By some estimates, at least one third of the cell cultures used for research are already infected with mycoplasma—sneaky prokaryotes that can infect cultures without researchers’ knowledge. Because they are small and slow-growing, mycoplasma can easily be missed. However, their presence can alter cultured cells’ metabolic processes and cause genetic changes or other intracellular damage that can wreak havoc with your results (or at best cause unexplained inconsistencies in your data). Even worse, rescuing infected cultures is difficult or impossible. Thus, when it comes to mycoplasma contamination, prevention is the best option. Taking some fairly straightforward precautionary measures will minimize your risk of mycoplasma-infected cells.
Buy certified mycoplasma-free cells
One of the most common causes of mycoplasma contamination is incoming cultures that are already infected. Once in the lab, the little critters can easily spread to all other cultures, and you suddenly have a culture crisis on your hands. The easiest way to avoid this is to decline “gifted” cultures from other labs. Instead, purchase new cultures from cell suppliers or repositories that can certify the cells as mycoplasma-free with rigorous testing. Examples of such repositories are the European Collection of Cell Cultures (ECACC) and the American Type Culture Collection (ATCC).
If you do accept cultured cells from another lab, be smart and quarantine them in a separate incubator while you test them for the presence of mycoplasma. You can perform the test yourself with one of several widely available do-it-yourself kits, or you can send a culture sample to a testing service (repositories usually offer this service too).
Test for mycoplasma routinely
Even without the introduction of new cells to a lab, mycoplasma can infect existing cultures via transmission from lab workers who are carrying it unknowingly (and possibly using poor cell-culture techniques; see below). Another route mycoplasma can take is from your cell-culture reagents straight to your cells. Sadly, the reagents you purchase for your cultures—media and enzymes, for example—may be contaminated with mycoplasma.
Although it is impossible to prevent contamination from people or reagents completely, routine mycoplasma testing can help you avoid disaster. Of course, testing your cultures routinely for the presence of mycoplasma does not in fact prevent an initial infection, but it can prevent the spread of the microbes from contaminated to healthy cultures if caught early. In addition to testing services to which you can send samples of your cultures, you can test them yourself with test kits like those mentioned above.
Mycoplasma tests come in several varieties. Unfortunately, the most accurate test (direct culturing, or the agar-and-broth method) takes three to four weeks. But other so-called indirect-type tests are reliable enough to use routinely and can be followed up with the more cumbersome direct test if any positives show up. These quicker indirect tests include fluorescent and biochemical assays based on enzymatic activity, PCR-based assays, immunoassays and nucleic acid hybridization assays.
Rely on good aseptic technique
In addition to contaminated incoming cultures, another main source of mycoplasma contamination is poor aseptic cell-culture technique by lab members. Although using good technique in the cell-culture hood is perhaps the simplest preventive measure against mycoplasma, it is also the most difficult to implement. Everyone makes mistakes, especially during routine, repetitive, sometimes mindless lab tasks. But nobody wants “lab police” watching people at the hood. A happy medium needs to be achieved between training, supervision and trust.
Insisting on high standards in cell-culture technique will go a long way toward protecting your cultures. Only lab members who are fully and recently trained in aseptic technique should use the hood. People trained elsewhere should demonstrate their routines and be monitored until it’s clear they are well versed in aseptic practices. It is also a good idea to make sure everyone in the lab fully understands the nature of mycoplasma, its potentially devastating effects and how severely it could affect the lab’s work.
Many labs still use antibiotics routinely, but this is increasingly viewed by cell culturists as irresponsible, because it contributes to the problem of antibiotic-resistant microbes. Instead, it is usually best to base your initial defense against mycoplasma on good aseptic practice by everyone involved in care of the cultures and to use antibiotics only as needed—when you have a suspected or confirmed infection.
Bank some cells, just in case
Finally, a wise preventive move is to bank some cultures by cryopreservation, just in case the dreaded contamination crisis does occur. This is especially important if you are using one-of-a-kind cell lines (in which case you have probably already banked them—right?). If mycoplasma rears its ugly head, or routine testing shows a positive result, the cleanest and surest remedy is to throw out all cultures, clean the incubators and hoods and start again. Instead of starting from scratch, hopefully you can just pull some of the same cells you were using prior to mycoplasma infection out of liquid nitrogen and be on your merry way. And of course, be sure your banked cells are mycoplasma-free.