How does a plaque assay work?

How does a plaque assay work?

In this assay, cell monolayers are infected with a low ratio of virus, such that sporadic cells become infected. An overlay of agarose keeps the cells stable and limits the spread of virus. When each infected cell produces virus and eventually lyses, only the immediately adjacent cells become infected.

How do you count plaque assay?

To determine the virus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used. Statistical principles dictate that when 100 plaques are counted, the sample titer will vary by plus or minus 10%.

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.

What is meant by plaque forming unit?

Plaque forming units (PFU) is a measure of concentration used in virology. The PFU is the concentration of viruses in a solution which are capable of lysing host cells and forming a plaque, or colony, in a culture.

How is a plaque formed?

Plaque forms when bacteria in your mouth mix with sugary or starchy foods, such as milk, juice, soft drinks, bread, pasta and fruit. These bacteria release acids that break down carbohydrates in food and drinks.

What is one unit of a virus?

A complete virus particle, known as a virion, consists of nucleic acid surrounded by a protective coat of protein called a capsid. These are formed from identical protein subunits called capsomeres. Viruses can have a lipid “envelope” derived from the host cell membrane.

How is viral infectivity measured?

Luciferase reporter assay. The luciferase reporter assay is commonly used to measure the infectivity of a viral strain. Here, the ratio μ = N/M of total infections over the number of plated cells is estimated by measuring the transcription activity of viral proteins (14, 15, 16).

Do all viruses form plaques?

Plaque assay is an extremely sensitive and easy method to determine the number of infectious viruses (plaque forming units, pfu) and hence the success of the purification of infectious viruses. However, not all virus systems produce plaques and the efficiency of the plating (EOP) can be very low.

Why do plaques stop increasing in size?

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.

How do you find the viral multiplicity of infection?

Multiplicity of infection (MOI) is a frequently used term in virology which refers to the number of virions that are added per cell during infection. If one million virions are added to one million cells, the MOI is one. If ten million virions are added, the MOI is ten. Add 100,000 virions, and the MOI is 0.1.

How do I know if I have Moi virus?

For figuring out the amount of virus you need to add for a certain MOI, use the formula: #cells * desired MOI= total PFU (or Plaque Forming Units) needed. Then use the formula: (total PFU needed) / (PFU/ml) = total ml of virus needed to reach your desired dose.

What is meant by multiplicity of infection?

For example, when referring to a group of cells inoculated with infectious virus particles, the multiplicity of infection or MOI is the ratio defined by the number of infectious virus particles deposited in a well divided by the number of target cells present in that well.

How do you calculate a viral titer?

Calculate the Stock Titer

1. # of cells at Transduction = Total number of cells in the culture when viral particles were added.
2. MOI = Derived from the chart above based on the percentage of transduced cells.
3. ml of Lentiviral Stock used for Transduction = The volume in ml of the virus added to the cells.

How do you calculate the amount of water needed to dilute a solution?

To make a fixed amount of a dilute solution from a stock solution, you can use the formula: C1V1 = C2V2 where: V1 = Volume of stock solution needed to make the new solution. C1 = Concentration of stock solution.

What does a dilution factor of 2 mean?

When a concentrated solution is diluted, the dilution factor may be expressed as the ratio of the concentration of stock solution to the concentration of the diluted solution. As another example, a 2-fold dilution is the same as a dilution factor of 2.

What is a 5% dilution?

Answer: 1:5 dilution = 1/5 dilution = 1 part sample and 4 parts diluent in a total of 5 parts. If you need 10 ml, final volume, then you need 1/5 of 10 ml = 2 ml sample. To bring this 2 ml sample up to a total volume of 10 ml, you must add 10 ml – 2 ml = 8 ml diluent.

What is a 1 to 20 dilution?

These two components proportionally combine to create a dilution. For example, a 1:20 dilution converts to a 1/20 dilution factor. Multiply the final desired volume by the dilution factor to determine the needed volume of the stock solution. In our example, 30 mL x 1 ÷ 20 = 1.5 mL of stock solution.