What is the principle of plaque assay?

The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell.

What is a plaque forming cell?

Two of the immune function assays, relying on antibody response, are the Plaque Forming Cell Assay (PFC) and the hemagglutination test. The PFC assay measures IgM producing cells and: is an indicator of cell proliferation. identifies the primary and secondary lymphoid organs.

What is a plaque assay in microbiology?

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.

Why do we use plaque forming units?

A plaque-forming unit (PFU) is a measure used in virology to describe the number of virus particles capable of forming plaques per unit volume.

What is the role of the agar in the plaque assay?

A widely used approach for determining the quantity of infectious virus is the plaque assay. After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel.

Why is the original phage solution diluted?

The first dilution should be a 1/100 dilution to minimize the volume of the original phage stock used.

What is hemolytic plaque assay?

The hemolytic plaque assay (Jerne and Nordin, 1963) detects antibody formation by single lymphocytes. This assay quantifies the number of lymphocytes secreting antibodies to a particular antigen and permits visualization of the antibody-forming cell’s morphology.

Why is the spleen used in the hemolytic plaque assay?

Why is the spleen used in the Hemolytic plaque assay? The spleen is used because it synthesizes antibodies in its white pulp and removes antibody-coated bacteria along with antibody-coated blood cells by way of blood and lymph node circulation.

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 is PFU per 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.

What causes the formation of plaques in a bacteriophage assay?

Initially the nutrients are plentiful so the bacteria grow rapidly and, since the MOI is low, the phage grow lytically. After several lytic cycles the local MOI increases and most of the cells are lysed, producing a plaque in the lawn of cells.

How do you identify a virus titer?

The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. 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.

What is the objective of the plaque assay?

The objective of the plaque assay is to enumerate and study individual antibody-forming cells, specially in situations where only a few such cells are present among millions of cells that do not release antibody.

Why is the plaque assay used in coliform?

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.

How to prepare overlay medium for plaque assay?

The virus is then allowed to attach to the cells for ~ 1 h at 28 °C and the overlay medium is prepared during the viral adsorption period. We prepare 125 mL bottles containing 50 mL of 2% (w/v) Seaplaque ® low melting temperature agarose (Cambrex) in water, then autoclave the bottles and allow the agarose to solidify.

What is the plaque assay value for baculovirus?

The cells will adhere more tightly to the plastic when seeded in the absence than when seeded in the presence of serum. While the cells are attaching, the serial 10-fold dilutions of the virus stock are performed, working under the assumption that a typical baculovirus stock will have a plaque assay titer of ~ 1 × 10 7 pfu/mL.