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Plaque Assay Method

An assay is a technique used to isolate and purify viruses and to to determine viral titers (the lowest concentration of virus that results in infection). A plaque assay, originally called a virological assay, was developed to measure and count the infectivity of bacteriophages. According to Biology-Online.org, it was later also applied to count mammalian viruses.

  1. The Facts

    • A plaque is a confluent, monolayer culture of cells. It forms as a result of infection of one of the cells by a single virus particle, according to Biology-Online.org. Once the cell is infected, the virus replicates and eventually kills the cell. Furthermore, the newly replicated virus particles spread, infect and kill the nearby cells.

    Function

    • In a plaque assay, the culture is first stained with a particular dye. This die stains only viable (live) cells, according to Biology-Online.org. As a result, the dead cells appear unstained in the culture, against the dyed background. Currently, the plaque assay is applied to detect cells that produce antibodies that destroy erythrocytes by hemolysis.

    Features

    • Erythrocytes are cells (also called red blood cells) that contain a hemoglobin and transport oxygen and carbon dioxide in the blood, from lungs and tissues. Hemolysis occurs when erythrocytes break open and release hemoglobin, according to Biology-Online.org. As a result, in the plaque assay, the basis is clear plaque and indicates that the red blood cells have been hemolysed by the antibodies.

    Method Part I

    • Ten-fold dilutions of a virus stock are prepared prior to performing a plaque assay and 0.1mL aliquiots are put on to the cell monolayers. This mixture is incubated for a period of time so that the virus has time to attack the cells. Furthermore, the monolayers are covered by a nutrient medium that contains a substance (usually agar), according to "Detecting Viruses: A Plaque Assay" by Vincent Racaniello. The nutrient medium forms a gel and after the culture is incubated, the original infected cells release viral progeny. The gel restrict the spread of the virus to surrounding cells, and as a result, the infectious particle produces a circular zone called a plaque. With time, the plaque grows to a size visible with the naked eye and dyes are used to enhance the viewing.

    Method Part II

    • The virus titer is calculated in plaque forming units, called PFU, per milliliter. They are counted and, to minimized error, only plates that contain between 10 and 100 plaques are counted. Furthermore, according to "Detecting Viruses: A Plaque Assay," for every 100 counted plaques the same titer will vary by plus or minus 10 percent. Therefore the estimated error of a plaque assay is about 10 percent, when performed correctly.

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