How is plaque-forming unit calculated?

Hey there! As a plaque supplier, I often get asked about how plaque - forming units are calculated. It's a bit of a technical topic, but I'll break it down in a way that's easy to understand.

First off, let's talk about what plaque - forming units (PFUs) actually are. In simple terms, PFUs are a measure used in virology to quantify the number of infectious virus particles in a sample. When a virus infects a layer of host cells grown in a culture dish, it causes the cells in the infected area to die. These dead cell areas are called plaques, and each plaque is assumed to have been formed by a single infectious virus particle.

So, how do we calculate PFUs? The most common method is the plaque assay. Here's a step - by - step breakdown of how it works.

Step 1: Preparation of the Virus Sample

We start by taking a sample that contains the virus. This could be a clinical sample, like a blood or tissue sample, or a sample from a virus culture. The sample is usually serially diluted. Serial dilution means that we take a small amount of the original sample and mix it with a larger volume of a diluent (usually a buffer solution). Then, we take a small amount of this first dilution and mix it with more diluent to make a second dilution, and so on.

For example, we might start with a 1:10 dilution. That means we take 1 part of the virus sample and mix it with 9 parts of the diluent. Then, we take 1 part of this 1:10 dilution and mix it with 9 parts of diluent to make a 1:100 dilution, and we can keep going to make even more dilute samples like 1:1000, 1:10000, etc.

Step 2: Inoculation of Host Cells

Next, we take a layer of host cells that are sensitive to the virus. These cells are usually grown in a culture dish. We add a small amount of each of the diluted virus samples to separate dishes with the host cells. The virus particles in the sample will attach to the host cells and start to infect them.

Step 3: Incubation

After inoculation, the dishes are incubated for a certain period of time. During this time, the virus will replicate inside the infected cells, and the infected cells will start to die, forming plaques. The incubation time depends on the type of virus. Some viruses may take a few hours to form plaques, while others may take several days.

Step 4: Counting the Plaques

Once the plaques have formed, we count them. We can do this by simply looking at the dish under a microscope or using a plaque - counting device. It's important to count only the dishes where the number of plaques is in a countable range. If there are too many plaques, they may merge together, and it will be difficult to accurately count them. Usually, we aim for dishes with between 30 and 300 plaques.

Step 5: Calculating the PFUs

To calculate the number of PFUs per milliliter (PFU/mL) of the original virus sample, we use the following formula:

PFU/mL=(Number of plaques counted)×(Dilution factor)÷(Volume of the virus sample added to the dish)

Let's say we counted 50 plaques in a dish that was inoculated with 0.1 mL of a 1:1000 dilution of the virus sample. The dilution factor is 1000, and the volume of the virus sample added to the dish is 0.1 mL.

PFU/mL = (50)×(1000)÷(0.1)=500000 PFU/mL

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References

• Flint, S. J., Enquist, L. W., Racaniello, V. R., & Skalka, A. M. (2009). Principles of Virology: Molecular Biology, Pathogenesis, and Control. ASM Press.
• Knipe, D. M., & Howley, P. M. (Eds.). (2013). Fields Virology. Lippincott Williams & Wilkins.