Mycoplasma Question - (Jan/27/2010 )

Hi Everyone,

Quick question, I've been running some disease testing on my cell lines to prep them for mouse injection and tumor formation. Running two types of mycoplasma tests (PCR from media after 5 days of continuous growth) and fluorescent microscopy test I have negative results. However we sent our cell line out for IMPACT profile testing (which isolates genomic DNA straight from frozen cell suspensions and PCR tests that way) and it came back positive. I'm not sure which tests are most reliable.

Does mycoplasma integrate into host genomes? If it does and if the cells were ever contaminated with mycoplasma, even if cleared with plasmocin/primocin treatment. Would it still test positive through genomic DNA PCR testing? So in actuality is it ever possible to rid a cell line of the effects of mycoplasma contamination? Based on my media/microscopy tests I believe the cell line is free of active mycoplasma, but I do know that it was contaminated at one point (through media PCR tests) and cleared using antibiotic treatments (again through media PCR tests). Would it invalidate my results to use this cell line in future experiments? It is a stable transfectant so it is not easily replaceable.

Thanks!

You could have a small but active contamination that is below your current detection limits (I think the PCR will work for greater than 1000 bacteria/ml, similar but more specific for the fluorescence).

cancergeek on Jan 27 2010, 05:26 PM said:

Dear Cancergeek,

I am sure that I have posted many times on this forum about Mycoplasma and the "detection Methods" available. Briefly:

a) The "Gold Standard test" is the Agar Growth test which is the most sensitive and reliable method available. IT IS FDA APPROVED and used for all human vaccines and drugs that are administered.

PCR IS NOT FDA APPROVED and is highly UNRELIABLE. For this reason, the ATCC (largest supplier of cell lines in the world), NO LONGER USE PCR AS A TEST FOR MYCOPLASMA. They only use/offer as a service Hoescht staining in combination with Agar growth test.

c) If a cell line is contaminated with mycoplasma no journal will allow these results to be published. Mycoplasma's affect nearly every aspect of the cells biology. The advice is always to:

i) REGULARLY TEST YOUR CELLS...preferably before novel transfection.
ii) USE THE RIGHT TEST
iii) QUARANTINE ANY CELLS COMING FROM SOURCES OTHER THAN FROM THE ATCC/ECACC
iv) Throw away any cells that are positive.
v) Even using so called "clean up procedures" i.e. very high concentration of antibiotics/antimycotics, WILL NOT REVERSE THE PHENOTYPIC CHANGES THAT OCCUR DUE TO INFECTION.


I am always amazed that researchers who use cells as their basic tools for experimental discovery, do not validate their cells before spending hundreds of working hours and tens of thousands of pounds on experiments.

Hopely reading this post will help you massively in the future

Kindest regards

Uncle Rhombus (now 31 years in the lab)