United States
Environmental Protection
Agency
Office of Research
and Development
Washington DC 45260
EPA 600/4-84/013 (N15)
April 2001
&EPA
USEPA Manual of
Methods for Virology
Chapter 15
April 2001
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April 2001
Chapter 15
TOTAL CULTURABLE VIRUS QUANTAL ASSAY1
1. Introduction
1.1 Scope
This chapter describes a quantal
method for assaying culturable human
enteric viruses from water matrices.
The assay differs from the plaque assay
described in Chapter 10 (December
1987 Revision) in that it is based upon
the direct microscopic viewing of cells
for virus-induced cytopathic effects.
The quantal method can be used to as-
say viruses concentrated from sewages,
effluents and waters using the methods
described in Chapters 5, 6 and 14 (Feb-
ruary 1999), Dahling and Wright
(1986b), Fout et al. (1996) or from
sludges and other solids (Chapter 7,
September 1989 Revision; Fout, 1999).
1.2 Significance
Quantal assays are less quantita-
tive but more sensitive than plaque as-
says. In plaque assays each plaque-
forming unit (PFU) is counted as devel-
oping from a single infectious virus. In
quantal assays a positive response may
be caused by one or more infectious vi-
rus particles and thus quantitation is ac-
complished through inoculation of rep-
licate samples and the use of most prob-
able number statistics (Chang et al.,
1958).
The disadvantage caused by the
loss in accuracy in the measurement of
infectious virus particles is often offset
in quantal assays by an increased sensi-
tivity in the assay's capacity to detect
infectious viruses (Morris and Waite,
1980). This increase is presumably due
to enhanced replication of some virus
strains under quantal assay conditions.
Detection of slow growing virus strains
is probably also enhanced by quantal
assays, because the cell monolayers stay
viable for longer periods of time.
1.3 Safety
Human pathogenic enteric viruses
can be present in surface or ground-
waters impacted by untreated or inade-
quately treated domestic wastes. The
presence of these viruses can cause hep-
atitis, gastroenteritis and numerous
other diseases. Laboratories performing
virus analyses are responsible for estab-
lishing an adequate safety plan and
must rigorously follow the guidelines on
decontamination and waste disposal
given in Chapter 2 (May 1991 Revi-
sion).
2. Apparatus, Materials, Media
and Reagents
Analytical Reagent or ACS grade
chemicals (unless specified otherwise)
and deionized or distilled reagent grade
water (dH2O) should be used to prepare
all media and reagents. The dH2O must
have a resistance of greater than 0.5
megohms-cm at 25 °C, but water with a
resistance of 18 megohms-cm is pre-
ferred.
Appendix 1 gives a list of potential
vendors for the apparatus, materials,
media and reagents used in this method.
Equivalent items from other vendors
may be substituted for those given in
the text.
2.1 Apparatus and Materials
2.1.1 Incubator capable of maintaining
the temperature of cell cultures at 36.5
±1°C.
2.1.2 Sterilizing filter — 0.22 |im
(Costar Product No. 140666).
Always pass about 10-20 mL of
1.5% beef extract, pH 7.0-7.5, through
the filter just before use to minimize
virus adsorption to the filter (see Sec-
tion 2.2.2).
2.1.3 Sterilizing filter unit —0.2 |im
(PGC Scientifics Product No. 33-8803-
42)
2.2 Media and Reagents:
2.2.1 Sodium hydroxide (NaOH) —
prepare 1 M and 5 M solutions by dis-
solving 4 g or 20 g of NaOH in a final
volume of 100 mL of dH2O, respec-
tively.
NaOH solutions are self-sterilizing
and may be stored for several months at
room temperature.
2.2.2 Beef extract, desiccated powder
(DifcoProductNo. 0115-17-3) —pre-
pare buffered 1.5% beef extract by dis-
solving 7.5 g of beef extract powder and
1.875 g of glycine (final glycine con-
centration = 0.05 M) in 450 mL of
dH2O. Adjust the pH to 7.0-7.5 with 1
or 5 M NaOH and bring the final vol-
ume to 500 mL. with dH2O. Autoclave at
121°C for 15 min and use at room tem-
perature.
Beef extract solutions may be
stored for one week at 4°C or for longer
periods at -20°C.
2.2.3 Iodine disinfectant, 0.5% — dis-
solve 5g of iodine in 1000 mL of 70%
ethanol.
2.2.4 Earle's Balanced Salt Solution
with 0.5% lactalbumen hydrolysate
(ELAH) — dissolve 5 g of lactalbumen
hydrolysate (Life Technologies Product
No. 11800) and the salts from a 1 L
packet of Earle's Balanced Salts (Life
'Prepared by G. Shay Fout, D.R. Dahling and R.S. Safferman
15-1
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April 2001
Technologies Product No. 81100) in a
total volume of 1 L of dH2O. Sterilize
through a sterilizing filter unit and store
at room temperature.
2.2.5 Prepare growth medium by sup-
plementing MEM/L-15 medium with
10% serum and antibiotics (see chapter
9 (January 1987 Revision) for recipes to
prepare medium and antibiotics; e.g.,
100 mL of serum, 1 mL of peni-
cillin-streptomycin stock, 0.5 mL of
tetracycline stock and 0.2 mL of fungi-
zone stock per 900 mL of MEM/L-15).
2.2.6 Sterile fetal calf, gamma globu-
lin-free newborn calf or iron-supple-
mented calf serum, certified free of vi-
ruses, bacteriophage and mycoplasma.
Test each lot of serum for cell
growth and toxicity before purchasing
in quantity. Serum should be held at
-20°Cfor long-term storage. Upon
thawing, each bottle must be
heat-inactivated in a waterbath set at
56 ±1 °Cfor 30 min and stored at 4°C
for short term use.
2.2.7 Prepare maintenance medium by
supplementing MEM/L-15 with antibi-
otics and 2% serum (20 mL of serum,
antibiotics as above for growth medium
and80mLofdH2O).
2.2.8 Prepare BGM cell culture test
vessels using standard procedures given
in Chapter 9 (January 1987 Revision).
The BGM cell culture line is cur-
rently the most widely used line for
detecting total culturable viruses from
water matrices. It is a continuous cell
line derived from African Green mon-
key kidney cells that was first described
for clinical use by Barron et al. (1970).
The use of BGM cells for recovering
viruses from environmental samples
and conditions for maximal sensitivity
for virus detection have been described
by Dahling et al. (1974), Dahling et al.
(1984), Dahling and Wright (1986a)
and given in Chapter 9 (January 1987
Revision). Although the line is highly
susceptible to many enteric viruses, -
other cell lines (e.g., RD, MA 104,
FRhK-4) may be used to increase detec-
tion sensitivity.
Stock cultures of BGM cells may be
maintained on roller bottles (Figure 15-
1). BGM cells to be used for virus de-
tection should be between passage num-
ber 117 (the earliest passage routinely
available) and 250.
/ .
Figure 15-1 BGM Roller Bottle
Culture
3. Sample Inoculation and CPE
Development
Wipe down surface areas with dis-
infectant before and after working with
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 facili-
ties used for no other purposes. Viruses
or other microorganisms must not be
transported, handled, or stored in
rooms used for cell culture transfer. It
is also recommended that a microbio-
logical biosafety cabinet be used to in-
oculate cell cultures with water sample
concentrates.
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 pas-
sage. Those older than seven days
should not be used.
3.1 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.
3.2 Decant and discard the medium
from cell culture test vessels. Wash the
test vessels with ELAH or growth medi-
um without serum using a wash volume
of at least 0.06 mL/cm2 of surface area.
Rock the wash medium over the surface
of each monolayer several times and
then decant and discard the wash me-
dium.
Do not disturb the cell monolayer.
The wash medium can be added to
flasks several hours before inoculation
and discarded just prior to inoculation.
3.3 Calculate the Inoculum Volume.
3.3.7 Determine the volume of the
original water or solids sample to be as-
sayed. It is recommended that this vol-
ume be > 1 L for sewage, > 100 L for
surface or recreational waters, > 1000 L
for finished drinking water or untreated
groundwaters or the volume which con-
tains >5 g of total sludge or other solids.
This volume is designated the Volume
of Original Water Sample Assayed
(D). Record the value of D on a Virus
Data Sheet (Appendix 2 gives an exam-
ple of a Virus Data Sheet that contains
information that should be recorded on
each sample tested).
In order to have sufficient sample
for assay and account for losses on the
sides of containers, problems with cyto-
toxicity, etc., the Volume of Original
Water Sample Assayed should repre-
sent about 33 to 66% of the total sam-
ple collected. Thus, the recommended
sample volumes for sewage are >2 L.
Recommended minimum sample vol-
umes for surface or recreational waters
are >200 L and > 1500 L for finished
drinking water or for untreated ground-
water. Recommended maximum sample
volumes for source and recreational
waters and finished waters/untreated
groundwaters are 300 L and 2000 L,
respectively.
3.3.2 Calculate an Assay Sample Vol-
ume.
(a) Calculate the Assay Sample Vol-
ume (S) for water and solids samples
using the formula:
S =
D
TSV
x FCSV
15-2
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April 2001
where TSV is the Total Sample Vol-
ume [i.e., the total volume in liters of
water or sewage sample passed through
a 1MDS filter (Chapter 14, February
1999) or through a negatively charged
filter (Chapters 5-6) or of virus concen-
trates from sludges, soils, sediments and
other solids (Chapter 7, September 1989
revision; Fout, 1999)]. FCSVisthe
Final Concentrated Sample Volume
[the volume in milliliters of the final
processed sample that will be assayed
for viruses. This is usually the concen-
trate from an organic flocculation con-
centration procedure (e.g., Chapter 14,
Step 4.2.7). The Assay Sample Vol-
ume is the volume of the final concen-
trated Sample that represents the vol-
ume of original water sample assayed.
Record the Assay Sample Volume onto
the Virus Data Sheet.
(b) Calculate the Assay Sample Vol-
ume for QC and PE samples using the
formula:
S = 0.4 x FCSV
3.3.3 Determine the Inoculum Volume
by dividing the Assay Sample Volume
by 20.
For ease of inoculation, a sufficient
quantity ofO. 15MNa2HPO4, pH7.0-
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 ml) = 2.84 ml of
sodium phosphate, pH 7.0-7.5 could be
added to 10.5 * 0.73 = 7.67 ml of con-
centrated sample. Each milliliter of the
resulting mixture will contain the re-
quired Inoculum Volume.
The Inoculum/Inoculation Volume
should be no greater than 0.04 mL/cm2
of the cell culture test vessel surface
area. If the Inoculum or Inoculation
Volume is greater than 0.04
mL/cm2,use larger culture vessels.
3.4 Inoculate each BGM cell culture
test vessel with an amount of assay con-
trol or water sample equal to the Inocu-
lum or Inoculation Volume and record
the date of inoculation on the Virus
Data Sheet (see Appendix 2).
Avoid touching either the cannula
or the pipetting device to the inside rim
of the cell culture test vessels to avert
the possibility of transporting contami-
nants to the remaining culture vessels.
3.4.1 Total Culturable Virus Assay
Controls
Run a negative and positive assay
control with every group of samples
inoculated onto cell cultures.
(a) Negative Assay Control: Inoculate
a BGM culture with a volume of sodium
phosphate, pH 7.0 - 7.5, equal to the
Inoculum or Inoculation Volume. This
culture will serve as negative control for
the tissue culture quanta! assay. If any
Negative Assay Control develops
cytopathic effects (CPE), all subsequent
assays of water samples should be halt-
ed until the cause of the positive result
is determined.
(b) Positive Assay Control: Dilute a
stock of attenuated poliovirus type 3 in
sodium phosphate, pH 7.0 - 7.5, to give
a concentration of 20 PFU per Inocu-
lum or Inoculation Volume. Inoculate
a BGM culture with an amount of di-
luted virus equal to the Inoculum or
Inoculation Volume. This control will
provide a measure for continued sensi-
tivity of the cell cultures to virus infec-
tion. Additional positive control sam-
ples 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 halt-
ed until the cause of the negative result
is determined.
3.4.2 Inoculation of Water or Solids
Samples
Samples from environmental waters
and solids may contain components that
are cytotoxic to cell cultures or may
have virus levels too high to quantitate
using undiluted samples. To avoid los-
ing the entire sample to these condi-
tions, this procedure calls for dividing
the Assay Sample Volume into two
equal subsamples and assaying one of the
subsamples one week after the first.
(a) Rapidly thaw one subsample, if
frozen, and inoculate an amount equal
to the Inoculum or Inoculation Vol-
ume onto each often cell cultures (see
Figure 15-2).
Hold a thawed subsample for no
more than 4 h at 4°C. Warm the
subsample to room temperature just
before inoculation. Freeze any remain-
ing portion of the first subsample at
-70°Cfor use with Step 3.4.2e.
Figure 15-2 Inoculation of 25 cm2
Flasks.
(b) Determine if cell death has oc-
curred due to cytotoxicity (see Figure
15-3). If the sample is cytotoxic, go to
Step 3.4.2e. If there is no evidence for
cytotoxicity, determine the number of
flasks that are positive for CPE.
Examine cells, especially during
the first three days, for the development
of cytotoxicity. Determine cytotoxicity
from the initial daily macroscopic ex-
amination of the appearance of the cell
culture monolayer by comparing the
negative control from Step 3.4. la and
the positive control from Step 3.4.1b
with the test samples from Step 3.4.2.
Cytotoxicity should be suspected when
the cells in the test sample develop CPE
within 24 h or before its development
on the positive control.
A small portion of the Final Con-
centrated Sample may by inoculated
onto cultures several days before inocu-
lating the first subsample as a control
for cytotoxicity.
(c) If at least three cell cultures are
negative for CPE (see Figure 15-3),
15-3
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April 2001
Inoculate 10 Cultures with Subsample 1
Treat for
Cytotox/cify
[Section 3.4.2e]
Are3
A
i
Negative?
0
< ,
Prepare 10 Cultures each of
Undiluted Subsample 2 and
Subsample 2 Diluted 5-Fold and
25-Fold
r
Prepare 10 Cultures each
of Undiluted Subsample 2
Figure 15-3 Subsample Decision Tree
thaw a second subsample and inoculate
an amount equal to the Inoculum or
Inoculation Volume onto each often
additional BGM cell cultures.
(d) If more than seven cultures are pos-
itive for CPE after seven days (see Fig-
ure 15-3), prepare and inoculate BGM
cell cultures with dilutions of the second
subsample.
(d. 1) Prepare a 1:5 dilution by adding
a volume equal to 0.1334 x Assay Sam-
ple Volume (amount "a") to a volume
of 0.15 M sodium phosphate (pH 7.0-
7.5) equal to 0.5334 x Assay Sample
Volume (amount "b"). If the more con-
venient Inoculation Volume is used,
determine amount "b" by multiplying
0.5334 x Assay Sample Volume x (In-
oculation Volume -^ Inoculum Vol-
ume).
(d.2) Mix the 1:5 dilution thoroughly
and then prepare a 1:25 dilution by add-
ing amount "a" of the 1:5 diluted sam-
ple to amount "b" of 0.15 M sodium
phosphate (pH 7.0-7.5).
(d. 3) Using an amount equal to the
Inoculum or Inoculation Volume,
inoculate ten cell cultures each with
undiluted subsample 2, subsample 2
diluted 1:5 and subsample 2 diluted
1:25, respectively.
Freeze the remaining portions of
the 1:25 dilution at -70°C until the sam-
ple results are known. If the inoculated
cultures are all positive, thaw the re-
maining 1:25 dilution and prepare
1:125, 1:625 and 1:3125 dilutions by
transferring amount "a" of each lower
dilution to amount "b " of sodium phos-
phate as described above. Inoculate 10
cultures each with the additional dilu-
tions 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 high-
est dilution tested is negative. Higher
dilutions can also be assayed along
with the initial undiluted to 1:25 dilu-
tions if it is suspected that the water to
be tested contains more than 500 most
probable number (MPN) of infectious
total culturable virus units per 100 L.
A maximum of 60 and 580 MPN
units per 100 L can be demonstrated by
inoculating a total of 20 cultures with the
undiluted Assay Sample Volume or
a total of 10 cultures each with undi-
luted sample and sample diluted 1:5
and 1:25, respectively.
(e) If the first subsample is cytotoxic,
prepare and inoculate BGM cell cul-
tures with dilutions of the first subsam-
ple remaining from Step 3.4.2a.
(e.l) Prepare 1:2, 1:4 and 1:8 dilutions
of the first subsample by serially dilut-
ing an amount equal to 1.3 to 5.3 times
the Inoculum or Inoculation Volume
with an equal volume of 0.15 M sodium
phosphate (pH 7.0-7.5). Inoculate up to
five cell cultures with each dilution us-
ing an amount equal to the Inoculum or
Inoculation Volume.
With most water samples a 1:2
dilution should be sufficient to overcome
cytotoxicity.
(e.2) If cytotoxicity is overcome by
dilution, dilute the second subsample by
the amount necessary to remove
cytotoxicity and inoculate a number of
cultures equal to ten times the dilution
factor used using the Inoculum or Inoc-
ulation Volume (e.g., if a 1:2 dilution
does not show cytotoxicity, inoculate 10
x 2 = 20 cell cultures).
If dilution fails to remove
cytotoxicity, the procedures described
in Chapter 8 (April 1986 revision) may
be used.
Remaining portions of the second
subsample should be frozen at -70°C to
prepare additional dilutions if required
due to high virus liters.
In addition to dilution, the chang-
ing of liquid maintenance medium at
the first signs of cytotoxicity may pre-
vent further development.
3.5 Rock the inoculated cell culture test
vessels gently to achieve uniform
distribution of inoculum over the sur-
face of the cell monolayers (Figure 15-
4). Place the cell culture test vessels on
15-4
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April 2001
a level stationary surface at room tem-
perature so that the inoculum remains
distributed evenly over the cell
monolayer.
cant clusters ofrounded-up cells over
and beyond what is observed in the
uninfected controls.
Figure 15-4 Distributing Inoculum F'9ure 15-5 CPE Examination
3.6 Continue incubating the inoculated
cell cultures for 80 -120 min to permit
viruses to adsorb onto and infect cells.
It may be necessary to rock the ves-
sels every 15-20 min or to keep them on
a mechanical rocking platform during
the adsorption period to prevent cell
death in the middle of the vessels from
dehydration.
3.7 Add liquid maintenance 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 possi-
bility of transporting contaminants to
the remaining vessels. The cultures
may be re-fed with fresh maintenance
medium after 4-7 days.
3.8 Examine each culture microscopi-
cally for the appearance of CPE daily
for the first three days (Figure 15-5)
and then every couple of days for a total
of 14 days.
CPE may be identified as cell dis-
integration or as changes in cell mor-
phology (compare Figure 15-7 with
Figure 15-6; Malherbe and Strickland-
Cholmley, 1980). Rounding-up of in-
fected cells is a typical effect seen with
enterovirus infections. However,
uninfected cells round-up during mito-
sis and a sample should not be consid-
ered positive unless there are signifi-
3.9 Freeze cultures at -70°C when more
than 75% of the monolayer shows signs
of CPE. Freeze all remaining negative
cultures, including controls, after 14
days.
-
Figure 15-6 Culture Without CPE
Figure 15-7 Culture With CPE
3.10 Thaw all the cultures to confirm
the results of the previous passage. Fil-
ter at least 10% of the medium from
each vessel that was positive for CPE or
that appeared to be bacterially contami-
nated through separate 0.22 |im steriliz-
ing filters (Figure 15-8). Then inocu-
late another BGM culture with 10% of
the medium from the previous passage
for each vessel, including those that
were negative. Repeat Steps 3.7-3.8.
Confirmation passages may be per-
formed in small vessels or multiwell
trays, however, it may be necessary to
distribute the inoculum into several ves-
sels or wells to insure that the Inocul-
um or Inoculation Volume is less than
or equal to 0.04 mL/cm2 of surface
area.
•I
Figure 15-8 Filtration of Positive
Samples
3.11 Score cell cultures that developed
CPE in both the first and second pas-
sages as confirmed positives.
Cultures that show CPE in only the
second passage must be passaged a
third time along with the negative con-
trols according to Steps 3.9-3.10.
Score cultures that develop CPE in
both the second and third passages as
confirmed positives.
Cultures with confirmed CPE may
be stored in a -70°C freezer for re-
search purposes or for optional
identification tests.2
4. Virus Quantitation
4.1 Record the total number of con-
firmed positive and negative cell cul-
tures for each subsample onto a Total
Culturable Virus Data Sheet (Appendix
3). Do not include the results of tests
forcytotoxicity!
4.2 Transcribe the number of inocu-
lated cultures and the confirmed num-
ber of positive cultures from the Total
2For more information see Chapter 12
15-5
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April 2001
Culturable Virus Data Sheet for each
subsample to the Quantitation of Total
Culturable Virus Data Sheet (Appendix
4). If dilutions are not required, add the
values to obtain a total undiluted count
for each sample.
4.3 Calculate the MPN/mL value (Mm),
the upper 95% confidence limit/mL
(CL^J and the lower 95% confidence
limit/mL (CLlm) using the total undi-
luted count from step 4.2 and an MPN
software program.3 If dilutions are re-
quired, calculate the MPN/mL value
and 95% confidence limits using only
the values from the second subsample.
Place the values obtained onto the
Quantitation of Total Culturable Virus
Data Sheet.
4.4 Calculate the MPN per 100 liter4
value (M;) of the original sample ac-
cording the formula:
M, =
each water sample according to the for-
mula:
D
where S equals the Assay Sample Vol-
ume and D equals the Volume of Orig-
inal Water Sample Assayed (the val-
ues for S and D can be found on the
Virus Data Sheet). Record the value of
M, onto the Virus Data Sheet.5
4.5
its.
Calculate the 95% confidence lim-
4.5.1 Calculate the lower 95% confi-
dence limit per 100 liter value (CL;) for
3A MPN software program (MPNV) can
be downloaded from
http ://www. epa. gov/microbes/
Virus liters may be expressed in terms
of MPN per volume other than 100 L by
substituting the "100" value in the
equations in Sections 4.4 - 4.5 with the
volume desired. Virus liters from solids
samples should be expressed on a per
gram basis (e.g., MPN/4 g).
5Use significant figures when reporting
all resulls Ihroughoul Ihe protocol (see
APHA, 1995, p. 1-17).
CL,
100 CLJS
where CLlm is Ihe lower 95% confidence
limil per millililer from Ihe Quanlila-
lion of Tola! Cullurable Virus Data
Sheet
4.5.2 Calculate Ihe upper 95% Confi-
dence Limil per 100 liter value (CLU)
according lo Ihe formula:
CL,, =
100 CL,J$
where CL^ is Ihe upper 95% confi-
dence limil per millililer from Ihe
Quanlilalion of Tola! Cullurable Virus
Dala Sheet
4.5.3 Record Ihe lower and upper lim-
ils per 100 liter values on Ihe Virus
Dala Sheel (see Appendix 2 and Figure
5.9).
Figure 15-9 Recording Values
4.6 Calculate Ihe lolal MPN value and
Ihe lolal 95% confidence limil values
for each QC and PE sample by multi-
plying the values per milliliter by S and
dividing by 0.4.
5. References
APHA. 1995. Standard Melhods for
the Examination of Water and
Waslewaler (A. D. Eaton, L. S.
Clesceri and A. E. Greenberg, ed),
19th Edition. American Public
Heallh Association, Washington,
D.C.
Barren, A. L., C. Olshevsky and M. M.
Cohen. 1970. Characteristics of
Ihe BGM line of cells from African
green monkey kidney. Archiv. Ge-
sam. Virusforsch. 32:389-392.
Chang, S. L., G. Berg, K. A. Busch, R.
E. Stevenson, N. A. Clarke and P.
W. Kabler. 1958. Application of
Ihe "mosl probable number" meth-
od for estimating concenlralion of
animal viruses by Ihe tissue culture
technique. Virology 6:27-42.
Dahling, D. R. and B. A. Wright
1986a. Optimization of Ihe BGM
cell line cullure and viral assay pro-
cedures for monitoring viruses in
Ihe environment Appl. Environ.
Microbiol. 51:790-812.
Dahling, D. R. and B. A. Wright
1986b. Recovery of viruses from
water by a modified flocculation
procedure for second-step concen-
lralion. Appl. Environ. Microbiol.
51:790-812.
Dahling, D. R., G. Berg and D. Berm-
an. 1974. BGM, a continuous
cell line more sensitive lhan pri-
mary rhesus and African green kid-
ney cells for Ihe recovery of viruses
from water. Heallh Lab. Sci.
11:275-282.
Dahling, D. R., R. S. Safferman and B.
A.Wright. 1984. Resulls of a sur-
vey of BGM cell cullure practices.
Environ. Internal. 10:309-313.
Foul, G.S., F.W. Schaefer III, J.W.
Messer, D.R. Dahling and R.E.
Sleller. 1996. ICR Microbial Labo-
oralory Manual. U.S. Environmen-
tal Protection Agency Publication
No. EPA/600/R-95/178. Office of
Research and Development, Wash-
ington, D.C.
Foul, G.S. 1999. Appendix H. Melhod
for Ihe recovery and assay of lolal
cullural viruses from sludge. In
Environmental Regulations and
Technology: Control of Palhogens
and Vector Attraction in Sewage
Sludge. U.S. Environmental Protec-
tion Agency Publication No.
EPA/625/R-92/013 (October, 1999
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April 2001
revision). Office of Research and
Development, Washington, D.C.
Morris, R., and W.M. Waite. 1980.
Evaluation of procedures for recov-
ery of viruses from water-II detec-
tion systems. Water Res. 14: 795-
798.
Appendix 1. VENDORS
The vendors listed below represent one
possible source for required products.
Equivalent items from other vendors
may substituted for items given in the
text.
Costar Corp.
7035 Commerce Circle
Pleasanton, CA 94588
(800)882-7711
Difco Laboratories
P.O. Box 331058
Detroit, MI 48232
(800) 521-0851 (Ask for a local dis-
tributor)
Life Technologies
8400 Helgerman Court
Gaithersburg, MD 20898
(800) 828-6686
PGC Scientifics
7311 Governors Way
Frederick, MD 21704
(800) 424-3300
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Appendix 2. Virus Data Sheet
VIRUS DATA SHEET
SAMPLE NUMBER:
ANALYTICAL LABORATORY NAME:
ANALYTICAL LABORATORY ADDRESS:
CITY: STATE: ZIP:
ANALYST NAME:
VOLUME OF
ORIGINAL WATER SAMPLE ASSAYED
ASSAY SAMPLE VOLUME (S):
INOCULUM VOLUME:
INOCULATION VOLUME (IF USED):
(D): L
mL
mL
mL
DATES ASSAYED BY CPE:
1st Passage
First
Second
Subsample:
Subsample:
MPN/IOO L1:
COMMENTS:
. . _ 3rd Passage
2nd Passage ,,,
(If necessary)
95% CONFIDENCE LIMITS/100 L
LOWER: UPPER:
'Calculate MPN/IOO L and Lower and Upper 95% Confidence Limits/100 L from the Quantitation of Total
Culturable Virus Data Sheet as described in Sections 4.4 - 4.5.
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Appendix 3. Total Culturable Virus Data Sheet
TOTAL CULTURABLE VIRUS DATA SHEET
SAMPLE #:
Sample
1st Passage
Neg. Cont.
Pos. Cont.
Undiluted
1:5 DiL
1:25 DiL
2nd Passage1
Neg. Cont
Pos. ConL
Undiluted
1:5 DiL
1:25 DiL
3rd Passage2
Neg. ConL
Pos. Cont.
Undiluted
1:5 DiL
1:25 DiL
Total Number of Replicates
First Subsample
Inoculated
Without CPE
With CPE
Second Subsample
Inoculated
Without CPE
With CPE
'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 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.
Comments:
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April 2001
Appendix 4. Quantitation of Total Culturable Virus Data Sheet
QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET
SAMPLE NUMBER:
Sample
Number
Replicates
inoculated
Number
with CPE
Undiluted Samples
Subsample 1
Subsamplc 2
Total Undiluted
Suhsample 2 results (Dilutions Required)
Undiluted
1:5 Dilution
1:25 Dilution
MPN/mL!
95% Confidence
Limits
Lower
Upper
'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 snbsample 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.
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