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#1 jacksonbear



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Posted 26 February 2013 - 06:46 AM

I have been titering a batch of HIV on TZMbl cells using B-Gal staining. I have calculated the TCID50 to be 5.6x10^4 for 100ul of virus, and calculated the FFU/ml to be 1.2x10^6. It is my understanding that the concentration of infectious particles (pfu/ml or ffu/ml) should be approximately 0.7(TCID50). Why am I getting a FFU/ml that is a log higher when TCID50 is adjusted for 1ml?

if it helps, this is what I did.

seeded TZMbl cells in quadruplicate in 96 well plate for 7 serial log dilutions starting with neat virus through 10^-6. The following day I diluted my virus by serial log dilution, and added 100ul of each viral dilution to TZMbl cells in quadruplicate. I infected the cells for 3hours, then washed off the virus, and incubated for 3 days. After 3 days I washed the cells, fixed them and stained with X-Gal for 2 hours. I then proceeded to count the number of blue foci in each well.

10^0 = all wells positive (too many to count)
10^-1 = all wells positive (too many to count)
10^-2 = all wells positive (too many to count)
10^-3 = 75, 86, 99, 122 blue cells/ well
10^-4 = 20, 10, 7, 20 blue cells/well
10^-5 = 0, 0, 0, 5 blue cells/well
10^-6 = 0, 0, 0, 0 blue cells/well

Thanks for any help/advice you can share.


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