This study outlines a method for quantifying viral titer using a plaque assay technique. The process involves infecting host cells with diluted virus samples and observing plaque formation as an indicator of viral replication.
Begin with a virus-containing sample obtained from an infected porcine cornea treated with an antiviral drug.
Perform a serial dilution of the sample to create a sequence of decreasing virus concentrations.
Optimum dilution prevents single-cell infection by multiple viruses.
Introduce the diluted samples to a host-cell monolayer and incubate.
The virus attaches to the host-cell receptor, enters the cell, and initiates its replication. The infected cell lyses, releasing virions.
Add methylcellulose, a viscous substance that confines the viruses to the nearby cells.
Subsequently, the viruses cause cell death and plaque formation in the infected region.
Add methanol to fix the cells.
Use crystal violet to stain the live cells.
Observe plaques as clear zones indicating infected cells and cell death, surrounded by crystal violet-stained uninfected cells.
Finally, count the number of plaques at the highest dilution to quantify the virus titer in the starting solution to determine the efficacy of the antiviral drug.
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.
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