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When there is no alternative to using untreated (not heat inactivated or irradiated) serum in a medium formulation, there is a risk of mollicute contamination. Improvements in filtration may reduce the risk, but also mean that when contamination occurs it can be very low grade and therefore more difficult to detect. A.laidlawii, one of the commonest serum contaminants, is also inhibited in growth by bovine serum.

Prior filtration, if not completely retaining mycoplasmas, should ensure that few viable units contaminate the final serum lot so any test procedure needs to detect contaminants in relatively large volumes.

The previously recommended ‘large volume culture test’ method taken from the findings of M.F. Barile and J. Kern (Proc. Soc. Ep. Biol. Med., 1971, 138, 432-437) who detected Mycoplasma arginini at an incubation temperature of 36°C and Acholeplasma laidlawii after six weeks incubation at 32°C, is cumbersome, costly and time-consuming. Bovine serum is also inhibitory for A.laidlawii growth. PCR testing is not suitable as it does not distinguish between DNA from viable contaminants and residues from non-viable organisms, additionally, the sample volumes used are inadequate to detect the expected low numbers.

As such, we have developed a new rapid serum test protocol, using 100mL samples of serum.


100 mL volumes of bovine serum are filtered through 0.1uM filters and the filters then transferred to 20mL ME liquid medium to test for the presence of mycoplasmas. Quality control testing of the liquid medium, and all solid medium lots used, includes M. Arginini, M.Bovis  and A. Laidlawii. Samples spiked with these organisms and passed through 0.1uM filters show evidence of growth in broth within 3 days of incubation. The broth + filter is incubated for 14 days with a subculture onto agar at 7 days if no growth is observed. Test results are available 14 days from initiation of the test.

The guidelines of Good Laboratory Practice are followed in all aspects of our work and copies of the documents used to record test progress and the quality control of media lots are included with the final report.



Where, for example, horse serum, pig serum or other serum sources require testing, we could include a mycoplasma species commonly found in the host animal for test media lot validation (e.g. M. equirhinis with horse serum).

We would be pleased to arrange a quotation for alternative test protocols, either as supplied by the client or designed to meet your needs.


Mycoplasma Experience scientists could advise serum manufacturers on the development and validation of new and improved procedures for testing sera for mycoplasma contamination.




1 X 100mL


3 x 100mL

(sampled at the start,

middle and end of the run process)