Persistent stable cell line (Bos23) Contamination... - (Jan/11/2011 )

The lab that I work in has had contamination problems many times in the past yet it appears (at least to me) to come and go cyclically. We switched from Pen/Strap to Gentamicin for our antibiotic in our media awhile back, we've had the cell culture room cleaned, we spray with an ethanol and benzalkonium chloride mixture, etc. Still, no matter what we do, we still get contamination, especially after transfections (we generally use Bosc23 cells). With our strongest magnification on our scope, we can see small, black, moving squares, so it seems to be bacterial as opposed to fungal, but at this point, we really don't know. This contamination issue has pushed me (and others) back a long time. Some think that it could be the instruments (i.e. pipettors, etc.), but we can't autoclave them (someone tried and they didn't work afterward). We've tried to clean things with bleach every now and then, but that still doesn't seem to do the trick.

Does anyone have advice?

My best guess would be that your frozen down cell lines are contaminated. If your frozen cells are contaminated, then every time you thaw them and start a new culture, you are carrying over the bacteria. Try reordering your cell line from a company. Companies are usually pretty good about avoiding contamination in their frozen cell lines.

I guess that it's possible that it could be the cell line, but considering that many times the cells are fine (and do not become contaminated), that seems a little strange. If it consistently happened, sure, but there have been many experiments (including my own) where everything went fine. In addition, it's not just the one cell line, but others often become contaminated as well (i.e. 3T3 cells).