Isolation of Mycoplasma hyorhinis and Mycoplasma fermentans from cell cultures

Abstract

A mycoplasma medium with boiled blood and sucrose (Herderschee medium), a commercially available (Difco) PPLO medium and an enriched trypticase soy broth medium were tested for their suitability for isolation and subcultivation of a fastidious strain of Mycoplasma hyorhinis (DBS 1050) and a M. fermentans strain (IM 1) isolated on Herderschee medium. The mycoplasma strains were maintained in VERO-cell cultures, for which M. fermentans proved to be very pathogenic. It was therefore, stored at −70°C after two passages in the cell culture. M. hyorhinis was inoculated in various VERO-cell passages and subsequently propagated in the cells. On Herderschee and Difco media M. hyorhinis strain DBS 1050 could always be isolated from the cell cultures one to four days after trypsinization of the cells. Recovery succeeded better from frozen (−70°C) cell suspensions than from supernatant cell culture medium, and also better after direct inoculation on agar medium than after inoculation in broth. Primary isolations on agar medium could nearly always be subcultivated. The concentration of organisms detected on agar medium at primary isolation and the facility of subsequent subcultivation in mycoplasma medium seemed to depend partly on the number of mycoplasma passages in the cell culture, and partly on the subline of the cell culture used for inoculation. M. fermentans strain IM 1 could be isolated on Herderschee medium and on Difco medium with sucrose from supernatant cell culture medium as well as from frozen cell suspensions and equally well from one to seven days after trypsinization of the cell culture, not just after trypsinization. Both mycoplasma strains were only detected after anaerobic incubation (5% CO2 in N2). They could be cultivated as well when 2·5% instead of 10% (w/v) yeast extract was incorporated in the media. The yeast extract could be boiled and heated up to 120°C without impairing its growth promoting quality. Therefore, nicotinamide adenine dinucleotide is dispensable for these two strains. Neither mycoplasma strain could be cultivated on enriched trypticase soy broth medium. The implication of these observations when testing cell cultures and live virus vaccines for contamination with mycoplasma are discussed.