infected cell line? - fungal contamination? (May/02/2006 )

Hi,

I am culturing a cell line and I have not been using antibiotics of antimycotics. I noticed that I am getting strange stringy things in my flasks. They almost look a bit like black shoe laces. They appear to be under my cells although sometimes they appear to be floating in the media. I threw away my last lot of cells because of this. I started culturing a new bactch on tuesday but the same thing has happened.

On friday I incubated some media on its own in a flask. When I looked at it today I could see the same thing and the media had changed colour slightly. I spoke to someone else in the lab about this and she said she wondered if it was small bits of plastic and that the change in media colour was simply down to incubation in co2. I am going to plate some of the media up on sab today to see if I get any growth. Has anyone else seen this? Could it be fungal contamination?

Have you thought about making a new batch of media and trying this again (media alone in flask in incubator)? The description doesn't sound too much like plastic bits to me, but it could be. A contamination often results in a pretty dramatic change in media color (often the media turns orange or yellow). So if that was the case then that wouldn't necessarily just be from the media being in the incubator in CO2.

I plated out the old media on Sab agar and modified dixons agar to look for fungal growth but nothing has grown on either plate. I also made up some fresh media on friday and put some in a flask on its own and incubated it over the weekend. The colour is not the same as that in my cell culture flasks but under the microscope I can see the same stringy bits.

My old bottle of media is pinker than the new bottle which is a more red colour. I don't know what to do or think? Could the stringy bits be some sort of cell debris? Maybe present in the FCS?

Should I discard the cells? I wanted to store some down today but I don't know if its worth it if I am only going to have to throw them away?

Most media turns a pinker color when opened a few times ... that's actually normal (so I wouldn't worry about that so much). Do your cells appear dead or damaged aroud the stringy bits and do the stringy bits cause the media to turn yellow at an unusually fast rate (i.e overnight)? If that's the case then I would definately think contamination. Otherwise I'm not too sure. Do your cells tend to grow closer to the stringy bits (i.e. do you notice a lot of your cells around those stringy bits and growing on them?)? If that's the case they could be just as your collegue in your lab suggested bits of the flask ... cells will tend to be drawn to those spots and grow on them ... if it was contamination it would be just the opposite the cells wouldn't be near that spot. I would keep my eye out for dead and damaged cells that would be a sure sign of contamination.