Hello I am a graduate student who has been having contamination issues for 3+ months now. Specifically in human embryonic stem cell culture. I have worked with these cells 2 years ago as an undergraduate and had no issues until recently. I have gone ~2 weeks without contamination but it always comes back. We have cleaned the incubators, hoods (recently re-certified), and I have thrown anything away that has been suspect. I have had other students watch me to catch any errors but nothing is out of the ordinary. The equipment I use like pipets, tips, etc. is all used by everyone but I am the only one experiencing contamination. I tried different lab coats but now I do not wear one at all and spray diligently. I scrub my arms with soap before putting on gloves and spray my arms with 70% ethanol. Once in the hood I do not stick my arms all the way in...usually just wrists.
I feed these cells everyday with filtered media. The ingredients for media is shared, like I said earlier, others haven't had any issues. It takes less than 16 hours for me to get contamination where the media (sometimes) turns yellow and always turns murky. The media becomes incredibly blurry and as the plate is being swirled you can see the murkyness mix. The pictures I have attached are H9 aMHC GFP cells on a mouse embryonic fibroblast feeder layer and the very next day where the stem cells are dead and a lot of cell debris. When contamination hits I usually thaw from a different vial/person/passage. I can usually go 1-2 passes before I get contamination again.
Any help ID-ing this would be greatly appreciated. Hopefully then I can figure out the source of the contamination.
If I can find a picture of the petri dish with the murky contamination I will upload.