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Mycoplasma contamination in cell cultures is shockingly common. It is estimated that 15-35% of continuous human or animal cell lines are contaminated. These bacteria are difficult to detect and can easily spread from one culture to another once inside the lab. Once mycoplasma has infected your cell cultures, the reliability and reproducibility of your research studies can be a serious challenge if left undetected. This shows the importance of routine testing to make sure you are working with uncontaminated cultures.
This article provides an introduction to mycoplasma, methods for detecting cell culture contamination, and the importance of testing your cell cultures.
Mycoplasma are one of the most common, yet elusive, contaminants of mammalian cell cultures. As the smallest known free-living organism, mycoplasma are a pervasive, parasitic species of highly-infectious bacteria. Mycoplasma species belong to the Mollicutes class, which includes Acholeplasma and Ureaplasma amongst others. Mollicutes lack a cell wall and thus can adopt various different shapes and because of its size, mycoplasma can’t be seen under standard microscopes.
Mycoplasma was first detected in cell culture in 1956, so why is it still such an issue? Detection is one challenge, but it is also more difficult to prevent and eliminate than other types of contaminants. It has a pliable membrane that makes it resistant to pressure, temperature, osmolality and dehydration. There are over 180 different species of mycoplasma with over 20 found as contaminants in cell culture. Mycoplasma can infect all types of eukarayotic cells. They have limited metabolic capabilities and they adapt and thrive in many cell culture environments by overtaking the cells, thriving in culture and going completely undetected.
Images represent examples of the most common mycoplasma species that are capable of infecting all cell culture labs. Regulatory guidance recommends that all cells be tested for the presence of mycoplasma on a routine basis.
There are six species that account for 95% of all detected mycoplasma contaminations in continuous cell line cultures: M. orale, M. arginini, M. fermentans, M. hyorhinis, M. hominis, and A. laidlawii1. The most common sources of mycoplasma originate from human, bovine, and swine with cross-contamination discovered by sharing cells in a lab, using contaminated animal sera, not wearing proper PPE, talking or sneezing, or handling more than one cell type at a time.
It has been found that 81% of lab techs are mycoplasma carriers, 38% of lab techs spread mycoplasma by sneezing, and 6% spread mycoplasma by merely talking. This highlights the importance of always wearing proper PPE, cleaning the lab regularly, and using high-quality cell culture media and reagents from reputable manufacturers that also test their products for mycoplasma.
Mycoplasma grow slowly compared to other bacteria and do not kill mammalian cells outright; instead, they use their subtle morphology to progressively cause increasingly adverse effects on cellular functions. Other ways mycoplasma impact cultures include:
Unfortunately, it can be challenging to reliably detect mycoplasma contamination. However, the earlier mycoplasma contamination is discovered, the simpler it is to treat. One reason is that mycoplasma contamination does not typically trigger the culture changes that are common with other types of bacterial or fungal contamination.
One of the only ways you can detect for contamination is to test your cell lines. There are several different methods to identify mycoplasma including direct staining, ELISA, DNA fluorochrome staining, biochemical methods, and PCR (chart below). PCR-based methods have been widely used as a powerful, highly sensitive, and a fast way for identifying mycoplasma contamination in cultures. While there are many PCR detection kits commercially available, many are not able to detect all species of Mycoplasma.
Gel electrophoresis results obtained following PCR reaction preparation and amplification. Eight total reactions, including six samples, one negative control, and one positive control were tested, each of which contain the internal control to rule out PCR inhibition (i.e. false negatives). As shown, Sample 4 produced a mycoplasma-positive band at 270bp in addition to the internal control band at 357bp, while Samples 1, 2, 3, 5, and 6 produced only internal control bands and are thus negative for mycoplasma. Image courtesy of WiCell Research Institute.
The earlier mycoplasma contamination is discovered, the simpler it is to treat. Captivate Bio offers an easy-to-use EZ-PCR™ Mycoplasma Detection Kit which is designed for the highly-sensitive routine screening and detection of over 90 different mycoplasma and other closely related species.
Routine testing for mycoplasma is an important quality control. Regulatory guidance requires that all products derived from mammalian cell culture be tested for the presence of mycoplasma, and many high-impact scientific journals now require that cell lines are verified to be mycoplasma-free.
To avoid contamination, testing should be carried out minimally every 2 weeks to 3 months, especially when shared incubator spaces are used, as well as prior to the incorporation of new cultures from outside sources.
Source: This article was originally written and posted on Cell Culture Dish. It has since been updated with new content. Main image source represents an example of Mycoplasma genitalium in Figure 1 of Daubenspeck JM, et al. 2020. Front. Microbiol. 11:585524.
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