>&EPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-84/013(R9)
January 1987
Revision
Research and Development
USEPA Manual of
Methods for Virology
Chapter 9
Revised January 1987
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January 1987
Chapter 9
Cell Culture Preparation and Maintenance
1. Introduction
This chapter outlines procedures
and media for culturing Buffalo green
monkey (BGM) kidney cells. BGM cells
are a continuous line derived from
African Green monkey kidney cells.
The characteristics of this line were
described by A. L. Barron, C.
Olshevsky, and M. M. Cohen in 1970.
Use of BGM cells for recovering vir-
uses from environmental samples was
described by D. R. Dahling, G. Berg,
and D. Berman in 1974. The media
and methods recommended in Chap-
ters 9 and 10 are the results of the
BGM cell line optimization studies by
D. R. Dahling and B. A. Wright, pub-
lished in 1986. The BGM cells can be
obtained by qualified laboratories from
the Virology Section, Environmental
Monitoring and Support Laboratory,
U.S. Environmental Protection Agency,
Cincinnati, Ohio 45268.
Chapter 9 is intended for the individ-
ual who is experienced in cell culture
preparation. Although the procedures
and media outlined in this chapter are
for use with the BGM cell line, the
method may be applied to the Madin
and Darby bovine kidney (MDBK) cell
line. The MDBK cell line has been
effectively used for the plaque assay of
reoviruses. The cytopathic effect of
reoviruses is slow to appear; thus,
human enteroviruses present in a
sample may interfere with their detec-
tion. Enterovirus interference is
avoided however with use of the
MDBK cells, since they do not support
the growth of these viruses. Cells of
the MDBK line may be obtained from
the American Type Culture Collection
{ATCC product no. CCL 22). This
method for cell culture preparation
and maintenance may also be used as
is or with minor modification for cells
other than BGM and MDBK. BGM
cells are highly susceptible to many
enteric viruses (Dahling et al., 1984;
Dahling and Wright, 1986); however,
these cells are not sensitive for detect-
ing all enteroviruses or certain other
viruses that may occur in environmen-
tal samples. Thus, to maximize the
number of viruses recovered from
environmental samples, several cell
lines may need to be used. Moreover,
viruses such as reovirus require
modification to the agar overlay
procedure. These modifications are
presented in Chapter 10.
2. Medium Preparation
2.1 Apparatus and Materials
2.1.1 Glassware, Pyrex glass, clear
(Corning Glass Works, or equivalent).
Storage vessels must be equipped
with airtight closures.
2.1.2 Magnetic stirrer and stir bars.
2.1.3 Autoclavable inner-braided
tubing with metal quick-disconnect
connectors or with thumb-screw-
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.
2.1.4 Positive pressure air or nitro-
gen 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 recommended by manufacturer.
2.1.5 Dispensing pressure vessel—
20-liter capacity (Millipore Corp., or
equivalent).
2.1.6 Disc filter holders—142-mm
or 293-mm diameter (Millipore Corp.,
or equivalent).
Use only pressure type filter holders.
2.1.7 Sterilizing filters—0.22-//m
pore size (Millipore Corp., GS series,
or equivalent).
2.1.8 Fiberglass prefilters (Millipore
Corp., AP15 and AP20, or equivalent).
Stack AP20 and AP15 prefilters and
O.22-fjm membrane filter into disc fil-
ter holder with AP20 prefliter on top
and 0.22-fjim membrane filter on bot-
tom.
2.1.9 Positively-charged cartridge
filter—10-inch (Zeta plus TSM, pro-
duct no. 45134-01-600P, AMF Cuno
Division, or equivalent).
2.1.10 Holder for cartridge filter
with adaptor for 10-inch cartridge
(type PL-1, product no. YY1601200,
Millipore Corp., or equivalent).
2.1.11 Culture capsule filter (pro-
duct no. 12140, Gelman Sciences Inc.,
or equivalent).
2.1.12 Cell culture vessels, Pyrex
borosilicate glass (Corning Glass
Works, or equivalent), soda or flint
glass prescription (Rx) bottles (Brock-
way, Inc., or equivalent), plastic flasks
(Falcon Tissue Culture Lab ware, Bee-
ton, Dickinson and Co., or equivalent),
disposable glass roller bottles (Bellco
Glass, Inc., or equivalent), or disposa-
ble plastic roller bottles (Corning Glass
Works, or equivalent).
Vessels (tubes, flasks, bottles) for
growth of cell cultures must be clear
glass or plastic to allow observation of
the cultures. Plastic vessels must be
treated by the manufacturer to allow
cells to adhere properly. Vessels for
cell cultures must be equipped with
airtight closures.
2.1.13 Screw caps, black with
rubber liners (thread finish no. 24-414
for 6-oz* prescription (Rx) bottles,
Brockway, Inc., or equivalent).
Caps for larger culture bottles usu-
ally supplied with bottles.
2.1.14 Roller apparatus (7730-
Series, Bellco Glass, Inc., or equival-
ent).
2.1.15 pH meter measuring to an
accuracy of at least 0.1 pH unit.
2.1.16 Incubator capable of main-
taining the temperature of cell cul-
tures at 36.5° ± 1°C.
2.1.17 Waterbath, equipped with
circulating device to assure even heat-
ing at 36.5° ± 1°C.
*Size is given in oz only when it is commercially
designated in that unit.
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January 1987
2,1.18 Light microscope, with con-
ventional light source, equipped with
lenses to provide 40X, 100X, and
400X total magnification.
2.1.19 Inverted light microscope
equipped with lenses to provide 40X,
100X, and 400X total magnification.
2.1.20 Pipettor syringes, sizes 2 mL,
5 mL and 10 mL (Cornwall-type, Bee-
ton, Dickinson and Co., or equivalent).
2.1.21 Pipetting machine (Brewer-
type, Curtin Matheson Scientific, or
equivalent).
2.1.22 Phase counting chamber
(hemocytometer) slide (product no.
168-501, Curtin Matheson Scientific,
or equivalent).
2.1.23 Conical centrifuge tubes,
sizes 50 mL and 250 mL.
2.1.24 Rack for tissue culture tubes
(product no. 2028, Bellco Glass, Inc.,
or equivalent).
2.1.25 Bottles, aspirator-type with
tubing outlet, size 2,000 mL.
Bottles for use with pipetting
machine.
2.1.26 Storage vials, size 2 mL.
Vials must withstand temperatures
to -70°C.
2,2 Media and Reagents
2.2.7 Fetal calf serum and GG-free
calf serum, filter-sterilized, heat-
inactivated at 56°C for 30 min, certi-
fied free of viruses, bacteriophage
and mycoplasma (GIBCO Laborato-
ries, or equivalent).
Test toxiclty of sample of serum on
cells before purchasing serum lot.
2.2.2 Trypsin, 1:250 powder (Difco
Laboratories, or equivalent) or trypsin,
1:300 powder (BBL, Becton, Dickin-
son and Co., or equivalent).
2.2.3 Sodium (tetra) ethylenediam-
ine tetraacetate powder (EDTA), tech-
nical grade (Fisher Scientific Co., or
equivalent).
2.2,4 Thioglycollate medium (Bacto
dehydrated fluid thioglycollate
medium, Difco Laboratories, or equi-
valent).
2.2.5 Water, distilled, deionized.
See Chapter 4.
2.2.6 Fungizone (amphotericin B, E.
R. Squibb and Sorjs, or equivalent),
Penicillin G and d|hydrostreptomycin
sulfate (Eli Lilly and Co., or equival-
ent), tetracycline (Pfizer, Inc., or
equivalent).
Use antibiotics of at least tissue
culture grade.
2.2.7 Eagle's minimum essential
medium (MEM) with Hanks' salts and
L-glutamine, without sodium bicarbo-
nate (product no. 410-1200, GIBCO
Laboratories, or equivalent).
2.2.8 Leibovitz's L-15 medium with
L-glutamine (product no. 430-1300,
GIBCO Laboratories, or equivalent).
2.2.9 Trypan bide (Sigma Chemical
Co., or equivalent).
Note: This chemical is on the EPA
list of proven or suspected carcino-
gens, i
2.2.10 Sodium bicarbonate
(NaHC03).
2.2.11
1 M.
Hydrochloric acid (HCI)—
2.2.12 Sodium hydroxide (NaOH)—
1 M.
2.2.13 Sodium chloride>(NaCI).
2.2.14 Dextrose.
2.2.15 Sodium phosphate dibasic
(Na2HP04-7H20).
2.2.16 Potassium chloride (KCI).
2.2.17 Potassium phosphate mono-
basic (KH2P04).
2.2.18 Ascorbic acid.
2.2.19 Dimethyl sulfoxide (DMSO).
3. Preparation of Cell Cul-
ture Media
3.1 Technique
3.7.7 Equipment care.
Carefully wash and sterilize equip-
ment used for preparing media before
each use. \
3.1.2 Disinfection of work area.
Thoroughly disihfect surfaces on
which medium preparation equipment
is to be placed.
3.1.3 Aseptic technique.
Use aseptic technique when prepar-
ing and handling media or medium
components.
3.1.4 Dispensing filter-sterilized
media.
To avoid post-filtration contamina-
tion, dispense filter-sterilized media
into storage containers through clear
glass filling bells in a microbiological
laminar flow hood. If hood is unavaila-
ble, use an area restricted solely to
cell culture manipulations.
3.2 General Procedures
3.2.7 Coding media.
Assign a lot number to each batch of
media or medium components
prepared.
3.2.2 Sterility test.
Test each tot of medium and
medium components to confirm steril-
ity before the lot is used for cell cul-
ture (see Section 4).
3.2.3 Storage of media and medium
components.
Store media and medium compo-
nents in clear airtight containers at
4°C or -20°C as appropriate.
3.2.4 Sterilization of NaHCO3-
containing solutions.
Sterilize media and other solutions
that contain NaHCOa by positive pres-
sure filtration.
Negative pressure filtration of such
solutions increases the pH and redu-
ces the buffering capacity.
3.3 Medium Preparation
3.3.7 Sources of cell culture media.
Commercially-prepared liquid cell
culture media and medium compo-
nents are available from several sour-
ces. 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 laboratory.
3.3.2 Procedure for preparation of
EDTA-trypsin.
The procedure described is for the
preparation of 10 liters of EDTA-
trypsin reagent. It is used to dislodge
cells attached to the surface of culture
bottles and flasks. This reagent, when
stored at 4°C, retains its working
9-2
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January 1987
strength for at least four months. The
amount of reagent prepared should be
based on projected usage over a four-
month period.
(a) Place three-inch stir bar into six-
liter flask.
(b) Add 30 g of trypsin (1:250) and two
liters of deionized distilled water to
flask.
(c) Place flask on magnetic stirrer and
mix trypsin rapidly for a minimum
of one hour.
Trypsin remains cloudy.
(d) Add four liters of deionized distilled
water to 20-liter clear plastic
carboy.
(e) Place three-inch stir bar into
carboy.
(f) Place carboy onto magnetic stirrer
and stir at a speed sufficient to
develop vortex while adding the
chemicals listed in steps (g)
through (I).
Each chemical does not have to
be completely dissolved before
adding the next one.
(g) NaCI— 80 g.
(h) EDTA—12.5g.
(i) Dextrose—50 g.
(j) Na2HPCv7H2O—11.5g.
(k) KCI—2.0 g.
(I) KHaPCu— 2.0 g.
(m) Add four more liters of deionized
distilled water to carboy.
Continue mixing after all chemi-
cals are completely dissolved.
(n) Add the two liters of trypsin from
step (c) to the prepared solution in
step (m) and mix for a minimum of
one hour.
(o) Adjust pH of the trypsin reagent to
7.5-7.7.
(p) 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 2.1.8. As an alternative,
use the described cartridge pref li-
ter in Section 2.1.9 and the cap-
sule sterilizing filter in Section
2.1.11.
(q) Test sterility of EDTA-trypsin rea-
gent in accordance with directions
given in Section 4.
(r) Store reagent in tightly stoppered
or capped containers at 4°C.
3.3.3 Procedure for preparation of
growth medium.
The procedure described is for prep-
aration of 10 liters of MEM/L-15
growth medium. The medium will be
supplemented with 10% fetal calf
serum and antibiotics (10 mL of
penicillin-streptomycin stock, 5 mL of
tetracycline stock and 2 mL of fungi-
zone stock per 10 liters of growth
medium) prior to addition of the BGM
cells.
(a) Place three-inch stir bar into 20-
liter carboy.
(b) Add four liters of deionized distilled
water to carboy.
(c) Place carboy on magnetic stirrer
and stir at a speed sufficient to
develop vortex.
(d) Add contents of a five-liter packet
of L-15 medium to carboy.
(e) Rinse medium packet with three
washes of 200 mL each of deio-
nized distilled water and add to
carboy.
(f) Mix until medium is evenly
dispersed.
L-15 medium may appear cloudy
as it need not be totally dissolved
before proceeding to step (g).
(g) Add three liters of deionized dis-
tilled water to carboy.
(h) Continue mixing at a speed suffi-
cient to develop vortex.
(i) Add contents of a five-liter packet
of MEM medium to carboy.
(j) Rinse medium packet with three
washes of 200 mL each of deio-
nized distilled water and add to car-
boy.
(k) Add 800 mL of deionized distilled
water and 7.5 g of NaHCOa to
carboy.
(I) Mix MEM/L-15 for an additional 60
min.
(m) Add medium to pressure can.
(n) Filter under positive pressure
through 0.22-um sterilizing filter
and collect in volumes appropriate
for the culturing of BGM cells.
(o) Collect sample of medium for ste-
rility testing in accordance with
directions given in Section 4.
(p) Store medium in tightly stoppered
or capped containers at 4°C.
Medium may be stored for peri-
ods of up to two months.
3.3.4 Procedure for preparation of
trypan blue solution.
The procedure described is for the
preparation of 100 mL of trypan blue
solution. It is used in the direct deter-
mination of the viable cell counts of
the BGM stock cultures. As trypan
blue is on the EPA suspect carcinogen
list, particular care should be taken in
its preparation and use so as to avoid
skin contact or inhalation. The wear-
ing of rubber gloves during prepara-
tion and use is recommended.
(a) Add 0.5 g of trypan blue to a 250-
mL flask.
(b) Add 100 mL of deionized distilled
water to the flask.
(c) Swirl flask until the trypan blue is
completely dissolved.
(d) Sterilize solution in accordance
with instructions given in Chapter
3, Section 2.1.
(e) Store in a screw-capped container
at room temperature.
3.3.5 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-fjm
membrane filters. It is important that
the recommended antibiotic levels not
be exceeded when planting cells as
the cultures are particularly sensitive
to excessive concentrations at this
stage.
Antibiotic stock solutions should be
placed in screw-capped containers
and stored at -20°C until needed.
Once thawed they may be re frozen;
however, to avoid repeated freezing
and thawing of these stock solutions
distribute them in quantities that are
sufficient to support a week's cell cul-
ture work.
(a) Preparation of penicillin-
streptomycin stock solution.
7776 procedure described is for
preparation often 1O-mL volume
penicillin-streptomycin stock solu-
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January 1987
t/ons at concentrations of
1.000,000 units of penicillin and
1,000,000 fjg of streptomycin per
10-mL unit. The antibiotic concen-
trations 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 of
penicillin G and dihydrostrepto-
mycin sulfate to a 250-mL flask
containing 100 mL of deionized
distilled water.
For penicillin supplied at 1435
units per mg, add 7 g of the antibi-
otic.
For streptomycin supplied at 740
mg per g, add 14 g of the
antibiotic.
(a.2) Mix contents of flask on mag-
netic stirrer until antibiotics are
dissolved.
(a.3) Sterilize antibiotics by filtra-
tion through 0.22-fjm membrane
filter.
(a.4) Dispense in 10-mL volumes
into screw-capped containers.
(b) Preparation of tetracycline stock
solution.
{b.1J 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 dis-
tilled water.
(b.2) Mix contents of flask on mag-
netic stirrer until antibiotic is dis-
solved.
(b.3) Sterilize antibiotic by filtration
through 0.22-um membrane filter.
(b.4) Dispense in 5-mL volumes
into screw-capped containers.
(c) Preparation of amphotericin B (fun-
gizone) stock solution.
(c.1) Add 0.125 g of amphotericin
B to a 50.-mL flask containing 25
mL of deionized distilled water.
(c.2) Mix contents of flask on mag-
netic stirrer until antibiotic is dis-
solved.
(c.3) Sterilize antibiotic by filtration
through 0.22-pm membrane filter.
(c.4) Dispense 2.5-mL volumes
into screw-capped containers.
4. Procedurejfor 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, rr/ycoplasma, and
microorganisms that possess fasti-
dious growth requirements or that
require living host Isystems 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 afteri sterility of the anti-
biotics, media andlmedium compo-
nents has been demonstrated.
4.1 Procedure for Verifying Sterility
of Small Volumes of Liquids
4.7.7 Inoculate 5 mL of the material
to be tested for sterility into 5 mL of
the thioglycollate broth.
4.1.2 Shake the mixture and incu-
bate at 36.5°±1°C.
4.1.3 Examine th|e inoculated broth
daily for seven days to determine
whether growth of contaminating
organisms has occurred.
Vessels that contain thioglycollate
medium must be tightly sealed before
and after medium is inoculated.
4.2 Procedure for Verifying Sterility
of Large Volumes of Liquids
4.2.7 Filter 50-1 (DO mL of the liquid
tested for sterility through a 47-mm
diameter, 0.22-//mipore size mem-
brane filter.
4.2.2 Remove filter from its holder,
and place filter on Surface of solidified
nutrient agar in a Petri dish.
Place filter face up on agar.
4.2.3 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.
4.3 Visual Evaluation of Media for
Microbial Contaminants
4.3.7 Incubate cell culture media
that contain NaHC03 at 36.5° ± 1 °C
for at least one wejek prior to use.
4.3.2 Visually examine the clarity of
the culture media.
A clouded condition that develops in
the media indicates the occurrence of
contaminating organisms.
4.3.3 Discard any media that lose
clarity.
5. Procedures for Prepara-
tion of Stock BGM Cell
Cultures
The BGM cell line grows readily on
the inside surfaces of plastic or glass
flat-sided vessels or round vessels. To
reduce the risk of contamination, cell
cultures should be prepared in con-
trolled facilities used for no other pur-
pose.
5.1 General Procedures
5.7.7 Pass and maintain BGM stock
cultures in 16-oz to 32-oz (or equival-
ent growth area) flat-sided, glass bot-
tles, in 150-cm2 plastic cell culture
flasks, or in 690-cm2 glass or 850-cm2
plastic roller bottles.
If available, roller bottles and roller
apparatus units are preferable to flat-
sided bottles or flasks for growing cells
because roller cultures require less
medium than flat-sided bottles per
unit of cell monolayer surface. For
growing cells in roller bottles, adjust
roller apparatus rotation speed to one-
half revolution per minute.
As a general rule, the BGM cell line
can be split at a 1:3 ratio. However, a •
more suitable inoculum is obtained if
low passages of the line (passages
100-150) are split at a 1:2 ratio and
higher passages (generally above pas-
sage 250) are split at a 1:4 ratio. To
plant 200 25-cm2 cell culture flasks
weekly from a low-level passage of the
line would require the preparation of
six roller bottles (surface area 690 cm2
each): two to prepare six roller bottles
and four to prepare the 25-cm2 flasks.
5.1.2 Except during handling opera-
tions, maintain BGM cells at 36.5° ±
1 °C in airtight cell culture vessels.
(a) Maintain rotation of roller bottles
so that cells are constantly bathed
in the growth medium.
(b) Maintain flat-sided cell culture bot-
tles or flasks that contain cells in a
stationary position with the flat
side (cell monolayer side) down.
5.7.3 To reduce shock to cells, warm
growth media and maintenance media
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January 1987
to 36.5° ± 1 °C before placing them on
cell monolayers.
5.1.4 Change medium on stock
BGM cell cultures on day three or
four. ',
Use growth medium described in
Section 3.3.3. Supplement the
medium with antibiotics and either 2%
or 5% fetal calf serum. Use the lower
percentage (maintenance medium) if
the cell monolayer is at least 95% con-
fluent. Add a sufficient amount of dei-
onized distilled water to the change
medium so as to compensate for the
volume difference between the 10%
fetal calf serum to be used initially in
the medium and the 2% or 5% serum
concentration which is to be used in
the change medium.
5.1.5 Before discarding, autoclave
all media that have been in contact
with cells or that contain serum.
Table 9-1. Guide for Preparation of BGM Stock Cultures
Vessel Type
16-oz** glass
flat bottles
32-oz glass
flat bottles
ISO-cm2 plastic
flat flask
690-cm2 glass
roller bottle
850-cmz plastic
roller bottle
Volume of EDTA-
Trypsin Used to
Remove Cells (ml.)
10
20
25
40
50
Volume of
Medium (mL)*
25
50
60
100
120
Final
Cell Count
per Bottle
2.5X1 0s
5.0 X 10s
6.0 X 10e
7.0 X 107
8.0 X JO7
*Serum requirements: growth medium contains 10% fetal calf serum; maintenance
medium contains 2-5% fetal calf serum.
Antibiotic requirements: penicillin-streptomycin stock solution, 1.0mL/liter; tetracycline
stock solution. 0.5 mL/liter; fungi zone stock solution, 0.2 mL/liter.
**Size is given in oz only when it is commercially designated in that unit.
6. Procedure for Passage of
BGM Cells
Pass stock BGM cell cultures at
approximately seven-day intervals.
6.1 General Procedure
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.
6.1.1 Add serum and antibiotics to
the stored growth medium (see Sec-
tion 3.3.3} on day BGM cells will be
subcultured.
6.1.2 Pour spent medium from cell
culture vessels, and discard the
medium.
To prevent splatter, a gauze-covered
beaker may be used to collect spent
medium.
6.1.3 Add to the cell cultures a
volume of warm EDTA-trypsin reagent
equal to 40% of the volume of medium
replaced. :
See Table 9-1.
To reduce shock to cells, warm
EDTA-trypsin reagent to 36.5° ±1°C
before placing it on cell monolayers.
Dispense the EDTA-trypsin reagent
directly onto the cell monolayer.
6.1.4 Allow EDTA-trypsin reagent to
remain in contact with the cells at
either room temperature or incubation
temperature of 36.5° ± 1°C until cell
monolayer can be shaken loose from
inner surface of cell culture vessel
(about five min).
If necessary, a sterile rubber police-
man (or scraper) may be used to physi-
cally remove the cell sheet from the
bottle. However, this procedure should
be used only as a last resort because
of the risk of cell culture contamina-
tion inherent in such manipulations.
The EDTA-trypsin reagent should
remain in contact with the cells no
longer than necessary as prolonged
contact can alter or damage the cells.
6.1.5 Pour the suspended cells into
centrifuge tubes or bottles.
To facilitate collection and resus-
pension of cell pellets, use tubes or
bottles with conical bottoms. Centrif-
uge tubes and bottles used for this
purpose must be able to withstand the
g-force applied.
6.1.6 Centrifuge cell suspension at
1,000 x sr for 10 min to pellet cells.
Do not exceed this speed as cells
may be damaged or destroyed.
6.1.7 Pour off and discard the super-
natant.
6.1.8 Suspend the pelleted cells in
growth medium.
Resuspend pelleted cells in suffi-
cient volumes of medium to allow tho-
rough mixing of the cells (to reduce
sampling error) and to minimize the
significance of the loss of the 0.5 mL
of cell suspension required for the cell
counting procedure. The quantity of
medium used for resuspending pel-
leted cells varies from 50 to several
hundred mL, depending upon the
volume of the individual laboratory's
need for cell cultures.
6.1.9 Perform a viable count on the
cell suspension according to proce-
dures in Section 6.2.
6.1.10 Dilute the cell suspension to
the appropriate cell concentration with
growth medium (see Section 3.3.3).
Calculate the dilution factor require-
ment using the cell count established
in Section 6.2.6 and the cell concen-
tration parameters given in Table 9-1.
6.1.11 Dispense the cell suspension
into cell culture vessels with either a
Cornwall-type syringe or Brewer-type
pipetting machine dispenser.
7"/7e volume of cell suspension that
must be used for a particular stock
culture vessel is listed in Table 9-1.
6.2 Procedure for Performing Viable
Cell Counts
With experience a fairly accurate
cell concentration can be made based
on the volume of packed cells. How-
ever, viable cell counts should be per-
formed periodically as a quality control
measure.
6.2.1 Add 0.5 mL of cell suspension
(or diluted cell suspension) to 0.5 mL
of 0.5% trypan blue solution in a test
tube.
To obtain an accurate cell count, the
optimal total number of cells per hem-
9-5
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January 1987
ocytometer section should be between
20 and 50. This range is equivalent to
between 6.0 x JO5 and 1.5 x 10B cells
per mL of cell suspension. Thus, a
dilution of 1:10 is usually required for
an accurate count of a cell
suspension.
6.2.2 Disperse cells by repeated
pipetting.
Avoid introducing air bubbles into
the suspension, because air bubbles
may interfere with subsequent filling
of hemocytometer chambers.
6.2.3 With a capillary pipette, care-
fully fill hemocytometer chambers on
one side of a slip-covered hemocy-
tometer slide.
Do not under or over fill the
chambers.
6.2.4 Rest slide on flat surface for
about one min to allow trypan blue to
penetrate cell membranes of nonvia-
ble cells.
6.2.5 Under 100X total magnifica-
tion, count the cells in the four large
corner sections and the center section
of the hamocytometer chamber.
Include in the count cells lying on
the lines marking the top and left mar-
gins of the sections, and ignore cells
on the lines marking the bottom and
right margins. Trypan blue is excluded
by living cells. Therefore, to quantify
viable cells, count only cells that are
clear in color. Do not count celts that
are blue.
6.2.6 Calculate the average number
of viable cells in each mL of cell sus-
pension by totaling the number of via-
ble cells counted in the five sections,
multiplying this sum by 4000, and
where necessary, multiplying the
resulting product by the reciprocal of
the dilution.
6.3 Procedure for Changing Medium
on Cultured Cells
Three to four days after seeding
with an appropriate number of cells,
monolayers normally become 95% to
100% confluent, and growth medium
becomes acidic. Growth medium on
confluent stock cultures should then
be replaced with maintenance
medium. If stock culture cell monolay-
ers have not reached 95% to 100%
confluency by this time and the
medium on these cultures has not
become acidic, then the medium
should not be changed until the mono-
layers reach 95% to 100% confluency.
If, three to four days after passage,
monolayers are not yet 95% to 100%
confluent and the ^medium in which
they are immersed has become acidic,
then the medium must be replaced
with fresh growth \medium supple-
mented with 5% fetal calf serum.
6.3.1 Pour spent medium from cell
culture vessels ana1 discard the spent
medium. '
6.3.2 Add to the cell culture vessels
a volume of fresh maintenance or
growth medium equal to the volume of
spent medium discarded.
7. Procedure for Preparation
of BGM Cell Cultures for
Virus Assay
i
This section is an extension of the
procedure described in Section 5.
BGM cell cultures planted for virus
assay are generally found to be at
their most sensitive level between the
third and sixth days, and cultures
older than seven days should not be
used. The maintenance medium
change recommended for the stock
cultures is not necessary in the prepa-
ration of these celts. However, prior to
inoculation with virus the cultures
should be washed\with Earle's bal-
anced salts with lactalbumin hydrolys-
ate (ELAHj maintenance medium that
contains no serum, (see Chapter 10).
Care must be taken to ensure that all
caps on bottles, flasks or tubes are
tight; otherwise, the gas seal will not
be complete and poor growth will
result.
\
7.1 Preparation of Cell Culture Bot-
tles or Flasks
7.7.7 Use cell suspension from Sec-
tion 6.1.8 to prepare cell cultures for
virus plaque assay.
7.7.2 Dilute the cell suspension to
the appropriate cell concentration with
MEM/L-15 growtn medium supple-
mented with 10%|fetal calf serum and
antibiotics (see Section 3.3.3).
Calculate the dilution factor require-
ment using the cell count established
in Section 6.2 and the cell concentra-
tion parameters given in Table 9-2.
7.1.3 Dispense jthe cell suspension
into cell culture vessels with either a
Cornwall-type syrjmge or a Brewer-
type pipetting machine dispenser.
The volume of cell suspension that
must be used for a particular virus
assay culture vessel is listed in Table
9-2. I
7.1.4 Place tightly-capped cell cul-
ture vessels that contain cells in a sta-
tionary position at 36.5° ± 1 °C with
the flat side down so that cell mono-
layers develop on the proper surface.
7.7.5 Conduct plaque assay for vir-
uses in accordance with instructions
given in Chaper 10.
7.2 Preparation of Cell Culture
Tubes
7.2.7 Use cell suspension from Sec-
tion 6.1.8 to prepare cell cultures for
plaque confirmation procedure (Chap-
ter 11) and virus identification (Chap-
ter 12).
7.2.2 Dilute the cell suspension to
the appropriate cell concentration with
growth medium (see Section 3.3.3).
Calculate the dilution factor require-
ment using the cell count established
in Section 6.2 and the cell concentra-
tion parameters given in Table 9-2.
7.2.3 Dispense the cell suspension
into cell culture tubes with a 2.0-mL
Cornwall-type syringe.
7.2.4 Place tightly-capped tubes in
tissue culture rack and incubate at 36°
± 1 °C statically for three days before
inoculating with viruses.
If tubes are to be held longer than 5
days, replace growth medium with
maintenance medium as directed in
Section 5.1.4. Cells may be held in
maintenance medium for an additional
five days.
7.2.5 Use the cell culture tubes to
pass viruses recovered from plaques
according to the technique described
in Chapter 11.
8. Procedure for Preserva-
tion of BGM Cell Line
An adequate supply of BGM cells
must be available to replace working
cultures that are used only periodically
or become contaminated or lose virus
sensitivity. Cells have been held at
-70°C for 15 years with a minimum
loss in cell viability.
8.1 Preparation of Cells for Storage
5.7.7 Prepare 80 mL of growth
medium as directed in Section 3.3.3.
The procedure described is for the
preparation of 100 cell culture vials.
Cell concentration per mL must be at
least 1 x 106.
Base the actual number of vials to
be prepared on usage of the line and
9-6
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January 1987
Table 9-2. • Guide for Preparation of Virus Assay Cell Cultures
Vessel Type
1 -oz** glass bottle
25-cm2 plastic flask
6-oz glass bottle
75-cmz plastic flask
16 -mm X 150 -mm tubes
Volume of
Medium* (mL)
4
10
15
30
2
Final Cell Count
per Bottle
9.0 X 10s
3.5 X10e
5.6 XW6
1.0X10''
4.0 X JO4
*Serum requirements: growth medium contains 10% fetal calf serum or 10% GG-free calf
serum.
Antibiotic requirements: penicillin-streptomycin stock solution, 1.0 mL/liter; tetracycline
stock solution, 0.5 mL/liter; fungizone stock solution, 0.2 mL/liter.
**S/ze is given in oz only when it is commercially designated in that unit.
the anticipated time interval require-
ment between cell culture start-up
and full culture production. At least
two vials are needed to start-up a new
working culture.
8.1.2- Add 10 mL of fetal calf serum
and 10 mL of DMSO to growth
medium.
8.1.3 Sterilize cell storage medium
by passage through an 0.22 yuni steril-
izing filter.
Collect sterilized medium in 250-mL
flask containing stir bar.
8.1.4 Harvest BGM cells from cell
culture vessels as directed in Sections
6.1.2 thru 6.1.7.
8.1.5 Suspend cells in the storage
medium prepared in Section 8.1.3.
Cell concentration per mL must be
at least 1 x,)0e.
8.1.6 Place flask containing sus-
pended cells on magnetic stirrer.
8.1.7 Mix contents of flask for 30
min. :
8.1.8 Perform viable cell count
according to procedure given in Sec-
tion 6.2.
If cell culture is below 1 x 10e cells
per mL, centrifuge cell suspension at
1,000 x g for 10 min to pellet cells,
resuspend cells in lesser volume of
storage medium and determine new
cell concentration.
8.1.9 Dispense 1 -mL volumes of cell
suspension into 2-mL vials.
8.2 Procedure for Freezing Cells
7776 freezing procedure requires
slow cooling of the cells with the opti-
mum rate of 1°C per min. Because a
controlled temperature freezer is
generally unavailable, the following
method is recommended as an alter-
native system.
8.2.1 Place vials in rack.
To allow for more uniform cooling.
wells adjoining each vial should
remain empty.
8.2.2 Place rack in refrigerator at
4°C.
8.2.3 Remove rack from refrigerator
after 30 min and immediately place in
-20°C freezer.
8.2.4 Remove rack from -20°C
freezer after 30 min and immediately
place in -70°C freezer.
8.2.5 Hold overnight at -70°C before
placing vials into boxes or other con-
tainers for long-term storage.
To prevent substantial loss of cells
during storage, temperature of cells
should be kept constant after -70°C
has been achieved.
8.3 Procedure for Thawing Cells
Cells must be thawed rapidly to
decrease loss in cell viability.
8.3.1 Add appropriate volume of
MEM/L-15 growth medium supple-
mented with 10% fetal calf serum and
antibiotics to either a 6-oz tissue cul-
ture bottle or 75-cm2 tissue culture
flask.
See Table 9-2 and Section 3.3.3.
8.3.2 Place vial into 36°C water
bath and agitate vigorously by hand
until all ice has melted.
5.3.3 Wipe vials with disinfectant
solution of 0.5% I2 in 70% ethanol
(Chapter 3, Section 3.1).
5.3.4 Add BGM cells to growth
medium prepared in Section 8.3.1.
5.3.5 Incubate BGM cells at 36.5° ±
8.3.6 Pour off growth medium after
incubation period of 18 h to 24 h.
8.3.7 Add fresh MEM/L-15 growth
medium supplemented with 10% fetal
calf serum and antibiotics.
8.3.8 Re-incubate BGM cells at
36.5° ± 1 °C for an additional five
days.
5.3.9 Pass and maintain stock BGM
cell cultures as directed in Section 5.
9. Bibliography
Barron, A. L., C. Olshevsky, and M. M.
Cohen. 1970. Characteristics of the
BGM Line of Cells from African
Green Monkey Kidney. Archiv. for
Die Gesamte Virusforschung.
32:389-392.
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.
11:275-282.
Dahling, D. R., R. S. Safferman, and B.
A. Wright. 1984. Results of a Survey
of BGM Cell Culture Practices.
Environ. International. 10:309-313.
Dahling, D. R. and B. A. Wright. 1986.
Optimization of the BGM Cell Line
Culture and Viral Assay Procedures
for Monitoring Viruses in the Envir-
onment. Appf. Environ. Microbiol.
51:790-812.
Eagle, H. 1959. Amino Acid Metabo-
lism in Mammalian Cell Cultures.
Science. 130:432-437.
Freshney, R. I. 1983. Culture of
Animal Cells: A Manual of Basic
Technique. Alan R. Liss, Inc., New
York, New York. 295 pp.
Hay, R. J. 1985. ATCC Quality Control
Methods for Cell Lines. American
Type Culture Collection, Rockville,
Maryland. 81 pp.
9-7
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January 198?
Leibovitz, A. 1963. The Growth and
Maintenance of Tissue-Cell Cultures
In Free Gas Exchange with the
Atmosphere. American J. Hygiene.
78:173-180.
Laboratory Manual in Virology. 1974.
Edition Two. Ontario Ministry of
Health, Toronto, Ontario, Canada.
375 pp.
Paul, J. 1975. Cell and Tissue Culture.
Fifth Edition. Churchill Livingstone,
Medical Division of Longman Group
Limited, London, Great Britain. 484
PP'
Rovozzo, G. C. and C. N. Burke. 1973.
A Manual of Basic Virological Tech-
niques. Prentice-Hall, Inc., Engle-
wood Cliffs, New Jersey. 287 pp.
Waymouth, C., R. G. Hamm, and P. J.
Chappie. 1981. The Growth
Requirements of Vertebrate Cells In
Vitro. Cambridge University Press,
Cambridge, Great Britain. 542 pp.
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