Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Your cells aren’t acting themselves—maybe they’re sluggish, aren’t doing what you expect them to or just plain don’t look right. But the culture medium is the right color and isn’t turbid, and you can detect no signs of contamination under the microscope. Are you crazy? Or is something lurking in your incubator that you can’t see—something like Mycoplasma?
Mycoplasma are diminutive bacteria that lack a cell wall, grow very slowly and can achieve densities of 107–108 organisms/ml of culture without being observable. Of the more than 190 species known to cause disease in plants and animals, eight are responsible for 95% of cell-culture contamination, ATCC microbiologist Cara Wilder said in a recent webinar [1]. Between 15% and 35% of continuous cell cultures are infected, often without anyone being the wiser.
Are your cultures among their numbers? Here's how to find out, and what you can do about contamination when it’s found.
Culture is golden
The gold standard for detecting Mycoplasma is a three-pronged approach sanctioned by the U.S. Food and Drug Administration (FDA), United States Pharmacopeia (USP) and their international counterparts. “This includes culture of the Mycoplasma in both agar and broth, as well as culture of the test sample in an indicator cell system,” says Marian McKee, principle scientist at BioReliance, part of SAFC, the custom manufacturing and services business unit of Sigma-Aldrich, which does testing for the biopharmacological and biologicals industries.
Some Mycoplasma grow better in soft agar, some grow better in a liquid suspension and some are obligatory parasites, requiring cells to grow in. “I’m not aware of Mycoplasma that won’t grow in one of the three,” McKee says. She adds, “The formulations have been around for a long time, chosen and designed to theoretically pick up all possible Mycoplasma. So you’re really getting a near 360° view of the sample.”
Yet these methods take time, and it may be a month or more before a culture can be given the all-clear.
Uncultured
There are faster ways to detect (at least signs) that Mycoplasma inhabit your culture. Perhaps the fastest, but least sensitive, method is to use a DNA-binding dye like Hoechst or DAPI, which cause nuclei to fluoresce under UV light. Mycoplasma should be easily differentiable from mitochondria simply by their brightness, but cell debris and detritus may make them more difficult to distinguish, so make sure you have the right controls, notes Donald Finley, market segment manager for research cell culture at Sigma-Aldrich.
The most sensitive nonculture tests are PCR-based assays and other nucleic acid tests (NATs), such as ELISA-hybridization assays, offered by a large variety of vendors. These generally target sequences coding for the 16S ribosomal RNA, which is highly conserved across the eight most common species, including M. hyorhinis, an obligate parasite not detectable by agar or broth culture methods. Yet nucleic acid methods are not designed to recognize some minority Mycoplasma species, and if you are unlucky enough to have one of these in your culture, the NAT will yield a false negative. Similarly, McKee warns that when using a NAT, “you may detect the presence of sequences of Mycoplasma [that are] residual sequences from a long-cleared or -removed contamination.”
Several enzyme-based tests on the market can recognize most if not all species. Lonza’s MycoAlert™ Mycoplasma Detection Kit, for example, uses a chemiluminescent reporter to detect a Mycoplasma-specific enzyme. InvivoGen’s PlasmoTest™ uses a reporter cell line engineered to express a natural receptor for Mycoplasma. “In the presence of Mycoplasma in a sample, the cells will secrete alkaline phosphatase,” explains sales and technical support manager Scott Vara. “We have detection media that will turn blue in the presence of alkaline phosphatase, and you get a nice colorimetric, visual change.” The advantage of such tests, Vara points out, is that they can be performed easily by anyone versed in cell culture.
Although several of these methods will pick up other organisms, as well, that shouldn’t be a problem in practice. In preparation for the tests, cultures often are grown for several days to near confluency in the absence of antibiotics, Vara says. A (non-Mycoplasma) bacterial or fungal contamination should be readily apparent under such conditions.
Trash talk
When Mycoplasma is detected, it’s usually best to bleach the culture and toss it in the trash. But that’s not always feasible. If you’re not in a regulated environment, with some time, patience and luck you may be able to save your culture.
Because Mycoplasma don’t have a cell wall and grow so slowly, typical antibacterial treatments won’t work, and “so you wind up using antibiotics that affect both the mammalian cells and the microbial cells to some degree,” Finley observes.
There are several different antibiotic options on the market for treating Mycoplasma, including macrolides that target protein synthesis and quinolones that target DNA replication. Vara recommends using a combination of two different antibiotics, like those found in InivoGen’s Plasmocin™, to lessen the chance for developing resistance.
Wilder cautions that antibiotics boast only about 66% to 85% efficiencies, and that treated cells may be altered in their genetic, antigenic and metabolic properties in the process.
Another option is a detergent like Minerva’s Mynox®, whose “activity is based on a biophysical mechanism, thus there is no development of resistant strains,” according to the manufacturer’s web site. “It’s pretty good. It works 80[% to] 90% of the time, but not 100% of the time. I don’t think anything does, that I know of,” remarks Finley, whose portfolio includes both antibiotics and the Mynox®-based LookOut® Mycoplasma Elimination Kit.
Of course, the best defense is a good offense, as they say, so your best bet is avoiding contamination in the first place. Keeping cell lines quarantined until they are verified safe, and testing periodically for contamination, especially if you’re gearing up for a major project or publication, is a good place to start. So is enforcing good aseptic technique; ensuring reagents are sterile; disinfecting incubators, hoods and surfaces; and allowing cell-line acquisitions only from reliable sources. Such commonsense practices can take you a long way toward a happier, healthier culture environment. But just in case, it’s good to know there are ways to detect contamination and perhaps to do something about it.
Reference
[1] Wilder, C., “Mycoplasma Detection – Protect your continuous cell cultures,” 2013. Available at http://www.atcc.org/Documents/Learning_Center/ATCC_Webinars_Recorded.aspx.
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