United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-84/013{R 10)
December 1987
Revision
Research and Development
&EPA
USEPA Manual of
Methods for Virology
Chapter 10
Revised December 1987
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December 1987
Chapter 10
Cell Culture Procedures for Assaying Plaque-Forming Viruses
1. Introduction
This chapter outlines procedures to
detect waterborne viruses by use of
the plaque assay system. Use of the
method has been described by Dul-
becco (1952), Dulbecco and Vogt
(1954), Hsiung and Melnick (1955)
and Dahling and Wright (1986). The
principle of the system is based simply
on allowing virus particles to adsorb to
the host cells, adding agar medium to
localize virus growth and a vital stain
such as neutral red to enhance visual-
ization of the cells. Localized lesions
(plaques) developing some days after
viral infection are then counted. These
counts are reported as plaque forming
units, whose number is proportionate
to the amount of virus inoculated. A
detailed method, verifying virus etiol-
ogy of a plaque fay "picking" followed
by passage in cell culture monolayer
with liquid medium overlay and then
observing the monolayer for develop-
ment of a cytopathic effect, is pre-
sented in the next chapter.
Chapter 10 is solely intended as
guidance for the investigator who is
preparing to isolate and enumerate
waterborne viruses utilizing either the
cell monolayer or the suspended cell
plaque assay technique. The proce-
dures outlined in this chapter are for
use with the Buffalo green monkey
(BGM) kidney cell line. The method
may also be applied to the Madin and
Darby bovine kidney (MDBK) cell line,
which has been effectively used for
the plaque assay of reoviruses, or
when deemed preferable to other cell
lines that may be used in the plaque
assay of these animal viruses. How-
ever, it should be noted that water-
borne viruses do not all form plaques.
Chapter 9, Section 7 provides the
procedures for preparation of cell cul-
tures used for virus assay in this chap-
ter. Cells should be planted three to
six days prior to their use in these
analyses.
The detection methodology pre-
sented here has focused on labora-
tories with a small-scale virus assay
requirement. Where the quantities of
cell cultures, media and reagents set
forth in this chapter are not sufficient
for processing the test sample concen-
trates, the prescribed measures may
be increased proportionately to meet
the demands of more expansive test
regimes.
2. Cell Monolayer Procedure
2.1 Sample Inoculation of Cell
Monolayer for Virus Assay
2.1.1 Apparatus and materials.
(a) Glassware, Pyrex glass, clear
(Corning Glass Works, or
equivalent).
Storage vessel must be
equipped with airtight closures.
(b) Cornwall syringe, or equivalent
with cannula or syringe needle.
A pipetting device will expedite
virus assay when large volumes of
cells, media or reagents must be
handled.
(c) Magnetic stirrer and stir bars.
(d) Waterbath set at 36.5 ± 1 °C.
(e) Sterilizing filter—0.22-/um pore
size with a 47-mm diameter to
sterilize stock antibiotic solutions
from Section 2.1.3 (Millipore
Corp., GS series, or equivalent).
The 47-mm sterilizing filter may
also be used for the verification of
medium and reagent sterility in
Section 2.4.
(f) Sterilizing filter—0.22-jt/m pore
size with a 142-mm diameter to
sterilize ELAH—Earle's base solu-
tion from Section 2.1.4 (Millipore
Corp., GS series, or equivalent).
Where the volumes of media
and reagents prepared have been
for large-scale viral analyses, the
use of a 293-mm diameter steril-
izing filter may be more
appropriate.
(g) Fiberglass prefilters for use with
sterilizing filters (Millipore Corp.,
>AP1 5 and AP20, or equivalent).
Stack AP20 andAP15 prefilters
and 0.22-um membrane filter into
disc filter holder with AP20 prefli-
ter on top and0.22-/jm membrane
filter on bottom.
(h) Membrane filter apparatus for ster-
ilization—47-mm diameter filter
holder and 50-mL slip tip syringe
(Millipore Corp., Swinnex filter,
product no. SX0047000, or equiv-
alent for filter holder only).
Disassemble Swinnex filter
holder. Place membrane filter and
prefilters in holder as instructed in
Section 2.1.1, Step (g).
(i) Disc filter holders—142-mm or
293-mm diameter (Millipore
Corp., or equivalent).
Use only pressure type filter
holders. Place membrane filter
and prefilters in holder as
instructed in Section 2.1.1, Step
(9).
(j) Autoclavable inner-braided tubing
with metal quick-disconnect con-
nectors or with thumbscrew-drive
clamps for connecting tubing to
equipment to be used under
pressure.
Quick-disconnect connectors
can be used only after equipment
has been properly adapted.
(k) Positively-charged cartridge fil-
ter—10-inch (Zeta plus TSM,
product no. 45134-01-600P, AMF
Cuno Division, or equivalent).
(I) Holder for cartridge filter with
adaptor for 10-inch cartridge (type
PL-1, product no. YY1601200,
Millipore Corp., or equivalent).
(m) Culture capsule filter (product no.
12140, Gelman Sciences Inc., or
equivalent).
(n) Positive pressure air or nitrogen
source equipped with pressure
gauge.
Pressure source, if laboratory
air line or pump, must be
equipped with oil filter. Deliver to
pressure vessel and filter holder
no more pressure than recom-
mended by manufacturer.
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December 1987
(o) Dispensing pressure vessel—5- or
20-liter capacity (Millipore Corp.,
or equivalent).
(p) Petri dish—50-mm diameter (Fal-
con Labware Division, or
equivalent).
(q) Incubator capable of maintaining
temperature of cell cultures at
36.5° ± 1 °C.
2.1.2 Media and reagents.
(a) ELAH—Earle's base, with 0.5% lac-
talbumin hydrolysate and without
NaHCOa (Hazleton/Kansas City
Biological, product no. DM-303, or
equivalent).
See Section 2.1.4, Step (h) for
options in sterilization of the
ELAH—Earle's base solution.
(b) Sodium bicarbonate (NaHCO3).
(c) Antibiotics—penicillin G, dihydro-
streptomycin sulfate, tetracycline
and amphotericin B (Sigma Chem-
ical Co., or equivalent).
Use antibiotics of at least tissue
culture grade. See Section 2.1.3
for preparation of stock antibiotic
solutions.
(d) Ascorbic acid.
(e) Water, deionized, distilled.
See Chapter 4.
({) Nutrient agar(Difco Laboratories,
or equivalent).
2.1.3 Procedure for preparation of
stock antibiotic solutions.
If not purchased in sterile form.
stock antibiotic solutions must be
filter-sterilized by the use of0.22-um
membrane fitters.
Antibiotic stock solutions should be
placed in screw-capped containers
and stored at -20°C until needed.
Once thawed they may be refrozen;
however, to avoid repeated freezing
and thawing of these stock solutions
distribute them In quantities that are
sufficient to support no more than a
week's virus assay work. Stock solu-
tions can be stored for up to 4 months.
Quantities prepared in steps (a), (b)
and(c) are sufficient for at least 100
liters of media.
(a) Preparation of penicillin-
streptomycin stock solution.
The procedure described is for
preparation often 10-mL volume
penicillin-streptomycin stock solu-
tions at concentrations of
1,000,000 units of penicillin and
1,OOO mg of streptomycin per 10-
mL unit. The antibiotic concentra-
tions listed in step fa. 1) may not
correspond to the concentrations
obtained from other lots or from a
different source.
(a.1) Add appropriate amounts
(within 5%) of penicilllin G
and dihydrostreptomycin sul-
fate to a 250-mL flask con-
taining 100 mL of deionized
distilled water.
For penicillin supplied at
1435 units per mg, add 7 g
of the antibiotic.
For streptomycin supplied
at 740 mg per g, add 14 g of
the antibiotic.
(a.2) Mix contents of flask on
magnetic stirrer until antibi-
otics are dissolved.
(a.3) Sterilize antibiotics by filtra-
tion through 0.22-/um mem-
brane filter.
(a.4) Dispense the penicilllin-
streptomycin stock in. 10-mL
volumes into screw-capped
containers.
(b) Preparation of tetracycline stock
solution.
The procedure described is for
preparation of ten 5-mL tetracy-
cline stock solutions at concentra-
tions of 0.125 g per 5 mL unit.
(b.1) Add 1.25 g of tetracycline
hydrochloride powder and
3.75 g of ascorbic acid to a
125-mL flask containing 50
mL of deionized distilled
water.
(b.2) Mix contents of flask on
magnetic stirrer until antibi-
otic is dissolved.
(b.3) Sterilize antibiotic by filtra-
tion through 0.22-//m
membrane filter.
(b.4) Dispense the tetracycline
stock in 5-mL volumes into
screw-capped containers.
(c) Preparation of amphotericin B (fun-
gizone) stock solution.
The procedure described is for
preparation often2.5-mL fungi-
zone stock solutions at concentra-
tions of 0.0125 g per 2.5 mL unit.
(c.1) Add 0.125 g of fungizone to
a 50-mL flask containing 25
mL of deionized distilled
water.
(c.2) Mix contents of flask on
magnetic stirrer until antibi-
otic is dissolved.
(c.3) Sterilize antibiotic by filtra-
tion through 0.22-um mem-
brane filter.
(c.4) Dispense the fungizone stock
in 2.5-mL volumes into
screw-capped containers.
2.1.4 Preparation of maintenance
medium.
(a) Determine the volume of ELAH—
Earle's base solution required.
The volume of ELAH—Earle's
base solution needed will be equal
to the volume of growth medium
used in propagation of cell cul-
tures for the plaque assay proce-
dure. Thus, see Table 9-2 in
Chapters to determine the
volume required.
The procedure described is for
preparation of 1 liter of ELAH—
Earle's base solution in a IX for-
mulation and will provide suffi-
cient media for at least sixty 6-oz
glass bottles or ninety 25-cm2
plastic flasks.
(b) Place a three-inch stir bar into a
two-liter flask.
(c) Add contents of a 1 -liter packet of
ELAH—Earle's base" reagent irtto
flask.
(d) Add 890 mL of warmed deionized
distilled water (50-60°C) to rea-
gent in flask.
(e) Rinse medium packet with three
washes of 20 mL each of warmed
deionized distilled water and add
to flask.
Note measure of deionized dis-
tilled water is 5% less than
desired total volume of reagent.
(f) Mix on magnetic stirrer until rea-
gent is completely dissolved.
(g) Add 2.25 g of NaHCO3 to the dis-
solved reagent and continue mix-
ing for an additional 15 min.
10-2
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December 1 987
(h) Filter reagent under pressure
through a disc filter stack.
•This sterilizing step requires the
use of preiliters in line before the
final sterilizing filter. Prepare filter
stack according to instructions in
Section 2.1.1. Step (g).
As an alternative, use the car-
tridge prefilter described in Sec-
tion 2.1.1, Step (k) and the cap-
sule sterilizing filter in Section
2.1.1. Step(m).
(i) Sterility testing.
Test each lot of reagent to con-
firm sterility (see Section 2.4).
(j) Store sterilized reagent in tightly
stoppered or capped container at
4°C.
Reagent may be stored for per-
iods up to two months.
(k) Add 1 ml of penicillin-
streptomycin stock, 0.5 mL of
tetracycline stock and 0.2 mL of
fungizone stock (Section 2.1.3) to
ELAH—Earle's base solution
immediately before washing of
cells.
2.1.5 Procedure for inoculating test
sample.
Cell cultures used for virus assay
are generally found to be at their most
sensitive level between the third and
sixth days after initiation. Those older
than seven days should not be used.
(a) Decant growth medium from pre-
viously prepared cell culture test
vessels.
To prevent splatter, a gauze-
", covered beaker may be used to
collect spent medium.
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.
(b) Discard growth medium.
(c) Replace discarded medium with an
equal volume of maintenance
medium on day cultures are to be
inoculated.
For BGM and MDBK cells use
antibiotic supplemented ELAH—
Earle's base solution from Section
2.1.4, Step (k). 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 the cell cul-
ture test vessel opposite the cell
monolayer.
(d) Identify cell culture test vessels by
coding them with an indelible
marker.
(e) Return cell culture test vessels to
36.5 ± 1°C incubator and hold
vessel at the temperature until the
cell monolayer is to be inoculated.
(f) Decant maintenance medium from
cell culture test vessels.
Do not disturb the cell
monolayer.
(g) Discard maintenance medium.
(h) Inoculate onto each cell monolayer
a volume of test sample concen-
trate appropriate for the cell sur-
face area of the cell culture test
vessels used.
Inoculum volume should be no
greater than 1 mL for each 40 cm2
of surface area. Use Table 10-1 as
a guide for inoculation size.
A void touching either the can-
nula or the pipetting device to the
inside rim of the cell culture test
vessels to avert the possibility of
transporting contaminants to the
remaining culture vessels.
(i) Rock inoculated cell culture test
vessels gently to achieve uniform
distribution of inoculum on sur-
face of cell monolayers.
(j) Place cell culture test vessels on a
level stationary surface at room
temperature (22-25°C) so that the
inoculum will remain distributed
evenly over the cell monolayer.
(k) Incubate inoculated cell cultures at
room temperature for 80 min to
permit viruses to adsorb onto and
infect cells.
(I) Proceed immediately to Section
2.2.
2.2 Agar Overlay Procedure for
Plaque Assay
If there is a likelihood that a test
sample will be toxic to cell cultures or
may be so darkly colored as to result in
inaccurate plaque counts, the cell
monolayer should be treated in ac-
cordance with the method described in
Chapter 8 (April, 1986 revision).
2.2.1 Apparatus and materials.
(a) Glassware, Pyrex glass, clear .
(Corning Glass Works, or
equivalent).
(b) Magnetic stirrer and stir bars.
(c) Autoclavable inner-braided tubing
with metal quick-disconnect con-
nectors,or with thumbscrew-drive
clamps for connecting tubing to
equipment to be used under
pressure.
Quick-disconnect connectors
can be used only after equipment
has been properly adapted.
(d) Positive pressure air or nitrogen
source equipped with pressure
gauge.
Pressure source, if laboratory
air-line or pump, must be
equipped with oil filter. Deliver to
pressure vessel and filter holder
no more pressure than recom-
mended by manufacturer.
(e) Dispensing pressure vessel—5- or
20-liter capacity (Millipore Corp.,
or equivalent).
(f) Waterbath set at 36° ± 1 °C.
Used for maintaining the
temperature of the overlay
medium.
See Section 2.2.5. Step (c).
Table 10-1. Guide for Virus Inoculation, Suspended Cell Concentration and Overlay
Volume of Agar Medium
Vessel Type
1 -oz* glass
bottle
25 -cm' plastic
flask
6-oz glass
bottle
75 -cm2 plastic
flask
Volume of Virus
Inoculum (mLJ
0.1
0. 1 -0.5
0.5-1.0
1.0-2.0
Volume of Agar
Overlay Medium (mL)
5
10
20
30
Total Numbers
of Cells
1 X 107
2X107
4X107
6X107
'Size is given in oz only when it is commercially designated in that unit.
10-3
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December 1987
(g) Waterbath set at 50° ± 1 °C.
Used for maintaining the agar
temperature.
See Section 2.2.6, Step (d).
(h) Incubator capable of maintaining
the temperatures of cell cultures
at 36.5° ± 1 °C.
(i) Sterilizing filter—0.22-um pore
size with a 142-mm diameter to
sterilize Medium 199 from Sec-
tion 2.2.3 and reagents in excess
of 1 liter volumes (Millipore Corp.,
GS series, or equivalent).
Where the volumes of media
prepared are for large-scale viral
analyses (20 liters or morel, the
use of a 293-mm diameter steril-
izing filter may be more appro-
priate. Use a 47-mm diameter fil-
ter (or sterilizing volumes of less
than 1 liter.
(j) Fiberglass prefilters for use with
sterilizing filters {Millipore Corp.,
AP15 and AP20, or equivalent).
Stack AP20 andAP15 prefilters
and 0.22-fjm membrane filter into
disc filter holder with AP20 prefli-
ter on top and 0.22-um membrane
filler on bottom.
{k) Disc filter holders—47-mm, 142-
mm or 293-mm diameter (Milli-
pore Corp., or equivalent).
Use only pressure type filter
holders. Place membrane filter
and prefilters in holder as
instructed in Section 2.2.1. Step
in.
(I) Positively-charged cartridge fil-
ter—10-inch (Zeta plus TSM,
product no. 45134-01-600P, AMF
Cuno Division, or equivalent).
(m) Holder for cartridge filter with
adaptor for 10-inch cartridge (type
PL-1), product no. YY1601200,
Millipore Corp., or equivalent).
(n) Culture capsule filter (product no.
12140, Gelman Sciences, Inc., or
equivalent).
(o) Petri dish—50-mm diameter (Fal-
con Labware Division, or
equivalent).
2.2.2 Media and reagents.
(a) Medium 199 prepared at a 2X con-
centration with Earle's salts,
0,05% lactalbumin and L-
glutamine and without phenol red
and NaHC03 (Grand Island Biolog-
ical Co., product no. 400-1100, or
equivalent).
(b) HEPES—1 M (Sigma Chemical Co.,
product no. H3375, or equivalent).
Prepare SO mL of a 1 M solution
of HEPES.
(c) GG-free newborn calf serum—
heat inactivated at 56°C for 30
min, certified free of viruses, bac-
teriophage and mycoplasma
(Grand Island Biological Co., prod-
uct no. 210-6400, or equivalent).
Procure at least one 100-mL
size bottle.
(d) Sodium bicarbonate (NaHCO3)—
7.5% solution.
Prepare 50 mL of a 7.5% solu-
tion of sodium bicarbonate. Steril-
ized by filtration through 0.22-um
filter.
(e) Magnesium chloride (MgCI2-
6H20)—1.0% solution.
Prepare 50 mL of a 1.0% solu-
tion of magnesium chloride.
(f) Sodium chloride (NaCI).
(g) Sodium phosphate dibasic
(Na2H PCv7H20).
(h) Sodium phosphate monobasic
(NaH2P04-H2O).
(i) Water, deionized, distilled.
See Chapter 4.
(j) Hydrochloric acid (HCI)—1 M.
Prepare 100 mL of a 1M solu-
tion of hydrochloric acid.
(k) Sodium hydroxide (NaOH)—1 M.
Prepare 100 mL of a 1M solu-
tion of sodium hydroxide.
(I) Pancreatin 1X powder (ICN Nutri-
tional Biochemicals, product no.
102557, or equivalent).
im) Neutral red solution—0.1%, 100
mL volume (Grand Island Biologi-
cal Co., product no. 630-5330, or
equivalent).
Procure one 100-mL bottle.
(n) Bacto skim milk (Difco Laborato-
ries, product no. 0032-01, or
equivalent).
Prepare 1OO mL of Bacto skim
milk in accordance with directions
given by manufacturer.
(o) GIBCO bacteriological agar (Grand
Island Biological Co., product no.
M00010B, or equivalent).
(p) Antibiotics—penicillin G, dihydros-
treptomycin sulfate, tetracycline
and amphotericin B (Sigma Chem-
ical Co., or equivalent).
Use stock antibiotic solutions
previously prepared in Section
2.1.3.
(q) Thioglycollate medium (Bacto de-
hydrated fluid thioglycollate
medium, Difco Laboratories, or
equivalent).
Prepare 100 mL of thioglycol-
late medium in accordance with
directions given by manufacturer.
(r) Nutrient agar (Difco Laboratories,
or equivalent).
Prepare 100 mL of nutrient agar
and pour 5 mL aliquots into petri
dishes for use in Section 2.4.2.
2.2.3 Preparation of Medium 199.
(a) Sources of cell culture media.
Commercially-prepared liquid
cell culture media and medium
components are available from
several sources. Cell culture
media can also be purchased in
powder form that requires only
dissolution in deionized distilled
water and sterilization.
Media from commerical sources
are quality controlled. However.
media can also be prepared in the
laboratory from chemicals. Such
preparations are labor intensive
and may be expensive but allow
quality control oLthe process by
the preparing laboratory.
The procedure described is for
preparation of 500 mL of Medium
199 at a 2X concentration. This
procedure will prepare sufficient
medium for at least fifty 6-oz glass
bottles or eighty 25-cm2 plastic
flasks.
(b) Place a three-inch stir bar into a
one-liter flask.
(c) Add contents of a 1 -liter packet of
Medium 199 into flask.
(d) Add 355 mL of deionized distilled
water to medium in flask.
(e) Rinse medium packet with three
washeS'Of 20 mL each of deion-
ized distilled water and add to
flask.
Note measure of deionized dis-
tilled water is 5% less than
desired final volume of medium.
10-4
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December 1987
(f) Mix on magnetic stirrer until
medium is completely dissolved.
(g) Filter reagent under pressure
through a disc filter stack.
This sterilizing step requires the
use of pref liters in line before the
final sterilizing filter. Prepare filter
stack according to instructions in
Section 2.2.1. Step (j).
As an alternative, use cartridge
pref liter described in Section
2.2.1, Step II) and the capsule
sterilizing filter in Section 2.2.1,
Step (n).
(h) Sterility testing.
Test each lot of medium to con-
firm sterility before the lot is used
for plaque assay procedure (see
Section 2.4).
(i) Store sterilized medium in tightly
stoppered or capped container at
4°C.
Medium may be stored for per-
iods of up to two months.
2.2.4 Preparation of pancreatin
solution for use in detecting reovirus.
(a) Add 0.03 g of NaH2 PCvH2O to a
250-mL flask.
(b) Add 100 mL of deionized distilled
water to the flask.
(c) Mix contents of flask on magnetic
stirrer until reagent is dissolved.
(d) Add 0.05 g of Na2H PCv7H2O to a
250-mL flask.
(e) Add 100 ml of deionized distilled
water to the flask.
(f) Mix contents of flask until reagent
is dissolved.
(g) Sterilize NaH2PO4 and Na2H PO4
stock solutions in accordance with
instructions given in Chapter 3,
Section 2.1.
(h) Cool to room temperature (22°-
25°C).
(i) Prepare phosphate buffer from the
stock solutions by mixing 16 mL of
NaH2 PO4 with 84 mL of Na2H POi
in a 250-mL flask.
(j) Place three-inch stir bar into a
2000-mL flask.
(k) Add 984 mL of deionized distilled
water to flask.
(I) Place flask on magnetic stirrer and
stir at a speed sufficient to
develop vortex.
(m) Add 16 mL of phosphate buffer
from Step (i) to the water.
Remaining phosphate buffer
solution may be stored at 4°C
until needed.
(n) Add 10 g of pancreatin from Sec-
tion 2.2.2, Step (I) and 8.5 g of
NaCI to the phosphate buffered
water.
(o) Continue mixing contents of flask
on magnetic stirrer until reagents
are dissolved.
(p) Check pH of pancreatin solution
and adjust to pH 7.5 if necessary.
(q) Filter solution under pressure
through a disc filter stack.
This sterilizing step requires the
use ofpref liters in line before the
final sterilizing filter. Prepare filter
stack according to instructions in
Section 2.2.1, Step (j).
As an alternative, use the car-
tridge pref liter described in Sec-
tion 2.2.1, Step (I) and the capsule
sterilizing filter in Section 2.2.1,
Step In).
(r) Sterility testing.
Test each lot to confirm sterility
before the lot is used for reovirus
recovery (see Section 2.4).
(s) Store sterilized pancreatin solution
in tightly stoppered or capped con-
tainer at-20°C.
Recommend solution be divided
into 50-mL aliquots. Quantities
prepared in Steps (a) through (s)
are sufficient for at least fifty 6-oz
glass bottles or eighty 25-cm2
plastic flasks.
Solution may be stored for peri-
ods of up to six months.
2.2.5 Preparation of overlay medium
for plaque assay.
The procedure described is for prep-
aration of 100 mL of over/ay medium
and will prepare sufficient media for at
least ten 6-oz glass bottles or twenty
25-oz plastic flasks when mixed with
the agar prepared in Section 2.2.6.
(a) Add 79 mL of Medium 199 (2X con-
centration from Section 2.2.3,
Step (i) and 4 mL of GG-free new-
born calf serum from Section
2.2.2, Step (c) to a 250-mL flask.
(b) Add the stock solutions listed in
Steps (b.1) through (b.7) to flask.
Start with Step (b. 1) and add in
the order listed, swirling to mix
after each addition.
{b.1) 6 mL-of NaHC03 from Sec-
tion 2.2.2, Step (d).
(b.2) 2 mL of MgCI2 from Section
2.2.2, Step (e).
(b.3) 3 mL of neutral solution
from Section 2.2.2, Step (m).
(b.4) 4 mL of 1 M HEPES from
Section 2.2.2, Step(b).
(b.5) 0.2 mL of penicillin-
streptomycin stock from Sec-
tion 2.1.3, Step (a).
(b.6) 0.1 mL of tetracycline stock
from Section 2.1.3, Step (b).
(b.7) 0.04 mL of fungizone stock
from Section 2.1.3, Step (c).
For preparation of overlay
medium for reovirus recov-
ery, the serum and the skim
milk from Section 2.2.7,
Step (a) are replaced with
2.8 mL of pancreatin solu-
tion and 3.2 mL of deionized
distilled water.
(c) Place flask with overlay medium in
waterbath set at 36° ± 1 °C.
2.2.6 Preparation of overlay agar for
plaque assay.
(a) Add 3 g of agar from Section 2.2.2,
Step (o) to a 250-mL flask.
(b) Add 100 mL of deionized distilled
water to flask.
(c) Sterilize agar in accordance with
instructions given in Chapter 3,
Section 2,1.
(d) Hold agar in waterbath set at 50° ±
2.2.7 Preparation of agar overlay
medium.
(a) Add 2 mL of skim milk from Section
2.2.2, Step (n) to overlay medium
prepared in Section 2.2.5.
(b) Mix equal portions of overlay
medium and agar by adding the
medium to the agar flask.
10-5
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December 1987
To prevent solidification of the
liquified agar, limit the portion of
agar overlay medium mixed to
that volume which can be
dispensed in 10 min.
2.2.8 Addition of overlay agar to cell
culture test vessels.
(a) To each cell culture test vessel, add
the volume of warm (42-46°C)
agar overlay medium appropriate
for the cell surface area of the
vessels used.
The volume of agar overlay
medium that is appropriate for a
particular virus assay culture ves-
sel is listed in Table 10-1.
To prevent disturbing cells with
the force of the liquid against the
cell monolayer, add agar overlay
medium to the side of the cell cul-
ture test vessel opposite the cell
monolayer.
(b) Place cell culture test vessel, mon-
olayer side down, on a level sta-
tionary surface at room tempera-
ture (22-25°C) so that the agar
will remain distributed evenly
before it solidifies.
Care must be taken to ensure
that all caps on bottles and flasks
are tight; otherwise, the gas seal
will not be complete and an
erroneous virus assay will result.
(c) Cover cell culture test vessels with
a sheet of aluminum foil, a tightly
woven cloth, or some other suita-
ble cover to reduce light intensity
and thus, prevent damage to the
cell monolayer.
Agar is fully solidified within 30
min,
(d) After 30 min invert cell culture test
vessels and incubate in the dark
at 36.5° ± 1 °C.
2.3 Counting Viral Plaques
2.3.1 Counting technique.
(a) Count, mark and record plaques in
cell culture test vessels on days
two, three, four, six, eight and
twelve after adding the agar over-
lay medium.
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 prefera-
ble. Virus titers are calculated
from total count.
(b) Examine cell culture test vessels on
day sixteen.
If no new plaques appear at 16
days, discard vessels; otherwise
continue to count, mark and
record plaques every two days
until no new plaques appear
between counts.
Note that most plaques will
appear within 1 week.
2.3.2 Calculation of virus liter.
(a) Calculate virus liter in plaque form-
ing units (PFU) for each virus-
conlaining sample concenlrale.
To determine the numbers of
PFU per mL in water, sewage
sludge, soil, or dredge spoil sam-
ple concentrate, multiply the
number of PFU by the reciprocal
of the inoculum volume.
If the inoculum volume was di-
luted, also multiply the number of
PFU by the reciprocal of the dilu-
tion made.
(b) 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 2.3.2. Step (a) by the con-
centration factor which is calcu-
lated by dividing the volume of the
original sample by the volume of
the sample concentrate. For soil.
digested dewatered sludge and
dredge spoil samples, correct for
water content and report in PFU
per gram of dry weight.
Dry weight is determined by
evaporating a given sample in a
weighed dish, drying in an oven at
104 ± 1°C to a constant weight
and then determining the increase
in weight over that of the empty
dish.
2.4 Procedure for Verifying Sterilily
of Liquids
There are many techniques availa-
ble for verifying the sterility of liquids
such as cell culture media and
medium components. Three tech-
niques, described below, are standard
in many laboratories. The capabilities
of these techniques, however, are
limited to detecting microorganisms
that grow unaided in the test medium
utilized. Viruses, mycoplasma, and
microorganisms that possess fastid-
ious growth requirements or that
require living host systems will not be
detected. Nonetheless, with the
exception of a few special contamina-
tion problems, the test procedures and
microbiological media listed below
should prove adequate. Do not add
antibiotics to media or medium com-
ponents until after sterility of the rea-
gents, media and medium components
has been demonstrated.
2.4.1 Procedure for verifying sterility
of small volumes of liquids.
(a) Inoculate 5 mL of the material to be
tested for sterility into 5 mL of thi-
oglycollate broth.
(b) Shake the mixture and incubate at
36.5° ±1°C,
(c) Examine the inoculated broth daily
for seven days to determine
whether growth of contaminating
organisms has occurred.
Vessels that contain thioglycol-
late medium must be tightly
sealed before and after medium is
inoculated. A clouded condition
that develops in the media indi-
cates the occurrence of contami-
nating organisms.
2.4.2 Procedure for verifying sterility
of large volumes of liquids.
(a) Filter 50-100 mL of the liquid '
tested for sterility through a 47-
mm diameter, 0.22-jUrri pore size
membrane filter.
(b) Remove filter from its holder, and
place filter on surface of solidified
nutrient agar in a Petri dish.
Place filter face up on agar.
(c) Incubate Petri dish at 36.5° ± 1 °C
and examine filter surface daily
for seven days to determine
whether growth of contaminating
organisms has occurred.
i
2.4.3 Visual evaluation of media for
microbial contaminants.
(a) Incubate cell culture media that
contain NaHC03 at 36.5° ± 1 °C
for at least one week prior to use.
(b) Visually examine the clarity of the
culture media.
A clouded condition that devel-
ops in the media indicates the
occurrence of contaminating
organisms.
(c) Discard any media that lose clarity.
10-6
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December T987
3. Suspended Cell
Procedure
The suspended cell technique for
enumeration of viruses from environ-
mental samples has the advantage of
detecting more viruses at a lower con-
centration than the cell monolayer
procedure. The suspended cell tech-
nique also requires less time because
plaque assay involves no prior planting
of the cells or medium changes.
Plaques are easier to pick because the
cells are suspended within the agar
layer and not on the surface of the
bottle or flask where they must be
scraped off, and the system is inde-
pendent of the surface area of the
plaque assay vessel. On the other
hand, the suspended cell technique
requires ten times more cells initially
than the cell monolayer procedure.
Suspended cells last only about one
week in the agar overlay as compared
to three weeks for cells used in the
monolayer technique. Moreover,
plaques in the suspended cell proce-
dure are not as well defined and thus
more difficult to count and because
the addition of pancreatin in the over-
lay would inhibit or retard cell growth
in this procedure, it cannot be used for
reovirus detection.
Where low numbers of indigenous
viruses are anticipated in a test sam-
ple (5 PFU or less per mL), the sus-
pended cell procedure should be used
by itself or together with the cell mon-
olayer procedure. For assaying sam-
ples with high viral concentrations,
the recommended method would be
the cell monolayer procedure.
If there is a likelihood that a test
sample will be toxic to cell cultures or
may be so darkly colored as to result in
inaccurate plaque counts, then only
the cell monolayer procedure should
be used and only after the cell mono-
layer is treated in accordance with the
method described in Chapter 8 (April,
1986 revision).
3.1 Sample Inoculation of Sus-
pended Cells for Virus Assay
3.1.1 Apparatus and materials.
(a) Glassware, Pyrex glass, clear
(Corning Glass Works, or
equivalent).
Storage vessel must be
equipped with airtight closures.
(b) Culture tubes, disposable, borosili-
cate glass—18 x 150 mm (Curtin
Matheson Scientific, product no.
339-333, or equivalent).
(c) Culture tube caps, slip-on-type—
18 mm diameter (VWR Scientific,
product no. 60879-162; or
equivalent).
(d) Test tube rack for culture tubes.
(e) Cornwall syringe, or equivalent
with cannula or syringe needle.
A pipetting device will expedite
virus assay when large volumes of
cells, media or reagents must be
handled.
(f) Magnetic stirrer and stir bars.
(g) Shaker, rocker platform style (Bel-
Ico Glass, Inc., product no. 7740-
20020, or equivalent).
(h) Incubator capable of maintaining
temperature of cells at 36.5° ±
3.1.2 Procedure for inoculating test
sample.
(a) Arrange culture tubes in series.
Number of culture tubes
required will be equal to the mL of
sample to be tested.
(b) Place stir bar in flask.
(c) Add cell suspension from Chapter
9, Section 6.1.8 to flask.
(d) Dilute cell suspension to the
appropriate cell concentration,
while mixing on magnetic stirrer.
See Table 10-1 for cell concen-
tration parameters. Dilute cell
suspension with growth medium
prepared in Chapter 9, Section
3.3.3.
Base cell dilution on results of
cell counts performed in Chapter
9, Section 6.2
(e) Add 1 mL of diluted cell suspension
to each tube.
A pipetting device will expedite
processing when large cell
volumes must be dispensed.
(f) Add into each tube a 1 -mL volume
of test sample concentrate.
(g) Swirl each tube gently to achieve a
uniform mixture.
(h) Place inoculated tubes in rack.
(i) Place test tube rack on,rocker plat-
form shaker housed in a 36° ±
1 °C incubator.
(j) Rock tubes at a rate of 16 oscilla-
tions per min for 60 min to permit
viruses to adsorb onto and infect
cells.
Complete preparation for agar
overlay procedure during 60 min '
incubation period.
(k) Before cell culture test vessels are
inoculated, identify vessels by
coding them with an indelible
marker.
3.2 Agar Overlay Procedure for
Plaque Assay
3.2.1 Apparatus and materials.
(a) Glassware, Pyrex glass, clear
(Corning Glass Works, or
equivalent).
(b) Magnetic stirrer and stir bars.
(c) Autoclavable inner-braided tubing
with metal quick-disconnect con-
nectors or with thumbscrew-drive
clamps for connecting tubing to
equipment to be used under
pressure.
Quick-disconnect disconnectors
can be used only after equipment
has been properly adapted.
(d) Positive pressure air or nitrogen
source equipped with pressure
gauge.
Pressure source, if laboratory
air line or pump,-must be
equipped with oil filter. Deliver to
pressure vessel and filter holder
no more pressure than recom-
mended by manufacturer.
(e) Dispensing pressure vessel—5- or
20-liter capacity (Millipore Corp.,
or equivalent).
(f) Waterbath set at 36° ±1°C.
Used for maintaining the
temperature of the overlay
medium.
See Section 3.2.5. Step (c).
(g) Wat.erbath set at 50° ±1°C.
Used for maintaining agar
temperature. '.:;'-
See Section 3.2.6, Step (d).
(h) Incubator capable of maintaining
the temperatures of cell cultures
at36.5°±1°C.
(i) Sterilizing filter—0.22-pm pore
size with a 47-mm diameter to
sterilize stock antibiotic solutions
from Section 3.2.3 (Millipore
10-7
-------
December 1987
Corp,, GS series, or equivalent).
The 47-mm sterilizing filter may
also be used for the verification of
medium and reagent sterility in
Section 3.4.
(j) Sterilizing filter—0.22-pm pore
size with a 142-mm diameter to
sterilize Medium 199 from Sec-
tion 3.2.4 (Millipore Corp., GS ser-
ies, or equivalent).
Where the volumes of media
prepared are for large-scale viral
analyses, the use of a 293-mm
diameter sterilizing filter may be
mora appropriate.
(k) Fiberglass prefilters for use with
sterilizing filters (Millipore Corp.,
APIS and AP20, or equivalent).
Stack AP20 and AP15 prefilters
and 0.22-nm membrane filter into
disc filter holder with AP20 prefil-
ter on top and0.22-um membrane
filter on bottom.
(I) Membrane filter apparatus for ste-
rilization—47-mm diameter filter
holder and 50-mL slip tip syringe
(Millipore Corp., Swinnex filter,
product no. SX0047000, or equiv-
alent for filter holder only).
Disassemble Swinnex filter
holder. Place membrane filter and
prefilters in holder as instructed in
Section 3.2.1. Step (k).
(m) Disc filter holders—142-mm or
293-mm diameter (Millipore
Corp., or equivalent).
Use only pressure type filter
holders. Place membrane filter
and prefilters in holder as
instructed in Section 3.2.1, Step
M.
(n) Positively-charged cartridge fil-
ter—1 0-inch (Zeta plus TSM,
product no. 45134-01-600P, AMF
Cuno Division, or equivalent).
(o) Holder for cartridge filter with
adaptor for 10-inch cartridge (type
PL-1. product no. YY1601200,
Millipore Corp., or equivalent).
(p) Culture capsule filter (product no.
12140, Gelman Sciences, Inc., or
equivalent).
(q) Petri dish—50-rnm diameter (Fal-
con Labware Division, or
equivalent)
3.2.2 Media and reagents.
(a) Medium 199 prepared at a 2X con-
centration with Garle's salts.
0.05% lactalbumin and L-
glutamine and without phenol red
and NaHCOa (Grand Island Biolog-
ical Co., product no. 400-1 1 00, or
equivalent).
(b) HEPES — 1 M (Sigma Chemical Co.,
product no. H-3375, or
equivalent).
Prepare 50 mL of a 1 M solution
of HEPES.
(c) GG-free newborn calf serum —
heat inactivated at 56°C for 30
min, certified free of viruses, bac-
teriophage and mycoplasma
(Grand Island Biological Co., prod-
uct no. 210-6400, or equivalent).
Procure at least one 100-mL
size bottle.
(d) Sodium bicarbonate (NaHCOs) —
7.5% solution.
Prepare 5O mL of a 7.5% solu-
tion of sodium bicarbonate. Steril-
ized by filtration through 0.22 -/urn
filter.
(e) Magnesium chloride (
6HZO)—1 .0% solution.
Prepare 50 mL of a 1.0% solu-
tion of magnesium chloride.
(f) Water, deionized distilled.
See Chapter 4.
(g) Hydrochloric acid (HCI)—1 M.
Prepare 100 mL of a 1M solu-
tion of hydrochloric acid.
(h) Sodium hydroxide (NaOH)—I'M."
Prepare JOO mL of a 1M solu-
tion of sodium hydroxide.
(i) Neutral red solution—0.1% (Grand
Island Biological Co., product no.
630-5330, or equivalent).
Procure one 100-mL bottle.
Sterilize by filtration through
0.22-/jm filter.
(j) Bacto skim milk (Difco Laborato-
ries, product no. 0032-01, or
equivalent).
Prepare 100 mL of Bacto skim
milk in accordance with directions
given by manufacturer.
(k) GIBCO bacteriological agar (Grand
Island Biological Co., product no.
MOOO10B, or equivalent).
(I) Antibiotics—penicillin G, dihydro-
streptomycin sulfate, tetracycline
and amphotericin B (Sigma Chem-
ical Co., or equivalent).
Use antibiotics of at least tissue
culture grade. See Section 3.2.3
for preparation of stock antibiotic
solutions.
(m) Ascorbic acid.
(n) Thioglycollate medium (Bacto de-
hydrated fluid thioglycollate
medium, Difco Laboratories, or
equivalent).
Prepare 100 mL of thioglycol-
late medium in accordance with
directions given by manufacturer.
(o) Nutrient agar (Difco Laboratories,
or equivalent).
Prepare 100 mL of nutrient agar
and pour 5 mL aliquots into petri
dishes for use in Section 3.4.2.
3.2.3 Procedure for preparaton of
stock antibiotic solutions.
If not purchased in sterile form,
stock antibiotic solutions must be
filter-sterilized by the use of0.22-fjm
membrane filters.
Antibiotic stock solutions should be
placed in screw-capped containers
and stored at -20°C until needed.
Once thawed they may be refrozen;
however, to avoid repeated freezing
and thawing of these stock solutions
distribute them in quantities that are
sufficient to support no more than a
week's virus assay work. Stock solu-
tions can be stored for up to 4 months.
Quantities prepared in Steps (a), (b)
and(c) are sufficient for at least 100
liters of media.
(a) Preparation of penicillin-
streptomycin stock solution.
The procedure described is 'for
preparation of ten 10-mL volume
penicillin-streptomycin stock solu-
tions at concentrations of
1,000,000 units of penicillin and
1,000 mg of streptomycin per 10-
mL unit. The antibiotic concentra-
tions listed in Step (a. 1) may not
correspond to the concentrations
obtained from other lots or from a
different source.
(a.1) Add appropriate amounts
(within 5%) of penicillin G
and dihydrostreptomycin sul-
fate to a 250-mL flask con-
taining 100 mL of deionized
distilled water.
For penicillin supplied at
1435 units per mg, add 7 g
of the antibiotic.
For streptomycin supplied
at 740 mg per g, add 14 g of
the antibiotic.
10-8
-------
December 1987
(a.2) Mix contents of flask on
magnetic stirrer until antibi-
otics are dissolved.
(a.3) Sterilize antibiotics by filtra-
tion through 0.22-//m
membrane filter.
(a.4) Dispense the penicillin-
streptomycin stock in 10-mL
volumes into screw-capped
containers.
(b) Preparation of tetracycline stock
solution.
The procedure described is for
preparation of ten 5-mL tetracy-
cline stock solutions at concentra-
tions of 0.125 g per 5 mL unit.
(b.1) Add 1.25 g of tetracycline
hydrochloride powder and
3.75 g of ascorbic acid to a
125-mL flask containing 50
mL of deionized distilled
water.
(b.2) Mix contents of flask on
magnetic stirrer until antibi-
otic is dissolved.
(b.3) Sterilize antibiotic by filtra-
tion through 0.22-//m mem-
brane filter.
(b.4) Dispense the tetracycline
stock in 5-mL volumes into
screw-capped containers.
(c) Preparation of amphotericin Bffun-
gizone) stock solution.
The procedure described is for
preparation of ten 2.5-mL fungi-
zone stock solutions at concentra-
tions of O.0125 g per 2.5 mL unit.
(c.1) Add 0.125 g of fungizone to
a 50-mL flask containing 25
mL of deionized distilled
water.
(c.2) Mix contents of flask on
magnetic stirrer until antibi-
otic is dissolved.
(c.3) Sterilize antibiotic by filtra-
tion through 0.22-^m mem-
brane filter.
(c.4) Dispense the fungizone stock
in 2.5-mL volumes into
screw-capped containers.
3.2.4 Preparation of Medium 199.
(a) Sources of cell culture media.
Commercially-prepared liquid
cell culture media and medium
components are available from
several sources. Cell culture
media can also be purchased in
powder form that requires only
dissolution in deionized distilled
water and sterilization.
Media from commercial sources
are quality controlled. However,
media can also be prepared in the
laboratory from chemicals. Such
preparations are labor intensive
and may be expensive but allow
quality control of the process at
the level of the preparing labora-
tory. The procedure described is
for preparation of 500 mL of
Medium 199 at a 2X concentra-
tion. This procedure will prepare
sufficient medium for at least fifty
6-oz glass bottles or eighty 25-
cm2 plastic flasks.
(b) Place a three-inch stir bar into a
one-liter flask.
(c) Add contents of a 1 -liter packet of
Medium 199 into flask.
(d) Add 355 mL of deionized distilled
water to medium in flask.
Note measure of deionized dis-
tilled water is 5% less than
desired total volume of medium.
(e) Rinse medium packet with three
washes of 20 mL each of deion-
ized distilled water and add to
flask.
(f) Mix on magnetic stirrer until
medium is completely dissolved.
(g) Filter reagent under pressure
through a disc filter stack.
This sterilizing step requires the
use of prefliters in line before the
final sterilizing filter. Prepare filter
stack according to instructions in
Section 3.2.1, Step (k).
As an alternative, use the car-
tridge pref liter described in Sec-
tion 3.2.1, Step (n) and the cap-
sule sterilizing filter in Section
3.2.1, Step Ip).
(h) Sterility testing.
Test each lot of medium to con-
firm sterility before the lot is used
for plaque assay procedure (see •
Section 3.4).
(i) Store sterilized medium in tightly
stoppered or capped container at.
4°C.
Medium may be stored for peri-
ods of up to two months.
3.2.5 Preparation of overlay medium
for plaque assay.
The procedure described is for prep-
aration of 100 mL of overlay medium
and will provide sufficient medium for
at least ten 6-oz glass bottles or
twenty 25-cm2 plastic flasks when
mixed with the agar prepared in Sec-
tion 3.2.6.
(a) Add 79 mL of Medium 199 (2X con-
centration) from Section 3.2.4,
Step (i) and 4 mL of GG-free new-
born calf serum from Section
3.2.2, Step {c} to a 250-mL flask.
(b) Add the stock solutions listed in
Steps (b.1) through (b.7) to flask.
Start with Step (b. 1) and add in
the order listed, swirling to mix
after each addition.
(b.1) 6 mL of NaHCOs from Sec-
tion 3.2.2, Step (d).
(b.2) 2 mL of.MgCI;, from Section
3.2.2, Step (e).
(b.3) 3 mL of neutral red solution
from Section 3.2.2, Step (i).
(b.4) 4 mL of 1 M HEPES from
'Section 3.2.2., Step(b).
(b.5) 0.2 mL of penicillin-
streptomycin stock from Sec-
tion 3.2.3, Step (a).
(b.6) 0.1 mL of tetracycline stock
from Section 3.2.3, Step (b).
(b.7) 0.04 mL of fungizone stock
from Section 3:2.3, Step (c).
(c) Place flask with overlay medium in
waterbath set at 36° ± 1 °C,
3.2.6 Preparation of overlay agar for
plaque assay.
(a) Add 3 g of agar from Section 3.2.2,
Step (k) to a 250-mL flask.
(b) Add 100 mL of deionized distilled
water to flask.
(c) Sterilize agar in accordance with
instructions given in Chapter 3,
Section 2.1.
(d) Hold agar in waterbath set at 50° ±
3.2.7 Preparation of agar overlay
medium.
10-9
-------
December 1987
(a) Add 2 mL of skim milk from Section
3,2.2, Step (j) to overlay medium
prepared in Section 3.2.5.
(b) Mix equal portions of overlay
medium and agar by adding the
medium to the agar flask.
To prevent solidification of the
liquified agar. limit the portion of
agar overlay medium mixed to
that volume which can be
dispensed in 10 min.
3.2.8 Procedure.
(a) Following incubation of test tubes
in Section 3,1.2, Step (j), add to
one tube at a time the volume of
warm (42-44°C) agar overlay
medium appropriate for the cell
surface area of the cell culture
test vessels to be used.
The volume of agar overlay
medium that is appropriate for a
particular virus assay culture ves-
sel is listed in Table 10-1.
(b) Immediately after adding agar to
tube, pour contents into cell cul-
ture test vessel.
(c) Tightly cap vessel.
(d) Swirl to mix contents of vessel.
(e) Place cell culture test vessel on a
level stationary surface at room
temperature (22-25°C) so that the
contents will distribute evenly
before it solidifies.
Care must be taken to ensure
that all caps on bottles and flasks
are tight; otherwise, the gas seal
will not be complete and an
erroneous virus assay will result.
(f) Cover cell culture test vessels with
a sheet of aluminum foil, a tightly
woven cloth, or some other suita-
ble cover to reduce light intensity
and thus, prevent damage to the
cell monolayer.
Agar is fully solidified within 30
min.
(g) Invert cell culture test vessels and
incubate in the dark at 36.5° ±
3.3 Counting Viral Plaques
3.3.1 Technique.
(a) Mark and record plaque numbers at
each counting period.
Virus titers are calculated from
total count.
(b) Count on days two, three, four, five
and seven after adding the agar
overlay medium.
Cells that are suspended in the
agar over/ay degenerate more
rapidly that those from'an over-
lay ed monolayer culture. Thus,
scheduling plaque counts for
longer than seven days is not
recommended.
Depending on the virus density
and virus types present in the
inoculate sample, a rescheduling
of viral counts at plus or minus
one day may be deemed prefera-
ble within the seven-day counting
period.
3.3.2 Virus enumeration.
(a) Calculate virus titer in plaque form-
ing units (PFU) for each virus-
containing sample concentrate.
To determine the numbers of
PFU per mL in water, sewage
sludge, soil, or dredge spoil sam-
ple concentrate, multiply the
number of PFU by the reciprocal
of the inoculum volume.
If the inoculum volume was di-
luted, also multiply the number of
PFU by the reciprocal of the dilu-
tion made.
(b) 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 2.3.2, Step fa) by the con-
centration factor which is calcu-
lated 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.
3.4 Procedure for Verifying Sterility
of Liquids
There are many techniques availa-
ble for verifying the sterility of liquids
such as cell culture media and'
medium components. Three tech-
niques, described below, are standard
in many laboratories. The capabilities
of these techniques, however, are
limited to detecting microorganisms
that grow unaided on the test medium
utilized. Viruses, mycoplasma, and
microorganisms that possess fastid-
ious growth requirements or that
require living host systems will not be
detected. Nonetheless, with the
exception of a few special contamina-
tion problems, the test procedures and
microbiological media listed below
should prove adequate. Do not add
antibiotics to media or medium com-
ponents until after sterility of the rea-
gents, media and medium components
has been demonstrated.
3.4.1 Procedure for verifying sterility
of small volumes of liquids.
(a) Inoculate 5 mL of the material to be
tested for sterility into 5 mL of thi-
oglycollate broth.
(b) Shake the mixture and incubate at
36.5°±1°C.
(c) Examine the inoculated broth daily
for seven days to determine
whether growth of contaminating
organisms has occurred.
Vessels that contain thioglycol-
late medium must be tightly
sealed before and after medium is
inoculated. A clouded condition
that develops in the media indi-
cates the occurrence of contami-
nating organisms.
3.4.2 Procedure for verifying sterility
of large volumes of liquids.
(a) Filter 50-100 mL of the liquid
tested for sterility through a 47-
mm diameter, 0:22-/urn pore size
membrane filter.
(b) Remove filter from its holder, arid
place filter on ssurface of solidified
nutrient agar in a Petri dish.
Place filter face up on agar.
(c) Incubate Petri dish at 36.5° ± 1 °C
and examine filter surface daily
for seven days to determine
whether growth of contaminating
organisms has occurred.
3.4.3 Visual evaluation of media for
microbial contaminants.
(a) Incubate cell culture media that
contain NaHCO3 at 36.5° ± 1 °C
for at least one week prior to use.
(b) Visually examine the clarity of the
culture media.
A clouded condition that devel-
ops in the media indicates the
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occurrence of contaminating
organisms.
(c) Discard any media that lose clarity.
4. Bibliography
Cooper, P. D. 1967. The Plaque Assay
of Animal Viruses, p. 243-311./n K.
Maramorosch and H. Koprowski
(eds.). Methods in Virology, Vol. 3.
Academic Press, New York, NY.
Dahling, D. R., G. Berg, and D. Ber-
man. 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.
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Dahling, D. R. and B. A. Wright. 1986.
Optimization of the BGM Cell Line
Culture and Viral Assay Procedures
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ronment. Appl. Environ. Microbiol.
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Dahling, D. R. and B. A. Wright.
Optimization of Suspended Cell
Method and Comparison with Cell
Monolayer Technique for Virus
Assays. In Preparation.
Dulbecco, R. 1952. Production of
Plaques in Monolayer Tissue Cul-
tures by Single Particles of an
Animal Virus. Proc. Natl. Acad. Sci
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Dulbecco, R. and M. Vogt. 1954.
Plaque Formation and Isolation of
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ruses. J. Exp. Med. 99:167-182.
Hsiung, G. D. and J. L. Melnick. 1955.
Plaque Formation with Poliomyelitis,
Coxsackie and Orphan (Echo) Vi-
ruses in Bottle Cultures of Monkey
Epithelial Cells. Virology. 1:533-535.
Lennette, E. H. and N. J. Schmidt
(eds.). 1979. Diagnostic Procedures
for Viral, Rickettsial and Chlamydial
Infections. 5th ed. American Public
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Laboratory Manual in Virology. 1974.
Edition Two. Ontario Ministry of
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375 pp. 5
Rovozzo, G. C. andC. N. Burke. 1973.
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