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Viral Titering-Plaque Assay Protocol

One of the most important procedures in virology is to measure the virus titer – the concentration of viruses in a sample. A widely used method for determining the quantity of infectious virus is the plaque assay. This technique was first used to calculate the titers of bacteriophage stocks. Renato Dulbecco modified this procedure for use in animal virology in 1952, and it has since been used for reliable determination of the titers of a number of different viruses.

During a plaque assay, a confluent monolayer of host cells is infected with a lytic virus of an unknown concentration which has been serially diluted to a countable range, typically between 5-100 virions. Then, infected monolayers are covered with an immobilizing overlay medium to prevent viral infection, which is from indiscriminately spreading through either the mechanical or convectional flow of the liquid medium during viral propagation. Although solid or semisolid overlays such as agarose, methyl cellulose or carboxymethyl cellulose (CMC) have traditionally been used, liquid overlays have become a more and more attractive alternative with the development of novel liquid overlays such as Avicel. Plaque assays utilizing liquid versus traditional overlays have several advantages because the overlay can be applied at room temperature, and application and removal is significantly easier. Since liquid overlays do not require warming, delicate and heat labile viruses may also prove easier to plaque.

After the initial infection and application of the immobilizing overlay, individual plaques, or zones of cell death, will start to develop as viral infection and replication are constrained to the surrounding monolayer. Infected cells will continue the replication-lysis-infection cycle, further propagating the infection, leading to increasingly distinct and discrete plaques. According to the viral growth kinetics and host cell used, a visible plaque will ordinarily form within 2-14 days. After fixing and staining the infected cellular monolayer, plaques are counted so that titer viral stock samples in terms of plaque forming units (pfu) per milliliter.

The workflow of plaque assay as follows:

1. Grow the host cells in wells with the recommended growth medium for the cell line. Allow the cells to reach the appropriate confluency.

2. Remove the growth medium and wash with Dulbecco's Phosphate-Buffered Saline (DPBS). Add diluted virus to each well, using multiple wells per dilution.

3. Incubate dishes for 1-2 hours to make viral adsorption.

4. Remove the inoculum and wash with basal medium.

5. Overlay the cells with overlay medium. Incubate for a length of time appropriate for infection. If applicable, remove overlay.

6. Observe the cell monolayers every day for the presence of foci or plaques.

7. In order to determine the virus titer, the plaques are counted. A log drop should be noted between serial dilutions and, depending on plate size, between 5-100 plaques counted, with a negative control used as a reference. Statistically samples will vary by 10% for every 100 plaques counted when comparing sample replicates.

Viral Titering-Plaque Assay Protocol

8. Calculate Viral Titer through counting the number of well isolated plaques. Then use the following formula to determine the titer (pfu/ml) of the viral stock:
No. of Plaques / (D x V) = pfu/ml
D = Dilution factor
V = Volume of diluted virus/well
Sample calculation:
- An average of 50 plaques formed in the 1:10,000 dilution wells.
- Volume of diluted virus added: 0.2ml
= 50/ (0.0001 x 0.2) = 2.5 x 106 pfu/ml

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