Addendum to HEPES/MES buffering of tissue culture media

Hinayana Bawagan bawagan at umdnj.edu
Fri Dec 10 14:55:15 EST 1993


     First of all, I would like to thank Joseph Lipsick, Bernard Murray, Fred,
Shaun Black  and Christian Belanger for their very helpful and prompt 
responses. 
     As a pilot experiment for investigating the effect of different pHs
on the infectivity of my virus, I plan to incubate the virus in the
medium with the appropriate pH for a period of 90 seconds.  I am using
1-ml virus stock and possibly 1 ml of the media with the desired pH during
the short incubation period.  In one paper in which a similar experiment was 
done, "the inoculum was exponentially diluted in BA-1 diluent, after the
incubation".  [ BA-1 (M-199 (GIBCO, Grand Island, NY), pH 7.6, 1% BSA,
0.35 g/liter sodium bicarbonate, 100 units/ml penicillin, 100 ug/ml
streptomycin, and 1 ug/ml Fungizone ].
     After incubation, wherein the particular pH has exerted its effect on
the envelope proteins of the virus, I should be able to bring all the
samples (2-ml inoculum) to the normal pH which is about 7 via some diluent
and then use the entire sample to infect monolayers of cells (1 hr) and then
let the infection go for 48 hrs in normal medium.  I harvest the virus and
assay for the titers.  The pH difference is only done in one step.
     My problem is the adjustment part.  If I have a 2-ml inoculum at  pHs
ranging from 4.5 to 7.5, how do I bring that volume to a pH of 7.0 with an
appropriate diluent? I have to work under the sterile hood. 
I don't quite understand "exponential dilution". 
I also have to keep the volume minimal.
     Looking forward to your most insightful suggestions.



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