Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

The overall goal of this procedure is to determine viral concentration through the quantification of infectious varion by counting discrete plaques in cell culture. This is accomplished by first plating cells to form a confluent monolayer for infection. The second step is to perform serial dilution of the unknown viral sample to be quantified.

Next, the confluent monolayers are infected using the serial dilution of the viral stock, followed by application of a immobilizing overlay. The final step is to fix and stain the plaques after the appropriate incubation time for the virus in question in order to count the discreet plaques. Ultimately, plaque assays can be used and modified in a number of different ways in order to determine the viral titers of an unknown viral sample.

The main advantage of this technique over existing methods, such as the semi-solid overlay, is that application and removal are far simpler in both high throughput and traditional applications. In addition, no heating of the reagents is required. The implications of this technique extend towards the research of many different lytic viruses as the use of a liquid overlay.

In a 96 well format allows for the rapid screening of novel therapeutic compounds, which can greatly decrease the long hours spent in the lab performing traditional plaque assays On the day prior to the assay plate, the appropriate cells for the virus in question in order to achieve 90 to 100%co fluency the day of infection. The stock solutions can also be prepared the day before the assay first prepare a fixing solution of 10%formaldehyde in distilled water by mixing 5.56 milliliters of 36%stock formaldehyde with 14.44 milliliters of distilled water. Then prepare the crystal violet stain consisting of 1%crystal violet in 20%ethanol diluted with distilled water.

Next, while working under sterile conditions, prepare the appropriate two x plaque media for the type of cells being used. Filter the solution through a 0.2 micron filter if any of the reagents are not sterile. To prepare a liquid overlay for use in the plaque assay at 2.4 grams of avy cell to a flask containing 97.6 milliliters of distilled water that is rapidly mixing with a stir bar.

Mix the solution at room temperature until it becomes too viscous for the stir bar. At this point, transfer the flask to a flask shaker and heavily agitated for 30 minutes to ensure even homogenization. Finally autoclave the solution at 121 degrees Celsius at 14 PSI for 30 minutes.

And when the program has finished, store sealed at room temperature for an aros overlay. Prepare a 0.6%agro stock solution in distilled water. Mix with a stir bar and autoclave to bring into solution or a carboxy methylcellulose overlay.

Prepare a 2%carboxy Methylcellulose solution in distilled water and mix than autoclave as before the day after plating. Use an inverted microscope to check the co fluency and viability of the cells prior to starting the assay. Ensure that the cells exhibit standard cellular morphology and approximately 90%co fluency.

Perform a tenfold serial dilution of the infectious samples in cellular growth media, varying the number of dilution required based on the expected titer of the virus in question. Also, prepare an uninfected control sample to independently ensure cellular viability and aid in plaque identification. Pipette the uninfected control and serial dilution into the wells of the plate, being careful to avoid disrupting the monolayer.

Use a sufficient volume of inoculum to cover the cells while keeping the volume as low as possible to maximize viral contact with the monolayer. Put the plates into the tissue culture incubator and infect the cells for 45 minutes to one hour every 20 minutes. Gently rock the plates to ensure even coverage and to prevent the cellular monolayer from drying during the infection.

Prepare the overlay working solution for liquid overlays. Mix warmed plaque media and 1.2 to 2.4%room temperature Stock avy cell in a one-to-one ratio to obtain a working solution of 0.6 to 1.2%avy cell overlay medium or AROS or CMC overlays mixed warmed plaque media, and a stock solution of either heated 0.6%AROS or 2%CMC in a one-to-one ratio. Then place in a 56 degree Celsius water bath, 30 minutes for temperature equilibration.

Ensure the solution is warm but not hot to the touch to prevent cell death and reduced viral titers. After the infection, apply the overlays to the infected monolayers after addition of the overlay Incubate plates at 37 degrees Celsius and 5%carbon dioxide for two to 14 days depending on the virus being analyzed to fix cells with a liquid overlay, pour off or aspirate the e cell overlay, then pipette 10%formaldehyde solution into the wells and fix at room temperature for 30 minutes to overnight or aros or CMC directly. Add the formaldehyde solution to the overlay for one hour to overnight at room temperature after fixation, discard the formaldehyde and then manually remove the semi-solid plugs where the aros with the spatula.

Rinse the aby cell plaques with water to remove residual overlay fixative prior to staining. To stain cover the cells with a minimal amount of crystal violet solution for approximately 15 minutes. Rock the plates if necessary to ensure even coverage.

Gently wash off the crystal violet stain with water once fixed, stained and dried store plex indefinitely for future analysis. Count the plaques in each well discounting wells with fewer than five or greater than 100 plaques. Take the average for any technical replicates of the same dilution.

Take note of plaque size and morphology. Determine the viral titer of the stock sample by taking the average number of plaques for a dilution and the inverse of the total dilution factor. This image shows Rift Valley fever virus plaque overlay comparisons utilizing 12 well plates bureaus were plated at 2.5 times 10 of the fifth cells in 12 well plates and infected with 200 microliters using the same serially diluted starting sample of MP 12.

After infection, 1.5 milliliter overlays of 0.3%aros 0.6%AVY, or 1%CMC were applied to directly compare the overlays. This histogram shows the results of plaque counts and tittering for all three overlays.Here. ViiV plaque overlay comparisons utilizing 12 well plates are shown bureaus were plated as before and infected with a volume of 200 microliters using the same serially diluted starting sample of the ViiV TC 83 vaccine strain after infection, 1.5 milliliter overlays of 0.3%aros 0.6%avy cell or 1%CMC were applied as before.

Again, the histogram shows the results of plaque counts and titering for all three overlays. This image shows an example of high throughput plaque overlays. A 96 well plate of VIRs plated at three times 10 of the fourth cells per well was infected with 50 microliters of inoculum using the same serially diluted starting sample of Rift Valley fever virus MP 12 for one hour in quad duplicate for the overlays 0.6 and 1.2%Final concentrations of avy cell were trialed in order to determine the feasibility and reproducibility of utilizing liquid overlays in a high throughput manner.

This histogram shows the titers determined from each overlay. While attempting this procedure, it's important to remember that every virus is different and optimizations including utilizing different cell types, incubation times and or growth medium may be necessary. After watching this video, you should have a good understanding of how to perform a basic plaque assay utilizing both liquid and semi-solid overlays.