Virus Plaque Assay: A Technique to Measure Infectious Herpes Simplex Virus via Quantification of Plaques Formed in Monolayer of Virus-Infected Cells

Remove the cell culture medium from one or two wells using a pipette. Carefully, add the diluted virus sample drop-wise to each monolayer of cells, drop-wise through the side of each well.

Repeat the process for every one or two wells until the entire plate is filled with the virus samples being plaqued. Gently rock the entire plate by hand.

Then, incubate the plate at 37 degrees Celsius for 1 hour to allow the virus adsorption. After every 10 minutes, remove the plate from the incubator, gently rock it again to spread the virus more evenly across each well, and then, place it back in the incubator.

To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells using a 1,000-microliter pipette tip, and discard the sample in a waste beaker.

Place 2.5 milliliters of a methylcellulose overlay on the plate, and place it back in the incubator. Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet.

After the two-day incubation, remove the methylcellulose overlay from one or two wells at a time, using a pipette, and place it in a waste beaker. Add approximately 2 milliliters of 1% crystal violet and 50% ethanol to each well. Incubate the plate with all wells filled with the stain for 30 minutes at 37 degrees Celsius.

At this point, disinfect the waste beaker, as demonstrated previously. Wash the plates vigorously with tap water, until the runoff is clear.

Ensure that there are approximately 10-fold differences in the number of plaques across each dilution series.