Mycoplasma - How to detect and treat mycoplasma contamination in culture and RNA? (Jul/21/2008 )

Hi everybody,
I am growing several cell lines and after having some problems with one new line which I got from another lab -slow growing and oddly shaped cells, which I discarded after 3 weeks of hopeless efforts to get them fit again - I have the feeling that the other cell lines I cultivate have been contaminated... they grow much slower and some also started to loose their shape. However I did not recognize and cloudy bacteria or fungi...
My suspecion is that I got a hidden mycoplasma contamination - do you have any experience which detection method works best (ELISA, PCR, DAPI?) and which reagents are best for treating the cells without affecting their health? Or should I discard all of them and start from the beginning. I already did some RNA extractions for NimbleGen-microarray analysis about 2 weeks ago when the cells still looked fine but already had the other cells in the incubator - can I test these RNAs for mycoplasma NAs before I start the microarrays
Looking forward to your suggestions and ideas...
Thank you!

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For fast initial check, I think the DAPI stain works well. Positive is positive, but negative can also be positive. PCR or ELISA can be used to be certain, and for checking negatives. I would go with whatever is cheaper/easier/faster to get.

I don't have any experience with trying to eradicate contamination. I think that no matter what you use, there is the fear that you got rid of the contamination by 99.99% and in time the contamination will just remanifest itself.

Did you talk to the other lab about the general growing and morphology of your cells?

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i would tell you how we test but i think rhombus would shout at me

however we've recently used plasmocin which cleaned it up a treat (pity they turned out not to be the cells we wanted - oh well)

dom

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Dear Senior scientist,

You need to use the Agar growth test as a definitive and gold standard, FDA approved method for detecting mycoplasme contamination. Hoescht staining will only show a massively contaminated culture, but again is FDA approved.
PCR is NOT FDA approved and has been dropped by the ATCC ( the world's largest cell line provider)as a method for mycoplasma detection.

The general rule is " if in doubt chuck them out". You can treat cells with a vast range of compounds....HOWEVER all these compounds will DEFINETLY affect your cells at all levels i.e. receptor expression, morphology, gene expression etc.

Most of what Judes states I completely disagree with, except for the point about never really knowing if the clean up has worked.

Hope this is useful........and Dom DO THE RIGHT TEST MATE.

kindest regards

Rhombus

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I was thinking of posting a reply for this yesterday, but I just knew that Rhombus wouldn't be able to resist this one!!
Good advice though, gotta love him

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Just "spreading the gospel" ........ I love you all as well !!!!!!!!!

Rhombus.

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I would go for a ELISA-Kit. It is sensitive fast and validated. I can recommend you the roche kit: "Mycoplasma Detection Kit" -it is easy to use and they have a positive control inside. If they are contaminated throw them away and hopefully you have a frozen backup in liquid N2...
If you do not have a replacement for the cells, use BM-Cyclin (also from roche applied sciences) to get rid of the contamination - the 2 times I used it, it was really successful.
And decontaminate your incubator... maybe it would be usefull to check your collegues cells as well. We have a monthly mycoplasma test of all our cell lines which is part of our routine labwork!!!!

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does this mean i need testing too?

lets hope i'm mycoplasma negative (depends on the test i 'spose )

dom

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This is interesting, as we use PCR (real-time) as a standard method for detecting mycoplasma contamination in our cultures (Ishikawa paper) .
Do you know the reasons why ATCC dropped the PCR approach? Not enough species detected, variable mycoplasma genome or something else?

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agar growth test? What is this?

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