Mycoplasma contamination is a type of biological contamination; however, it differs from regular bacterial, viral, or fungal contamination because mycoplasma is extremely difficult to detect. Mycoplasma are simple bacteria that lack a cell wall, and they are considered the smallest self-replicating organism. It is because of their extremely small size (typically less than 1 µm), that mycoplasma contamination is difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often no visible signs of infection. 

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Causes of mycoplasma contamination in cell culture

Like other forms of biological contamination, mycoplasma is spread through various channels, usually arising from contaminants already present in your laboratory or working area. Through improper handling or a lack of adherence to aseptic techniques, mycoplasma contamination can be difficult to detect while becoming increasingly pervasive.

Learn more about how to prevent mycoplasma contamination with aseptic techniques 


Detecting mycoplasma contamination

Some slow growing mycoplasma may persist in culture without causing cell death, but they can alter the behavior and metabolism of the cells in the culture.

Chronic mycoplasma infections might manifest themselves with decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures; however, the only assured way of detecting mycoplasma contamination is by testing the cultures periodically using fluorescent staining, ELISA, PCR, immunostaining, autoradiography, or microbiological assays.

Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with mycoplasma (panels B and C)

Figure 1. Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with mycoplasma (panels B and C). The cultures were tested using the MycoFluor Mycoplasma Detection Kit, following the kit protocols. In fixed cells, the MycoFluor reagent has access to the cell nuclei, which are intensely stained with the reagent, but the absence of fluorescent extranuclear objects indicates that the culture is free from mycoplasma contamination (panel A). In fixed cells infected with mycoplasma, the MycoFluor reagent stains both the nuclei and the mycoplasma, but the intense relative fluorescence of the nuclei obscures the mycoplasma on or near the nuclei. However, the mycoplasma separated from the bright nuclei are readily visible (panel B). In live cells, the MycoFluor reagent does not have access to the nuclei, but readily stains the mycoplasma associated with the outside of cells (panel C). The images were obtained using 365 nm excitation and a 100/1.3 Plan Neoflaur® (Zeiss) objective lens coupled with a 450±30 nm bandpass filter.


Removing mycoplasma from cell cultures

If you detect mycoplasma contamination in a cell culture before it causes cellular deterioration, it can be treated with antibiotics. However, because of their lack of a cell wall, mycoplasma are resistant to common antibiotics that target the area, such as penicillin.

Thermo Fisher Scientific offers several antibiotics suitable for removing mycoplasma from cell cultures. Always use caution when deciding when to use an antibiotic for your cultures as persistent use can result in antibiotic resistant strains.


Preventing mycoplasma contamination

There are many efforts one can take to minimize and prevent mycoplasma contamination within a cell culture lab. Mycoplasma contamination is best prevented by following proper aseptic techniques in the cell culture lab. Through proper sterilization procedures and handling, donning of PPE, and diligent aseptic technique, mycoplasma contamination can be severely limited. Though as a last line of defense, when handling culture media, serum, additives, and buffers, you can also opt for a sterilization filter that has a pore size of 0.1 μm to enable maximum protection.

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