Survey of Oxidase-Positive and -Negative Bacteria Using a Quantitative Kovacs Oxidase Test

Abstract

A manometric assay system employing ascorbate and N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD) was used to quantitate terminal oxidase activity in bacterial non-proliferating whole cells. A wide variety of physiologically diverse bacteria, all of which were grown heterotrophically, was tested by this assay. For this survey study, 79 bacterial strains, which represented 34 genera, were used. Turbidimetrically standardized resting (non-proliferating) cell suspensions were prepared from cells harvested at the late logarithmic growth phase; all cells were grown under identical nutritional conditions. The TMPD oxidase activity obtained quantitatively correlated exceptionally well with results of the standard Kovacs oxidase test. In fact, the increased sensitivity of this quantitative assay allowed for further reclassification within the two major divisions of Kovacs oxidase-positive and -negative groups. Groups I and II contained all of the oxidase-positive microorganisms and the bacteria listed in group I had the highest TMPD oxidase rates, the Qo2 values (microliters of O2 consumed per hour per milligram [dry weight] at 30 C) ranging from 393 to 2, 164. The organisms listed in group II still had moderately high TMPD oxidase activity, the Qo2 values ranging from 27 to 280. All oxidase-negative bacteria fell into groups III and IV. Bacteria in group III had low but still measurable TMPD oxidase rates, the Qo2 values ranging from 3 to 33, whereas the bacteria found in group IV were inert and unable to oxidize TMPD. A grouping analysis allowed for the resolution of that point which separates oxidase-positive from oxidase-negative bacteria. This point, for non-proliferating cells, was found to be an absolute TMPD oxidation Qo2 value of 33 (after correcting for the endogenous rate by subtraction) and a Qo2 (TMPD/endogenous) ratio of 5; the latter parameter indicated that the uncorrected TMPD oxidation Qo2 value had to be five times greater than the rate for endogenous respiration. All Kovacs oxidase-positive organisms were found to have TMPD oxidase Qo2 values greater than these two metabolic parameters, whereas all Kovacs oxidase-negative organisms had lower values.