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.
Mycoplasma refers to any of over 200 species of tiny (0.2-0.8 µm diameter) bacteria that commonly invade cell cultures, and may even persist unrecognized for some time. They can come from sources as mundane as other cells introduced to the lab via media, sera, or lab personnel. Thus it is important to test your cell lines for mycoplasma regularly. Consequences to your cells of mycoplasma infestation include changes in growth rates, morphology, gene expression, metabolism, and viability.
Even though most lab contamination can be blamed on as few as 8 main culprit species, these differ enough that no one test can find them all. And the little blighters are hardy – most antibiotics commonly used in cell culture are ineffective against mycoplasmas. For example, mycoplasmas lack a cell wall, which is the key to penicillin’s success. Flawless cell culture technique is one of the best preventative tools, as well as testing to ascertain which cultures are clean, and which cultures are infected – so you can take quick and aggressive action before the infestation spreads. Here is an overview of common methods used to detect these pesky bugs in your cultured cells.
Agar-and-broth procedure
The so-called gold standard test for the most accurate detection of mycoplasma is the culture isolation method, also known as the agar and broth procedure. In this test, a sample of your suspected cultures is added to agar plates that have been set up as absolute mycoplasma heaven. The idea is that you give them everything they could ever want, so that any mycoplasmas present in your culture at the time of sampling will grow like crazy and be readily apparent on your agar test plates. This is why a negative result will (nearly always) be a true negative, hence the gold standard label. Alas, there are two downsides to this otherwise reliable detection method: the long wait time (the test takes at least 4 weeks for definitive colony growth); and the fact it detects most, but not all, mycoplasma species.
If you can’t or don’t want to wait that long, or perhaps want answers while you wait for the results of the culture isolation test, you can try the DNA, PCR, or enzymatic methods of detection. A caveat with these is that none of them detects all species, and they have lower sensitivity than the culture isolation method, so it’s best to use two of these methods for a confident result.
DNA detection
The DNA method of detection takes several days, because you grow a sample of your suspected cell cultures with indicator cells – usually Vero cells, which have a large cytoplasmic area. Cells are then stained for DNA with a fluorescent dye (such as Hoechst dye), and mycoplasmas that had been in your cell sample will be visible as fluorescent spots, or granules, surrounding the nucleus of the indicator cells. A benefit of this method is that it detects the mycoplasma strain M. hyorhinis, which is not reliably detected by the gold-standard culture isolation method.
PCR method
The M. hyorhinis strain is also picked up by another method of detection – the PCR method. This is the quickest (only a few hours), but also the least sensitive, mycoplasma assay. In this method, you perform PCR on samples of your suspected cell cultures, using primers specific for mycoplasma DNA, usually mycoplasma’s 16S rRNA genes. Running the PCR product on a gel shows the presence of mycoplasma DNA by band(s) of distinct sizes. This method detects the majority of mycoplasma species, but using another method to verify is prudent.
Enzymatic and ELISA methods
Other forms of mycoplasma detection are also available. In the enzymatic method, you add a sample of your suspected cell culture to a substrate that converts ADP to ATP in the presence of mycoplasmal enzymes. The test reagent also contains another compound that functions as a read-out, such as the enzyme luciferase, which uses the ATP to produce light – signaling the presence of mycoplasma infection in your cultures. Another alternative detection strategy relies on the widely-used ELISA format, which can indicate the presence of mycoplasma using either antibodies, or labeled probes for the mycoplasma’s 16S rRNA genes.
Test regularly
As a general rule of thumb, mycoplasma testing should be accomplished (endured?) every 1-3 months, with regularity being the key. A lab that relies on cultures, but cannot spare the people-power to conduct regular tests, might consider one of the many high-quality mycoplasma testing services available today.
The image at the top of the page is from Life Technologies' MycoFluor Mycoplasma Detection Kit.