Keeping Tabs on Cultured Cells

THE CULTURE WITHIN:Photo courtesy of Drs. C.C. Uphoff and H.G. Drexler, DSMZ-German Collection of Microorganism and Cell Cultures, Braunschweig, GermanyHeLa cell line infected with Mycoplasma hyorhinis. Scanning electron micrograph of critical point-dried cell culture grown on cover slips. Note the loop- and rod-like mycoplasmas attached to the host cell membrane. Smaller web-like structures on the cell surface represent microvilli of the cell. Original magnification 3,000×.Mycoplasmas have

Mar 14, 2005
Lissa Harris(

Photo courtesy of Drs. C.C. Uphoff and H.G. Drexler, DSMZ-German Collection of Microorganism and Cell Cultures, Braunschweig, Germany

HeLa cell line infected with Mycoplasma hyorhinis. Scanning electron micrograph of critical point-dried cell culture grown on cover slips. Note the loop- and rod-like mycoplasmas attached to the host cell membrane. Smaller web-like structures on the cell surface represent microvilli of the cell. Original magnification 3,000×.

Mycoplasmas have been called the crabgrass of cell cultures.1 Hard to detect, and even harder to eliminate, these simple intracellular bacteria frequently contaminate cell culture lines, wreaking havoc on cell function and metabolism without raising obvious warning flags such as changes in turbidity or pH.

Labs often don't test their cell lines adequately for mycoplasma, in part because the methods for detecting contamination historically have been time-consuming and difficult to interpret, and sending cells to a specialized testing service can be prohibitively expensive. But in recent years, kits for easy, rapid mycoplasma detection have become available and dramatically reduce the time and technical expertise required to perform the screening.

Members of the class Mollicutes, mycoplasmas are simple, tiny prokaryotes that lack the cell wall found in other bacteria. More than 90 species of mycoplasma have been identified, many of which cause disease in humans and livestock, and more than 20 of which have been found in contaminated cell lines. But the vast bulk of mycoplasma contamination is caused by a few usual suspects: Acholeplasma laidlawii, Mycoplasma arginini, M. fermentans, M. hominis, M. hyorhinis, and M. orale account for 90–95% of infections.

Mycoplasmas generally attach themselves to cell membranes but can sometimes penetrate into the cell's interior, where they are even more difficult to eliminate. Once present in a cell line, mycoplasma contamination can affect everything from metabolism to morphology to protein synthesis.

"You can't count on your results being valid if you have an organism in your cells that's affecting the way they grow and metabolize," says Lisa Masterson, a product manager at Sigma Aldrich of St. Louis, which carries Hoechst staining and PCR kits for mycoplasma detection ($478 for 100 tests and $142 for 25 tests, respectively).

Testing is important, as the effects of mycoplasma infection can affect research parameters in subtle ways, says Leota Terri, a research specialist at the cell center services facility at the University of Pennsylvania Medical School, which provides low-cost mycoplasma screening to Penn researchers. "It is a good idea to periodically test a cell line to see if it's contaminated. If they're kind of sickly, that's a time to suspect contamination. But they don't always act sickly, and can yet be contaminated, so it's best to be certain," says Terri.

Contamination commonly stems from sloppy lab technique resulting in cross-contamination, the use of contaminated materials, or direct infection from the researcher. Estimates in the literature of how many continuously cultured cell lines are contaminated with mycoplasma vary widely, ranging from about 15% to an alarming 80%.2

"If you've had cultures for years, it would behoove any lab once or twice a year, or when you get a new line, to screen just for peace of mind," says Masterson. "There's a huge window to introduce those sorts of contaminants."



HeLa cells stained for actin (red), eukaryotic nuclei (blue), and Mycoplasma fermentans genomic DNA (green, revealed by fluorescence in situ hybridization analysis)

The most basic mycoplasma screening method is indirect culture of cells on slides, followed by imaging with a fluorescent DNA dye such as Hoechst stain. The technique requires no special equipment other than a fluorescence microscope, and several kits are available that include stain, slides, and mounting medium. But it takes several days to grow the cells on a slide, and a trained eye to distinguish the organisms. The Hoechst stain is an interchelating dye that binds to all DNA, so any stray fragments of DNA or other contaminants in the sample can be mistaken for mycoplasma infection.

"They're very, very small. They're smaller than gram-negative bacteria. It looks like a grain of pepper. You can't recognize any organelles. There's no typical morphological characteristic that lets you know that it's a mycoplasma," says Masterson. "It can be somewhat of a subjective test. We supply some positive control slides so scientists have some idea of what they're looking at."

Direct-culture testing, in which the supernatant is cultured on agar, is more conclusive. In direct culture, mycoplasmas form distinctive "fried-egg"-shaped colonies that can be readily identified. But the direct culture method requires several weeks of incubation, and the common M. hyorhinis is finicky about what it will grow on, in most cases preferring the nourishing company of your research cells to a plate of agar.

Many cell culture facilities that offer mycoplasma-testing services, including the American Type Culture Collection, still use indirect and direct-culture methods. The FDA requires direct culture for screening products that will be injected into human subjects.

"The gold standard is still the microbiological technique," says Hans Drexler, head of the department of human and animal cell cultures at the German Collection of Microorganisms and Cell Cultures in Braunschweig. He has written several papers on the topic of mycoplasma detection and elimination.

But for a lab that does its own mycoplasma testing, he says, the time involved in using the standard methods, and the subjectivity of the results yielded, may be prohibitive. Enter the latest crop of mycoplasma detection kits, which borrow from a variety of toolkits in life sciences to deliver quick and objective results.


The use of PCR to amplify and detect mycoplasmal DNA has steadily increased in popularity since the technique was invented in the late 1980s, and a variety of PCR-based kits for mycoplasma detection are now available. While sometimes yielding false-positive or false-negative results, PCR is by far the most sensitive rapid-detection technique, able to detect as little as a single mycoplasma cell in culture, and to detect cells that are dead or have been temporarily suppressed by treatment with antibiotics.

Proper experimental controls can help to ensure that problems with the PCR process itself will not yield misleading results. Using nested PCR, which relies on two sets of primers that target partially overlapping sequences, is another way to improve the specificity and sensitivity of PCR.

Drexler wholeheartedly recommends PCR as the detection method of choice for researchers untrained in the classical direct-culture technique. "The PCR method is comprehensive and very sensitive," he says, "and a PCR machine is usually available. Everybody has a PCR machine."

Other rapid-detection kits require various instruments that might represent a significant expense for a lab that screens only occasionally. MycoProbe, a kit released last year by Minneapolis-based R&D Systems, uses a hybridization capture-probe procedure that targets the 16S rRNA sequence of the eight most common mycoplasmas. The assay is performed using a colorimetric plate reader ($350 for a 96-test kit).

A popular new kit, MycoAlert from Cambrex Bioproducts of East Rutherford, NJ, ($104 for a 10-test kit, $494 for a 100-test kit), relies on a metabolic pathway common to all mycoplasmas to create ATP. The kit uses the firefly enzyme luciferase and a luminometer to measure ATP levels before and after the addition of the pathway-stimulating reagent. The two readings yield a ratio that indicates the presence or absence of contamination.

"We don't see false positives. There's not many things that interfere with luciferase. It's a very, very robust assay," says Andrew Paine, a product manager for Cambrex BioScience in Nottingham, UK. "It's as simple as two reagent additions. The whole assay takes 15 minutes to do."

The University of Pennsylvania's cell culture facility recently switched from Hoechst staining to more rapid testing methods, using Cambrex's MycoAlert kit and a single-step PCR kit developed by Minerva Biolabs called VenorGeM ($142 for a 25-test kit). Released in 2002 by manufacturer Sigma-Aldrich, VenorGeM is one of the more broad-ranging PCR kits, targeting at least 25 species of mycoplasma.

Running the two tests side-by-side, says Terri, is a good way to ensure you have the best results. "Sometimes you get two different answers for one sample by using PCR and MycoAlert," she says. "The PCR test is very sensitive, but it tests for the eight most common strains. The MycoAlert is a little less sensitive, but it doesn't miss the various strains."

Paine noted that with PCR, there's a tradeoff between the range of mycoplasma detection and the accuracy of the test. "The more species your PCR detects, the less stringent it is, and the more false positives you'll get," he says. The European Collection of Animal Cell Cultures, says Paine, is currently in the process of comparing MycoAlert's effectiveness to that of PCR and direct culture.


In cell culture, as in most endeavors, an ounce of prevention is worth a pound of cure. Once a cell line is contaminated with mycoplasma, the options typically are not good: Throw it away, or spend a great deal of time and money eliminating the infection. Standard antibiotics commonly used to treat yeast and bacterial contamination are generally ineffective against mycoplasma.

If you cannot bear to discard a culture, Drexler recommends the use of mycoplasma-specific antibiotics such as tetracyclines or quinolones, which he has found in his own research to be highly effective, although such treatment is also stressful to the cultured cells. Certain antibiotics are sometimes promoted as preventive measures, but Drexler cautions against that. "We don't use them preventively, because then your culture might develop resistance. We say, if you have contamination, treat it, and then take out the antibiotics."


Some labs that offer mycoplasma detection services also offer decontamination, but the price tags are steep: Bionique, in Saranac, NY, charges at least $1,680 per sample, as compared to $130 for Hoechst staining. A more radical technique is to inject contaminated cells directly into a host organism, and allow the animal's immune system to tackle the infection.

The best solution, Drexler says, is to use good laboratory technique, quarantine cell lines whose infection status is unknown, maintain stocks of clean cell lines in storage, and check cells often.