Recently, mycoplasma contamination in one cell line was detected in our institution. Unfortunately, the cells were not checked regularly for mycoplasma and this contamination dates back to at least 6 months (revealed after checking the previously Hoechst-stained cell sample images). Now we will start a deep-cleaning of everything; the hood, the incubator, the room, the water bath etc. Then we want to check whether our frozen cell culture stocks are contaminated as well and my main question is about this part.
How long (days, weeks or subcultures) should the cells from our frozen stocks should be continued in culture to be ready for mycoplasma testing? If it is lightly contaminated, I assume it should need some time to be detected. And would you recommend using Pen-Strep in the complete medium through this process (we always supply the cells with Pen-Strep unless it is stated not to be supplied).
It would be very helpful to read your comments and experiences on the issue. Thanks in advance.
I have used cells after a single passage for dapi based Mycoplasma detection. However since both Dapi and Hoechst based detection are not super sensitive, and can only detect high levels of contamination, you might want to try myco detection kits for an inspection of this sorts. Pen-strap does not inhibit mycolasma contamination as its primarily an antibiotic and antimycotic, so I don't think its presence in you culture media would make any difference. you could try using Mycoplasma-Ex for eliminating myco in you important cell lines if there is a heavy contamination.
As mentioned in the first post pen-strep isnt really required as it will have no effect on mycoplasma infections. Our laboratory uses DAPI and Hoechst staining and usually leave the cells for 3 splits/passages before analysis. Hope that helps!
Try growing your cells out of antibiotic for at least three passages before testing for mycoplasma. When you take the media for testing the cells should have been in the media for a few days.
I agree with Sandra in saying that it would be sensible to grow cells out of antibiotic for several passages. Mycoplasma species vary in behaviour and there are six different species that commonly contaminate cell cultures (Barile et al 1973, Ann NY Acad Sci 225:251-264). M. fermentans can be difficult to detect and is known to be slow to recover from freezing (Volokhov et al 2008, PMID 18606798). So taking a few passages to be sure sounds like a wise course to me. Pen will have no effect on mycoplasma but strep may be inhibitory so I would definitely avoid any antibiotics.
- Badges
- Science method