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Mycoplasma Detection

Mycoplasma is a kind of bacteria that lacks a cell wall around their cell membrane. It is the most common contaminants of cell culture in laboratories. According to the statistics, 15% to 35% of continuous cell cultures are contaminated by mycoplasma. Mycoplasma contaminants are usually from untested cells, contaminated materials, infected donor tissues, or laboratory researchers. Typical routes of infection include generation of aerosols during pipetting, use of same media bottles, and handling of more than one cell type at the same time. In general, cross-contamination is the primary source of mycoplasma during experiments. Mycoplasma grows slowly and does not kill the cells directly, but it affects various cellular parameters. For example, mycoplasma can inhibit the cell growth, change the cell membrane antigenicity, inhibit the cell metabolism, disrupt the nucleic acid synthesis, and increase the cell sensitivity to inducers of apoptosis. Thus, mycoplasma can seriously impact the reliability, reproducibility, and consistency of experimental results, which are major problems for basic research and the manufacture of biologics.

Mycoplasma Detection at Creative Animodel
Creative Animodel has developed several methods for mycoplasma detection that are complied with guidelines of multiple global regulatory authorities. Product-specific method development and custom detection services are also available. Mycoplasma detection methods at Creative Animodel include but are not limited to:

PCR method
The PCR method utilizes specific nucleic acid sequence of mycoplasma as primers. The PCR amplification is performed using the primers and nucleic acid extracts of cell cultures to be tested. Although rather fast and inexpensive, the PCR method requires stringent assay-setup to avoid the risk of false positives or negative results.            
Fluorescent DNA staining
The test is performed with the fluorescent DNA-binding dye. The murine cell line 3T6 (ATCC CCL96) is used as the indicator cell line on which most mycoplasma strains are expected to grow. Cell cultures to be tested are added to cover slips with indicator cells. After DNA staining process with 0.001% solution of bisbenzimide in PBS, the specimens are examined under illumination with a high-pressure mercury lamp. The microscopic slide is examined for the presence of extranuclear fluorescent particles of characteristic appearance.

Hybridization assay
The cell pellet to be tested is collected by centrifugation and added with the 3H-labelled DNA probe. Hydroxylapatite solution containing 0.02% sodium azide is added to the pellet. After centrifugation, the supernatant is decanted and saved. The pellet is washed with the buffered solution containing 0.02% sodium azide. The supernatant is saved again. Scintillation solution is added to the supernatant. The radioactivity of the hydroxyapatite pellet containing the probe with any hybridized rRNA is counted in a scintillation counter. A hybridization greater than or equal to 0.4% is considered diagnostic of mycoplasma contamination.

Creative Animodel has experienced scientists in the field of cell culture and mycoplasma detection. We can provide reliable detection results within rapid turnaround time and at favorable price. If you have any specific needs, please feel free to contact us.

Reference:
1. Benisheva, T.; Loewer, J. Comparison of three methods for the detection of mycoplasms in cell cultures. Biotechnology & Biotechnological Equipment. 2014, 8(4):42-45.

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