Virus plaque assay principle

Plaque assay is one of the widely used approaches for determining the quantity of infectious virus in a sample.

Only viruses that cause visible damage to cells can be assayed in this way. Plaque assay was first developed to calculate the titers of bacteriophage stocks. Currently, its modified procedure is being used for the determination of titer of many different animal viruses too. When a suspension of an infective phage e. T4 phage is spread over the lawn of susceptible bacterial cells e.

Escherichia coli , the phage attaches the bacterial cell, replicate inside it, and kills it during its lytic release. Lysis of the bacteriophage is indicated by the formation of a zone of clearing or plaque within the lawn of bacteria. In the absence of lytic phage, the bacteria form a confluent lawn of growth. Each plaque corresponds to the site where a single bacteriophage acted as an infectious unit and initiated its lytic cycle.

The spread of infectious phage from the initially infected bacterial cell to the surrounding cells results in the lysis of the bacteria in the vicinity, eventually forming the plaque that is large enough to be visible to the naked eye. Plaques do not continue to spread indefinitely. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high.

Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric.

These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations.

Next, this molten bacterial-phage agar mix is spread over a more solid, concentrated agar nutrient medium which is already solidified on a Petri dish. On incubation at room temperature, the low concentration agar-phage-bacteria broth also solidifies to form a soft agar overlay. Here, the bacterial cells can derive additional nutrients from the bottom layer and should rapidly multiply to produce a confluent lawn of bacteria.

However, as phage particles are also present in the soft layer, these will infect and replicate their genetic material within the bacteria, culminating in cell lysis, which releases multiple progeny. These phage progeny then infect the neighboring cells, as the semi-solid state of the bacteria-phage layer restricts their movement to more distantly located host cells. This cycle of infection and lysis continues over multiple rounds, killing a large number of bacteria in a localized area.

The effect of the neighboring cells being destroyed, is to produce a single circular clear zone, called a plaque, which can be seen by the naked eye, effectively amplifying the bacteria lytic activity of the phage and enabling their enumeration.

The number of plaques on a Petri dish are referred to as Plaque-Forming Units, or PFUs, and, providing the initial bacteriophage concentration was sufficiently dilute, should directly correspond to the number of infective phage particles in the original sample. This technique can also be used for characterization of plaque morphology, to aid in identification of phage types, or to isolate phage mutants.

In this lab, you will learn how to perform the plaque assay for enumerating phages, using the T7 phage of E. First, identify a suitable medium for the culturing of the host bacterial cells and the bacteriophage. Here lysogeny broth, or LB medium, was used to culture E.

Now, weigh out four grams of pre-formulated LB powder in three sets and then transfer one set of weighed dried media into each bottle.

Add milliliters of water to the first bottle. Mix the contents using a magnetic stir bar. Then, using a pH meter and constant stirring, bring the final pH to 7. Repeat the water addition and pH adjustment for the other two remaining bottles, as well. Now, weigh out three grams of agar powder and add it to the second bottle to make a 1. Finally, weigh 1. The broth condition in bottle one does not need an agar addition. Cap the bottle semi-tightly and then, sterilize the media by autoclaving at degrees Celsius for 20 minutes.

Once complete, remove the media bottles from the autoclave and immediately twist the bottle caps to close them fully to prevent contamination. When the LB-Bottom agar reaches approximately 45 degrees Celsius, transfer it to the work bench. Next, add microliters of sterile one molar calcium chloride to the molten bottom agar to make a final concentration of 2. Gently swirl the bottle to mix. Then, set out seven clean Petri dishes. Label each dish on the bottom with the media name and preparation date.

Then, pour 15 milliliters of the bottom agar into each of the seven Petri dishes. Allow the plates to set for a few hours or overnight at room temperature.

Once set, the culture plates can be stored at four degrees Celsius for several days if needed, upside down to minimize condensation. Transfer the Petri dishes from the four degrees Celsius refrigerator to a 37 degrees Celsius incubator one hour before the assay. The day before the assay is to be preformed, the E. Here, 10 microliters of E. Place the bacteria to grow overnight in a shaking incubator set to 37 degrees Celsius at RPM.

Then, on the day of the assay, remove the bacterial culture from the incubator. Seed a fresh 10 milliliters of fresh LB broth with 0. Place these cells to grow into a shaking incubator set to 37 degrees Celsius at RPM. Next, use a spectrophotometer to check when this culture reaches log phase growth, indicated by an optical density of 0.

Once the OD reaches this level, stop the incubation by transferring the cell culture to the bench. They are now ready to be used for phage overlay assay. Phage titers can vary exponentially across different phage types and samples.

So in order to count them effectively, they should be diluted to generate a wide range of phage concentrations. On the day of the assay, generate a series of phage dilutions ranging from one tenth to one millionth concentrations, following a fold dilution technique. To obtain statistically significant and accurate data, perform the serial dilution in triplicate.

Next, melt the solidified LB-top agar using a microwave. Then, place it in a water bath that is preset at 45 degrees Celsius for one hour. After one hour, collect the Petri dishes containing the bottom agar layer from the incubator. Label the plates with phage concentration and assay date. Then, set out seven clean test tubes. Label each test tube with the serial phage dilution number and designate one as control. When the LB-top agar reaches 45 degrees Celsius, transfer it to the working bench.

Now, add microliters of one molar calcium chloride to the milliliter agar to make a final concentration of 2.

Next, add 35 milliliters of LB-top agar and four milliliters of bacterial suspension to a sterile conical tube. Gently swirl to evenly distribute the cells but avoid shaking to prevent foaming.

Now, aliquot five milliliters of this bacteria- top agar mix to each of the seven test tubes. Then, transfer microliters of the serially diluted bacteriophage samples and control media, which should be simply media with no bacteriophage, to the respectfully labeled test tubes.

Swirl the mixture gently to ensure proper mixing. Gently transfer five milliliters of bacteriophage mix onto the respective Petri plate. Evenly spread the mix throughout the whole surface by gently swirling the Petri plate.

Once all of the Petri plates are layered with the mix, allow solidification of the top layer by incubating at room temperature for 15 minutes. After completion of these steps, repeat the process for the second and then the third sets of the Petri dishes using the remaining two sets of phage dilutions. Seal each dish with parafilm and incubate at room temperature for 15 minutes. Place the culture plate upside down at a suitable temperature for 24 hours or until plaques develop. Here, plates were placed in a 37 degrees Celsius incubator for one day, a stimulating growth condition for E.

Plaques will appear after one to five days of incubation, depending on the bacterial species, incubation conditions, and the choice of medium.