The Stealthy Invader
What Causes a Mycoplasma Contamination?
Apr 26, 2024 | Informative Articles

Featured in this article:
• Main sources of mycoplasma in cell culture
• Experimental Study – See how easily mycoplasma can spread in your lab
Main sources of mycoplasma in cell culture
Mycoplasma contamination poses a significant threat to cell culture research, can endanger scientific data, delay research projects and jeopardise the safety of biopharmaceutical products. Mycoplasma can enter cell cultures through various sources. The largest percentage of Mycoplasma found in contaminated cell cultures is of human origin. Other species are of bovine and porcine origin.
After reading this article you have learned about the frequency and sources of mycoplasma contamination in cell cultures and understand the ways in which cells get contaminated.
Laboratory Personnel
The major source of mycoplasma contamination is assumed to be the laboratory personnel because the largest percentage of mycoplasma found in cell culture are of human origin. M. orale, M. fermentans, and M. hominis account for more than half of all mycoplasma infections in cell cultures and physiologically are found in the human oropharyngeal tract [1], [2].
By sneezing, coughing or simply talking aerosols are generated that carry various mycoplasma species and can contaminate cells directly or any lab reagents (e.g. cell culture medium and supplements), lab consumables (e.g. flasks, plates and falcons) or equipment (e.g. pipettors, water baths and incubators). Furthermore, street clothes and dirty lab coats are a major source of dust and airborne particles containing mycoplasma.
The illustration gives an overview of the colonization of humans with microorganisms (not only mycoplasma).
lightbulb The major source of mycoplasma contamination is assumed to be the human operator. Since mycoplasma are part of the human microbial flora cell cultures can get contaminated easily.
Cross-Contamination
Once you have a mycoplasma-infected cell culture in your lab, it can spread among all other cultures via cross-contamination. Operator-induced contamination is a multifaceted problem. Mycoplasmas are spread by using laboratory equipment, media, or reagents that have been contaminated by previous use in processing mycoplasma-infected cells. Also incubators, water baths and other devices are often shared among multiple users and cell culture experiments, increasing the risk of cross-contamination between different cell lines.
It was shown, that in laboratories with contaminated cells, most or all cultures are positive containing the same mycoplasma species [4]. This is due to the ease of droplet generation during handling of cell cultures, the high concentration of mycoplasmas in infected cultures, and the prolonged survival of mycoplasma cells.
lightbulb Mycoplasma-infected cell lines are themselves the single most important source for further spreading of the contamination.
Media, Sera and Other Reagents

Cell culture media and sera serve as essential components in supporting cell proliferation and viability. However, these reagents can also introduce mycoplasma contamination into cell cultures. Media and sera sourced from animal-derived materials, such as fetal bovine serum (FBS), are susceptible to mycoplasma contamination. The high incidence of bovine mycoplasma species, predominantly A. laidlawii and M. arginini, underlines this assumption.
Trypsin solutions, that are used in cell passaging to detach adherent cells, are provided by swines and represent a major source of M. hyorhinis.
Whereas sera products were a primary source of infection (with reported contamination rates of 18 to 40 %) in the 1960s and 1970s, today, sera and media obtained from reputable manufacturers are rarely the source of mycoplasma contamination [1]. However, it is still the responsibility of the end user to verify that the products they purchase have been adequately filtered, tested and certified as mycoplasma-free.
lightbulb FBS and trypsin are a major source of animal-derived mycoplasma contamination with species of bovine or porcine origin being involved.
Incubators and Water Baths

Water baths and humidified incubators represent further potential contamination sources by offering ideal environmental conditions for microbial growth. Many species of bacteria can proliferate in tepid water, rapidly reaching high titers.
Also surfaces within the incubator, including shelves, walls, and door seals, can harbor mycoplasma contamination. Improper cleaning and disinfection practices can allow mycoplasma to persist on these surfaces and serve as a source of ongoing contamination.
Incubators are often shared among multiple users and cell culture experiments, increasing the risk of cross-contamination between different cell lines. Incubators are equipped with fans and air currents. Mycoplasma-contaminated aerosols generated within the incubator can settle onto the surface of cell culture dishes, leading to contamination. Poor airflow patterns or ventilation in the incubator can exacerbate airborne transmission of mycoplasma.
lightbulb
Incubators and water baths
offer ideal environmental conditions for microbial growth.
Airborne Particles and Aerosols

Aerosols and airborne particles represent a significant yet often overlooked source of mycoplasma contamination in cell culture laboratories.
Laboratory design, ventilation systems, airflow patterns as well as street clothes and dirty lab coats can influence the dispersion and deposition of aerosols and airborne particles. Poorly ventilated or overcrowded laboratories may have higher concentrations of airborne contaminants, increasing the risk of mycoplasma contamination.
Airborne microorganisms settle down in still air. As a result, the air in a sealed, draft-free room or laboratory is nearly free of biological contaminants. However, as soon as people enter the room, particles that have settled down will be easily resuspended.
Equipment and activities within the laboratory, such as pipetting and vortexing devices, centrifuges, refrigerators/freezers and sonicators can generate microbial particles and aerosols containing mycoplasma. These aerosols can remain suspended in the air for extended periods and travel considerable distances, facilitating the spread of contamination.
lightbulb Aerosols and airborne particles can contaminate multiple cell cultures within the same laboratory or between different laboratories.
Experimental Study – See how easily mycoplasma can spread in your lab
McGarrity conducted a study to investigate the spread of mycoplasmas in a laminar flow hood during routine cell subculturing. He infected a cell culture with mycoplasma. After trypsinization in the laminar flow hood, live mycoplasmas from various surfaces, including the flask, hemocytometer, pipettor, and the outside of the pipette discard pan, were isolated.

Remarkably, live mycoplasmas could still be recovered from the laminar flow hood's surface even four to six days later. A clean culture, subcultured weekly in the same hood following the work with the mycoplasma-infected cells, tested positive for mycoplasma contamination after just six weeks. These findings demonstrate how easily and rapidly mycoplasma can spread and show the risk of contaminating other cultures in the laboratory due to the introduction of a single infected culture [5].
lightbulb By understanding the mechanisms of contamination and implementing robust prevention and control strategies, laboratories can minimize the risk of mycoplasma and ensure the integrity of cell culture experiments.
Stay tuned for more articles about the ways in which cells are contaminated, explore detection methods and find out about prevention and removal strategies!
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[1] Nikfarjam L, Farzaneh P. Prevention and detection of Mycoplasma contamination in cell culture. Cell J. 2012 Winter;13(4):203-12. Epub 2011 Dec 22. PMID: 23508237; PMCID: PMC3584481. [2] Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology. 2002 Jul;39(2):75-90. doi: 10.1023/A:1022913015916. PMID: 19003295; PMCID: PMC3463982. [3] WHO Guidelines on Hand Hygiene in Health Care: First Global Patient Safety Challenge Clean Care Is Safer Care. Geneva: World Health Organization; 2009. 5, Normal bacterial flora on hands. Available at: https://www.ncbi.nlm.nih.gov/books/NBK144001/ [4] McGarrity, G. J., H. Kotani, and H. Butler. Mycoplasmas: molecular biology and pathogenesis. Eds Maniloff J., McElhaney RN, Finch L., Baseman JB Washington: ACM 1992 (1992): 906. [5] McGarrity, G. J. Spread and control of mycoplasmal infection of cell cultures. In Vitro. 1976 Sep;12(9):643-8. doi: 10.1007/BF02797464. PMID: 1034618.