Mycoplasma testing

Mycoplasma are the smallest (0.2 - 2µm in diameter) and most unusual of the prokaryotes.  ECACC can test your cell lines, cell culture media and reagents to determine if mycoplasma is present. The effects of mycoplasma contamination on cell lines can be wide ranging and are often underestimated. These include:

• alteration of growth rate
• induction of morphological changes
• chromosomal aberrations
• altered cell metabolism
• decreased viability upon resuscitation of frozen ampoule.
• induction of transformation, apoptosis, cytokines, signal transduction and oxidative radicals
• macrophage activation
• inhibition of antigen presentation

In addition, Mycoplasma can also interfere with membrane receptor function and penetrate host cells.

All laboratories carrying out cell culture should test for mycoplasma contamination on a regular basis and many researchers choose to use our service to ensure they are working with mycoplasma free cell cultures. We recommend that a combination of methods is used to determine whether a sample is free of mycoplasma.  This is because the different methods have different levels of detection sensitivity and some strains of mycoplasma do not grow in vitro. These can be detected by DNA stain and PCR.

Find out how to submit samples for mycoplasma testing

Three different mycoplasma detection methods are routinely used by our scientists:

1. PCR

The ECACC PCR detection assay identifies mycoplasma positive samples using primers developed inhouse for screening purposes and is useful for detecting contamination with the non-cultivable strains of M.hyorhinis.

The PCR method is useful for screening a large number of samples quickly. Results are obtained within a few hours. The presence of contaminant mycoplasma is easily detected using agarose gel electrophoresis by verifying the bands of amplified DNA fragments from test samples against both positive and negative controls.

2. Indirect Hoechst Stain

This test is used to detect mycoplasma by Hoechst stain using an indicator cell line (Vero ECACC 84113001) and meets the requirements of the FDA Points to Consider 1993.

The test sample is inoculated onto Vero cells which have been grown on coverslips in tissue culture dishes. Both negative and positive controls (Mycoplasma hyorhinis NCTC 10112 and Mycoplasma orale NCTC 10130) are also set up. All samples are then incubated for 3-5 days after which the samples are fixed to the coverslip and stained using the fluorescent dye (Hoechst 33258). The fixed samples are then examined under UV epi fluorescence at x1000 magnification. Positive samples are identified by their characteristic particulate or filamentous pattern of fluorescence on the cell surface or if contamination is heavy in the surrounding area. Negative samples should demonstrate no evidence of fluorescing DNA outside of the cell membrane against the fluorescing Vero cell nuclei.

3. Culture Isolation

Often regarded as the gold standard method for mycoplasma detection, the culture isolation detection method uses a combination of selective growth media and incubation conditions to positively enrich for any mycoplasma present in a sample. Most mycoplasma contaminants can be detected by growth on standardised agar, with the exception of certain strains of M.hyorhinis.

Colonies of mycoplasma exhibit a distinctive "fried egg" morphology when viewed under a plate microscope. The assay requires a 28 day test interval before a definitive result can be obtained. This test meets the requirments of the FDA Points to Consider 1993.

Mycoplasma Detection Methods and Prices 

We also offer a sterilty testing service.