VIROMED LABORATORIES

NOTE:
The following is an excerpt from ViroMed’s Clinical Laboratory Testing Services & Products
Catalog. Page references within the text reproduced here may refer to other sections of
the catalog not part of this file.

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CLINICAL LABORATORY

Testing Services
& Products

PHONE 952.563.3300
800.582.0077

FAX FOR ADMINISTRATION 952.939.4215

FAX FOR LABORATORIES 952.939.4012

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ADDRESS ViroMed Laboratories

6101 Blue Circle Drive
Minneapolis, MN 55343

WEBSITE www.viromed.com

Information effective as of January 31, 2001

CLTSP0101A
 USING CELL CULTURES IN THE CLINICAL LABORATORY
Specimen Selection
Guidelines for the selection, collection and transportation
of specimens is provided in the "Specimen Guide" section
of this catalog. See especially the "Specimen Selection
for Viral Cultures" on Pages S-6 and S-7.
Viruses and the Cell Cultures Used for Their Isolation
A chart on Pages U-2 and U-3 of this section lists viruses,
the cell cultures commonly used for their isolation and the
characteristic cytopathic effect (CPE).
Illustrations of CPE produced by viruses in cell culture can
be found Page U-9 of this section.
U-1
Diagnostic Techniques Utilizing Cell Cultures
The assay descriptions provided in this section are
intended as a guide to well-established, standard
techniques for the detection of viruses using cell cultures.
Conventional Tube Culture
The fact that many viruses, when inoculated into cells,
cause a characteristic cytopathic effect (CPE), is the
guiding principle of conventional tube cultures.
Appropriate cell cultures, chosen for their susceptibility to
the suspected virus, are inoculated with prepared
specimens. The cultures are then incubated and
observed, and viral replication may be detected, in the
case of most isolates, by the development of cytopathic
effects and/or hemadsorption.
See assays for:
• Cytopathic Effect (Page U-4).
• Hemadsorption (Page U-4).
• Neutralization (Page U-5).
Detection of Viral Antigens
A concerted effort to provide the clinical laboratory with
more rapid means of isolating and identifying viruses from
clinical specimens has been accomplished in many
instances through methods that detect viral antigens by
using specific monoclonal antibodies. These methods rely
on the ability of virus-specific antibodies to bind to viral
antigens. With the availability of a wide range of antigen-
specific monoclonal antibodies, detection of viruses may
be accomplished with a higher degree of sensitivity and
specificity than was previously possible.
The antibodies are the key components in a number of
different test formats, including immuno-fluorescence
(IFA), enzyme-linked immunoassay (ELISA), and latex
agglutination (LAA).
See Shell Vial Assay (Page U-6).
 VIRUSES, CELL CULTURES COMMONLY USED FOR THEIR ISOLATION,
AND CHARACTERISTIC CYTOPATHIC EFFECT (CPE)

Virus Cell Cultures Characteristic CPE

Adenovirus A549, HEK293, HeLa, Tightly associated clusters or lattice-like

HEp-2, MRC-5, SF, WI-38 larrangements of enlarged, refractile,
rounded cells.

Coxsackie A Rhesus monkey kidney, Highly refractile, rounded cells throughout

LLC-MK2, MRC-5, RD, SF, monolayer or in loose clusters.
WI-38

Coxsackie B Rhesus monkey kidney, Highly refractile, rounded cells throughout

BGMK, HeLa, HEp-2, monolayer or in loose clusters.
LLC-MK2, Vero

Cytomegalovirus MRC-5, SF, WI-38 Plump, rounded cells in elongated

foci parallel to long axis of cells.

Echovirus Rhesus monkey kidney, Highly refractile, rounded cells throughout

LLC-MK2, MRC-5, SF, WI-38 monolayer or in loose clusters.

Herpes Simplex Rabbit kidney, A549, HeLa, Grape-like clusters of rounded, refractile
HEp-2, Mink lung, MRC-5, cells with or without syncytia. Early
RD, SF, Vero, WI-38 CPE is focal but progresses throughout
monolayer.

Influenza A, B Rhesus monkey kidney, Varies. CPE may be absent or appear

LLC-MK2, MDCK as vacuolated, granular or non-specific
cell degeneration. Rounded refractile
cells are more frequently associated
with Type B.

Measles Rhesus monkey kidney Syncytia appear as large multinucleated

refractile areas. Nuclei may encircle
granular mass of giant cell. Extensive
vacuolization may also be present.

Mumps Rhesus monkey kidney, Variable, with increased rounding,

LLC-MK2, Vero granularity, progressive degeneration
and syncytia.

Parainfluenza Rhesus monkey kidney, Variable, with increased rounding,

LLC-MK2 granularity, progressive degeneration.
Syncytia are associated with parainfluenza
type 2 and 3 viruses.

U-2
 VIRUSES, CELL CULTURES COMMONLY USED FOR THEIR ISOLATION,
AND CHARACTERISTIC CYTOPATHIC EFFECT (CPE)

Virus Cell Cultures Characteristic CPE

Poliovirus Most fibroblast and Highly refractile, rounded cells with

heteroploid cell lines rapid destruction of monolayer.

Rhinovirus MRC-5, WI-38 Highly refractile, rounded cells throughout

monolayer or in loose clusters.

RSV HeLa, HEp-2, MRC-5,WI-38 Syncytia appear as large,

multinucleated refractile areas.

Rubella BHK-21, BS-C-1, LLC-MK2, CPE may be produced in serially

RK 13, SIRC propagated cell lines but may require
several passages to develop.
Early CPE -- Foci of rounded cells which
progress to scattered rounded cells
with loss of detachment to tube surface.

Varicella Zoster A549, MRC-5, SF, WI-38 Foci of small, rounded, refractile cells
with or without syncytia. Cytoplasmic
strands between infected cells, as
well as granularity, may be prominent
as CPE progresses.

CHLAMYDIA

C. pneumoniae H292, HeLa 229, HEp-2 CPE -- Not relevant.

The presence of Chlamydia is usually
C. psittaci BGMK, McCoy determined by staining methodologies.

C. trachomatis BGMK, McCoy

U-3
 DIAGNOSTIC TECHNIQUES UTILIZING CELL CULTURES
CONVENTIONAL TUBE CULTURE
Cytopathic Effect
Virus replication in susceptible cell culture produces the
degenerative morphologic changes referred to as
cytopathic effects (CPE), which may appear as a rapid
and extensive rounding of the cells or as a slowly
progressive change involving only discrete foci.
Certain viruses produce such characteristic CPE that a
presumptive identification can be made on this basis.
Preliminary identification of specific viruses is usually
dependent upon the type of CPE, the length of time
required for CPE to develop, and the type of cell culture
in which CPE appears.
Hemadsorption
Certain viruses – by virtue of viral-specific proteins
(hemagglutinins) expressed on the surface of infected
cells – are able to agglutinate erythrocytes. The
hemadsorption procedure takes advantage of this
property. In the procedure, a suspension of guinea pig
or chicken erythrocytes is added to an inoculated cell
culture, and after an incubation period, the culture is
examined microscopically for evidence of adherence of
erythrocytes to the infected cells. If hemadsorption is
detected, the specimen can be reported to contain a
hemadsorbing agent. Further studies using virus-
specific antisera that selectively block hemadsorption
may then be performed to precisely identify the
hemadsorbing virus.
U-4
PROCEDURE
1. Select tube of appropriate cell culture, with confluent
or nearly confluent monolayer, and remove
maintenance medium.
2. Inoculate cells with 0.2ml of the specimen.
3. Refeed tube with 1-2ml of maintenance media.
4. Incubate tube at 35-37°C. (Medium should cover the
monolayer, but should not reach as far as the tube's
cap or neck.)
5. Observe the inoculated culture microscopically for
the cytopathic effect(s) of viral replication.
(See Page U-9 for illustrative examples of typical viral CPE.)
PROCEDURE
1. Remove maintenance media from tubes previously
inoculated with patient specimen and from
uninoculated control tubes.
2. Wash the cell monolayers three times, using 2ml of
Hanks' balanced salt solution (HBSS) for each wash.
3. Add 2ml of HBSS.
4. Add 0.2ml of 0.4% washed guinea pig erythrocytes
to each of the cell cultures.
5. Incubate tubes at 4°C for 15 minutes. (Tube caps
should be tightened and tubes placed in a slanted
position. Medium should cover the monolayers, but
should not reach as far as the tubes' caps or necks.)
6. Rock the tubes gently and observe them
microscopically for evidence of hemadsorption.
7. Then, incubate the tubes at room temperature 15-30
minutes and observe them again microscopically for
evidence of hemadsorption.
8. Tubes in which erythrocytes adhere to the cell
monolayer (while the control tubes remain negative)
are considered positive for hemadsorption.
Note: Non-specific hemadsorption may occur, but may
be distinguished from specific hemadsorption by
comparing the degree of hemadsorption of the
uninoculated control tubes with tubes inoculated with
the patient specimen.
 DIAGNOSTIC TECHNIQUES UTILIZING CELL CULTURES
CONVENTIONAL TUBE CULTURE
Neutralization
Viral neutralization, defined as the loss of infectivity
through reaction of the virus with specific antibody, may
be used either to identify an isolate or to measure the
antibody response to a virus. The test has a high
degree of immunological specificity that makes it the
standard method for identifying certain viruses,
specifically identification of adenoviruses and
enteroviruses. To perform the neutralization test, virus
and specific antibody are incubated together and then
inoculated into cell cultures. If the virus is not
neutralized, it may be detected by the presence of
cytopathic effects (CPE) or hemadsorption. If the
antibody is specific for the virus, however, the virus will
be neutralized and will not produce CPE or
hemadsorption.
U-5
PROCEDURE
1. Using a viral dilution that contains 100 TCID50 , mix
0.3ml of the virus with 0.3ml of specific antiserum.
2. In a separate tube, mix 0.3ml of the virus with 0.3ml
of maintenance media. This will serve as the virus
control.
3. Shake the mixtures and incubate both at 36-37°C for
2 hours.
4. Inoculate each of 2 cell culture tubes with 0.2ml of
the virus-serum mixture and inoculate each of 2
separate cell culture tubes with 0.2ml of the virus
control mixture.
5. To determine the number of TCID50 actually present
in the test, add 0.1ml of serially diluted virus (100
TCID50 , 10 TCID 50 , 1 TCID 50 , 0.1 TCID50 ) to each of
2 tubes.
6. Add 0.2ml of maintenance media to 2 uninoculated
cell culture tubes. These serve as negative cell
controls.
7. Incubate tubes at 36-37°C and observe daily.
Results are usually apparent on day 3 or 4 with final
readings on day 6 or 7.
8. Findings: The negative cell controls should show no
CPE. The virus control should show CPE. The virus-
serum tube – if the virus has been neutralized by the
specific antiserum – should show no CPE.
 DIAGNOSTIC TECHNIQUES UTILIZING CELL CULTURES
DETECTION OF VIRAL ANTIGENS
Shell Vial Assay for the Rapid Detection
of Viral Infection
The shell vial assay is a spin-amplification technique
that significantly decreases the length of time needed
to detect positive cultures. The assay makes possible
the rapid diagnosis of many viral infections through the
use of cell cultures propagated on coverslips contained
in flat-bottomed shell vials. (Cell cultures seeded in
wells of microtiter plates may be substituted for shell
vials.) Viral antigens may be detected within 24 hours
after inoculation. Even though cytopathic effects have
not had time to develop, viral antigens – synthesized in
the early stages of replication – may be detected when
the cell cultures are stained with virus-specific
fluorescein-labeled monoclonal antibodies and read
under a fluorescence microscope. The viruses to
which this rapid detection method may be applied
include herpes simplex, measles, cytomegalovirus,
adenovirus, varicella zoster viruses, influenza,
parainfluenza, and respiratory syncytial virus.
U-6
PROCEDURE
Inoculation
1. Determine the cell cultures to be inoculated on the
basis of suspected virus(es).
2. Remove and discard the medium from the shell vial
cell cultures to be inoculated.
3. For blood cultures, inoculate each of three vials with
0.2 ml of the WBC preparation (combined mono-
nuclear and PMN cells). For all other specimens,
inoculate each of two vials with 0.2 ml of specimen.
4. Inoculate 2 separate vials with 0.2 ml of main-
tenance medium. These will serve as negative
controls. Also inoculate 2 other vials with the
appropriate virus control. These will serve as positive
controls.
5. Centrifuge the vials at 700 x g for 1 hour at room
temperature.
6. After centrifugation, add 1.0 ml of maintenance
medium to each vial.
7. Incubate at 35-37°C for 16 to 24 hours or longer.
The duplicate vials may be stained at the same time
or at different times.
Harvest and fixation
1. Aspirate the medium from the cultures.
2. Wash each monolayer two or three times by
sequentially adding and removing 1 to 2 ml of PBS
(pH 7.2 to 7.6) for each wash cycle.
3. Add 1 to 2 ml of chilled acetone to each vial, and fix
monolayers for 20 minutes.
4. Aspirate acetone, and allow the reagent to evaporate
completely.
5. Proceed with immunostaining of the monolayers.
Alternatively, the fixed monolayers may be stored at
this point for staining at a later time. Recap the vials
or seal them with Parafilm, and store at 4°C for up to
5 days or at -70°C for longer periods.
[Continued]
 DIAGNOSTIC TECHNIQUES UTILIZING CELL CULTURES
DETECTION OF VIRAL ANTIGENS
PROCEDURE [Continued]
Immunofluorescence staining
1. Determine the optimal dilutions of the antibody
reagents unless these are designated by the
manufacturer.
2. Add 100 to 150 µl of the appropriate antibody
reagent to each shell vial. Be sure that the entire
cell culture monolayer is covered, and do not allow
the monolayers to dry once the staining procedure
has been started.
3. Incubate the vials for 30 minutes at 35 to 37°C (or
as specified by the reagent manufacturer) in a moist
chamber, or cover the vials with moistened towels.
4. After incubation, wash the monolayers twice by
sequentially adding and removing 1 to 2.0 ml of
PBS for each wash cycle. Antibody does not need
to be aspirated before the first washing.
5. When using an FITC-labeled (direct) primary
antibody, proceed to step 8.
6. When using an unlabeled (indirect) primary
antibody, add 150 µl of FITC-labeled antibody to
each vial, and incubate as described in step 3.
7. Wash all monolayers twice with PBS as described
in step 4. Alternatively, the last wash may be with
distilled water to eliminate residual salt.
8. Allow stained monolayers to air dry completely.
9. Using slightly bent fine-tipped forceps, remove the
coverslips from the shell vials and transfer them to
labeled microscope slides. Mount coverslips cell
side down onto a drop of buffered glycerol.
10. Examine the entire monolayer at x200. Observe
questionable areas at x400 to differentiate between
specific and non-specific staining.
Gleaves, C.A., T. F. Smith, E. A. Shuster, and G. R. Pearson. 1984. Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by
using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbiol. 19: 917-919.
Gleaves, C.A., T. F. Smith, E. A. Shuster, and G. R. Pearson. 1985. Comparison of standard tube and shell vial cell culture techniques for the detection of
cytomegalovirus in clinical specimens. J. Clin. Microbiol. 21: 217-221.
U-7
RESULTS
Controls:
Uninfected monolayers (negative controls)
should show typical cellular morphology with no
specific and only minimal nonspecific fluorescence.
The positive control should reveal cells with
intense (3+ to 4+) apple green nuclear and /or
cytoplasmic fluorescence. The cellular pattern of
fluorescence depends on several factors, including the
type of virus and the specificity of the antibody, or
antibodies, in the immunoreagent. For example,
staining with a monoclonal antibody directed against
CMV early nuclear protein will be confined to a
homogeneous nuclear fluorescence. However, both
nuclear and cytoplasmic staining will result when this
antibody is used in combination with one directed
against late CMV proteins.
Specimens:
• A negative culture is one exhibiting no specific
fluorescence and minimal or no nonspecific staining.
• A positive culture is one exhibiting characteristic
specific staining of the nucleus, cytoplasm, or both,
depending on the virus and immunoreagent.
• An unsatisfactory culture is one that does not
show characteristic specific fluorescence and exhibits
either intense nonspecific fluorescence or excessive
destruction of the monolayer by specimen toxicity or
contamination.
U-8
U-9