Hi,

do I get you right that you mean the serum used for medium supplementation (not e.g. patient samples)?

If so, you can try to directly take some of the FCS, incubate it at 100°C for a few minutes and directly use a small volume (inhibitory effect) as template in PCR. For medium supernatants in heavily infected cultures, this works perfectly, however if you only have a low mycoplasma titer in the serum, you may not detect them with adequate sensitivity.

A more elaborate approach would be to supplement a knowingly mycoplasma-negative indicator line with your serum sample, incubate it for an extended period (weeks) in antibiotic-free medium and run a standard protocol on those cells.

Finally, if you do not use highly expensive supplements, it is in general not a bad idea to completely discard all opened reagents and start from zero just to be safe. It would be very frustrating if an infection occured again after several weeks only because you have overlooked something.

Thanks Patrick, you understand what I mean, I don't know if the FCS is contaminated, and now all of the cell lines of my institute have mycoplasma so I can´t use any knowingly mycoplasma-negative indicator line. For this reason I have to take the first option.

Do have you any protocol you can share with me to evaluate the mycoplasma titer in serum used for medium supplementation by PCR or can you suggest me some papers where I can find that?

Again, Thanks for your answere

Hi,

generally, most PCR approaches can only give you a qualitative answer and in theory it should able to detect even a single bacterial genome, however especially when you apply a crude sample preparation by boiling, this is most likely not the case. If you only have a few mycoplasma particles in your FCS, it may test negative, however even a single viable particle will eventually lead to a infected culture.

We use a protocol modified from this publication: Article Uphoff, C.C. & Drexler, H.G. Comparative PCR analysis for de...

We always got results consistent with those from DAPI staining, however I am pretty sure that both methods will not give you positive results for fresh (say 1-2 weeks old) infections, or those supressed (but not cured!) by standard antibiotics. For those reasons it is highly recommended to incubate cells in antibiotic-free medium to first enrich myoplasma for reliable detection.

I think the best solution in your situation would be to get a clean indicator line (e.g. from a cell bank, the particular cell line should not matter), inoculate/complement the medium with your serum, cultivate the cells for at least two weeks without antibiotics and run a standard protocol on those cultures. Be sure to include appropriate controls to exclude the possibility that your lab equipment (laminar flow hood, incubator, water bath, ...) is the source of the infection.

Thank Patrick, I´ll read the paper and I´ll take your suggestions into account. Thanks again for your help.