1. Seeding of the Vero cells

1. The day before initiating the plaque assay, trypsinize Vero cells and resuspend them in regular Dulbecco's Modified Eagles Medium (DMEM) and supplement as per standard cell culture methodology. Resuspend the trypsinized cells in 10 mL of DMEM per T-75 flask of the confluent Vero cells (~10⁷ cells).
   NOTE: DMEM is used for plaque assays instead of alpha-MEM because it slightly slows the cell growth rate and favors better plaque assay results.
2. Count the cells by one's preferred method (e.g., a standard hemocytometer with Trypan Blue exclusion).
3. Seed the cells at 4 x 10⁶ cells/plate; for HSV-2, use 6-well plates with 2.5 mL of DMEM (with supplements) per well; and for HSV-1, use 12-well plates with 1.25 mL of DMEM (with supplements) per well.
   NOTE: The number of cells should be constant regardless of the plate used, though the size of the wells matters concerning accuracy in the plaque assay. It is essential to get cells evenly distributed, which can be most effectively accomplished by moving the plate back and forth, not in a circular fashion.
4. Allow the cells to grow overnight at 37 °C/5% CO₂ in a humidified incubator.
   NOTE: Humidity is maintained with a pan of distilled water containing an algicide in the bottom of the incubator.

2. Sample dilution

1. Complete the sample dilution and the addition of the virus to cells in one lab session.
   CAUTION: HSV-1 and -2 are infectious for humans and must be handled under proper biosafety containment (BSL-2). Keep all materials that come into contact with the virus separate and disinfect with a quaternary agent at 1/256 dilution (see Table of Materials), iodophor, bleach, or strong ionic detergent (e.g., SDS) before removing them from the biosafety cabinet.
2. The day after seeding of the Vero cells, serially dilute each virus sample to be plaqued in 1x PBS; typically use 10-fold dilutions for the most precise tracking.
3. Make these dilutions through 10^-4 (for low concentrations of viruses) or through 10^-9 (for expectations of higher titer samples) with at least 1 mL of each sample remaining for the plaque assay itself.
4. Keep all the viral dilutions on ice (no longer than 1 h) until ready to add these diluted virus samples to the cells.

3. Addition of virus to the cells

1. Remove the cell culture medium from one or two wells by a pipette.
   NOTE: Do not use an aspirator because it removes too much liquid from the cell monolayer and dries the cells out. One crucial consideration is ensuring that the cell monolayer remains hydrated throughout the entire course of the experiment. If the cells dry out, they will wash off the plate in the final step and generate unusable data. Therefore, process only one or two wells at a time to reduce this possibility.
2. Carefully add the diluted virus sample (100-400 µL and 50-200 µL of virus sample are used for 6-well and 12-well plates, respectively) dropwise to each monolayer, adding the drops down 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.
   NOTE: Adding the drops to the center of the well may inadvertently slough cells off the substrate.
3. Gently rock the entire plate by hand to ensure the PBS that contains the virus covers the entire monolayer of cells in each well, then place the plate in a CO₂ incubator at 37 °C to allow the virus to adsorb.
4. Every 10 min within 1 h, 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.
   NOTE: Each experiment will dictate the number of replicates and which dilutions are used; the reader's preference dictates that decision.
5. To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells with 1000 µL pipette tips and place it in a waste beaker.
   NOTE: Again, this procedure is conducted with only one or two wells at a time to maintain the hydration of the cell monolayer.
6. Place 2.5 mL of a methylcellulose overlay (dissolve 5% methylcellulose w/w in 100 mL of PBS, autoclave for 15 min at 121 °C and 15 psi on a liquid cycle, then add 375 mL of DMEM plus 25 mL of FBS).
7. Once an entire plate contains overlay, place the plate back in the incubator to allow growth for two days.
   NOTE: If the virus and cells are allowed to grow for three days, even in DMEM plus supplements, a substantial loss of cells in the monolayer may result, thereby compromising the final data.
8. Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet.

4. Staining for plaques

1. After the two-day incubation, remove the methylcellulose overlay by a pipette, again one or two wells at a time, and place it in a waste beaker.
2. Add ~2 mL of 1% crystal violet (see Table of Materials) in 50% ethanol to each well to stain the plaques.
   NOTE: It is not essential to remove every last drop of the overlay.
3. Incubate the plate with all wells filled with the stain for 30 min at 37 °C. At this point, disinfect the waste beaker as above in step 3.8.
4. Wash the plates vigorously with tap water until the runoff is clear.
   NOTE: A gentle stream of water is not vigorous enough; full pressure from a lab sink fixture is required.
5. At this point, ensure that there are approximately 10-fold differences in the number of plaques across each dilution series.
6. Allow the plates to dry overnight upside down, after which the individual plaques may be counted.