How to Perform a Plaque Assay

Summary

The plaque assay is a crucial technique in virology that measures infectious virus particles by observing their ability to infect and kill susceptible cells. The method involves infecting cultured cells with virus samples, applying a semi-solid overlay to limit virus spread, and then counting the resulting plaques, which are zones of infected cells. This process helps determine the virus concentration in plaque-forming units per milliliter. The plaque assay remains one of the most reliable and accurate methods for studying infectious viruses, despite being over a century old, showcasing its enduring value in scientific research.

Takeaways

• 😀 The plaque assay is a reliable method to measure infectious virus particles in a sample.
• 😀 It involves adding viruses to a layer of susceptible cells and observing how they infect and kill these cells.
• 😀 A semi-solid overlay is used in the plaque assay to limit virus spread, causing each infectious particle to form a localized plaque.
• 😀 Plaques are visible zones of infected cells that appear due to virus-induced cell destruction.
• 😀 The plaque assay works best with viruses that cause visible damage to cells, such as herpes virus 1.
• 😀 The process begins with cultivating cells to form a confluent monolayer, providing a sheet of cells susceptible to infection.
• 😀 Serial dilution of the virus sample is done to determine the optimal concentration for plaque formation.
• 😀 The virus is incubated on the cells for about an hour, and the excess virus is washed off before adding the semi-solid overlay.
• 😀 After incubation, plaques form in the monolayer, each representing the progeny of a single infectious virus particle.
• 😀 The virus concentration in the original sample is calculated based on the number of plaques counted and the dilution factor.
• 😀 The plaque assay remains one of the most accurate methods for measuring infectious viruses, even after over a century of use.

Q & A

What is the plaque assay and why is it important in virology?

The plaque assay is a method used to measure the amount of infectious virus particles in a sample. It is crucial in virology because it quantifies only infectious virus particles, which are capable of infecting host cells and forming plaques.

How does the plaque assay work to measure infectious viruses?

In a plaque assay, viruses are added to a layer of susceptible cells, and the virus infects and kills these cells, forming localized zones of cell destruction called plaques. These plaques are counted to determine the virus concentration in the original sample.

What role does the semi-solid overlay play in the plaque assay?

The semi-solid overlay, such as agarose or methyl cellulose, restricts the movement of the virus, preventing it from spreading too far and ensuring that each infectious virus particle infects only neighboring cells. This results in distinct plaques.

What are plaques, and how are they formed?

Plaques are clear zones that form in a monolayer of cells due to virus-induced cell death. Each plaque originates from a single infectious virus particle, and their formation allows scientists to quantify the virus.

Why is it important to select the correct dilution for the plaque assay?

Choosing the right dilution ensures that the plaques are clearly visible and countable. Too many plaques will overlap and make counting difficult, while too few plaques may lead to unreliable results.

How are the plaques counted in a plaque assay?

Plaques are counted manually once they have grown large enough to be visible. To ensure accuracy, the plaques are counted on replicate plates, and the average number is used to calculate the virus concentration.

What is the formula used to calculate the virus concentration in the original sample?

The formula for calculating the virus concentration is based on the number of plaques counted, the dilution factor, and the volume of the virus added. The result is expressed as plaque-forming units (PFU) per milliliter.

What is the difference between plaque-forming units (PFU) and viral particles?

Plaque-forming units (PFU) refer to infectious virus particles capable of forming plaques, while viral particles include both infectious and non-infectious particles. Not all viral particles will form plaques due to factors that influence infectivity.

What is an alternative to the plaque assay for viruses that do not form plaques?

For viruses that do not form plaques, the focus-forming assay is used. This method involves using virus-specific antibodies to detect clusters of infected cells, known as foci, and quantify them using fluorescence or enzyme-linked detection.

Why is the plaque assay still considered one of the most reliable methods for measuring virus infectivity?

The plaque assay remains one of the most reliable methods for measuring infectious viruses because it provides a direct measure of virus infectivity through visible plaques, and it has been proven effective over more than a century of use.