Does anyone know the best method for detection of Mycoplasma contamination in the cell culture system? Also what could be the best approach (drug etc.) to avoid mycoplasma contamination?
PCR on culture supernatant, when negative analyse your cell extract in PCR.
Through away always contaminated cells and used media, sterilize incubator and put new steril water....forget about antibiotica
PCR on culture supernatant, when negative analyse your cell extract in PCR.
Through away always contaminated cells and used media, sterilize incubator and put new steril water....forget about antibiotica
Gerold is completely right. There are a lot of Mycoplasma Detection Kits available, all based on a simple PCR. Another way would be a DAPI staining, but I don't recommend this, because it is not that convenient and meaningful.
Hi,
we use a modified PCR-based test based on this paper: http://www.ncbi.nlm.nih.gov/pubmed/8118618
Works fine for us and is much more economic than ready-to-use kits. However, I expect the detection limit to be higher.
For prevention, a good sterile technique is essential. If you should do so, don't prepare your medium from powder and screen your culture frequently until you are sure that your sterile technique works. Standard antibiotics like pen/strep can indeed disguise infections, since most mycoplasma strains are resistant to most of them. If you dont use antibiotics like us, you see if something got into your media or cultures within days, since most normal contaminating bacteria will overgrow your cells.
Gerold Untergasser, Dogan Demircioglu and Patrick Heinrich...Thanx, got it....How about using plasmocin? Is it OK to be used in the cell culture system to avoid such type of contamination?
I would not recommend to use it routinely, despite the manufacturer says it is ok. One thing is that fluoroquinolones also act on eukaryotic topoisomerases (with much lower affinity, but could still be a problem), the other is the fact that you might introduce resistances in potential contaminants, especially if you use low concentrations. As far as I heard, it works for decontamination, but be sure to re-test regularly for several weeks while cultivating in antibiotic-free medium. We tried to cure our cultures with sparfloxacin, but it seems a few mycos escaped treatment, so the cultures became positive again after a few weeks. With new cell stocks and revised sterile technique, we never had a problem with mycos again.
Our lab is using the VenorGeM Mycoplasma Detection Kit by Minerva Biolabs. This works pretty fine for us and we are regularly (every three months or so) performing a screen for mycoplasma. Some time ago we had an contamination in our cell culture and after identifying the problematic cells we tried to decontaminate them using a Mycoplasma Removal Agent. Unfortunately this agent basically killed most of the treated cells. The best solution - i guess - is discarding the contaminated cultures and going back to a hopefully uncontaminated stock of cells.
I totally agree with you, dear Jonasz, but the very best way is to avoid such contaminations. The main source is our mouth (Mycoplasma orale). That's why, I really don't like to talk,when working with cell lines. Following this and all other rules of perfect aseptic work, I could avoid contaminations for 2 years now.
Lipnam Lo ,·Patrick Heinrich , Jonasz Weber ·and Dogan Demircioglu Thank you all for your nice explaination. This is in fcat a serious problem we are facing.....But I hope your suggestions would help us alot to overcome this problem.
We have a routine DAPI staining protocol for myco testing in our lab. It only takes about 20 minutes after everyone in the lab plates cells they are using into 6 well plates, and they can be visualized using a fluorescent microscope. A lot less cost and time than pcr for a few cell lines to test, but with DAPI, you can get false negatives, while with PCR you can get false positives.
We also dont generally try to remove the contamination unless its an important irreplacable cell line. We find that attempting to treat requires at least two treatment courses, which takes 6 weeks, and many times the cells are still contaminated. The best policy is good sterile technique, which i find that almost every lab i visit, someone isn't following even if the rest of the lab is. In this case one bad apple can rot the basket, so training everyone and having them pass some sort of competence test important.
Also dont use routine antibiotics for cell culture. It masks contamination and poor sterile technique. Usually mycoplasma contamination occurs at the same time as bacterial contamination from saliva. Bacterial contamination is very obvious, but if the media contains antibiotics, it kills the bacteria but not the more sneaky mycoplasma, making it seem like the cells are still clean.
If you want to outsource, then I would send the cells to Multiplexion. They can do Authentication and contamination testing (Mycoplasma, interspecies and viral contaminations) at very affordable prices. http://www.multiplexion.de/en/
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