I am curious about ways/tests that can be routinely use to detect cell culture contamination. As per my knowledge low level contamination by bacteria and fungi would not yield any physically observable changes. This is also true for mycoplasma contamination. Growth rate of cells also depends on so many other factors. I would like to know what type of test/strategies, you guys use to check each contamination and time frame for each test (e.g. every two months ).
I appreciate your input here.
Thanks
Very good question. Prolonged tissue culture often leads to the accumulation of contamination with mycoplasma. This can be contained by periodic treatment with antibiotics eg -cipro but is not a cure. There are PCR based kits which can detect contamination but they are not cheap and it is likely most people forget about the issue until it becomes a problem. If you have a big lab with many different cell lines, then it is worthwhile doing mass screening periodically, for most of us it is best to treat cells for mycoplasma at the start of an exercise and limit the number of passages / media changes before discarding and starting afresh. Biological sterility does not equal "PCR" sterility, so even PCR for bacterial DNA can give rise to positives from for eg serum in the absence of any living organism.
Very good question. Prolonged tissue culture often leads to the accumulation of contamination with mycoplasma. This can be contained by periodic treatment with antibiotics eg -cipro but is not a cure. There are PCR based kits which can detect contamination but they are not cheap and it is likely most people forget about the issue until it becomes a problem. If you have a big lab with many different cell lines, then it is worthwhile doing mass screening periodically, for most of us it is best to treat cells for mycoplasma at the start of an exercise and limit the number of passages / media changes before discarding and starting afresh. Biological sterility does not equal "PCR" sterility, so even PCR for bacterial DNA can give rise to positives from for eg serum in the absence of any living organism.
Lonza has a mycoplasm contamination kit called Mycoalert that can be used.
Thanks Ian and Minh.
How about Fungi and bacteria ? Do you guys know anything about Cell Culture Contamination Detection Kit from LIfe Technologies (for fungi and bacteria)
and PlasmoTest™ from Invivogen?
In additio to periodic Mycoplasma tests using commercially available kits, it helps not to keep the same line in culture for endless passages, as the likelihood of contamination increases with time in culture. This is because Mycoplasma often derives from operators, who carry M. hominis in their airways. One helpful straty is to keep a master cell bank from Mycoplasma-free stock. From a vial of the master cell bank, derive a working cell bank (also Mycoplasma free). This can consist in several dozen frozen vials. Periodically discard old cultures and start fresh from a vial of working cell bank. This is the strategy used by biotech companies, who must keep their cell lines free of any contaminants if they use them to produce biopharmaceuticals. This strategy also has the advantage of minimizing the likelihood of clonal selection in vitro, which can change the characteristics of your cell line.
A number of companies have developed antibody, antibody+antibiotic ( fluoroquinolone) (Sigma, Roche, Abserotec etc) kits for "elimination" of mycoplasma. One drawback is that they often have cytotoxic effects and can reduce growth rates considerably and lead to clonal outgrowth as mentioned by Lucio.
http://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_96_1_p48-51.pdf
Re fungal infections - it is almost never worth trying to combat it, but start afresh and limit the possible spread of infection further. Generally you do not need a kit to tell you if a culture is fungally infected - observation is often sufficient. Addition of Amphotericin B may be useful in preventing infection in the first instance, but is less good at total elimination of infection. Any organism that can form spores is often very difficult to eliminate by antibiotic treatment alone.
Mycoplasma is not visible and carried over during multiple passages despite good lab t/c practice. Fungal infections in my experience are "de-novo" infections due to sterility failures.
Hi Nipuna, I'm considering using the culture contamination kit from life technologies for my experiments. Have you tried it?
Hi Golnaz,
Sorry for the delay in replying. I did not try any contamination treatment kit. I realized that I don't have that much of a problem with fungi and bacteria. I only culture one cell line for about 40 passages. Then start new.
- Badges
- Science method