Mycoplasma contamination of cell cultures is a widespread problem for both basic research and industrial production. Surveys have shown incidences ranging from 5% to 85% of all cell culture examined. In infected cell culture the number of mycoplasma frequently far exceeds, often by 1000-fold, the number of cultured cells. With as 5 mycoplasmas per mammalian cell in a culture the effects can be rather drastic.

The name “mycoplasma” encompasses all species included in the class Mollicute, i.e. the genera Mycoplasma, Acholeplasma, Spiroplasma, Anaeroplasma and Ureaplasma. Normally, mycoplasmas live commensally or parasitically in man, animals or plants. 6 species namely M. orales, M. hyorhinis, M. fermentans, M. salivarum and A. laidlawii account for more than 95% of mycoplasma contaminants in cell culture. Mycoplasma are the smallest self-replicating prokaryotes with a diameter of 0.1-0.8 µm. In some cases their genome is only a quarter of that of E. coli.  

Most mycoplasmas produce microscopic colonies (50-600 µm) with their characteristic “fried-egg” appearance. Because of the absence of a rigid cell wall , mycoplasmas are pleiomorphic and extremely flexible in shape, varying from spherical or pear-shaped cells, to branched-filamentous or helical cells.  

Incidence of mycoplasma species (%)

Laboratory personnel:

• M. hominis 1.8 %
• M. fermentans 1.8 %
• M. salivarum 9.8 %
• M. orale 32.3 %

Bovine (e.g. serum):

• M. argini 9.7 %
• A. laudlawii 16.7 %

Porcine (e.g. trypsin):

• M. hyorhinis  

• Reduced rate of cell proliferation
• Reduced saturation density
• Rounded, degenerated cells
• Agglutination during growth in suspension
• pH reduction  

A detection by microscope is not possible.
Even 10⁸ mycoplasmas/ml remain undetected with microbiological methods.

Most used methods are:

• DNA-fluorochrome staining
• ELISA
• PCR and or /ELISA-PCR

Mycoplasma species can be identified with specific antisera, monoclonal antibodies or sequencing of the 16rRNA-gene (PCR-product). It is recommended to use at least two different detection methods to ensure reliable results.

Be careful using PCR since PCR products are also detectable if mycoplasma are not alive. To circumvent this problem please use also an immunological method to test the viability.

For reliable PCR use a positive control (cloned target sequence and diluted), a negative control (PCR without DNA), “spike” control (sample to be tested spiked with mycoplasma or positive control to check the refurbishment of the DNA-extraction method)  

• Strictly remain “Good Laboratory practice”
• Swab the work surface with 70% alcohol
• Run the laminar flow for at least 15 minute before and after work
• Clean hood regulary
• Wash hands and wear gloves
• Sterilize instruments
• Avoid mouth pipetting
• Obtain cell lines from reputable cell banks
• Check the cell lines with PCR and ELISA
• Test all animal-derived media components with PCR and ELISA

Mycoplasma can pass through standard filters. For efficient removal of mycoplasma three different methods are used:

UV irradation

Gamma irradiation

Treatment with mycoplasma-specific antibiotics (MycoKill AB: P11-006)

Detection of Mycoplasma with PCR