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Obviously the best way to avoid mycoplasma contamination is to avoid it in the first place. In our case, however, it is not possible to avoid. We are culturing viruses and tissues out of human nasal swabs, washes, and biopsies.

As you can imagine, anytime you are pulling something out of someone's nose, it is quite often full of mycoplasma. I've reviewed current examples of mycoplasma elimination, but found the section in Current Protocols in Molecular Biology to be the most helpful.

Indeed, we found that checking with a PCR based kit for common lab mycoplasma contaminants often missed detection of mycoplasma, because it did not include "clinically relevant" strains. Kits which did include these species clearly detected mycoplasma, in addition to Hoechst stain confirming their presence.

Now the question is there a good way to remove them? We've tried serial dilutions, elimination kits (also MycoZap), titrated doxycycline and enrofloxacin doses, and even mouse passage.

Of these mouse passage was probably the most effective, but not desirable because it might induce additional confounding factors. The kits had fair results, but 5 passages post treatment mycoplasma were again detectable via Hoechst stain (PCR detection persisted through out).

The dox and enrofloxacin may have produced better results at higher concentrations, but cell toxicity became a serious issue. I was wondering if anyone had examples of combination methods for cleaning primary cells and/or virus stocks (been grown on more standard immortalized cell lines) for a more complete treatment method.

Just as note on our hands, we regularly check our cell lines, and are in fact maintaining a mycoplasma free tissue culture system. Further, as proof of concept, we were able to clean a culture that was known to be infected with M. hominis with kits.

It doesn't seem worth the effort, but is there any evidence to suggest typing down to the species would be worth it in the eradication effort? Each donor could conceivably be providing a different contaminate.

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  • $\begingroup$ did you try using a combination of dox/enrofloxacin? Also, did you try using an iron-free medium during enrofloxacin treatment? enrofloxacin kills by chelating iron, so the less iron in the medium the better (however the cytotoxicity may also increase) $\endgroup$ – Thawn May 27 '16 at 14:55
  • $\begingroup$ As far as I know Doxicyclin is a bacteriostatic antibiotic not a bactericide which in your case could be the reason why some mycoplasma survived and started to grow again at the end of treatment. As a potential solution, you could try (combinations) of other bactericide antibiotics that are effective against mycoplasma. $\endgroup$ – Thawn May 27 '16 at 14:55
  • $\begingroup$ @Thawn Dox/Enrofloxacin were used sequentially (in both orders), but we found the direct combination too toxic. EMEM was tried, which has minimal iron. $\endgroup$ – Atl LED May 27 '16 at 15:12
  • $\begingroup$ @Thawn I believe combinations of antibiotics are used in the commercial kits. $\endgroup$ – Atl LED May 27 '16 at 15:17
  • $\begingroup$ I guess then I am out of ideas, sorry $\endgroup$ – Thawn May 27 '16 at 17:23

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