Tetramethyl-p-phenylenediamine (TMPD) oxidase activity and cytochrome distribution in the genus

Kovacs' [1] qualitative test for N,N,N',N',-tetramethyl- p-phenylenediamine (TMPD) oxidase activity is a fundamental test in the identification of bacteria [2,3]. TMPD (E~ = +0.26 V at pH 7.0) acts as an artificial electron donor to the terminal portion of aerobic respiratory systems [4], and the ability to oxidise TMPD, reduced by ascorbate, has been widely used as a quantitative assay of terminal oxidase activity [5]. In a wide range of heterotrophic bacteria there is a close correlation between their response to the Kovacs test and their ascorbate-TMPD oxidase activity measured manometrically [6]. The inability to oxidise ascorbate-TMPD or a negative response to the Kovacs test, however, does not necessarily indicate that an organism lacks an aerobic respiratory system and a terminal oxidase. For example, species belonging to the family Enterobacteriaceae are oxidase- negative yet form aerobic respiratory systems[5,7]