solution to a weird problem - pH - Adjusting pH in cell culture media (Dec/20/2010 )

Few days back I had posted a problem on this forum that my CHO cells are dying coz of some mysterious reason and the media appears lemon green in color and stinks

Although i suspected contamination, it was actually a pH issue - it was around 6.5 making the media very acidic and hence the cells failed to adhere and died

We buy commercial Hyclone Ham's F-12 media and supplement it with 10% FBS, 1% PS, 1X Bufferall, Geneticin, 100mM NaOH and 10mg per ml Hemin - this leads to a pH of 6.5. This is weird coz i never remember adjusting pH for commercially bought media anywhere - normally the pH is always mainted at 7.4.

Moreover, I'm scared to take freshly prepared media out from the hood coz our pH meter is a lab across and I fear contamination. I normally filter sterilize the media after pH adjustment but will this suffice? My post-doc who made this protocol for media preparation refuses to believe that pH needs to be adjusted but I have checked the pH and its always acidic. I HAVE to adjust the pH unless i'm doing something stupidly wrong here. Coz once i make the media, its golden in color. The moment i add a few drops of NaOH - it becomes pink.

Give a look at my media constituents? Is something wrong? And will taking my media bottle out to the adjacent lab, adjusting pH and re-sterilizing it by filtration still risk contamination?

SuHas

I agree with your post-doc. You should not have to adjust the pH of your media if you're buying it from a commercial source. New media will look a little more golden than media that has been sitting open for a while because of absorbed CO2. Unless these are very precious cells just throw them out and thaw new ones.