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

Table 10.2 An example of a quantal assay for virus infectivity.

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:

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