Mycoplasma contamination is a pervasive issue in cell culture laboratories, often going undetected unlike more visible microbial infections. Understanding mycoplasmas and their effects is fundamental for researchers using cell cultures. Their presence can lead to unreliable experimental outcomes, making identification and control a significant concern for scientific reproducibility.
Defining Mycoplasma and Its Impact on Cell Cultures
Mycoplasmas are among the smallest known free-living bacteria, distinguished by lacking a rigid cell wall. This makes them resistant to common antibiotics like penicillin, which target cell wall synthesis. Their small size (0.2 to 0.8 micrometers) also allows them to pass through standard sterilization filters, making exclusion from reagents difficult.
Once established in a cell culture, mycoplasmas can significantly alter the physiology and metabolism of host cells. They compete for essential nutrients like amino acids, fatty acids, sugars, and nucleic acid precursors, leading to nutrient depletion. This competition can result in slowed cell growth rates, and in some cases, induce apoptosis or cell death. Mycoplasmas can also induce chromosomal aberrations, including translocations and deletions, and alter gene expression profiles within infected cells. Such widespread cellular changes can compromise the validity of experimental results.
Common Sources of Contamination
Mycoplasma contamination originates from several sources within the laboratory. Laboratory personnel are a frequent source, as mycoplasma species are found on human skin and in the respiratory tract. Poor aseptic technique, such as improper handling of cultures or sneezing and coughing near open dishes, can easily introduce these bacteria into sterile cell lines.
Contaminated biological reagents are another significant source. Fetal bovine serum (FBS) is a notable vector for mycoplasma transmission due to its origin and processing. Other cell culture additives and media components can harbor mycoplasmas if not properly screened and sterilized. Cross-contamination from infected cell lines is another common route of spread within a laboratory. Mycoplasmas can be easily transferred between cultures via aerosols, shared reagents, or even reused pipet tips, quickly jeopardizing an entire cell culture collection.
Detection Techniques
Identifying mycoplasma contamination requires specific methods, as these bacteria are too small to be seen with standard light microscopy and do not cause visible turbidity. DNA staining is a common approach, using dyes like DAPI or Hoechst 33258 that bind to DNA. When observed under a fluorescence microscope, mycoplasma DNA appears as small, extranuclear fluorescent particles, distinct from the larger host cell nuclei. This method is relatively quick but requires expertise in interpretation and may not detect low levels of contamination.
PCR-based assays offer a highly sensitive and specific detection method by amplifying mycoplasma-specific DNA sequences. These assays can detect even minute amounts of bacterial DNA, making them suitable for early detection and routine screening. Results are typically obtained within a few hours, providing a rapid assessment of contamination status. ELISA (Enzyme-Linked Immunosorbent Assay) kits detect specific mycoplasma antigens or antibodies produced by host cells in response to infection. While generally less sensitive than PCR, ELISA kits can be adapted for high-throughput screening and are relatively straightforward to perform.
Elimination and Prevention Strategies
Elimination
Upon detecting mycoplasma contamination, the most recommended action is often to discard the affected cell culture and restart with a verified clean stock. This approach minimizes the risk of persistent contamination and protects the integrity of ongoing research. However, if a cell line is irreplaceable due to its unique properties or extensive previous work, various elimination strategies can be employed.
Treatment options include specific anti-mycoplasma antibiotics, such as quinolones, tetracyclines, or macrolides, which target unique mycoplasma metabolic pathways or DNA replication. These treatments typically involve a multi-day regimen followed by several rounds of testing to confirm eradication. Success rates can vary, and prolonged antibiotic use may impact cell behavior.
Prevention
Prevention remains the most effective strategy against mycoplasma contamination. Strict adherence to aseptic technique in all cell culture procedures is important, including regular cleaning of biosafety cabinets and avoiding shared reagents or equipment between different cell lines. Regularly testing all cell stocks, particularly new cell lines from external sources, is also a preventative measure. Quarantining new cell lines and reagents until their mycoplasma-free status is confirmed can prevent widespread contamination within a lab. Utilizing certified, pre-screened reagents, such as mycoplasma-tested fetal bovine serum, further reduces the risk of introducing these stealthy contaminants.