Quick Answer: How Do Plaques Form During A Phage Overlay Assay?

What causes the formation of plaques in a bacteriophage assay?

The basis of plaque assay is to measure the ability of a single infectious virus to form a “plaque” on a concurrent monolayer culture cells.

A plaque is developed as a part of infection of one cell by a single virus particle that is followed by the replication of that virus, and finally, the death of the cell..

Which is the best method to determine bacteriophage concentration in a sample?

A widely used method for determining phage concentration in a sample takes advantage of this lytic activity. In this technique, the phage from the sample is mixed with bacteria and soft agar. This mixture is poured onto Petri dishes with regular agar as a substrate, and the top layer forms an overlay.

How do you cook top agar?

Microbiology – Plating in Top AgarWarm plates to room temperature before use. … Prepare top agar as the appropriate liquid medium with 0.7% agar. … Melt top agar in the microwave completely. … Allow agar to cool to 48°. … Add cells and phage to a 13 mm yellow-capped tube in the 48° temperature block.Remove the cap and add 2.5 mL molten top agar.More items…•

How many viruses are needed to form a plaque?

Most viruses follow one-hit kinetics, i.e., one virus is enough to form a plaque. There are some viruses, though, that follow two-hit kinetics. When you do this dose response curve, you get a curve such as the blue line here. And this is because for these viruses you need two virus particles to form a plaque.

What is the agar overlay method and why can’t you just plate bacteriophage on a NA plate?

Active and infectious bacteriophage particles. It’s important to use hard agar with soft agar overlay because The hard agar underneath the soft agar overlay is where you make a lawn streak of your bacteria. Since phage can only grow in the presence of bacteria, this is the only way you can visualize plaques.

How is phage titer calculated?

For example, if the plate you selected was the 10^-5 plate, you would multiply 1570 by 10^5 to get 157000000. This final number is your phage titer, and represents the number of viruses per ml of your original culture.

What is the purpose of the serial dilution in the plaque assay?

What is the purpose of the seial dilution in the plaque assay? To dilute the original sample enough so that when plated, a countable plate can be produced.

Why do phage plaques not continually grow?

After several lytic cycles the local MOI increases and most of the cells are lysed, producing a plaque in the lawn of cells. As the cell lawn becomes saturated, the rate of cell growth slows down and, since lysis requires rapid metabolism, the plaque stops increasing in size.

What is an infectivity assay?

The infectivity assay is used to titrate virus-containing clarified culture supernatant fluids to determine the 5O%-tissue culture infective dose (TCIDSO) of HIV-1 per ml of original fluid. … This assay can be modified for use with different viral isolates and different cell types.

How do you calculate Moi?

It is related to pfu by the following formula: Multiplicity of infection (moi) = Plaque forming units (pfu) of virus used for infection / number of cells.

What is the agar overlay method?

This technique allows you to produce a homogeneous lawn of bacteria within a thin layer of agar across the surface of a plate. The melted agar/bacterial suspension is mixed and poured evenly across the top of an agar plate and allowed to solidify. …

What are the three methods used to cultivate viruses?

Cultivation of viruses can be discussed under following headings: Animal Inoculation. Inoculation into embryonated egg….Types of cell culturePrimary cell culture: … Diploid cell culture (Semi-continuous cell lines): … Heteroploid cultures (Continuous cell lines):

What is PFU ml?

The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle.

How is dilution factor calculated?

For example, a 1:5 dilution (verbalize as “1 to 5” dilution) entails combining 1 unit volume of solute (the material to be diluted) + 4 unit volumes of the solvent medium (hence, 1 + 4 = 5 = dilution factor).

What influences plaque growth?

In general, plaque size increases as the velocity of phage diffusion increases. The diffusion rate is dependent on certain phage properties e.g. phage dimensions and whether the phage aggregates. It is also dependent on the concentration of agar in the overlay layer.

Why is it necessary to have a top and a bottom agar layer in the plaque assay?

The bottom layer solid layer is crucial for an even bacteria lawn, which the phage will grow on. The softer, semi-solid media allows for (like Victor mentioned) better diffusion, or an easier medium for the phage to interact with the bacteria. In my experience you can add indicator reagents to either layer.

What is plaque assay technique?

The plaque assay can be used to purify a clonal population of virus or to determine viral titer as plaque-forming units per ml (pfu/ml) so that known amounts of virus can be used to infect cells during subsequent work. … An overlay of agarose keeps the cells stable and limits the spread of virus.

Do all viruses form plaques?

Plaque assay is limited to only a subset of animal viruses that can lead to cell lysis, forming plaques on the monolayer of cells in a cell culture plate. In fact, many animal viruses do not form plaques on the monolayer, but nonetheless induce a discernible CPE.