Viral Titer Calculator

A plaque assay counts infection events, not particles. Diluted virus is adsorbed onto a confluent cell monolayer and covered with agarose or methylcellulose, which stops released virions from diffusing across the dish so that spread is limited to neighbouring cells. Each focus of killed cells becomes a plaque visible after fixing and staining with crystal violet, and because plaque number varies linearly with dilution, a single infectious particle is sufficient to start one. The titre is the mean plaque count divided by the product of plated volume and dilution, giving plaque forming units per mL of undiluted stock. Counts between about 10 and 100 per plate are used, since plaque counts are Poisson distributed and the standard deviation is the square root of the mean, so 40 plaques carry roughly 16 percent sampling error. Infectivity is far lower than particle number for most animal viruses, with particle to PFU ratios commonly between 10 and several thousand, so PFU/mL and physical genome copies are not interchangeable. TCID50 measured by endpoint dilution relates to PFU by a Poisson factor of about 0.7. Multiplicity of infection is PFU divided by cell number, and Poisson statistics mean an MOI of 1 leaves about 37 percent of cells uninfected, so an MOI of 3 to 5 is needed for near-synchronous infection. Viruses that do not lyse cells, or that spread poorly under an overlay, cannot be titrated this way.

Plaque counting began with bacteriophage. Felix d'Herelle, working at the Institut Pasteur in Paris, reported in 1917 that filtrates from dysentery patients produced clear circular spots on lawns of Shigella, and he recognized that counting those spots gave a direct measure of the number of infectious units in a filtrate. For three decades the technique remained a bacteriophage method, because animal viruses were assayed in whole animals or embryonated eggs, where results came back as death or survival and had to be converted to a 50 percent endpoint by the interpolation formulas of Reed and Muench, published in 1938, or of Karber. Renato Dulbecco adapted the plaque assay to animal viruses at the California Institute of Technology, publishing in 1952 a method in which western equine encephalomyelitis virus produced countable plaques on monolayers of chick embryo fibroblasts under an agar overlay. With Marguerite Vogt he extended it to poliovirus the following year. This gave animal virology a clonal, quantitative assay and made single particle genetics possible, work recognized when Dulbecco shared the Nobel Prize in Physiology or Medicine in 1975. The plaque assay remains the reference titration method, with fluorescent focus and TCID50 assays used where plaques are absent or slow to form.