new Virus culture techniques

Virus culture
techniques
By Dr. Shariq Ahmed

Definition of Virus
 Acellular organisms which consist of nucleic acid core
surrounded by a protein coat , and which obligately
replicate inside host cells using host metabolic machinery
and ribosomes to form a pool of components which
assemble into particles called VIRIONS, which serve to
protect the genome and to transfer it to other cells.

Viruses are different from other
microbes
1. Smaller than other organisms
2. Carry genetic information in the form of DNA or
RNA, but not both
3. Obligate intracellular parasites.
• Depend totally on their host cells for their existence.
• Extremely difficult to get good insight of them in natural
conditions, because the internal characteristics of the host
cells are likely to interfere with the observations.
• Due to these reasons, it has been found desirable that
viruses are cultivated or grown in the laboratory itself.

River’s Postulates
• T.M. River, 1937
• Modified from Koch’s Postulates (proof of bacterial diseases)
1. Isolate virus from diseased hosts.
2. Cultivation of virus in host cells.
3. Proof of filterability.
4. Production of a comparable disease when the cultivated virus
is used to infect experimental animals.
5. Reisolation of the same virus from the infected experimental
animal.
6. Detection of a specific immune response to the virus.

Indications for lab diagnosis of viral infection
•If rubella is diagnosed in the first trimester of pregnancy, abortion is
recommended
•If a baby is borne of an HbsAg positive mother ,abortion is recommended
For proper management
of certain diseases
•for which antiviral chemotherapy is available (herpes viruses)
Diagnosis of diseases
caused by viruses
•For hepatitis B & HIV virus helps to prevent spread of these viruses
Screening of blood
donors
•To initiate appropriate control measures
Early detection of
epidemics
5

Laboratory Diagnosis of Viral
diseases
1. Microscopy
2. Demonstration of virus antigen
3. Isolation of virus
4. Serological methods
5. Molecular Methods

Microscopy
• Examine specimen for viruses
Electron Microscope
• Labeled antibody
Immuno-electron
microscopy
• Fluorescent tag bound to Fc region of Ab
Immunofluorescence
• Histological appearance of affected cells
• Inclusion bodies
Light microscope
7

Electronmicrographs
Adenovirus Rotavirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)

2. Demonstration of viral antigens
Precipitation in gel eg HBsAg
Immunofluorescence
Counter Immunoelectrophoresis (CIEP)
Radioimmunoassay
Enzyme Linked Immunosorbent Assay (ELISA)

IF staining of rabies
infected brain cells

3. Isolation of virus
 Laboratory animals
 Fertilized Hen’s Egg
 Organ/Tissue/Cell
Culture

4. Serologic procedures
• If the antibody titer in the convalesent-phase serum
sample is at least 4-fold higher than the titer in the
acute-phase serum sample, the patient is considered
to be infected.
• In certain viral diseases, the presence of IgM
antibody is used to diagnose current infection
• Other nonspecific serologic tests are available

• Complement fixation：
• Hemagglutination inhibition
• Neutralization
• Immunofluorescence ( direct or indirect)
• Latex agglutination
• In situ EIA
• ELISA
• RIA

5. Molecular methods
DNA/RNA Probes
Polymerase chain reaction
Insitu DNA Hybridisation
---provide rapid, sensitive and specific
information about the presence of viruses in
clinical samples

CULTIVATION OF VIRUSES
 Purpose
1. to isolate and identify viruses in
clinical specimens
2. to prepare viruses for vaccines
3. to do detailed research on viral
structure, multiplication cycles,
genetics, and effects on host cells.
 Bacteriophages – cultivation and
identification is simple and easy , due to the
simplicity of the host cells
 Animal viruses- more difficult & expensive

VIRUS CULTURE
• As viruses are obligate intracellular
parasites, they cannot be grown on
any inanimate culture medium

1. Animal Inoculation
 Humans -The earliest method for the
cultivation of viruses causing human
diseases was inoculation into human
volunteers
Reed & colleagues used human for their
pioneer work on Yellow fever
-due to serious risk involved , used only when
no other method is available & when virus
is harmless

Monkey – for the isolation of polio virus by
Landsteiner & Popper
- have limited application in virology due
to their cost & risk to handlers
Non human primates
Mice – pioneered by Theiler
- most widely employed animal
- Infant (Suckling ) Mice – susceptible to
Coxsackie & Arboviruses , which do not grow in
any other system
- routes- IC, SC, IP, IN

• Suckling mice are required for the recovery of some
coxsackie A enteroviruses, and suckling mice, suckling
hamsters, rabbits, mosquitoes, or ticks are needed for some
arboviruses.
• Primates are the most sensitive systems for the hepatitis
viruses, with ducks, woodchucks, and mice as alternatives
for hepatitis B virus and mice and piglets for hepatitis E
virus.

• The growth of the virus in inoculated
animals is indicated by death or disease
or visible lesions
The viruses are identified by testing for
neutralization of their pathogenicity for
animals, by standard antiviral sera.

DISADVANTAGES
• Immunity may interfere with viral growth
• Animal may harbour latent viruses.
• Cost & Maintenance
• Individual variations
• Difficulty in choosing of animals for particular
virus

2. Embryonated eggs
• The Embryonated hen’s egg was first used for
cultivation of viruses by Good Pasteur and the
method was further developed by Burnet (1931).
• For all practical purposes they all themselves
behave as tissue cultures.
• The process of cultivation of viruses in
embryonated eggs depends on the type of egg
which is used.
• The egg used for cultivation must be sterile and
the shell should be intact and healthy.
23

Burnet as director of the hall
institute, 1944-1965
• F.M. Burnet in the
laboratory in the early
1950's, was
experimenting on
influenza virus
genetics, using the
developing hen's egg.

Burnet
Wins Nobel Prize
Burnet was facilitated
by the award of the
1960 Nobel Prize to
him and Peter Medawar
for the discovery of
immunological
tolerance, a discovery
in immunology of minor
importance compared
with the clonal
selection theory.

Advantages of fertile eggs
• Fertile chicken eggs provide a
convenient, space-saving
incubator for many kinds of
animal viruses. Different
viruses can be injected into an
egg at different sites and the
egg can be easily observed for
viral replication throughout
the development of the
chicken embryo.
• ease of handling,
production of large amounts
of infectious amniotic or
allantoic fluids, and lack of
antibody production by the
host which might hinder
virus identification.

Advantages
• Sterile : free from bacteria
and many latent viruses
• Free from specific and non
specific factors of defense
• Cost- much less
• Maintenance-easier
• Readily available

• The chick embryo is a versatile host system in diagnostic
virology, although its use has been diminished by the
development of alternate culture and direct detection
methods.
• It is still vital to the isolation of some influenza viruses,
and the size and appearance of pocks on the chorioallantoic
membrane (CAM) afford a quick diagnostic criterion for
poxviruses.
• Disadvantages include the need to obtain sterile eggs of
known age (certified germ-free eggs can be virtually
unobtainable in some countries) and the need to eliminate all
viable bacteria, yeasts, and mycoplasmas from the patient’s
specimen before inoculation.

• Fertile eggs must be incubated at 37C in 40–70
percent humidity and rotated about every 4 h to
keep the embryo floating in the center of the egg.

Candling Eggs
• Candling is the process of
holding a strong light above
or below the egg to observe
the embryo. A candling lamp
consists of a strong electric
bulb covered by a plastic or
aluminum container that has
a handle and an aperture.
The egg is placed against
this aperture and
illuminated by the light. If
you do not have a candling
lamp, improvise. Try using a
torch.

• The embryo which is seen as a dark
shadow must be alive , as demonstrated by
its spontaneous movements and by the well
defined shadow of blood vessels in the
chorioallantois.
• The air sac can be seen at the rounded
pole of the egg and can be outlined for the
later guidance, by the pencil marking on
the shell.

Marking the inoculation site
 Hold the blunt end of the egg
against the aperture of the
candling lamp and note the position
of the eye of the embryo &
movement of blood vessels.
 Turn the egg a quarter turn away.
 Draw a line on the shell marking
the edge of the air sac.
 shell is cut with a rotating drill at
seleted site

• inoculation through the hole on the
shell using fine needle
• Seal the site
• Incubate at 35-36 degree celsius

Structure and Utility of Fertilized
Egg
Dr.T.V.Rao MD 35

1. Chorioallantoic membrane
(CAM):
• poxvirus- variola
- vaccinia
• Herpes simplex virus
• Virus replication produces visible lesions
(pocks)
• 12 days incubated eggs are taken.
• Circle of 12mm dia is marked over the area
of densest opacity (no blood vessels) &
shell is cut along it to expose CAM.

• The shell is drilled over the centre of the air sac so as to make a
small perforation through the shell membrane into the sac.
• A drop of normal saline is now deposited on the exposed shell
membrane and the membrane under the drop is split along the line
of the fibre by means of a dissecting needle.
• Suction with a rubber teat is next applied to opening in the air sac
and as a result the chorioallantois recedes from the shell
membrane.
• The opening in the shell membrane is then enlarged with a capillary
pipette held vertically the inoculum (0.05 ml) is dropped into the
space that is the chorio allantoic membrane.
• Opening is sealed with adhesive tape 1 inches wise.

Viral culture in eggs: Some viruses, such as influenza viruses,
are grown in embryonated chicken eggs

Harvesting
• The inoculated egg is incubated for as long as necessary at
the optimum temperature for the particular virus been
cultivated.
• The egg is then placed on a cotton wool pad moistened with
antiseptics in an open petridish, the seal is then removed
and the edge of the opening is flamed with bunsen burner.
• The shell is broken down to the level of the displaced
choriom allantois , which is then separated by cutting
scissors and carefully removed, spread out in buffered
saline in a petridish.
• Examined for pocks against a black background.

Herpes virus lesion on the
chorioallantoic membrane

2. Allantoic cavity
• Simplest method with a larger yield.
• Allantoic inoculation is employed for growing
the influenza virus for vaccine production.
• Other allantoic vaccines include Yellow fever
(17D strain), and rabies (flury strain) vaccines.
• Duck eggs are bigger and have a longer incubation period than hen’s egg.
• provide a better yield and were used for the preparation of the inactivated
non-neural rabies vaccines.

method
• Eggs incubated for 10-11 days.
• The outline of the air sac pencilled and a point on the shell
is marked where the chorioallantois is well developed but
without large vessels.
• A small is cut through the shell and shell membrane over
the centre of the air sac and a small groove is drilled at the
marked point on the shell.
• The egg is placed on the triangular stand with groove
uppermost and the inoculation is made by injecting to a
depth of 2mm, 0.1 ml- 0.2 ml, through the groove with a
tubercular syringe.
• The opening is sealed with melted paraffin.

harvesting
• Incubation is carried out for two days and the egg is then
refrigerated for 2 to4 hrs to kill the embryo and obviate
bleeding in later manipulation.
• For withdrawl of the allantoic fluid, the sac over the air sac
is drilled and removed, the shell membrane and
chorioallantois in the floor of the air sac are cut away.
• The fluid of the underlying allantoic sac can then be
aspirated with a capillary pipette.

3. AMNIOTIC
INOCULATION
• Primary isolation of inluenza viruses
• Eggs used are 13-14 days incubation
• Circle of 3cm dia removed with its shell from air sac.
• With forceps inserted through CAM, amniotic sac is pulled &
inoculation is then made.
• A tuberculin syringe containing 0.05-0.25 ml of inoculum and a
0.1ml of air.
• Air is to ascertain if a bubble forms under the amnion, and if so
the inoculation is complete. Opening is sealed with adhesive tape.

harvesting
• The egg is incubated for 3-5 days atr 36’ C.
• The shell is then removed down to the level of the receded
chorio-allantois and the latter is cut away.
• The allantoic fluid is drained off and the amnion is picked up
with the forceps, and amniotic fluid aspirated with capillary
pipette.

4. YOLK SAC ROUTE
• - mammalian viruses
- chlamydiae
- rickettsiae
•Not suited for avian viruses

method
• Eggs incubated for 5-9 days.
• At the rounded pole of the egg over the air sac a small
groove is drilled, and the yolk sac is inoculated with a
inoculum of 0.1-0.5ml by a syringe and needle of 12-14
gauge, 3-3.5 cm long.
• Opening is sealed.
• Harvesting- incubation is then carried out and the egg is
then candled daily.
• The yolk sac is removed when the embryo dies.
• The shell is drilled and separated over the air sac and the
shell membrane and chorioalantois is cut away.
• The contents of egg are turned on petridish, and since sac
membrane itself contain a large amount of virus, this is
retained for further investigations.

 The signs of viral growth include
- death of the embryo,
- defects in embryonic development,
- localized areas of damage in the membranes,
resulting in discrete opaque spots called pocks
 The embryonic fluid and tissue can be prepared
for examination with an electron microscope
 - Some can also be detected by their ability to
agglutinate red blood cells or by their reaction
with an antibody of known specificity that will
affix to its corresponding virus, if it is present.

Specialised uses- How vaccines
are produced in eggs??
• In egg culture, flu viruses
are injected into chicken
egg embryos, where they
multiply. After several days
of incubation a machine
opens the egg and harvests
the virus, which is then
purified and chemically
killed.
• On average it takes one or
two eggs to produce a
single dose of annual flu
vaccine.

How the reassortmant vaccines
for influenza produced in eggs
• The egg is inoculated with
a mixture of the epidemic
influenza virus strain
(red) and a standard
strain (green) that can
replicate in chicken eggs.
Both strains replicate
themselves, but as they
do so their genetic
material becomes mixed,
producing hybrid viruses
known as reassortants

Eggs are used in mass scale development
of vaccines
Dr.T.V.Rao MD 54

Egg Allergies and Vaccines
• No suitable cell culture
system exists and egg
inoculation is the
method of
choice. Influenza virus
vaccines are still
cultivated in eggs, and
hence people with egg
allergies cannot
tolerate the influenza
vaccines.
55

3. Tissue culture
• Cultivation of bits of tissues and organs in
vitro were used by physiologists & surgeons
for the study of morphogenesis & wound
healing.
• First application in virology was by Steinhardt
in 1913 – maintained the vaccinia virus in
fragments of rabbit cornea

Major development in cell culture
technique
• Discoveries greatly enhanced the usefulness of
cell cultures for virologists and scientists
1. Use of antibiotics made it possible to prevent
bacterial contamination
2. Use of proteolytic enzymes (e.g. trypsin) can free
animal cells from surrounding tissues without
injuring freed cells
3. Use of chemically defined culture media.

• The turning point which made tissue culture the
most important method in cultivation of viruses
was the demonstration by Enders , Weller ,&
Robbins that polio virus , till then considered a
strictly neurotropic virus, could be grown in
tissue culture of non neural origin.
• Since then almost every human virus has been
grown in tissue culture

Types of tissue culture
1. Organ Culture
 Small bit of organs can be maintained in vitro for
days & weeks preserving their original
architecture & function.
 Isolation of some viruses which appear to be
highly specialised parasites of certain organs.
 Tracheal ring organ culture – corona virus

2. Explant Culture
• Fragments of minced tissue can be grown as
“explants” embedded in plasma clots
• Cultivated in suspension
• Originally known as ‘tissue culture’
• Seldom employed
• Adenoid tissue cultures - adenoviruses

3. Cell Culture
• Routinely employed for growing viruses
• Tissues are dissociated into component cells
by trypsin and mechanical shaking
• Cells filtered through sterile gauge , washed
by centrifugation in cold growth medium, &
finally suspended in a GM to the desired
concentration

CELL CULTURE MEDIA
• Most of the basic culture media used today are chemically
defined, but must be supplemented with 1–20 percent
serum.
• This is usually obtained from calf fetuses and must be
carefully checked for contamination by viruses, mycoplasma,
bacteria, and specific viral antibodies.
• Serum-free media is also commercially available for
specialized applications (e.g. vaccine development).
• Cells generally grow well at pH 7.0–7.4. Phenol red is often
used as an indicator in the medium, changing progressively
from purple at pH 7.8 through red at pH 7.4 to yellow at pH
6.5

• Culture media require careful buffering to stabilize the pH
under all conditions. The CO2 level is critical and can be
monitored by a gas analyzer. As little as a 1percent increase
can result in cell death.
• Medium components should be prepared with endotoxin-
free water.

• Media, media components, and reagents are
sterilized by autoclaving if they are heat-stable
(e.g. water, salt solutions, amino acid
hydrolysates) or by membrane filtration if they
are not (e.g. protein or sugar solutions).
• The type of membrane is important. Cellulose
acetate membranes are best for applications
involving low protein binding, and cellulose nitrate
membranes for general purpose filtration.

• Enable most cell types to multiply with a division time of
24-48 hrs
• Dispensed in bottles, tubes , petridishes
• Cells adhere to the glass surface & on incubation , divide
to form a confluent monolayer sheet of cells covering the
surface within a week
• Cell culture tubes may be incubated in a sloped horizontal
position, either as “stationary culture”
• May be rolled in special “roller drums” to provide better
aeration
• Fastidious viruses grow only in such roller cultures

Preparation of cell
culture
• Tissues from young or embryonic animals give better results
than those from adults. The tissues are first minced into 3-
mm pieces and the cells disaggregated by one or more
treatments with a solution of 0.22 percent trypsin and 0.02
percent versene.
• The cells are then filtered through sterile gauze, washed by
centrifugation in cold growth medium (GM), and finally
suspended in GM to the desired concentration.
• The concentration of viable cells is determined by counting
a sample diluted in trypan blue, which selectively stains
dead or damaged cells. Tubes or flasks are seeded at
approximately 3 * 105 viable cells/ml.

Types of cell cultures
• Based – origin
-chromosomal characters
-number of generations
• 3 types - primary
-diploid
-continuous

1 . Primary cell cultures
• Normal cells freshly taken from the body & cultured
• Capable of only limited growth in culture (5-10
divisions )
• Cannot be maintained in serial cultures
• These cells have a normal diploid chromosomal
complement- prefered for vaccine production
• Eg. Rhesus monkey kidney cell culture, human amnion
cell culture and chick embryo fibroblast cell culture.

2. Diploid cell cultures
• Cells of single type (euploid) that retain the original
diploid chromosome number & karyotype during serial
subcultivation for a limited number of divisions
• 50 serial passages – stop dividing
• k/a semicontinuous cell lines
• Use- for isolation of Fastidious pathogens and viral
vaccine production.
• For eg. Rabies vaccine is produced by cultivation of fixed
rabies virus in WI- 38 human embryonic lung cell strain.
• Other eg .MRC-5, HFL 1.

3. Continuous cell lines
• Cells of single type (aneuploid or heteroploid),
lines have a chromosome number that is not an
exact multiple of the haploid number.
• Derived from cancer cells
• Capable of continuous serial cultivation
indefinitely
• maintained by serial subcultivation or stored at -
70°C for use when necessary
• (e.g. HEp-2, HeLa, LLC-MK2).

Cell cultures
Primary cell cultures
1. Rhesus monkey kidney
2. Human amnion
3. Chick embryo fibroblast
Diploid cell cultures
1. WI-38 Human embryonic lung cell
strain
2. HL-8 Rhesus embryo cell strain

 Continuous cell lines
1.HeLa Human carcinoma of cervix
2.HEP-2 Human epithelioma of larynx
3. KB Human carcinoma of nasopharynx
4. McCoy Human synovial carcinoma
5. Detroit-6 Sternal marrow
6. BHK-2 Baby Hamster kidney
7. Vero African green monkey kidney cells
8. MDCK Dog kidney
9. A549 human lung carcinoma
10. LLC-MK2 Rhesus monkey kidney
11. BGM buffolo green monkey kidney
12. RD Human rhabdomyosarcoma cell line

• HeLa and HEp2- used for herpes simplex virus,
adenovirus, poliovirus and some coxsackie viruses.
• Special clones of HeLa cells (eg. HeLa ‘Bristol’ or
‘Ohio’ – susceptible to respiratory synctial virus,
some rhinoviruses.
• BHK21- arbovirus
• RK13 and BHK21 –rubella virus
• RD cells- for Coxsackie A viruses.

Insect cell lines
• Continuous cultures derived from
-mosquitoes (Aedes aegypti & A. albopictus)
-SF9 ovary-fall army worm ( Spodoptera frugiperda)
• SF9 line is highly susceptible to infection with baculovirus –
used for baculovirus expression vectors
• Mosquito lines – suspension cultures for the replication of
many mosquito borne viruses
• Have special media & subculturing requirements

Suspension cultures
• Suspension cultures can be prepared from many mouse and
human leukemias and ascites tumors.
• They are incubated on shakers or in roller drums or allowed
to settle to the bottom of a large culture flask, such as a
roller bottle.
• Alternatively, a spinner apparatus is available that forces
normally adherent cells to grow in suspension in a special
medium.

Shell vial culture technique
• Is a modification of the conventional cell culture
technique for rapid detection of viruses in vitro.
• Contain a cell monolayer grown on a coverslip in a 1-dram vial
• Vial incubated with the specimen & centrifuged at low speed
(700g for 45 min)
• Which apparently distorts the cell surface & renders it
more susceptible to viral attachment

• After 1–3 days of incubation, irrespective of any cytopathic
effects (CPE), the coverslip is washed briefly, and tested
by immunofluorescence assay (IFA), nucleic acid
hybridization,
• Shell vial cultures have been successfully used as a rapid
test for adenovirus, Cytomegalovirus (CMV), varicella-
zoster virus (VZV),respiratory syncytial virus (RSV), and
other respiratory viruses.

MICROCULTURES
• Microcultures are cost-efficient systems of growing
popularity, having overcome earlier problems with
crosscontamination between wells, over-oxygenation, and
toxic plastics.
• Microcultures in 24- to 48- and 96-well plates with sealable
lids (or tightly fitting lids for CO2 incubators) are now
commonly used in a variety of viral assays.
• In 96-well microtiter plates with flat well bottoms,
microcultures are ideal for neutralization tests, tissue
culture enzyme immunoassay (EIA), monoclonal antibody
testing, and many screening assays.

Mixed Cell Culture
• Mixed cultures of different cell types combined in a single
vial have been used successfully to grow a broad range of
viruses.
• A commercial product, Mixed Fresh-Cells, available from
Diagnostic Hybrids, Inc. (Athens,OH, USA), combines two
compatible cell lines optimized for growing particular groups
of viruses, e.g. R-Mix for respiratory viruses, E-Mix for
enteroviruses and H&V-Mix for herpesviruses.
• For example, R-Mix consists of a mixed monolayer of mink
lung cells (Mv1Lu) and human adenocarcinoma cells (A549)
that have been shown in clinical studies to support the
detection of influenza A and B viruses, RSV, adenovirus, and
parainfluenza viruses 1, 2, and 3.

Genetically Engineered
Cell Lines
• Genetically engineered cell lines offer potential improvements in
our ability to grow viruses and to rapidly detect virus infection.
• Stable transformation of specific virus receptors for HIV-1 and
EBV into previously nonpermissive cell lines has been shown to
permit growth of these viruses.
• Genetically modified cells that express an easily measured
reporter enzyme after infection with a specific virus have also
been developed.
• For example, a transgenic baby hamster kidney cell line (BHKIC
BHKICP6LacZ) that contains multiple copies of a virus-inducible
HSV promoter sequence linked to the reporter gene LacZ
developed by Stabell and Olivo (1992) has been used successfully
for diagnosis of infections with HSV types 1 and 2.

Problems with cell
culture
• Long period (50days–CMV) required for result.
• Often very poor sensitivity, sensitivity depends on a
large extent on the condition of the specimen.
-Susceptible to bacterial contamination.
-Susceptible to toxic substances which may be present in the
specimen.
-Certain viruses don’t grow or grow slowly
-Other techniques for detecting viral infection more cost effective
-Successful culture depends on viability of virus in specimen

Quality control
• Contamination by bacteria & fungi is an inherent
problem of cell culture
• Can be detected by appearance of turbidity or color
changes due to changes in the pH of the medium or
more definitively by microscopic examination of the
culture
• Contamination with mycoplasmas & viruses is more
difficult to detect As they -
• Do not cause changes in turbidity or pH changes & may
not cause a CPE
• Are able to pass through filters used for sterilization
of media

• Primary rhesus monkey kidney (MK), primary
African green monkey kidney (AGMK), and
primary bovine embryonic kidney cell cultures
are particularly notorious for adventitious virus
contamination.
• A variety of methods have been described for
detection of the mycoplasma species most
commonly found in mammalian cell culture,
including microbiological culture, DNA staining
with fluorescent dyes, mycoplasmal enzymatic
assays, IFA, and EIA, hybridization probes, and
polymerase chain reaction (PCR) assay.

Commercial testing services are widely available for
mycoplasmas and viruses and commercial kits, such as the
MycoTect test (Invitrogen), MycoSensor PCR Assay
(Stratagene), and Mycoplasma Detection Kit (Roche Applied
Science) are available that can be incorporated into a
quality control program.
• Contamination by other cells lines can also occur – intrusion
of Hela cells into many other lines giving the most problems

Preventive measures
• Working with only one cell line at a time
• Keeping bottles of medium , trypsin etc separate for each
cell line
• Allowing a resting period of approx 30 min between
manipulation of different cell lines in a biological safety
cabinet
• Decontaminating cabinet surfaces before introducing
another cell line to the work area

PROPAGATION OF
CELLS
• Two method- physical method and enzymatic method
• 1- Physical method- scraping, used when enzymatic removal
may be toxic to the cells or may destroy receptors or other
important cell surface molecules.
• The GM is discarded, the cells are physically scraped from
the vessel surface with a disposable cell scraper into fresh
GM, and the cells are then dispersed by vigorous pipetting
to obtain a suspension of single cells.
• The cells are counted and appropriately diluted for passage
to new flasks or other vessels.

• Cell counts - best performed in the presence of a vital stain, which
is excluded by living cell membranes and is incorporated only into
dead cells.
• In the trypan blue vital stain procedure, a sample of the cell
suspension is mixed with 0.4 percent dye in physiological saline,
placed in the counting chamber of a hemocytometer, and the cells
counted at 100 magnification.
• Trypan blue and erythrosin B are the dyes most commonly used
for dye exclusion tests.

• 2. Enzymatic method- a chemical method of detaching adherent
cells from the culture vessel with enzymes, such as trypsin,
pronase, or collagenase, together with chelating agents, such as
versene (tetrasodium ethylenediaminetetraacetic acid (EDTA)).
• After washing twice with phosphate-buffered saline (PBS), the
monolayer is covered with a solution of 0.05 percent trypsin and
0.53 mM versene and placed at room temperature with cell surface
down until the cells detach. After vigorously pipetting to break up
clumps, the cells are resuspended at the desired concentration in
fresh GM.
• Cells in suspension cultures are propagated by adding a sample to
sufficient GM to give the desired concentration.

MAINTENANCE OF
CULTURES
• Once cells have been passaged, maintenance is necessary to
encourage their viability by feeding them with fresh amino acids,
vitamins, glucose, and other metabolic constituents.
• The initial cell concentration and the metabolic rate of the cells
will determine the feeding schedule.
• Feeding is done with a maintenance medium (MM), which is the
regular medium with reduced serum content (1–2 percent serum vs.
10–20 percent used in GM).
• MM may hold untransformed cell lines at a single cell layer for an
extended period, even 2–3 weeks, because it tends not to
stimulate mitosis.

• Transformed (heteroploid) epithelial cells, such as HeLa and HEp-
2, metabolize rapidly and should be subcultured twice a week.
• Diploid fibroblast cells, such as MRC-5 and WI-38, metabolize
slowly and require subculturing only once a week, with a feeding at
least once during that week.

STORAGE OF CELLS
• Cells may be stored by cryopreservation. Cells are packed
by lowspeed centrifugation and resuspended in a freeze
medium containing serum and dimethylsulfoxide (DMSO)
• One-ml volumes are dispensed into cryovials, frozen slowly
at approximately 1C per min, preferably in a controlled-rate
freezer, and stored in liquid nitrogen (165C).
• Cells are recovered from storage by rapidly thawing in a
37C water bath, diluting the cells with the appropriate
medium, and placing them in a cell culture flask.
• The medium is replaced after 24 h to remove all traces of
DMSO.

Detection of viral growth in cell culture
1.Cytopathic effect
• The term "cytopathic effect" (CPE) is frequently applied to
virus-induced morphological changes in cellular cultures that
are visible by light microscopy.
• cytopathogenic viruses
• The most efficient way to demonstrate cellular changes is by
staining (hematoxylin &Eosin)

Examples of Cytopathic
Effects of Viral Infection
• Nuclear shrinking
(pyknosis)
• Proliferation of nuclear
membrane
• Vacuoles in cytoplasm
• Syncytia (cell fusion)
• Margination and breaking
of chromosomes
• Rounding up and
detachment of cultured
cells
• Inclusion bodies

• Crenation of cells & degeneration of cell sheet-
enteroviruses
• Multinucleate giant cells (Syncytium formation) – measles
virus, respiratory synctial virus, HIV.
• Discrete focal degeneration – herpes virus
• Large granular clumps (bunches of grapes), rounding and
aggregation- adenovirus
• Cytoplasmic vacuolation – SV40
• Inclusion bodies-

Cytopathic effect (cpe)
Adenovirus Herpes virus

Syncytium formation in cell
culture caused by RSV (top),
and measles virus (bottom).

Inclusion bodies
• Inclusion bodies are virus-specific intracellular globular
masses which are produced during replication of virus in
host cells.
• They can be demonstrated in virus infected cells under
light microscope after fixation & staining
• Eg: Negri bodies in rabies .
98

Inclusion bodies
• Intranuclear or cytoplasmic aggregates of
products of viral replication
-virus particles ready for release
-over production of particular viral protein
-some aberrant cellular structures –clumped
chromatin
• Can be seen in stained preparations under light
microscope

• Cytoplasmic / nuclear / both
• Acidophilic / basophilic
• Single / multiple
• Large / small
• Round / irregular
• Intracytoplasmic
vaccinia - guarneri bodies
rabies - negri bodies
fowl pox - bollinger bodies
molluscum contagiosum - molluscum bodies

• Intranuclear
Cowdry type A- herpes virus
- yellow fever
virus
Cowdry type B – polio virus
- adenovirus
 basophilic - adenoviruses

Negri
bodies
Negri
bodies
neuron
102

2. Metabolic inhibition
• In normal cell cultures, medium turns acidic due to cellular
metabolism.
• Cell metabolism is inhibited when viruses grow in cell
culture.
• There is no acid production which can be made out by the
color of indicator (phenol red) in the medium.

3. Hemadsorbtion:
• Some orthomyxoviruses ( influenza) &
paramyxoviruses ( parainfluenza , measles , mumps )
code for red cell agglutinins which are incorporated
into the cell membrane during infection
• So that erythrocytes adhere to the cells
• This adherence of erythrocytes to the cells is known
as Hemadsorption
• Can be used to recognise infection with non-
cytocidal viruses & early stage of cytocidal viruses

Hemadsorbing viruses
• Orthomyxoviruses (influenza) and some
paramyxoviruses (parainfluenza, measles,
mumps)
• Insert viral glycoproteins (haemaglutinin)
into host cell membrane.
Promotes attachment of RBC of certain
species (e.g guinea pig) to cell membrane.

Hemadsorbtion
add red blood cells
www.freelivedoctor.com

Haemadsorption
Syncytial formation caused by mumps virus and
haemadsorption of erythrocytes onto the surface of
the cell sheet.

Hemadsorption of erythrocytes to cells infected with influenza viruses, mumps virus, parainfluenza viruses, or togaviruses.
These viruses express a hemagglutinin on their surfaces, which bind erythrocytes of selected animal species.
Hemadsorption

4. Interference
• Multiplication of one virus in cell usually inhibits
the infection with a second virus called the
challenge virus when added to the culture
• Can be used for detection of the growth of a non-
cytopathogenic virus in cell culture

Interference
• Rubella virus growing in monkey
kidney cells inhibits infection with
echovirus 2.

5. Transformation
• Oncogenic viruses induce cell transformation &
loss of contact inhibition
• Growth appears in piled up fashion producing
“Microtumours”
• Herpesviruses, adenoviruses, papovaviruses,
hepadnavirus, retroviruses

Transformation
loss of contact inhibition

Focus formation by transforming viruses
Focus assay. Monolayers of the NIH3T3 mouse fibroblast cell line were infected with Maloney murine sarcoma virus. The top two panels
show photomicrographs of uninfected cells (left) and a single virus-induced focus (right). The bottom two panels show stained dishes of
uninfected (left) and infected (right) cells. Foci are clearly visible as darker areas on the infected dish.

6. Immunofluorescence
• Cells from virus infected cultures can be stained
with fluorescein-conjugated antiserum and seen
under fluorescence microscope for virus
antigens
Positive
immunofluorescence
test for rabies virus
antigen

7. Detection of
enzymes
• The virus isolates can be identified
by detection of viral enzymes –
reverse trancriptase in retroviruses
, in the culture fluid.

Follow all the Biosafety
Considerations
 All procedures involving cell
lines should be performed in
a class II biological safety
cabinet under strict aseptic
conditions, both to minimize
the risk of contaminating
cells with bacteria, yeasts
and mycoplasmas and to
protect the worker.

• Cell lines should be considered potentially
hazardous .
• Although continuous cell lines are assumed
to be free of infectious agents, they may
in fact harbour latent viruses that could
infect the worker.

Viral assay
• Virus content of a specimen can be
assayed in 2 ways
-total virus particles
-infectious virions
2 methods used for total particle
enumeration
1. Electron microscopy
2. Hemagglutination

Electron microscopy
• Negative staining – virus particles in a
suspension can be counted directly
• Suspension is mixed with a known
concentration of latex particles
• Ratio between virus & latex particles -
virus count

Hemagglutination
• Determination of hemagglutinating titres is a
convenient method of quantification of
hemagglutinating viruses
• Not a very sensitive indicator of the presence
of small amounts of virus
• Approx 107 influenza viruses are required to
produce macroscopic agglutination of a
convenient quantity of chicken erythrocytes
(0.5ml of 0.5 % suspension)

Hemagglutination assay
• method for detection & assay of influenza virus
• When red cells are added to serial dilutions of a
viral suspension , the highest dilution that
produces haemagglutination provides
hemagglutination titre.
• Red cells which are not agglutinated settle at the
bottom (button) ,agglutinated cells are seen spread
into shield like pattern.

• As hemagglutination is specifically
inhibited by antibody to virus,
hemagglutination inhibition
provides a convenient test for
antiviral antibody.
• Parainfluenza, measles, rubella, rabies,
reovirus

Hemagglutination
RBC

Titer = 32 HA units/ml
Hemagglutination test: method
1:8
1:2 1:2
1:2
1:2
1:2
8 16 32 64 128 256
virus
serial dilution
mix with red
blood cells
side view
top view

Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as
indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row
contain no virus. Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the
bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity.
Hemagglutination assay: influenza virus

ASSAY OF INFECTIVITY
1.Quantitative: actual no. of infectious particles in
inoculum.
a.Plaque assay in monolayer cell culture
b.Pock assay on chick embryo CAM
2.Quantal assays: indicate presence/absence of
infectious viruses (LD50 or ID50)

 Young actively growing bacteria ( in log phase)
 Effective for the viruses which can infect monolayer cells and for
viruses which can break down the cells
 STEPS
- A dilution of a suspension containing the virus material is mixed in a
small amount of melted agar with the sensitive host bacteria.
- The mixture is poured on the surface of a nutrient agar plate
- Plate is allowed to solidify
- Incubated at 37 degree C for 18-24 hrs
- Host bacteria form a lawn of confluent growth
- Plate is observed for plaque formation
Plaque assay technique

• Plaque assay
technique for
quantification of
bacterial viruses.

 Plaque ?
 The basis is that one viral particle infects one cell,
is replicated and the cell lyses. The nearby cells are
infected and a ‘plaque’ of dead cells is formed over
time.
Zone of cell death/ a clear area in a bacterial lawn culture where
viruses have lysed host cells
HOW TO IDENTIFY
TEMPERATE PHAGE?
–cloudy plaque

 Basis of plaque formation:
 Plaque assay – also to calculate
number of phages present.
 The titer of a phage suspension,
is determined by counting the
number of plaques that form from
a given volume of suspension.
 Phage titer is expressed as
plaque forming units (PFU) per
milliliter (ml).
pfu/ml * measurement of the
number of viable, infectious
bacteriophage

• The number of plaque-forming units is
almost always lower than direct counts by
microscopy
– Inactive virions
– Conditions not appropriate for
infectivity

Quantal assays
• Can be carried out in animals ,eggs, or tissue culture.
• Endpoints used for titration are death of animal, production
of haemagglutinin in allantoic fluid or appearance of CPE
• The titre is expressed as “50% of infectious dose”( ID-50)
/ml
• Indicates highest dilution of inoculum that produces effect
in 50% of animals,eggs or cell cultures inoculated

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