Virus Quantification using plaque assay

Summary

This video demonstrates the process of quantifying virus concentration using a plaque assay. The method involves infecting a monolayer of cells with a virus and preventing the infection from spreading using a semi-solid medium. The resulting viral plaques are counted manually, and the number of infective units is calculated, yielding the plaque-forming units (PFU/ml). The procedure includes virus dilution, cell infection, agarose overlay preparation, and final plaque visualization using crystal violet. The entire process takes 3 to 14 days, depending on the virus, and helps in determining viral titer and purifying virus populations.

Takeaways

• 😀 Emerging infectious diseases are often caused by viruses, and quantifying virus concentration is important for research and development.
• 😀 The plaque assay is a standard method for determining virus concentration by observing the formation of plaques in infected cell monolayers.
• 😀 A plaque is a clear area in the monolayer where cells have died due to viral infection, and its size helps in estimating the viral titer.
• 😀 The plaque assay process involves infecting cells with a virus at various dilutions to observe how the virus spreads and forms plaques.
• 😀 The cells are prepared in a 6-well plate, incubated at 37°C for 24 hours until 90-100% confluent before viral inoculation.
• 😀 The virus sample is diluted in culture medium without FBS, which could interfere with the viral infection process.
• 😀 A serial 10-fold dilution of the virus is made, and then the diluted virus is added to the wells with cells to infect them.
• 😀 After viral infection, an agarose solution (0.3% in culture medium) is used to overlay the cells, preventing the virus from spreading uncontrollably.
• 😀 The infected cells are incubated for 60 to 84 hours at 37°C to allow plaques to form, which represent areas of infected and dead cells.
• 😀 After incubation, cells are fixed using formaldehyde, and plaques are stained with crystal violet to make them visible and countable.
• 😀 The number of plaques is counted under a microscope and used to calculate the viral titer in terms of PFU/ml, representing the number of infectious virus particles in the sample.

Q & A

What is the purpose of virus quantification in the context of the plaque assay method?

The purpose of virus quantification is to determine the concentration of infectious virus particles in a sample by counting the number of plaques formed in a cell monolayer. This helps in assessing the virus's infectivity and its potential effects on a host.

What is the standard method used to determine viral concentration in the plaque assay?

The standard method used to determine viral concentration is the plaque assay, where a monolayer of cells is infected with the virus, and plaques (areas of cell death) are counted to calculate the virus concentration.

How does the plaque assay method work to determine virus concentration?

In the plaque assay, the virus infects a fixed cell monolayer, forming plaques as infected cells die and spread the infection. The number of plaques is counted manually, and the result is used to calculate the plaque-forming units per milliliter (PFU/mL), which represents the number of infectious viral particles.

Why is Fetal Bovine Serum (FBS) excluded from the culture medium when diluting the virus?

FBS is excluded from the culture medium when diluting the virus because it can affect viral infection. The culture medium without FBS ensures that the virus can infect the cells without interference from the serum components.

What is the significance of the agarose overlay in the plaque assay?

The agarose overlay prevents the virus from spreading indiscriminately to neighboring cells. It helps contain the viral infection in localized areas, allowing the formation of distinct plaques that can be easily counted under a microscope.

What is the role of crystal violet staining in the plaque assay?

Crystal violet staining is used to visualize the plaques formed by the virus. After fixing the infected cells, the crystal violet stains the cells, making the plaques visible as distinct areas of cell death, which can then be counted to determine viral concentration.

How long does the plaque assay typically take to complete?

The plaque assay typically takes between 3 to 14 days to complete, depending on the virus being analyzed. This includes incubation and observation periods for plaque formation.

What is the significance of incubating the plates at 37°C for 60-84 hours?

Incubating the plates at 37°C for 60-84 hours allows sufficient time for the virus to infect the cells, spread, and form visible plaques. This incubation period is crucial for accurate plaque formation and virus quantification.

How is the virus diluted for the plaque assay?

The virus is diluted in culture medium without FBS in a series of steps. A 120 μL sample of the virus is added to the first two wells, followed by a 10-fold serial dilution to achieve varying concentrations for the assay.

Why is PBS used to wash cells before adding the virus?

PBS (phosphate-buffered saline) is used to wash the cells before adding the virus to remove any residual medium or impurities. This ensures that the virus is introduced directly to the cells without interference, helping to maintain the accuracy of the assay.