Sidebar: I have plaques on my wall to commemorate the virus plaque assay

Welcome my fellow friends to a new short sidebar series! Over the next couple of weeks, I (Manolya Sag) Deanna Kim, and Jamie Lee will be going over some of the most common laboratory techniques many scientists are using to answer questions and make discoveries related to SARS-CoV-2. Many of the techniques we will be explaining have been mentioned in our research re-hashed posts. But we wanted to make sure you fully understand the science and mechanisms behind these common experimental techniques. So, welcome everyone to: 

How do they do that? Your basic guide to virology lab techniques-Part 1.

Today, we will be focusing on a technique known as virus titering. In a virology lab, scientists perform virus titers to quantify just how much virus there is from a sample we collect. Because of this, it’s a pretty important technique to know1

There are many different techniques out there used to quantify the virus concentration. Some of these are specific to a certain virus, whereas other techniques are pretty universal for all viruses, like the plaque assay. We will mainly focus on how plaque assays work as they are most often brought up in many SARS-CoV-2/COVID-19 research papers.

The plaque assay was first used to quantify bacteriophages (viruses that can only infect bacteria), but in the 1950s it was redesigned to be able to quantify and determine the concentration of animal viruses, like SARS-CoV-23.

So, we know that a plaque assay can allow scientists to figure out how much virus they have in a sample. But how does a plaque assay work? Well, let’s help our new scientist friend, Marybelle, do her very own plaque assay in the lab, shall we?

Before Marybelle starts working with her virus samples, she first has to grow cells on an agar plate. Depending on what virus she’s working with, any number of different cell lines can be used. What’s most important is that cell line she uses must be susceptible to infection (or else, the experiment won’t work ☹). For example, in a paper published in Science, scientists used Vero E6 cells (find out what kind of cells these are: here), for SARS-CoV-2 virus titer determination6. Marybelle also should make sure that when she’s growing her selected cells on an agar plate, only a monolayer (mono meaning 1, so one layer) of cells grows.  Now Marybelle has her multiple plates of susceptible cells all ready to go (after a few days of growing and caring for them), the next step will be to take the virus sample and create a series of 10-fold dilutions. One of the best ways to show Marybelle and you what this means is by using a handy dandy visual aid. Take a look at figure 1 to fully understand and see how we make 10-fold dilutions of the virus sample we will be using

Figure 1. When we make a 10-fold serial dilution of our viral sample, we first fill test tubes with 0.9 mL of a reagent used for mixing. Now we take 0.1mL of our original sample and add it into the first test tube. This makes the test tube 1.0 mL in volume and we dilute the virus sample by 1 10-fold. Next we take 0.1mL from that test tube and add it to the next test tube to dilute the virus sample once again. We continue to do this serial dilution until we dilute the original virus sample to 0.000001 the original concentration! We have to do this as the original virus sample is too concentrated and we won’t be able to create plaques in our plates.

Once Marybelle creates her series of diluted virus samples, she can take 0.1 mL (or 100 uL) of the sample and spread it onto one of her susceptible cell plates she prepared earlier. She can do this for each dilution she prepared and can then incubate the cells (in warm temperatures) for a bit. After some time, when Marybelle takes a look at her plates she may see a circular zone of infected cells (known as plaques). This zone indicates that the virus infected these cells and spread within the neighbouring cells. Essentially, the virus can infect the cells which causes them to die and create a large hole we can see. (Figure 2). 

Figure 2. An example of a petri plate with plaques. After a monolayer of cells is grown on the plate and we infect them with our diluted viral stock, we can see plaques develop as cells die from viral infection.

This means Marybelle can now count how many plaques developed in each plate. Depending on which dilution factor of virus Marybelle used to infect her plate, a number of different plaques will form. For example, Marybelle may count 30 plaques on one plate, and 80 plaques on another. 

A plate with few plaques means a highly diluted form of virus was used for infection. On the other hand, a plate with a lot of plaques means that plate was infected with a more concentrated form of the virus. After Marybelle counts all the plaques from all her plates she will have to calculate the concentration of the actual virus sample she used. In virology we calculate this value using plaque-forming units (PFU)/mL. 

So, when Marybelle considers the dilution factor she used and determines the numbers of plaques in her plate, she can easily determine the PFU/mL of the viral sample. See figure 3 for a quick math example on how we determine the PFU/mL. 

Figure 3. An example for determining the virus sample concentration.

That’s awesome, we showed Marybelle how to do a plaque assay to determine viral concentration and now you understand how scientists do it too! 

The plaque assay is considered to be a scientist’s best and most accurate method for determining concentration of a virus sample1,4. However, while a viral plaque assay is very accurate, it is also a technique that takes time and patience 5. At a time like this, sometimes that wait is not favourable. But worry not, there are so many other methods of viral quantification methods out there that scientists have been using! One of these methods is reverse-transcription quantitative (some scientists call this real-time) polymerase chain reaction (RT-qPCR) which can give us the concentration of an RNA virus (like SARS-CoV-2) in a sample. To learn more about how RT-qPCR works, tune in next time for part 2 of How do they do that? Your basic guide to virology lab techniques! 

1. Racaniello, V. Detecting viruses: the plaque assay. Virology blog: about virues and viral disease (2009).

2. Virus Titration – an overview | ScienceDirect Topics.

3. Dulbecco, R. & Vogt, M. Some problems of animal virology as studied by the plaque technique. Cold Spring Harb. Symp Quant Biol 18, 273–279 (1953).

4. Baer, A. & Kehn-Hall, K. Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems. J. Vis. Exp. JoVE (2014) doi:10.3791/52065.

5. Smither, S. J. et al. Comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity. J. Virol. Methods 193, 565–571 (2013).

6. Shi, J. et al. Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS–coronavirus 2. Science (2020) doi:10.1126/science.abb7015.

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