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
ECACC: European collection of cell culture
ATCC: American Type culture collection

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. Standard Virus Isolation from Samples
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

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
6 cell lines
6 cell lines
Sample 1
(4 dilutions)
Sample 2
Sample 3 4
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
Monolayer of uninfected Hep-2 cells (x20)
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
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.