Adjusting pH of RPMI/ bicarbonate containing media
Posted 05 November 2012 - 09:32 AM
The problem I'm having is that I want my medium to contain bicarbonate, but I don't want it to buffer the medium. Is there a way/ alternative media to include bicarb without it doing this?
Posted 05 November 2012 - 11:54 AM
Is this problem in the incubator or bottles out on the bench/in the fridge? If in the incubator - check the CO2 content, the bicarb buffering system requires CO2 to keep the pH stable at around 7.4.
the problem I'm having is that after a few hours (about 8-9) the pH has been reasserted from my desired pH, and I want to do incubations of up to 24 hours.
Posted 05 November 2012 - 03:22 PM
Carbonate-bicarbonate is stable at pH about 9.6-10. At so low pH is quite close to the pKa1 of carbonate system (6.37) so you are losing bicarbonate as CO2, that's why the pH changes
Posted 06 November 2012 - 03:23 AM
I'm only doing slight adjustments as well, to pH 6.7 and 7.
Would a sealed system be required do you think then to maintain the pH? Or is it not possible to make medium with HCO3- in it that won't rebufferer itself?
Posted 06 November 2012 - 11:49 AM
How much bicarb did you add per litre?
Posted 06 November 2012 - 06:59 PM
Posted 07 November 2012 - 01:58 AM
I've considered maybe trying to seal the plates I've been using (96-well plates) with parafilm to see if they would be able to maintain the pH better.
Edited by RossM, 07 November 2012 - 01:58 AM.
Posted 07 November 2012 - 11:49 AM
Posted 07 November 2012 - 12:59 PM
I think it's this gradual change over time that is stopping me from being able to copy the experiment I read of in the paper.
Posted 07 November 2012 - 03:15 PM
Much more detailed preparation:
That's because of the disociation rate, if you don't have enough pCO2 you won't be able to make stable acidic medium using bicarbonate. I really think that it will be impossible without increasing the CO2 concentration
apart from acidic pH's were kept in a 7% CO2 environment (something I don't have access to right now).