Mycoplasma contamination is a widespread and reoccurring problem for many cell culture systems in life science research and the pharmaceutical industry. Mycoplasmas can grow to high titers in culture media without exhibiting typical bacterial contamination signs such as turbidity. Their effects on cultured cells include altered metabolism, slowed proliferation, and chromosomal aberrations. In short, Mycoplasma contamination compromises the data validity of affected cell lines and of results in life science research. This makes Mycoplasma testing and thus the maintenance of contamination-free cell cultures fundamental to cell-based research and to the manufacturing of goods where consumer health is a major concern.
Mycoplasmas are among the smallest known bacteria, being able to pass filters with pore sizes of 0.2 µm. These microorganisms grow aerobically or under facultative anaerobic conditions.
Mycoplasmas are either parasitic or saprophytic. Several species, for example M. pneumoniae, are pathogenic, causing pneumonia and other respiratory disorders in humans. M. genitalium is presumably involved in pelvic inflammatory diseases. As bacteria that lack a cell wall, mycoplasmas are not susceptible to penicillins or other antibiotics that act on this structure. To stabilize their cytoplasmic membranes most mycoplasmas require sterols, which they take up from their environment, usually as cholesterol from their animal hosts. Many Mycoplasma strains are also tolerant to a variety of other antibiotics.
The sources of Mycoplasma contamination in the laboratory and in manufacturing are very challenging to control. Some species are found on human skin and can be introduced to cultures through poor aseptic practices. Additionally, mycoplasmas may be introduced via contaminated supplements, such as fetal bovine serum or, most commonly, by transmission from other contaminated cell cultures. Once a cell culture contains mycoplasmas, these microbes can quickly spread and contaminate other areas of the lab by aerosols and particulates generated during culture handling. Strict adherence to good aseptic laboratory practices is key, and routine testing of cultures is highly recommended for successful control of mycoplasma contamination in manufacturing areas.
Biopharmaceutical facilities that use eukaryotic cells for the production of vaccines have to test their cell banks and virus seed lots as well as bulk vaccines for contamination with Mycoplasma. Species from this bacterial genus infect eukaryotic cells, disrupting their growth and metabolism. When mycoplasmas interfere with vaccine production, this may affect protein quality and yields, and more importantly cause side effects in patients who are administered the final vaccine product.
Bioreactor contamination with mycoplasmas can lead to significant loss of time, materials and revenue unless identified early in the manufacturing process. Without rigorous testing routines, Mycoplasma contamination of biopharmaceutical production batches is extremely difficult to discover because the presence of mycoplasmas does not generally lead to pH changes or visual turbidity in the culture media. The compendial Mycoplasma test method involves both culture and indicator cell tests for detection. If only two media are used for analysis, it is recommended to use FREY and FRIIS media in combination.
It is important to test biopharmaceuticals, vaccines, cell cultures and virus cultures for mycoplasmas at various points during quality control. The three most common test methods are:
* On days after inoculation, subculture each liquid medium by inoculating 0,2 mL on at least 1 plate of each solid medium. Repeat the procedure between the 6th and 8th day, again between the 13th and 15th day and again between the 19th and 21st day of the test. Observe the liquid media every 2 or 3 days and if a color change occurs, subculture.
Note: In addition, one positive and one negative control per solid and liquid medium is required, with an incubation time of 14 days.
We provide a complete portfolio of ready-to-use liquid and solid culture media required for detecting mycoplasmas according to European Pharmacopoeia 6.1 (2.6.7.) and USP 35 (63). Without any further preparation steps, our ready-to-use Mycoplasma culture media ensure constant product quality and complete traceability with lot specific certificates of analyses.
Due to their ingredients, Mycoplasma media generally have far shorter shelf lives than most other culture media for microbiological quality control. The shelf lives of our ready-to-use Friis, Frey, and Hayflick liquid and solid media have been extended to 4 months for the agar plates and 6 months for the broths. Experience our complete range of ready-to-use culture media with a longer shelf life to make Mycoplasma testing easier.
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