Plaque Assays: Study with Video Lessons, Practice Problems & Examples

Plaque assays are essential for quantifying bacteriophages, which are obligate intracellular parasites. This technique involves diluting phage samples to control plaque formation, where each plaque represents a plaque-forming unit (PFU), indicating phage concentration. The titer, or original concentration of phage particles, is determined by counting the plaques formed in a bacterial lawn after phage infection. Understanding plaque formation and its implications in virology is crucial for studying phage behavior and applications in phage therapy.

Plaque Assays

In this video, we're going to introduce plaque assays. And so since bacteriophages or phages are obligate intracellular parasites that cannot replicate outside of a host cell, cultivation of the appropriate cells is actually required for studying these phages. And so a plaque assay is a specific laboratory technique or method that is used to determine the concentration of phage particles in a sample by the formation of plaques. Now, plaques are defined as clear areas in a layer of cultured cells that indicate cell death from phage infections. And so each plaque that's present represents a plaque forming unit or a PFU for short. And so each plaque forming unit or each PFU represents a single phage particle. And so the number of plaques can be used to represent the number of phage particles.

Now, dilutions of the phage sample are going to be used to control the number of PFUs on the plate in order to determine the titer. And so the titer is really just the concentration of phage particles in the original sample, or in other words, in the undiluted sample. And so if we take a look at our image down below, we can get a better understanding of these plaque assays. And so the plaque assays once again are going to be used to determine the titer or the concentration of bacteriophages. And so notice on the far left over here, we have the first step where we have a lawn of bacteria growing on a petri dish, and so there are bacteria growing all over this petri dish. And so then what we do is we introduce the bacteriophage, phage, which is going to infect the bacteria. And so we introduce the bacteriophage, and you could see here the phage is able to carry out a phage infection. And the infected bacteria, we know ultimately with the lytic cycle is going to end up killing or lysing the cells.

And so the infected bacteria are ultimately going to lyse and die, and when they die, they create these plaques, which are again these clear areas that represent cell death. And so if you take a look at our image down right here, notice that there is a plaque forming right here, which is basically a cleared out area that does not have cell growth because the cells have died from the phage infection. And so this here would represent the formation of a plaque. And so zooming into this region, this little zone here, that has cell death is going to represent the plaque. And so it's going to have infected bacteria that have lysed. And so these infected bacteria lysed, and so they create these plaques.

Now, ultimately, the number of plaques, when there are multiple plaques that form, each plaque that forms represents a plaque forming unit or a PFU for short. So one plaque represents 1 PFU, and each PFU or each plaque forming unit represents 1 phage. And so the number of plaques can be used to represent the number of phages. And so it can be used to determine the titer or the concentration of phage particles in the original sample. And so what you'll notice here is that through the formation of multiple plaques as you see here, the number of plaques can be used to determine the number of phages and the titer of the sample.

And so this here concludes our brief introduction to plaque assays, and we'll be able to get a little bit of practice applying these concepts as we move forward in our course. So I'll see you all in our next video.

A clear area against a confluent "lawn"of bacteria is called a:

• A. Phage.
• B. Pock.
• C. Cell growth.
• D. Plaque.
• E. Rash.

The approximate viral concentration of a sample is known as:

• A. Quantal assay.
• B. Endpoint assay.
• C. The titer.
• D. The lysate assay.

Which of the following statements accurately describes viral plaques?

• A. Each plaque is presumed to arise form a single lytic virus particle.
• B. Plaques are formed only by defective viral particles.
• C. All bacteriophages produce plaques that have exactly the same size and appearance.
• D. Plaques are only formed by animal viruses.e) Two or more of the above answers are correct.
• E. Two or more of the above answers are correct.

Here’s what students ask on this topic:

What is a plaque assay and how is it used to determine phage concentration?

A plaque assay is a laboratory technique used to quantify bacteriophages, which are viruses that infect bacteria. In this method, a diluted phage sample is introduced to a lawn of bacteria on a petri dish. The phages infect the bacteria, leading to cell lysis and death, forming clear zones called plaques. Each plaque represents a plaque-forming unit (PFU), which corresponds to a single phage particle. By counting the number of plaques, scientists can determine the titer, or concentration, of phage particles in the original sample. This technique is crucial for studying phage behavior and applications in phage therapy.

What is the significance of plaque-forming units (PFUs) in a plaque assay?

Plaque-forming units (PFUs) are a critical measure in plaque assays, representing individual phage particles capable of infecting and lysing bacterial cells. Each PFU corresponds to a single plaque, a clear area on a bacterial lawn where cells have been lysed by phage infection. By counting the number of plaques, researchers can determine the concentration of phages in a sample, known as the titer. This quantification is essential for understanding phage dynamics, optimizing phage therapy, and conducting various virological studies.

How do you calculate the titer of a phage sample using a plaque assay?

To calculate the titer of a phage sample using a plaque assay, follow these steps: 1) Perform serial dilutions of the phage sample. 2) Plate each dilution on a bacterial lawn and incubate. 3) Count the number of plaques formed on each plate. 4) Select a plate with a countable number of plaques (typically 30-300). 5) Use the formula: $\frac{P}{V\times D}$, where P is the number of plaques, V is the volume of the diluted phage sample added to the plate, and D is the dilution factor. This calculation gives the titer, or concentration, of phage particles in the original sample.

Why are serial dilutions important in plaque assays?

Serial dilutions are crucial in plaque assays to control the number of plaques formed on a bacterial lawn. Without dilution, the phage concentration might be too high, leading to overlapping plaques that are difficult to count accurately. By diluting the phage sample, researchers can achieve a countable number of plaques (typically 30-300 per plate), ensuring precise quantification of phage particles. This step is essential for determining the titer, or concentration, of phages in the original sample, which is vital for various virological studies and applications.

What are the steps involved in performing a plaque assay?

Performing a plaque assay involves several steps: 1) Prepare a bacterial lawn on a petri dish. 2) Perform serial dilutions of the phage sample. 3) Mix a small volume of each diluted phage sample with the bacterial culture. 4) Pour the mixture onto the bacterial lawn and spread evenly. 5) Incubate the plates to allow phage infection and plaque formation. 6) After incubation, count the number of plaques on each plate. 7) Calculate the titer of the phage sample using the formula: $\frac{P}{V\times D}$, where P is the number of plaques, V is the volume of the diluted phage sample, and D is the dilution factor.