Effects of storage methods on the recovery of Mycoplasma species from milk samples.

Mycoplasma species are fastidious microorganisms causing mastitis in dairy cows. Storage by freezing milk samples affects their viability. The purpose of this study was to compare the effect of alternative storage methods on their recoverability. In Experiment I, mycoplasma counts from fresh milk samples were compared to those same samples stored for 1, 3, and 5 days at refrigerated (5 degrees C) temperatures. Experiment II was done to compare the mycoplasma counts of fresh milk samples with those stored frozen (-20 degrees C) with addition of 0%, 10%, 30% and 50% glycerol (v/v). Two strains of each of 5 species: M. bovis, M. californicum, M. bovigenitalium, M. canadense and M. alkalescens, were selected and inoculated into bulk tank milk free of this pathogen. Compared to those in fresh milk samples, counts were approximately reduced by: 0.3 log(10)CFU/ml in 5 day refrigerated milk (P<0.05) and by 1.0 log(10)CFU/ml in milk frozen without glycerol (P<0.05). Addition of glycerol (10% and 30%, v/v) to milk samples increased the number of recovered Mycoplasma by up to 0.4 log(10)CFU/ml in frozen milk samples (P<0.05). No significant interactions were detected between either Mycoplasma species or starting concentration and survival as effected by storage method. Refrigerating milk samples for 5 days and freezing milk samples lowers the number of recovered Mycoplasma species. The addition of glycerol to achieve 10% and 30% v/v solutions improves the recovery of Mycoplasma species from frozen milk samples. To maximize detection of this pathogen, fresh milk samples should be cultured without storage.

Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster.

AIMS: To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas. METHODS AND RESULTS: Growth, substrate preferences and tetrazolium reduction were assessed for 18 strains of Mycoplasma bovigenitalium and Mycoplasma ovine serogroup 11. NBT reduction was detectable in 1 h with 10(8) CFU ml(-1). Use of alpha-ketobutyrate, lactate and pyruvate to support growth and NBT reduction were correlated: pyruvate was preferred and lactate was used by only four of the 18 strains. Selected members of the Mycoplasma mycoides cluster were also assessed and monotetrazoles tested as alternatives to NBT. The NBT method was applied to a further 19 species. CONCLUSIONS: This simple and reproducible method requires only small amounts of cells, enabling routine assessment of substrate use within 1 h, and the rapid assignment of numerous mycoplasmas to one of six physiological groups. The four physiological groups of M. bovigenitalium and Mycoplasma serogroup 11 strains were indistinguishable from each other, which supports the view that these belong to the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific substrate-utilization patterns by mycoplasmas can be obtained rapidly and reliably. The method has potential as a large-scale semi-automated procedure to monitor numerous strains and substrates simultaneously.