Mycoplasma contamination. - (Dec/14/2008 )

Hey

Everybody knows that mycoplasma contaminations are very common in cell cultures, and so mine are... lol
I would like some advise how to avoid, mycoplasma infections, how to detect ( PCR tests are good?) and if possible a protocol to decontamination of cells with mycoplasma.


Thank you in advance!

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Fix and stain cells with Hoescht 22358 is the gold standard, it is the most reliable and consistent detection method. PCR works but gives a lot of false negatives, and contamination of the reagents with amplified DNA can give false positives. Also, keeping a positive control is difficult (do you want to be culturing mycoplasma in your lab?).

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There are treatments available for ridding your cells of mycoplasma. They aren't cheap- we used them only for precious cell lines (usually we throw cells and media away, clean incubator etc thoroughly and start again). The treatment I've used for mycoplasma is called BM cyclin - its 2 compounds that you cycle between over a month or so, untill your mycoplasma is gone.
BM cyclin is from Roche Bioscience. I'm sure there are others, but this one definately worked for us.

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hi,

the gold standard for mycoplasm detection is the direct culture technique (detection of mycoplasma colonies after incubation in broth and on agar) together with the hoechst staining (e.g. 33258). thats because not every mycoplasma strain grows in culture (e.g. M. hyorhinis). the problem with the DNA staining is that it is quite insensitive and you need experienced interpretation of the results (danger of false positives due to cell debris). but there is also a ready reagent from Invitrogen availabe (MycoFluor).

PCR is often used but be quite sceptical especially when using commercial kits, moreover they do not detect every species. always double check your samples with another test.

the easiest and fastest test i know is the MycoAlert from Lonza, but i don't have any experience with that.

Recommended mycoplasmacidal agents by the ATCC:

 BM-Cyclin, (Roche, catalogue no. 799050)

 Mycoplasma Removal Agent (MRA), (MP Biomedicals, catalogue no.
093050044)

 Ciprofloxacin, (Bayer, catalogue no. 89-001)

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Good post Ned Land.

The only area which I disagree with you is the "clean up of cell lines". The phrase which we use is:

"If in doubt......chuck them out"

The main reasons for this is because:

i) Mycoplasma infection will put SELECTION pressure on your cells i.e. only those cells that are more resistant to infection will survive.

ii) The cells will change and MAY NOT REVERT BACK TO THEIR ORIGINAL PHENOTYPE.

iii) Can you be 100% sure that the clean up has worked.

iv) The "clean up agents" also have OTHER EFFECTS ON THE CELLS...so further selection pressure.

Kindest regards


Rhombus

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Hey friends!!

Thank you very much for replying me !!

I think i will throw everything out and start over again. I work in virology lab and my experiments are involved with immune response in viral infection, then mycoplasma infection can change drastically these response. Some papers has describle interferon pathways in mycoplasma infection and this terrible for me.
other bad point is that high concentration of antibiotics and antimycotics has related with inhibition of viral infection, but i'm not sure, some literature has describle. What do you think?
My cells line are: c6/36, LLC-MK, Vero, U937 and HepG2.

Thank you in advance again!!! Regards!

Reis, V.P.

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Hi, mycoplasma contaminations are unfortunately very frequent. To avoid them the best thing is to use gloves during your work and pray not to have other plates contaminated in the incubator. to decontaminate the cells I use cyclin treatment, it will take you around three weeks. I attach the protocol of BM cyclin. I hope you could have good results¡¡¡¡

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