Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

To quantify the amount of virus collected from each cornea, prepare a log₁₀ one-fold dilution of the virus in serum-free medium in microcentrifuge tubes until a dilution of 1 x 10^-8 is reached.

After aspirating the growth medium from each well of cells, transfer 1 milliliter of each virus dilution, starting at 1 x 10^-3 to the plated cell monolayers, and place the infected cells in the cell culture incubator for two hours. At the end of the incubation, gently wash each well 2 times with PBS before adding 2 milliliters of methylcellulose-laden medium to each well for a 72-hour incubation, or until the formation of plaques can be observed.

Upon plaque observation, slowly add 1 milliliter of methanol to the corner of each well for a 15-minute incubation at room temperature. At the end of the incubation, slowly aspirate the contents from each well without disturbing the cell monolayer.

Next, label each well with 1 milliliter of crystal violet working solution, taking care that all of the cells are covered for a 30-minute incubation protected from light. At the end of the incubation, discard the solution and dry the wells on a sheet of absorbent paper. Then, count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution 3 times.