
Solutions B -1
Autoclavable Glassware, Plasticware, and Equipment B - 1
Chlorine Sterilization B - 2
Procedure for Verifying Sterility of Liquids B - 2
Media and Reagents B - 2
Verifying Sterility of Small Volumes of Liquids B - 2
Visual Evaluation of Media for Microbial Contaminants B - 3
Contaminated Materials B - 3
Appendix C: Bibliography and Suggested Reading C -1
Appendix D: Vendors - D -1
Appendix E: Data Sheets E - 1
Appendix F: Examples of Calculations F - 1
Example 1 F -1
Example 2 F - 8
IV
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About This Document
This bench-top guide is intended for use by laboratory
personnel analyzing water samples for the presence of
culturable viruses in accordance with the Information
Collection Requirements Rule. It provides details of the pro-
cedure presented in the accompanying video. To further asso-
ciate the steps of the procedure with the training video, this
manual is illustrated with stills taken directly from the video.
Several graphic conventions are used throughout the manual
to differentiate steps or draw attention to important informa-
tion:
A step icon is used to denote each step in the virus
monitoring protocol. These steps also are illustrated
in the training video.
Important information, and valuable tips which are not
part of the published protocol, are denoted by this icon.
In addition, explanatory information is provided in ital-
ics throughout the document.
EPA recommends that laboratory analysts and technicians
watch the training video and read this manual in order to be-
come familiar with the virus monitoring protocol. The video
concludes with a summary of the analytical procedure. Ana-
lysts and technicians also can use the manual as a bench-top
reference during the execution of the procedure.
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Acknowledgments
This bench-top guide and the accompanying training
video were developed by the U.S. Environmental Pro-
tection Agency (EPA), Office of Water, Office of Ground
Water and Drinking Water (OGWDW), Cincinnati, Ohio. Jim
Walasek of OGWDW's Technical Support Division (TSD) was
the project manager. Shay Fout, Ph.D. of the Office of Re-
search and Development's National Exposure Research Labo-
ratory (NERL) wrote the text of the guide. He was the princi-
pal technical advisor in the preparation of the video, and was
responsible for the photomicrography used in both the guide
and the video. Frank Schaefer, Ph.D. of NERL also served as
technical advisor on the project. Ken Mayo of The Cadmus
Group, Inc. in Waltham, Massachusetts edited, designed, and
coordinated production of the guide.
The photographs used in this guide come from the accompa-
nying training video. The video was taped at the U.S. EPA
laboratories in Cincinnati, Ohio. Melissa Godoy of Impact
Video Productions in Cincinnati was the producer, and Gra-
ham Spencer and Steve Willis were the editors. Donna Jensen
of Cadmus acted as analyst during the taping, and Michelle
Thomas was the technician. Ms. Thomas is a participant at
EPA in a post-graduate research program administered by the
Oak Ridge Institute for Science and Education.
VI
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Foreword
The Virus Monitoring
Protocol
The surface water treatment rule (40 CFR
Part 141) established the maximum
contaminant level for enteric virus in pub-
lic water systems by requiring that systems using
surface water or ground water under the influence
of surface water reduce the amount of virus in
source water by 99.99 percent.
The rule requirements are currently met on the ba-
sis of treatment alone (e.g., disinfection and/or fil-
tration), and thus the degree of actual protection
against waterborne viral disease depends upon the
source water quality. Utilities using virus-free
source water or source water with low virus levels
may be overtreating their water, while utilities us-
ing highly contaminated water may not be provid-
ing adequate protection.
To determine more adequately the level of protec-
tion from virus infection and to reduce the levels
of disinfection and disinfection by-products, where
appropriate, EPA is requiring all utilities serving a
population of at least 100,000 to monitor their
source water for viruses monthly for a period of
18 months. Systems finding greater than one in-
fectious enteric virus particle per liter of source
water must also monitor their finished water on a
monthly basis. The authority for this requirement
is Section 1445(a)(l) of the Safe Drinking Water
Act, as amended in 1986.
This Virus Monitoring Protocol was developed by
virologists at the U.S. Environmental Protection
Agency and modified to reflect consensus agree-
ments from the scientific community and com-
ments to the draft rule. The procedures contained
herein do not preclude the use of additional tests
for research purposes (e.g., polymerase chain re-
action-based detection methods for noncytopathic
viruses).
The concentrated water samples to be monitored
may contain pathogenic human enteric viruses.
Laboratories performing virus analyses are re-
sponsible for establishing an adequate safety
plan and must rigorously follow the guidelines
on sterilization and aseptic techniques given in
Appendix B.
Analytical Reagent or ACS grade chemicals (un-
less specified otherwise) and reagent-grade water
should be used to prepare all media and reagents.
The reagent-grade water must have a resistance
greater than 0.5 megohms-cm at 25°C, but water
with a resistance of 18 megohms-cm is preferred.
Water and other reagent solutions may be avail-
able commercially. For any given section of this
protocol only apparatus, materials, media and re-
agents which are not described in previous sec-
tions are listed, except where deemed necessary.
The amount of media prepared for each part of the
protocol may be increased proportionally to the
number of samples to be analyzed.
VII
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Chapter 1
Introduction
Under the U.S. Environmental Protection Agency's
(EPA's) Information Collection Requirements Rule
(ICR), public water systems that use surface water,
or ground water under the influence of surface water, and that
serve 100,000 persons or more must monitor their source wa-
ter for viruses and other pathogens. If more than one patho-
gen per liter of source water is found, systems also will have
to monitor their finished water for pathogens and for other
water quality indicators.
EPA will use the information collected under the ICR when
considering possible changes to the Surface Water Treatment
Rule (SWTR) and when developing drinking water regula-
tions covering disinfectants and disinfectant by-products. If
the SWTR is amended, information collected under the ICR
also will help utilities comply with new and updated require-
ments.
Laboratory Approval Requirements
To make comparing results from different systems easier, EPA
requires that all laboratories approved to analyze pathogens
monitored under the ICR use the same analytical procedures.
To qualify for approval to analyze water samples for viruses,
a laboratory must (1) analyze satisfactorily a set of perfor-
mance evaluation (PE) samples of viruses at various concen-
trations, (2) meet personnel requirements for at least one prin-
cipal analyst, and (3) pass an on-site evaluation by EPA.
Cell Culture Preparation
A lab must maintain separate rooms for preparing cell cul-
tures and for processing virus cultures. If separate rooms
are not available, then Class II biological safety cabinets
must be used to prevent contamination when preparing
cell cultures.
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CHAPTER 1
INTRODUCTION
Personnel Requirements
Principal Analyst/Supervisor
A qualified, experienced microbiologist must be available to
act as principal analyst/supervisor. This person oversees the
entire analysis of water samples for the presence of viruses.
He or she must have a bachelor's degree in life sciences, plus
three years of experience in both cell culture and animal virus
analyses.
Analyst
The analyst performs at the bench level and is involved in all
aspects of the analysis, including sample collection, filter ex-
traction, sample processing, and assay. This person must have
two years of full-time college course work in the life sciences,
or at least six months' bench experience in cell culturing and
animal virus analyses.
Technician
The technician extracts the filter and processes the sample, but
does not perform tissue culture work. This person must have
at least three month's experience in filter extraction of virus
samples and sample processing.
For More Information
More information about the procedures presented in this docu-
ment is available from the:
ICR Coordinator
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
26 West Martin Luther King Drive
Cincinnati, OH 45268
You can also call EPA's Safe Drinking Water Hotline at
1 800 426-4791.
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Chapter 2
Sample Processing
Quality Control and Performance
Evaluation Samples
Quality control (QC) samples, containing 1 mL of
attenuated poliovirus type 3 of known
concentration per vial, and performance evaluation
(PE) samples consisting of 1MDS filters containing
attenuated poliovirus type 3 at unknown concentrations, will
be prepared by an ICR contractor and shipped to analysts seek-
ing approval.
As part of the initial approval process, each analyst participat-
ing in the ICR virus monitoring program must successfully
analyze PE samples. After initial approval, each analyst must
successfully analyze one QC sample set per sample batch and
new PE samples every month. A QC sample set is composed
of a negative and a positive QC sample. A sample batch con-
sists of all the ICR samples that are analyzed by an analyst
during a single week. Each sample batch and its associated
QC sample set must be assigned a unique batch number. QC
samples do not have to be processed during weekly periods
when no ICR samples are processed. QC and PE data should
be sent directly to EPA as specified in the final rule.
Gloves and a lab coat should always be worn when-
ever handling virus and water samples that may con-
tain virus.
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CHAPTER 2
SAMPLE PROCESSING
QC Samples
Negative QC Sample
Elute a sterile 1 MDS filter in a sterile cartridge hous-
ing according to the Elution Procedure given below.
Process the eluate using the Organic Flocculation and
Total Culturable Assay procedures that follow.
Positive QC Sample
Place 40 liters of reagent-grade water in a sterile
polypropylene container (Cole-Parmer Product No.
G-06063-32) and add 1 mL of QC stock of attenuated
poliovirus containing 200 PFU/mL.
y
•A QC sample with a titer of 200 PFU/mL will be sup-
plied for the QC tests described in this section. The
titer of QC sample may be changed before the start or
during the testing phase of the ICR. Analysts must use
these samples as supplied and not attempt to adjust the titer to
200 PFU/mL. A high titer QC sample will also be shipped to
each analyst so that laboratories can develop their own inter-
nal QC programs. The high titered sample is not to be used for
the QC tests described in this section.
Mix and pump the solution through a standard appara-
tus containing a 1MDS filter.
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CHAPTER 2
SAMPLE PROCESSING
Process and analyze the 1MDS filters containing QC
samples using the Elution, Organic Flocculation and
Total Culturable Virus Assay procedures given below.
PE Samples
Process and analyze PE samples according to the Elution, Or-
ganic Flocculation and Total Culturable Virus Assay proce-
dures of this protocol and according to any additional proce-
dures supplied with the samples.
Sample Arrival
The cartridge filters must arrive from the utility refrigerated,
but not frozen. Filters should be refrigerated upon arrival at
the laboratory and eluted within 72 hours of the start of the
sample collection.
When the sample arrives, record its condition on the Sample
Data Sheet. (See Appendix E.)
Also note the sample volumes as recorded on the Sample Data
Sheet at the utility. Between 200 and 300 liters of source
water, or 1,500 - 1,800 liters of finished water, must have
been sampled. Samples that do not fall within these ranges
should not be analyzed. More than one 1MDS filter may be
submitted for the same sample, however, in order to meet the
minimum sample volume requirements.
Elution Procedure
Apparatus and Materials
• Positive pressure air or nitrogen source equipped with a pres-
sure gauge.
If the pressure source is a laboratory air line or pump, it
must be equipped with an oil or air filter.
• Dispensing pressure vessels, 5- or 20-liter capacity (Millipore
Corp. Product No. XX67 OOP 05 and XX67 OOP 20).
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CHAPTER 2
SAMPLE PROCESSING
• pH meter with combination-type electrode and an accuracy
of at least 0.1 pH unit.
• Autoclavable inner-braided tubing with screw clamps or
quick connects for connecting tubing to equipment.
• Magnetic stirrer and stir bars.
Media and Reagents
• Sodium hydroxide (NaOH). Prepare 1 M and 5 M solutions
by dissolving 4 g or 20 g of NaOH hi a final volume of 100
mL of reagent-grade water, respectively.
NaOH solutions may be stored for several months at room
temperature.
• Beef extract V powder (BBL Microbiology Systems Prod-
uct No. 97531). Prepare buffered 1.5% beef extract by dis-
solving 30 g of beef extract powder and 7.5 g of glychie
(final glycine concentration = 0.05 M) in 1.9 liters of re-
agent-grade water. Adjust the pH to 9.5 with 1 or 5 M NaOH
and bring the final volume to 2 liters with reagent-grade
water. Autoclave at 121°C for 15 minutes and use at room
temperature.
When sterilizing the flask of beef extract, be sure to
put the flask in a pan of water before placing the flask
and the pan in the autoclave. This will ensure even
heating of the flask and will help keep the flask from
breaking.
Beef extract solutions may be stored for one week at 4 °C or
for longer periods at -20°C.
Screen each new lot of beef extract prior to use in the or-
ganic flocculation concentration procedure to determine
whether virus recoveries are adequate. Perform the screen-
ing by spiking 1 liter of beef extract solution -with 1 mL of a
diluted QC sample containing 200 PFU/mL. Assay the spiked
sample according to the Organic Flocculation and Total
Culturable Virus Assay procedures given below. Use a single
passage with undiluted sample and sample diluted 1:5 and
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7:25 along with an equivalent positive control. The mean
recovery of poliovirus for 3 trials should be at least 50%.
Procedure
CHAPTER 2
SAMPLE PROCESSING
Attach sections of braided tubing (sterilized on inside
and outside surfaces with chlorine and dechlorinated
with thiosulfate as described in Appendix B) to the
inlet and outlet ports of a cartridge housing module
containing a 1MDS filter to be tested for viruses.
If a prefilter was used, keep the prefilter and cartridge housing
modules connected. Attach the tubing to the inlet of the prefilter
module and to the outlet of the cartridge housing module.
Place the sterile end of the tubing connected to the
outlet of the cartridge housing module into a sterile
2-liter glass or polypropylene beaker.
Connect the free end of the tubing from the inlet port
of the prefilter or cartridge housing modules to the
outlet port of a sterile pressure vessel. Connect the in-
let port of the pressure vessel to a positive air pres-
sure source.
Add pressure to blow out any residual water from the cartridge
housing(s) into a sterile 2-liter glass or polypropylene beaker.
Discard any residual water that collects in the beaker,
and use another sterile beaker for the remainder of this
step and for the steps that follow.
Open the vent/relief valve to release the pressure.
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CHAPTER 2
SAMPIE PROCESSING
Remove the top of the pressure vessel and pour 1,000
mLof buffered 1.5% beef extract (pH 9.5), prewarmed
to room temperature, into the vessel.
Replace the top of the pressure vessel and close its vent/relief
valve.
Acceptable alternatives to the use of a pressure vessel include:
1) the use of a peristaltic pump and sterile tubing to push the
beef extract through the filter, and 2) the addition of beef ex-
tract directly to the cartridge housing and the use of positive
pressure to push the beef extract through the filter.
Open the vent/relief valve(s) on the cartridge housing(s)
and apply sufficient pressure to purge trapped air from
them.
Close the vent/relief valve(s) as soon as the buffered beef ex-
tract solution begins to flow from it.
D
Make sure to wear gloves and to place a gauze pad
treated with iodine over the cartridge housing vent/
relief valve for this procedure.
Turn off the pressure and allow the solution to contact the
1MDS filter for one minute.
Wipe up spilled liquid with laboratory disinfectant. Carefully
observe alternative housings without vents to ensure that all
trapped air has been purged.
Increase the pressure to force the buffered beef extract
solution through the filter(s).
You can use the pressure vessel's relief valve to con-
trol the beef extract's rate of flow. Use of the relief
valve provides finer control over the flow rate than
does controlling the air pressure at the source.
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The solution should pass through the 1MDS filter slowly to
maximize the elution contact period. When air enters the line
from the pressure vessel, elevate and invert the filter housing
to permit complete evacuation of the solution from the filters.
CHAPTER 2
SAMPLE PROCESSING
Turn off the pressure at the source and open the vent/
relief valve on the pressure vessel.
Pour the buffered beef extract from the 2-liter beaker
back into the pressure vessel.
Replace the top of the pressure vessel and close its vent/relief
valve.
Repeat Steps 5 and 6.
Turn off the pressure at the source and open the vent/
relief valve on the pressure vessel. Thoroughly mix
the eluate.
If archiving is not required, and if the optional coliphage assay
is not performed, measure the volume of the eluate and record
it on the Virus Data Sheet as the Eluate Volume Recovered.
Take the Total Sample Volume from the Sample Data Sheet
and record it as the Adjusted Total Sample Volume on the Vi-
rus Data Sheet.
If archiving is required, or if the optional coliphage assay will
be performed, adjust the pH of the eluate to 7.0-7.5 with 1M
HC1. Measure the volume of the adjusted eluate and record it
onto the Virus Data Sheet as the Eluate Volume Recovered.
Determine the amount of sample to be removed for archiving
by multiplying the Eluate Volume Recovered by 0.1. Record
this amount onto the Virus Data Sheet as the Volume of Eluate
Archived. Place a volume equal to this amount into a separate
container. Freeze the archive sample and ship it to the ICR
Laboratory Coordinator, U.S. EPA, Office of Ground Water
and Drinking Water, 26 West Martin Luther King Drive, Cin-
cinnati, OH 45268.
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CHAPTER 2
SAMPLE PROCESSING
Determine the amount of sample to be used in the coliphage
assay by multiplying the Eluate Volume Recovered by 0.035.
Place a volume equal to this amount into a separate container
and store at 4°C. Multiply the recorded Total Sample Volume
from the Sample Data Sheet by 0.9 if an archive sample is
taken, by 0.965 if a coliphage sample is taken, or by 0.865 if
both an archive sample and a coliphage sample are taken.
Record the amount on the Virus Data Sheet as the Adjusted
Total Sample Volume.
M
All freezing of sample and cell cultures throughout this
protocol should be performed rapidly by placing ves-
sels in a freezer at -70°C or below, or in a dry ice-
alcohol bath. Frozen samples and cell cultures should
also be thawed rapidly. This may be done by placing vessels in
a 37°C water bath, but vessel caps must not be immersed and
vessels should be removed from the water bath as soon as, or
just before, the last ice crystals melt.
Proceed to the Organic Flocculation Concentration Pro-
cedure immediately. If the Organic Flocculation Con-
centration Procedure cannot be undertaken immedi-
ately, store the pH-adjusted eluate at 4°C for up to 24
hours, or for longer periods at -70°C.
Organic Flocculation Concentration
Procedure
Apparatus and Materials
• Refrigerated centrifuge capable of attaining 2,500 - 10,000
x g and screw-capped centrifuge bottles with 100 to 1,000
mL capacity.
Each bottle must be rated for the relevant centrifugal force.
• Sterilizing filter — 0.22 |im Acrodisc filter with prefilter
(Gelman Sciences Product No. 4525).
Samples containing a lot of debris may require a sterilizing
filter stack. Prepare sterilizing filter stacks using 0.22
10
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CHAPTER 2
SAMPLE PROCESSING
pore size membrane filters (Millipore Corp. Product No.
GSWP 47 00) stacked with fiberglass prefilters (Millipore
Corp. APIS 47 00 and AP20 47 00).
Stack the prefilters and 0.22 /urn membrane in a disc filter
holder (Millipore Corp. Product No. SXOO 47 00) with the
AP20 prefilter on top and 0.22 jjim membrane filter on bot-
tom. Disassemble the filter stack after each use to check the
integrity of the 0.22 pn filter. Refliter any media filtered with
a damaged stack.
Always pass about 10 - 20 mL of beef extract, pH 7.0-7.5
(prepared as above, without pH adjustment), through the
filter just prior to use. This step will reduce virus adsorption
onto the filter membranes.
Media and Reagents
• Sodium phosphate, dibasic (Na2HPO4 • 7H2O) — 0.15 M,
pH 9.0-9.5.
Dissolve 40.2 g of sodium phosphate in a final volume of
1,000 mL. The pH should be checked to ensure that it is be-
tween 9.0-9.5 and adjusted with NaOH, if necessary. Auto-
clave at 121 °Cfor 15 minutes.
• Sodium phosphate, dibasic (Na2HPO4 • 7H2O) — 0.15 M,
pH 7.0-7.5.
Dissolve 40.2 g of sodium phosphate in 900 mL. Adjust the
pH to 7.0-7.5 with 1MHCL Bring to 1,000 mL. Autoclave at
121 °Cfor 15 minutes.
Procedure
Minimize foaming (which may inactivate viruses) throughout
the procedure by stirring or mixing no faster than necessary to
develop a vortex.
Place a sterile stir bar into the beaker containing the
buffered beef extract eluate from the cartridge filter(s).
Place the beaker on a magnetic stirrer, and stir at a
speed sufficient to develop a vortex.
11
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CHAPTER 2
SAMPLE PROCESSING
Insert a combination-type pH electrode into the beef
extract eluate. Slowly add 1M HC1 to the flask, while
moving the tip of the pipette in a circular motion away
from the vortex to facilitate mixing. Continue adding
1 M HC1 until the pH of the beef extract reaches 3.5 ± 0.1.
Continue to stir slowly for 30 minutes at room temperature.
The pH meter must be standardized at pH 4 and 7. Electrodes
must be sterilized before and after each use as described in
Appendix B.
A precipitate will form. If pH falls below 3.4, add 1 MNaOH
to bring it back to 3.5 ± 0.1. Exposure to a pH below 3.4 may
result in some virus inactivation.
Remove the electrode from the beaker, and pour the
contents of the beaker into a centrifuge bottle.
Cap the bottle and centrifuge the precipitated beef ex-
tract suspension at 2,500 x g for 15 minutes at 4°C.
Remove and discard the supernatant.
To prevent the transfer of the stir bar into a centrifuge bottle,
hold another stir bar or magnet against the bottom of the bea-
ker while decanting the contents. The beef extract suspension
will usually have to be divided into several centrifuge bottles.
Balance the centrifuge bottles aseptically with beef
extract or normal saline before placing them in the
centrifuge whenever centrifugation is called for.
Place a stir bar into the centrifuge bottle that contains
the precipitate.
Add 30 mL of 0.15 M sodium phosphate.
Place the bottle on a magnetic stirrer, and stir slowly until the
precipitate has dissolved completely.
12
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CHAPTER 2
SAMPLE PROCESSING
The precipitate may be partially dissipated with a spatula be-
fore or during the stirring procedure. It also may be dissolved
by repeated pipetting, or by shaking at 160 rpmfor 20 minutes
on an orbital shaker, in place of stirring. When the centrifuga-
tion is performed in more than one bottle, dissolve the precipi-
tates in a total of 30 mL and combine into one bottle. If the
precipitate is not completely dissolved before proceeding, sig-
nificant virus loss may occur in Step 5. Since virus loss may
also occur by prolonged exposure to pH 9.0-9.5, laboratories
which find it difficult to resuspend the precipitate may dissolve
it initially in 0.15 M sodium phosphate that has been adjusted
topH 7.5 with 1 MHCl. If this variation is used, the pH should
be readjusted to 9.0-9.5 with 1 M NaOH after the precipitate
is completely dissolved and mixed for 10 minutes at room tem-
perature before proceeding to Step 5.
Check the pH and readjust to 9.0-9.5 with 1 M NaOH,
as necessary.
Remove the stir bar and centrifuge the dissolved pre-
cipitate at 4,000 - 10,000 x g for 10 minutes at 4°C.
Remove the supernatant and discard the pellet.
Adjust the pH of the supernatant to 7.0-7.5 with 1 M HC1.
To remove microbial contamination, load the supernatant into
a 50-mL syringe and force it through a sterilizing filter pre-
treated with beef extract. (Laboratories may use other ap-
proaches to remove contamination, but their effectiveness must
be documented.)
Record the final volume of supernatant (designated the Final
Concentrated Sample Volume, or FCSV) on the Virus Data
Sheet (Appendix E).
If the sterilizing filter begins to clog badly, empty the loaded
syringe into the bottle containing the unfilteredprecipitate, fill
the syringe with air, and inject air into the filter to force any
residual sample from it. Continue the filtration procedure with
another filter.
13
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CHAPTER 2
SAMPLE PROCESSING
Determine the volume of sample that must be assayed.
This volume is at least 100 liters for source water or
1,000 liters for finished water, and is designated the
Volume of Original Source Water Sample Assayed (D).
Record the value of D on the Virus Data Sheet. Calculate the
Assay Sample Volume (S) for source and finished water samples
using the formula:
S =
D
ATSV
xFCSV
where ATSV is the Adjusted Total Sample Volume from the
Virus Data sheet. The Assay Sample Volume is the volume of
the Final Concentrated Sample that represents 100 liters of
source water or 1,000 liters of finished water.
D
'Analytical laboratories assaying more than the required
volume must use the actual volume to be assayed in
the calculation. See Appendix F for examples of the
calculations used throughout this protocol.
Record the Assay Sample Volume on the Virus Data Sheet.
Prepare a subsample (subsample 1) containing a volume 0.55
times the Assay Sample volume.
Prepare a second subsample (subsample 2) containing a vol-
ume that is 0.67 times the Assay Sample volume. Divide the
Final Concentrated Sample from QC and PE samples into two
equal subsamples. Calculate the Assay Sample Volumes for
these samples by multiplying FCSV by 0.4. Label each sub-
sample with appropriate sampling information for identifica-
tion.
Hold any portion of the sample that can be assayed within 24
hours at 4°C and freeze all other portions at -70°C.
Final Concentrated Samples, subsamples, PE and QC samples
processed to this point by a laboratory not doing the virus as-
say must be immediately frozen at -70°C and then shipped on
dry ice to the laboratory approved for the virus assay within
24 hours. Samples which can be rapidly transported between
laboratories can be held at 4 °C as long as the samples can be
transported and assayed within 24 hours.
14
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Chapter 3
Total Cultivable Virus Assay
Quanta! Assay
Apparatus and Materials
• Incubator capable of maintaining the temperature of cell
cultures at 36.5 ± 1°C.
• Sterilizing filter — 0.22 p,m (Costar Product No. 140666).
To minimize virus adsorption to the filter, always pass about
10 - 20 mL of 1.5% beef extract, pH 7.0-7.5, through the
filter just prior to use.
Media and Reagents
• Prepare BGM cell culture test vessels using standard proce-
dures.
BGM cells are a continuous cell line derived from African
Green monkey kidney cells and are highly susceptible to many
enteric viruses (Dahling et al, 1984; Dahling and Wright,
1986). The characteristics of this line were described by
Barron et al. (1970). The use of BGM cells for recovering
viruses from environmental samples was described by
Dahling et al. (1974). For laboratories with no experience
with virus recovery from environmental samples, the media
and procedures described by Dahling and Wright (1986)
and given in Appendix A are recommended for maximum
sensitivity.
The U.S. Environmental Protection Agency will supply an
initial culture of BGM cells to all laboratories seeking ap-
proval. Upon receipt, laboratories must prepare an adequate
supply of frozen BGM cells using standard procedures to
15
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CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
replace working cultures that become contaminated or that
lose virus sensitivity. A procedure for Preservation of the
BGM Cell Line is given in Appendix A.
Sample Inoculation and CPE Development
Cell cultures used for virus assay are generally found to be at
their most sensitive level between the third and sixth days after
their most recent passage. Those older than seven days should
not be used.
Identify cell culture test vessels by coding them with
an indelible marker.
Return the cell culture test vessels to a 36.5 + 1°C
incubator and hold at that temperature until the cell monolayer
is to be inoculated.
Decant and discard the medium from cell culture test
vessels. Wash the test vessels with balanced salt solu-
tion or maintenance medium without serum using a
wash volume of at least 0.06mL/cm2 of surface area.
Rock the wash medium over the surface of each monolayer
several times, and then decant and discard the wash medium.
Do not disturb the cell monolayer.
Determine the Inoculum Volume by dividing the As-
say Sample Volume by 20. Record the Inoculum Vol-
ume onto the Virus Data Sheet.
For ease of inoculation, a sufficient quantity of 0.15M sodium
phosphate, pH 7.0-7.5, may be added to the Inoculum Volume
to give a more usable working Inoculation Volume (e.g., 1.0
mL). For example, if an Inoculum Volume of 0.73 mL is to be
placed onto 10 vessels, then 10.5 x (1-0.73) = 2.84 mL of the
sodium phosphate could be added to 10.5 x 0.73 = 7.67 mL of
16
-------
subsample. Each milliliter of the resulting mixture will con-
tain the required Inoculum Volume.
The Inoculum, or Inoculation, Volume should be no greater
than 0.04 mL/cm2 of surface area. If the Inoculum, or Inocula-
tion, Volume is greater than 0.04 mL/cm2, use larger culture
vessels.
CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Inoculate each BGM cell culture test vessel with an
amount of assay control or water sample equal to the
Inoculum, or Inoculation, Volume and record the date
of inoculation on the Sample Data Sheet.
Avoid touching either the cannula or the pipetting device to
the inside rim of the cell culture test vessels to avert the possi-
bility of transporting contaminants to the remaining culture
vessels.
Negative Controls
Inoculate a BGM culture with a volume of sodium phosphate,
pH 7.0-7.5, equal to the Inoculum, or Inoculation, Volume.
These cultures will serve as negative controls for the tissue
culture quanta! assay. If any Negative Assay Control develops
cytopathic effects (CPE), all subsequent assays of water
samples should be halted until the source of the positive result
is determined.
Positive Controls
Dilute attenuated poliovirus type 3 (from the high titered QC
.stock) in sodium phosphate, pH 7.0-7.5, to give a concentra-
tion of 20 PFU per Inoculum or Inoculation Volume. Inoculate
a BGM culture with an amount of diluted virus equal to the
Inoculum, or Inoculation, Volume. This control will provide a
measure for continued sensitivity of the cell cultures to virus
infection. Additional positive control samples may be prepared
by adding virus to a small portion of the final concentrated
sample and/or by using additional virus types. If any Positive
Assay Control fails to develop CPE, all subsequent assays of
water samples should be halted until the source of the negative
result is determined. It may be necessary to thaw and use an
earlier passage of the BGM cell line supplied by EPA.
17
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CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Water Samples
Rapidly thaw subsample 1, if frozen, and inoculate
an amount equal to the Inoculum, or Inoculation,
Volume onto each of 10 cell cultures. If there is no
evidence of cytotoxicity and if at least 3 cell cultures
are negative for CPE after 7 days (see below), thaw
subsample 2 and inoculate an amount equal to the
Inoculum, or Inoculation, Volume onto each of 10
additional cultures.
y
Subsample 1 Decision Tree
Inoculate 10 Cultures with Subsample 1
No
If dilutions are not required and the Inocu-
lum Volume is not adjusted for surface area,
inoculate subsamples 1 and 2 onto a total of 20 cul-
tures (10 replicates per subsample).
Hold a thawed subsample no more than 4 hours at 4 °C. Warm
ihe subsample to room temperature just before inoculation.
i
Proceed with
Standard Assay
Yes
^
r
Treat for
Cytoxicity
A small portion of the Final Concentrated Sample
may by inoculated onto cultures several days before
inoculating subsample 1 as a control for cytotoxic-
ity.
If cytotoxicity is not a problem and more than 7
cultures are positive for CPE after 7 days, prepare 5-
and 25-fold dilutions of subsample 2. To prepare a
1:5 dilution, add a volume equal to 0.1334 times the
Assay Sample Volume (amount "a") to a volume of
0.15 M sodium phosphate (pH 7.0-7.5) equal to
0.5334 times the Assay Sample volume (amount "b").
After mixing thoroughly, prepare a 1:25 dilution by adding
amount "a" of the 1:5 diluted sample to amount "b" of 0.15 M
sodium phosphate (pH 7.0-7.5). A working Inoculation Vol-
ume may be prepared from undiluted subsample 2 and from
subsample 2 diluted 1:5 and 1:25 as described in Step 3. Using
an amount equal to the Inoculum, or Inoculation, Volume, in-
oculate 10 cell cultures each with undiluted subsample 2, sub-
sample 2 diluted 1:5 and subsample 2 diluted 1:25, respec-
tively. Freeze the remaining portions of the 1:25 dilution at
-70°C until the assay results are known.
If all the inoculated cultures are all positive, thaw the re-
maining 1:25 dilution and prepare 1:125, 1:625, and 1:3125
Standard Assay
No
\
Prepare 1 0 Cultures each of
Subsample 2
•^Undiluted
> Diluted 5-Fold
*-Diluted 25-Fold
Yes
/
Prepare 1 0 Cultures of
Undiluted Subsample 2
18
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CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
dilutions by transferring amount "a" of each lower dilution to
amount "b" of sodium phosphate as described above. Inocu-
late 10 cultures each with the additional dilutions and freeze
the remaining portion of the 1:3125 dilution.
Continue the process of assaying higher dilutions until at least
one test vessel at the highest dilution tested is negative. Higher
dilutions can also be assayed along with the initial undiluted
to 1:25 dilutions if it is suspected that the water to be tested
contains more than 500 most probable number (MPN) of in-
fectious total culturable virus units per 100 liters.
If subsample 1 is cytotoxic, then five cell cultures
should be inoculated with Final Concentrated Sample
using the same volume required for subsample 1 and
the procedures described in the Reduction of Cyto-
toxicity in Sample Concentrates section below. If
these procedures remove cytotoxicity, inoculate sub-
sample 2 using the procedures for removal of cyto-
toxicity and 10 cultures each with undiluted sample,
sample diluted 1:5, and sample diluted 1:25 as de-
scribed above. If the procedures fail to remove cyto-
toxicity, write for advice on how to proceed from the
ICR Laboratory Coordinator, U.S. EPA, Office of
Ground Water and Drinking Water, Technical Support Divi-
sion, 26 W. Martin Luther King Drive, Cincinnati, OH 45268.
A maximum of 60 and 580 MPN units per 100 liters can be
demonstrated by inoculating a total of 20 cultures with the
undiluted Assay Sample Volume from source water or a total
of 10 cultures each with undiluted sample and sample diluted
1:5 and 1:25, respectively.
QC and PE Samples
Prepare 5- and 25-fold dilutions of subsample 1 for each QC
or PE sample, as described above. Inoculate 10 cultures each
with undiluted subsample and subsample diluted 1:5 and 1:25
using an amount of inoculum equal to the Inoculum, or Inocu-
lation, Volume.
Use subsample 2 only as a backup for problems with the analy-
sis of subsample 1.
Cytotoxicity Treatment Decisions
No
1
\
Prepare and Treat 10
Cultures each of Subsample 2
for Cytotoxicity
•^Undiluted
> Diluted 5-Fold
> Diluted 25-Fold
19
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CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Rock the inoculated cell culture test vessels gently to
achieve uniform distribution of inoculum over the sur-
face of the cell monolayers.
Place the cell culture test vessels on a level stationary surface
at room temperature so that the inoculum will remain distrib-
uted evenly over the cell monolayer.
Continue incubating the inoculated cell cultures for
80-120 minutes to permit viruses to adsorb onto and
infect cells.
// may be necessary to rock the vessels every 15-20 minutes or
to keep them on a mechanical rocking platform during the ad-
sorption period to prevent the death from dehydration of cells
in the middle of the vessels.
After returning the cell cultures to the laboratory, add
liquid maintenance medium (see Item 2 of Vessels and
Media for Cell Growth in Appendix A for recom-
mended medium) and incubate at 36.5 + 1°C.
Warm the maintenance medium to 36.5 ±1 °C before placing
it onto cell monolayers. Add the medium to the side of the cell
culture vessel opposite the cell monolayer. Avoid touching any
pipetting devices used to the inside rim of the culture vessels to
avert the possibility of transporting contaminants to the re-
maining vessels.
'A Cornwall syringe or other large pipetting device may
be used to add the medium.
20
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CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Examine each culture microscopically for the appear-
ance of cytopathic effects (CPE) daily for the first 3
days and then every couple of days for a total of 14
days.
CPE may be identified as cell disintegration or as changes in
cell morphology. Rounding-up of infected cells is a typical ef-
fect seen with enterovirus infections. However, uninfected cells
round up during mitosis, and a sample should not be consid-
ered positive unless there are significant clusters ofrounded-
up cells over and beyond what is observed in the uninfected
controls. Photomicrographs demonstrating CPE appear in the
reference by Malherbe and Strickland-Cholmley (1980).
Freeze cultures at -70°C when more than 75% of the
monolayer shows signs of CPE.
Freeze all remaining negative cultures, including con-
trols, after 14 days.
Thaw all the cultures to confirm the results of the pre-
vious passage.
Filter through separate 0.22 jam sterilizing filters at least
10% of the medium from each vessel that was positive for CPE
or that appeared to be bacterially contaminated.
Then inoculate another BGM culture with 10% of the medium
from the previous passage for each vessel, including those that
were negative. Repeat Steps 7 and 8.
21
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CHAPTER 3
TOTAt. CULTURABLE VlRUS ASSAY
Confirmation passages may be performed in small vessels or
mitltiwell trays. However, it may be necessary to distribute the
Inoculum into several vessels or wells to ensure that the vol-
ume of inoculum is less than or equal to 0.04 mL/cm2 of sur-
face area.
Score cultures that developed CPE in both the first and
second passages as confirmed positive cultures.
Cultures that show CPE in only the second passage
must be passaged a third time along with the negative controls
according to Steps 9 and 10.
Score cultures that develop CPE hi both the second and third
passages as confirmed positive cultures.
Cultures with confirmed CPE may be stored in a-70 °C freezer
for research purposes or for optional identification tests.
D
For more information, see Chapter 12 (May 1988 re-
vision) of Berg et al. (1984).
Virus Quantitation
Record the total number of confirmed positive and
negative cultures for each subsample onto the Total
Culturable Virus Data Sheet (Appendix E). Do not
include the results of the tests for cytotoxicity!
Transfer the number of cultures inoculated and the
confirmed number of positive cultures from the Total
Culturable Virus data sheet for each subsample to the
Quantitation of Total Culturable Virus data sheet.
If dilutions are not required, add the values to obtain a total
undiluted count for each sample.
Calculate the MPN/mL value (Mm) and 95% confidence limits
using the total undiluted count and the computer program sup-
plied by the U.S. EPA.
22
-------
If dilutions are reuired, calculate the MPN/mL value and 95%
confidence limits from the subsample 2 values.
Record these values on the Quantitation of Total Culturable
Virus data sheet.
CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Calculate the MPN per 100 liter value (ML) of the origi-
nal water sample according to this formula:
Mi =
100 MmS
D
where S equals the Assay Sample Volume and D equals the
Volume of Original Water Sample assayed. (The values for S
and D can be found on the Virus Data Sheet.) Record the value
of ML on the Virus Data Sheet.
Calculate the lower 95% confidence limit 100 liter
value (CLt) for each water sample according to the
formula:
CL =
100 CLlmS
D
where CLlm is the lower 95% confidence limit per milliliter
from the Quantitation of Total Culturable Virus data sheet.
Calculate the upper 95% confidence limit per 100 liter value
(CLu) according to the formula:
p. ^100 CLumS
D
where CLum is the upper 95% confidence limit per milliliter
from the Quantitation of Total Culturable Virus data sheet.
Record the limit per 100 liter values on the Virus Data Sheet.
Calculate the total MPN value and the total 95% con-
fidence limit values for each QC and PE sample by
multiplying the values per milliliter by S and dividing
by 0.4.
23
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CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Reduction of Cytotoxicity in Sample
Concentrates
The procedure described in this section may result in a signifi-
cant liter reduction and should be applied only to inocula known
to be toxic.
Media and Reagents
• Washing solution.
Dissolve 8.5 g of NaCl in a final volume of 980 mL of
reagent-grade water. Autoclave the solution at 121 °C for
15 minutes. Cool to room temperature. Add 20 mL of se-
rum to the sterile salt solution. Mix thoroughly.
Store the washing solution at 4°C for up to 3 months, or at
-20°C.
Procedure for Cytotoxicity Reduction
Decant and save the inoculum from inoculated cell
culture vessels after the adsorption period (Step 5 of
Sample Inoculation and CPE Development).
Add 0.25 mL of the washing solution for each cm2 of cell sur-
face area into each vessel.
Warm the washing solution to 36.5 + 1°C before placing on
the cell monolayer. Add the washing solution to the side of the
cell culture vessel opposite the cell monolayer. Avoid touching
any pipetting devices used to the inside rim of the culture ves-
sels to avert the possibility of transporting contaminants to the
remaining vessels.
The inocula saved after the adsorption period should be stored
at -70 °Cfor subsequent treatment and may be discarded when
Cytotoxicity is successfully reduced.
24
-------
CHAPTER 3
TOTAL CULTURABLE VIRUS ASSAY
Gently rock the washing solution across the cell mono-
layer a minimum of two times.
Decant and discard the spent washing solution with-
out disturbing the cell monolayer.
It may be necessary to rock the washing solution across the
monolayer more than twice if the sample is oily and difficult to
remove from the cell monolayer surface.
Continue with Step 7 of the procedure for Sample In-
oculation and CPE Development.
If this procedure fails to reduce cytotoxicity with a particular
type of water sample, backup samples may be diluted 1:2 to
1:4 before repeating the procedure. This dilution requires that
two to four times more culture vessels be used. Dilution alone
may sufficiently reduce cytotoxicity of some samples without
washing. Alternatively, the changing of liquid maintenance
medium at the first signs of cytotoxicity may prevent further
development.
Determine cytotoxicity from the initial daily macroscopic ex-
amination of the appearance of the cell culture monolayer by
comparing the negative and positive controls from Step 4 of
the procedure for Sample Inoculation and CPE Development
with the test samples from that step). Cytotoxicity should be
suspected when the cells in the test sample develop CPE prior
the positive control.
25
-------
-------
Appendix A
Cell Culture Preparation and
Maintenance
Preparation of Cell Culture
Medium
General Principles
1. Equipment care — Carefully wash and steril-
ize equipment used for preparing media before
each use.
2. Disinfection of work area — Thoroughly dis-
infect surfaces on which the medium prepara-
tion equipment is to be placed.
3. Aseptic technique — Use aseptic technique
when preparing and handling media or medium
components.
4. Dispensing filter-sterilized media — To avoid
post-filtration contamination, dispense filter-
sterilized media into storage containers through
clear glass filling bells in a microbiological
laminar flow hood. If a hood is unavailable,
use an area restricted solely to cell culture
manipulations.
5. Coding media — Assign a lot number to and
keep a record of each batch of medium or me-
dium components prepared. Place the lot num-
ber, the date of preparation, the expiration date,
and the initials of the person preparing the
medium on each bottle.
6. Sterilization of NaHCO3-containing solutions
— Sterilize media and other solutions that con-
tain NaHCO3 by positive pressure filtration.
Negative pressure filtration of such solutions in-
creases the pH and reduces the buffering capacity.
7. Antibiotic solutions prepared in-house must be
filter sterilized with 0.22 p,m membrane filters.
It is important that the recommended antibi-
otic levels not be exceeded during the planting
of cells, as cultures are particularly sensitive
to excessive concentrations at this stage. Anti-
biotic stock solutions should be placed in
screw-capped containers and stored at -20°C
until needed. Once thawed, they may be refro-
zen; however, repeated freezing and thawing
of these stock solutions should be avoided by
freezing them in quantities that are sufficient
to support a week's cell culture work.
Apparatus and Materials
1. Glassware, Pyrex (Corning Product No. 1395).
Storage vessels must be equipped with airtight
closures.
2. Disc filter holders — 142 mm or 293 mm di-
ameter (Millipore Product No. YY30 142 36
andYY30293 16).
Use only positive pressure type filter holders.
3. Sterilizing filter stacks — 0.22 |im pore size
(Millipore Product No. GSWP 142 50 and
GSWP293 25). Fiberglass prefilters (Millipore
APIS 142 50 or APIS 293 25, and AP20 142
50orAP2029325).
A-1
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
Stack AP20 andAPlSprefilters and 0.22
membrane filter in a disc filter holder with
AP20 prefilter on top and 0.22 pn membrane
filter on bottom.
Always disassemble the filter stack after use to
check the integrity of the 0.22 ^m filter. Refilter
any media filtered with a damaged stack.
4. Positively charged cartridge filter — 10-inch
(Zeta plus TSM, Cuno Product No. 45134-01-
600P). Cartridge housing with adaptor for
10-inch cartridge (Millipore Product No. YY16
01200).
5. Culture capsule filter (Gelman Sciences Prod-
uct No. 12170).
6. Cell culture vessels — Pyrex, soda or flint
glass, or plastic bottles and flasks or roller
bottles (e.g., Brockway ProductNo. 1076-09A,
1925-02; Corning Product No. 25100-25,
25110-75,25120-150,25150-1750).
Vessels must be made from clear glass or plas-
tic to allow observation of the cultures and must
be equipped with airtight closures. Plastic ves-
sels must be treated by the manufacturer to
allow cells to adhere properly.
7. Screw caps, black with rubber liners
(Brockway Product No. 24-414).
Caps for larger culture bottles usually are sup-
plied with the bottles.
8. Roller apparatus (Bellco Glass Product No.
7730).
Required only if roller bottles are used for
maintenance of stock cultures.
9. Waterbath set at 56 ± 1°C.
10. Light microscope, with conventional light
source, equipped with lenses to provide 40X,
100X, and 400X total magnification.
11. Inverted light microscope equipped with lenses
to provide 40X, 100X, and 400X total magni-
fication.
12. Phase contrast counting chamber
(hemocytometer) (Curtin Matheson Scientific
ProductNo. 158-501).
13. Conical centrifuge tubes — 50 and 250 mL ca-
pacity.
14. Rack for tissue culture tubes (Bellco Product
No. 2028).
15. Bottles, aspirator-type with tubing outlet —
2,000 mL capacity.
Bottles for use with pipetting machine.
16. Storage vials — 2 mL capacity.
Vials must withstand temperatures to -70°C.
Media and Reagents
1. Sterile fetal calf, gamma globulin-free newborn
calf or iron-supplemented calf serum, certified
free of viruses, bacteriophage, and myco-
plasma.
Test each lot of serum for cell growth and tox-
icity before purchasing. Serum should be stored
at -20 °Cfor long-term storage. Upon thawing,
each bottle must be heat-inactivated in a
waterbath set at 56+1 °Cfor 30 minutes and
stored at 4 °Cfor short-term use.
2. Trypsin, 1:250 powder (Difco Laboratories
ProductNo. 0152-15-9) ortrypsin, 1:300 pow-
A-2
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
der (Becton Dickinson Microbiology Systems
Product No. 12098).
3. EDTA (Fisher Scientific Product No. S657-
500).
4. Fungizone (amphotericin B, Sigma Product
No. A-9528), penicillin G (Sigma Product No.
P-3032), streptomycin sulfate (ICN
Biomedicals Product No. 100556), tetracycline
hydrochloride (ICN Biomedicals Product No.
103011).
Use antibiotics of at least tissue culture grade.
5. Eagle's minimum essential medium (MEM)
with Hanks' salts and L-glutamine, without
sodium bicarbonate (Life Technologies Prod-
uct No. 410-1200).
6. Leibovitz's L-15 medium with L-glutamine
(Life Technologies Product No. 430-1300).
7. Trypan blue (Sigma Chemical Product No. T-
6146).
8. Dimethyl sulfoxide (DMSO; Sigma Chemical
Product No. D-2650).
Media Preparation Recipes
The conditions specified by the supplier for stor-
age and expiration dates of commercially avail-
able media should be strictly observed.
1. Procedure for the preparation of 10 liters of
EDTA-trypsin.
The procedure described is used to dislodge
cells attached to the surface of culture bottles
and flasks. This reagent, when stored at 4°C,
retains its working strength for at least four
months. The amount of reagent prepared should
be based on projected usage over four months.
Step a. Add 30 g of trypsin (1:250) or 25 g of
trypsin (1:300) to 2 liters of reagent-grade wa-
ter in a 6 liter flask containing a 3-inch stir bar.
Place the flask on a magnetic stirrer and mix
the trypsin solution rapidly for a minimum of
1 hour.
Trypsin remains cloudy.
Step b. Put four liters of reagent-grade water
and a 3-inch stir bar into a 20-liter clear plastic
carboy. Place the carboy on a magnetic stirrer
and stir at a speed sufficient to develop a vor-
tex while adding the following chemicals: 80 g
NaCl, 12.5 g EDTA, 50 g dextrose, 11.5 g
Na2HPO4 • 7H20,2.0 g KC1, and 2.0 g KH2PO4.
Each chemical does not have to be completely
dissolved before adding the next one.
Step c. Add four more liters of reagent-grade
water to the carboy and continue mixing until
all the chemicals are completely dissolved.
Step d. Add the two liters of trypsin from Step
2a to the solution from Step 2c and mix for a
minimum of one hour. Adjust the pH of the
EDTA-trypsin reagent to 7.5 - 7.7.
Step e. Filter the reagent under pressure
through a filter stack and store the filtered re-
agent in tightly stoppered or capped contain-
ers at 4°C.
The cartridge prefilter (Item 4 of Apparatus and
Materials) can be used in line with the culture
capsule sterilizing filter (Item 5) as an alter-
native to a filter stack (Item 3).
Procedure for the preparation of 10 liters of
MEM/L-15 medium.
A-3
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
3.
Step a. Place a 3-inch stir bar and four liters of
reagent-grade water into a 20-liter clear plas-
tic carboy.
Step b. Place the carboy onto a magnetic stir-
rer. Stir at a speed sufficient to develop a vor-
tex and then add the contents of a 5-liter packet
of L-15 medium to the carboy. Rinse the me-
dium packet with three washes of 200 mL each
of reagent-grade water and add the rinses to
the carboy.
Step c. Mix until the medium is evenly dis-
persed.
L-15 medium may appear cloudy as it need not
be totally dissolved before proceeding to the
next step.
Step d. Add 3 liters of reagent-grade water to
the carboy and the contents of a 5-liter packet
of MEM medium to the carboy. Rinse the
MEM medium packet with 3 washes of 200
mL each of reagent-grade water and add the
rinses to the carboy. Add 800 mL of reagent-
grade water and 7.5 g of NaHCO3 and con-
tinue mixing for an additional 60 minutes.
Step e. Transfer the MEM/L-15 medium to a
pressure can and filter under positive pressure
through a 0.22 |im sterilizing filter. Collect the
medium in volumes appropriate for the cultur-
ing of BGM cells (e.g., 900 mL in a 1-liter
bottle) and store in tightly stoppered or capped
containers at 4°C for up to 2 months.
Procedure for preparation of 100 mL of trypan
blue solution.
The procedure is used in the direct determina-
tion of the viable cell counts of the BGM stock
cultures. As trypan blue is on the EPA suspect
carcinogen list, particular care should be taken
in its preparation and use so as to avoid skin
contact or inhalation. The wearing of rubber
gloves during preparation and use is recom-
mended.
Step a. Add 0.5 g of trypan blue to 100 mL of
reagent-grade water in a 250 mL flask. Swirl
the flask until the trypan blue is completely
dissolved.
Step b. Sterilize the solution by autoclaving at
121 °C for 15 minutes and store in a screw-
capped container at room temperature.
4. Preparation of 100 mL of penicillin-streptomy-
cin stock solution containing 100,000 units/mL
of penicillin and 100,000 jig/mL of streptomy-
cin.
Step a. Add 10,000,000 units of penicillin G
and 10 g of streptomycin sulfate to a 250 mL
flask containing 100 mL of reagent-grade wa-
ter. Mix the contents of the flasks on a mag-
netic stirrer until the antibiotics are dissolved.
Step b. Sterilize the antibiotics by filtration
through a 0.22 jim membrane filter and dis-
pense in 10 mL volumes into screw-capped
containers.
5. Preparation of 50 mL of tetracycline stock so-
lution.
Step a. Add 1.25 g of tetracycline hydrochlo-
ride powder and 3.75 g of ascorbic acid to a
125 mL flask containing 50 mL of reagent-
grade water. Mix the contents of the flask on a
magnetic stirrer until the antibiotic is dissolved.
Step b. Sterilize the antibiotic by filtration
through a 0.22 Lim membrane filter and dis-
pense in 5 mL volumes into screw-capped con-
tainers.
A-4
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
6. Preparation of 25 mL of amphotericin B
(fungizone) stock solution.
Step a. Add 0.125 g of amphotericin B to a 50
mL flask containing 25 mL of reagent-grade
water. Mix the contents of the flask on a mag-
netic stirrer until the antibiotic is dissolved.
Step b. Sterilize the antibiotic by filtration
through a 0.22 Jim membrane filter and dis-
pense in 2.5 mL volumes into screw-capped
containers.
Preparation and Passage of BGM
Cell Cultures
A microbiological biosafety cabinet should be used
to process cell cultures. If a hood is not available,
cell cultures should be prepared in controlled fa-
cilities used for no other purposes. Viruses or other
microorganisms must not be transported, handled,
or stored in rooms used for cell culture transfer.
Vessels and Media for Cell Growth
1. The BGM cell line grows readily on the inside
surfaces of glass or specially treated, tissue
culture grade plastic vessels. Flat-sided, glass
bottles (16 to 32 oz. or equivalent growth area),
75 or 150 cm2 plastic cell culture flasks, and
690 cm2 glass or 850 cm2 plastic roller bottles
are usually used for the maintenance of stock
cultures. Flat-sided bottles and flasks that con-
tain cells in a stationary position are incubated
with the flat side (cell monolayer side) down.
If available, roller bottles and roller apparatus
units are preferable to flat-sided bottles and
flasks because roller cultures require less me-
dium per unit of cell monolayer surface area
than do flat-sided bottles. Roller apparatus ro-
tation speed should be adjusted to one-half
revolution per minute to ensure that cells are
constantly bathed in growth medium.
2. Growth and maintenance media should be pre-
pared on the day they will be needed. Prepare
growth medium by supplementing MEM/L-15
medium with 10% serum and antibiotics (100
mL of serum, 1 mL of penicillin-streptomycin
stock, 0.5 mL of tetracycline stock and 0.2 mL
of fungizone stock per 900 mL of MEM/L-15).
Prepare maintenance medium by supplement-
ing MEM/L-15 with antibiotics and 2% or 5%
serum (20 or 50 mL of serum, antibiotics as
above for growth medium and 80 or 50 mL of
reagent-grade water, respectively). Use main-
tenance media with 2% serum for CPE devel-
opment.
General Procedure for Ceil Passage
Pass stock BGM cell cultures at approximately
seven day intervals using growth medium.
Stepl
Pour spent medium from cell culture vessels, and
discard the medium.
A gauze-covered beaker may be used to collect
spent medium to prevent splatter. Autoclave all
media that have been in contact with cells or that
contain serum before discarding.
Step 2
Add a volume of warm EDTA-trypsin reagent
equal to 40% of the volume of medium replaced
to the cell cultures.
See Table 1 for the amount of reagents required
for commonly used vessel types. Warm the EDTA-
trypsin reagent to36.5±l°C before placing it onto
cell monolayers.
Step 3
Allow the EDTA-trypsin reagent to remain in con-
tact with the cells at room temperature until the
cell monolayer can be shaken loose from the inner
A-5
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
surface of the cell culture vessel (about five min-
utes).
The EDTA-trypsin reagent should remain in con-
tact with the cells no longer than necessary to pre-
vent cell damage.
Step 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.
Centrifiige tubes and bottles used for this purpose
must be able to withstand the g-force applied.
StepS
Centrifuge cell suspension at 1,000 x g for 10 min-
utes to pellet cells. Pour off and discard the super-
natant.
Do not exceed this speed as cells may be damaged
or destroyed.
Step 6
Suspend the pelleted cells in growth medium (see
Item 2 of Vessels and Media for Cell Growth) and
perform a viable count on the cell suspension ac-
cording to the Procedure for Performing Viable Cell
Counts section below.
Rcsuspend pelleted cells in a sufficient volume of
medium to allow thorough mixing of the cells (to
reduce sampling error) and to minimize the sig-
nificance of the loss of the 0.5 mL of cell suspen-
sion required for the cell counting procedure. The
quantity of medium used for resuspending pelleted
cells varies from 50 to several hundred milliliters,
depending upon the volume of the individual
laboratory's need for cell cultures.
Step?
Dilute the cell suspension to the appropriate final
cell concentration with growth medium and dis-
pense into cell culture vessels with a pipet, a
Cornwall-type syringe, or a Brewer-type pipetting
machine dispenser.
Calculate the dilution factor requirement using the
cell count and the cell and volume parameters
given in Table 1 for stock cultures and in Table 2
for virus assay cultures.
As a general rule, the BGM cell line can be split at
a 1:3 ratio. However, a more suitable inoculum is
obtained if low passages of the line (passages 100-
150) are split at a 1:2 ratio and higher passages
(generally above passage 250) are split at a 1:4
ratio. To plant 200 25-cm2 cell culture flasks weekly
from a low-level passage of the line would require
the preparation of 6 roller bottles (surface area of
690 cm2 each): the contents of 2 to prepare the
next batch of 6 roller bottles, and the contents of
the other 4 to prepare the 25-m2 flasks.
StepS
Except during handling operations, maintain BGM
cells at 36.5 ± 1°C in airtight cell culture vessels.
Step 9
Replace growth medium with maintenance me-
dium containing 2% serum when cell monolayers
become 95 to 100% confluent (usually three to four
days after seeding with an appropriate number of
cells). Replace growth medium which becomes
acidic before the monolayers become 95 to 100%
confluent with maintenance medium containing
5% serum. The volume of maintenance medium
should equal the volume of the discarded growth
medium.
Procedure For Performing Viable Cell
Counts
Stepl
Add 0.5 mL of cell suspension (or diluted cell sus-
pension) to 0.5 mL of 0.5% trypan blue solution in
a test tube.
A-6
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
Table 1
Guide for Preparation of BGM Stock Cultures
Vessel Type
16 oz. glass flat bottles
32 oz. glass flat bottles
75 cm2 plastic flat flask
1 50 cm2 plastic flat
flask
690 cm2 glass roller
bottle
850 cm2 plastic roller
bottle
Volume of EDTA-
Trypsin Used to
Remove Cells (ml)
10
20
12
24
40
48
Volume of Medium
(ml)a
25
50
30
60
100
120
Total Number of
Cells to Plate per
Vessel
2.5 x106
5.0 x106
3.0 x106
6.0 x106
7.0 x106
8.0 x107
aSerum requirements: growth medium contains 10 % serum; maintenance medium contains 2-5%
serum. Antibiotic requirements: penicillin-streptomycin stock solution, 1.0 ml/liter; tetracycline
stock solution, 0.5 ml/liter; fungizone stock solution, 0.2 ml/liter
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 6.0x10* and 1.5 x 106 cells per mL of cell
suspension. Thus, a dilution of 1:10 (0.5 mLof cells
in 4.5 mL of growth medium) is usually required
for an accurate count of a cell suspension.
Step 2
Disperse cells by repeated pipetting.
Avoid introducing air bubbles into the suspension,
because air bubbles may interfere with subsequent
filling of the hemocytometer chambers.
Step 3
With a capillary pipette, carefully fill a
hemocytometer chamber on one side of a slip-cov-
ered hemocytometer slide. Rest the slide on a flat
surface for about one minute to allow the trypan
blue to penetrate the membranes of nonviable cells.
Do not under- or overfill the chambers.
Step 4
Under 100X total magnification, count the cells in
the four large corner sections and the center sec-
tion of the hemocytometer chamber.
Include in the count cells lying on the lines mark-
ing 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 viable cells, count only
cells that are clear in color. Do not count cells that
are blue.
A-7
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
Table 2
Guide for Preparation of
Virus Assay Cell Cultures
Vessel Type
1 02. glass bottle
25 cm2 plastic flask
6 oz. glass bottle
75 cm2 plastic flask
1 6 mm x 150 mm tube
Volume of
Medium* (ml)
4
10
15
30
2
Final Cell Count
per Vessel
9.0 x105
3.5 x 10s
5.6 x106
1.0x107
4.0 x 1 04
"Serum requirements: growth medium contains 10 % serum. Antibiotic
requirements: penicillin-streptomycin stock solution, 1.0 ml/liter; tetracy-
cline stock solution, 0.5 ml/liter; fungizone stock solution, 0.2 ml/liter
StepS
Calculate the average number of viable cells in each
mL of cell suspension by totaling the number of
viable cells counted in the 5 sections, multiplying
this sum by 2,000, and where necessary, multiply-
ing the resulting product by the reciprocal of the
dilution.
Procedure for Preservation of
BGM Cell Line
An adequate supply of frozen BGM cells must be
available to replace working cultures that are used
only periodically, that become contaminated, or
that lose vims sensitivity. Cells have been held at
-70°Cfor more than 15 years with a minimum loss
in cell viability.
Preparation of Cells for Storage
The procedure described is for the preparation of
100 cell culture vials.
Cell concentration
must beat least 2xltf
per mL.
The actual number of
vials to be prepared
should be based upon
line usage and the an-
ticipated time interval
requirement between
cell culture start-up
and full culture pro-
duction.
Step I
Prepare cell storage
medium by adding 10
mL of DMSO to 90
mL of growth medium
(see Item 2 of Vessels
and Media for Cell
Growth). Sterilize the
resulting cell storage medium by passage through
a 0.22 u,m sterilizing filter.
Collect sterilized medium in a 250-mL flask con-
taining a stir bar.
Step 2
Harvest BGM cells from cell culture vessels as
directed in Steps 1 to 5 of General Procedures for
Cell Passage. Count the viable cells as described
above and resuspend them in the cell storage me-
dium at a concentration of at least 2 x 106 cells per
mL.
StepS
Place the flask containing suspended cells on a
magnetic stirrer and slowly mix for 30 minutes.
Dispense 1 mL volumes of cell suspension into
2-mL capacity vials.
A-8
-------
APPENDIX A
CELL CULTURE PREPARATION AND MAINTENANCE
Procedure for Freezing Cells
The freezing procedure requires slow cooling of
the cells with the optimum rate of-1 °Cper minute.
A slow cooling rate can be achieved using the fol-
lowing method or by using the recently available
freezing containers (e.g., Nalge Product No. 5100-
0001) as recommended by the manufacturers.
Stepl
Place the vials in a rack and place the rack in a
refrigerator at 4°C for 30 minutes, then in a -20°C
freezer for 30 minutes, and finally in a -70°C
freezer overnight. The transfers should be made as
rapidly as possible.
To allow for more uniform cooling, wells
adjoining each vial should remain empty.
Stepl
Rapidly transfer vials into boxes or other
containers for long-term storage.
To prevent substantial loss of cells during
storage, the temperature of cells should be kept
constant after -70°C has been achieved.
Procedure for Thawing Cells
Cells must be thawed rapidly to decrease loss in
cell viability.
Stepl
Place vials containing frozen cells into a 36.5 ±
1°C water bath and agitate vigorously by hand un-
til all ice has melted. Sterilize the outside surface
of the vials with 0.5 % L, in 70% ethanol.
Step 2
Add BGM cells to either 6 oz. tissue culture bottles
or 25 cm2 tissue culture flasks containing an ap-
propriate amount of growth medium (see Table 2).
Use two vials of cells for 6 oz. bottles and one vial
for 25 cm2 flasks.
StepS
Incubate BGM cells at 36.5 ± 1°C. After 18 to 24
hours replace the growth medium with fresh growth
medium, then continue the incubation for an addi-
tional 5 days. Pass and maintain the new cultures
as directed above.
A-9
-------
-------
Appendix B
Sterilization and
Disinfection
General Guidelines
1. Use aseptic techniques for handling test wa-
ters, eluates, and cell cultures.
2. Sterilize apparatus and containers that will
come into contact with test waters and all so-
lutions that will be added to test waters unless
otherwise indicated. Thoroughly clean all items
prior to final sterilization using laboratory stan-
dard operating procedures.
3. Sterilize all contaminated materials before dis-
carding.
4. Disinfect all spills and splatters.
Sterilization Techniques
Solutions
Sterilize all solutions, except those used for cleans-
ing, standard buffers, hydrochloric acid (HC1), so-
dium hydroxide (NaOH), and disinfectants by au-
toclaving them at 121°C for 15 minutes.
The HCl and NaOH solutions and disinfectants
used are self-sterilizing. When autoclaving buff-
ered beef extract, use a vessel large enough to ac-
commodate foaming. Place the vessel in a pan of
water to ensure even heating and to avoid break-
age of the vessel.
Autoclavable Glassware, Plasticware,
and Equipment
Water speeds the transfer of heat in larger vessels
during autoclaving and thereby speeds the steril-
ization process. Add reagent-grade water to ves-
sels in quantities indicated in the table below. Lay
large vessels on their sides in the autoclave, if pos-
sible, to facilitate the displacement of air in the
vessels by flowing steam.
1. Cover the openings into autoclavable glass-
ware, plasticware, and equipment loosely with
aluminum foil before autoclaving. Autoclave
at!21°Cforonehour.
Glassware may also be sterilized in a dry heat
oven at a temperature ofl 70 °Cfor at least one
hour.
2. Sterilize stainless steel vessels (dispensing
pressure vessel) in an autoclave at 121°C for
30 minutes.
Quantity of Reagent-Grade Water to be
Added to Vessels During Autoclaving
Vessel Size (L)
2 and 3
4
8
24
54
Water (ml)
25
50
100
500
1,000
. B-1
-------
APPENDIX B
STERILIZATION AND DISINFECTION
3.
Vent-relief valves on vessels so equipped must
be open during autoclaving and closed imme-
diately when vessels are removed from the au-
toclave.
Presterilize 1MDS filter cartridges andprefilter
cartridges by wrapping the filters in Kraft pa-
per and autoclaving at 121°C for 30 minutes.
4. Sterilize working instruments, such as scissors
and forceps, by immersing them in 95% etha-
nol and flaming them between uses.
Chlorine Sterilization
Sterilize pumps, plasticware (filter housings), tub-
ing that cannot withstand autoclaving, and ves-
sels that are too large for the autoclave by chlori-
nation. Prefilters, but not 1MDS filters, may be
presterilized with chlorine as an alternative to
autoclaving. Filter Apparatus modules should be
disinfected by sterilization and then cleaned ac-
cording to laboratory standard operating proce-
dures prior to final sterilization.
Media and Reagents
1. 0.1% chlorine (HOC1) — add 19 mL of house-
hold bleach (Clorox, The Clorox Co.) to 981
mL of reagent-grade water and adjust the pH
of the solution to 6-7 with 1 M HC1.
Procedures
Ensure that the solutions come in full contact with
all surfaces when performing these procedures.
1. Sterilize filter apparatus modules, injector tub-
ing, and plastic bags for transporting injector
tubing by recirculating or immersing the items
in 0.1 % chlorine for 30 minutes. Drain the chlo-
rine solution from objects being sterilized.
Dechlorinate using a solution containing 2.5
mL of 2% sterile sodium thiosulfate per liter
of sterile reagent-grade water. Rinse with ster-
ile reagent-grade water.
2. Thoroughly rinse pH electrodes after each use
to remove particulates. Sterilize before and af-
ter each use by immersing the tip of the elec-
trode in 0.1% chlorine for at least one minute.
Dechlorinate the electrode as in the step above.
Procedure for Verifying Sterility of
Liquids
Do not add antibiotics to media or medium com-
ponents until after their sterility has been demon-
strated. The BGM cell line used should be checked
every six months for mycoplasma contamination
according to test kit instructions. Cells that are
contaminated should be discarded.
Media and Reagents
1. Mycoplasma testing kit (Irvine Scientific Prod-
uct No. T500-000). Use as directed by the
manufacturer.
2. Thioglycollate medium (Difco Laboratories
Product No. 0257-01-9). Prepare broth medium
as directed by the manufacturer.
Verifying Sterility of Small Volumes of
Liquids
Step 1
Inoculate 1 mL portions of the material to be tested
for sterility into tubes containing 9 mL of
thioglycollate broth by stabbing the inoculum into
the broth. Incubate at 36.5 ±1°C.
Step 2
Examine the inoculated broth daily for seven days
to determine whether growth of contaminating or-
ganisms has occurred.
B-2
-------
ApPENDfX 6
STERILIZATION AND DISINFECTION
Containers holding the thioglycollate medium must
be tightly sealed before and after the medium i,
inoculated.
Visual Evaluation of Media for
Microbial Contaminants
Stepl
Incubate either the entire stock of prepared media
or aliquots taken during preparation which repre-
sent at least 5% of the final volume at 36.5 ±1°C
for at least one week prior to use.
Step 2
Visually examine and discard any media that lose
clarity.
A clouded condition that develops in the media
indicates the occurrence of contaminating organ-
isms.
Contaminated Materials
1. Autoclave contaminated materials for 30 min-
utes at 121 °C. Be sure that steam can enter
contaminated materials freely.
2. Many commercial disinfectants do not ad-
equately kill enteric viruses. To ensure thor-
ough disinfection, disinfect spills and other
contamination on surfaces with either a solu-
tion of 0.5% iodine in 70% ethanol (5 g I2 per
liter) or 0.1 % chlorine. The iodine solution has
the advantage of drying more rapidly on sur-
faces than chlorine, but may stain some sur-
faces.
B-3
-------
-------
Appendix C
Bibliography and Suggested
Reading
ASTM. 1992. Standard Methods for the Exami-
nation of Water and Wastewater (A. E.
Greenberg, L. S. Clesceri and A. D. Eaton,
ed), 18th Edition. American Public Health
Association, Washington, D.C.
Barren, A. L., C. Olshevsky and M. M. Cohen.
1970. Characteristics of the BGM line of cells
from African green monkey kidney. Archiv.
Gesam. Virusforsch. 32:389-392.
Berg, G., R. S. Safferman, D. R. Dahling, D.
Berman and C. J. Hurst. 1984. U.S. EPA
Manual of Methods for Virology. U.S. Envi-
ronmental Protection Agency Publication No.
EPA/600/4-84/013, Cincinnati, OH.
Chang, S. L., G. Berg, K. A. Busch, R. E.
Stevenson, N. A. Clarke and P. W. Kabler.
1958. Application of the "most probable num-
ber" method for estimating concentration of
animal viruses by the tissue culture technique.
Virology 6:27-42.
Crow, E. L. 1956. Confidence intervals for a
proportion. Biometrika. 43:423-435.
Dahling, D. R. and B. A. Wright. 1986. Optimi-
zation of the BGM cell line culture and viral
assay procedures for monitoring viruses in the
environment. Appl. Environ. Microbiol.
51:790-812.
Dahling, D. R. and B. A. Wright. 1987. Com-
parison of the in-line injector and fluid
proportioner used to condition water samples
for virus monitoring. J. Virol. Meth. 18:67-
71.
Dahling, D. R., G. Berg and D. Berman. 1974.
BGM, a continuous cell line more sensitive
than primary rhesus and African green kid-
ney cells for the recovery of viruses from
water. Health Lab. Sci. 11:275-282.
Dahling, D. R., R. S. Safferman and B. A.
Wright. 1984. Results of a survey of BGM
cell culture practices. Environ. Internat.
10:309-313.
Eagle, H. 1959. Ammo acid metabolism in mam-
malian cell cultures. Science. 130:432-437.
EPA. 1989. Guidance manual for compliance
with the filtration and disinfection require-
ments for public water systems using surface
water sources. Office of Drinking Water,
Washington, D.C.
Freshney, R. I. 1983. Culture of Animal Cells:
A Manual of Basic Technique. Alan R. Liss,
New York, NY.
Hay, R. J. 1985. ATCC Quality Control Meth-
ods for Cell Lines. American Type Culture
Collection, Rockville, MD.
C-1
-------
APPENDIX C
BIBLIOGRAPHY AND SUGGESTED READING
Hurst, C. J. 1990. Field method for concentrat-
ing viruses from water samples, pp. 285-295.
In G. F. Craun (ed.), Methods for the Investi-
gation and Prevention of Waterborne Disease
Outbreaks. U.S. Environmental Protection
Agency Publication No. EPA/600/l-90/005a,
Washington, D.C.
Hurst, C. J. 1991. Presence of enteric viruses in
freshwater and their removal by the conven-
tional drinking water treatment process. Bull.
W.H.O. 69:113-119.
Hurst, C. J. and T. Goyke. 1983. Reduction of
interfering cytotoxicity associated with waste-
water sludge concentrates assayed for indig-
enous enteric viruses. Appl. Environ.
Microbiol. 46:133-139.
Katzenelson, E., B. Fattal and T. Hostovesky.
1976. Organic flocculation: an efficient sec-
ond-step concentration method for the detec-
tion of viruses in tap water. Appl. Envkon.
Microbiol. 32:638-639.
Laboratory Manual in Virology. 1974. 2nd Ed.
Ontario Ministry of Health, Toronto, Ontario,
Canada.
Leibovitz, A. 1963. The growth and maintenance
of tissue-cell cultures in free gas exchange
with the atmosphere. Amer. J. Hyg. 78:173-
180.
Lennette, E. H. and N. J. Schmidt (ed.). 1979.
Diagnostic Procedures for Viral, Rickettsial
and Chlamydial Infections, 5th ed. American
Public Health Association, Washington, D.C.
Malherbe, H. H. and M. Strickland-Cholmley.
1980. Viral Cytopathology. CRC Press. Boca
Raton, FL.
Morris, R. and W. M. Waite. 1980. Evaluation
of procedures for recovery of viruses from
water—II detection systems. .Water Res.
14:795-798.
Paul, J. 1975. Cell and Tissue Culture. 5th Ed.
Churchill Livingstone, London, Great Brit-
ain.
Payment, P. and M. Trudel. 1985. Influence of
inoculum size, incubation temperature, and
cell culture density on virus detection in en-
vironmental samples. Can. J. Microbiol.
31:977-980.
Rovozzo, G. C. and C. N. Burke. 1973. A
Manual of Basic Virological Techniques.
Prentice-Hall, Englewood Cliffs, NJ.
Sobsey, M. D. 1976. Field monitoring techniques
and data analysis, pp. 87-96. In L. B. Baldwin,
J. M. Davidson and J. F. Gerber (eds.), Virus
Aspects of Applying Municipal Waste to
Land. University of Florida, Gainesville, FL.
Sobsey, M. D. 1980. Poliovirus concentration
from tap water with electropositive adsorbent
filters. Appl. Environ. Microbiol. 40:201-210.
Thomas, H. A., Jr. 1942. Bacterial densities from
fermentation tube tests. J. Amer. Water Works
Assoc. 34:572-576.
Waymouth, C., R. G. Ham and P. J. Chappie.
1981. The Growth Requirements of Vertebrate
Cells In Vitro. Cambridge University Press,
Cambridge, Great Britain.
C-2
-------
Appendix D
Vendors
The vendors listed below represent one possible source for required products. Other vendors may
supply the same or equivalent products.
American Type Culture Collection
12301 Parklawn Dr.
Rockville, MD 20852
(800) 638-6597
Baxter Diagnostics, Scientific Products Div.
1430 Waukegan Rd.
McGaw Park, IL 60085
(800) 234-5227
BBL Microbiology Systems: products may be
ordered through several major scientific supply
houses
Bee ton Dickinson Microbiology Systems
250 Schilling Circle
Cockeysville, MD 21030
(410) 771-0100 (Ask for a local distributor)
Bellco Glass
340 Edrudo Rd.
Vineland, NJ 08360
(800) 257-7043
Brockway: products may be ordered through
Continental Glass & Plastics
Cincinnati Valve and Fitting Co.
3710 Southern Ave.
Cincinnati, OH 45227
(513) 272-1212
Cole-Parmer Instrument Co.
7425 N. Oak Park Ave.
Niles, IL 60714
(800) 323-4340
Continental Glass & Plastics
841 W. CermakRd.
Chicago, EL 60608
(312)666-2050
Corning: products may be ordered through most
major scientific supply houses
Costar Corp.
7035 Commerce Circle
Pleasanton, CA 94588
(800)882-7711
Cuno, Inc.
400 Research Parkway
Meriden, CT 06450
(800)243-6894
Curtin Matheson Scientific
P.O. Box 1546
Houston, TX 77251
(713) 820-9898
D-1
-------
APPENDIX D
VENDORS
DEMA Engineering Co.
10014 Big Bend Blvd.
Karkwood,MO63122
(800) 325-3362
Difco Laboratories
P.O. Box 331058
Detroit, MI 48232
(800) 521-0851 (Ask for a local distributor)
Fisher Scientific
711 Forbes Ave.
Pittsburgh, PA 15219
(800) 766-7000
Gelman Sciences
600 S. Wagner Rd.
Ann Arbor, MI 48103
(800) 521-1520
ICN Biomedicals
3300 Hyland Ave.
Costa Mesa, CA 92626
(800) 854-0530
Irvine Scientific
2511 Daimler Street
Santa Ana, CA 92705
(800) 437-5706
Life Technologies
P.O. Box 68
Grand Island, NY 14072
(800) 828-6686
Millipore Corp.
397 Williams St.
Marlboro, MA 01752
(800) 225-1380
Nalge Co.
P.O. Box 20365
Rochester, NY 14602
(716) 586-8800 (Ask for a local distributor)
Neptune Equipment Co.
520 W. Sharon Rd.
Forest Park, OH 45240
(800) 624-6975
OMEGA Engineering, Inc.
P.O. Box 4047
Stamford, CT 06907
(800) 826-6342
Plast-o-matic Valves, Inc.
1384 Pompton Ave.
Cedar Grove, NJ 07009
(201) 256-3000 (Ask for a local distributor)
Parker Hannifin Corp.
Commercial Filters Div.
1515 W. South St.
Lebanon, IN 46052
(317)482-3900
Ryan Herco
2509 N. Naomi St.
Burbank,CA 91504
(800)848-1141
Sigma Chemical
P.O. Box 14508
St. Louis, MO 63178
(800) 325-3010
United States Plastic Corp.
1390 Neubrecht Rd.
Lima, OH 45801
(800) 537-9724
Watts Regulator
Box 628
Lawrence, MA 01845
(508)688-1811
D-2
-------
Appendix E
Data Sheets
SAMPLE NUMBER:
UTILITY NAME:
UTILITY ADDRESS:
CITY:
STATE:
ZIP:
SAMPLER'S NAME:
WATER TEMPERATURE:
TURBIDITY:
NTU
WATER pH:
ADJUSTED WATER pH:
THIOSULFATE ADDED:
(CHECK)
.YES
NO
INIT. METER READING:
date:
(CHECK UNITS)
time:
gallons ft3
FINAL METER READING:
date:
(CHECK UNITS) __ gallons __ ft3
time:
TOTAL SAMPLE VOLUME:
liters
(Final-Initial meter readings x 3.7854 (for readings in gallons)
or x 28.316 (for readings in ft3))
SHIPMENT DATE:
CONDITION ON ARRIVAL:
COMMENTS:
E-1
-------
APPENDIX E
DATA SHEETS
! : • " ,':"; " • •' VIRUS DATASHEET ' ' ]' \ . /; .
, * v° • ' ' ,. " /- 1 >
SAMPLE NUMBER:
ANALYTICAL LABORATORY NAME:
ANALYTICAL LABORATORY ADDRESS:
CITY: STATE: ZIP:
ADJUSTED TOTAL SAMPLE VOLUME (ATSV)1: L
DATE ELUTED:
ELUATE VOLUME RECOVERED :
VOLUME OF ELUATE ARCHIVED
DATE CONCENTRATED:
TIME:
mL
mL
TIME:
FINAL CONCENTRATED SAMPLE VOLUME (FCSV): mL
ASSAY SAMPLE VOLUME (S):
mL
VOLUME OF ORIGINAL WATER SAMPLE
ASSAYED (D) L2
INOCULUM VOLUME:
DATES ASSAYED 1st Passage
BY CPE:
Subsample 1:
Subsample 2:
MPN/100 LITERS3:
COMMENTS:
mL
2nd Passage 3rd Passage
(If necessary)
95% CONFIDENCE LIMITS
LOWER: UPPER:
ANALYST:
'Enter the Total Sample Volume from the Sample Data Sheet times 0.965 if a coliphage sample is
taken, times 0.9 if archiving is required, tunes 0.865 if a coliphage sample is taken and archiving
is required, or times 1.0 if a coliphage sample is not taken and archiving is not required.
2This value must be at least 100 liters for source water and 1,000 liters for finished water.
3Value calculated from the Quantitation of Total Culturable Virus form as described in the Virus
Quantitation section of Part 3.
E-2
-------
APPENDIX E
DATA SHEETS
K^f^'ii^^^^^^^^^i^^^^&^
SAMPLE #:
Sample
1st Passage
Neg. Cont.
Pos Cont.
Undiluted
1:5 DiL
1:25 DiL
2nd Passage1
Neg. Cont.
Pos. Cont.
Undiluted
1:5 DiL
1:25 DiL
3rd Passage2
Neg. Cont.
Pos. Cont.
Undiluted
1:5 DiL
1:25 DiL
Total Number of Replicates
Inoculated
Subsample 1
Without
CPE
With CPE
Subsample 2
Inoculated
Without
CPE
With CPE
^T^T^M™?^1 fr°m each lst PassaSe vessel> including controls, must be repassaged for conformation. The terms
Undiluted, 1:5 Dilution" and "1:25 Dilution" under the 2nd and 3rd Passage headings refer to the original sample
dilutions for the first passage. If higher dilutions are used, record the data from the three highest dilutions for the 1st
passage. If higher dilutions are used, record the data from the three highest dilutions showing positive results and place
the actual dilution amount in the sample column.
Samples that were negative on the first passage and positive on the 2nd passage must be passaged a third time for
conformation. If a third passage is required, all controls must be passaged again.
E-3
-------
APPENDIX E
DATA SHEETS
1 QUANTITATION OF TOTAL CULTPRABLE VIRUS ;, '"J.*{,
SAMPLE NUMBER:
Sample
Number
Replicates
Inoculated
Number
with CPE
Undiluted Samples
Subsample 1
Subsample 2
Total Undiluted
Subsample 2 results (Dilutions Required)
Undiluted
1:5 Dilution
1:25 Dilution
MPN/mL1
95% Confidence
Limits
Lower
Upper
JUse the values recorded in the Total Undiluted row to calculate the MPN/mL result and
confidence limits when dilutions are not required. If dilutions are required, base the
calculation upon the values recorded in the Undiluted, 1:5 Diluted, and 1:25 Diluted
rows for subsample 2. If higher dilutions are used for subsample 2, record the data from
the three highest dilutions showing positive results and place the actual dilution amount
in the sample column. The MPN/mL and 95% Confidence Limit values must be
obtained using the computer program supplied by the U.S. EPA.
E-4
-------
Appendix F
Examples of Calculations
Example 1
A source water sample of 211.98 liters was col-
lected at the Sampleville Water Works on May 5,
1995 and shipped by overnight courier to CEPOR
Laboratories. CEPOR Laboratories processed the
sample on May 2, 1995. After elution, the pH of
the beef extract V eluate was adjusted to 7.3 with
1 M HC1. The volume of the preadjusted eluate,
980.0 mL, was recorded. Volumes of 34.3 mL
(980 x 0.035) and 98.0 mL (980 x 0.1) were re-
moved for the Coliphage Assay (see Section IX
of the ICR Microbial Laboratory Manual) and for
archiving, respectively. An Adjusted Total Sample
Volume (ATS V) was then calculated by multiply-
ing 211.98 liters x 0.865. An adjusted volume of
183 liters was recorded on the Virus Data Sheet.
The sample was immediately processed by the
Organic Flocculation Concentration Procedure.
Following centrifugation at 4,000 xg, the superna-
tant was adjusted to pH 7.3 and passed through a
sterilizing filter. A Final Concentrated Sample
Volume (FCSV) of 28.0 mL was obtained.
The Assay Sample Volume was calculated using
the formula:
ASSAY SAMPLE VOLUME (S) =
D
ATSV
xFCSV
where D is the Volume of Original Water Sample
Assayed (i.e., 100 liters for source water or 1000
liters for finished water) and ATS V is the Adjusted
Total Sample Volume from the Virus Data Sheet.
Thus the Assay Sample Volume for Sampleville-
01 is:
S =
100 liters
183 liters
x28.0 ml = 15.3 ml
The 15.3 mL is the volume of the Final Concen-
trated Sample which must be inoculated onto tis-
sue culture and which represents 100 liters of the
source water.
Two subsamples were prepared from the Final
Concentrated Sample. Subsample 1 was pre-
pared by placing 0.55 x 15.3 mL = 8.4 mL into a
separate container. Subsample 2 was prepared by
placing 0.67 x 15.3 mL = 10.2 mL into a third con-
tainer. Although only 0.5 x 15.3 = 7.65 mL (repre-
senting 50 liters of source water) must be inocu-
lated onto tissue culture flasks for each subsample,
the factor "0.55" was used for subsample 1 to ac-
count for unrecoverable losses associated with re-
moving a subsample from its container. The factor
"0.67" was used for subsample 2 to account for
losses associated with the container and to provide
additional sample for the preparation of dilutions,
if required.
Subsample 2 and the remaining portions of the
Final Concentrated Sample were frozen at -70°C.
The Inoculum Volume was calculated to be 15.3
mL •«- 20 = 0.76 mL per flask. To make the
inoculuation procedure more convenient, it was
decided to use an Inoculation Volume of 1.0 mL.
F-1
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
To do this, 10.5 x (1.00-0.76) = 2.52 mL of so-
dium phosphate, pH 7.3, was added to 10.5 x 0.76
= 7.98 mL of subsample 1. One milliliter of di-
luted subsample 1 was then inoculated onto each
of ten 25-cm2 flasks of BGM cells at passage 123.
A negative control was prepared by inoculating a
flask with 1.0 mL of sodium phosphate, pH 7.3. A
positive control was prepared by inoculating a
flask with 1.0 mL of sodium phosphate, pH 7.3,
containing 20 PFU/mL of attenuated poliovirus
type 3. Following adsorption, 9.0 mL of mainte-
nance medium was added and the cultures were
incubated at 36.5°C. These cultures and those de-
scribed below were observed for CPE as described
in the protocol, and positive cultures were frozen
when 75% of a flask showed signs of CPE.
On May 9, five flasks inoculated with subsample
1 and the positive control showed signs of CPE.
Since fewer than eight flasks inoculated with sub-
sample 1 showed CPE, 10 additional 25-cm2 flasks
of BGM cells at passage 124 were inoculated with
1.0 mL each of subsample 2 diluted for inocula-
tion as described above. Another negative control
and positive control were also prepared and in-
oculated.
By May 16, a total of seven flasks inoculated with
subsample 1 showed signs of CPE. The flasks that
had not been previously frozen were now frozen
at -70"C, and then all flasks were thawed. Several
milliliters of fluid from each of the eight positive
flasks (seven samples plus the positive control)
were passed through a sterilizing filter. Twelve
flasks of BGM cells at passage 125 were inocu
lated with 1.0 mL of the supernatant from either
negative cultures or from filtered positive cultures.
By May 23, a total of five flasks from subsample
2 showed signs of CPE. All flasks were frozen,
thawed, and then passaged as described for sub-
sample 1 using BGM cells at passage 126.
By May 30, only six flasks from the second pas-
sage of subsample 1 and the positive control
showed CPE. Thus one culture from the first pas-
;age failed to confirm in the second pass, and a
value of 6 was recorded in the Number of Repli-
cates with CPE column of the Total Culturable
Virus Data Sheet. The flasks were then discarded.
On June 6, seven flasks (the five original plus two
new flasks) from the second passage of subsample
2 demonstrated CPE. The two new flasks and con-
trols were frozen at -70°C, thawed, and passaged a
third time as described above using BGM cells at
passage 127. All other flasks were discarded.
By June 12, the positive control and the two third-
passage flasks had developed CPE. All flasks were
discarded at this time. (The flasks would have been
examined until June 20 if at least one had remained
negative.) A value of 7 was recorded into the Num-
ber of Replicates with CPE column of the Total
Culturable Virus Data Sheet.
The MPN software program supplied by the U.S.
EPA was used to calculate the MPN/mL and 95%
confidence limit values. "I. SIZE OF INOCULUM
VOLUME (mL)" on the main screen was changed
from 1 to 0.76. "A. PROCEED WITH DATA IN-
PUT" was pressed followed by "ENTER" to over-
write the existing output file. Alternatively, "NO"
could have been entered and the output file re-
named. The number of positive replicates, "13,"
was then entered. Following the calculation by the
program, the MPN and 95% Confidence Limit
values were recorded onto the Quantitation of To-
tal Culturable Virus data sheet. The program was
exited by pressing "I. EXIT THE PROGRAM."
The MPN per 100 liter value (ML) was calculated
according to the formula:
1 00 MmS 1 00 x 1.38 x 1 5.3 _
D
100
F-2
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
The Lower 95% Confidence Limit per 100 liter
was calculated according to the formula:
1 00 CLimS _ 1 00 x 0.70 x 1 5.3
D
100
,__
- 1 U. /
where Cllm is the lower 95% confidence limit per
milliliter from the Quantitation of Total Culturable
Virus data sheet.
The Upper 95% Confidence Limit per 100 liter
(CLu) was calculated according to the formula:
n. 100 CLumS 100x2.27x15.3
ULu = r = = 34.7
D
100
where CLum is the upper 95% confidence limit per
milliliter from the Quantitation of Total Culturable
Virus Data Sheet.
F-3
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
11111!
'!1! 11
i SAMPLE DATA
SAMPLE NUMBER:
Sampleville-01
UTILITY NAME:
Sampleville Water Works
UTILITY ADDRESS:
CITY: Sampleville
1 Water Street
STATE: OH
ZIP: 45999
SAMPLER'S NAME: Mr. Brian Hall
WATER TEMPERATURE:
23.5°C
TURBIDITY: 3.6 NTU
WATER pH: 7.8
ADJUSTED WATER pH: NA
THIOSULFATE ADDED:
(CHECK)
YES
XNO
INIT. METER READING: 6048.10
date: 5/1/95
(CHECK UNITS) X gallons __ ft3
time: 9 am
FINAL METER READING: 6104.10 (CHECK UNITS) X. gallons __ ft3
date: 5/1/95 time: 9:30 am
TOTAL SAMPLE VOLUME:
211.98
liters
(Final-Initial meter readings x 3.7854 (for readings in gallons)
or x 28.316 (for readings in ft3))
SHIPMENT DATE: 5/1/95
CONDITION ON ARRIVAL: Cold/Not frozen
COMMENTS:
F-4
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
SAMPLE NUMBER: SAMPLEVILLE-01
ANALYTICAL LABORATORY NAME: CEPOR LABORATORIES
ANALYTICAL LABORATORY ADDRESS: 42 RUECKERT ST.
CITY: CINCINNATI STATE: OH ZIP: 45219
ADJUSTED TOTAL SAMPLE VOLUME (ATSV)1:
183 L
DATE ELUTED: 5/2/95
TIME: 10 am
ELUATE VOLUME RECOVERED:
980 mL
VOLUME OF ELUATE ARCHIVED
98.0 mL
DATE CONCENTRATED: 5/2/95
TIME: 1 pm
FINAL CONCENTRATED SAMPLE VOLUME (FCSV): 28.0 mL
ASSAY SAMPLE VOLUME (S):
15.3 mL
VOLUME OF ORIGINAL WATER SAMPLE
ASSAYED (D)
100
INOCULUM VOLUME:
0.76 mL
DATES ASSAYED
BY CPE:
1st Passage
2nd Passage
3rd Passage
(If necessary)
Subsample 1:
5/2/95
5/16/95
Subsample 2:
5/9/95
5/23/95
6/6/95
MPN/100 LITERS3:
21
95% CONFIDENCE LIMITS
LOWER: 11 UPPER:
35
COMMENTS:
ANALYST: B.G. Moore
^nter the Total Sample Volume from the Sample Data Sheet times 0.965 if a coliphage sample is
taken, times 0.9 if archiving is required, times 0.865 if a coliphage sample is taken and archiving
is required, or times 1.0 if a coliphage sample is not taken and archiving is not required.
2This value must be at least 100 liters for source water and 1,000 liters for finished water.
3Value calculated from the Quantitation of Total Culturable Virus form as described in the Virus
Quantitation section of Part 3.
F-5
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
" " TOTAL OOLTURABLE VIRUS DATA SHEET: ' - " ' • "' ^ '
> / / * / (
* r f / / /
SAMPLE #: Samplevffle-01
Sample
1st Passage
Neg. Cont.
Pos Cont.
Undiluted
1:5 Dil.
1:25 Dil.
2nd Passage1
Neg. Cont.
Pos. Cont.
Undiluted
1:5 Dil.
1:25 Dil.
3rd Passage2
Neg. Cont.
Pos. Cont.
Undiluted
1:5 Dil.
1:25 Dil.
Total Number of Replicates
Subsample 1
Inoculated
1
1
10
1
1
10
Without
CPE
1
0
3
1
0
4
With CPE
0
1
7
0
1
6
Subsample 2
Inoculated
1
1
10
1
1
10
1
1
2
Without
CPE
1
0
5
1
0
3
1
0
0
With CPE
0
1
5
0
1
7
0
1
2
»A portion of medium from each 1st passage vessel, including controls, must be repassaged for conformation. The terms
"Undiluted," "1:5 Dilution" and "1:25 Dilution" under the 2nd and 3rd Passage headings refer to the original sample
dilutions for the Grst passage. If higher dilutions are used, record the data from the three highest dilutions for the 1st
passage. If higher dilutions are used, record the data from the three highest dilutions showing positive results and place
the actual dilution amount in the sample column.
'Samples that were negative on the first passage and positive on the 2nd passage must be passaged a third time for
conformation. If a third passage is required, all controls must be passaged again.
F-6
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
tek;ff&f4^£^^^
SAMPLE NUMBER:
Sample
Sampleville-01
Number
Replicates
Inoculated
Number
with CPE
Undiluted Samples
Subsample 1
Subsample 2
Total Undiluted
10
10
20
6
7
13
Subsample 2 results (Dilutions Required)
Undiluted
1:5 Dilution
1:25 Dilution
MPN/mL1
1.38
95% Confidence
Limits
Lower
0.70
Upper
2.27
'Use the values recorded in the Total Undiluted row to calculate the MPN/mL result and
confidence limits when dilutions are not required. If dilutions are required, base the
calculation upon the values recorded in the Undiluted, 1:5 Diluted, and 1:25 Diluted
rows for subsample 2. If higher dilutions are used for subsample 2, record the data from
the three highest dilutions showing positive results and place the actual dilution amount
in the sample column. The MPN/mL and 95% Confidence Limit values must be
obtained using the computer program supplied by the U.S. EPA.
F-7
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
Example 2
A source water sample of 200.63 liters was col-
lected at the Sampleville Water Works on June 5,
1995 and shipped by overnight courier to CEPOR
Laboratories. CEPOR Laboratories processed the
sample on June 6,1995. After elution, the pH was
adjusted to 7.3. A volume of 985 mL of pH-ad-
justed eluate was obtained and 34.5 mL
(985 x 0.035) was removed for the Coliphage As-
say (see Section IX of the ICR Microbial Labora-
tory Manual). Archiving was not required. An
Adjusted Total Sample Volume of 194 liters
(200.63 x 0.965) was recorded on the Virus Data
Sheet.
The sample was immediately processed by the
Organic Flocculation Concentration Procedure.
Following centrifugation at 4,000 xg, the superna-
tant was adjusted to pH 7.3 and passed through a
sterilizing filter. A Final Concentrated Sample
Volume (FCSV) of 32.0 mL was obtained, giving
an Assay Sample Volume (S) for Sampleville-02
of:
S =
100.00 liters
194 liters
x 32.0 ml = 16.5 ml
Subsample 1 was prepared by placing 0.55 x 16.5
mL =9.1 mL into a separate container. Subsample
2 was prepared by placing 0.67 x 16.5 mL =11.1
niL into a third container. Subsample 2 and the
remaining portions of the Final Concentrated
Sample were frozen at -70°C.
Subsample 1 was inoculated onto each of 10 25-
cm2 flasks of BGM cells at passage 127 using an
Inoculum Volume of 16.5 mL * 20 = 0.82 mL per
flask. A negative control was prepared by inocu-
lating a flask with 0.82 mL of sodium phosphate,
pH 7.3. A positive control was prepared by in-
oculating a flask with 0.82 mL of sodium phos-
phate, pH 7.3, containing 25 PFU/mL (20 PFU,
0.82 mL) of attenuated poliovirus type 3. Following
.dsorption, 9.18 mL of maintenance medium was
added, and the cultures were incubated at 36.5°C.
On June 13, nine flasks inoculated with subsample
1 and the positive control showed signs of CPE.
After thawing subsample 2, a 1:5 dilution was
prepared by mixing 0.1334 x 16.5 = 2.20 mL of
subsample 2 with 0.5334 x 16.5 = 8.80 mL of so-
dium phosphate, pH 7.3. A 1:25 dilution was pre-
pared by mixing 2.20 mL of the 1:5 dilutions with
8.80 mL of sodium phosphate, pH 7.3. Ten 25-cm2
flasks of BGM cells at passage 128 were then in-
oculated with 0.82 mL each of undiluted sub-
sample 2. Ten flasks were inoculated with 0.82
mL each of subsample 2 diluted 1:5, and 10 flasks
were inoculated with 0.82 mL each of subsample
2 diluted 1:25. Another negative control and posi-
tive control were also prepared and inoculated.
By June 20, all 10 flasks inoculated with sub-
sample 1 showed signs of CPE and were
repassaged as described in example 1.
By June 27, all 10 flasks inoculated with undiluted
subsample 2 had developed CPE. Eight flasks in-
oculated with the 1:5 dilution of subsample 2 and
four flasks inoculated with the 1:25 dilution of
subsample 2 demonstrated CPE. All flasks were
passaged again as described for example 1.
By July 5, all 10 flasks from the second passage of
subsample 1 were confirmed as positive and were
discarded.
By July 11, all 10 flasks inoculated with the sec-
ond passage of undiluted subsample 2 had devel-
oped CPE. The eight positive flasks from the first
passage of the 1:5 dilution of subsample 2 were
positive in the second passage. Three flasks inocu-
lated with the second passage of the 1:25 dilution
of subsample 2 remained positive.
F-8
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
The MPN software program supplied by the U.S.
EPA was used to calculate the MPN/mL and 95%
confidence limit values. After the main screen ap-
peared, "G. NUMBER OF DILUTIONS" was
changed from 1 to 3. "H. NUMBER OF REPLI-
CATES PER DILUTION" was changed from 20
to 10, and "I. SIZE OF INOCULUM VOLUME
(mL)" was changed from 1 to 0.82. "A. PROCEED
WITH DATA INPUT" was pressed followed by
"ENTER" to overwrite the existing output file. The
number of positive replicates per dilution, "10, 8,
and 3," was entered with the values separated by
spaces. Following program calculations, the MPN/
mL and 95% confidence limit values/mL were re-
corded onto the Quantitation of Total Culturable
Virus data sheet. The program was exited by press-
ing "I. EXIT THE PROGRAM."
The MPN per 100 liter value (ML) was calculated
according to the formula:
IVlmS 100.00 x 10.15 x 16.5
D
100.00
The Lower 95% Confidence Limit per 100 liter
j) was calculated according to the formula:
1 00 CI_lmS_ 100.00 x 5.04 x 16.5
D
100.00
„„ „
— o3.2
where Cl,m is the lower 95% confidence limit per
milliliter from the Quantitation of Total Culturable
Virus data sheet.
The Upper 95% Confidence Limit per 100 liter
(CLu) was calculated according to the formula:
100CLumS 100.00x18.25x16.5
D
100.00
= 301
where CLum is the upper 95% confidence limit per
milliliter from the Quantitation of Total Culturable
Virus data sheet.
F-9
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
(i1 II 111 III I ll II II I ( Illlllllll (Hill i t i 11
SAMPLE DATA SHEET
SAMPLE NUMBER:
Sampleville-02
UTILITY NAME:
Sampleville Water Works
UTILITY ADDRESS:
CITY: Sampleville
1 Water Street
STATE: OH
ZIP: 45999
SAMPLER'S NAME: Mr. Brian Hall
WATER TEMPERATURE:
26.5°C
TURBIDITY: 2.3 NTU
WATER pH: 7.7
ADJUSTED WATER pH: NA
THIOSULFATE ADDED:
(CHECK)
YES
XNO
INIT. METER READING: 6129.3 (CHECK UNITS)
date: 6/1/95 time: 8:30 am
X. gallons ft3
| FINAL METER READING: 6182.3
date: 6/5/95
(CHECK UNITS)
time: 9:00 am
X gallons _ ft3
TOTAL SAMPLE VOLUME:
200.63
liters
(Final-Initial meter readings x 3.7854 (for readings in gallons)
or x 28.316 (for readings in ft3))
SHIPMENT DATE: 6/5/95
CONDITION ON ARRIVAL: Cold/Not frozen
COMMENTS:
F-10
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
';*:-?,; i?-;^,* ?ig§f ;- ir':^tS35v-y K^qKi^atv ;> "A-p-»> -^ »4F''£} ;?^:o;;; K;> ww.:: v- J.JM^
jr^V^^kS*-.1^^*:^
l%^?nft;u^£fC4QM^
SAMPLE NUMBER:
ttSS^iltH^©^
SAMPLEVILLE-02
ANALYTICAL LABORATORY NAME:
CEPOR LABORATORIES
ANALYTICAL LABORATORY ADDRESS: 42 RUECKERT ST.
CITY: CINCINNATI STATE: OH ZIP: 45219
ADJUSTED TOTAL SAMPLE VOLUME
DATE ELUTED: 6/6/95
ELUATE VOLUME RECOVERED:
(ATSV)1: 194 L
TIME: 9:50 am
985 mL
VOLUME OF ELUATE ARCHIVED
DATE CONCENTRATED: 6/6/95
0 mL
TIME: 1 pm
FINAL CONCENTRATED SAMPLE VOLUME (FCSV): 32.
ASSAY SAMPLE VOLUME (S):
16.
0 mL
5 mL
VOLUME OF ORIGINAL WATER SAMPLE
ASSAYED (D) 100.00 L2
INOCULUM VOLUME:
DATES ASSAYED
BY CPE:
Subsample 1:
Subsample 2:
MPN/100 LITERS3:
1st Passage
6/6/95
6/13/95
167
COMMENTS:
0.82 mL
2nd Passage
6/20/95
6/27/95
3rd Passage
(If necessary)
95% CONFIDENCE LIMITS
LOWER: 83 UPPER: 301
ANALYST: E.G. Moore
JEnter the Total Sample Volume from the Sample Data Sheet times 0.965 if a coliphage sample is
taken, times 0.9 if archiving is required, times 0.865 if a coliphage sample is taken and archiving
is required, or times 1.0 if a coliphage sample is not taken and archiving is not required.
2This value must be at least 100 liters for source water and 1,000 liters for finished water.
3Value calculated from the Quantitation of Total Culturable Virus form as described in the Virus
Quantitation section of Part 3.
F-11
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
TOTAL CULTURABLE VIRUS DATA SHEET
! .,'<''*'
SAMPLED: SampleviUe-02
Sample
1st Passage1
Neg. Cont.
Pos Cont.
Undiluted
1:5 Dil.
1:25 Dil.
2nd Passage1
Neg. Cont.
Pos. Cont.
Undiluted
1:5 Dil.
1:25 Dil.
3rd Passage2
Neg. Cont.
Pos. Cont.
Undiluted
1:5 Dil.
1:25 Dil.
Total Number of Replicates
Subsample 1
Inoculated
1
1
10
1
1
10
Without
CPE
1
0
0
1
0
0
With CPE
0
1
10
0
1
10
Subsample 2
Inoculated
1
1
10
10
10
1
10
10
10
10
Without
CPE
1
0
0
2
6
1
0
0
2
7
With CPE
0
1
10
8
4
0
1
10
8
3
'A portion of medium from each 1st passage vessel, including controls, must be repassaged for conformation. The terms
"Undiluted," "1:5 Dilution" and "1:25 Dilution" under the 2nd and 3rd Passage headings refer to the original sample
dilutions for the Grst passage. If higher dilutions are used, record the data from the three highest dilutions for the 1st
passage. If higher dilutions are used, record the data from the three highest dilutions showing positive results and place
the actual dilution amount in the sample column.
'Samples that were negative on the first passage and positive on the 2nd passage must be passaged a third time for
conformation. If a third passage is required, all controls must be passaged again.
F-12
-------
APPENDIX F
EXAMPLES OF CALCULATIONS
SAMPLE NUMBER:
Sampleville-02
Sample
Number
Replicates
Inoculated
Number
with CPE
MPN/ml1
95% Confidence
Limits
Lower
Upper
Undiluted Samples
Subsample 1
Subsample 2
Total Undiluted
10
NA
10
10.15
5.04
18.25
NA
Subsample 2 results (Dilutions Required)
Undiluted
1:5 Dilution
1:25 Dilution
10
10
10
10
8
JUse the values recorded in the Total Undiluted row to calculate the MPN/mL result
and confidence limits when dilutions are not required. If dilutions are required, base
the calculation upon the values recorded in the Undiluted, 1:5 Diluted, and 1:25
Diluted rows for subsample 2. If higher dilutions are used for subsample 2, record the
data from the three highest dilutions showing positive results and place the actual
dilution amount in the sample column. The MPN/mL and 95% Confidence Limit
values must be obtained using the computer program supplied by the U.S. EPA.
F-13
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