Virus titration by plaque assay:
1. Prepare confluent monolayers of cells (Hep-2 for Polio
virus) in 24 well plates (Nunc).
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 (once in 15 min).
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 min 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 9
Dilution of virus 1 x 105
Volume of inoculum 0.2 ml
Virus titre = 9 x 1x105 x 1/5 pfu per ml
= 4.5 x 106
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