What is a plaque forming assay?
The plaque assay is a well established method for measuring virus concentration as it relates to infectious dose. The assay relies on determining the number of plaque forming units (pfu) created in a monolayer of virus-infected cells.
Who developed the plaque assay?
It is remarkable that DMEM, which was developed in the 1950s, is still used these days. More relevantly, in fact, Renato Dulbecco first established the plaque assay for animal viruses.
What is the difference between a plaque and a colony?
Colony refers to a cluster of particular bacteria developed in a medium. Plaque refers to a clear zone, produced by a Phage which is formed by lysis of the bacterial cells in the medium.
How many viruses does it take to form one plaque?
For most animal viruses, one infectious particle is sufficient to initiate infection. This conclusion can be reached by studying the relationship between the number of infectious virus particles and the plaque count. A linear relationship means that one infectious particle can form a plaque.
How are viral titers determined?
Traditionally, viral titers are quantified by the number of formed plaques, foci, or individually infected cells. The plaque formation assay (PFA) and focus formation assay (FFA) are typically counted with the naked eye or microscopy (light/fluorescence).
How is bacteriophage plaque formed?
Plaques are clear zones formed in a lawn of cells due to lysis by phage. To avoid this, phage are often preadsorbed to cells under conditions that do not allow phage growth (e.g. low temperature) then shifted to permissive conditions to induce all of the phage to develop at the same time.
What type of media was used for the plaque assay?
This assay is based on a microbiological method conducted in a plate. Specifically, a confluent monolayer of host cells is infected with the virus at varying dilutions and covered with a semisolid medium, such as agar, to prevent the virus infection from spreading indiscriminately.
How are viral plaques formed?
A viral plaque is a visible structure formed after introducing a viral sample to a cell culture grown on some nutrient medium. The virus will replicate and spread, generating regions of cell destruction known as plaques.
What is extended XC plaque assay used for?
The extended XC plaque assay is utilized to detect infectious ecotropic murine retroviruses (E-MLV). E-MLV infects only cells of mouse and rat origin. Cells from the mouse embryo cell line, SC-1, are inoculated with the sample, and passaged to amplify any low level of virus present.
What are the basic plaque assay principles and how are they modified?
Basic plaque assay principles can also be adapted and modified in a number of different ways, such as in the use of focus forming assays (FFAs). FFAs do not rely on cell lysis and counterstaining to detect plaque formation, but rather employ immunostaining techniques to directly detect intracellular viral proteins through tagged antibodies.
What is a plaque assay for lytic disease?
During a plaque assay, a confluent monolayer of host cells is infected with a lytic virus of an unknown concentration that has been serially diluted to a countable range, typically between 5-100 virions.
Why are plaque assays used to determine viral titers?
The advantage of using plaque assays to determine viral titers lies in their ability to quantitate the actual number of infectious viral particles within the sample. As multiple virions could potentially infect a single cell, the terminology of units versus virons is used during plaque titrations 1,2.