- Mycoplasma contamination is a common and challenging problem in cell culture laboratories, but the following measures can substantially reduce the risk of contamination.
- The most effective measure against mycoplasma contamination is to block the entry of mycoplasma to the cell culture lab. This requires a comprehensive understanding of the sources of mycoplasma contamination, as well as the specific properties of mycoplasma that allow them to escape detection and regular sterilization procedures. Good knowledge coupled with practicing good aseptic techniques can prevent mycoplasma contamination.
- Some of the features of mycoplasma such as very small size and lack of cell wall allow them to escape regular microscopic inspection and filter sterilization procedures. In addition, they do not cause drastic changes in culture medium pH (change in color of phenol red-containing medium) or generate turbidity, usually seen in case of bacterial and yeast contamination. In addition, common cell culture antibiotics are also largely ineffective against mycoplasma.
- In absence of visible signs, mycoplasma-contaminated cultures are handled with other uninfected cell lines in the same workspace (laminar flow hood, incubator etc), which results in spreading of mycoplasma to other cell lines.
- A good knowledge of mycoplasma sources is extremely important. One of the major sources of mycoplasma is the newly arrived mycoplasma-contaminated cell lines from other labs. Such cell culture needs to be handled in a quarantine zone unless they are tested negative for mycoplasma. Purchasing mycoplasma-free cell lines from reputable cell banks is a good alternative if you do not have a quarantine zone.
- Cells directly isolated from tissues/organs of animals can also be a potential source of mycoplasma-contamination. Such culture should be handled in a separate area and requires dedicated incubators and laminar flow hood.
- Lab workers can also be a potential source of mycoplasma contamination. Mycoplasma species which can colonize the human oropharyngeal tract have been recovered from contaminated cell culture. Talking, sneezing, or coughing generate aerosol which can cause mycoplasma contamination. Discussion while handling cells in laminar flow hood or in an incubator should be strictly prohibited.
- Cell culture reagents and disposables such as culture medium can also be a source of mycoplasma contamination. Earlier serum used to be the major source of mycoplasma contamination. Since now many suppliers provide mycoplasma-tested serum, it is no longer a major source of contamination.
- Practicing good aseptic techniques is key to prevent any type of contamination including mycoplasma. Proper training of lab personnel, especially master students and junior researchers, should be done before they are allowed to use the main cell culture lab. In addition, lab personnel must maintain good hygiene, wear lab coats and personal protective equipment while handling cell culture.
- Short-term experiments such as transient transfection or growing cells on coverslips can also be a source of mycoplasma contamination. Often researchers do not strictly adhere to aseptic techniques for such experiments. Reagents used in these experiments can also be a source of contamination. In addition, extensive use of antibiotics further compromise in aseptic techniques and indirectly cause spreading of mycoplasma. All procedures which involve cell culture lab equipment and incubators must be performed with extreme care by researchers. Furthermore, when using reagents with low sterility for experiential needs, always properly clean the laminar flow hood with 70% ethanol and turn the uv-light on for 20 min after use.
- Lab equipment especially faulty laminar flow hood, incubator or dirty water bath can also be the source of mycoplasma contamination. Any spillage of medium in the cell culture lab must be cleaned immediately. Cell culture labs must be dust-free, insect-free and animal-free. Cleaning of the cell culture lab including equipment especially water bath, laminar flow hood and incubator must be done regularly.
- Identifying contaminated culture as quickly as possible is also essential to prevent mycoplasma from spreading to other cultures.
- Since mycoplasma contamination requires special procedure to detect, a regular detection program must be employed by the laboratory head.
- Once the contamination is detected, the contaminated culture as well as reagents used to maintain this culture such as culture medium must be discarded immediately.
- Mycoplasma-free cryopreserved stocks are crucial in case of detection of mycoplasma contamination.
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