Depending on the viral growth kinetics and host cell used, a visible plaque will normally form within 2-14 days. Infected cells will continue the replication-lysis-infection cycle, further propagating the infection, resulting in increasingly distinct and discrete plaques. As liquid overlays do not require warming, delicate and heat labile viruses may also prove easier to plaque.Īfter the initial infection and application of the immobilizing overlay, individual plaques, or zones of cell death, will begin to develop as viral infection and replication are constrained to the surrounding monolayer. Plaque assays utilizing liquid versus traditional overlays have several advantages as the overlay can be applied at room temperature, and application and removal is significantly easier. While solid or semisolid overlays such as agarose, methyl cellulose or carboxymethyl cellulose (CMC) have traditionally been used, liquid overlays have become an increasingly attractive alternative with the development of novel liquid overlays such as Avicel 5– 7. Infected monolayers are then covered with an immobilizing overlay medium to prevent viral infection from indiscriminately spreading through either the mechanical or convectional flow of the liquid medium during viral propagation. While advances in technologies and techniques continue to refine and alter the landscape plaque assays continue to represent the gold standard in determining viral concentrations for infectious lytic virons 1,4.ĭuring 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. While alternative means for viral quantification have since been developed and adapted, such as immunoassays, fluorescence and transmission electron microscopy, tunable resistive pulse sensing (TRPS), flow cytometry, recombinant reporter systems, and quantitative reverse transcription polymerase chain reaction (qRT-PCR), these methods fail to identify and quantitate replication competent virons 1,3. This technique was first adapted and modified from phage assays, which had previously been used to calculate titers of stock bacteriophages in plant biology 1,2. It was not until the advent of the plaque assay in 1952 that a means to quantitatively and qualitatively calculate animal viral titers was first developed 1,2. The accurate isolation and quantification of viable viral samples has consistently been an ongoing research goal in virology. Liquid overlays may also prove more sensitive than traditional overlays for certain heat labile viruses. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium.
#BLOOD OVERLAYS HOW TO#
Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture.
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