I am confused about the calculation of TCID50/ml values for HIV virus. I am using 10,000 cells/well of TZMbL cells and 2 days of infection in in presence of 10microG/ml concentration of DEAE, as per the standard protocol and the virus was diluted in series with dilution factor 4. Attached excel sheet is the calculation made by me for a sample.
I want to know.
1. What to put in the box "Dilution per ml" ? (There is a dilution factor asked in a different box, which is “4”, so what does “dilution per ml” mean?) (I thought it doesn’t matter if it is 100microL or 1 L, dilution remains the same)
2. I guess the cut off factor of 2 should be fine to know 50% infection. But, should it be different?
Please let me know.
As a cutoff, we use mean of background + (2,5 x SD of background) = 98.7% confidence interval.
As x̅ ± 2σ roughly = 95% confidence interval, we go a bit beyond that.
With 2x mean, you are a bit stringent for your cutoff.
As the equation for the TCID50 is not in the H43 cell, it is difficult to see if the equation used is correct.
Good luck,
Thank you Michel. Now I know why cutoff of 2.5 is suggested.
The cells I used were TZMbL cells.
In the H43 cell in the excel document, there should be the equation to calculate the TCID50: Something like M=xk+d[0,5-(1/n)(r)] where xk is the largest dilution used (11), d is the interval between dilution (1), n is the number of wells per dilutions (4) and r is the number of wells that have values lower than x̅ ± 2,5σ. The attached protocol from ACTG is well done and explains how the TCID50 should be calculated. Good luck!
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