Virus titration by using the plaque assay

1. Prepare confluent monolayers of cells (vero cell) in 24 well plates. 2. Prepare serial 10-fold dilutions (101 to 107 ) of virus in chilled maintenance medium (MEM, with 1% serum). 3. Remove culture medium and add 0.2ml of virus inoculum, starting from the highest dilution. Ensure that a film of medium completely covers the cell sheet. 4. Incubate the plate at 37 C for 1 hour with intermittent rocking of the plate. 5. Remove the inoculum, preferably with a pipette and then add 1.5 ml of agarose overlay medium (growth medium with 0.3% agarose and 2.5% FCS). 6. Ensure that the overlay medium has spread evenly over the monolayer, leave at room temperature for 10 mins and then incubate at 37 C . 7. Examine the monolayers daily, starting from second day of incubation. 8. Once the plaques have developed, usually by the fourth day post inoculation, count the number of plaques at each dilution, remove the agarose overlay and gently wash the monolayer with PBS. 9. Stain the plate with 0.1% crystal violet solution and count the plaques again. 10.Estimate the virus titre as a plaque forming units per ml (pfu /ml) as follows by counting the number of plaques at an appropriate dilution. For example : Number of plaques produced Dilution of virus Volume of inoculum Virus titre = =

9 1 x 10 0.2 ml 9 x 1x105 x 1/5 pfu per ml 4.5 x 106

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