MEF culture contamination? - Contaminated cell culture? (Jul/17/2010 )

Think this might be a yeast infection in my culture of mouse embryonic fibroblasts. Not growing fast enough to change the pH of the media (based on color). The image attached is taken at 100x (10x obj + 10x scope). MEFs appear to be okay, but I need to get rid of the infection. Can't afford to lose this cell line. Any insight into what I'm likely dealing with and how to get rid of the infection would be helpful. The media I'm using is DMEM supplemented with 5% FBS and penicillin streptomycin and fungisone. Should I try upping my fungisone concentration?

It does look like yeast. Fungizone should deal with it, but it will take some time and you need to be aware that the drug also affects human cells to some extent. Make sure you change the medium frequently to reduce the amount of yeast in the culture, making the drug more effective.

You could try limited dilution - plate a whole heap of cells out in 96 well plates at different concentrations and see if you can get some wells which are free of the yeast - then grow these up from there.

Given that MEFs have a limited amount of passages before they are no longer any good (ours are ok to P4, unless you've immortalised them of course) I would say your best bet is to make them again. Apart from anything else, now that your cells have been contaminated this may have affected how they behave and so aren't any good for experiments anyway.