by Jeffrey M. Perkel
It’s 5pm: Do you know how clean your eukaryotic cell cultures are? They may not be as pristine as you think.
Your culture dishes could be infected with Mycoplasma, tiny bacteria that thrive in cell culture environments. Published rates vary—the brochure for R&D Systems’ MycoProbe™ Mycoplasma Detection Kit cites a frequency of Mycoplasma contamination ranging from 4% to 92%—but according to Michael Anderson, manager of new technologies at R&D Systems, the anecdotal rate is more like 10% to 15%.
To be clear, there is no one Mycoplasma; the name actually refers to a family of related bacterial species with dozens of members. The vast majority of cases, though, can be traced to just eight or so different species: M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium and Acholeplasma laidlawii.
The impact of Mycoplasma detection can be profound. Like microbial ninjas, these cellular stealth warriors are invisible, resistant to most common antibiotics and produce no telltale changes in growth media. Yet they may be sabotaging your research, quietly changing growth characteristics, viability or rates of DNA and protein synthesis in ways that have nothing to do with your experimental treatments.
“You are manipulating the cells, but … is your manipulation causing the differences, or is it Mycoplasma?” says Mimi Ly, product manager for mutagenesis, cloning and protein expression at Agilent Technologies, Stratagene Products Division.
As a result, for those who are serious about cell culture, Mycoplasma detection and Mycoplasma detection kits are serious business. Fortunately, a wide range of products for the detection and removal of Mycoplasma contamination is available. Although the Agar and Broth assay may be the gold standard for detection, PCR-based assays continue to gain in popularity because of their relative speed and scalability. ELISA and enzyme-based approaches also exist. Most researchers trash contaminated cultures, but if necessity drives you to find a method for removal of that Mycoplasma contamination, there are products specific to that aim, as well. Below we review the relative merits of each approach and offer recommendations for selecting the Mycoplasma detection kit that will work best for your research.
The Gold Standard
The U.S. Food and Drug Administration specifies two tests for Mycoplasma contamination in cell lines being used for vaccine production.[1] In the Agar and Broth Media Procedure, the bacterial growth medium is inoculated with a test sample. Samples are periodically removed, plated on agar and incubated for two weeks. The presence of fried-egg-shaped colonies indicates contamination, says Don Finley, market segment manager for Mycoplasma detection products at Sigma-Aldrich. This assay, says Finley, is “the gold standard” for Mycoplasma detection and is both sensitive and specific (that is, it produces very few false positives). But, Finley adds, it’s exceptionally slow – the complete test requires at least 28 days – and not all Mycoplasma strains are detected (in particular, M. hyorhinis).
The other FDA-approved assay, the “DNA Fluorescence Method,” involves incubating an indicator cell line called Vero with test samples, waiting three to five days, and staining with Hoechst DNA stain. Contaminated cultures produce nucleus-associated punctate fluorescence. According to Finley, this assay is relatively fast compared with the Agar and Broth Media Procedure but is also somewhat less sensitive. And as fast as it is, it’s still far slower than other assay methods, like PCR.
PCR-based Assays
The most common assays for academic labs rely on PCR. These assays have the advantage of speed—they can be completed in just a few hours—as well as high sensitivity and throughput. According to Finley, these kits are popular for their speed, but convenience is also a factor; after all, “most labs have a PCR machine of some sort.”
Generally speaking, these tests detect sections of the 16S Mycoplasma rRNA gene using primers at loci that are well conserved across the class Mollicutes (which includes Mycoplasma) but not well conserved in less-related bacteria. (At least one test, Agilent’s Mycoplasma Plus PCR assay, is based on a different bacterial gene.) As a result, these tests tend to be sensitive to a wide range of species. According to Christian Weilke, manager for new product marketing at Roche Applied Science, the biotech industry-focused MycoTOOL PCR test is “thoroughly validated” for 11 species of interest to U.S. and European regulatory agencies; this test can detect “over 120 further Mycoplasma species,” with good specificity against non-Mollicutes bacteria, as well. The American Type Culture Collection (ATCC)’s new Universal Mycoplasma Detection Kit can detect more than 60 strains, and Sigma-Aldrich’s new qPCR-based test can detect 33.
Sensitivity also can be impressively high for PCR assays. According to Weilke, the MycoTOOL assay has a detection limit of “less than one CFU [colony-forming unit] per milliliter.” The Universal Mycoplasma Detection Kit boasts a sensitivity of “2.5 to 25 fg of target DNA, corresponding to 4 to 40 genome copies per assay.” On the other hand, says Finley, PCR-based assays are prone to false positives. “None of the methods is perfect,” he says, “but if you use a combination of two, you should be pretty good.”
Both endpoint and quantitative RT-PCR-based tests are available, the latter enabling real-time reaction monitoring. In addition to those mentioned above, kits include the LookOut® Mycoplasma PCR detection kit (Sigma-Aldrich); the Venorgem Mycoplasma Detection Kit, PCR-based (Sigma-Aldrich); the Mycoplasma Plus PCR Primer Set (Agilent Technologies); and the MycoSensor PCR assay kit (Agilent Technologies). The qPCR assays include the LookOut Mycoplasma qPCR Detection Kit (Sigma-Aldrich) and the MycoSensor qPCR kit (Agilent Technologies).
Assay Alternatives
Other assays for Mycoplasma detection include both ELISA- (Roche's Mycoplasma Detection Kit) and enzyme activity-based (Lonza's MycoAlert® Mycoplasma Detection Kit) tests.
R&D Systems’ MycoProbe Mycoplasma Detection Kit is essentially a hybrid; it’s a DNA-based ELISA assay. Cell lysates are incubated with two probes, both of which target the bacterial 16S rRNA gene. The probes are a biotin-labeled capture probe and a digoxigenin-labeled detection probe. The DNA complexes are then captured on a streptavidin-coated microtiter plate, incubated with an anti-digoxigenin alkaline phosphatase and developed.
According to Anderson, who helped develop the test, the goal was to create an ELISA-based assay with PCR sensitivity for researchers who might be uncomfortable with (or unable to run) PCR. “We took the approach of a basic ELISA user, a straightforward plate-based assay that people were used to,” he says.
InvivoGen’s PlasmoTest™ offers yet another option. This live cell assay features a cell line expressing the innate immune receptor, Toll-like receptor-2 (TLR2). When TLR2 binds Mycoplasma, it kicks off a signaling cascade that results in NF-kappaB activation. That, in turn, activates an inducible reporter gene, which expresses a secreted form of alkaline phosphatase. Finally, that enzyme reacts with a specially formulated growth medium, turning it from pink to blue. According to Scott Vara, sales and support manager at InvivoGen, the impetus for developing this assay was that InvivoGen’s scientists wanted something that anyone could use, even if they lacked a PCR machine. “We wanted something colorimetric that people could see with their naked eye,” he says.
Testing Regimen
Each lab is different, and there’s no easy fix to controlling Mycoplasma contamination. That said, routine testing is critical, says Finley, if for no other reason than it’s easier to clean infected cultures that are caught early. Some labs test monthly, others more frequently. According to Robin Rothrock, market development director for cell biology and biostandards at ATCC, “Our market research reveals that most scientists test lines on a quarterly basis.” Each lab must decide on the best regimen for its staff and their needs. But one time when it is absolutely essential to test is when receiving new lines from an outside source. “Mycoplasma can spread from cell to cell in the incubator,” says Vara, “so any contaminated line can contaminate other cells.”
Elimination: To Clean or Not to Clean
Generally speaking, if you find a contaminated culture, the smart move is to throw it out. “Our recommendation is, if you discover that your cultures are contaminated, that you autoclave [them], clean your hood, clean your incubator and start over,” says Rothrock. Sometimes, though, you have cells that are just too precious—you may not have a frozen stock, for instance, or that stock also may be contaminated.
In that case, a few antibiotic treatment options are available, including Plasmocin™ and Plasmocure™ from InvivoGen and the LookOut Mycoplasma Elimination Kit from Sigma-Aldrich. Each of these products uses a mixture of different antibiotics to clear the infection. Plasmocin, for instance, contains agents that attack both the bacterial protein synthesis and DNA synthesis machinery. (Other products, including InvivoGen’s Primocin™ and Normocin™ and Sigma-Aldrich’s LookOut DNA Erase, help to maintain a Mycoplasma-free work environment after you’ve eliminated the contamination.)
According to their manufacturers, these formulations generally can clear infections quickly, within weeks. But they definitely aren’t foolproof, a reflection of the fact that every strain, and every host, is different. If you choose to try to salvage your culture, be sensible, says Rothrock, and make sure the contamination doesn’t spread in the meantime: “[Users will] need to quarantine it from the rest of their lab and just be extraordinarily mindful of their cell culture technique. That’s tough to do.”
Reference:
[1] FDA, “Draft Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Starting Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases,” 2006.
The image at the top of the page was taken from Lonza's webpage for Mycoplasma Detection and Removal