cell culture contamination - (Aug/07/2006 )

Hello,
I have some primary cell cultures that might be infected with yeast. There are big round cells with a very round nucleus in the middle that occasionaly are dividing. I used a protocol to try to get rid of them. Here is the link:
http://www.geocities.com/mesentsev/Bibliot...tamination.html
After 4 days they are still there, however there seems to be no growth (increase in number) and the media is not cloudy, but looks very acidic (light orange). Does anyone have any idea what these big round cells are? Thanks.

Sorry the link does not show up...Here it is:
Washed cells 3x with PBS
incubated with fungizone 5 min in incubator
washed
incubated with trypsin 5 min in incubator
washed
fed with media without antibiotics and fungizines

Yeasts tend to clump together in small balls and have quite a distinctive appearance. In my experience they can be tolerated at low concentration in cell culture so long as they don't become too dense. The media will appear acidic and eventually become quite cloudy as their numbers increase.

My advice would be to wash the culture with PBS a couple of times to remove as much of the contamination as possible and then add fresh media and fungizone to a final concentration of around 1ug/ml. You will need to keep fungizone in the culture to keep the infection at bay.

If you have an unaffected backup its easier to ditch your culture and continue with one that isn't contaminated.

I agree with karyotyper's last comment...... chuck the cells away and start again. Fungizone has many effects on the cells and should be avoided at all costs. I worked with Primary endothelial cells in the 1980's and it had a profound effect on the cells ability to generate and release Nitric Oxide. As we were mainly interested in this vasodilator we had to resist using Fungizone in any of our cultures. We did not look at any other markers,receptors, enzymes secondary messengers etc but I am sure they would have been effected as well.
The art of experimentation is to keep things as simple as possible, and the cell culture adage is

" If in doubt, CHUCK IT OUT "