Mycoplasma: Tools for Detection, Rescue and Prevention

Mycoplasma’s behavior has proven subversive too. They usually don’t overgrow your cells—because of their small size, it’s possible that you could have an infection without knowing it, with infections not being visible to the eye. Nonetheless, some claim that if you know the normal, healthy behavior of your cells, you will be able to tell if they are infected with mycoplasma. As Ren-He Xu, senior scientist at the WiCell Research Institute says, “it is easy to tell by the appearance of the cell culture and difficulty to passage the cells if [they are] contaminated.”

The anatomy of mycoplasma is also confounding. The traditional antibiotics used in cell culture do not work against mycoplasma, because these drugs typically attack the integrity of cell walls, which mycoplasma lack. This is particularly alarming given the high incidence of mycoplasma infections—recently estimated at up to 30% of lab cultures, with some citing even higher numbers. Says Hans Drexler, head of the Human and Animal Cell Line Department at the German Collection of Microorganisms and Cell Cultures, “We are experts on mycoplasma, not because we like them so much, but because about 25-30% of the cultures submitted to our cell lines bank were infected.” Given the potential experimental consequences of mycoplasma infection, these numbers are sobering when it comes to trusting the results of work done in culture. Mycoplasma can have dire effects on cell metabolism, potentially skewing data without the investigators’ knowledge. So what’s a scientist to do?

Xu takes clean techniques to an extreme. His lab doesn’t use antibiotics to avoid possible interference in his human embryonic stem cell cultures, so “precautions against any contamination including mycoplasma all rely on a super-clean cell culture facility and well-trained experimenters.” Xu particularly recommends filtering the lab air and all media, UV radiation of hoods, cleaning gloves with alcohol before use and the hoods after use, and “routine cleaning of cell incubators and the use of distilled water for [humidification] in the incubators.” But most importantly, says Xu, he relies on “careful handling of the cells.”

However frequently you decide to test, you have a number of choices for the testing method, whether PCR, biochemical assays, ELISA, or imunofluorescence. Xu says that methods based on “RT-PCR can often detect non-obvious mycoplasma contamination with clean controls.” Most PCR-based testing kits use primers to amplify the mycoplasmal 16S and/or 23S ribosomal RNA genes—a band on a gel run with the PCR product indicates a positive result. One example is Stratagene’s new MycoSensor™ QPCR Assay Kit, which detects mycoplasma using real-time quantitative PCR in about two hours. It’s designed with internal controls to reduce false positives and false negatives. Other PCR-based kits are offered by Sigma-Aldrich (the VenorGeM® Mycoplasma PCR Detection Kit) and Talron Biotech (the i-Myco Mycoplasma Detection Kit).

For those wanting a simple and quick test, try a biochemical assay that relies on the activity of mycoplasmal enzymes to react with a substrate, catalyzing the conversion of ADP to ATP. Measurement of ATP before and after the reaction indicates whether mycoplasma is present. Cambrex currently offers its MycoAlert™ Mycoplasma Detection Assay, purported to take less than 20 minutes and to be reliable even with low levels of contamination.

If you are more comfortable using oligonucleotide hybridization techniques, you might try R&D Systems’ MycoProbe™ Mycoplasma Detection Kit. This system targets the eight most common mycoplasmas with labeled oligonucleotide probes that hybridize to the 16S ribosomal RNA. A probe labeled with alkaline phosphatase, along with a colored substrate solution, indicates the presence of mycoplasma. R&D Systems claims that this test takes approximately 4.5 hours.

Staining with fluorescent antibodies is the method of choice for MP Biomedicals (formerly ICN Biomedicals) in their ImmuMark MycoTest. MP Biomedicals claims that the monoclonal antibody used in their test, CCM-2, is specific for the mycoplasma species that account for over 96% of culture contamination. A fluorescently labeled secondary antibody is included in the kit for detection.

No, no! you say? What if you don’t have any uninfected cells in the freezer, or starting over is simply unfeasible for other reasons? Take heart, there are last-ditch options—namely, antibiotics to which even mycoplasma are susceptible, although treatment with any antibiotic incurs the risks of complications such as antibiotic resistance, cellular toxicity, or alteration of cellular phenotypes. Plasmocin, offered by Cayla and InvivoGen, is a common recourse for mycoplasma infection, and works by interfering with mycoplasma protein synthesis and DNA replication. Other antibiotics used against mycoplasma include ciprofloxacin, BM-Cyclin, and Mycoplasma Removal Agent (made by MP Biomedicals). Another option for treatment is the Mynox® Mycoplasma Elimination Reagent from Sigma-Aldrich, a non-antibiotic alternative that Sigma claims is toxic for mycoplasma but safe for cells.