It is well known that a significant percentage of cell cultures are contaminated with mycoplasma. The extent of this contamination problem has been surveyed many times over the last three decades and found to vary from 10% to 70% in mammalian cultures. It is thus safe to say that the chances of cultures in your lab being contaminated with mycoplasma now or in the future are moderate to high.

Don’t despair though. While preventing mycoplasma contamination is difficult, it is not impossible. We recently queried four experts to learn more about how you can protect your cell cultures from mycoplasma contamination and, if they do end up contaminated, what your best course of action is.

Our expert panel includes Maryellen de Mars, Ph.D., vice president, standards resource center at the ATCC (American Type Culture Collection); Dr. Jürgen Becker, product manager at Promocell; Amanda Capes Davis, Ph.D., founding manager of CellBank Australia and Chair of the International Cell Line Authentication Committee (ICLAC); and Mike Mortillaro, Ph.D., owner, Bulldog Bio.

Biocompare (BC): What method(s) do you recommend for mycoplasma detection and why?

de Mars: Mycoplasma contamination is a serious concern for cell culturists because these bacteria can significantly affect the physiology and viability of cell lines. Therefore, the rapid and accurate identification of mycoplasma species is essential. Currently, three detection methods are used routinely: (1) the direct culture method, which uses both agar and broth; (2) the indirect method, which incorporates the Hoechst DNA stain; and (3) PCR-based testing, which typically uses universal primers that are specific to the 16S rRNA region.

To ensure that any possible contaminants are identified, both the direct and indirect methods should be performed simultaneously. Together, these methods can detect most known culturable and nonculturable mycoplasma species. Each test includes positive and negative controls, and complete testing requires four to five weeks.

PCR-based testing provides a quick and sensitive method for detecting mycoplasma contaminants in cell culture through the amplification of conserved target regions in the mycoplasma genome. Samples that are positive for mycoplasma contamination are easily recognized by a distinct PCR product of a defined size. This method is very accurate and rapid, with results typically available in less than one working day. At ATCC, we provide services and a kit for mycoplasma detection.

Becker: I would definitely choose a PCR-based detection method because it is the most sensitive and typically covers all mycoplasma strains relevant for cell culture contamination. Moreover, this method not only detects viable mycoplasmas but also dead mycoplasmas, giving you better information on the degree of contamination. The PCR-based assays are clearly more robust and reliable than enzyme-based assays, which strongly depend on the viability and cell metabolism (i.e., the cell culture conditions)—giving rise to false-negative results. For more information, see Mycoplasma Test Kits from PromoCell.

Mortillaro: We find that, when including the proper internal controls, a simple assay using PCR and visualizing on an agarose gel is very inexpensive and accurate without a big investment for a real-time PCR machine. Bulldog offers several PCR detection kits. 

Capes-Davis: It’s easy to be overwhelmed by all the different choices. I suggest starting by looking at the kits or methods that relate to your area of expertise—for example, if you are a molecular biologist, then PCR-based methods are your obvious choice. You need a method you can repeat easily, so it makes sense to go for your testing comfort zone if possible. For any kit or method, you need to know the number of species detected and the sensitivity of the method. Applying these two criteria will show which of the various methods or kits is the best choice for you.

The term “mycoplasma contamination” can be misleading because it’s not caused by a single entity. There are many different species that can cause mycoplasma-type contamination, including related species such as Acholeplasma laidlawii.

BC: What is the best way to eliminate mycoplasma contamination? When should you just discard the culture?

de Mars: Mycoplasma contamination in cell culture is often unavoidable. Since mycoplasma-infected cells are considered a source of contamination and can infect additional cultures in the lab, eliminating the contamination should occur as quickly as possible. ATCC recommends discarding all mycoplasma-contaminated cells and obtaining new starting materials whenever possible.

When cell cultures are too valuable or difficult to replace, there are chemical agents that can be used to eliminate mycoplasma infection. A variety of fluoroquinolones, tetracyclines, pleuromutilins, and macrolides have demonstrated strong antimycoplasma properties in cell culture. For example, BM-Cyclin and Plasmocin™ are often applied to mycoplasma-contaminated cells. BM-Cyclin, which is a combination of two antibiotics (tiamulin and minocycline), inhibits bacterial protein synthesis. Plasmocin, which is strongly active against mycoplasma species, contains two bactericidal components that affect protein synthesis and DNA replication, respectively.

Becker: If you cannot simply discard your cell culture and restart the experiment with a mycoplasma-free culture, we recommend our Mycoplasma-EX Kit, which contains a component that directly and selectively kills mycoplasmas very fast and efficiently (almost 100% elimination)—and does not simply inhibit mycoplasma growth as most antibiotics do. Antibiotics are admittedly cheaper, but they have the disadvantage that treatments take a long time, mycoplasma resistance may occur, and the cultured mammalian cells themselves might be altered and impaired by antibiotics.

Mortillaro: The feedback we receive from our customers is that they typically dispose of infected cultures and decontaminate the areas in which these cultures were used. Multiple-antibiotic treatments can help, but are not always successful. In some cases, it is easy for the mycoplasma contamination to re-establish. Starting with seed cultures that are not contaminated is the best way to minimize exposure. These seed cultures can be tested early on for mycoplasma.

Capes-Davis: Discarding the culture is always your first and best outcome. Cell lines can be contaminated with more than one mycoplasma species. Also, treatment is toxic and may kill or alter the behavior of the cells. Mycoplasma can become resistant to antibiotics and often recurs after treatment, which may be due to an intracellular reservoir—many species invade their host cells, e.g., Mycoplasma penetrans (given that name for a good reason!). Only treat the culture if you have no other option.

If the cells are irreplaceable, you need a regime of antibiotics that works against mycoplasma. Penicillin and streptomycin do not work—penicillin acts against the rigid bacterial wall, which is not present in mycoplasma species. There are some excellent publications on mycoplasma eradication from Cord Uphoff and his colleagues at the DSMZ, so you can look at their methods and data to help you decide. Be careful to quarantine infected cultures from your other cell lines, and continue to test afterward to pick up any recurrences.

BC: Can you suggest some best practices to avoid contamination as well as stop existing contamination from spreading?

de Mars: The best practices for avoiding contamination and preventing the spread of existing contamination include keeping a documented history of your cell line, following cell culture best practices, and routine testing. When possible, always use certified or registered cell lines that are confirmed to be negative for mycoplasma contamination. To both avoid and control the spread of mycoplasma during cell culture, we recommend the following laboratory practices:

  • Only work with one cell line at a time. Aerosols and operator error are two of the most common means of transferring mycoplasma contamination.
  • Always wear clean personal protective equipment (PPE) and adopt appropriate aseptic techniques.
  • Use a vertical laminar flow hood that is isolated from other standard cell culture facilities. Disinfect inner surfaces of the hood between each use. Clean incubators that store cultures on a regular schedule.
  • Avoid the indiscriminate use of antibiotics. This practice can permit repeated lapses in aseptic technique, will mask low-grade contaminations, and may lead to the emergence of resistant organisms.
  • Promptly autoclave infected cultures, media, and reagents to avoid contamination of clean cell lines.

Becker: We recommend good laboratory practice, i.e., working under conditions where infection of cell culture and spreading of contamination are avoided. Cell culture labs should have respective standard operating procedures (SOPs) in place and train all personnel already working in the lab or joining the group. Use reliable, validated disinfectants regularly to disinfect workbenches, laminar flows, water baths, etc. For example, 70% ethanol is, not a safe, reliable disinfectant. Our PromoCidal or Spore-EX are highly efficient, thoroughly validated and non-irritant, non-corrosive surface disinfectants. And don’t forget that water baths and the water in CO2 incubators can harbor mycoplasmas. Use reliable water stabilizers/cleaners such as  Aquaguard to further reduce contamination risks.

Test existing cell cultures (e.g., from former lab members) as well as cell cultures coming into the lab from outside (cell banks, other research groups) for mycoplasma contamination before starting to work with them. Test cell cultures regularly for mycoplasma contamination; mycoplasma-free cultures can be contaminated at any time, for instance, by the lab staff itself or contaminated media components. If mycoplasma contamination is detected, discard the cell culture, or try to eliminate contamination (if you can not discard the contaminated cells). Try to identify and eliminate the source of mycoplasma contamination. More information on keeping your lab free of microbial contamination can be found on our website.

Mortillaro: Avoiding seed cultures that are already contaminated with mycoplasma is the first and most important step. You can also disinfect using various reagents. We recommend MycoClean™. We also offer an elegant approach to physically isolate (essentially quarantine) your cell culture plates during growth/incubation and transport from other cell dishes and the general cell culture environment that may be contaminated—FastGene Cell Culture Protective Trays.

Capes-Davis: The best way to avoid contamination is to separate cultures from one another. In the early days, mycoplasma contamination was linked to particular reagents, e.g., trypsin or serum. Today, the culprits are likely to be the other cell lines that you or your colleagues are growing in the same room. Keep separate supplies of media and other reagents for each cell line, and never handle them in the biosafety cabinet at the same time. Wipe down the work surface and run the cabinet for a little while between cell lines.

You should be aware that mycoplasmas survive in dried form very nicely—back in 2001, a lab looked at the viability of dried mycoplasma, and they showed that many common species in cell culture remained viable after 168 days. So it is important to wipe down your work surface with 70% ethanol or a similar disinfectant and continue your testing so you can deal with the positive cell lines as they arise.

BC: How often should testing be done? Are there specific times/occasions it should be done?

de Mars: There is no limit to the number of times one should perform mycoplasma testing, but it is preferable to screen cultures on a regular schedule. Routine testing helps ensure that the experiments performed in your lab are verifiable and reproducible. According to best practices, there are specific occasions when testing should occur. Mycoplasma testing should be performed upon the receipt of an initial vial of cells, particularly if it is from an unreliable source; this should occur before a master cell bank is generated. Mycoplasma testing should then be repeated when a master cell bank is expanded to a working cell bank. If cells are isolated from primary tissue, it may be necessary to use antibiotics in the primary culture. Antibiotics should be removed as soon as possible, and the culture should be tested for mycoplasma after at least two passages in the absence of antibiotics.

Becker: Test cell cultures before starting your experiments. We recommend testing of cell cultures on a monthly basis as well as before cryopreservation. Test each incoming cell culture immediately.

Mortillaro: Guidelines should be dictated by an individual lab’s requirements and related to past contamination history as well as the number of new seed stocks and personnel being added. Usually we see our customers test all cultures anywhere from once a week to once a year. The worst habit is to test stocks after they exhibit signs of contamination, because at that point experimental results can be questioned and cross-contamination issues become more complicated.

Capes-Davis: Test regularly rather than stressing about exactly how often it should be done. If you test for mycoplasma regularly, you will remove the highest-risk samples and then you are just ensuring that no new contaminants arrive to disturb the status quo. Always test cell lines when they arrive in the lab, and make sure you test before you publish so you can report what you have done and confirm that cells are mycoplasma-free.

Additional resources

Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.

Effect of mycoplasma infection on pyruvate dehydrogenase complex activity of normal and pyruvate dehydrogenase complex-deficient fibroblasts.

Interaction of mycoplasma with host cells.

Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive.

Treatment of mycoplasma contamination in cell cultures with Plasmocin