Detection of Microbial and Viral
Contaminants in Cell Lines
The presence of microbial contaminantsmbacteria,
fungi, mycoplasma, or protozoamin cell cultures seriously
compromises virtually all research or production
work involving culture technology. Although many
contamination events are overt and readily apparent,
others are insidious and more difficult to detect. Similarly,
viral infection may be obvious if cytopathogenesis
is affected, but many viruses do not induce a drastic
alteration in host cells and some are present in latent
This article provides representative test protocols
suitable for detecting most microbes and many viruses
that might be expected in cell culture systems. The perspective
is that of staff operating a national cell culture
The following media and reagents are from Difco:
Bacto Sabouraud dextrose-broth (Cat. No: 0382-1);
Bacto fluid thioglycollate medium (Cat. No. 0256-01);
beef extract (Cat. No. 0131); brain-heart infusion (BHI)
broth (Cat. No. 0037-016); neopeptone (Cat. No. Bl19);
proteose peptone (Cat. No. 3); YM broth (Cat. No.
0711-01); nutrient broth (Cat. No. 003-01); Bacto yeast
extract (Cat. No. 012701); trypsin, 1:250 Difco certified
(Cat. No. 0152-15); and blood agar base (Cat. No. 0045-
01). Trypticase soy broth powder (Cat. No. 01-162),
trypticase (Cat. No. Bl1770), mycoplasma broth base
(Cat. No. 11458), and mycoplasma agar base (Cat.
No. 11456) are from Becton-Dickinson Microbiology Systems (formerly BBL). Fresh defibrinated rabbit
blood (Cat. No. 82-8614) and sheep blood are from
Editek, and North American Biologicals provided
Diamond's TP-S-1 broth base powder (Cat. No. 73-
9502) and Diamond's TP-S-1 vitamin solution (40X,
Cat. No. 72-2315). American Hoechst is the source of
bisbenzamide fluorochrome stain (Cat. No. 33217), and
oil-free, dry annealed aluminum foil is from Reynolds
Aluminum (Cat. No. 1235-0). Unless specified otherwise,
cell culture media (various) and sera are from
ATCC, Sigma, GIBCO-BRL, or Hyclone.
Template-primers poly(rA) 9 poly(dT)12-18
7878) and poly(dA)-poly(dT)12-18
(Cat. No. 7868) are
from P-L Biochemicals, and [methyl
triphosphate ([3H]TTP, carrier-free, specific activity
50-60Ci/mmol, 1.0mCi/ml, Cat. No. NET 221X) is
from Dupont New England Nuclear. The scintillation
counter fluid (Betafluor, Cat. No. LS-151) was purchased
from National Diagnostics.
The following items are required in screening for
c-type viruses such as HIV and HTLV:
Rneasy minikit, Qiagen (Cat. No. 74104)
SuperScript double-stranded cDNA synthesis kit,
Invitrogen (Cat. No. 11917-010)
Platinum Taq DNA polymerase, Invitrogen (Cat. No.
Sybr Green I, Molecular Probes, Inc. (Cat. No. S7563)
DNA standard and HIV and HTLV positive cell lines,
ATCC (Cat. No. 53069 CRL-8993 and CRL 8543)
Standards, primers, and probes:
A pUC18 vector containing a 9.9-kb insert from
HIV-standard DNA is used as standard DNA for
HIV (ATCC, Manassas, VA, ATCC No. 53069). For
GAPDH and HTLV, polymerase chain reaction (PCR) products containing the primer and probe
sequence for both targets have been cloned into the
Primers and probes include:
In most cases, Sigma or VWR supplied general
laboratory chemicals and solvents plus such items as
instruments and bacteriological or cell culture glassand
The following additional specialty items are
required: Leighton tubes (Cat. No. 3393) from Costar;
cellulose filters, 0.45 and 0.22µm (Cat. Nos. HATF 14250
and GSTF 14250, respectively) from Millipore; GasPak anaerobic
systems (Cat. No. 60465) from Becton-Dickinson
Microbiology Systems; embryonated chicken eggs from
SPAFAS; egg candlers (Cat. No. C6372N-50001) from
Nasco; egg drills, cutters, and moto tool (Cat. No. 9826-
00) from Cole-Parmer; stainless-steel sterilizing pans
(Cat. No. 2065-5) from Orem; and adjustable microliter
pipettes and Pipetman (Cat. No. P-20 D/P-200D) from
Rainin. Reference microbes, cell lines, and viruses are
from the American Type Culture Collection (ATCC): Pseudomonas aeruginosa
(e.g., ATCC 14502), Micrococcus
(e.g., ATCC 14344), Escherichia coli
ATCC 4157), Bacteroides distasonis
(e.g., ATCC 8503), Penicillium notatum
(e.g., ATCC 8537), Aspergillus niger
(e.g., ATCC 34467), Candida albicans
(e.g., ATCC 10231),
influenza virus (e.g., ATCC VR-95 or VR-810), Newcastle
disease virus (e.g., ATCC VR-108 or VR-109), and Rous sarcoma
virus (e.g., ATCC VR-140 or VR-724). The
Gen-Probe kit is available from Fisher (Cat. No.
GP1591) and the mycoplasma PCR kit is from ATCC
(Cat. No. 90-1001K).
A. Bacteria and Fungi
Tests for sterility are performed routinely at ATCC
on all culture media used, on cultures submitted from the community, on cultures at various stages during
the accessioning process, and on all seed and distribution
species, micrococci, and E.
are common bacterial isolates, whereas Penicillium
, and Candida
species are common fungal
and yeast contaminants.
1. Preparation of Media Solutions
- Sabouraud dextrose broth: Dissolve 30g dehydrated
powder in 1000 ml distilled water and dispense
10-ml aliquots into each of one hundred 16 × 150-mm
test tubes. Cap each tube loosely and sterilize in an
autoclave for 15min at 151b pressure (121°C on slow
exhaust. After removing the tubes from the autoclave,
press down caps securely and store at room temperature
until used. Caps of differing colors may be used
to permit ready identification.
- Nutrient broth with 2% yeast extract: Dissolve 8g
of nutrient broth powder plus 20g of Bacto yeast
extract in 1000ml distilled water and dispense 10-ml
aliquots into each of one hundred 16 × 150-mm test
tubes. Cap each tube loosely, sterilize, and store as
described for solution 1.
- Thioglycollate medium: Suspend 29.8 g dehydrated
powder in 1000 ml distilled water in a 3-liter flask and
heat to boiling to dissolve the powder completely.
Dispense 10-ml aliquots of the thioglycollate medium
into each of one hundred 16 × 150-mm test tubes;
cap each tube loosely. Sterilize in the autoclave as
described for solution 1. After removing the tubes from
the autoclave, press down caps securely and store in
the dark at room temperature. This medium changes
color in processing. As it dissolves it turns red or gold
depending on the amount of dissolved oxygen. After
autoclaving it is clear and gold in color, like nutrient
broth. After cooling, the top layer of medium oxidizes
and the indicator in the upper portion of the tube turns
pink or red. The fluid should not be used if the indicator
has changed to a red color in the lower third of
- Trypticase soy broth: Suspend 30g powder in
1000ml distilled water and mix thoroughly and warm
gently until solution is complete. Dispense 10-ml
aliquots of die trypticase soy booth into each
of one hundred 16 x 150-mm test tubes; cap each
tube loosely and sterilize and store as described for
- BHI broth: Suspend 37 g powder in 1000 ml of distilled
water, dissolve completely, and dispense 10-ml
aliquots of BHI into 16 × 150-mm test tubes. Cap each
tube loosely. Sterilize in the autoclave for 15min at
151b pressure on slow exhaust. After autoclaving,
press down caps securely, cool, and store at 4°C.
- YM broth: Dissolve 21 g powder in 1000ml distilled
water and dispense 10-ml aliquots of the broth
into each of one hundred 16 × 150-mm test tubes; cap
each tube loosely. Sterilize and store as described for
- Blood agar plates: Suspend 40 g blood agar base in
950ml cold distilled water and heat to boiling to dissolve
the powder completely. Sterilize in the autoclave
for 15min at 151b pressure on slow exhaust. When the
sterile blood agar base is cooled to 50°C, add 5% (50
ml) of pretested, fresh, defibrinated rabbit blood and
mix by swirling. Dispense aseptically to 9-cm plates
and store at 4°C. The rabbit blood is pretested for sterility
by inoculating 0.5-ml aliquots into BHI broth and
YM broth and onto blood agar base plates with subsequent
incubation at 25° and 37°C. Negative results
in 48 to 72 h are usually sufficient to permit use of the
3. Inoculation and Incubation of Test Samples
- Using an inverted microscope, equipped with
phase-contrast optics if possible, examine cell culture
vessels individually. Scrutiny should be especially vigorous
in cases where large-scale production is involved.
- Check each culture first using low power. After
moving the cultures to a suitable isolated area, remove
aliquots of fluid from cultures that are suspect; retain
these for further examination. Alternatively, autoclave
and discard all such cultures.
- Prepare wet mounts using drops of the test fluids
and observe under high power.
- Prepare smears, heat fix, and stain by any conventional
method using filtered solutions.
- Examine under oil immersion for microbial
- Consult Freshney (2000) for further details.
- After cryopreservng stocks of cells (Hay et al.,
2000), retrieve and thaw about 5% of the ampoules
from liquid nitrogen or vapor storage. Pool and mix
the contents of the ampoules from each cryopreserved
lot using a sterile 1-ml disposable pipette. It is recommended
that antibiotics not be included in media used
to prepare stocks of cells for preservation. If antibiotics
are used, the pooled suspension should be centrifuged
at 2000g for 20min and the pellet should be resuspended
in antibiotic-free medium. A series of three
such washes with antibiotic-free medium prior to
testing eliminates traces of antibiotics that could
- From each pool, inoculate each of the following
with a minimum of 0.3 ml of the test cell suspension:
(a) two blood agar plates, (b) two tubes of thioglycollate
broth, (c) two tubes of trypticase soy broth, (d) two
tubes of BHI broth, (e) two tubes of Sabouraud broth,
(f) two tubes of YM broth, and (g) two tubes of nutrient
broth with 2% yeast extract.
- Incubate test plates and broths as follows. (a)
Blood agar plates: one at 37°C under aerobic conditions
and one at 37°C anaerobically (a BBL Gaspak
anaerobic system is convenient for the latter). (b) Tubes
of thioglycollate broth, trypticase soy broth, BHI broth,
Sabouraud broth, YM broth, and nutrient broth with
yeast extract: one each at 37°C and one each at 26°C under aerobic conditions. (c) Incubate and examine
periodically for 14 days the tubes of thioglycollate,
trypticase soy broth, BHI broth, and blood agar plates.
(d) Observe the tubes of Sabouraud broth, YM broth,
and nutrient broth with yeast extract for 21 days before
concluding that the test is negative. Contamination is
indicated if colonies appear on solid media or if any of
the liquid media become turbid.
- Repeat any components of the test series that
are positive initially to confirm the presence of a
- Autoclave and discard any contaminated cultures
or ampule lots.
Of the seven media employed, trypticase soy, BHI,
blood agar, and thioglycollate are suitable for detecting
a wide range of bacterial contaminants. Sabouraud
broth, YM broth, and nutrient broth with yeast extract
will support growth of fungal contaminants. Stock
media and incubation conditions used can be tested
with the following ATCC control strains: P. aeruginosa,
M. salivarius, E. coli, B. distasonis, P. notatum, A. niger,
and C. albicans.
Table I summarizes this recommended
Although this test regimen permits detection of
most common bacterial and fungal organisms that
grow in cell cultures, we have experiences with at least
one very fastidious bacterial strain that initially
escaped observation. This was present in nine different
cell cultures from a single clinical laboratory in the
United States submitted for testing and expansion
under a government contract. The organism grew
extremely slowly but could be detected after 3 weeks
of incubation with cell cultures without antibiotics and
with no fluid changes. Samples so developed were
inoculated into sheep blood agar plates and New York
City broth (ATCC medium 1685). The organism could be observed during a subsequent 6-week incubation
period at 37°C
The bacteriology department at ATCC determined
suitable culture conditions for this microorganism and
tentatively identified it as a Corynebacterium. Antibiotic
sensitivity tests revealed bacteriostasis with some
compounds, but no bactericidal antibiotics have yet
This incident emphasizes the critical importance
of diligent testing of cell cultures for contaminant
microorganisms. By combining protocols such as those
described here with procedures discussed later (e.g.,
fluorescent or nucleic acid probes for mycoplasma and
viruses), one can be more certain that clean cell cultures
are available for experimentation.
Mycoplasmal contamination of cell cultures has
been established as a common occurrence that is
capable of altering normal cell structure and function.
Mycoplasmas have been shown to inhibit cell metabolism
and growth, alter nucleic acid synthesis, affect
cell antigenicity, induce chromosomal alterations,
interfere with virus replication, and mimic viral
actions. Basically, the growth of mycoplasma in cell
cultures can be detected either by a direct microbiological
agar culture procedure or by indirect procedures
using staining, biochemical methods, or nucleic
acid hybridization techniques (McGarrity, 1982; Hay et al
., 1989, 1992, 2000).
In testing cell cultures for contamination, both
direct and indirect procedures should be employed.
The indirect method employed most frequently at the
ATCC was originally described by Chen (1977). It requires the bisbenzamide DNA fluorochrome staining
procedure plus an indicator cell. This adaptation is
described here with slight modifications.
Duplicate screening techniques are generally recommended
for rigorous cell line testing. Alternative
methods to those included here include nucleic acid
hybridization and a new technique involving the
polymerase chain reaction. Details are available
elsewhere (Hu and Buck, 1993; ATCC website
In screening cell lines for mycoplasma contamination,
it is important to include positive controls in
order to be assured that the test systems being used are
optimal. Special precautions, however, are necessary
when working with such material. The handling of
mycoplasma cultures should be done at the end of a
particular test and, when possible, in an isolated area
using a biohazard-type hood. All equipment used in
manipulations involving control cultures should be
collected and sterilized immediately by autoclaving.
More detailed accounts of the measures necessary to
detect and prevent the spread of contamination can be
found elsewhere (Uphoff and Drexler, 2001; Freshney,
1. Direct Method
The procedures used in preparing and pretesting
the following culture media should be standardized,
and the final pH should be adjusted to 7.2 to 7.4. Both
media are prepared in quantities to be utilized within
3 to 4 weeks. The quality of the major components of
the media may vary from batch to batch in the degree
of toxicity and in their ability to support the growth of
mycoplasma. The growth-promoting properties of
each new lot of freshly prepared broth and agar media are determined by making inoculations using ATCC
23206, Acholeplasma laidlauii,
and ATCC 23838, Mycoplasma arginini.
In addition, the horse serum, like
all sera employed for cell culture work, is screened for
mycoplasmal contamination. Briefly, a 100-ml aliquot
of the serum being tested is used as the serum
supplement for 400ml of broth medium. The cultures
are incubated aerobically at 37°C for 4 weeks and are
observed for turbidity and change in pH. Subcultures
to agar plates and inoculations onto Vero indicator cultures
for the indirect test are performed weekly during
the incubation period. In testing samples in which bacterial
and/or fungal contaminations may be prevalent,
penicillin and thallium acetate are added to the basic
medium. Penicillin is added to the stock solution (step
a below) to provide a final concentration of 500 U per
milliliter. Thallium acetate is added to the basic media
(step b below) to provide a final concentration of
a. Preparation of Stock Solution.
freshly distilled water, add 50g dextrose and 10g
L-arginine HCl. Mix the ingredients at 37°C until
dissolved. Bring the final volume up to 1000-ml. Sterilize
the solution by filtration using a 0.22-µm filter,
dispense into 100-ml aliquots, and store at-70°C until
b. Preparation of Mycoplasma Broth Medium.
Add 14.7 g mycoplasma broth base and 0.02g phenol
red to 600 ml water, heat to dissolve. Sterilize the solution
by autoclaving for 15min at 121°C using a slow
exhaust cycle and allow the broth mixture to cool to
room temperature. Aseptically add 200ml horse
serum, 100 ml yeast extract (15 %), and 100 ml thawed
stock solution (step a). Mix the solution completely.
Dispense 10-ml aliquots of the broth medium into
sterile test tubes and cap. Store broth tubes at 5°C and
use within 3 to 4 weeks.
c. Preparation of Mycoplasma Agar Medium.
Add 23.8 g mycoplasma agar base to 600ml water. To
dissolve, bring solution to a boil and sterilize the solution
by autoclaving for 15min at 121°C using a slow
exhaust cycle. Place the sterilized medium in a water
bath at 50°C Place 200ml horse serum, 100ml yeast
extract (15%), and 100ml stock solution (step a) in a
water bath at 37°C Allow the components to equilibrate
at these respective temperatures. Aseptically add
the horse serum, yeast extract, and stock solution to
the medium; mix well. Proceed immediately to dispense
10-ml aliquots in 60 × 15-mm petri dishes. Add
the fluid as quickly as possible in order to eliminate the problem of the agar solidifying before the medium
is dispensed completely. Stack the agar plates into
holding racks, wrap in autoclave bags to minimize
dehydration, and store at 5°C Use within 3 to 4
NOTE This procedure involves a total incubation time of about 35
days for both broth and agar cultures. This schedule is advisable
for detecting lower levels of mycoplasma contamination that otherwise
might be scored as false negatives.
d. Inoculation of Test Sample.
Select a cell culture
that is near confluency and has not received a fluid
renewal within the last 3 days. Remove and discard all
but 3 to 5 ml of the culture medium. Scrape a portion
of the cell monolayer into the remaining culture
medium using a sterile disposable scraper. For suspension
culture systems, take the test sample directly
from a heavily concentrated culture that has not
received a fresh medium supplement or renewal
within the last 3 days. Samples for testing can also be
taken directly from thawed ampules that have been
stored in the frozen state.
Inoculate 1.0ml of the test cell culture suspension
into a mycoplasma broth culture. Inoculate 0.1 ml of
the test sample onto an agar culture plate. Incubate the
broth culture aerobically at 37°C Observe daily for the
development of turbidity and/or shift in pH. Incubate
the agar plate anaerobically at 37°C in a humidified
atmosphere of 5% CO2
-95% nitrogen. After 5 to 7 days
of incubation, and again after 10 to 14 days, remove a
0.1-ml sample from the broth culture and inoculate
a new agar plate. Incubate these plates anaerobically
Microscopically examine the agar plates weekly
for at least 3 weeks for mycoplasma colony formation
and growth before considering them to be negative.
Observe the plates at 100 to 300 magnification using an
The positive differentiation of mycoplasma colonies
on agar plates, as opposed to air bubbles, tissue culture
cells, and pseudocolonies, can be accomplished by
subculturing a small section (1 cm2
) of the suspicious
area of the agar culture into a new broth culture (for
examples, see Freshney, 2000).
2. Indirect Method (Staining for DNA)
The bisbenzamide stain concentrate (step a below)
should be examined routinely for contamination.
Sterilization by filtration diminishes the quality of
fluorescence, and fresh stock needs to be prepared
periodically. The pH of the mounting medium (step b
below) is critical for optimal fluorescence and should
also be monitored routinely.
Continuous cells lines, such as ATCC.CCL 81, Vero,
African green monkey kidney or ATCC.CCL 96, 3T6
mouse fibroblast, have been used very effectively as
indicator cells in the indirect DNA-staining procedure.
The use of transformed cell lines is not recommended
because they produce large amounts of nuclear background
fluorescence, which interfere with the interpretation
of the results.
Utilization of the indicator cell with the DNAstaining
procedure provides two major advantages.
First, the indicator cell line supports the growth of the
more fastidious mycoplasma species. Second, both
positive and negative control cultures are readily available
for direct comparisons with the culture samples
a. Preparation of Stain Concentrate.
To 100 ml of
Hanks' balanced salt solution without sodium bicarbonate
or phenol red, add 5.0mg bisbenzamide fluorochrome
stain and 10mg thimerosol. Mix thoroughly
using a magnetic stirrer for 30 to 45 min at room temperature.
The stain is heat and light sensitive. Prepare
the concentrate in a brown amber bottle wrapped completely
in aluminum foil. Store aliquots at -20°C These
are stable for about 1 year.
b. Preparation of Mounting Medium.
citric acid, 27.8ml 0.2M
and 50 ml glycerol and adjust pH of mixture to
5.5. Store in a cold room at 5°C.
c. Preparation of Indicator Cell Cultures and
Inoculation of Test Samples.
Aseptically place a
glass coverslip (previously sterilized) into each 60 ×15-
mm culture dish. Dispense 3ml Eagle's minimum
essential medium with Earle's salts, 100 U/ml penicillin,
and 100µg/ml streptomycin plus 10% bovine
calf serum into each culture dish. Make certain that
each glass coverslip is totally submerged and not floating
on top of the medium.
Prepare a single cell suspension of ATCC.CCL 81,
the African green monkey kidney cell line Vero, in this
medium at a concentration of 1.0 × 105
3T6 murine line (ATCC.CCL 96) can be used instead.
Inoculate 1 ml of the cell suspension into each culture
dish and incubate the cultures overnight in a 5% CO2
95% air incubator at 37°C. Examine the cultures microscopically
to verify that the cells have attached to the
glass coverslip. Number the top of each culture dish
for identification purposes to record the test sample
inoculated. Add 0.5 ml of culture medium to each of
two cultures for negative controls and 0.2 to 0.5ml
of each test sample to each of two culture dishes. Add
0.5 ml ATCC 29052, M. hyorhinis
, to each of two cultures
for positive controls. Alternatively, a known
infected cell line can be used. Return the cultures to the
incubator and allow to incubate undisturbed for
d. Fixing, Staining, and Mounting Coverslips.
To prepare the staining solution:
- Add 1.0ml of stock concentration (step a) to 100
ml Hanks' balanced salt solution without sodium
bicarbonate and phenol red.
- Prepare in a brown amber bottle wrapped in
- Mix thoroughly for 30 to 45 min at room temperature
using a magnetic stirrer.
Remove cultures from the incubator and aspirate
the medium from each dish. Add 5 ml of a 1:3 mixture
of acetic acid:methanol to each culture dish for 5 min.
Do not allow the culture to dry between removal of the
culture medium and addition of the fixative. Aspirate
each culture dish and repeat the fixation step for 10
min. Aspirate the fixative and let the cultures air dry.
Add 5 ml of the staining solution [step d (1-3)] to each
culture dish; cover and let stand at room temperature
for 30min. Aspirate the stain and rinse each culture
three times with 5 ml distilled water.
After the third rinse, aspirate well so that the glass
coverslip is completely dry. Let air dry if necessary.
Place a drop of mounting fluid (step b) on a clean glass
slide. Use forceps to remove the glass coverslip containing
the specimen from the culture dish and place
face up on the top of the mounting fluid. Add a second
drop of mounting fluid onto the top of the specimen
coverslip and cover with a larger clean coverslip.
Lower both coverslips onto the mounting fluid in such
a way as to eliminate trapped air bubbles. Label each
slide to identify the specimen being tested and record
Observe each specimen under oil immersion,
including both the positive and the negative controls,
by fluorescence microscopy at 500X. A blue glass
excitation filter (BG12 for Zeiss microscopes) is used
in combination with a No. 50 barrier filter. Small
fluorescing particles indicate mycoplasmal DNA and
Alternative molecular methods readily available in
kit form should also be considered. The PCR-based
method for mycoplasma detection (Harasawa et al
1993; Hu and Buck, 1993) in use at the ATCC requires
primers based on the DNA sequences in 16S and 23S
mycoplasmal rRNA. These amplify DNA from all of
the common mycoplasma found in cell cultures to
levels easily detected after gel electrophoresis and ethidium bromide staining. Advantages of the method
include speed and sensitivity, as well as the ability to
detect and identify species of most of the common
mycoplasma known to infect cell cultures. Furthermore,
it does not suffer from interpretation difficulties .
associated with some of the Hoechst or DAPI-stained
preparations. Levels of sensitivity compare favorably
with the Hoechst stain. Sample sizes need consideration.
Detailed methodologies are provided with the
kits (see the ATCC website for more details).
The overall frequency of infection of cell cultures
with protozoans is low but the incidence may be
higher if one is working with tissues such as human
clinical material and animal tissues such as kidney
or colon. The small limax amoebae belonging to the Acanthamoeba
) genus are ubiquitous in
nature and have been isolated from cells and tissues in
culture in a significant number of laboratories. Jahnes
et al. (1957) first reported spontaneous contamination
of monkey kidney cells in culture by such free-living
amoebae. The organisms have also been detected as
occasional contaminants in such diverse cell lines as
dog lymphosarcoma (LS30), HeLa, chick embryo
fibroblast-like, and Chang liver cells (Holmgren, 1973).
In some cases, protozoans are demonstrably cytopathic
in cell culture.
Observation, cytological examination, and attempts
at isolation are required in the detection of protozoan
contaminants. These techniques are suitable for detecting
many of the most common flagellates and amoeboid
protozoans, including species of the genera Acanthamoeba, Giardia, Leishmania, Naegleria, and Trypanosoma
(for more details, see Hay et al
1. Preparation of Solutions and Protozoan Media
2. Preparation of Cell Culture Samples for
Inoculation into Protozoan Media
- Trypsin-EDTA: Combine 2.5g trypsin (1:250
Difco certified), 0.3 g EDTA, 0.4 g KCl, 8.0 g NaCl, 1.0 g
glucose, 0.58g NaHCO3, and 0.01 g phenol red in 1
liter double-distilled water, sterilize by filtration (0.22-
µm Millipore filter), and store at-40°C.
- Hanks" balanced salt solution without divalent
cations: Combine 8.0 g NaCl, 0.4 g KCl, 0.05 g Na2HPO4,
0.06 g KH2PO4, and 0.02 g phenol red in 50ml doubledistilled
water to dissolve chemicals; then bring
volume to 100 ml. Autoclave on slow exhaust for 15
min, adjust pH to 7.2 to 7.4 with sterile 0.4N NaOH,
and store at 4°C.
- Giemsa stock solution: For stock solution of stain,
combine 40ml glycerol, 65 ml absolute methanol, and 1.0g Giemsa powder. Filter two or three times and
store at 4°C.
- Price's buffer (IOX): Combine 6.0g Na2HPO4, 5.0 g
KH2P04, and 1.0 liter distilled water. Before use dilute
buffer with distilled water to 1X.
- Price's Giemsa stain: Dilute Giemsa stain stock
3:97 with 1X buffer. After staining, discard unused
- ATCC medium No. 400, Diamond's TP-S-1 medium
for axenic cultivation of Entamoeba (ATCC medium No.
400): Dissolve one packet of Diamond's TP-S-1 broth
base powder in 875 ml distilled water, adjust pH to 7.0
with 0.4N NaOH, and filter through Whatman No. 1
paper. Sterilize at 120°C for 15 min. Aseptically add 100
ml inactivated (56°C for 30min) bovine serum and 25
ml Diamond's TP-S-1 vitamin solution (40X, North
American Biologicals), and aseptically dispense 13ml
per sterile test tube. Some commercial lots of
Diamond's TP-S-1 medium have been shown to be
toxic to Entamoeba. To test for toxicity, subculture Entamoeba through three to five passages.
- Locke's solution: Combine 8.0g NaCl, 0.2 g NaCl,
0.2 CaCl2, 0.3 g KH2PO4, 2.5 glucose, and 1.0 liter distilled
water and autoclave the solution for 20min at
- Diphasic blood agar medium (ATCC medium No.
1011): Infuse 25.0 g beef extract in 250 ml distilled water
by bringing to a rapid boil for 2 to 3 min while stirring
constantly. Filter through Whatman No. 2 filter paper
and add 10.0 g Difco neopeptone, 2.5 g NaC1, and 10.0
g agar. Heat to boiling and filter through Whatman No.
2 paper, make up volume to 500ml with distilled
water, and adjust pH to 7.2 to 7.4. Autoclave for 20 min
at 121°C, cool mixture to 50°C aseptically add 30%
sterile, defibrinated rabbit blood (Editek) to whole
mixture, and dispense in sterile tubes and slant. After
the slants have set, cover with 3.0ml sterile Locke's
- PYb medium (ATCC medium No. 711): Combine 1.0
g Difco proteose peptone, 1.0g yeast extract, 20.0g
agar, and 900.0 ml distilled water. Prepare and sterilize
separately each of the following stock solutions and
add to the basal medium as indicated to avoid precipitation:
CaCl2 (0.05M), 4.0ml; MgSO4.7H20 (0.4M),
2.5 ml; Na2HPO4 (0.25 M), 8.0 ml; and KH2PO2 (0.25 M),
32ml. Make the volume to 1 liter, check that the pH is
at 6.5, and sterilize by autoclaving for 25 min at 120°C.
Pour into petri dishes and allow to solidify.
- Brain-heart infusion blood agar (ATCC medium No.
807): For the agar component, dissolve 37.0 g Difco BHI
broth and 18.0 g agar in 1 liter boiling water. Dispense
5.0ml solution per tube (16 × 125mm) and sterilize for
25 min at 121°C. Cool to 48°C. Add 0.5 ml per tube of
sterile, defibrinated rabbit blood and slant. After slants have set, cover with 0.5 ml BHI broth (1.0 liter distilled
water and 37.0 g BHI broth) with sterilization by autoclaving
at 121°C for 25 min.
- Leishmania medium (ATCC medium No. 811):
Combine 1.2g sodium citrate, 1.0g NaCl, and 90.0ml
distilled water. Dispense 1.0 ml per tube, autoclave for
25min at 121°C, and cool. Add 1.0ml defibrinated,
lysed rabbit blood solution (prepare by mixing equal
parts of whole rabbit blood and sterile distilled water
and freezing and thawing twice).
- NTYG medium (ATCC medium No. 935):
Combine 5.0 g trypticase, 5.0 g yeast extract, 10.0 g
glucose, and 1.0 liter distilled water. Dispense 10.0ml
per test tube and sterilize. Just before use, add
0.2ml dialyzed, heat-activated bovine serum and
0.1ml defibrinated sheep blood. Protozoan growth
media retain stability for at least 3 months if maintained
at 4°C with the exception of ATCC medium
400, which maintains stability for 2 to 4 weeks.
3. Preparation of Culture Cells for Giemsa Staining
- Rapidly thaw a frozen ampoule of the sample in
a water bath at 37°C.
- Aseptically open the ampoule. Continue to use
- Transfer 0.8ml of the concentrated cell suspension
from the ampoule into a T-25 flask. Save 0.2 ml of
the suspension for Giemsa staining (step 3 below).
- Add 7 ml of the appropriate cell culture medium
to maintain the culture.
- Incubate at 37°C until the monolayer becomes
confluent (3 to 5 days depending on the cell line).
Examine the culture microscopically during this incubation
period for the presence of (a) movement (i.e.,
motile cells), (b) intracellular contaminants, and (c)
- Transfer the supernate from the confluent test
cell culture to a sterile 15-ml plastic centrifuge tube
and retain at room temperature for use in step 12.
- Rinse the cell monolayer (T-25 flask, step 5) with
5ml Ca"- and Mg'- free Hanks' saline and discard
- Add 2ml 0.25% trypsin-EDTA solution to the
T-25 flask and incubate at 37°C for 10rain.
- Add 7 ml of cell culture medium to the T-25 flask
and aspirate gently to obtain a single-cell suspension.
- Dispense aliquots (0.5ml) of the trypsinized
single-cell suspension to the following ATCC protozoan
growth media: (a) ATCC medium No. 400 (for Entamoeba, Giardia); (b) ATCC medium No. 711 with Enterobacter aerogenes (for Acanthamoeba) [use a wire loop to streak medium No. 711 with E. aerogenes (ATCC
15038) 48h before use]; (c) ATCC medium Nos. 807,
811, and 1011 (for trypanosomatids); and (d) ATCC
medium No. 935 (for Naegleria).
- Incubate samples for 7 to 10 days at 35°C and
examine microscopically for the presence of flagellate,
cyst, and trophozoite forms of protozoa.
- Prepare five wet mounts for each test cell monolayer
using the supernate collected in step 6. Examine
microscopically with phase contrast for the presence of
motile and nonmotile protozoans.
- Aseptically add 1.5 ml of the appropriate culture
medium to a sterile Leighton tube containing a
- Dispense 0.2ml of the original cell suspension
(sample preparation just earlier) into the Leighton tube
and incubate at 37°C until the culture is confluent.
- Remove the coverslip from the Leighton tube, fix
with absolute methanol for I min, and airdry.
- Stain for 10min with Price's Giemsa, rinse with
tap water, and mount the coverslip to a glass slide
- Examine the slide; use low power (20X) for scanning
and high power (100X) for close examination.
It is recommended that positive controls be
included. For example, if cultured cells of the upper
respiratory tract are being used, Acanthameba castellanii
(ATCC 30010) or Naegleria lovaniensis
can be used as positive controls. A. castellanii
from human clinical material. N. lovaniensis
TS, another nonpathogenic strain of amoebae, was isolated
from a Vero cell culture at passage 120. Entamoeba
(ATCC 30042), the common pathogen
causing amoebic dysentery, or the nonpathogenic Entamoeba invadens
(ATCC 30020) can be used for
positive controls if cells are being isolated from the
intestinal tract. E. histolytica
is a human isolate, and E.
strain PZ is a snake isolate.
The methods described are suitable for the detection
of most common protozoan genera (i.e., limax
amoebae) that could survive in association with cells
in culture. Because cysts and trophozoites closely
resemble damaged tissue cells, their presence as occasional
contaminants can remain unnoticed. However,
cells in cultures infected productively with amoebae of
the genus Acanthamoeba
frequently become granular and gradually progress to complete disintegration.
The time elapsed depends on the inoculum size and
whether cysts or the motile trophozoites predominate
in the inoculum. The cytopathic effect of amoebic contaminants
has been reported, and in some cases the
responsible agent has been mistakenly identified as
viral in origin. Therefore, frequent observation of the
cell culture is particularly stressed when examining for
parasitic protozoan contaminants.
The possible presence of other genera (i.e., Entamoeba
or trypanosomatids) should be considered
not only in experimental studies involving primary
tissues, but also with work requiring development or
utilization of cell lines. The only known case of an isolation
other then an amoeboid protozoan occurred in
the isolation of a trypanosomatid from liver tissue.
The particular animal and tissue employed provide
valuable clues as to the type of protozoan contaminant,
the specific media, and staining procedures required.
Of the various tests applied for detection of adventitious
agents associated with cultured cells, those for
endogenous and contaminant viruses are the most
problematical. Table II lists representative problem
viruses. Development of an overt and characteristic
cytopathogenic effect (CPE) will certainly provide an
early indication of viral contamination; however, the
absence of a CPE definitely does not indicate that the
culture is virus free. In fact, persistent or latent infections
may exist in cell lines and remain undetected
until the appropriate immunological, cytological,
ultrastructural, and/or biochemical tests are applied.
Unfortunately, separate tests are necessary for each
class of virus and for specific viruses. Additional host
systems or manipulations, e.g., treatment with halogenated
nucleosides, may be required for virus activation
and isolation (Aaronson et al
., 1971). Common screening methods or tests for specific virus classes are
listed in Table III.
Without such screens, latent viruses and viruses
that do not produce an overt CPE or hemadsorption
will escape detection. Some of these could be potentially
dangerous for the cell culture technician. For
example, Hantaan virus, the causative agent of Korean
hemorrhagic fever, replicates in tumor and other cell
lines. Outbreaks of the disease in individuals exposed
to infected colonies of laboratory rats have been
reported separately in five countries. An incident of
transmission during passage of a cell line was confirmed
in Belgium. As a result of these findings, cell
lines expanded in this laboratory were screened using
an indirect immunofluorescent antibody assay (LeDuc et al
., 1985) and were found to be negative.
Substantial concern over laboratory transmission of
the human immunodeficiency viruses is also evident.
Cases of probable infection during processing in U.S.
laboratories have been described. One, for example,
was presumed due to parenteral exposure and another
to work with highly concentrated preparations (Weiss et al
., 1988). In the latter circumstance, strict adherence
to biosafety level 3 containment and practices is essential.
A more detailed discussion of safety precautions
for work with cell lines in general is provided elsewhere
ATCC cell lines from selected groups have been
screened for HIV-1 using PCR amplification followed
by a slot-blot test for envelope and GAG sequences
(Ou et al
., 1988). The oligonucleotide primer pairs
and SK 68/69 plus
or SK 70
were used. Human cell lines of T-cell, monocytemacrophage,
brain and nervous system, B-cell, and
gastrointestinal origin plus an array of other primate
lines have been examined to date. Only those already
known to be infected with HIV-1 have been positive.
Additional viruses that could present a substantial
health hazard to cell culture technicians include, for
example, hepatitis and cytomegaloviruses. Rapid
PCR-based tests for these have been described [e.g., Ulrich et al
. (1989) and Cassol et al
Other viruses that may present problems generally
in cell culture work include ectromelia virus, the
causative agent of bovine viral diarrhea (BVDV), and
Epstein-Barr virus (EBV). [See also Bolin et al
Harasawa et al
. (1994), and Hay et al
. (2000) for testing
methodology and further discussion.]
It should be emphasized at the outset that the following
protocols represent an expedient compromise
established at ATCC to monitor for readily detectable
viruses associated with cell lines. Egg inoculations
plus select cocultivations and hemadsorption tests
were included in addition to routine examinations for
CPE using phase-contrast microscopy. Similar general
tests are recommended by government agencies in
cases where cell lines are to be used for biological
production work (Code of Federal Regulations on
Animals and Animal Products, 9 CFR 113.34-113.52
revised Jan. 1, 1978; Code of Federal Regulations on
Food and Drugs, Subchapter F on Biologics, 21
b-c, revised April 1, 1979; IABS, 1989; Lubiniecki
and May, 1985). Procedures for reverse transcriptase
assays to detect oncogenic viruses are also being
applied at the ATCC for selected cell lines.
Because endogenous and most exogenous retroviruses
produce no morphological transformation or
cytopathology in infected cells, the production of such
viruses by cell cultures is generally undetectable
except by serological or biochemical means. At ATCC
the concentration of particulate material from culture
supernates and assay for viral RNA-directed DNA
polymerase (RDDP) provide sensitive and reliable
means for detecting retrovirus production by cultured
One or more of the following procedures is currently
being applied to all cell lines accessioned for the
ATCC repository. Tests for specific viruses may be
applied through collaborations as described earlier.
1. Examination of Established Cultures for Overt
Cytopathogenic Effect or Foci
2. Application of the Hemadsorption Test
- Hold each flask or bottle so that light is transmitted
through the monolayer and look for plaques,
foci, or areas that lack uniformity. If frozen stocks of
cells are to be examined, pool and mix the contents of
about 5% of the ampoules from each lot using a
syringe with a cannula. Establish cultures for morphological
examinations and for tests in the following
sections using progeny from such pooled populations.
- Using an inverted microscope equipped with
phase-contract optics wherever possible, examine cell culture vessels individually, paying special attention to
any uneven areas in gross morphology observed in
step 1. Check first using low power. If the cell line
is suspect, subculture taking the appropriate safety
precautions. Prepare coverslip cultures for further
examination. Alternatively, autoclave and discard all
suspect cultures. (Stainless-steel collection and sterilizing
pans for this purpose can be obtained from the
Orem Medical Company.)
- Remove fluid from coverslip cultures that require
additional study. Treat with neutral buffered formalin
or other suitable fixative. Prepare a wet mount and
examine under high power. [Consult Rovozzo and
Burke (1973), Hsuing et al. (1994), and Yolken et al.
(1999) for examples of cytopathogenic effects and
3. Egg Preparation
- Establish test cultures in T-25 flasks using an
inoculation density such that the monolayers become
confluent in 48 to 72 h.
- Prepare washed red blood cell suspensions on
the day the test is to be performed. Pack the erythrocytes
from 5ml of the purchased suspensions by
centrifugation at 100g and resuspend in 35 ml Hanks'
saline without divalent canons. Repeat three times and
resuspend the final pellet to yield a 0.5% suspension
(v/v) of red blood cells in saline.
- Remove the culture fluid and rinse the test monolayers
with 5 ml Hanks' saline minus divalent cations.
- Add 0.5ml each of the suspensions of chick,
guinea pig, and human type O erythrocytes from step
2. Then place the flask with monolayer down at 4°C for 20 rain.
- Observe macroscopically and microscopically
under low power for clumping and adsorption of red
blood cells to the monolayer.
- Repeat steps 2-4 on all test cultures not exhibiting
hemadsorption before recording a negative result.
[A suitable positive control can be established by
infecting a flask of rhesus monkey kidney cells with
0.2 ml of undiluted ATCC VR-95 (influenza virus strain
A/PR/8/34) 48 to 72h before testing.]
4. Egg Inoculations
- Drill a small hole in the egg air sac (blunt end)
using the electric drill (Cole-Parmer) and a 1/16-in.
burr-type bit or an 18-gauge needle in this and subsequent
operations; work with sterile instruments. Swab
areas of the shell to be drilled with 70% ethanol before and after each manipulation. The drill bits may be
placed in 70% ethanol before use.
- Using the candling lamp (Nasco), locate the area
of obvious blood vessel development and, at a central
point, carefully drill through the shell, leaving the shell
- Place 2 or 3 drops of Hanks' saline on the side
hole and carefully pick through the shell membrane
with a 26-gauge syringe needle. The saline will seep in
and over the chorioallantoic membrane (CAM) to facilitate
its separation from the shell membrane.
- Apply gentle suction to the hole in the air sac
using a short piece of rubber tubing with one end to
the mouth and the other pressed to the blunt end of
the egg. Use the candling lamp to monitor formation
of the artificial air sac over the CAM.
- Seal both holes with squares of adhesive or
laboratory tape and incubate the eggs horizontally at
37°C. Standard cell culture incubators and walk-in
rooms are entirely adequate for egg incubations. Highhumidity
or air/CO2 boxes are not satisfactory.
5. Cocultivation Trials
- Obtain suspensions of test cells in the appropriate
growth medium and adjust the concentration such
that 0.2 ml contains 0.5 to 1 × 107 cells.
- Remove the seal from side holes in the embryonated
eggs and inject 0.2ml of the cell suspension
onto the CAM of each of 5 to 10 eggs.
- Using the candling lamp, examine the embryos
1 day after adding the cell suspension; discard any
embryos that have died. Repeat the examination periodically
for 8 to 9 days.
- If embryos appear to be viable at the end of the
incubation period, open the eggs over the artificial air
sac and examine the CAM carefully for edema, foci, or
pox. Check the embryo itself for any gross abnormalities
such as body contortions or stunting.
- In cases in which viral contamination is indicated,
repeat steps 1-4 both with a second aliquot of
the suspect cells and with fresh fluid samples from
eggs in which the embryos have died or appear abnormal.
Positive controls may be established by inoculating
eggs with influenza virus, Newcastle disease virus,
and/or Rous sarcoma virus.
- Select two appropriate cell lines for cocultivation
with each cell line to be tested. The lines chosen will
depend on the species from which the test cell line
originated. For example, for a human cell line, one could cocultivate with ATCC CCL 75 (WI-38), ATCC
CCL 171 (MRC-5), or primary human embryonic
kidney (HEK) cells. A cell line from a second species
of choice in this example could be ATCC CCL 81 (Vero)
originating from the African green monkey.
- Inoculate a T-75 flask with 106 cells from each
line in a total of 8 ml of an appropriate growth medium.
In some cases, the inocula may have to be adjusted in
an attempt to maintain both cell populations during
the cocultivation period. For example, if a very rapidly
proliferating line is cocultivated with a test line that
multiplies slowly, the initial ratio of the former to the
latter could be adjusted to 1:10. Similarly, the population
that multiplies slowly might have to be reintroduced
to the cocultivation flasks if it were being
overgrown by the more rapidly dividing cells.
- Change the culture fluid twice weekly and subcultivate
the population as usual soon after it reaches
- Examine periodically for CPE and hemadsorption
over a 2- to 3-week period at minimum, using
procedures described earlier.
Viral isolates may be identified through standard
neutralization (hemadsorption inhibition, plaque inhibition,
hemagglutination inhibition) or complement
fixation tests. The ATCC virology department retains
and distributes antisera to many viral serotypes, and
identification can be accomplished readily.
6. Reverse Transcriptase Assays
Positive serological assays for retrovirus antigens in
cells and cell packs indicate that a retrovirus genome
is present, but these assays do not indicate whether
release of progeny virus particles is occurring. It has
been found that the concentration of particulate material
from culture supernates and the assay for viral
RDDP (Baltimore, 1970; Temin and Mizutani, 1970)
provide a sensitive and reliable means for detecting
retrovirus production by cultured cells.
a. Preparation of Cell Cultures
. Cell cultures to
be examined for the production of retrovirus should be
cultured by the methods and in the media that are
optimal for the particular cells. It is important that the
cells be in good condition and not undergoing degeneration
b. Processing of Culture Fluid
- When adherent cell cultures are about 50 to 60%
confluent, or when suspension cultures are at a cell
density about 50% of the maximum, completely
replace the medium and reincubate the cultures.
- Harvest fluid approximately 24 h after feeding.
c. Assay of RNA-Directed DNA Polymerase Activity.
- Collect culture medium aseptically.
- Clarify medium by centrifugation at 1000 to
3000g for 10rain at 4°C. Decant and save the clarified
supernates and discard sedimented materials.
- The clarified medium contains 0.15M NaCl;
add 5.0M NaCl to a final concentration of 0.5 M NaCl.
Calculate the volume of 5.0M NaCl according to the
formula 0.15(V1) + 5.0(V2) = 0.5 (1/1 + V2), where 1/1 is the
volume of clarified culture fluid and V2 is the volume
of 5.0M NaCl to be added. Mix well. If the medium
becomes cloudy after adding NaCl, centrifuge at 10,000 g for 10rain and save the supernate.
- To 2 volumes of clarified supernate containing
0.5M NaCl, add 1 volume 30% PEG 6000 in 0.5M
NaCl. Mix well.
- Allow precipitation to occur for at least 1 h while
holding in wet ice. At this point samples may be held
overnight at 4°C if necessary.
- Centrifuge at 7000g for 10 min.
- Decant and discard the supernates.
- Drain the pellets thoroughly while holding at
- Resuspend the pellets in 50% (v/v) buffer A [0.05 M Tris-HCl, pH 7.5, 0.1M KCl, 0.5mM EDTA, 10mM
dithiothreitol, 0.05% (v/v) Triton X-100, and 50%
glycerol]. Care must be taken to ensure that pellets
are completely resuspended.
- Store resuspended pellets at-20°C Aliquots are
used for RDDP assays.
This procedure is based on that of Gallagher and
- Stock mix: 0.5% (v/v) Triton X-100, 1.13M KCI.
- Template-primer solutions (P-L Biochemicals). Mix
A: Combine 1 mg/ml poly(rA)-poly(dT)12-18s with 0.01 M Tris-HCl, pH 7.5, and 0.1M NaCl. Mix B: Combine
1 mg/ml poly(dA)-poly(dT)12-18 with 0.1M NaCl and
0.01 M Tris-HCI, pH 7.5.
- Working mixtures of template-primer solutions: Mix
stock mix and template-primer solutions in 3:2 (v/v)
- Reaction cocktail: Evaporate 250 µl [3H]TTP
(carrier-free, New England Nuclear 221-X) to dryness
under vacuum. Redissolve in 720/µl H2O before
adding the following components (volumes given are
for 10 tubes): 1.0M Tris-HCl, pH 7.8 (40µl), 0.2M
dithiothreitol (40µl), 0.01 M MnCI2 (50µl). Add MnCI2 last (just before initiating reactions).
- Distribute culture medium concentrates and
positive and negative control samples into siliconized
10 × 75-mm assay tubes. (a) For positive controls,
use concentrates prepared from culture media of
cell cultures known to be producing retroviruses.
(b) For negative controls, use buffer A-glycerol
- Add appropriate template-primer mix to each
tube and mix with a vortex mixer. Hold tubes in wet
ice for 15 min.
- Initiate reactions by adding reaction cocktail to
each tube and mixing. Allow reactions to proceed for
30min in a 37°C water bath.
- At the end of incubation period, remove tubes to
an ice bath; terminate reactions by adding 25B1 0.1M
EDTA per tube (Sethi and Sethi, 1975).
- Spot 100µl from each tube onto appropriately
numbered DE-81 filters. Allow liquid to soak into
- Wash batches of filters with gentle manual
swirling in at least 10ml (per filter) of 5% (w/v)
Na2HPO4 . 7H20. Repeat for a total of six washes (Sethi
and Sethi, 1975).
- Wash twice with distilled H20 and twice with
95% ethanol and arrange filters on cardboard covered
with absorbent paper. Dry thoroughly under a heat
- Place each filter in a separate numbered scintillation
vial, add 10 ml PPO-POPOP scintillation cocktail
(Betafluor) to the vial, and count in a liquid scintillation
counter (Beckman LS-3133) using a tritium
A number of precautions must be observed in the
interpretation of the results obtained in this assay. If
the cultures to be tested are very heavy and undergoing
autolysis, a large amount of cellular DNA-directed
DNA polymerase may be associated with microsomal
particles in the culture medium. These particles are
concentrated by the polyethylene glycol procedure just
as virus particles are. Because cellular DNA polymerases
do not exhibit an absolute specificity for
a DNA template, a certain level of [3H]TMP
H]thymidylate) incorporation directed by an RNA
template will result from cellular polymerase activity.
If a high level of DNA-directed cellular polymerase
activity is present in medium concentrates, the (sometimes
high) degree of incorporation by these enzymes
can mask true RDDP activity, which may be present.
Consequently, it is important that media be collected
from healthy, actively growing cultures.
It must be remembered that enzymes that catalyze
the polymerization or terminal addition of [3
with a poly(rA) 9 oligo(dT) template-primer are not
exclusively viral (Harrison et al
., 1976). poly(rA)
DD-CC oligo(dT) is generally employed because the
activity of retroviral RDDP is usually greater with that
template-primer than it is when measured by the
incorporation of dGMP (deoxyguanylate) directed by
poly(rC) 9 oligo(dG1) or by methylated derivatives of
the poly(rC) template; however, DNA synthesis
directed by poly(rC) 9 oligo(dG) is more specific for
viral enzyme. Consequently, medium concentrates
that show incorporation of [3
H]TMP with the poly(rA)
template should be tested for the incorporation of
H]dGMP directed by a poly(rC) template.
Incorporation of isotopic precursors into macromolecular
form is generally detected by the precipitation
of macromolecules with trichloroacetic acid after the enzymatic reaction is terminated. Although background
levels of radioactivity may be somewhat
higher by the use of adsorption to and elution from
ion-exchange filter paper, the ion-exchange procedure
obviates the need for a filtration manifold, which is
generally employed for acid precipitation. Also, the
batch method employed allows many more samples to
be processed efficiently.
7. A Rapid PCR-Based Procedure for Detecting the
Presence of HIV and HTLV RNA in ATCC Cell Lines
An additional high-throughput method has been
adopted to screen for HIV and HTLV RNA in ATCC
cell lines using quantitative PCR. Priorities include
lines, which might be expected to support growth of
these viruses, namely T cells, macrophages and monocytes,
lines from the brain and nervous system, lines
of gastrointestinal origin, selected human hybridomas,
Notes and Results
- Total RNA is extracted from 106 cells of selected
cell lines (Qiagen, Valencia, CA).
- After quantitative and qualitative analyses using
the Bioanalyser 2100, 500ng of RNA is reverse transcribed
in the presence of a mixture of random hexamers
and oligo(dT). For real-time PCR (ABI PRISM
700, sequence detector, Foster City, CA), 12.5ng of
initial RNA (0.5 µl of cDNA reaction) is used.
- Specific primers and TaqMan probes are used
individually to measure levels of HIV-1, HTLV-1, and
GAPDH transcripts. For HIV-1 and HTLV-1, the Sybr
Green I dye assay is also used.
Both the SybrGreenI dye (Molecular Probes,
Eugene, OR) and TaqMan assays for HIV-1 and HTLV-
1 show an amplification signal. GAPDH transcripts,
used as an internal control, are detectable in all the cell
lines tested. Every sample is run on triplicates, and an
average is calculated. Absolute quantities of HIV,
HTLV, and GADPH are calculated using the standard
DNA method as described in User Bulletin 2 (PE
Applied Biosystems, Foster City, CA).
We have used two different detection methods: Sybr
Green I dye or the TaqMan probe method. Sybr Green
I is a double-stranded DNA-binding dye, which, when
added into the PCR mix, binds to the amplicon, the
double-stranded DNA fragment produced during
PCR. As the PCR progresses, more amplicons are
created. Due to the binding of Sybr Green I dye to all
double-stranded DNA, the increase in fluorescence
is proportionate to the amount of PCR product. In
addition to forward and reverse primers, TaqMan technology uses a sequence-specific oligonucleotide
labeled with fluorescent dye at the 5' end and a
quencher dye at the 3' end. While the probe is intact,
the proximity of the quencher dye reduces the fluorescence
emitted by the reporter dye by fluorescence
resonance energy transfer (FRET) through space. If the
target sequence is present, the probe anneals downstream
from one of the primer sites and is cleaved by
the 5' nuclease activity of Taq
DNA polymerase during
primer extension (Figs. 1A and 1B). The cleavage of the
probe separates the reporter dye from the quencher
dye, increasing the reporter dye signal (Fig. 1C). Additional
cleavage of the probe occurs at every cycle,
resulting in an increase of the fluorescent signal proportional
to the amount of the amplicon. Thus the
presence of the TaqMan probe enables detection of the
specific amplicon as it accumulates during PCR cycles.
|FIGURE 1 Description of 5' nuclease assay. (A) Sequence-specific
primers and dual-fluorescence TaqMan probe anneal to complementary
sequences in the DNA template. Due to frequency resonance
energy of transfer emission of the fluorescence dye (reporter)
is reduced significantly by the presence of the proximal quencher.
(B) Due to Taq polymerase activity, primer extension and synthesis
of a complementary strand occur. (C) While extending, due to its 5'
nuclease activity, the Taq polymerase cleaves the TaqMan probe and
enables the release of a fluorescent signal by the reporter.
|FIGURE 2 Real-time PCR assays for the detection and quantitation of HIV. Amplification plots (A) and
standard curves (B) generated for the quantitation of HIV. (C) Amplification plots of unknown templates
(total RNA from human cell lines).
HIV- and HTLV-specific primers and probes were
designed to avoid false-negative samples due to
nucleotide variation in the target sequence (Desire
2001; Schutten, 2000; Bisset, 2001). Blast search of
GenBank indicated that the probe and primer set used
in these analyses will detect the majority of HIV-1
subtypes and HTLV-I. Total RNA was extracted from
frozen cell pellets for all the cell lines used in the test.
For CRL-8993, total RNA was used from both frozen and fresh cells as a comparison. During RNA extraction,
DNase I was added to avoid contamination of
RNA extracts with genomic DNA. Extracted RNA was
evaluated using the 2100 Bioanalyzer (Agilent Technologies,
Wilmington, DE). Although the amount of
total RNA extracted from frozen cells is significantly
lower than the total RNA extracted from fresh cells, the
RNA extracted from frozen cells is sufficient for several
RT-PCR assays and no RNA degradation was
observed. Total RNA (300ng) was reverse transcribed
in a 20-µ reaction using Superscript (Invitrogen, Carlsbad,
CA) and 1 µl of the cDNA product was used for
real-time PCR. We have used both Sybr Green and
TaqMan methods to quantitate and detect HIV-1 and
HTLV-I viruses in human cell lines. For absolute quantitation,
known amounts of plasmid DNA containing
full-length or partial sequences of HIV and HTLV were
used in parallel with the unknown templates. PCR was
performed using the same set of primers and the
TaqMan probe for both standard and sample cDNA.
Amplification plots and standard curves were generated
for HIV (Fig. 2A) and HTLV (data not shown).
The absolute amounts of HIV and HTLV for each
sample were calculated based on the standard curves
(Figs. 2B and 2C). We have also analyzed levels of
GAPDH as an endogenous control, and standard
curves for GAPDH were generated using plasmid
DNA containing a GAPDH cDNA (Fig. 3). Absolute
amounts of GAPDH were also calculated based on the
standard curve method.
|FIGURE 3 Real-time PCR assays for the quantitation of endogenous GAPDH transcripts as an internal
control. Amplification plots (A) and standard curves (B) generated for the quantitation of GAPDH.
(C) Amplification plots of endogenous GAPDH from each specific cell line.
|FIGURE 4 Detection and quantitation of HIV, HTLV, and
GAPDH transcripts based on real-time PCR analyses.
Quantitation of copies of viral HIV and HTLV, as
well as quantitation of endogenous GAPDH, is shown
in Table V and Fig. 4. Sybr Green assays were run for
the quantitation of HIV, HTLV, and GAPDH, analyses
were performed using the standard curve method, and
similar results were obtained (data not shown). Therefore,
if primers are well designed and PCR conditions
are such that do not generate nonspecific amplicons or
primer dimers, testing for the presence of viral RNA
using Sybr Green I dye could be less expensive and as
effective as the TaqMan assay. As shown in Figs. 3 and
4, HIV and HTLV viral RNA was detected only in CRL-
8993 and CRL-8543 cell lines. CRL-8993 is a lymphoid
cell line reportedly positive for HIV, and CRL-8543
is also reported to contain HIV and HTLV viruses.
CRL-8993 was used as a control, and total RNA was
extracted from both fresh culture and frozen cells. As
shown in Fig. 4, the difference in absolute copies of
either GAPDH or HIV transcripts between the two
RNA extracts is insignificant, suggesting that total
RNA extracted from frozen cells could be used effectively
for RT-PCR analyses.
In summary, we have established a quick and accurate
method for the detection of HIV-1 and HTLV-I viruses. Given the high sensitivity of real-time PCR,
very low amounts of total RNA are needed for PCR
analyses. Therefore, RNA can be extracted directly
from frozen cell pellets and particular cell lines can be
tested for the presence of viral RNA prior to cultivation.
Real-time PCR offers the possibility of detecting
and quantitating as low as 10 copies of viral DNA.
IV. GENERAL COMMENTS
The microbial contamination and viral infection of
cell lines are still extremely serious problems. Mycoplasmal infection has been especially well
studied, and the incidence of problems has been
documented through government-funded programs.
Screening results reported within the past two decades
showed that as many as 4 to 33% of cultures tested
were infected with one or more species of mycoplasma
(Hay et al., 1989). It is absolutely imperative that cell lines
used in research or production work be tested routinely
for such adventitious infection. The comparative
cost in time and materials is extremely small. Rewards
in terms of research or production reliability are
Testing for viral infection is more problematical in
that it is expensive and multiple tests are required to
provide even a limited degree of assurance on freedom
from infection. We recommend consideration of
screening on a case-by-case basis depending on anticipated
use for the line, funding available, and a riskversus-
benefit analysis. Of course, a potential health
hazard for cell culture technicians is a major concern.
Finally, it is also critically important to verify the
identity of cell lines employed. Hukku et al. (1984)
documented the incidence of cross-contamination of
cell lines reporting misidentifications in excess of 35%.
Thus, a reasonably rigorous authentication
pro-gram must include not only reliable tests to
ensure an absence of microbial infections (including
mycoplasma), but also cell species verification.
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