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This experiment introduces the titration of viral infectivity (TCID50 determination). It helps to understand several common methods for the determination of viral infectivity and to grasp the procedure, calculation method and meaning of TCID50 for the amount of infection in half cell cultures.
LD50 (50% lethal dose): animal or chicken embryo is used to detect.
Half chicken embryo infective dose EID50 (egg 50% infective dose): using chicken embryo to detect.
Half of the cell culture infective dose TCID50 (50% tissue culture infective dose): using cells to detect.
Plaque forming unit (PFU): detected by cells.
Trypsin, growth fluid, viral fluid (PRV).
1 bottle of full-grown monolayer cells.
(1) Reed-Muench method
| Dilution of viral fluid | Number of CPE holes | No CPE hole number | Cumulative | Percentage of occurrence of CPE holes | |
| CPE hole number | No CPE hole number | ||||
| 10-1 | 8 | 0 | 27 | 0 | 100 |
| 10-2 | 8 | 0 | 19 | 0 | 100 |
| 10-3 | 7 | 1 | 11 | 1 | 91.6 |
| 10-4 | 3 | 5 | 4 | 6 | 40 |
| 10-5 | 1 | 7 | 1 | 13 | 0 |
| 10-6 | 0 | 8 | 0 | 21 | 0.7 |
CPE：Cytopathic effect
Distance ratio = (percentage of lesions above 50% - 50%) / (percentage of lesions above 50% - percentage of lesions below 50%)
＝（91.6-50）/（91.6-40）
＝ 0.8
lgTCID50 = distance ratio × difference between the logarithms of dilutions higher than the logarithm of the dilution of 50% lesion rate.
=0.8× (-1) (-3)
=-3.8
TCID50=10-3.8/0.1 ml
Meaning: Inoculation of 100 µl of this virus at a dilution of 103.8 can cause lesions in 50% of the cells.
(2) Karber Method
| Dilution of viral fluid | Number of holes in which CPE appears | Rate of occurrence of CPE holes |
| 10-1 | 8 | 8/8=1 |
| 10-2 | 8 | 8/8=1 |
| 10-3 | 7 | 7/8=0.875 |
| 10-4 | 3 | 3/8=0.375 |
| 10-5 | 1 | 1/8=0.125 |
| 10-6 | 0 | 0/8=0 |
lgTCID50=L-d(s-0.5)
L: Logarithm of the highest dilution
D: Difference between logarithms of dilution
S: Sum of positive pore ratios
lgTCID50=-1-1× (3.375-0.5)
=-3.875
TCID50=10-3.875/0.1 ml
Meaning: Inoculation of 100 µl of this virus at a dilution of 103.875 can cause lesions in 50% of the cells.
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