&EPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-84/013
April 1986
Revision
Research and Development
USEPA Manual of
Methods for Virology
Chapter 8
Revised April 1986
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April 198$
Chapter 8
Method for Reduction of Cytotoxicity of Sample Concentrates
This method may be used for the
reduction of cytotoxicity in the
recovery of viruses from surface
waters, waste waters, drinking waters,
ground waters, sludges, soils and
dredge spoils found toxic to
mammalian cells used for the assay of
enteroviruses. The method may also
be used for deeply colored sample
concentrates that, if left on the cell
monolayer, would result in inaccurate
plaque counts. The procedure may
result in less than usual recovery of
viruses- A titer reduction of about 30%
may be anticipated; thus, the
procedure is to be applied only if there
is a livelihood that inocula will be toxic
to cell cultures, or may be so darkly
colored as to obscure plaques.
Use aseptic techniques and sterile
materials and apparatus only. Sterilize
all contaminated materials before
discarding them (See Chapters 2 and
3).
1. Virus Recovery from
Samples
1.1 Process samples containing
large amounts of solids, such as
sludge, soil and dredge spoil using the
procedures described in Chapter 7.
1.2 Process water and wastewater
samples by following the VIRADEL
procedure described in Chapters 5 and
6.
2. Storage of Sample
Concentrates
2.1 Maintain samples at 4°C if
samples can be processed within eight
hogrs.
2.2 Store samples immediately at
-70°C if processing cannot be
undertaken within eight hours.
3. Predetermine Cytotoxicity
of Sample Concentrate
Pretest all samples which may be
cytotoxic.
3.1 Following the instructions in
Sections 5.3.1 thru 5,3.13, inoculate
one cell culture monolayer with each
of the sample concentrates requiring
testing.
In the absence of experience in
identifying sample cytotoxicity, it is
advisable also to process an
uninoculated cell culture which will
serve as a control in later comparisons
to determine any reduction in
sensitivity or survival of the
mammalian cells due to the toxicity of
the samples.
3.2 Incubate cell cultures at room
temperature (22-25°C) for 80 min.
3.3 Add overlay medium to bottle(s)
in accordance with the instructions
given in Sections 6.1 and 6.2 of this
chapter.
3.4 Invert cell culture bottles and
incubate for three days in the dark at
36.5° + 1°C.
3.5 Examine monolayer for
cytotoxicity.
Determine cytotoxicity by
macroscopic examination of the
appearance of the cell culture
monolayer. Cytotoxicity should be
suspected when the agar color is more
subdued, generally yellow to yellow- >
brown. This change in color results in
a mottled or blotchy appearance
instead of the evenly diffused color
observed in "healthy" cell monolayers.
Also viral plaques may be difficult to
distinguish from the surrounding
monolayer.
4. Processing of Deeply
Colored Sample Concentrates
4.1 Identify color interference on the
basis of past experience or from
results obtained in the pretesting of
sample concentrates (Section 4.2).
4.2 Predetermine color interference
from sample concentrates.
In the absence of experience, it is
advisable to predetermine whether the
presence of a particularly prominent
color in a virus assay sample will
interfere with subsequent plaque
counting. To pretest the sample
concentrate, add a portion of
concentrate equal to that of the
inoculum to a single cell culture bottle.
It is also advisable to process an
uninoculated cell culture that will
serve as a control in later comparisons
with the test samples. Immediately add
overlay medium to the bottle(s) in
accordance with the instructions given
in Sections 6.1 and 6.2 of this chapter.
Invert the cell culture bottles and
incubate for 30 min in the dark at
36.5° ± 1°C. Examine the bottles for
clarity of the agar overlay medium.
4.3 Process colored sample
concentrate that may interfere with
" virus assay by following the procedure
used for reduction in sample toxicity
(Section 5).
5. Reduction of Toxicity of
Sample Concentrate
5.1 Apparatus and Materials
5.1.1 Cornwall syringe or
equivalent with cannula or syringe
needle.
A pipetting device will expedite virus
assay when large numbers of cell
cultures must be treated.
5.1.2 Magnetic stirrer and stir bars.
5.1.3 Cell culture bottles.
See Chapter 9 for the preparation of
cell culture bottles. Chapter 9 outlines
procedures using the Buffalo Green
Monkey (BGM) kidney cells. These
cells are very sensitive to many
enteroviruses; however, cells other
than BGM may also be used.
5.1.4 Waterbath set at 36.5 ± 1 °C.
5.2 Media and Reagents
5.2.1 Sodium chloride (NaCI).
5.2.2 GG-free newborn calf serum
(Grand Island Biological Co., or
equivalent).
5.2.3 ELAH—Earle's base, with 0.5%
lactalbumin hydrolysate and without
NaHCOa (Kansas City Biological, cat.
no. DM-303, or equivalent).
8-1
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April 1986
5.2,4 Washing solution.
(a) To a flask containing an
appropriate volume of deionized
distilled water, add sufficient NaCI to
result in a final concentration of
0.85%.
The volume of NaCI solution
required will depend on the number of
bottles to be processed end the cell
surface area of the bottles used for
plaque assay. For determining volume,
see Sect on 5.3.14.
(b) Mix contents of flask on magnetic
stirrer at speed sufficient to dissolve
salt.
(c) Autoclave the salt solution at
121°Cfor15min.
(d) Cool salt solution to room
temperature.
(o) Add 2% (volume/volume) GG-free
newborn calf serum to salt solution.
(f) Mix on a magnetic stirrer at a speed
sufficient to uniformly suspend the
serum.
(g) Store the washing solution at 4°C.
Although the washing solution may
be stored at 4°C for an extended time
period. It is advisable to prepare
solutions on a weekly basis, thereby
lessening the possibility of microbial
contamination.
5.3 Procedure
5.3. 1 Decant growth medium from
cell culture bottles previously prepared
in accordance with Instructions given
in Chapter 9.
The medium is changed from one to
four hours before cultures are to be
inoculated and carefully decanted so
as not to disturb the cell monolayer.
5.3.2 Discard medium.
5.3.3 Replace discarded medium
with an equal volume of maintenance
medium.
For BGM cells use ELAH—Earle's
base solution without serum as
maintenance medium.
To reduce shock to cells, warm
maintenance medium to 36.5 ±1°C
before placing on cell monolayer.
To prevent disturbing cells with the
force of the liquid against the cell
monolayer, add maintenance medium
to the side of cell culture bottle
opposite the cell monolayer.
5.3.4 Return cell culture bottles to
36.5 ± 1°C incubator and hold at that
temperature until the bottles are to be
inoculated.
5.3.5 Identify culture bottles by
coding them with an indelible marker.
5.3.6 Decant maintenance medium
from cell culture bottles.
Do not disturb the cell monolayer.
5.3.7 Discard medium.
5.3.8 Inoculate onto each cell
monolayer a volume of test sample
concentrate appropriate for the cell
surface area of the cell culture bottles
used.
Inoculum volume should be no
greater than 1 mL for each 40 cm2 of
surface area.
5.3.9 Rock inoculated cell culture
bottles gently to achieve uniform
distribution of inoculum on surface of
cell monolayers.
5.3.10 Place cell culture bottles on a
leveled stationary surface at room
temperature (22-25°C) so that the
inoculum will be distributed evenly
over the cell monolayer.
5.3.7 7 Incubate inoculated cell
cultures for 80 min to permit viruses to
adsorb onto and infect cells.
5.3.72 Decant inoculum from each
cell culture bottle.
5.3.73 Discard inoculum.
5.3.14 Into each cell culture bottle,
add the volume of washing solution
appropriate for the cell surface area of
the bottles used.
Add 0.25 mL of washing solution for
each cm2 of cell surface area. Use
washing solution as prepared in
Section 5.2.4.
To reduce thermal shock to cells,
warm washing solution to 36.5 ±1°C
before placing on cell monolayer.
To prevent disturbing cells with the
force of the liquid against the cell
monolayer, add washing solution to
the side of the cell culture bottle
opposite the cell monolayer. Also,
avoid touching either the cannula or
syringe needle of the pipette or the
pipetting device to the inside rim of the
cell culture bottles to avert the
possibility of transporting
contaminants to the remaining culture
bottles.
5.3.15 Rock the washing solution
gently across the cell monolayer a
minimum of two times.
It may be necessary to gently rock
washing solution across the
monolayer more than twice if sample
is deeply colored or oily and difficult to
remove from the cell monolayer
surface.
5.3.16 Decant spent washing
solution in a manner that will not
disturb the cell monolayer.
5.3.77 Discard washing solution.
5.3.18 Proceed immediately to
Section 6.
6. Plaque Procedure for
Titrating Viruses
6.1 Preparation
6.1.1 Apparatus and Materials
(a) Waterbath set at 36° ± 1 °C.
Used for maintaining the
temperature of the overlay medium.
See Section 6.1.2 (a) to (h).
(b) Waterbath set at 50° ± 1 °C.
Used for maintaining the agar
temperature. See Section 6.1.2 (i).
(c) Incubator capable of maintaining
the temperature of cell cultures at
36.5°±1°C.
6.1.2. Media and Reagents
(a) 2X Medium 199 with Earle's salts
(Grand Island Biological Co., cat. no.
400-1100, or equivalent).
(b) HEPES (Sigma Chemical Co., cat.
no. H-3375, or equivalent).
(c) GG-free newborn calf serum
(Grand Island Biological Co., cat. no.
210-6400, or equivalent).
(d) Magnesium chloride (MgCIa).
(e) Sodium bicarbonate (NaHCOs).
(f) Neutral red, biological stain (Fisher
Scientific Co., cat. no. N-129, or
equivalent). ^
(g) Bacto skim milk (Difco
Laboratories, cat. no. 0032-01, or ,
equivalent).
(h) Antibiotics—penicillin G,
dihydrostreptomycin sulfate,
amphotericin B and tetracycline
hydrochloride (Sigma Chemical Co., or
equivalent).
8-2
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(i) GIBCO bacteriological agar (Grand
Island Biological Co., cat. no.
M00010B, or equivalent).
6.2 Procedure
6.2.1 Mix equal portions of overlay
medium and agar.
For BGM cells use the following or
equivalent ingredients for the final
agar overlay medium: 39.5% of a 2X
Medium 199 with Earle's salts, 2% of
a 1M solution of HEPES, 2% of GG-
free newborn calf serum, 3% of a 7.5%
solution of NaHCOs, 1% of a 1%
solution ofMgC/2, 1.5% of a 0.1%
solution of neutral red, 1% of a 1%
solution of Bacto skim milk and 50% of
a 3% suspension of GIBCO
bacteriological agar. For each 1 mL of
overlay medium prepared, supplement
with 100 units of penicillin G, 100 fig
of dihydrostreptomycin sulfate, 1 fjg of
amphotericin B and 0.0125 mg of
tetracycline hydrochloride.
6.2.2 To each cell culture bottle, add
the volume of warm (42-46°C) agar
overlay medium appropriate for the
cell surface area of the bottles used.
For each cm2 of cell surface area,
add 0.5 mL of agar over/ay medium.
To prevent disturbing cells with the
force of the liquid against the cell
monolayer, add agar overlay medium
to the side of the cell culture bottle
opposite the cell monolayer.
6.2.3 Place cell culture bottle,
monolayer side down, on a leveled
stationary surface at room
temperature (22-25°C) so that the agar
will distribute evenly before it
solidifies.
Agar is fully solidified within 30 to
60 min.
6.2.4 Cover cell culture bottles with a
sheet of aluminum foil, a tightly woven
cloth, or some other suitable cover to
reduce light intensity and thus,
prevent damage to the cell monolayer.
6.2.5 Invert cell culture bottles and
incubate in the dark at 36.5° + 1°C.
6.3 Counting Viral Plaques
6.3.1 Count, mark and record
plaques in cell culture bottles on days
two, three, four, six, eight and twelve
after overlaying.
Counting procedure described is for
enteroviruses. Depending on the virus
density and virus types present in the
inoculated sample, rescheduling of
virus counts at plus or minus one day
may be deemed preferable.
Virus titers are calculated from total
count.
6.3.2 Examine cell culture bottles on
day sixteen.
If no new plaques appear after 16
days, discard cell culture bottles;
otherwise continue to count, mark and
record plaques every two days until no
new plaques appear between counts.
6.3.3 Calculate virus liter in plaque
forming units (PFU) for each virus-
containing sample concentrate.
To determine the number of PFU per
mL in water, sewage sludge, soil, or
dredge spoil sample concentrate,
multiply the number of PFU by the
reciprocal of the inoculum volume. If
the inoculum volume was diluted, also
multiply the number of PFU by the
reciprocal of the dilution made.
6.3.4 Calculate virus content of
original sample.
To obtain virus content of the
original sample in terms of PFU per
mL, multiply the product from Section
6.3.3 by the concentration factor
which is calculated by dividing the
volume of the original sample by the
volume of the sample concentrate. For
soil, digested de watered sludge and
dredge spoil samples, correct for water
content and report in PFU per gram of
dry weight.
7. Bibliography
Berg, G., D. Berman, and R. S.
Safferman. 1982. A method for
concentrating viruses recovered
from sewage sludges. Can. J.
Microbiol. 28:553-556.
Berg, G., and D. R. Dahling. 1980.
Method for recovering viruses from
river water solids. Appl. Environ.
Microbiol. 39:850-853. .
Berman, D., G. Berg, and R. S.
Safferman. 1 981. A method for
recovering viruses from sludges. J.
Virol. Methods 3:283-291.
Brashear, D. A., and R. L. Ward. 1982.
A comparison of methods for
recovering indigenous viruses from
raw wastewater sludge. Appl.
Environ. Microbiol. 43:1413-1418.
Dahling, D. R., G. Berg, and D.
Berman. 1974. BGM, a continuous
cell line more sensitive than primary
rhesus and African green kidney
cells for the recovery of viruses from
water. Health Lab. Sci. 11:275-282.
Dahling, D. R., and B. A. Wright. 1986.
Optimization of the BGM cell line
culture and viral assay procedures
for monitoring viruses in the
8-3
April 1986
environment. Appl. Environ.
Microbiol. 51:790-812.
Farrah, S. R., P. R. Scheuerman, and
G. Bitton. 1981. Urea-lysine method
for recovery of enteroviruses from
sludge. Appl. Environ. Microbiol.
41:455-458.
Goddard, M. R., J. Bates, and M.
Butler. 1981. Recovery of
indigenous enteroviruses from raw
and digested sewage sludges. Appl.
Environ. Microbiol. 42:1023-1028.
Hurst, C. J., and T. Goyke. 1983.
Reduction of interfering cytotoxicity
associated with wastewater sludge
concentrates assayed for indigenous
enteric viruses. Appl. Environ.
Microbiol. 46:133-139.
Katzenelson, E., B. Fattal, and T.
Hostovesky. 1976. Organic
flocculation: an efficient second-step
concentration method for the
detection of viruses in tap water.
Appl. Environ. Microbiol. 32:638-
639.
Lund, E., and C.-E. Hedstrom. 1966.
The use of an aqueous polymer
phase system for enterovirus
isolations from sewage. Am. J.
Epidemiol. 84:287-291.
Nielsen, A. L., and B. Lydholm. 1 980.
Methods for the isolation of virus
from raw and digested wastewater
sludge. Water Res. 14:175-178.
Sattar, S. A., and J. C. N. Westwood.
1976. Comparison of four eluents in
the recovery of indigenous viruses
from raw sludge. Can. J. Microbiol.
22:1586-1589.
Sattar, S. A., and J. C. N. Westwood.
1 979. Recovery of viruses from field
samples of raw, digested, and
lagoon-dried sludges. Bull. World
Health Org. 57:105-108.
Turk, C. A., B. E. Moore, B. P. Sagik,
and C. A. Sorber. 1980. Recovery of
indigenous viruses from wastewater
sludges, using a bentonite
concentration procedure. Appl.
Environ. Microbiol. 40:423-425.
Ward, R. L, and C. S. Ashley. 1 976.
Inactivation of poliovirus in digested
sludge. Appl. Environ. Microbiol.
31:921-930.
U.S. GPO: 1986-6I46-116M0605
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