TCID₅₀

The TCID₅₀  is defined as that dilution of virus required to infect  50% of a series  of replicate  inoculated cell cultures, and  like the  plaque assay  it relies  on  the  presence and detection of CPE. Host cells are grown in confluent healthy monolayers, usually in the wells of a multi-well (24-, 48- or 96-well) tissue culture plate, to which aliquots of virus dilutions are added. The use of such plates allows between 4 and 12 replicates of each virus dilution, established in a single row or column of the plate. During incubation the virus replicates and releases progeny virions into the culture medium of each well, which in turn infect other healthy cells in the monolayer. The CPE is allowed to develop over a period of days, at which time the cell monolayers are observed microscopically, directly or following fixing and/or staining. Each well is scored for the presence or absence of CPE, and marked as positive or negative, accordingly. The numbers of positive wells at each dilution tested are used to calculate the TCID₅₀, which represents the dilution of virus (and hence is a measure of original virus titer) that would give CPE in 50% of the monolayers (wells) inoculated.

               The worked example below is derived from simulated data represented in Figure and Table 1:

The titer is therefore 10^7.17  TCID₅₀ per unit  vol^-1.

Figure . Diagrammatic representation of a TCID₅₀  assay.  Cell monolayers in a 48-well tissue culture  plate are infected with virus at increasing dilutions (i.e. six replicate wells are infected with each  dilution  in the  range 10^-2  to 10^-9). Following incubation, all wells are examined for the presence (+) or absence (blank) of CPE.

            Table 1.  Data from 48-well plate  used  to calculate TCID50 (see  Figure  and text for calculation)