Hi all,
So recently my lab is experiencing a low-grade contamination that keeps carrying over to other incubators. We have run decons on our incubators every time they show up, have switched to new medium, have switched out materials, labcoats, have used pen-strep and anti-anti, have discarded our contaminated cultures in bleach, have gone to extra measures with our PPE (face masks, hair masks, etc.) and are STILL finding a small (visible at 40,000X) circular, slow growing little bugger.
I've tried culturing them in LB broth, no success, although they seem to like the media we grow our stem cells in (mTeSR, E8). We have also tried culturing our media and it has come out clean every time...so the media isn't coming in with the contamination...
I think it may be possible that our frozen stocks were contaminated before being frozen down but am unsure, since they seem to be so slow growing, it's difficult to identify any contamination right after being thawed. Usually they are seen up to a week after culture...
Anyhow, does anyone have any other advice that helped them get rid of a particularly stubborn contaminant?
you may want to test for Mycoplasma. verify your stocks as they may be contaminated you just didnt notice. antibiotics are not recomended for daily use as they mask contamination. you are on the right track for cleaning measures. I would recomend alternating sanitizers and adding some peroxide based ones. also a good air cleaner device will help a lot in removing airborne contamination.
We use normocin and plasmocin from invivo gen. The normocin is 1:500 dilution, and plasmocin 20-50 ug/ml
I also was thinking in mycoplasma...we add mycoplasma removal agent to some of our culture media to prevent initial infections or to remove it from infected cell cultures. Good luck!
If ALL culture that you are working with got contamination and if you are using the same hood each time it might be contamination of hood's airfilter system..
Thank-you all for your answers. I had received a new low passage freshly thawed dish of cells a couple of days ago and left it in a freshly deconned incubator for a couple of days. The person I received them from has a reputation of great aseptic technique so I believe it is coming from the water baths that they are being thawed in, or the contaminant was frozen down with the cells. Either way, we are a little bit closer to getting rid of this stubborn thing.
Couldn't that be some kind of cellular debris, microvesicles or exosomes shed continuously by the cells?
Vladimir, it certainly could be, but on some occasions, the debris is moving independently of the motion seen in the rest of the dish. It's unfortunately really difficult to tell. We are going to run some further analysis.
- Badges
- Science method