tissue culture and virology, tissue culture for virology, cytopathic effect,(CPE), plaque assay,

1. TISSUE CULTURE AND
VIROLOGY
Supervision of:
DR.GHULAM MUHAMMAD
Presented By:
NOMAN HAFEEZ KHOSA
Subject:
Cell and tissue culture technology

2. Tissue Culture
• Since the discovery by Enders (1949) that
polioviruses could be cultured in tissue, cell culture
has become a very useful and convenient method for
isolating viruses in vitro.
• A single cell culture can be used to cultivate a broad
spectrum of viral agents.
• Viral culture also facilitates the production of high
tittered virus which can be used in:
• Antibody testing
• Viral characterization
• or molecular analysis

3. Monolayer Cell Cultures
• Most diagnostic virology laboratories use monolayer
cell cultures to propagate viruses
• The main advantage of using monolayer cultures is
the ease with which the infected cultures can be
monitored microscopically
• Many viruses present themselves in cell culture by
producing degenerative changes in the cells, the so-
called cytopathic effect (CPE)
• The CPE is often characteristic of a specific virus
and this allows the experienced observer to make a
presumptive diagnosis based on the type of CPE
present on the monolayer

4. Transport Medium
• Enveloped viruses are particularly labile
• Viruses such as respiratory syncytial
virus, influenza and the herpes viruses,
may lose infectivity if they are not
adequately protected
• Rapid transportation to the laboratory
under the proper conditions can greatly
enhance effective isolation
• Viruses should be transported to the
laboratory in the appropriate transport
medium (viral transport medium) which
can be bought commercially or made up
in Lab.

5. Isolation of Viruses in Cell Culture
• The ability to culture viruses successfully in the
laboratory depends on a number of important factors
which include:
• The sensitivity of the cells used
• The viability of the virus
• The type of specimens sent to the laboratory
• The stage of the patients illness when the specimen is taken
• and the way they are processed
• The culture conditions
• Even when all these considerations are taken into
account, not all viruses can be cultured
• There are certain viruses that are very difficult to
grow or require very specialized culture conditions.

6. Isolation of Viruses in Cell Culture
• However, most of the more common human pathogenic
viruses can be cultured relatively easily provided the
proper conditions are satisfied
• A wide variety of virus-sensitive cell lines are available
either commercially or through one of the national or
international cell bank collections such as ATCC &
ECACC

7. Standard Virus Isolation from Samples
• Seed cell suspensions into culture vessels using
freshly made medium
• For viral isolation it is usual to prepare at least three
different cell types for inoculation to increase the
chances of isolation
• While some cell lines have a broad range of viral
susceptibility, no single cell line is sensitive to every
virus

8. • Vessels are incubated and allowed to reach 90%
confluent
• 0.2 ml of freshly prepared specimen is inoculated
into each vessel in duplicate
• Incubation at 37°C
• Some viruses (influenza, parainfluenza) need to be
cultured at lower temperatures
• Examine the cultures daily for CPE
Standard Virus Isolation from Samples

9. Micro titer Method of Virus Isolation
from Samples
• Using this method six cell lines are seeded in
suspension on 96 micro titter plates thereby
improving the sensitivity of virus isolation
• Up to four specimens can be inoculated with each
plate
• The cell lines selected for micro titer plate work
should represent a broad range of viral
susceptibility
• The plates are monitored daily for CPE using an
inverted microscope

10. Microtiter Method of Virus Isolation from Samples
Sample 1
(4 dilutions)
Sample 2
(4 dilutions)
Sample
3
Sample
4
6 cell lines 6 cell lines

11. Identification of Virus Isolates
• Development of characteristic CPE in cell culture is often useful in
making a presumptive identification of the viral isolate
• This identification would also be based on the specimen source
and the cell type in which the virus has grown
• However, final identification of the viral isolate needs to be
confirmed
A. Monolayer of uninfected Hep-2 cells (x20)
B. Hep-2 cells infected with respiratory syncytial virus showing typical
syncytial cytopathic effect (x40)

13. The upper left panel shows uninfected cells, and the other panels show the
cells at the indicated times after infection. As the virus replicates, infected
cells round up and detach from the cell culture plate. These visible changes
are called cytopathic effects.

14. Cytopathic Effect (CPE)
• Uninfected cells are adherent, they normally grow
flat and stuck down firmly on the tissue culture
flask
• After infection with rhinovirus, the cells change
shape, becoming round and more refractile
(brighter) under phase contrast microscopy
• Some infected cells detach from the tissue culture
flask and float in the medium

15. Identification of Virus Isolates
• Not all viruses will produce a CPE and some
viruses are slow to grow
• Immunoassay techniques can also allow early
detection of viral replication prior to the formation
of a CPE and allow more rapid viral diagnosis.

16. Plaque Assay
• Based on the ability of infectious virus
particles to form small areas of cell lysis or
foci of infection on the cell monolayer
• This is achieved by first adsorbing the virus
onto a confluent cell monolayer and then
overlaying the monolayer with agar
• The overlay medium restricts the spread of secondary infection so
that only areas of the cell monolayer adjacent to the initially
infected cells will become infected and form plaques or small
areas of CPE
• These plaques can then be counted and the viral titer calculated
• Plaque assays can be carried out in 24-well cell cluster plates or
cell culture plates

17. Plaque Assay

19. Thank
you

20. Plaque assay- additional notes
• Plaque assay
• Viral Plaques of Herpes Simplex Virus
• Plaque-based assays are the standard method used to
determine virus concentration in terms of infectious dose. Viral
plaque assays determine the number of plaque forming
units (pfu) in a virus sample, which is one measure of virus
quantity. This assay is based on a microbiological method
conducted in petri dishesor multi-well plates. Specifically, a
confluent monolayer of host cells is infected with the virus at
varying dilutions and covered with a semi-solid medium, such
as agar orcarboxymethyl cellulose, to prevent the virus
infection from spreading indiscriminately. A viral plaque is
formed when a virus infects a cell within the fixed cell
monolayer. The virus infected cell will lyse and spread the
infection to adjacent cells where the infection-to-lysis cycle is
repeated.

21. Plaque assay- additional notes(2)
• The infected cell area will create a plaque (an area of
infection surrounded by uninfected cells) which can
be seen visually or with an optical microscope.
Plaque formation can take 3 – 14 days, depending on
the virus being analyzed. Plaques are generally
counted manually and the results, in combination with
the dilution factor used to prepare the plate, are used
to calculate the number of plaque forming units per
sample unit volume (pfu/mL). The pfu/mL result
represents the number of infective particles within the
sample and is based on the assumption that each
plaque formed is representative of one infective virus
particle.