mycoplasma contamination through liquid nitrogen storage - (Aug/05/2006 )

Although this is apparently not a new subject (was on one year ago), it still is a big enigma for me; some people say that mycoplasma can travel from an infected vial in liquid nitrogen to other (non-infected) vials in the same vessel, be it through gas or liquid phase.
How on earth is this possible, give the fact that in your vial in liquid nitrogen there is only a clump of deep (-150-196 C) -frozen medium, serum, DMSO and other watery bits like cells (including mycoplasma)? I was taught that from any frozen solution not even a single atom can escape, so how can an entire organism achiev this?
We are about to combine our primary cells with cell lines, and the fact that entire institutes strictly separate these makes me feel a bit uneasy...
Any comment will help, thanks!

Laura

between what you just described and alien adoption, I would take the later at no time.

well i think that a mycoplasma contamination occurs more by other stuff air, contamination of incubators or hood rather in liquid nitrogen (but i don't want to store in liquid N2 contaminated culture too )

My old lab got some contaminated cell stocks in liquid nitrogen. And it spread to all the other cell stocks. We had to get rid of the entire stuff as liquid nitrogen seemed to have entered the cryovials somehow. Its quite puzzling.

Dear All,

I look after our Institutes cell repository and also worked for 18 years in Industry again looking after cell repository's. This is what I teach all staff in respect to Mycoplasma control and possible cross contamination.

a) When using cell lines or Primary cells ALWAYS do mycoplasma testing. PCR identification of Mycoplasma's IS A WASTE OF TIME. All vaccines and drugs are tested using the direct culture method in combination with Hoescht Staining. They are FDA approved and as yet PCR is not.

If you get a positive CHUCK THEM AWAY.

c) Quarantine all cells that come into your labs. The only ones that can go straight into a negative area are the cells purschased from the ATCC or the ECACC... they do the correct testing.

d) Mycoplasma contamination comes from 3 main areas... Foetal Cal/Bovine Serum, the SCIENTIST/TECHNICIAN and from the culture room environment. Always use GLP and good Asceptic technique.

e) ALWAYS STORE CELL LINES SEPERATELY FROM PRIMARY CELLS AND TISSUE. Scolix mentions cross contamination which is more common than people think. Research groups generally store everything together because they think they cannot afford to have 2/3 seperate repositories....this is a false economy.


Remember that Mycoplasma contaminattion is a major problem with an estimated 30-40% of cells being used that are POSITIVE. It is relatively easy to irradicate the problem. At the moment our Institute has a 4% rate for positives. These are ALWAYS in cells obtained from other academic research groups and never from commercial companies, again in an attempt to save money.
ALSO REMEMBER THAT YOU CANNOT PUBLISH ANY DATA FROM CELLS THAT HAVE BEEN TESTED AS POSITIVE AS IT WILL BE REJECTED BY ALL JOURNALS.

The above information has been collected through 30 years of cell culture experience in both Industry and Academia. In all this time we have never had a mycoplasma problem.

Mycoplasma can cause cell contamination in liquid phase of liquid nitrogen. Liquid nitrogen may enter the cryovials and may work as a carrier among the cryovials. You can possibly get more information from the companies producing cryogenic equipment.

I don't know of contamination in vapor phase, if anyone knows how this happens I wil be glad to learn.

I remain unconvinced.

On one hand, I guess there is a reason why you want to place vials above liquid N2 to prevent explosion (and possible contamination afterward ?!), and dip the whole vial in ETOH, or spread with ETOH before open it to further limit such possibility.

However, on the other hand, as Laura pointed out, the vials are typically frozen at -80 C before placed in N2 tank, they are rock solid already, even if N2 deposited in the vial evaporates, it will be vapor that escapes only, not with macroplasm that are trapped in solid ice. At this point, there should be a positive pressure inside the vial due to N2 evaporation that will prevent any cross contamination.