Mycoplasma are a unique type of bacteria, distinct from most others due to their lack of a rigid cell wall. These microscopic organisms commonly contaminate cell cultures, presenting a persistent and often undetected problem in research laboratories and biopharmaceutical production. Their presence significantly compromises experimental reliability and can lead to misleading scientific conclusions. Addressing mycoplasma contamination is a significant concern for maintaining the integrity of cell-based research.
Understanding Mycoplasma’s Nature and Effects
Mycoplasma are among the smallest self-replicating bacteria, ranging from 0.1 to 0.8 micrometers in size. Unlike most bacteria, they lack a peptidoglycan cell wall, making them naturally resistant to many common antibiotics, such as penicillin and streptomycin, which target cell wall synthesis. This unique biological feature allows them to pass through standard filtration membranes designed to remove larger bacteria, making them particularly challenging contaminants.
Their presence in cell cultures can induce a range of detrimental effects on host cells. Mycoplasma compete with host cells for metabolic precursors, like arginine and pyrimidines, disrupting cellular metabolism and reducing ATP production. This competition leads to altered cell growth rates, including reduced proliferation, and can cause changes in cell morphology. Prolonged contamination can also result in chromosomal aberrations, changes in gene expression profiles, altered protein synthesis, and even induce apoptosis, ultimately skewing experimental outcomes.
How Mycoplasma Enters Cell Cultures
Mycoplasma primarily enter cell cultures through a few common routes within the laboratory environment. A significant source is contaminated biological reagents, with animal-derived components such as fetal bovine serum, trypsin, and other media supplements frequently implicated. These products may carry mycoplasma from their source animals, introducing the contaminants directly into sterile cultures.
Cross-contamination between cell lines is another prevalent pathway for mycoplasma spread. This can occur through shared media bottles, pipettes, or other instruments used sequentially across different cell lines. Aerosolized particles generated during routine handling, such as uncapping flasks or pipetting, can also carry mycoplasma from an infected culture to a clean one.
Laboratory personnel can inadvertently introduce mycoplasma into cultures. Poor aseptic technique, like talking over open cell culture dishes or inadequate hand hygiene, allows mycoplasma to transfer to the sterile environment. Rigorous adherence to aseptic protocols during all cell culture manipulations is important.
Identifying Mycoplasma Contamination
Detecting mycoplasma contamination in cell cultures is important for maintaining experimental integrity. One widely used method is direct DNA staining, employing fluorescent dyes like Hoechst 33258 or DAPI. These dyes bind to DNA, allowing visualization of mycoplasma as small, extranuclear fluorescent particles under a fluorescence microscope. This technique offers a quick assessment but requires experienced personnel to accurately interpret the results.
PCR-based detection methods are sensitive and specific, capable of identifying mycoplasma even at low contamination levels. This technique amplifies specific regions of mycoplasmal ribosomal RNA genes, providing rapid results within a few hours. Its sensitivity makes it a preferred method for routine screening and for confirming the absence of mycoplasma in newly acquired cell lines.
Microbiological culture remains a reliable method for mycoplasma detection due to its reliability in identifying viable organisms. This method involves inoculating cell culture supernatant onto specialized mycoplasma-specific agar or broth media. While dependable, it requires a longer incubation period, ranging from two to four weeks, to allow for sufficient growth of the slow-growing mycoplasma. The appearance of characteristic “fried egg” colonies on agar plates serves as a confirmation of contamination.
Strategies for Control and Elimination
Preventing mycoplasma contamination is the most effective approach to maintaining healthy cell cultures and reliable experimental results. Strict adherence to aseptic technique is important, involving consistent use of biosafety cabinets, sterile reagents, and dedicated equipment for cell culture work. Regular testing of all cell lines, especially upon receipt from external sources or before cryopreservation, helps identify contamination early.
Establishing quarantine procedures for new cell lines is also recommended; these lines should be isolated and tested for mycoplasma before being introduced into the main cell culture area. Routine cleaning and disinfection of incubators, water baths, and biosafety cabinets with appropriate agents, such as 70% ethanol or specialized disinfectants, further reduce the risk of spread. Implementing these preventative measures lowers the likelihood of contamination.
If contamination occurs, elimination strategies can be attempted, though prevention is always preferred due to potential cell damage. The use of specific antibiotics, such as quinolones (e.g., ciprofloxacin) or tetracyclines, can target mycoplasma. It is important to distinguish between mycoplasmacidal agents, which kill the bacteria, and mycoplasmastatic agents, which inhibit their growth. Treatment involves multiple passages with the antibiotic, followed by a washout period to remove residual drugs. After any treatment, repeated retesting over several passages is necessary to confirm eradication, as dormant mycoplasma can reactivate.