Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Since the dawn of cell culture, scientists have been contending with the plague of mycoplasma contamination. Mycoplasma (class Mollicutes) are primitive bacteria so tiny that even sterile filtration cannot remove them. They are invisible under a light microscope. Unlike most other bacteria, mycoplasma lack a cell wall, so antibiotics commonly used in cell culture—such as penicillin and streptomycin, which act by disrupting cell walls—have no effect. Other antibiotics, such as neomycin, tetracycline and gentamycin, can halt mycoplasma growth, but this effect is “restricted to specific mycoplasma species and only suppresses mycoplasma growth,” says Andrea Toell, senior product manager in pharma and biotech for Lonza. “Once treatment is stopped, contamination will recur.”
Given the widespread yet unpredictable effects of mycoplasma on cells in culture, researchers have every reason to avoid contamination [1]. Mycoplasma can skew your data before you even know it’s there, so it is smart to test cultures routinely. But what happens if you get a positive result? Here are some tips from those in the know.
Toss or treat?
Many who have wrangled with mycoplasma advise researchers to toss infected cultures in the biohazard bin immediately (perhaps autoclaving them first). But what if the cultures are precious? Are there alternatives?
Yes and no. “The first choice is always to go back to a fresh, uncontaminated stock,” says Don Finley, market segment manager for research cell culture at Sigma-Aldrich. Yes, it will cost extra time to get the new cultures up and running, but it’s worth it when you consider the insidious effects of a mycoplasma infection. And who knows how that could affect your data.
Sometimes, though, there is no clean stock in the freezer. And there are other reasons to treat rather than toss, too. For example, “there might be situations where starting from scratch would cause a big issue for the progress of your research, such as if the cell type is hard to access, hard to isolate or grows very slowly,” says Toell. Precious or rare cultures, such as human primary cells excised from tumors, also may warrant treatment. “First and foremost is the value of the cell line,” says Joanna Rowe, laboratory manager at AbD Serotec (a Bio-Rad company). “If the cell line is unique or perhaps produces a protein that is unique, such as a hybridoma producing a monoclonal antibody, then there is every [reason] to try to eliminate mycoplasma.”
Antibiotic treatments
Despite the impotence of many standard antibiotics, some antibiotics do show substantial mycoplasma-fighting abilities, such as those from the tetracycline, macrolide and quinolone families. Tetracyclines and macrolides block mycoplasma’s protein synthesis by interfering with ribosome translation, and quinolones inhibit replication of mycoplasmic DNA. Individual antibiotics available today include: AbD Serotec’s Mycoplasma Removal Agent; AppliChem’s Myco-1, Myco-2 and Myco-3; and PromoKine’s BIOMYC-1, BIOMYC-2 and BIOMYC-3.
But no single antibiotic can kill 100% of the mycoplasma in a culture. One popular remedy is to treat with two antibiotics (usually two that differ in their mechanisms of action). This gives the bacteria very little chance to bounce back, if any manage to survive the first antibiotic. Examples of double-antibiotic remedies include: Genlantis’ MycoGONE Mycoplasma Antibiotics Cocktail; InvivoGen’s Plasmocin™ and Plasmocure™; and Roche’s BM-Cyclin.
Not all antibiotics have the same effect on contaminated cultures. Consider, for example, the two remedies offered by InvivoGen. The company recommends that researchers try Plasmocin first, according to Rachael Gray, research and development supervisor at InvivoGen, as it eliminates up to 95% of mycoplasma commonly found in cell culture. On the other hand, “Plasmocure is a little harsher on the cells, but it will cure everything else,” she says. As Plasmocure interrupts mitochondrial respiration, it tends to slow cellular growth, Gray says. “It can take a little while for your cells to bounce back. But typically, antibiotics shouldn’t have a [lasting] negative effect on your cells.”
Multicompound treatments
One drawback to using two antibiotics to treat mycoplasma is that—depending on the product—it may take up to a month of cycling from one antibiotic to the other with successive cell passages to complete the treatment. A potentially faster alternative is combining an antibiotic with an undisclosed reagent that kills mycoplasma cells directly (usually by disrupting the integrity of the bacterial membrane or interfering with bacterial metabolism). These are two-step treatments: Step one is the reagent, which kills most of the mycoplasma in a few hours or less, followed by the antibiotic, which kills the remaining mycoplasma (this usually takes up to two weeks).
Combination remedies include AppliChem’s Myco-4, Minerva Biolabs’ Mynox® Gold, PromoKine’s Mycoplasma-EX and Sigma-Aldrich’s LookOut® Mycoplasma Elimination Kit. Another example is Lonza’s MycoZap™ Mycoplasma Elimination Reagent, which “can eliminate detectable mycoplasma contamination in as few as four days by using a combination of antibiotic and antimetabolic agents,” says Toell.
A note about antibiotics
Mixed observations are reported in the literature regarding the safety of antibiotics for cultured cells, though those may have pertained more to earlier generations of the drugs [2]. It is true, though, that cell health can sometimes suffer while you’re trying to rid cultures of mycoplasma. “The cells may not survive, or they may change sufficiently that they no longer express the same proteins they did previously,” says Rowe. However, other research has shown no lasting changes in cells during treatment with antibiotics that were effective at eliminating mycoplasma infections [3, 4].
One thing is certain, however: The detrimental effects of mycoplasma are clear, so if you can’t toss your cultures, you must treat them. Remember to test often. And brush up on your sterile technique.
References
[1] Drexler, HG, Uphill, CC, “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention,” Cytotechnology, 39(2):75-90, 2002. [PubMed: 19003295]
[2] Kuhlmann, I, “The prophylactic use of antibiotics in cell culture,” Cytotechnology, 19:95-105, 1995. [PubMed: 22359010]
[3] Uphoff, CC, Drexler, HG, “Elimination of mycoplasmas from infected cell lines using antibiotics,” Methods Mol Biol, 731:105-114, 2011. [PubMed: 21516401]
[4] Romorini L, et al., “Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth,” PLOS ONE, 8(7):e70267, 2013. [PubMed: 23936178]