Cytochrome oxidase analysis of Bacillus species

Organisms of the genus Bacillus were surveyed for cytochrome oxidase activity using a quantitative colorimetric N,N,N',N'-tetramethyl- p-phenylenediamine (TMPD) oxidase assay. This assay allowed for the calculation of cytochrome oxidase specific activities and provided a quantitative method for evaluating the taxonomic position of Bacillus strains. Three oxidase reacting groups were readily identified; (a) the TMPD oxidase positive group; (b) the oxidase negative group; and (c) the TMPD oxidase indeterminate group. This latter group of Baci1lus strains oxidized TMPD at rates that were slightly higher than the autooxidation rate. This oxidase survey analysis confirmed that Bacillus species are a highly heterogenous taxonomic group. Thirty-five type strains, representing 26 species (9 representing duplicate cultures obtained from different sources) were tested for TMPD-dependent cytochrome oxidase activity using both standardized whole cells and sonically 'opened' cells. Sonically 'opened' cells were used to examine the effects of cellular permeability of TMPD. Cells analyzed from most Bacillus type strains showed only minor permeability effects relating to TMPD oxidation. In no case was an oxidase positive reaction induced (with an oxidase negative strain) by sonically 'opening' cells and exposing the membranes-bound cytochrome oxidase to the TMPD-dye reagent. [...]