Fungal Contamination - What are my options? (May/02/2011 )

I have a flask of cells – A549 lung cancer cells in DMEM + 10% FBS – that we have been exposing to a novel anti-cancer agent for the last 3 months at very low levels in order to see if we can get a cell line to become resistant to the new drug and then look at gene expression to see how it differs from cells that aren’t resistant to the anti-cancer agent. However, starting last week ( 6 days ago) the flask has become contaminated with what I’m thinking is either yeast or fungus. The medium is slightly turbid, with no noticeable color change from normal. Over the weekend the contamination grew to the point where visible white specks can be seen hanging in suspension in the medium. Furthermore, treating with pen/strep caused no change in the contamination, neither did gentamycin nor ampho-B – all at their recommended concentrations.

When the contamination was first noted it was at a low enough level that it wasn’t interfering with cellular proliferation and as long as the cells were washed and sub-cultured every day it was manageable. However, over the weekend the contamination has increased significantly and the cells are starting to lose their morphology. A small white speck began to grow on the top portion of the culture flask – giving me further evidence that this is most likely caused by a fungus.

I’m at a total loss as for what to do now since nothing seems to be helping clear it up. I would rather leave discarding the culture entirely and starting over as an absolute last resort since that will delay us 2-3 months but it looks like that might be the only option.

i guess it is better to discard as the cells must have had changed/differentiated (as you said morohological change is evident)-even if you try for your drug now, it might not be worthwhile as they are not original anymore and would create a bias...that is frustrating to throw them, but this seems like the best and only option...moreover its not safe to keep them

Alright, that is what I was thinking. Thank you for confirming it. I disposed of the cells and am going to clean the incubator and everything else before starting them up again.

I agree. The best thing was to throw them out.

When you repeat this again, I would have a backup flask running at the same time, and I would freeze down some of the cells periodically so you don't have to go back to square one should you have contamination again.
I would probably freeze a vial every 2 weeks or so, when you are doing a split.

Good luck