What should we do to eradicate mycoplasma presence from our lab and samples?
I agree with Katleen, for anything not precious or expensive: throw it away.
For more precious samples we have used Plasmocin (http://www.invivogen.com/plasmocin), which you can add to the culture medium over roughly 2 weeks to remove mycoplasma. It's not particularly cheap and even though it works quite well, sometimes mycoplasma comes back. Regular testing is definitely recommended. I have the feeling that in some cells it just supresses mycoplasma very strongly but doesn't completely get rid of it (however, this is just my feeling).
also something to bear in mind is that if you have detected mycoplasma in any of your samples, it can also be in the incubator, tissue culture hood etc. and infect additional cells. Strict cleaning is therefore recommended and some labs even have a dedicated hood and incubator for cells of "unknown mycoplasma status" to limit spreading. I hope this helps. Good luck
For anything that is not rare or very expensive: throw it away.
If that is not an option (rare, primary cultures), you can try curing with an antibiotic that is effective against mycoplasmas, so a macrolide, tetracycline or fluoroquinolone. As some mycoplasmas are intracellular, they will be very impossible to remove from a cell culture. There are also specific mycoplasma removal agents, but they are also based on one or a combination of the antibiotics mentioned above. You will need to treat quite long and succes is not guaranteed. If you do manage to cure the culture, you will need to test regularly to make sure the mycoplasma is gone and stays gone.
I agree with Katleen, for anything not precious or expensive: throw it away.
For more precious samples we have used Plasmocin (http://www.invivogen.com/plasmocin), which you can add to the culture medium over roughly 2 weeks to remove mycoplasma. It's not particularly cheap and even though it works quite well, sometimes mycoplasma comes back. Regular testing is definitely recommended. I have the feeling that in some cells it just supresses mycoplasma very strongly but doesn't completely get rid of it (however, this is just my feeling).
also something to bear in mind is that if you have detected mycoplasma in any of your samples, it can also be in the incubator, tissue culture hood etc. and infect additional cells. Strict cleaning is therefore recommended and some labs even have a dedicated hood and incubator for cells of "unknown mycoplasma status" to limit spreading. I hope this helps. Good luck
Thanks to all. We found mycoplasma positive in our FBS, media and cell storage.
It's a disaster. We are throwing them away.
But besides of this we need to ensure the whole laboratory decontamination from mycoplasma. Can anyone suggest any procedure, protocol to ensure decontamination and prevention of further mycoplasma contamination in laboratory area?
One of the most important things to do is identify the likely source of the mycoplasma to avoid recontamination. Identifying the species via specialists at ECACC would be a good first step. They could offer advice on source. In general the usual sources are simply reagents or cells already contaminated. Obviously at some point these came from a human or animal source, but direct contamination is rarer simply because this would so often be accompanied by bacterial or fungal co-contamination. This is yet another reason to avoid the casual use of antibiotics in culture such as penicillin, streptomycin and fungizone etc. If you have contamination with mycoplasma or microbes, it's best to know as soon as possible! Antibiotics slow the detection of either. Anyway, make fresh purchases of reagents / cells with 'likely source' in mind and a quarantine lab available. Testing the reagents, (media etc) directly is no good alone as mycoplasma will only grow inside cells. These therefore need to be tested via co-culture with cells guaranteed to be clean. Testing the cells 2-3 weeks after 2-3 days contact with test reagents should be a viable measure. Make sure you test the final aliquots that you store. Provided you purchase from a reputable supplier, standard reagents should at least be delivered mycoplasma free, (no harm in checking though!).
Here are a few tips to eliminate mycoplasma already present, (after of course disposing of all previous reagents and cells). 1) Get in a specialised company to decontaminate labs and hoods with peroxide or EtO etc. 2) Eliminate the water in incubators etc, clean up with the most powerful compatible cleaners, restock with clean water. 3) Check cabinets for flow and filter integrity, (replace as necessary). 4) Clean up / autoclave pipette guns, liquid dispensers etc. Replace all filters in them. 5) Review operator practices with advice from ECACC in mind.
My experience Iberysay that You can try to remove mycoplasmata with antibiotics like ciprofloxacin, chlortetracycline and tylosin.
I had many problems with mico, tried to cipro, MicoKill, and plasmocin, but none worked. The only reagent that worked really well in my system was BM Clyclin from Roche, however I agree that the best option is to routinely test the cells by PCR as Hoechst can give false negative. good luck
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