United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-84/013(R T1)
March 1988
Revision
Research and Development
»EPA
USEPA Manual of
Methods for Virology
Chapter 11
Revised March 1988
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March 1988
Chapter 11
Virus Plaque Confirmation Procedure
1. Introduction
This chapter describes a procedure
that may be used for confirming virus
plaques in cell cultures adhering to
glass or plastic surfaces or in cells
suspended in overlay agar. Although
the procedures and reagents are for
use with the Buffalo green monkey
kidney (BGM) cell line, they may also
be used for cells other than BGM.
Where large numbers of plaques are
observed and confirmation of each
plaque is not practical, select no less
than ten plaques per sample. Select
only plaques that are well separated.
The virus plaque confirmation
procedure may also be used to obtain
sufficient virus quantities from plaque
isolates with which to identify the iso-
lated viruses in Chapter 12.
Use aseptic techniques and sterile
materials and apparatus only. Sterilize
all contaminated materials before dis-
carding them (see Chapters 2 and 3).
2. Recovery of Virus from
Plaque
2.1 Apparatus, Materials and
Reagents
2.1.1 Pasteur pipettes, disposable,
cotton plugged—229 mm (9 inches)
tube length.
Flame pipette gently about 2 cm
from end of tip until tip bends to
approximate angle of 45°. Place
pipettes into a 4-liter beaker, cover
beaker with aluminum foil, and dry
heat sterilize for not less than 1 hour
at170°C.
2.1.2 Rubber bulb—1 mL capacity.
2.1.3 Cell culture in tubes.
See Chapter 9 (January, 1987 revi-
sion). Section 7.2 for preparation of
cell culture tubes.
2.1.4 Tissue culture roller appara-
tus—1 /5 rpm speed (product no. TC-1,
New Brunswick Scientific, or
equivalent).
2.1.5 Culture tube drum for use with
roller apparatus (product no. ATC-
TT16, New Brunswick Scientific, or
equivalent).
2.1.6 ELAH—Earle's base with 0.5%
lactalbumin hydrolysate and without
NaHCO3(Hazleton Kansas City Biologi-
cal, product no. DM-303, or equiva-
lent) supplemented with antibiotics
(dihydrostreptomycin sulfate, penicillin
G, tetracycline and amphotericin B;
Sigma Chemical Co., or equivalent).
ELAH—Earle's base solution supple-
mented with antibiotics and serum
(from Section 2.1.8) is employed as a
maintenance medium in the cell cul-
ture tubes used for virus plaque con-
firmation testing (see Step (a) in Sec-
tion 2.2.2).
ELAH—Earle's base solution supple-
mented with antibiotics, but without
serum is used as a storage medium, if
plaque sample material must be stored
before confirmation procedure is com-
pleted (See Step (c) in Section 2.2.2).
Whenever possible, plaque sample
material should be inoculated onto a
cell culture immediately, because stor-
age of such sample material even at
-70°C may result in some reduction in
confirmation counts.
Only small volumes of the antibiotic
supplemented ELAH—Earle's base
solution are required. Employ stock
antibiotic and ELAH—Earle's base
solutions prepared for use in Chapter
JO {December, 1987 revision). If
unavailable, see Chapter 10, Section
2.1.3 lor procedure for preparation of
stock antibiotic solutions and Section
2.1.4 for preparation of ELAH—Earle's
base solution and for supplementation
of ELAH—Earle's base solution with
antibiotics. Remaining reagents may
be stored for subsequent use. Store
antibiotic stock solutions at -2O°C and
ELAH—Earle's base solution at 4°C for
periods no greater than 4 and 2
months, respectively. Reagents should
be held in tightly stoppered or capped
containers.
2.1.7 Vial, screw-capped (with
rubber insert)—3.7 mL(1 dram)
capacity.
Place 1 mL of antibiotic supple-
mented ELAH—Earle's base solution
(storage medium) from Section 2.1.6
in the screw-capped vial.
2.1.8 Fetal calf serum, filter-
sterilized, heat inactivated at 56°C for
30 min, certified free of viruses, bacte-
riophage and mycoplasma (GIBCO
Laboratories, or equivalent).
2.2 Procedure
2.2.1 Procedure for obtaining vi-
ruses from plaque.
Decision to test plaque mater/a/ for
viruses immediately or to store mate-
rial at -70°C for later testing must be
made before proceeding further.
(a) Place rubber bulb onto upper end of
pasteur pipette.
(b) Remove screw-cap or stopper from
plaque bottle (if plaque is in petri
dish, raise cover from dish suffi-
ciently to allow entry into dish).
(c) Squeeze rubber bulb on upper end
of pasteur pipette to expel air.
(d) Penetrate agar directly over edge of
plaque with tip of pasteur pipette.
(e) Gently force tip of pipette through
agar to surface of vessel, and
scrape cells from edge of plaque.
If cells are present as a mono-
layer on the surface of the vessel,
surface must be repeatedly
scratched and gentle suction ap-
plied to insure that virus-cell-agar
plug enters pipette. If cells are
suspended in the agar, scraping of
vessel surface with pipette is
unnecessary.
(f) Aspirate plug from plaque into
pipette.
(g) Remove pipette from plaque bottle
(or petri dish).
(h) Replace and tighten down screw-
cap or stopper on plaque bottle (if
plaque is in petri dish, replace
cover on dish).
If sample is to be tested in cell
culture immediately, proceed to
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March 1988
Section 2.2.2, Step (b.3J. If sample
must be stored, proceed to Sec-
tion 2.2.2. Step (c.2).
2.2.2 Procedure for inoculating vir-
uses obtained from plaques onto cell
cultures.
(a) Cell culture processing.
If at all feasible, use a laminar
flow hood while processing cell
cultures. Otherwise, use an area
restricted solely to cell culture
manipulations. Viruses or other
microorganisms must not be
transported, handled, or stored in
cell culture transfer facilities.
(a.1) Add fetal calf serum to the
antibiotic supplemented
ELAH—Earle's base solution
(see Section 2.1.6) for a final
concentration of 5% on day
samples will be tested.
(a.2) Pour spent medium from cell
culture tubes and discard the
medium.
To prevent splatter, a
gauze-covered beaker may
be used to collect spent
medium.
(a.3) Replace discarded medium
with a 2-mL volume of the
5% serum-antibiotic supple- .
mented ELAH—Earle's base
solution (maintenance
medium) from Step (a.1}.
To reduce shock to cells,
warm the 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 the maintenance
medium to the side of cell
culture test tube opposite
the cell monolayer.
(b) Procedure for samples tested
immediately.
(b.1) Prepare cell culture tube in
accordance with instructions
given in Steps (a.1) through
(a.3).
(b.2) Remove cap from cell culture
tube.
(b.3) Place tip of pasteur pipette
containing virus-cell-agar
plug from Section 2.2.1,
Step (h) into the mainte-
nance medium in cell culture
tube.
Tilt cell culture tube as
necessary to facilitate proce-
dure and avoid scratching
cell sheet with pipette.
(b.4) Force Virus-cell-agar plug
from pasteur pipette into the
maintenance medium by
gently squeezing rubber
bulb.
Squeeze bulb repeatedly to
wash contents of pipette into
the maintenance medium.
(b.5) Withdraw pipette from cell
culture tube, replace and
tighten down screw-cap on
tube, and discard pipette.
(b.6) Place cell culture tube in
drum used with tissue cul-
ture ro|ler apparatus.
(b.7) Place in drum three addi-
tional culture tubes which
have not been inoculated
with agar sample.
These tubes will serve as
negative controls.
(b.8) Incubate cell culture at
36.5° ± 1 °C, while rotating
tube at a speed of 1 /5 rpm.
(b.9) Examine cells daily micro-
scopically for 1 week, start-
ing with day 3, for evidence
of cytopathic effects (CPE).
CPE may be cell disinte-
gration or changes in cell
morphology. Rounding-up of
infected cells is a typical
effect seen with enterovirus
infections.
Incubation of BGM cells in
roller apparatus for periods
greater than 1 week is not
recommended as cells under
these conditions tend to die-
off if held longer.
Recovered virus (enterovi-
ruses) preparations may be
stored'In a -70° C freezer.
Procedures are given in
Chapter 12 for identification
of confirmed viruses. If iden-
tification of confirmed vir-
uses is to be directly under-
taken, see Chaper 12 for
sample requirement. Store
remaining sample portion at
-70°C until no longer
needed.
(c) Procedure for samples to be stored
at -70°C before testing.
(c.1) Remove cap from vial con-
taining storage medium.
(c.2) Place tip of pasteur pipette
containing virus-cell-agar
plug from Section 2.2.1, Step
(h) into vial.
(c.3) Force virus-cell-agar plug
from pasteur pipette into
storage medium by gently
squeezing rubber bulb.
Squeeze bulb repeatedly to
wash contents of pipette into
storage medium.
(c.4) Withdraw pipette from vial,
replace and tighten down
screw-cap onto vial, and dis-
card pipette.
(c.5) Store vial at -70°C.
(c.6) Place at -70°C three addi-
tional vials containing stor-
age medium that have not
' been inoculated with agar
sample.
These vials will be Used to
prepare the negative
controls.
When confirmation is to be
completed proceed to Step
(c.7).
(c.7) Prepare cell culture tube in
accordance with instructions
given in Steps (a.1) through
(a.3).
(c.8) Thaw sample quickly in
warm water (30-37°C).
(c.9) Remove cap from cell culture
tube.
(c.10) Remove cap from storage
vial containing thawed
sample.
(c.11) With a 1 -ml pipette,
transfer entire contents of
vial containing sample into
cell culture tube.
Tilt cell culture tube as
necessary to facilitate
procedure and avoid
scratching cell sheet with
pipette.
Place tip of pipette into
the maintenance medium
in cell culture tube.
Squeeze bulb repeatedly
to wash into the mainte-
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March 1988
nance medium any of the
remaining test sample.
(c.12) Withdraw pipette from cell
culture tube, replace and
tighten down screw-cap on
tube, discard pipette and
sample vial.
(c.13) Place cell culture tube in
drum used with tissue cul-
ture roller apparatus.
(c.14) With a 1-mL pipette,
transfer entire contents of
vials serving as negative
controls into cell culture
tubes.
Transfer contents of vial
in accordance with instruc-
tions given in Step (c. 11).
(c.15) Place the three cell culture
tubes in drum used with
tissue culture roller
apparatus.
(c.16) Incubate cell culture at
36.5° ± 1 °C, while rotating
tube at a speed of 1 /5 rpm.
(c.17) Examine cells daily micro-
scopically for 1 week, start-
ing with day 3, for evidence
of cytopathic effects (CPE).
CPE may be cell disinte-
gration or changes in cell
morphology. Rounding-up
of infected cells is a typical
effect seen with enter ovi-
rus infections.
Incubation of BGM cells
in roller apparatus for peri-
ods greater than 1 week is
not recommended as cells
under these conditions tend
to die-off if held longer.
Recovered virus (entero-
viruses) preparations may
be stored in a -7O°C
freezer.
Procedures are given in
Chapter 12 for identifica-
tion of confirmed viruses. If
identification of confirmed
viruses is to be directly
undertaken, see Chapter 12
for sample requirement.
Store remaining sample
portion at -70°C until no
longer needed.
3. Bibliography
Dahling, D. R. and B. A.;Wright. 1986.
Optimization of the BGM Cell Line
Culture and Viral Assay Procedures
for Monitoring Viruses in the Envi-
ronment. Appl. Environ. Microbiol.
57:790-812.
Dahling, D. R., G. Sullivan, and R. S.
Safferman. Factors Affecting the
False Positive Phenomenon of Virus
Plaque Picking Procedures. In
preparation.
Kedmi, S. and B. Fattal. 1981. Evalua-
tion of the False-Positive Enteroviral
Plaque Phenomenon Occurring in
Sewage Samples. Water Research
75:73-74.
11-3
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