The differentiation of Brucella species by substrate specific tetrazolium reduction

Abstract

In the development of a tetrazolium reduction assay to replace substrate stimulated oxygen uptake for the identification of Brucella species, nine tetrazolium salts were evaluated. Only the more readily reduced compounds (MTT and INT) detected increased metabolic activity with the more fastidious, slow growing strains and with B. suis strains on L-arginine and DL-ornithine. The assay was optimised with MTT. MTT reduction profiles offered with the medium on which the cells were grown. Cells grown on TSA gave profiles more similar to the published respirometric results than those grown on SDA. The optimal substrate concentration was 0.84 g l-1 and prolonged (> 3 h) exposure to substrate was necessary before adding MTT. MTT concentration was not critical and the OD was proportional to the MTT concentration between 0.03 and > 0.5 g l-1. MTT reduction was linear for 60 min after its addition. The reaction was, therefore, stopped after 60 min by adding formaldehyde solution. The optimised assay was evaluated with 71 strains of Brucella, representing all the species and biovars of the genus. Each strain was assigned to its previously identified species and sub-groups were defined.