Virology Guide

Virology Guide
Table of Contents
This guide contains general technical information for viral growth, propagation, preservation, and application. Additional information on
viral culturing can be requested from ATCC Technical Services at tech@atcc.org or can be found in A Manual of Basic Virological Techniques.¹
Getting Started with an ATCC Viral Strain.............3 Biosafety...........................................................................................19

Product Sheet.................................................................................... 3 Disposal of Infectious Materials.....................................................19
Viral Taxonomy .................................................................................. 3
Viral Authentication and Viability Testing............ 20
Viral Replication................................................................................. 3
Viability Testing................................................................................20
Preparation of Propagation Host and Reagents............................. 3
Viral Authentication........................................................................21
Opening Glass Ampoules Containing Frozen Material.................. 4
Initiating Frozen Cultures................................................................. 4 Viral Applications.................................................... 22
Cell Transformation.........................................................................22
Viral Replication and Propagation............................6
Phage Therapy..................................................................................22
Propagation Host Range................................................................... 6
Nanotechnology...............................................................................22
Viral Propagation............................................................................... 6
Recombinant Vectored Vaccines....................................................23
Propagation of Common Viruses and Chlamydia........................... 8
Viral Titering....................................................................................... 9 Glossary................................................................... 24
Growth Media for Tissue Culture-adapted Viruses... 12 Appendix................................................................. 25
Media for Culturing Propagation Hosts........................................12
References............................................................... 26
Media Formulations.........................................................................12
Media Supplements.........................................................................12 ATCC Reagents for Virus Expansion .....................27
Preservation............................................................ 14
Cryopreservation.............................................................................14
Lyophilization...................................................................................16
Preservation of Specific Strains...................................................... 17
Special Hazards................................................................................18
Biosafety and Disposal............................................ 19
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GETTING STARTED WITH AN ATCC VIRAL STRAIN
ATCC viral strains are predominantly shipped either frozen on dry ice in plastic cryopreservation vials, or as lyophilized materials within
glass ampoules or serum vials. Upon receipt of frozen material, either thaw and transfer the viral agent to an appropriate propagation
host, or briefly store the frozen material between -70°C and -80°C to allow time to seed host cells. However, please note that the viabil-
ity of some materials may decline at temperatures above -120°C. Upon receipt of freeze-dried strains, reconstitute cultures with sterile,
double-distilled water and add the rehydrated material to an appropriate propagation host. If this is not possible, store the vials in liquid
nitrogen vapor phase (below -120°C).
PRODUCT SHEET
A product sheet that contains detailed information on the production host and recommendations for infection are provided for each
ATCC virology strain. This product sheet, as well as additional information, can be found on the ATCC website or can be requested from
the ATCC Technical Service Department.
V I R A L TA XO N O M Y
Viruses are placed into taxonomic groups based on characteristics including morphology,
genome type, and host organism. Viral agents can significantly vary in size, often ranging
between 20 and 300 nanometers in diameter. They also vary in structure, including helical,
icosahedral, prolate, enveloped, and complex morphologies.
In addition to unique morphological structures, viruses also vary in genomic structure.

Unlike other microorganisms that have double stranded DNA as genomic material, viral
genomes can be composed of double-stranded DNA, single-stranded DNA, double-stranded
RNA, or single-stranded RNA. Single-stranded RNA viruses can be further described as posi-
tive sense, negative sense, or ambisense. For more detailed information on the various
morphological and genomic types, please refer to the glossary.
Changes in taxonomy or further analysis of viral strains may lead to a change in nomencla-
ture. Taxonomic nomenclature as well as the common name can be found on the product Influenza ultra-structure courtesy of Jordan Douglas,
CDC
sheet. Further information on viral nomenclature is available from the International
Committee on Taxonomic Viruses.
V I R A L R E P L I C AT I O N
Viruses are pathogenic intracellular organisms requiring living cells in order to multiply. The virus life-cycle can be divided into three major
steps: attachment, assembly, and release. Generally, infection is established when the virus binds to a specific cellular receptor and enters
the host cell. Upon cellular entry, host proteins are recruited to assist with viral replication. Once viral structural proteins are generated, new
viruses assemble within the cell. Depending on the nature of the viral agent, the replication and assembly process can vary in cellular loca-
tion and process. Following viral assembly, new infectious particles either remain cell-associated or exit the cell via lysis or virus shedding.
P R E PA R AT I O N O F P R O PA G AT I O N H O S T A N D R E A G E N T S
In advance, prepare the propagation host and associated reagents necessary for viral propagation. Information for the preparation of
these products is available on the provided product sheet.
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OPENING GLASS AMPOULES
C O N TA I N I N G F R O Z E N M AT E R I A L FREEZE-DRIED PREPARATIONS
OVERVIEW Tip
Insulator potentially hazardous and should
All cultures should be considered
Cotton plug
be opened by individuals trained in microbiological techniques. Work Borosilicate glass
Outer vial (soft glass)
should only be carriedInner
out vial
in facilities with containment require-
Freeze-dried
ments appropriate for the biosafety pelletlevel of the cultures. The Freeze-dried virus
Cotton
handling or opening of glass ampoules containing virus material
Desiccant with indicator
must be performed in a biological safety cabinet. Ensure that all
empty vials are sterilized before disposal.
1 Disinfect the outside of the ampoule with freshly prepared 70% These preparations may be enclosed in a thin skin of
Heat theortip
ethanol ofitthe
dip outer
into a beaker of freshly prepared 70% ethanol. cellulose; this skin must be removed (either with a sharp
vial in a flame blade or by soaking in water for a few minutes). Score the
2 To recover the material from the glass ampoule, score the neck ampule once briskly with a sharp file about one inch
of the ampoule with a sterile, small file. from the tip.
3 Wrap the ampoule within several folds of a sterile towel or
gauze to dry residual ethanol.
4 Working in a biological safety cabinet, hold the vial upright and
snap open the vial. Ensure that your gauze does not become too
wet with ethanol, or ethanol could be sucked into the culture
when the vacuum is broken. Propagate the virus immediately.
Squirt a few drops of water
on the hot tip to crack glass
I N I T I A T I N G F R O Z E N C U LT U R E S
TISSUE CULTURE-ADAPTED STRAINS
Disinfect the ampule with alcohol-dampened gauze
1 In advance, prepare the cell growth medium for growing the
host cell line. Additionally, prepare the virus growth medium
for virus propagation as noted on the product sheet. Viral
growth medium is usually supplemented with a lower
percentage of serum than cell growth medium, often ranging
Strike with
between file ordepending on the virus (See NOTE 1). Ensure
2-10%
pencil
that to remove
both the celltip
and viral growth media are equilibrated for
temperature and pH.
2 Initiate the recommended production host, and grow the
monolayer to an appropriate confluency.
3 Prior to thawing the frozen virus stock, check the virus titer Wrap gauze around the ampule, and break at the scored area.
Care should be taken not to have the gauze too wet, or
listed on the lot specific certificate of analysis to determine the
alcohol could be sucked into the culture when the vacuum is
amount of virus needed. Thaw the vial of frozen virus via gentle broken. Rehydrate material at once.
agitation in a water bath set at 37°C. Thawing will be rapid
Remove insulation and inner vial with
(approximately 2 minutes
forceps, gently raise cottonorplug
until all ice crystals have melted).
4 Remove the vial from the water bath and decontaminate the
outer surface using 70% ethanol. Follow strict aseptic
conditions in a biological safety cabinet for all further
manipulations.
5 Prepare the viral stock in base medium (without serum) unless
specified.
6 Remove the cell growth medium from the cell culture, wash
the monolayer of cells once or twice with phosphate buffered
saline (PBS), and inoculate with the prepared viral stock to
provide an optimal multiplicity of infection (MOI) as indicated NOTE 1
on the product sheet, if applicable.
Viral growth medium prepared for tissue culture-adapted strains
of Influenza should not be supplemented with Fetal Bovine
Serum (FBS).
7 Propagate the virus according to the recommended infection conditions as described on the product sheet. For additional
information, refer to the chapter Viral Growth and Propagation.
STRAINS PROPAGATED IN CHICKEN EGGS

1 In advance, prepare chicken eggs by incubating eggs under the recommended temperature and atmospheric conditions.
Additionally, all eggs should be candled to ensure viability and properly developed embryos. Healthy embryos will have an air sac,
well-developed blood vessels, and observable movement.
2 Prior to thawing the frozen viral preparation, check the certificate of analysis for lot specific titer information. Thaw the frozen virus
vial via gentle agitation in a water bath set at 37°C. Thawing will be rapid (approximately 2 minutes or until all ice crystals have melted).
3 Remove the vial from the water bath and decontaminate the outer surface using 70% ethanol. Follow strict aseptic conditions in a
biological safety cabinet for all further manipulations.
4 Inoculate eggs using the inoculation procedure described in the chapter Viral Growth and Propagation.
INITIATING LYOPHILIZED CULTURES

1 Open vial according to enclosed instructions. Instructions are also available on the ATCC website, www.atcc.org, within the
instructional guide, “Reviving Freeze-Dried Microorganisms.”
2 Aseptically add an appropriate volume of sterile double-distilled water at room temperature. Mix well.
3 Dilute the sample in base medium (without serum) unless specified, and inoculate host cells to provide an optimal MOI as indicated
on the product sheet, if applicable.
VIRAL REPLICATION AND PROPAGATION
P R O PA G AT I O N H O S T R A N G E
For tissue culture-adapted strains, the appropriate selection and processing of cell cultures is important for successful viral isolation,
titer, and infectivity.² Typically, viruses can only infect a limited number of hosts, known as the host range. This is best explained by a “lock
and key” mechanism as certain proteins on the viral surface must fit specific receptor sites on the host cell surface. When using ATCC viral
strains, the recommended host is indicated on the provided product sheet. For customer convenience, ATCC Cell Biology holdings include
cell lines for the propagation of viruses (Table 1, Appendix).
Table 1: Examples of propagation hosts for tissue culture-adapted viruses

ATCC® number Product name ATCC® number Propagation host
53592™ Chlamydophila pneumonia* CCL-23™ HEp-2
VR-129B™ Encephalomyocarditis CCL-81™ Vero
VR-1492™ Human herpesvirus 4 (Epstein-Barr virus) N/A B95-8
VR-260™ Humans herpesvirus 1 CCL-81™ Vero
VR-734™ Human herpesvirus 2 CCL-81™ Vero
VR-1367™ Human herpes 3 CCL-171™ MRC-5
VR-538™ Human herpesvirus 5 CCL-171™ MRC-5
VR-93™ Human parainfluenza CCL-7.1™ LLC-MK2 Derivative
VR-26™ Human respiratory syncytial virus CCL-23™ HEp-2
VR-283™ Human rhinovirus 16 CRL-1958™ H1-HeLa
VR-95™ Influenza A virus (H1N1) N/A SPF CE
VR-1469™ Influenza A virus (H1N1) CCL-34™ MDCK
VR-1520™ Influenza A (H1N1) CCL-34™ MDCK
VR-1679™ Influenza A virus (H3N2) CCL-34™ MDCK
VR-1535™ Influenza B virus CCL-34™ MDCK
VR-1583™ JC polyomavirus CRL-1651™ COS-7
*For historical reasons and because of similar requirements for handling, Chlamydia and Rickettsia are included in this guide
V I R A L P R O PA G AT I O N
PROPAGATION IN CELL CULTURE
A number of ATCC viruses are propagated in cell culture. Typically, propagation hosts are grown in tissue culture vessels (such as T flasks)
using media and reagents specified for the host cell line. Most cell lines are seeded the day prior to setting up an infection and should not
be seeded more than two days in advance, nor passaged more than 9 times prior to infection. In addition to setting up cells for infection,
negative control cells should also be set up to monitor cellular health.
1 Identify the recommended propagation host as indicated on the product sheet. Plan to use the propagation host.
2 Prepare the cell growth medium for growing the host cell line.
3 Prepare the virus growth medium as recommended on the product sheet.
4 One to two days prior to inoculation seed the host cells. Be sure to include a vessel that will not be inoculated with virus to serve as a
negative control.
5 Allow cells to reach the appropriate confluency. (See NOTE 2) NOTE 2
6 Prior to thawing the frozen virus stock check the virus titer The required confluency of the host cell line will differ between
listed on the certificate of analysis. Quickly thaw the virus in a viruses.
37°C water bath.
7 Dilute the virus stock in the appropriate volume of viral growth medium.
8 Remove the cell growth medium from the cell culture flasks.
9 Inoculate the diluted virus to provide an optimal MOI as indicated on the product sheet.
10 Incubate tissue cultures under the appropriate temperature and atmospheric conditions for the recommended incubation period.
11 After the recommended incubation time period, check the flask for cytopathic effects (CPE) when applicable.
Propagation of Feline infectious peritonitis virus (ATCC VR-2004™) in CRFK cells (ATCC CCL-94™). The panel on the left are uninfected CRFK
cells. The panel on the right are infected CRFK cells exhibiting CPE.
PROPAGATION IN CHICKEN EGGS

Chorioallantoic Amniotic
Several ATCC virus holdings, such as influenza, are propagated in cavity
membrane
Chorioallantic membrane
embryonated chicken eggs. There are many advantages to cultur- inoculation
Shell
ing in eggs, including easy care, quick viral replication, and an
inherently aseptic environment. When culturing in embryonated
eggs, ensure that eggs are viable, have properly developed blood Amniotic
inoculation
vessels and air sacs and are obtained from pathogen-free flocks. Allantoic
inoculation A i r
Depending on the virus, different inoculation routes and/or organs sac
may be used to cultivate the agent. These include the allantoic Yolk sac
cavity, the amniotic cavity, the chorioallantoic membrane (CAM),

and the yolk sac (Table 2). Below, we describe the procedures for
Shell
allantoic cavity, amniotic cavity, and CAM inoculation. For the follow- membrane Yolk sac
inoculation
Allantoic
ing procedures, ATCC recommends performing viral inoculation and Albumin
cavity
harvest within a biological safety cabinet. For additional informa-
tion on the inoculation or propagation of embryonated eggs, contact ATCC technical services.
A Allantoic Cavity Inoculation
1 For allantoic cavity inoculation, most viruses require 10-day old embryos. While candling the eggs, draw a pencil line around the
air sac. Using an 18.5 gauge needle, poke a hole into the air sac.
2 Using a 1.0 mL syringe fitted with a 22.5 gauge needle, inject the viral inoculum through the hole, into the allantoic cavity, being
careful not to stick the embryo.
3 Seal the hole with tape or wax.
4 Incubate the inoculated eggs under conditions recommended for viral replication. Candle eggs 12-18 hours after inoculation and
discard eggs that are non-viable.
5 To harvest allantoic fluid, refrigerate eggs for at least 2 hours post-incubation. Once the embryo is no longer viable, open the egg
by tapping on the shell just above the air sac until the shell breaks.
6 Use sterile scissors to cut away the shell around the air sac.
7 Aspirate the allantoic fluid with a syringe or a pipette. Usually 5 mL can be harvested from each egg; this is considered infectious material.
B Amniotic Cavity Inoculation
1 While candling the eggs, make a small hole in the side of the egg. Create the hole slowly as it is easy to accidently stick the embryo.
2 Using a 1.0 mL syringe, insert the needle and syringe containing the viral inoculum into the egg until the amniotic sac moves
slightly. The needle is then thrust through the membrane and the fluid is injected slowly.
3 Immediately seal the hole in the shell with tape or wax.
4 Incubate the inoculated eggs under conditions recommended for viral replication.
5 Candle eggs 12-18 hours after inoculation and discard eggs that are non-viable.
6 To harvest amniotic fluid, refrigerate eggs for at least 2 hours post-incubation. Once the embryo is no longer viable, open the egg
by tapping on the shell just above the air sac until the shell breaks.
7 A needle and syringe must be used to aspirate the fluid. The needle may be inserted directly into the amniotic sac, or the embryo
may be carefully removed from the egg and placed in a petri dish. Typically, one embryo will yield about 1.0 mL of fluid.
C Chorioallantoic Membrane (CAM) Inoculation
1 Mark an area free from large blood vessels on the side where the embryo is located and the area over the air sac.
2 Make one hole on the side and one over the air sac. Puncture the shell taking care not to damage the CAM.
3 Gently apply suction to the hole over the air sac. Do this over an egg candler to see the CAM drop and the new air sac form.
4 Add 1.0 mL of the viral inoculum on the dropped membrane and rotate the egg to distribute the inoculum over the membrane.
5 Seal the hole with nail polish.
6 Candle inoculated eggs daily. Any embryos that die within 24 hours should be discarded.
7 To harvest, refrigerate eggs for at least 2 hours. Once the embryo is no longer viable, make a tape handle over the area of
inoculation. To do so, cut a piece of tape, without joining either end, join the middle of the piece together leaving both ends free
to adhere to the egg shell with a middle piece that is no longer sticky, forming a handle sticking up from the egg shell.
8 Cut off the top half of the eggshell, including the infected area, and gently remove the CAM, which is attached to the shell.
9 Place the infected CAM in a tissue culture dish with PBS, spread the membrane flat against the dish, and place the dish on a dark
surface to facilitate counting of pocks.
Table 2: Examples of ATCC viruses propagated in embryonated chicken eggs

Product name Egg age (days) Inoculation route Incubation (days) Optimal temperature (°C) Death of embryo
Influenza A virus 10-11 Allantoic 2-3 33-35 -
Influenza B virus 10-11 Allantoic 2-3 33-35 -
Sendai virus 10-11 Allantoic 2-3 35-37 +
Rabies virus 7 Yolk 9-10 36.5 -
P R O PA G AT I O N O F C O M M O N V I R U S E S A N D C H L A M Y D I A
HUMAN HERPESVIRUS 4 (EPSTEIN-BARR VIRUS)
Epstein-Barr virus (EBV) is a universal human pathogen commonly transmitted via saliva.³ Primary infections among adolescents and
young adults result in mononucleosis, a condition characterized by fever and the swelling of lymph tissues. Following infection, EBV
remains in a latent stage in most adults. In rare cases, the virus may reactivate and contribute to neoplastic disorders such as Burkitt’s
lymphoma or post-transplant lymphoproliferative disorder (PTLD).⁴ To propagate EBV, the virus is often harvested from tumors growing
on patients with Burkitt’s lymphoma, or obtained from a biological resource center such as ATCC, and further grown in human lympho-
blastoid cells.
ATCC number: VR-1492™, VR-603™, VR-602™
Recommended Host: Human lymphoblastoid cells
Preparation: Infected tissue culture with DMSO and FBS
Incubation: 5-15 days at 37°C
Atmosphere: 5% CO₂ in an air atmosphere
Effect: Polykaryocyte formation and cell swelling
Image of Influenza courtesy of Dr. Erskine L
INFLUENZA Palmer and ML Martini, CDC
Influenza viruses are highly contagious, enveloped, airborne pathogens that cause acute febrile illness, fatigue, and respiratory infection.
There are three types of influenza including influenza A, B, and C, which are distinguished by the presence of specific, core nucleopro-
teins. Of these viral types, influenza A is considered the most pathogenic. To propagate influenza, the virus is often cultured in the allantoic
fluid of embryonated chicken eggs. However, some strains are propagated in amniotic fluids or have been adapted for growth in tissue
culture.
ATCC number: VR-1469™, VR-95™, VR-1520™, VR-544™, VR-897™, VR-1679™, VR-1807™, VR-1811™, VR-1813™
Recommended Host: Pathogen-free embryonated chicken egg, 10-11 days old, or MDCK cells (ATCC CCL-34)
Preparation: Infected chicken egg allantoic fluid, infected tissue culture
Incubation: 2-3 days at 34°C for influenza propagated in chicken eggs (CE), 33-35°C for influenza propagated in tissue culture (TC).
Harvest when CPE has progressed throughout the monolayer.
Atmosphere: An air atmosphere for CE, 5% CO₂ atmosphere for TC. CO₂ level is determined by media formulation.
Effect: Hemagglutination of chicken red blood cells, cell rounding sloughing in tissue culture.
RESPIRATORY SYNCY TIAL VIRUS

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children,
resulting in pneumonia and bronchiolitis. In adults, RSV often manifests symptoms similar
to the common cold. This virus is commonly transmitted when droplets containing the virus
are aerosolized by coughing or sneezing.
ATCC number: VR-1540™, VR-1540P™, VR-26™, VR-1580™, VR-1803™
Recommended Host: HEp-2 (ATCC CCL-23) and HeLa (ATCC CCL-2™) cells
Preparation: Infected tissue culture fluid and cell lysate
Incubation: 5-12 days at 37°C Image of Respiratory Syncytial Virus cour-
Atmospherte: 5% CO₂ in an air atmosphere tesy of Dr. Craig Lyerla, CDC
Effect: Syncytia formation
CHLAMYDIA
Species within the Chlamydia genus are obligate intracellular bacterial pathogens commonly transmitted through sexual activity. Common
symptoms associated with Chlamydia species include conjunctivitis, pelvic inflammatory disease, and infection of the reproductive organs.
These bacterial species are commonly categorized with viruses as they have similar propagation properties. Often it is recommended
that the Chlamydia stock be sonicated first before inoculating cells, and infection may be enhanced by centrifugation after inoculation.
Follow the specific instructions on the product sheet for preparing the cells.
ATCC number: VR-123™, VR-346™, VR-347™, VR-573™, VR-879™, VR-885™, VR-886™, VR-887™
Recommended host: McCoy cells (ATCC CRL-1696™)
Preparation: Infected tissue culture fluid and cell lysate
Incubation: 2-3 days at 35-37°C
Atmosphere: 5% CO₂ in an air atmosphere
Effect: Intracellular inclusion bodies visualized by fluorescent staining with genus or species specific conjugated monoclonal antibodies.
VIRAL TITERING
PLAQUE ASSAY
Calculating viral titer is necessary in order to determine viral infectivity. This can be determined
by a plaque assay or through calculating the infectious dose. The plaque assay was initially devel-
oped to count and measure the infectivity level of bacteriophages. This technique has since
been modified for use in animal virology, and has been a reliable determination of titer for a
number of tissue culture-adapted viruses. The basis of this assay is to measure the ability of a
single infectious virus to form a plaque on a cell culture monolayer. A plaque is developed as
part of the viral infection cycle, where following viral replication, the host cell dies.⁵
1 Grow the host cells in wells with the recommended growth medium for the cell line.
Allow the cells to reach the appropriate confluency.
Plaque assay of a tissue culture-adapted
2 Remove the growth medium and wash with Dulbecco’s Phosphate-Buffered Saline
viral strain.
(DPBS). Add diluted virus to each well, using multiple wells per dilution.
3 Incubate dishes for 1-2 hours to allow for viral adsorption.
4 Remove the inoculum and wash with basal medium, if applicable. NOTE 3
5 Overlay the cells with overlay medium. Incubate for a length of This is a general procedure used to determine the potency of a
time appropriate for infection. Remove overlay, if applicable. virus. Please refer to the following references for more details
6 Observe the cell monolayers daily for the presence of foci or on this procedure: A Manual of Basic Virological Techniques,
plaques. published by Prentice-Hall Inc.,¹ or Virology Methods Manual,
published by Academic Press. (1996).⁵
7 Count the number of plaques on plates with 20 or more
plaques. The average amount of plaques per Petri dish
multiplied by the virus dilution gives the number of plaque forming units (PFU) per volume of inoculum. This number may be
expressed as the titer of the virus. (See NOTE 3)
TISSUE CULTURE INFECTIOUS DOSE (TCID)
Viral titer can be determined in vitro by calculating the infectious dose. For tissue culture-adapted strains, this calculation is ascertained
through an endpoint dilution assay in cell culture. The most reproducible endpoint of the dilution assay is the dilution of the virus that will
produce a pathological change in 50% of the cell cultures inoculated. This number is expressed as 50% the infectious dose, or TCID₅₀, which
is analogous to the calculation for lethal dose 50. The accuracy of this method is related to the number of replicates at each dilution.⁵
1 Prepare a cell culture plate with the recommended cell line for viral propagation (preferably the same cells that the virus being
tested was grown in).
2 Incubate the plate under the appropriate conditions for cell growth until cells reach an optimum density for infection.
3 Prepare viral dilutions in base medium.
4 Remove cell growth medium from plate, the monolayer may be washed with remove any inhibitory agents.
5 Inoculate at least 3 wells with each dilution. Be sure to inoculate wells with base medium to serve as negative controls. Use a fresh,
sterile pipette for each dilution.
6 Allow the plate to incubate for 1 to 2 hours under conditions suitable for virus adsorption.
7 Add viral growth medium and incubate the plate under conditions suitable for the virus and observe all wells daily. Record results for
each well daily.
8 The endpoint is determined when the CPE or immunofluorescence assay (IFA) read-out appear the same per dilution for 3 separate readings.
9 The titer is calculated using the method of Reed and Muench.⁶ A titer expressed as 10(3.0)TCID₅₀/0.2 mL in 3 days in XXXX cell line
may be translated as: 0.2 mL of virus diluted at 1:1000 will infect 50% of the cells in 3 days when using XXXX cell line. (See NOTE 3)
The TCID₅₀ can be converted to plaque forming units (PFU) using the Poisson distribution. This conversion is an estimate based on the
rationale that the limiting dilution, which would infect 50% of the cell layers challenged, would be expected to produce a single plaque
in a cell monolayer. However, ATCC recommends that the actual number of PFUs be determined empirically.
To estimate PFU from TCID₅₀, the Poisson distribution can be applied; P(o) is the proportion of negative tubes and ‘m’ is the mean number
of infectious units per volume (PFU/mL), P(o)=e(-m). For any titer expressed as TCID₅₀, P(o)=0.5. Thus, e(-m)=0.5 and m= -ln 0.5, which is
≈0.7. Therefore, one could multiply the TCID₅₀ titer (per mL) by 0.7 to predict the mean number of PFU/mL. For example, one can assume
that material with a TCID₅₀ of 1x10⁵ TCID₅₀/mL will produce approximately 0.7x10⁵ PFU/mL. When applying this calculation, remember
that the estimated mean will only be valid if the changes in the protocol required to visualize plaques do not alter viral expression as
compared to conditions used to determine TCID₅₀.
CHICKEN EMBRYO INFECTIOUS DOSE (CEID)

For viruses normally propagated in chicken eggs, such as influenza virus, viral titer is calculated as the chicken embryo infectious dose
(CEID). Viral cultures are serially diluted and are used to inoculate embryonated chicken eggs. Following incubation, allantoic fluid is
harvested from each egg at all dilutions and virus titer is determined by the appearance of hemagglutination. A positive hemagglutina-
tion reaction indicates the virus is present at that dilution.
1 Order embryonated chicken eggs of the proper age, 8-12 days old depending on the virus. When the eggs arrive, place them in an
incubator at an appropriate temperature with a dish of water close by to provide moisture to prevent the eggs from drying out.
2 When you are ready to inoculate, candle the eggs to locate the
air sac and embryo. Using a pencil, outline the air sac area and NOTE 4
indicate where the embryo’s eye is located. In a biological A viable egg has a series of small blood vessels within the allan-
safety cabinet, punch a hole into the shell within the defined toic membrane, which can be seen during candling. Additionally,
air sac area, on the opposite side of the egg from the embryo’s the embryo’s eye will look like a black spot, which will move.
eye. Inoculate the allantoic fluid of each egg with the serial
dilution of virus. Seal the hole with nail polish and incubate the eggs near a pan of water at the proper temperature as recom-
mended on the provided product sheet. (See NOTE 4)
3 Candle the eggs 24 hours post-inoculation to check for viral-induced death. Place any of the dead eggs into a biohazard bag, seal the
bag with autoclave indicator tape and refrigerate to await autoclaving. Return the rest of the eggs to the incubator.
4 Following the recommended incubation period, harvest the virus. Depending upon the virus, death of the embryo may have
occurred. By this time, a viable embryo will have grown much larger, will be more active, and the network of blood vessels will be
more visible. A dead embryo will not move and the egg may or may not be as transparent and the blood vessels will have mostly
disappeared. For viable embryos, sacrifice the embryo prior to viral harvesting by placing the egg at 4°C for at least 2 hours.
5 Prepare a 0.5% suspension of chicken red blood cells using phosphate buffered saline
(PBS) while the infected eggs are chilling in a biological safety cabinet. Use sterile
forceps to break the top of the shell open and pull back the allantoic membrane. Place
all egg fragments, including parts of the shell into a double biohazardous materials
bag. Harvest the allantoic fluid from each egg by using a pipette to collect the fluid
into a different well of a rounded-bottom 96-well plate for each egg, being sure to use
a new pipette for each dilution.
6 Add a 1:1 ratio of the 0.5% red blood cell suspension to allantoic fluid in each tube. Mix
by gently tapping and allow the red blood cells to settle to the bottom of the wells.
After 30-45 minutes at room temperature, read the hemagglutination assay by
recording the presence of a button of cells or the presence of a lattice formation at Hemagglutination assay depicting Influenza
the bottom of the tube. A negative result is seen by the button of red blood cells at B virus (ATCC VR-295™) at dilutions varying
the bottom of the rounded well. A positive result is seen by the formation of the from 10¯⁹ to 10¯¹.
lattice formation at the bottom of the rounded well. Once the results are properly Left column = Negative control
recorded, place the plate in a biohazard bag to await proper decontamination. Columns 2-10 = Dilutions 10¯⁹ to 10¯¹
7 The end point is taken to be the highest dilution of virus suspension that produces a Column 11 = Positive control
positive result. HA = 1:640 means that the virus was titered by a hemagglutination
assay (HA) and the endpoint for the assay was a dilution of 1:640. (See NOTE 3)
GROWTH MEDIA FOR TISSUE CULTURE-ADAPTED
VIRUSES
M E D I A F O R C U LT U R I N G P R O P A G A T I O N H O S T S
For the propagation of tissue culture-adapted viruses, it is important to grow and maintain the host cell line in an appropriate medium. The
recommended media for all host cells is indicated on the product sheet for the host cell line and can also be found online at www.atcc.org.
Cell culture media are complex mixtures of salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones,
and trace elements. The requirements for these components vary among cell lines. Carbohydrates are supplied primarily in the form of
glucose. In some instances, glucose is replaced with galactose to decrease lactic acid build-up, as galactose is metabolized at a slower
rate. Other carbon sources include amino acids (particularly L-glutamine) and pyruvate.
In addition to nutrients, the medium helps maintain the pH and osmolality in a culture system. The pH is maintained by one or more buff-
ering systems; CO₂/sodium bicarbonate, phosphate, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) are the most common.
Sera will also buffer a complete medium. Phenol red, a pH indicator, is added to the medium to colorimetrically monitor changes in pH.
Media commonly used in the propagation of tissue culture-adapted strains include the following:
Eagle’s Minimum Essential Medium (EMEM) ATCC’s modification of EMEM (ATCC 30-2003) contains Earle’s balanced salt solution, non-es-
sential amino acids, L-glutamine, and sodium pyruvate. It is formulated with a reduced sodium bicarbonate concentration (1,500 mg/L)
for use with 5% CO₂. Because EMEM is a simple medium, it is often fortified with additional supplements or higher levels of serum.
Dulbecco’s Modified Eagle’s Medium (DMEM) has roughly twice the concentration of amino acids and four times the amount of vitamins
as EMEM, as well as ferric nitrate, sodium pyruvate, and some supplementary amino acids (though not all nonessential amino acids). The
original formulation contained 1,000 mg/L of glucose, but in the more commonly used variations this amount was increased to 4,500
mg/L. ATCC DMEM (ATCC 30-2002) has 4,500 mg/L of glucose and a reduced sodium bicarbonate concentration (1,500 mg/L) for use with
5% CO₂.
RPMI-1640 ATCC’s RPMI-1640 (ATCC 30-2001) was modified to

contain higher amounts of glucose (4,500 mg/L), sodium pyruvate,
and HEPES buffer. It also contains a reduced concentration of sodium
bicarbonate (1,500 mg/L) for use with 5% CO₂.
Leibovitz’s L-15 Medium (ATCC 30-2008) is formulated for free gas

exchange with atmospheric air. The standard sodium bicarbonate/
CO₂ buffering system is replaced by a combination of phosphate
buffers, free-base amino acids, higher levels of sodium pyruvate,
and galactose. A CO₂ and air mixture is detrimental to cells when
using this medium for cultivation. However, cell cultures can be
grown in CO₂ incubators with L-15 medium provided there is no
exchange between the air in the culture vessel with that of the incu-
bator (ie, caps of flasks are tightly closed).
M E D I A F O R M U L AT I O N S
The formulations for media used by ATCC can be found online. Please note that there are cell lines in the collection used for viral propa-
gation that may require media that is not currently sold by ATCC.
MEDIA SUPPLEMENTS
The growth media recommended for host propagation may require the addition of components not already available in the base medium.
These components may include hormones, growth factors, or serum.
After supplements have been added to the base medium, the shelf life of the medium should be determined on a case-by-case basis.
Media containing serum, antibiotics, and/or antimycotics tend to degrade faster than base media alone. Media containing supplements
should not be frozen as this may cause certain compounds to precipitate out of solution; media should be stored at 2°C to 8°C. For addi-
tional information regarding the preparation, storage, or usage of specific additives, contact your local supplier or consult with the
manufacturer’s product information sheet.
SERUM
Serum can serve as a source of growth factors, proteins, vitamins, hormones, carbohydrates, lipids, amino acids, minerals, and trace elements.
The exact composition of serum is unknown and varies from lot to lot, although lot-to-lot consistency has improved in recent years.
Sera from fetal bovine sources are commonly used to maintain cell cultures in preparation for viral infection. Fetal serum is a rich source
of growth factors and is appropriate for the growth of fastidious cells. It is often supplied at a concentration of 2-10%. (See NOTE 5)
Unfortunately, naturally derived products from animals, such as

sera, may contain adventitious microorganisms. All reputable suppli- NOTE 5
ers routinely test their products for infectious viruses by several For viral inoculation, ATCC recommends decreasing the percent-
methods including fluorescent antibody labeling, cytopathic effect, age of serum to 2% as it can interfere with viral attachment. This
and hemadsorption. These products are also screened for the stan- can vary from strain to strain, and is not applicable for influenza
dard microbial contaminants such as bacteria, fungi, and viruses.
mycoplasma. To reduce the risk of any possible contamination, ATCC
recommends that all serum should be triple filtered through 0.1 µm
sterile filters before use.
ATCC offers the following types of animal sera:

Fetal Bovine Serum (also known as fetal calf) – ATCC 30-2020
Fetal Bovine Serum qualified for embryonic stem cells – ATCC SCRR-30-2020
Iron-supplemented Calf Bovine Serum – ATCC 30-2030
These products are rigorously tested for adventitious infective agents and sourced only from U.S. herds. Further, each lot is tested for its
ability to support cell culture growth and is the same sera used in ATCC labs.
A Storage
Do not store serum at temperatures above -20°C for any length of time. Avoid any repeated freeze-thaws by dispensing and storing
sera in aliquots. Additionally, ensure that sera are stored away from direct light.
B Thawing
The following procedure is used to thaw serum:
1 Place the frozen serum in a refrigerator at 2°C to 8°C overnight.
2 Put the bottles in a 37°C water bath and gently agitate occasionally to mix the solutes that tend to concentrate at the bottom of
the bottle.
Do not keep the serum at 37°C any longer than necessary to thaw it, and do not thaw the serum at higher temperatures. Thawing
serum in a bath above 40°C without mixing may lead to the formation of a precipitate inside the bottle.
C Turbidity and precipitates
All sera may retain some fibrinogen. Because external factors may initiate the conversion of fibrinogen to fibrin, flocculent material
or turbidity may be observed after the serum is thawed. The presence of this material does not alter the serum’s performance. If the
presence of flocculent material or turbidity is a concern, it can be removed by filtration through a 0.45 µm filter.
A precipitate can form in serum when incubated at 37°C or higher for prolonged periods. This is often mistaken for microbial contam-
ination. This precipitate may include crystals of calcium phosphate, but this does not alter the performance of the serum as a supplement.
Heat inactivation of sera can also cause the formation of precipitates.
ADDITIVES AND CRYOPROTECTANTS

To preserve viral strains by cryopreservation, ATCC may use a mixture of FBS and DMSO (ATCC 4-X) or glycerol. Some viruses require a
specialized mixture of cryoprotectants or additives. For more information, refer to the cryopreservation section of the chapter entitled
Preservation.
PRESERVATION
There is no single procedure that is optimal for the preservation of all animal viruses. Both the type of agent and recommended growth
conditions of the culture influence the method of cryopreservation, and the determination of these ideal conditions may require some
experimentation. There are many advantages of preservation that far outweigh the required investment in equipment and reagents for
continuous in vitro expansion. These advantages include:
Overall safety of viral stocks against loss due to equipment failure or contamination by other microbial organisms.
Elimination of time, energy, and material costs associated with the maintenance of viral strains not currently in use.
Insurance against phenotypic drift due to genetic instability and/or selective pressures.
Creating a standard reagent that can be used for a series of experiments.
The methodologies used at ATCC have proven successful for a large number of organisms. Currently, ATCC only uses cryopreservation
methods to preserve viral strains. However, several viral strains in the collection are available as freeze-dried (lyophilized) cultures. The
basic methodology for the cryopreservation and lyophilization of viruses are described in the subsequent sections. For historical reasons
and because of similar requirements for handling and preservation, Chlamydia and Rickettsia species are also included here.
C R YO P R E S E R VAT I O N
OVERVIEW
Freezing a viral suspension often results in a decrease in viability and titer.¹¹,¹² For cell-associated pathogens, such as Chlamydia and
Rickettsia, as the suspension is cooled below the freezing point, ice crystals begin to form and the concentration of solutes in the suspen-
sion increases resulting in damage of host cell structures. This can be minimized if water within the cells is allowed to escape by osmosis
during the cooling process; a slow cooling rate, generally -1°C to -10°C per minute, facilitates this progression. However, as host cells lose
water, they shrink in size and will quickly lose viability if they surpass a minimum threshold volume. The addition of cryoprotectant agents,
such as glycerol or dimethylsulfoxide (DMSO), will mitigate these effects.¹³,¹⁴
The viability of viruses that are not cell-associated is best maintained by rapid freezing. In this method, samples are quickly frozen in a
dry ice slurry and stored in liquid nitrogen vapor or within a mechanical freezer at -80°C.
Overall, there are numerous factors that can affect the viability of recovered viruses. These critical parameters can include the compo-
sition of the cryoprotectant and the viral titer. For cell-associated viruses, obtain optimal cell viability upon recovery by modifying the
cryopreservation procedure for each viral strain. Contact ATCC for more information on the cryopreservation of viral strains.
FREEZE MEDIUM
Glycerol and DMSO are the most common cryoprotectant agents. To preserve viral strains, ATCC commonly uses a mixture of FBS and
DMSO or glycerol. When employing these cryoprotectants, use only reagent-grade DMSO or glycerol. Store both in aliquots protected
from light. ATCC offers DMSO (ATCC 4-X) that has been thoroughly tested for use.
Some viruses require specialized preservation methods. Several representative strains with unique preparations are listed in Table 3;
however, these examples and methods may not be applicable to all members of the group. Overall, the optimum formulations for indi-
vidual viral strains must be determined empirically.
Table 3: Examples of frozen preparations

ATCC number Product name Preparation
VR-1™ Human adenovirus 1 Infected culture medium
VR-343™ Japanese encephalitis virus Infected culture medium diluted 1:1 with calf serum
VR-977™ Human herpesvirus 5 Infected culture medium (EMEM + 10% FBS + 7% DMSO)
VR-897™ Influenza A virus (H1N1) Infected allantoic medium
VR-955™ Human respiratory syncytial virus Infected culture medium (EMEM + 2% FBS)
VR-129B™ Encephalomyocarditis virus Infected culture medium (MEM + 2% FBS)
VR-838™ Raccoonpox virus Infected culture medium (EMEM + 2% FBS)
VR-156™ Vaccinia virus Infected culture medium (L-15 + 2% FBS + L-glut)
VR-659™ Rous sarcoma virus Infected culture medium (M199)
VR-137™ Rabies street virus 10% infected mouse brain suspension in PBS + 10% horse serum
VR-612™ Rickettsia akari Infected yolk sac diluted 1:1 in sucrose-phosphate glutamate
EQUIPMENT
A Cryopreservation vials
There are two materials to choose from for cryopreservation vials: glass or plastic. Glass vials are more difficult to work with: they need
to be sterilized before use, they need to be sealed with a hot flame, and they can be difficult to open. However, they are considered
fail-safe once properly sealed.
If cryopreservation in glass ampoules is not possible, plastic vials can be used. Plastic vials come in two varieties: those with an inter-
nal thread and silicone gasket, and those with an external thread. Vials with an internal-thread were the first commercially available,
but have some disadvantages over the external-thread version. For example, while the silicone gasket provides an excellent seal, it
needs to be tightened just right; the vial will leak if the seal is too tight or too loose.
B Controlled-rate freezing chambers
For cell-associated viruses, there are several means to achieve a cooling rate of -1°C per minute. The best method involves the use of
a computer controlled, programmable electronic freezing unit (such as Thermo Scientific™ CryoMed™ Freezers), which rigorously main-
tains this rate of cooling. This is the method used exclusively at ATCC. Such equipment is relatively expensive and necessary for only
the most sensitive strains.
A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 24 hours in a mechanical freezer at
-70°C or colder. There are several commercially available freezing chambers, which achieve a cooling rate very close to the ideal -1°C
per minute (CoolCell® LX; ATCC ACS-6000). Alternatively, the vials can be placed into a polystyrene box, with 15 mm (3/4 inch) thick
walls and 1 L capacity that is packed with paper, cotton wool, or foam peanuts for insulation.
LIQUID NITROGEN FREEZING STORAGE

The ultra-low temperatures (below -120°C) required for long-term
storage can be maintained by specialized electric freezers, or more
commonly, by liquid nitrogen freezers. There are two basic types of
liquid nitrogen storage systems: immersing vials in the liquid or hold-
ing vials in the vapor phase above the liquid. The liquid-phase system
holds more nitrogen and thus requires less maintenance. However,
there is always a chance that some liquid will enter improperly
sealed vials, which may then explode when retrieved. For this reason,
ATCC strongly recommends storage in the vapor-phase.
Vapor-phase storage systems create a vertical temperature gradient within the container. The temperature in the liquid nitrogen at the
bottom will be -196°C, whereas the temperature at the top will vary depending upon the amount of liquid nitrogen at the bottom and
the length of time the container is opened. To ensure the safe storage of cells, maintain sufficient levels of liquid nitrogen in the container
so that the temperature at the top is -120°C or colder. All storage systems should be equipped with temperature alarms.
CRYOPRESERVATION PROCEDURE
A Uncontrolled Freezing
The viability of most viruses is best maintained by fast freezing after harvesting. Dispense the material into ampoules, place the
ampoules in a rack, and dip in a dry ice/ethanol bath. Store the frozen material in liquid nitrogen vapor or in a mechanical freezer at
-80°C.
B Controlled Freezing
To preserve cell-associated pathogens, such as Chlamydia and Rickettsia, a controlled freeze is preferred.
1 Incubate the viral agent under the recommended conditions until a maximum yield is obtained (maximum cytopathic effect if
applicable).
2 Add a cryoprotective agent to the viral suspension. For many viral strains, this will be a mixture of FBS and DMSO or glycerol.
Some strains may require specialized conditions, examples of these can be found in Table 1.
3 Within a biological safety cabinet, dispense 0.5 to 1.0 mL of the above mixture into sterile plastic cryovials or glass ampoules.
Store the filled glass ampoules on ice prior to sealing. Glass ampoules can be sealed using a gas-oxygen torch, pulling the neck of
the ampoules as it is rotated in the flame. Following sealing, disinfect glass ampoules by spraying with disinfectant.
4 Place the vials into a pre-cooled (4°C), controlled-rate freeze chamber and place the chamber in a mechanical freezer at -70°C (or
colder) for at least 24 hours. Alternately, use a pre-cooled (4°C) programmable freezer unit set to cool the vials at -1°C per minute
until a temperature below -40°C is achieved and then set the temperature to abruptly drop to -120°C.
5 Quickly transfer the vials to a liquid nitrogen or -120°C freezer. Frozen material will warm up above -50°C.
6 Record the location and details of the freeze.
7 After 24 hours at -120°C, remove one vial, appropriately restore the viral strain, and determine the viability and sterility.
RECOVERY OF CRYOPRESERVED VIRUSES

1 In advance, grow the recommended production host in a prepared culture vessel that contains growth medium equilibrated for
both temperature and pH.
2 Remove the vial of frozen virus from the liquid nitrogen freezer and thaw by gentle agitation in a 37°C water bath.
3 Thaw the strain rapidly until all ice crystals have melted (approximately 2 minutes).
4 Remove the vial from the water bath and decontaminate it by dipping in or spraying with 70% ethanol. Follow strict aseptic
conditions in a biological safety cabinet for all further manipulations.
5 Unscrew the top of the vial and inoculate the propagation host with the recommended infection conditions as described on the
supplied product sheet.
6 Examine the cultures after an appropriate length of time.
LY O P H I L I Z A T I O N
OVERVIEW
Freeze-drying is a process where water and other solvents are removed from a frozen product via sublimation.¹⁵ Sublimation occurs when
a frozen liquid goes directly to a gaseous state without entering a liquid phase. The freeze-drying process results in a stable, readily rehy-
drated product. This process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most
water, and secondary drying to remove bound water.
During the initial freezing process, ice crystals begin to form and the concentration of solutes in the suspension increases. The method
used during freezing can greatly affect the ability to freeze-dry the material. Slow cooling rates are recommended as this will result in
the formation of vertical ice crystal structures and allow for more efficient water sublimation from the frozen product.
Freeze-dried products are hygroscopic and must be protected from moisture during storage. Additionally, these products are sensitive
to other factors including oxygen and temperature, which can significantly decrease the shelf life. It is important to store freeze-dried
material in a manner that protects the product from exposure to moisture and oxygen and at refrigerated temperatures (4°C).
Generally, the lyophilization of viruses is not currently used by ATCC. This is because the methods for lyophilization vary depending on
the type of virus, and some viruses cannot be successfully preserved by freeze-drying due to loss of viability. Strongly cell-associated
viruses, including certain members of the herpes virus family, such as varicella-zoster, lose viability outside the host. Therefore, these
viruses cannot be preserved by this method. Though ATCC does not currently freeze-dry any virus preparations, it was done fairly exten-
sively in the past. The methods of lyophilization that were previously used by ATCC are described below. For additional information, contact
ATCC technical services.
EQUIPMENT
A Lyophilization Vials
For the storage of lyophilized viruses, ATCC uses sterile funnel-tipped glass ampoules (Wheaton) and glass serum vials. Generally, during
the lyophilization process, material is freeze-dried in a glass ampoule, disinfected, and sealed.
B Lyophilization Apparatuses
For the lyophilization of viruses, ATCC previously employed a commercial freeze-dryer. In this freeze-drying procedure, samples are
mixed with a suitable preservative, dispensed into the appropriate ampoule, and allowed to slowly freeze into a solid mass. The preser-
vative used for lyophilization can vary between viral agents; several examples are listed below in Table 4. Once frozen, samples are
lyophilized within a freeze-drying system (Virtis Genesis®, Millrock® Max 85, and LD 85).
During the primary drying phase, water is removed from the frozen product via sublimation. This is accomplished through the use of
a vacuum pump, which allows water molecules to migrate from the frozen product and condense on a moisture trap called a condenser.
For this to be possible the temperature of the condenser must be colder than the product temperature; the difference in these tempera-
tures will affect the rate of sublimation. When primary drying is complete, all residual moisture is removed by directly heating the
product. During this secondary drying phase, water must be desorbed to a residual moisture content of 1% or less. This process requires
a low pressure, low condenser temperature system. Once dried, the ampoules are properly sealed and stored at refrigerated tempera-
tures (4°C).
Table 4: Examples of freeze-dried preparations
ATCC number Product name Preparation
VR-343™ Japanese encephalitis virus Infected primary hamster kidney cells + 50% calf serum
VR-90™ Colorado tick fever virus Infected mouse brain (10% suspension in normal saline) diluted 1:1 in rabbit serum
VR-897™ Influenza A virus Infected allantoic fluid + 10% sucrose
STORAGE AND VIABILIT Y OF LYOPHILIZED STRAINS

To maximize the recovery of viable cells, viral cultures must be in optimum condition before the lyophilization process.¹⁶ Viruses should
be propagated under the recommended conditions for infection as indicated on the product sheet.
Because lyophilized products are hygroscopic, they must be stored under moisture-free conditions. Additionally, other factors such as
oxygen content and temperature can affect the shelf-life of freeze-dried strains. Oxygen can chemically react with the product, nega-
tively affecting culture viability. This reactivity is directly proportional to storage temperature. Therefore, lyophilized products should
be stored long-term in liquid nitrogen.
LYOPHILIZATION PROCEDURE
1 Grow the virus according to usual procedures.
2 For freeze-drying viral suspensions, glucose, skim milk, or Sucrose-Phosphate-Glutamate-
Albumin (SPGA) should be added before dispensing. The lyophilization of some viruses
may require specific preservation preparations; examples are listed in Table 4.
3 In a biological safety cabinet, dispense the suspension into either sterile funnel-
tipped ampoules or serum vials. Plug the ampoules loosely with slotted silicon rubber
stoppers, and place the ampoules in a freeze-dryer tray. Set an acrylic plate on top of
the stoppers.
4 Freeze-dry the material in a commercial freeze-dryer. Hold the material at -30°C for
18-48 hours, depending on the additive. Then raise the shelf temperature to 10°C per hour until the product temperature is 25°C.
5 When the cycles are complete, backfill the system with sterile nitrogen and press the caps into place. Disinfect the outside of the
ampoules and return them to the biological safety cabinet.
6 Seal the vials. Funnel-tipped ampoules will need to be sealed by an oxygen-gas torch.
RECOVERY OF LYOPHILIZED CELLS

1 To rehydrate freeze-dried strains, add sterile double-distilled water.
2 Mix well, dilute with culture medium, and add the virus to the propagation host cell culture.
3 Incubate strains under the recommended temperature and atmospheric conditions.
P R E S E R VAT I O N O F S P E C I F I C S T R A I N S
All viral strains can be cryopreserved and maintained in liquid nitrogen vapor, whereas only some strains can by lyophilized. In preparation
for cryopreservation or lyophilization, viral strains should be grown under optimal conditions. This can include growth in chicken eggs or
within tissue culture. Below, we provide information on the preservation of common viral strains.
ADENOVIRUSES
The less labile virus suspensions, such as human adenovirus, are
harvested in their culture media then dispensed into ampoules for
freezing.
HERPESVIRUSES
Certain members of the herpes family, such as cytomegalovirus or
varicella-zoster, lose viability outside the host. Therefore, keep
infected cells intact prior to preservation.
VIRUSES GROWN IN CHICKEN EMBRYOS

Viruses harvested from chicken eggs as allantoic fluid or yolk sac
preparations can be frozen or lyophilized. The high-protein content
of the egg serves as a cryoprotectant, and other additives may not
be necessary. Image of Adenovirus courtesy of Dr. G. William Gary, Jr., CDC
VIRUSES GROWN IN VIVO
Viruses grown in vivo and harvested in organ suspensions are preserved by freezing. Add sterile 50% glycerol or 10-50% serum as a
cryoprotectant.
SPECIAL HAZARDS
Care must be taken during the cryopreservation or lyophilization of animal viruses. Problems such as contamination, breakage of glass
ampoules during handling and storage, dispersal of freeze-dried virus when opening glass ampoules, and the handling of liquid nitrogen
must all be considered. To prevent contamination and the dispersal of viruses, aseptic technique must be followed. This can include the
decontamination of all equipment and vials as well as performing all preparations in a biological safety cabinet. Additionally, protective
clothing should be worn during preparation to prevent contamination as well as to guard against harm due to contact with liquid
nitrogen.
BIOSAFETY AND DISPOSAL
BIOSAFET Y
The need for precautions when experimenting with viral cultures depends upon the source and nature of the biological material, the
experimental procedure, and the laboratory/containment conditions. Since every situation is different, the risks need to be identified for
each individual strain and the appropriate precautions need to be taken before any work begins.
More information on risk assessment and precautions can be found in the Center for Disease Control (CDC) publication Biosafety in
Microbiological and Biomedical Laboratories.¹⁷ The text of this publication is available in its entirety online at www.cdc.gov.
A biosafety level (BSL) is assigned to each viral strain for the purposes of packaging for safe shipment. ATCC follows federal biosafety
guidelines and takes several factors into consideration when assessing a potential hazard, and in some cases the ATCC assigned biosafety
level is more restrictive. Generally, ATCC only ships and stores viruses with a biosafety level assignment of 1, 2, or 3.
BIOSAFET Y LEVEL 1
Work involving well-characterized viral strains not known to consistently cause disease in immunocompetent adult humans.
Work can be conducted on the bench top using aseptic technique, no special containment equipment or facility is required.
BIOSAFET Y LEVEL 2
Work involving viral strains that pose a moderate hazard to health adult humans.
Work should be conducted in designated biological safety cabinets within laboratories with restricted access.
BIOSAFET Y LEVEL 3
Work involving indigenous or exotic agents that may cause serious or potentially lethal disease via inhalation.
Work should be conducted in biological safety cabinets localized inside a specialized BSL-3 containment facility within laboratories
with restricted access.
As the recipient of an ATCC virus, take into account not only the nature of the material but also the manipulations employed during its
handling when assessing the potential laboratory risk. Keep in mind that there will be situations where the intended use of an agent may
require more stringent precautions than associated with the assigned biosafety level.¹⁷
D I S P O S A L O F I N F E C T I O U S M AT E R I A L S
All viral cultures, stocks, and potentially infectious materials need to be properly decontaminated prior to disposal. The written method
for proper decontamination should be available in the laboratory and BSL facility. Several methods of sterilization include use of an auto-
clave, chemical disinfection, incineration, or any other validated decontamination method. More information on the disposal of viral
cultures can be found in the Center for Disease Control (CDC) publication Biosafety in Microbiological and Biomedical Laboratories.¹⁷
VIRAL AUTHENTICATION AND VIABILITY TESTING
When preparing a viral strain for distribution, ATCC performs numerous quality control (QC) assays to guarantee that the product is of
the highest standard before it reaches the consumer. All viral strains grown in-house undergo viability testing as well as genotypic exam-
inations to ensure that the strain identification is accurate and the viral agent is sustainable. Described below are several tests used by
ATCC for the authentication and viability testing of animal viruses.
VIABILIT Y TESTING
CY TOPATHIC EFFECTS (CPE)
For tissue-culture adapted viruses, viability is primarily monitored by observing host cell appearance. During the synthesis of viral compo-
nents, the host cell undergoes morphological and biochemical changes. These deviations often result in degenerative changes known as
cytopathic effect. The degree of visible cell damage varies between
NOTE 6
viral species and host cell lines. Some viruses cause little to no CPE
in host cells, while others can destroy the host cell population. During early viral infection, cells may become rounded or appear
Characteristic CPE is best observed by analyzing cultures daily.¹⁸ more refractive. More severe CPE include focal degeneration,
(See NOTE 6) vacuolization, cellular fusion, or host cell death.
Propagation of Human herpes virus 1 (ATCC VR-260™) in Vero cells (ATCC CCL-81™). The left panel depicts uninfected CCL-81 cells. The
right panel depicts infected CCL-81 cells exhibiting CPE.
HEMAGGLUTINATION
Many viruses can attach to molecules present on the surface of red
blood cells. At certain concentrations, a viral suspension may cause Direct Hemagglutination
red blood cells to clump together. This natural phenomenon, known Positive Reation:
as hemagglutination, prevents red blood cells from settling out of
suspension and has been adapted for use in viability testing and
CEID viral titering. In a hemagglutination assay, chicken red blood
cell suspensions are incubated with a serially diluted virus and moni- Hemagglutinating virus Red blood cells Agglutination
tored for the formation of a red blood cell lattice. The end-point is
Negative Reation:
taken to be the highest dilution of the virus suspension that
produces a positive result.
HEMADSORPTION ASSAY
Non-hemagglutinating
The phenomenon of hemadsorption is dependent on the attach- Red blood cells No agglutination
virus
ment of red blood cells to the surface of cell monolayers infected
Image of Hemagglutination assay courtesy of Dr. F.T. Forrester., CDC
with enveloped, hemagglutinin-producing viruses. This natural
process can be adapted as a general procedure to determine viral
potency. In a hemadsorption assay, a red blood cell suspension is incubated with an infected cell culture. If the cell monolayer is infected
with a hemagglutinin-producing virus, hemagglutinin is inserted into the cell plasma membrane during viral reproduction in preparation
for viral maturation. It is at these modified areas of the cell surface that red blood cells will specifically bind. Thus, hemadsorption is indic-
ative of the presence of viruses that produce hemagglutinin, such as influenza, measles, mumps, and parainfluenza.
VIRAL INFECTIVIT Y
Viral infectivity is often measured by calculating titer. Depending on the required host for viral propagation, infectivity can be analyzed
using a plaque assay or by determining the infectious dose in tissue culture or embryonated chicken eggs. The basis of these assays is to
measure the ability of an infectious viral suspension to produce a pathological change in the host environment. For additional informa-
tion on plaque assays or calculating infectious dose, refer to the chapter entitled “Viral Replication and Propagation.”
V I R A L AU T H E N T I C AT I O N
SEQUENCING
The ATCC Virology collection utilizes sequencing to authenticate
our holdings as they are accessioned or come up for replenishment.
ATCC sequences approximately ≥500 bp of the viral genome that is
characteristic of the species or virus type.
ENZYME LINKED IMMUNOSORBANT ASSAY

All viruses have at least one unique surface antigen. Through an
enzyme-linked immunosorbant assay (ELISA), these antigens can
be used as a biological marker to authenticate a viral species. There
are two forms of this assay, the direct and indirect ELISA. Both assays
employ enzyme-conjugated monoclonal detection antibodies that
tightly bind a specific viral antigen. With a direct ELISA, one is test-
ing for the presence of the virus, while an indirect ELISA assay
detects specific viral antibody rather than the virus itself. The pres-
ence of a specific virus is analyzed by assessing the conjugated
enzyme activity via incubation with a substrate that is converted into a measurable colored product. When using an ELISA for authenti-
cation purposes, a colorimetric change indicates the viral sample was correctly identified.
VIRAL APPLICATIONS
C E L L T R A N S F O R M AT I O N
Cancerous cells emerge due to discrete changes in the cellular genome. These genomic deviations can arise due to viral transformation.
During the infection of non-permissive cells, viral genetic material can become covalently integrated into the host genome. If viral inte-
gration induces the aberrant expression of a proto-oncogene, cell growth can become unrestricted, resulting in an immortal cell state.
This natural process has been adapted by cell biologists for use in the immortalization of mammalian cells in tissue culture. Immortalized
cell lines offer the possibility of an inexhaustible supply of cells, allowing for more consistent and reproducible research. Viral genes such
as the simian virus 40 (SV40) T antigen have been used as a simple, reliable agent for the immortalization of a variety of cell types.¹⁹,²⁰
SV40 is believed to induce immortalization by suppressing the transcription of p53, a tumor suppressor protein responsible for initiating
apoptosis following cell damage.
In addition to their use in cell immortalization, viral strains can be used to introduce nucleic acids into cells. This process, termed transduc-
tion, allows scientists to analyze the function of specific genes, to examine the effects of gene silencing, or to introduce therapeutic genes.
PHAGE THERAPY
Bacteriophages are viruses that infect bacterial cells, often result-
ing in the lysis and subsequent death of the bacterial host. Since
the early 20 th century, European scientists have taken advantage of
this natural process to treat bacterial infections in a process known
as phage therapy. Because the host range of a bacteriophage is very
narrow, often specific to one species, they are considered more
effective than antibiotic treatment and result in less harm to the
human microflora.²² Though phage therapy has many benefits, it
was abandoned by many countries due to the antigenic properties
and unpredictable behavior of bacteriophages in addition to the
wide-spread production and use of antibiotics. However, with the
recent emergence of antibiotic-resistant bacteria, scientists are
now forced to re-evaluate the beneficial properties of the applica-
tion of phage therapy.²³
NANOTECHNOLOGY
Cancer treatments have commonly employed surgery, radiation, chemotherapy, and hormone therapy.²⁴ Though these treatments have
helped millions of patients go into remission, they come at a cost. Most cancer treatments attack both healthy and cancerous cells, often
leaving patients nauseous, fatigued, and immunocompromised. To combat these negative side effects, viral nanoparticles are being engi-
neered and analyzed as a potential form of drug delivery.
Viral nanoparticles are viruses whose genomic material has been removed and replaced with therapeutic drugs. Because viruses have
naturally evolved the ability to cross the host cell membrane, they are an ideal candidate for drug packaging and targeted delivery. To
minimize toxic side effects, infection, and induced immune response, human viruses are not used in the production of viral nanoparticles.
Rather, these drug delivery systems are engineered from plant, insect, and animal viruses.²⁵ Plant viruses, such as the Cowpea mosaic
virus, are ideal candidates for nanotechnology as they are easy to produce in large quantities, can self-assemble around a nanoparticle,
and hold a substantial volume of cancer drugs.²⁶
One of the major benefits to using viral nanoparticles for drug delivery is that targeting molecules can be easily attached to the viral
surface, allowing for the directed binding to cancer cells. This specific binding prevents the harm or death of surrounding healthy cells,
thus reducing possible side effects. Though viral nanoparticles have the potential to be a beneficial treatment, there are several compli-
cations associated with their use as a drug delivery system including immune rejection and potential toxicity. Regardless, viral nanoparticles
have the capability to revolutionize the treatment of cancer, creating a safe and specific form of drug delivery.
R ECO M B I N A N T V EC T O R E D VAC C I N E S
Vaccines are the most effective prophylactic tool for the prevention of disease. Viral-based vaccines are traditionally employed as live
attenuated viruses or as chemically inactivated viruses. Though these types of preparations have been shown to induce protective immu-
nity in animal models, incomplete viral attenuation or inactivation poses a serious risk to those who are vaccinated.²⁷,²⁸ Recombinant
vectored vaccines, however, offer a live-vaccine approach that does not employ the complete pathogen. Instead, genetic material from
the microbial target strain is inserted into the genome of harmless viral strains, allowing for the safe expression of microbial antigens.
Newcastle disease virus (NDV), a negative-sense RNA avian virus, is a common template used for the development of recombinant vectored
vaccines. Not only does NDV grow to high titers in many cell lines and eggs, it can elicit a strong immune response in vivo, is harmless to
humans, and is commercially available. Additionally, NDV replicates in the cytoplasm of infected cells, thus eliminating the problem of
genetic integration.²⁹ To create a viral vaccine vector from NDV, genes encoding foreign target proteins are inserted into the NDV genome
through recombination. Upon vaccination with the live virus, the foreign target protein is expressed within the host cell cytoplasm where
it is then available for processing by the cellular antigen-processing machinery for immune presentation. As a result, cellular immunity
is activated and neutralizing antibodies are generated.³⁰-³²
GLOSSARY
Ambisense. A term applied to single-stranded RNA viral genomes. Part of the nucleotide sequence is positive-sense and part is
negative-sense.
Complex morphology. A virus that possesses a capsid that is neither truly helical nor icosahedral. This morphology may have additional
structures such as a protein tail or a complex outer wall.
Cytopathic effect (CPE). CPE refers to the degenerative changes in association with viral infection.
Enveloped morphology. Viral envelopes are commonly derived from portions of the host cell membranes that have incorporated viral
glycoproteins. Envelopes surround the viral capsid and assist in viral entry of host cells.
Helical morphology. Viral morphology composed of a single type of capsomer stacked around a central axis to form a helical structure.
Hemagglutination. This term refers to the agglutination, or clumping, of red blood cells.
Icosahedral morphology. Viral structures built from repeated identical protein subunits to form a nearly spherical structure with rota-
tional symmetry.
Multiplicity of infection (MOI). The ratio of agents (eg, viruses, bacteriophages, bacteria) to infection targets (eg, propagation host).
Negative sense. Negative-sense RNA (3’ to 5’) forms the complementary strand and must be converted to positive-sense by an RNA poly-
merase prior to translation.
Non-permissive. Being or relating to a cell or environmental condition that does not support the replication of a virus or bacteriophage.
Positive sense. Positive-sense RNA (5’ to 3’) signifies that a particular sequence can be directly translated into protein.
Production host. The host strain used for the propagation of viruses.
Prolate morphology. Viral morphology composed of a cylinder with a cap at either end forming an elongated icosahedron structure along
a fivefold axis. This is a common arrangement of bacteriophage heads.
Proto-oncogene. This term defines a normal gene that can become an oncogene due to mutations or increased expression. These genes
commonly code for proteins involved in the regulation of cell growth or differentiation.
Sense. Sense is a concept used to compare the polarity of nucleic acids.
Transduction. The process by which genetic material is introduced into a cell by a virus or viral vector.
Transformation. This term refers to the cellular alteration resulting from the uptake, integration, and expression of exogenous genetic
material taken up from the environment.
APPENDIX
Table 5: Propagation hosts and recommended basal media
ATCC number Product description ATCC number Media
CCL-22™ MDBK (NBL-1) 30-2003 Eagle’s Minimum Essential Medium
CCL-34™ MDCK (NBL-2) 30-2003 Eagle’s Minimum Essential Medium
CRL-1590™ SL-29 30-2002 Dulbecco’s Modified Eagle’s Medium
CCL-10™ BHK-21 30-2003 Eagle’s Minimum Essential Medium
CCL-61™ CHO-K1 30-2004 F-12K Medium
CRL-1573™ 293 (HEK-293) 30-2003 Eagle’s Minimum Essential Medium
CCL-2™ HeLa 30-2003 Eagle’s Minimum Essential Medium
CCL-23™ Hep-2 30-2003 Eagle’s Minimum Essential Medium
CCL-171™ MRC-5 30-2003 Eagle’s Minimum Essential Medium
CCL-75™ WI-38 30-2003 Eagle’s Minimum Essential Medium
CRL-1721™ Sf9 30-2001 RPMI-1640 Medium
CRL-1651™ COS-7 30-2002 Dulbecco’s Modified Eagle’s Medium
CCL-81™ Vero 30-2003 Eagle’s Minimum Essential Medium
CRL-1586™ VERO C1008 [Vero 76, clone E6, Vero E6] 30-2003 Eagle’s Minimum Essential Medium
CRL-1587™ VERO 76 30-2002 Dulbecco’s Modified Eagle’s Medium
CCL-7.1™ LLC-MK2 Derivative 30-2003 Eagle’s Minimum Essential Medium
CL-160™ DBS-FRhL-2 30-2003 Eagle’s Minimum Essential Medium
CCL-33™ PK(15) 30-2003 Eagle’s Minimum Essential Medium
REFERENCES
1 Rovozzo, G. & Burke, C. A Manual of Basic Virological Techniques (Prentice-Hall, 1973).
2 Leland, D. & Ginocchino, C. Role of Cell Culture for Virus Detection in the Age of Technology. Clin Microbiol Rev 20, 49-78 (2007).
3 Cohen, J.I. Epstein-Barr virus infection. N Engl J Med 343, 481-92 (2000).
4 Gotoh, K. et al. Replication of Epstein-Barr virus primary infection in human tonsil tissue explants. PLoS One 6, e25490.
5 Mahy, B. & Kangro, H. Virology Methods manual (Academic Press, 1996).
6 Reed, L. & Muench, H. A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27, 493-497 (1938).
7 McLimans, W. (eds. Rothblat, G. & Cristofalo, V.) (Academic Press, New York, 1972).
8 Shipman, C., Jr. Evaluation of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as a tissue culture buffer. Proc Soc Exp Biol
Med 130, 305-10 (1969).
9 Giandomenico, A.R., Cerniglia, G.E., Biaglow, J.E., Stevens, C.W. & Koch, C.J. The importance of sodium pyruvate in assessing damage
produced by hydrogen peroxide. Free Radic Biol Med 23, 426-34 (1997).
10 Jacoby, W. & Pasten, I. (Academic Press, New York, 1979).
11 Mazur, P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 14, 251-72 (1977).
12 Mazur, P. Cryobiology: the freezing of biological systems. Science 168, 939-49 (1970).
13 Fahy, G.M. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology 23, 1-13 (1986).
14 Meryman, H.T. Cryoprotective agents. Cryobiology 8, 173-83 (1971).
15 Rowe, T.W. Optimization in freeze-drying. Dev Biol Stand 36, 79-97 (1976).
16 Heckly, R. Principles of preserving bacteria by freeze-drying. Dev Ind Microbiol 26, 379-395 (1985).
17 United States Department of Health and Human Serves, C.f.D.C., and National Institutes of Health. Biosafety in Microbiological and
Biomedical Laboratories (U.S. Government Printing Office, HHS Publication No. (CDC) 21-1112, Washington DC, 2009).
18 Enders, J.F. Cytopathology of virus infections: particular reference to tissue culture studies. Annu Rev Microbiol 8, 473-502 (1954).
19 Jha, K.K., Banga, S., Palejwala, V. & Ozer, H.L. SV40-Mediated immortalization. Exp Cell Res 245, 1-7 (1998).
20 Kirchhoff, C. et al. Immortalization by large T-antigen of the adult epididymal duct epithelium. Mol Cell Endocrinol 216, 83-94 (2004).
21 Mancheno-Corvo, P. & Martin-Duque, P. Viral gene therapy. Clin Transl Oncol 8, 858-67 (2006).
22 Miedzybrodzki, R. et al. Clinical aspects of phage therapy. Adv Virus Res 83, 73-121.
23 Kropinski, A.M. Phage Therapy - Everything Old is New Again. Can J Infect Dis Med Microbiol 17, 297-306 (2006).
24 Institute, N.C. (2011).
25 Singh, P., Destito, G., Schneemann, A. & Manchester, M. Canine parvovirus-like particles, a novel nanomaterial for tumor targeting. J
Nanobiotechnology 4, 2 (2006).
26 Franzen, S. & Lommel, S.A. Targeting cancer with ‘smart bombs’: equipping plant virus nanoparticles for a ‘seek and destroy’ mission.
Nanomedicine (Lond) 4, 575-88 (2009).
27 Brown, F. Review of accidents caused by incomplete inactivation of viruses. Dev Biol Stand 81, 103-7 (1993).
28 Nathanson, N. & Langmuir, A.D. The Cutter Incident. Poliomyelitis Following Formaldehyde- Inactivated Poliovirus Vaccination in the
United States during the Spring of 1955. Ii. Relationship of Poliomyelitis to Cutter Vaccine. Am J Hyg 78, 29-60 (1963).
29 Huang, Z., Elankumaran, S., Panda, A. & Samal, S.K. Recombinant Newcastle disease virus as a vaccine vector. Poult Sci 82, 899-906
(2003).
30 Huang, J.L. et al. High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen. J Med
Virol 65, 553-60 (2001).
31 Krishnamurthy, S., Huang, Z. & Samal, S.K. Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a
foreign gene results in growth retardation and attenuation. Virology 278, 168-82 (2000).
32 Nakaya, T. et al. Recombinant Newcastle disease virus as a vaccine vector. J Virol 75, 11868-73 (2001).
ATCC REAGENTS FOR VIRUS EXPANSION
Table 6: ATCC cell lines for virus expansion
ATCC number Product name Organism Tissue source
CCL-22™ MDBK Bovine Kidney
CCL-34™ MDCK (NBL-2) Canine Kidney
CRL-1590™ SL-29 Chicken Embryo
CCL-10™ BHK-21 Hamster Kidney
CCL-61™ CHO-K1 Hamster Ovary
CRL-1573™ 293 (HEK-293) Human Kidney
CRL-1573.3™ HEK-293.2sus Human Kidney
CCL-2™ HeLa Human Cervix
CCL-23™ Hep-2 Human Cervix
CCL-171™ MRC-5 Human Lung
CCL-75™ WI-38 Human Lung
CRL-1721™ Sf9 Insect Ovary
CRL-1651™ COS-7 Monkey Kidney
CCL-81.5™ Vero-SF-ACF Monkey Kidney
CCL-81™ Vero Monkey Kidney
CRL-1586™ VERO C1008 [Vero 76, clone E6, Vero E6] Monkey Kidney
CRL-1587™ VERO 76 Monkey Kidney
CCL-7.1™ LLC-MK2 Derivative Monkey Kidney
CL-160™ DBS-FRhL-2 Monkey Lung
CCL-33™ PK(15) Pig Kidney
Table 7: ATCC media product list

ATCC number Product name Volume
30-2002 Dulbecco's Modified Eagle's Medium (DMEM) 500 mL
30-2006 DMEM:F12 Medium (1:1 Ratio) 500 mL
30-2003 Eagle's Minimum Essential Medium (EMEM) 500 mL
30-2004 F12K Medium 500 mL
30-2005 Iscove's Modified Dulbecco's Medium (IMDM) 500 mL
30-2008 Leibovitz's L-15 Medium 500 mL
30-2007 McCoy's 5A Medium, Modified 500 mL
30-2001 RPMI-1640 Medium 500 mL
Table 8: Animal sera product list

ATCC number Product name Volume
30-2020 Fetal Bovine Serum 500 mL
30-2030 Calf Bovine Serum 500 mL
Table 9: Cell culture supplements
ATCC number Product name
Amino Acid Solutions

30-2214 L-Glutamine Solution, 200 mM
Subculture Reagents
30-2101 Trypsin EDTA Solution
30-2103 Non-Enzymatic Cell Dissociation Solution
30-2104 Soybean Trypsin Inhibitor
Buffers, Stains & Water

30-2200 Dulbecco's Phosphate Buffered Saline (DPBS)
Cryopreservation Reagents and Tools

4-X Dimethylsulfoxide (DMSO)
30-2600 Serum-Free Cell Freezing Medium
ACS-6000 CoolCell LX Alcohol-free Freezing Container
Table 10: Cell proliferation assays and mycoplasma detection

ATCC number Product name
30-1010K ATCC MTT Cell Proliferation Assay
30-1011K XTT Cell Proliferation Assay Kit
30-1012K Universal Mycoplasma Detection Kit
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Getting Started with an ATCC Viral Strain.............3 Biosafety...........................................................................................19

Biosafety and Disposal............................................ 19

In addition to unique morphological structures, viruses also vary in genomic structure.

STRAINS PROPAGATED IN CHICKEN EGGS

INITIATING LYOPHILIZED CULTURES

Table 1: Examples of propagation hosts for tissue culture-adapted viruses

PROPAGATION IN CHICKEN EGGS

cavity, the amniotic cavity, the chorioallantoic membrane (CAM),

Table 2: Examples of ATCC viruses propagated in embryonated chicken eggs

RESPIRATORY SYNCY TIAL VIRUS

CHICKEN EMBRYO INFECTIOUS DOSE (CEID)

RPMI-1640 ATCC’s RPMI-1640 (ATCC 30-2001) was modified to

Leibovitz’s L-15 Medium (ATCC 30-2008) is formulated for free gas

Unfortunately, naturally derived products from animals, such as

ATCC offers the following types of animal sera:

ADDITIVES AND CRYOPROTECTANTS

Table 3: Examples of frozen preparations

LIQUID NITROGEN FREEZING STORAGE

RECOVERY OF CRYOPRESERVED VIRUSES

STORAGE AND VIABILIT Y OF LYOPHILIZED STRAINS

RECOVERY OF LYOPHILIZED CELLS

VIRUSES GROWN IN CHICKEN EMBRYOS

ENZYME LINKED IMMUNOSORBANT ASSAY

Sense. Sense is a concept used to compare the polarity of nucleic acids.

Table 7: ATCC media product list

Table 8: Animal sera product list

Amino Acid Solutions

Buffers, Stains & Water

Cryopreservation Reagents and Tools

Table 10: Cell proliferation assays and mycoplasma detection

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