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I suspect mycoplasma is affecting the growth rate of my culture. How can I test for it? Product FAQ
There are several options. Our first recommendation is to use the Invitrogen MycoFluor Mycoplasma Detection Kit.
Mycoplasmas are small, self-replicating prokaryotes (0.3 - 0.8 mm diameter), that lack a cell wall and have the ability to adsorb onto host cells. Mycoplasma is one of the most serious forms of cryptic contamination and its presence is not detected unless appropriate tests are made or until some aspect of cell behavior is noticed to have changed. Between 15 and 50% of cell lines submitted to cell banks are contaminated with mycoplasma. Mycoplasma spreads readily among cell lines via reagents and media, the operator and the work surface.
The presence of mycoplasma may invalidate the results obtained with that culture. The presence of mycoplasma-infected cultures can result in the shut-down of the entire laboratory until the infection can be eliminated, whereupon complete restocking is required. The origin of contamination is usually traced to mycoplasma present in animal (bovine) serum or to human oral mycoplasma transferred by droplet infection during cell culture. The simplest test for the detection of mycoplasma in cultures is the use of a fluorescent dye which binds directly to DNA causing fluorescence (e.g. Hoechst 33258) which can be seen by fluorescence microscopy. Mycoplasma positive cells will show intense fluorescent spots on the plasma membranes or show filaments which may be absorbed onto the cells. Uncontaminated cells show only brightly fluorescent cell nuclei. The technique is rapid (less than 30 minutes), but requires heavy contamination (10E6 mycoplasma/ml) to produce a clear positive result. If however, the suspect cells are co-incubated for 2-4 days with an "indicator" cell line (such as 3T3) which is particularly suitable for demonstration of positive staining, then sensitivity can be substantially increased. Microbiological culture techniques are available that operate at a greater sensitivity, but it can take up to 21 days to obtain a result, a positive control is needed, and the result may require expert interpretation.
A variety of PCR-based methods are available, some of which have been utilized as commercially available detection kits. It is recommended to use a combination of DNA staining and a PCR-based method once every 3 months for all growing cultures in the laboratory and for every new cell line as it enters the laboratory. In addition, all Master and Working Cell Banks should be tested at the time of freezing. Quality control and good working practice will reduce potential problems. It is important that frozen stocks are created immediately after testing and re-tested before distribution. If cells are cultured for more than 3 months after testing, they should be re-tested. Regulatory bodies now insist that cell cultures used for the production of reagents for diagnostic kits or therapeutic agents are free from mycoplasma infection. Also, some scientific journals have the policy of requiring statements from authors that the culture work reported in those journals is carried out with mycoplasma-free cells. Normally, when contamination with mycoplasma is apparent, the recommendation would be to discard the cultures and start again. If necessary, and only if the contamination is not extensive, then it is often possible to rescue the cells by treatment with one of the commercially available antibiotics. This must only be considered for a remedial action, not as a routine supplement to growth media (and thereby a substitute for good cell culture practice).
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