I am working with breast cancer cells. I recently got mycoplasma contamination . I checked all my stocks and discarded the mycoplasma positive stocks and expanded/ froze the mycoplasma negative stocks. But now when I check the mycoplasma negative stocks second time, they show positive. None of the other users in lab (they have different cell lines) got mycoplasma positive. How is it possible that the cells always test negative the first time and positive the second time ? I am using PCR based method, which says cells should be kept in culture for atleast a week. So, I am following the protocol of the kit. If they really are positive, they should test positive the first time. And if I am the one contaminating by handling then they shouldnot test negative the very first time. I am perplexed.
Rather than the PCR kit I would check microsopically to see if this is a false positive. Plate some cells that are known to be contaminated on glass coverslips, fix and stain with DAPI. View at 60 or 100x. if there is mycoplasm you should see tons of little blue specks in the cells cytoplasm but not on the glass. The little blue specs are mycoplasms nuclei. This will tell you for sure if you have bacteria in your cells. Also, it can the the infection some time to get going too. But in case the PCR is giving you false positives, look under the scope to see evidence of the bacteria actually being present
Rather than the PCR kit I would check microsopically to see if this is a false positive. Plate some cells that are known to be contaminated on glass coverslips, fix and stain with DAPI. View at 60 or 100x. if there is mycoplasm you should see tons of little blue specks in the cells cytoplasm but not on the glass. The little blue specs are mycoplasms nuclei. This will tell you for sure if you have bacteria in your cells. Also, it can the the infection some time to get going too. But in case the PCR is giving you false positives, look under the scope to see evidence of the bacteria actually being present
Mycoplasma contamination is easily checked by DAPI staining. Mycoplasma exhibits numerous and much smaller particles in the cytoplasm than nuclei. When you find the little blue specks in the cells stained with DAPI, you should use PLASMOCIN (2599401,Invivogen).
Thank you so much for your answers Victoria Wu and Go J Yoshida . I will try microscopy and see if it is false positive.
Hi Richa,
This happened to me some time ago, we discovered that some of us in the lab were putting 2 microliters of sample (supernatant of cells cultured without antibiotics for 4 days) in the PCR reaction. While others in our group were using 1 microliter. We saw that some samples analyzed with only 1 microliter showed a false negative result.
Dear Richa,
I don't think it is very likely you contaminate them by handling at all, and especially not very single time anew. My guess is you got an infected culture in the first place and all you stocks are contaminated - maybe only slightly. The detection kits are tricky and I would believe you can easily get false-negatives there. Cells have to be in culture for a while and they should be quite confluent hand have had the last change of media at least 24h ago, better 48 or 72hrs. Also the DAPI/hoechst method may be a safer option.
Treating them is really a pain, but if it's a sturdy cell line may still be successful. Still, if it is a common cell line I'd just try to get a new stock from somewhere.
Also consider that there may be a possibility of cross contamination in N2 tanks (quite under debate though) - this may be interesting to you: http://www.protocol-online.org/biology-forums/posts/18841.html
Good luck!!
Dear Richa,
As mentioned by others DAPI staining will be a more confirmative test. Mycoplasma contamination had been shown to cause genetic abnormalities in different cells. So it's advisable to get new stock of cells. If the contamination load is not high you can try treating them with Sparfloxacin in media. We have used it for treating some sturdy cell lines that had mycoplasma. Treatment for a week with this antibiotic cleans the cells of Mycoplasma if the load is not very high.
Dear Richa,
I would treat the cells with normocin and then keep it at prophylactic levels. Some info here: http://www.invivogen.com/plasmocin
Also, we are treating our primary cells with primocin and using it in place of pen/strep because it is active against both Gram+ and Gram- bacteria, mycoplasma and fungi. I think it could be used in tumor cells (don't see why not). Here is some info: http://www.invivogen.com/primocin
Anyways, get a new stock of cells if that is a possible option.
Good luck!
Jackie
HI Richa,
I would also recommend a better test. Both Lonza (MycoAlert Plus) and Biotool (Rapid myco kit) sell non-PCR based kits that test for the presence of myco metabolites in the media. They are more sensitive and also test for ALL species of myco, where as your PCR test is only for a few subtypes. DO NOT use the Plasmocin as a prophylactic for routine growth, this can mask low level infections. Some say you can "cure" mycoplasma using this antibiotic but we never do this. It rarely works and the cells are most certainly genetically modified from the mycoplasma infection.
Primary cells can be very difficult to work with. We do use a 2X concentration of ciprofloxacin (similar to Plasmocin) in our primary culters for the frist few weeks to try and ward off the growth of myco in the new cells.
Kind regards,
Steve
There are Mycoplasma Elimination Cocktails available online that you could use. I used one before (add to complete media) and it worked for my cell cultures.
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