## The relation between dilution endpoint and infectious units of virus

Quantal endpoints are simply a measure of dilution of infectious virus, but they relate to the average number of PFUs in the aliquot. An example of a quantal assay is shown in Fig. 10.8. An HSV stock was diluted as shown and equal aliquots were added to individual wells of 48-well culture plates. Evidence of virus infection (CPE) is shown by the black wells. For the titration, one can construct a table such as Table 10.2, and from the tabulated data, one can make the graph shown in Fig. 10.9.

In the graph, one can estimate that a dilution at which 50% of the wells would be infected is about 4 X 103; therefore, the TCID50 was 4 X 103 in the original sample. More accurate measures of the ID50 of a virus stock can be obtained by using statistical methods such as the method of Reed and Muench, which is described in a variety of basic statistical texts.

Although ID50 is a measure of dilution, an ID50 unit is directly related to PFU; 1 ID50 unit measures a dilution required to ensure that 50% of the aliquots in that dilution have infectious virus in them. This will only occur if there are 0.7 PFU (average) per aliquot, or 7 PFUs in 10 ml in the above example.

This finding follows from certain rough arithmetical considerations: If a certain X number of PFUs per milliliter in the original concentration was diluted by a factor D so that each animal

 Sample dilution Log dilution No. of infected wells Total no. of wells % Infected None 0 100 1 00 100 1/1000 3 39 48 81 1/10,000 4 14 48 29 1/100,000 5 3 48 6 1/1000,000 6 1 1 00 0
of stock wells Experiment 1 Experiment 2
 1/1,000 81% (39/48) 1/10,000 29% (14/48) 1/100,000 6% (3/48)

Fig. 10.8 Quantal (endpoint dilution) assay of HSV in tissue culture wells. Replicate cultures of rabbit skin fibroblasts were grown to a density of about 5 X 104 cells per well of a 24-well tissue culture plate. Aliquots of the indicated stock virus dilutions were pipetted into the cultures and the plate was incubated for 48 hours and then developed with a stain that indicates black for virus-infected cells. Any well that received at least 1 PFU of virus stained black (two separate experiments are shown). The percentage of positive (infected) wells is shown at each dilution.

or tissue culture well has a 50% probability of being infected with a PFU, then the final concentration of virus defines a type of multiplicity of infection (call it m) where the probability of a positive infection is 50%. This value (m) should have the dimensions of units of infectivity in a standard volume (here 1 ml). Then: