Manuel and Holger,

Thanks very much. I suspect you are right that it is mycoplasma and will try your protocol. I hesitated to spend the money on a kit to test for mycoplasma but we can easily buy primers and make our own kit with your protocol.

I have experienced the same phenomena in cancer cells a few months ago.

If cells are comtaminated by micoplasma and you don't know its origin, I recommend a reagent, Zelshield. It can be possible to keep your cells, just adding to the media.

Our laboratory has recently implemented a pcr protocol to detect mycoplasma contamination based in an article published by Young et al. 2010. The method is rapid, easy of implement and cheap, and it has proved very useful in our lab. (Mycoplasma can be detected even in frozen cells).

http://www.ncbi.nlm.nih.gov/pubmed/20431538

Dear Richard,

I am not sure mycoplasma could be visible in phase contrast microscopy. This is why it is often nicknamed the "invisible killer"...

Anyway, bacterial contamination can result in what you described. I experienced a couple of these, and they usually appear 2-3 days after harvesting/seeding the cell cultures. They can look as dots or as moving sticks if they have flagella.

I have once been contaminated with super-delayed bacterial growth that would only become visible after 4 days.

The number one criteria to determine whether your culture are contaminated or not, is the appearence of the medium. It should turn from transparent to opaque with bacteria growing in it.

Best wishes,

Valerian

PS: If you have DAPI-pictures of mycoplasma, I would be glad to see them!

Dear Richard,

As far as I am aware, mycoplasma is not visible in phase contrast. I think what you are getting is some sort of bacterial contamination and sometimes it takes time for the bacteria to stabilize and start growing, especially after thawing or procedures like that. But, once they start growing, they will fill the whole culture in a couple of hours. There are PCR and chemiluminescent based mycoplasma detection kits. Please follow such a detection kit. Also, throw away all current stocks of fbs, media and cell culture reagents, wipe the laminar hood with 10%bleach followed by 70%ethanol and start afresh.

Best

So we traced the source to a contaminated bottle of FBS and that eliminated the problem. I am having trouble convincing the folks in the lab to do the PCR top determine if it was mycoplasma or bacterial now that the problem is gone. But based upon other comments I suspect it was slow growing bacteria, just an odd type I have not experienced in 30 years of cell culture. I have saved the PCR protocol for testing for mycoplasma as I know it will be useful later, if not now.

thanks,

R