Development of a miniaturized nitrate reduction test for the identification of oral bacteria

Abstract

A miniaturized nitrate reduction test (MNRT) for oral bacteria was developed and its reliability compared with a conventional nitrate reduction test (CNRT). In the MNRT 100 [mu]l aliquots of freshly grown heavy suspension of various oral bacterial species, in physiological saline, were added to equal volumes of 0.1% filter-sterilized KNO3 solution in distilled water in wells of transparent plastic plates. Duplicate plates were incubated aerobically or anaerobically at 35[deg]C for 12-15 h. At the end of the incubation period the test was performed by adding either a trace amount of a non-liquid reagent (mixture of -(+)-tartaric acid, sulfanilic acid and 1-naphthylenediamine dihydrochloride, 10:1:1, wt/wt) or conventional liquid reagents A and B (sulfanilic acid and N,N-dimethyl-1-naphthylamine). In the conventional nitrate reduction test (CNRT), tubes of a basal anaerobic broth were inoculated with the same bacterial species used for MNRT, and the nitrate reduction tests performed after anaerobic incubation of the cultures for 4-6 days. Several hundred anaerobic and facultative bacterial isolates belonging to genera Veillonella, Bacteroides, Fusobacterium, Selenomonas, Actinomyces and Capnocytophaga were characterized by MNRT and CNRT. Analysis of the data showed that MNRT and CNRT systems were comparable. In the MNRT system Veillonella parvula and Selenomonas sputigena were capable of reducing nitrate only under anaerobic conditions. Actinomycetes reduced the nitrates under aerobic and anaerobic conditions, while all black-pigmented Bacteroides, Fusobacterium and Capnocytophaga species did not reduce nitrate. These findings suggest that the MNRT is reliable, rapid and may be conveniently used in clinical or research laboratories with a heavy microbiological work load.