Rapid cytochemical demonstration of cytochrome oxidase activity in pathogenic bacteria

To develop and assess a cytochemical technique for the light microscopical detection of oxidase activity in pathogenic bacteria.

Live bacterial cells were deposited on to aminopropylsilane treated glass coverslips by centrifugation, dried, then reacted with either 1% (w:v) n,n,n',n'-tetramethyl-p-phenylene diamine (TPD) or 5 mM diaminobenzidine (DAB) at 37 degrees C. The preparations were mounted in 50% glycerol and assessed by brightfield microscopy. An optimised DAB procedure (5 minutes of drying at 37 degrees C and 10 minutes of reaction time) was applied to 44 strains of organisms commonly associated with infections in man and to two fresh positive blood culture containing Gram negative bacilli.

TPD gave no discernible localised reaction product and was not investigated further. With DAB, oxidase positive cells (brown) were clearly differentiated from oxidase negative cells (colourless) even in mixed preparations. The DAB technique correctly assigned 18 oxidase positive isolates (seven genera), 26 oxidase negative isolates (eight genera), and the organisms present in the two freshly positive blood cultures to their appropriate oxidase reactivity as defined by the standard macroscopic TPD technique.

The cytochemical reaction seems to be a reliable indicator of the macroscopic oxidase test. It permits determination of oxidase reactivity at an early stage in the assessment of clinical material when infecting organisms can be demonstrated by microscopy. Further development of this and related cytochemical techniques could permit the provision of microbiological information that would be relevant to patient management, well in advance of conventional techniques.