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METHODS OF CULTIVATION OF VIRUSES
METHODS OF CULTIVATION OF VIRUSES
Since viruses are obligate intracellular parasites they have to be grown in living cells. There are three systems for their cultivation:
- Tissue culture.
- Embryonated eggs.
- Intact animals.
1– Tissue Culture
Pieces of animal or human tissues are trypsinized to get separate cells. These are grown in presence of growth medium containing serum, on glass or plastic tubes, bottles or plates with a flat side. Amono layer or sheet of cells is formed on the flat side of the contains within few days.
Viruses are inoculated on the monolayer. There are three types of tissue culture.
- Primary cell lines: These are prepared from organ fragments e.g. monkey kidney. Such cells can only divide for several passages ( 4-6 ) and then degenerate.
- Human diploid cell lines: These are usually fibroblasts derived from human embryo tissues. They have diploid number of chromosomes. They grow rapidly and can be sub cultured up to about 50 passages in culture e.g. human embryo lung tissues.
- Continuous cell lines: These are derived from tumbler cells and they can divide indefinitely e.g. Hela cells which are derived from carcinoma of the cervix.
Detection of virus replication in cell culture
1- Cytopathogenic effects (CPE): These are changes in cells that can be observed microscopically:
a- cell death and detachment from the glass surface is produced by many viruses e.g. poliovirus.
b- Rounding and grape – like cluster formation is produced by adeno virus.
c- Syncytium or multinucleate giant cell formation are characteristic of measles or mumps.
d- Cell transformation: The cell lose the property of contact inhibition present in normal cells and pile up to form foci of malignantly transformed cells e.g. when infected with tumor viruses.
2- Plaque formation: Plaques are virally infected areas in tissue culture monolayer. They can be seen by the naked eye as unstained areas when using vital stains.
3– Inclusion bodies: These are intranuclear or intracytoplasmic structures or intracytoplasmic structures.
Which may appear in virus infected cells and can be seen by the light microscope. They are often the site of virus replication. There presence is of diagnostic value e.g. the ” Negri bodies ” in nerve cells of rabid animals.
4- Haem absorption: when RBCs are added to infected cells they will appear as losettes or clumps an the areas where the virus is growing. This is useful in haemagglutinating viruses e.g. influenze virus.
5- Fluorecent – antibody staining: Infected cell sheets on cover slips or microtiter plates may be treated with fluorescien labeled specific antibody and examined for positive fluorescence.
6– Inter ference: In some viruses which do not produce CPE their growth can be proved by their ability to interfere with the growth of mother CPE producing virus.
7- Detection of viral antigens by serology: soluble antigens which diffuse in the nutrient medium or those released after freeing and thawing of tissue culture cells, can be detected by any serologic method including complement fixation, haemagglutination… etc.
8- Neutralization tests: Neutralization of the effects of virus on tissue culture by specific antisera can be used to identify and type the virus isolated.
2- Embryonated Eggs
Different viruses can grow in various cavities of Embryonated eggs or in the developing embryo it self. The age of the embryo used and the site of inoculation vary according to the virus inoculated.
Choriallantoic membrance inoculation is used in pox and herpes viruses. The influenza virus can readily grow in the amniotic sec and in the respiratory cells of the embryo.
3 – Intact Animal
METHODS OF CULTIVATION OF VIRUSES
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