Problem Maintaining pH in Culture Media - (Apr/13/2009 )

Hi Everyone,

I have a problem maintaining the pH in my stock bottles of culture media.

I use Leibovitz (L-15) media, which I prepare from powder, adjust to pH 7.6, filter and then store in the fridge in glass bottles. After about a week or two of storage, the pH of media in these bottles drops to about 7.0-7.2. I'm pretty sure the filtering is not altering the pH as I've measured the pH before and after filtering and it remains relatively constant, and the media does not appear to become contaminated by storage in the fridge (e.g. it does not become cloudy or have any microorganisms swimming in it).

Does anyone have any idea why this is happening? The change in pH really affects the health of my cells.

Thank you for any advice!

do note that temperature does affect pH. Are you making the pH readings at the same temperature?

rustyshackleford on Apr 13 2009, 04:45 PM said:

You say that you do not see any contamination, but you cant always see this.
Have you allready tested it?

Another idea could be that the pH drops due a reaction in your medium caused by some sort of lightreaction. You say you store them in the fridge, but sometimes you open the fridge thus light can fall onto it.
Another point: you adjust the pH and then store it, but the agar itself is still "alive": chemical reactions can still happen and this cause a change in pH, however this should not cause a real deep drop in pH , I suposse its buffered?)
And also : do not forget that a change in temperature could also effect the pH, however this is mostly when autoclaving agar or so that you need to keep this in mind, I do not know if putting this media in the fridge could cause such a drop. I do not know what the effect might be.
Or also like preneseblue said: you need to measure the pH at the same temperature.

But I do not know the general specifics of this medium, but the things I named could cause changes in pH.

A slightly leaky bottle could do this - especially for media that are formulated to pH at about pH 7.0 for cell culture when first made up. The minor leak in the lid would allow diffusion of gas in and out of the bottle resulting in a change of pH from the measured to a higher pH as the CO₂ content changes.

Thank you everyone for your replies!

I initially pH the media at room temperature and when I suspect that a bottle has "gone", I let it warm up to room temperature before I check the pH, so I don't think it is a temperature issue.

As for light being an issue, yes, the bottles do become exposed to light when we open the fridge. I will try covering the bottles with foil to see if it helps.

Its possible that the bottles may be leaky...we inherited them from another lab, so they are pretty old. I will try to use newer, tighter bottles.

In the meantime, I will just continue to monitor pH and re-adjust when necessary.

Thank you again for your ideas!