Mycoplasma, a genus of bacteria, represents a pervasive and often undetected contaminant in various scientific and medical environments. These microorganisms pose a significant threat, particularly within research laboratories where cell cultures are routinely utilized. Their presence can severely compromise the integrity and reliability of experimental results, leading to misleading data and wasted resources. Beyond research, mycoplasma can also affect animal health and biopharmaceutical production, making their control a broad concern. Addressing mycoplasma contamination requires a focused approach, encompassing both effective eradication methods for existing issues and robust prevention strategies to safeguard future work.
Understanding Mycoplasma’s Unique Challenges
Mycoplasmas present distinct challenges for detection and elimination due to several biological characteristics. Unlike most bacteria, they lack a rigid cell wall, which renders them naturally resistant to common antibiotics like penicillin that target cell wall synthesis. Their exceptionally small size (0.15 to 0.3 micrometers) allows them to pass through standard sterilization filters (0.22 µm) and makes them invisible under a conventional light microscope.
Mycoplasmas are known for their stealthy nature, often growing to high densities in cell cultures without causing visible turbidity or overt signs of contamination. They can integrate into host cell membranes and metabolism, competing for essential nutrients and altering cell physiology, gene expression, and even chromosomal integrity. This allows contamination to persist and spread unnoticed for extended periods.
Strategies for Mycoplasma Eradication
Eliminating existing mycoplasma contamination requires specific and targeted approaches. Discarding heavily contaminated cell cultures or reagents is often the most straightforward and reliable method, especially if the materials are not irreplaceable. This step prevents further spread within the laboratory environment.
For valuable cell lines or materials that cannot be discarded, antibiotic-based treatments are frequently employed. However, due to the absence of a cell wall, antibiotics like penicillin and cephalosporins are ineffective against mycoplasma. Instead, specific classes of antibiotics, such as quinolones (e.g., ciprofloxacin, levofloxacin, moxifloxacin), tetracyclines (e.g., doxycycline), and macrolides (e.g., azithromycin, erythromycin, clarithromycin), are used.
These antibiotics target cellular processes that mycoplasma rely on, such as protein synthesis (macrolides and tetracyclines) or DNA replication (quinolones). Some commercially available treatments combine antibiotics with different mechanisms of action to enhance efficacy and reduce the development of resistance. A treatment regimen involves adding the antibiotic to the culture media for one to two weeks, followed by a period without antibiotics and subsequent re-testing to confirm eradication.
Heat inactivation is an effective physical method for eliminating mycoplasma from equipment and certain media. Autoclaving (e.g., 121°C for at least 20 minutes) is suitable for sterilizing laboratory equipment, glassware, and some heat-stable media. Dry heat sterilization (e.g., 165-170°C for two hours) can also inactivate mycoplasma. Incubators can be disinfected using heat cycles, such as 180°C dry heat for three hours or 90°C moist heat for nine hours.
Chemical disinfectants are important for decontaminating laboratory surfaces and equipment. Solutions such as 70% ethanol, 1% sodium hypochlorite (bleach), phenolic disinfectants, and specialized mycoplasma-killing reagents are effective. Regular cleaning and disinfection of work surfaces, biosafety cabinets, and incubators, along with prompt cleanup of spills, are essential for environmental control. Vaporized hydrogen peroxide (VHP) and peracetic acid are also known to be effective against mycoplasma.
Physical removal through filtration can be used for media and reagents, but it has limitations. While 0.1 µm filters can reduce mycoplasma presence, their small size and flexibility mean complete removal cannot always be guaranteed. Therefore, filtration should be considered a risk mitigation step rather than a sole eradication method for contaminated liquids.
Preventing Future Mycoplasma Contamination
Proactive measures are essential to prevent mycoplasma contamination. Strict adherence to aseptic techniques during all laboratory procedures is important. This includes proper hand hygiene, wearing personal protective equipment like clean lab coats and gloves, and maintaining a clean work area. Avoiding activities such as talking, sneezing, or coughing near open cultures, which can generate aerosols, further minimizes the risk of introducing contaminants.
Routine mycoplasma testing of cell lines, reagents, and even personnel is an important preventative step. Various detection methods are available, including PCR-based assays, DNA staining (e.g., Hoechst or DAPI), and traditional microbiological culture methods. PCR is often favored for its sensitivity and speed, while a combination of methods can provide comprehensive results. Regular testing helps to quickly identify and address any new contamination.
Quarantine and thorough screening of all new cell lines and reagents before introducing them into the main laboratory environment are essential. New materials should be isolated in a dedicated area until they are confirmed to be mycoplasma-free. This prevents potentially contaminated incoming materials from compromising existing clean cultures. Using certified mycoplasma-free reagents and media from reputable suppliers is also a valuable practice.
Implementing dedicated equipment for different cell lines or projects can minimize cross-contamination risks. This includes using separate biosafety cabinets, incubators, and even aliquots of media and reagents. Regularly cleaning and maintaining shared equipment, such as incubators and water baths, with appropriate disinfectants also contributes to a cleaner environment.
Laboratory design and airflow patterns can also influence contamination control. Facilities with controlled ventilation systems and appropriate airflow can help reduce the dispersion of airborne particles and aerosols, thus minimizing the spread of mycoplasma. While facility design alone cannot eliminate all risks, it supports overall contamination prevention efforts within a laboratory setting.