Dear all,
Hello! I have had issues with Hek293T cells maintenance for quite awhile whereby my Hek cells die after passages 6 or 7. I've recently started trouble shooting to find out the source of contamination.
I discovered white spots in my co2 incubator as well as white fuzzy substances in the water bath tray of the incubator. Are these signs of possible fungal contamination? I've also inoculated a 12 well plate with DMEM (only), DMEM (only) + FBS, DMEM (used for Hek293T cells maintenance) + FBS and DMEM (used for Hek293T cells maintenance) [ALL WITHOUT ANY HEK293T CELLS!!!] and incubated in the potentially contaminated incubator to determine wether the contamination was due to the incubator or the media/serum we've been using in the lab. Upon looking under the microscope, I saw thread/string like structures. I've checked on it a couple of days later and saw more of these structures under the microscope as well as some other structures that I am not able to recognise. Are those resulting from a potential fungal contamination?
Please help! I've attached some images I've took as well!!
Don't try to salvage anything if you can help it.
Discard all opened media and aliquots of regents. Follow all decontamination procedures for the incubator, hood and your lab.
You can try to treat precious cells with anti-microbial kits, but ensure you do this in a dedicated incubator and working space. It might not work.
Good luck
Sebastian and Chinedu provided some good advice. Additionally you can apply anti-fungal agents to the water bath (there are commercially available agents specifically designed for CO2 incubators).
Also if the cell aliquots I assume you keep in liquid nitrogen have been frozen under the same hood while the infection was already there, they might already be infected and need to be discarded as well. If you can get cells frozen elsewhere it would make more sense to use those.
Others have mentioned it but the major sources of contamination in cell culture are, in fact, the water bath and the incubator. Both are at 37°C and can have some microbial life even with "only" water. Try decontaminating those two (some incubator also have UV decontamination). After that, use water + some kind of antibacterial/fungal agents. The most important thing is to be sure to remove all of the water after getting a container from a water bath. Dry them thoroughly with a white paper towel before spraying them my ethanol. Be sure to spray the neck of the bottle as well (make sure that they are never in contact with the water in the water bath.
Also, check if your the only one having contaminated cultures.
If not, is there anybody that is making bread outside the lab? I have see some media contamination with yeast that others used at home to make bread. Pretty rare, but not impossible.
You could also change the filter in the pipetman; there is always a chance that someone didn't pay enough attention.
Hope it helps!
As you said the white fuzzy substances were seen in the water bath tray of the incubator, you can fumigate the incubator with KMNO4 and formaldehyde and check it again for contamination, which may be because of spilling of media or as discussed above if the water was not sterilized properly.
Add copper sulfate along with water keeping for humidity.
If it is indeed a fungal contamination, you could try incubator decontamination method. If it is not enough, I would try with diluted formaldehide over weekend (there are protocols in the web). The problem with fungal contamination is that fungal spores could disseminate through the CO2 tubes and remain there for longer. The UV from inbubator decontamination programm could not killed spores in that place.
We reached that conclusion after suffered for fungal contamination in an incubator for long. We tried to clean it up, sterilizing the selfs and the bath, contra-con programs, etc... and the contamination always returns. Finally, we tried to decontaminate twice with formaldehide diluted overweekend. And we finally succeed.
Ester Molina - wouldn't leaving formaldehyde for so long cause similar problems to what Sebastian Schmitt
described after fumigation?- Badges
- Science method