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Diagnostic Procedure
Antigen Detection ELISA
Cytopathic Effect in
Tissue Culture
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Isolation of Virus from Field Samples in Tissue Culture
1. Label the tissue culture tubes or flasks to be inoculated with the details of the virus sample(s) to be passaged, e.g., sample identification, cell type, passage level and date.
2. Use additional tissue culture tubes (or flasks) of the same batch as control cultures for comparative observation, i.e., uninoculated.
3. Discard the growth media into a sterile beaker using aseptic techniques.
4. Rinse the cell sheet carefully with a small volume of pre-warmed (37°C) Eagle's MEM and discard.
5. Add an aliquot of the 10% (w/v) epithelial suspension to the tubes (or flask) e.g., 0.2 ml/tube, 1 ml/flask.
6. Rinse outside of tubes (or flasks) in disinfectant solution and water.
7. Leave flat (monolayer at bottom) at 37°C for 30 minutes to allow any virus present in suspension to adsorb to cells.
8. Add sterile Eagle's MEM (containing antibiotics but not serum) in sufficient volume to cover cell monolayer, e.g., 2 ml/tube, 15 ml/4 oz flask.
9. Rinse the outside of tubes (or flasks) in disinfectant solution and water and return to 37°C, flasks kept stationary, roll tubes continuously.
10. Observe daily for cytopathic effect (CPE in tissue culture).
11. Once CPE is complete, harvest the tissue culture supernatant fluid into a centrifuge tube and centrifuge at maximum rpm for 10 minutes in a bench centrifuge.
12. Tip clear supernatant fluid into a fresh, sterile container and take an aliquot (minimum of 1.4 ml) for the antigen detection ELISA. The remainder of the viral suspension can be stored at 4°C until a result from ELISA is obtained.
Passage of Tissue Culture Grown Virus
13. If first passage tissue cultures are negative for CPE 48 hours after inoculation, harvest the supernatant fluid as in 11, re-inoculate fresh cultures (second passage) as in 1 to 10 (although maintenance media can be added to cultures prior to inoculation, thus making the virus adsorption stage unnecessary) and observe for a further 48 hours for evidence of CPE.
The above procedure can be used to amplify (grow) field isolates for virus characterisation and as a source of antigen for antibody detection and quantitation.
Sample Storage
1. Upon test completion, original material is stored if required at -20°C.
2. If positive tissue culture supernatant fluids are to be stored, mix with di-ethyl ether (5:1), and leave for a minimum of 2 hours or overnight at 4°C. Centrifuge ether-treated suspensions at approximately 1000 g (2-3000 rpm) for 10 min in a bench centrifuge and remove the ether by carefully pipetting off from the surface of the supernatant fluid. Add an equal volume of sterile glycerol to the remaining viral supernatant fluid, mix well and store in 1-2 ml aliquots in cryotubes at -20°C.
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