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How can I keep my culture medium from becoming alkaline while in the bottle?

I work in a research laboratory where we feed our cell cultures with media such as Ham's F12 or DMEM, both pH-regulated with CO2/bicarbonate. When working in the hood, I try to minimize the length of time the medium bottle is open, as the dissolved carbonic acid blows off as CO2 as long as it's open. Despite my precautions, the medium starts looking pretty purple by the time I'm nearing the end of the bottle. It worries me that my cells are undergoing considerable stress when they're fed with alkaline medium; I know it will absorb CO2 once back in the incubator, but I'd much rather that the medium pH was more physiological from the start. I don't like the idea of adding acids or even more bicarbonate to the medium, and don't know if bubbling CO2 through the medium would be practical or aseptic. Does anybody have a solution that they've found works for them?

Public Comments

1. I know this doesn't help answer your question any, but that seems like an odd problem. I don't remember ever seeing the DMEM in our lab turning color (becoming alkaline) during use like you describe. Even old bottles that had been used repeatedly never did that. Are you making your own media or are you purchasing it? We always bought it, maybe that makes a difference?
2. Unopened bottles of medium never turn alkaline. It seems CO2 escapes when you open the bottle too often. Try to buy smaller bottles, like 500 ml. Some people add HEPES buffer to their medium. It never concerns me. The fresh medium changes to neutral pH after a short while inside the CO2 incubator. You may try to warm up your bottle of medium with cap loose in the CO2 incubator instead of water bath.
3. Some things to consider: Use smaller bottles. You're going to lose a lot of C02 to the head space. There are no great mechanisms to get CO2 into the bottle without risking contamination. You could put the bottle in the incubator (or an alternate chamber), loosely capped, to get some CO2 into the bottle. You don't need to bubble. CO2 is heavier than air. All you have to do is get it into the bottle without carrying in airborne contaminants.