The smallest creatures on earth can cause huge problems in your cell culture. And many scientists don’t even realize that there is an invader hiding among their HEK 293, HeLa or self-made cell lines. “That’s the difficult thing about mycoplasma, you can’t see them, even with a microscope,” explains Dr. Tobi Limke, Scientific Application Manager at PromoCell. “You have to be looking for that specific contaminant to find it.” Statistics from the resource institute German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung für Mikroorganismen und Zellkulturen; DSMZ) show that every fourth cell culture is affected.
Mycoplasma are a group of bacteria without a rigid cell wall from the class of Mollicutes. These microbes are regarded as the smallest self-replicating organisms. This capability makes the bacterium unique, and sets mycoplasma apart from the even smaller group of viruses. Most of the time, tiny mycoplasma remain in the shadows. “The most significant hint pointing to their presence is the slower growth of your cells,” says Limke. In most cases, inexperienced researchers in particular won’t pick up on that.
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“But your results are affected. They are not as reproducible and comparable as results from the ’clean’ cell lines of your colleagues,” emphasizes Limke. Cell mass from the bacteria can constitute up to a third of total DNA, and one half of the protein within the culture.
Mycoplasma Contamination is a Threat to Your Scientific Data
Since it is hard to pinpoint exactly when mycoplasma infiltrated your plates and flasks, they threaten a large amount of your scientific data. A major problem is that “many labs are not regularly testing for mycoplasma,” says Limke. This results in a lot of time and money being wasted in advance, and even more resources are needed later, as scientists try to fight the bacteria.
According to Limke, it could take months for a lab to get back on track after a mycoplasma contamination. Such an event is often the starting point for a regular testing schedule, however. More and more scientists are realizing how crucial mycoplasma-tested cell lines are for valid results.
High-Impact Publications: Clean Cell Lines Only
The entire scientific community is rethinking mycoplasma contaminations. High-impact factor journals either strongly recommend, or even require, regular cell testing. “For example, if you are planning to publish your results in Nature you have to prove the cells you used are free of mycoplasma,” advises Limke. But prevention starts even earlier. Limke says it’s better not to wait for a contamination, or your first publication in a respected journal, before establishing quality-control measures. “My first advice to a young scientist is to be on guard for any signs of bacterial and fungal impurities, and especially mycoplasma contaminations.” An important aspect for every lab is the prevention strategy at hoods, water baths, and incubators. Many people use and share that equipment, and there’s obviously a higher risk of cross-contamination.
Awareness is increasing, but particularly in academia, some labs still underestimate the threat of mycoplasma to their scientific work. Limke summarizes: “They are playing a risky game. I’ve seen researchers in industry and academia dealing with a mycoplasma contamination, and it’s always very painful.”
Within cell culture, mycoplasma have found their biological niche. “The conditions just fit perfectly,” says Limke. When they have infiltrated one lab, bacteria will move on to the next cell culture lab in the building. So if you are facing contamination, you had better act fast and resolutely.
How to Deal With Mycoplasma Contamination
Prevention is the easiest way to keep your cell lines clean. If there is a suspicion, “you should use at least two methods for diagnosis. The gold standard approach is the agar plate assay; however, this approach takes at least four weeks for results – mycoplasma grow very slowly,” says Limke. “In addition to this method, use a rapid and sensitive PCR-Assay to get accurate results more quickly.” PromoCell’s PCR-kits contain primers specific for the 16S-rRNA-sequences of well-known cell culture contaminants such as Mycoplasma orale, M. hyorhinis and M. arginini, for example.
If you were not able to prevent mycoplasma from entering your cell culture, there are still ways to handle the problem. “This is incredibly important if you are working with cell lines that were developed in your lab and can’t be repurchased,” emphasizes Limke. In such a scenario, scientists will have to eliminate mycoplasma and “cure” their cells.
Secret weapons used by researchers include special antibiotics, because mycoplasma are resistant to standard antibiotics such as penicillin. PromoCell’s BIOMYC-solutions contain antibiotics that are suitable for dealing with mycoplasma. The reagents inhibit the growth of the contaminants, but more extensive treatment may be needed if you want to achieve a long-term effect. “PromoCell’s Mycoplasma-EX is a great option to explore, as it can kill mycoplasma directly and give you a chance to save your cell line,” says Limke.
With vigilance and quality control, you can keep mycoplasma out of your cell cultures. And prevention is the easiest way to stop the smallest creatures on earth from causing big trouble.