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.
OBJECTIVE
To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas.


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.


CONCLUSIONS
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.
Lin
Y-C
YC
Department of Life Sciences, King's College London, Franklin, London, UK.
Agbanyim
C N M
CN
Miles
R J
RJ
Nicholas
R A J
RA
Kelly
D P
DP
Wood
A P
AP
D001431:Bacteriological Techniques; D001483:Base Sequence; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D045646:Mycoplasma bovigenitalium / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009176:Mycoplasma mycoides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D021521:Ribotyping; D013379:Substrate Specificity; D013778:Tetrazolium Salts / Q000378:metabolism*
D013778:Tetrazolium Salts