Yeast Contamination - Need advice on dealing with chronic yeast contamination (Sep/08/2008 )

We grow HeLas and Cos7s in our lab & lately we've been having a massive problem with contamination. Our technique's pretty good, I think, and we've been growing the cells in gentomycin, penicillin & streptomycin. I sterile-filter every bottle of media I use & work in a BSL2 hood. Some of our HeLas were transfected with GFP & RFP centrin, and are grown in Blastocidin in addition to the three other antibiotics. So despite all these precautions, we're getting yeast contamination.

And the oddest thing is, the yeast will affect some flasks but not others! I'll split HeLas from the same flask into three flasks & then one flask will have yeast & the other two won't. I've have one well, in a 6-well plate, massively contaminated while the well right next to it is fine.

Could it be our incubator? We recently wiped it down with 70% ethanol & autoclaved the shelves. I UV the hood every day, use sterile, single-use pipettes, ethanol my gloves, spray down everything that goes into the hood with ethanol, never open the flasks outside the hood & yet the yeast returns.

At this point we're ordering new batches of HeLas & Cos 7s, but would like to prevent this from recurring. Any suggestions?

Thanks

Swapna

I've been told that ethanol isn't an effective method for killing fungi (in my mind, it makes sense that they'd have at least some resistance to a natural waste product). Though standard practice, wiping things down with ethanol simply helps remove the fungi from surfaces much as you could use water to rinse grime off dishes. If you truly want to decontaminate equipment such as incubators, you should probably use a topical fungicide rather than ethanol.

In my limited experience, water baths and incubators tend to be the major sources of contamination. You mentioned the incubator, but if you haven't already you should decontaminate the water bath as well. In our lab, we add a commercial fungicide for water baths into the water after every water change, and we try to clean it once a month.