TCID50

• The TCID50 assay measures infectious cytocidal virions. The host cells are grown
in confluent monolayer to which various dilutions of the virus are added. The
virus replicates and the progeny virus that is released into the supernatant fluid is
free to infect any other cell. The cytopathological damage is allowed to develop
usually over a period of days (depending on the given virus and the cells). At that
time the cells are observed microscopically and the results - presence or absence
of CPE - are recorded
• The TCID50 is defined as that dilution of virus required to infect 50% of the cell
cultures inoculated.
• Once CPE is obtained and the results are recorded, the virus infectivity titers are
calculated using the Reed and Muench method for the determination of the 50%
endpoint
PROCEDURE

 100µL of cell suspension will be seeded into a 96 well plate (~5000 cells/well).
 Plate will be incubated for 24 hour at 37 °C with 5% CO2.
 After incubation cell monolayer will be observed under microscope for 80% confluency.
 After observation growth media will be replaced with different dilution of sample/virus.
 Test sample/virus sample will serially diluted (10 fold) with infection media upto 10 -8.
• After treatment incubate the plates at 37°C in the 5 % CO2 incubator and monitor
by microscopic observation the wells daily for 4 days.
• Record the gradual development of CPE in the wells in the following manner:
0 - No CPE
1 - Partial CPE (less than 50% of the cells exhibit CPE)
2 - About 50% of the cells exhibit CPE
3 - About 75% of the ceils exhibit CPE
4 - The monolayer is totally destroyed

 Calculate the TCID50 counting all the wells with 1-4 CPE as positive.
 Calculation

Difference of logarithms = (% CPE next above 50% - 50) / (%CPE next above 50% - (% CPE next below 50%)
Difference of logarithms = (66.7-50) / (66.7-14.3)
Difference of logarithms = 16.7/ 52.4= 0.31
Log10 50% end point titre = titre next above 50% (i.e., sample dilution having % CPE next above 50%) - (Difference of
logarithms X log dilution factor)
TCID50= -6 + (0.3 X -1) = -6.31
= 10-6.31
This is the end-point dilution, namely the dilution that will infect 50% of the test
units inoculated. The reciprocal of this number yields the titer in terms of infectious
dose per unit volume. If the inoculum added to an individual test unit was 0.1 ml, the
titer of the virus suspension would therefore be:

106.31 TCID50/0.1ml = 10 x 10 6.31

TCID50/ml = 10 7.31 TCID50/ml

TCID50/ml of the virus = 10 7.31