The Cell Culture Test

MYCOPLASMAS: THE CELL BIOLOGISTS HIDDEN ENEMY

Cell cultures provide an ideal cultural environment for contaminating mycoplasmas which, once an infection is established, can spread rapidly to other cell lines in the laboratory. Mycoplasmas have been recognised as common contaminants of mammalian cell cultures for many years and they should not be overlooked as potential contaminants of insect or plant cell lines.

Whilst mycoplasmas, unlike bacterial or fungal contaminants, may not reveal their presence they can have a profound effect on cell cultures. To help you appreciate how mycoplasma infection could affect your work, Mycoplasma Experience has prepared a microfilm sheet of over 200 selected references on cell culture infection with mycoplasmas. (This is available on request and may be read under a microscope at low magnification if a viewer is unavailable).

Regular testing for mycoplasmas is the only way to be sure of working with mycoplasma-free cell cultures leading to confidence in the quality and validity of data obtained.

TESTING FOR MYCOPLASMAS

A combination of direct culture on mycoplasma media and an indirect, or non-cultural, method is generally recommended for reliable examination of cell cultures for mycoplasma infection. Direct culture, if properly controlled, is widely accepted as being the most sensitive method for their detection; the indirect test is required for detection of mycoplasma strains which do not grow on conventional media formulations.

The existence of "non-cultivable" strains of M. hyorhinis as cell culture contaminants was recognised in 1973 (H.E. Hopps et al. Ann. N.Y. Acad. Sci. 1973, 225, 265-276) and these strains are now identified as M. hyorhinis cultivar a . Subsequently fastidious strains of

M. orale (M. Merkenschlager et al. Immunology 1988, 63, 125-131) and M. fermentans (A. A. Polak-Vogelzang et al. J. Biol. Stand. 1980, 4, 243-254), both of human origin, have been identified.

Where non-cultural methods are employed for mycoplasma detection the inclusion of adequate controls is as important as the quality control testing of media used for mycoplasma isolation. A number of non-cultural techniques have been developed, the most widely used being DNA fluorochrome staining. The sensitivity of the test (10⁵ colony forming units/ml, if applied directly to the test culture; M. Thangavelu et al. Hum. Genet., 1979, 47, 199-202) is much improved by co-cultivation of the test sample with a stable cell culture for several days prior to staining, but the failure of the infecting mycoplasma to cytadsorb can be a hindrance to detection. Some common cell culture contaminants, including M. orale, show little or no cytadsorption.

Extensive media development by Mycoplasma Experience scientists has resulted in media which support the growth of M. hyorhinis cultivar a as well as many mycoplasma species recognised as difficult to culture including fastidious strains of M. orale and M. fermentans. (The strain of M. orale encountered by Merkenschlager is used routinely for quality control of media). Use of these media for cell culture testing eliminates the need for additional testing methods and permits detection of low level infection of both fastidious and less culturally demanding strains independently of their capacity to cytadsorb.

TESTING WITH MYCOPLASMA EXPERIENCE

After secure transportation from your laboratory under controlled conditions, the cell culture test is conducted as follows - samples are inoculated into both liquid and solid media, the liquid being subcultured onto solid medium after seven and fourteen days incubation. To ensure sensitivity the test continues for 28 days but, as many common mycoplasma species grow rapidly in the media, positive results would be likely within seven days.

Each lot of medium used in your test will be tested with low numbers of M. pneumoniae (human pathogen), M. orale (human throat commensal, cell culture isolate), M. hyorhinis cultivar a (porcine origin, cell culture isolate) and M. synoviae (avian pathogen, presently unique in its requirement for NAD). Additionally, media constituents are tested for their ability to support two, serum derived, common cell culture contaminants M. arginini and Acholeplasma laidlawii.

IDENTIFICATION

Identification can aid in tracing the source of infection and thus help avoid further outbreaks. Identification of isolates is an integral part of the cell culture test and will be initiated as soon as an isolation is confirmed. Although most mycoplasma infection of cell cultures is caused by a limited number of species, many less common species have been reported, infection with more than one mycoplasma species can occur and there is the possibility of isolating novel species.

REPORTING

On completion, a report is issued together with copies of the test worksheet and copies of media validation sheets which record the QC testing of all media lots used in the test.

Mycoplasma Experience

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