Nitrate reduction test

Nile Blue agar was prepared as described (Luellen and Schroth, 1994 (link)). A representative of each bacterial isolate was plated and incubated for 48 to 72 h at 30°C. The plates were then viewed under UV light to detect putative PHB production based on the fluorescence of the colonies. Nitrate reduction test was purchased from Sigma and conducted following the instructions provided by the manufacturer. A representative of each bacterial isolate was inoculated at high density, and tubes were sealed with liquid paraffin to create oxygen-poor conditions and incubated at 30°C up to 5 days.

Four suspected Neisseria spp. isolates were grown on GC agar plates (Oxoid) with Kellogg’s [29 (link)] and 5 % Fe(NO₃)₃ supplements at 37 °C with 5 % CO₂. Control species for India ink staining, Escherichia coli and Bacillus subtilis, were grown on nutrient agar (Oxoid) and incubated at 37 °C. Blood agar (Oxoid) included 7 % defibrinated horse blood (Oxoid) to test for haemolytic activity. API NH strips (bioMérieux) to identify the species were used according to the manufacturer’s instructions. The Nitrate Reduction Test (Sigma) was performed according to manufacturer’s instructions to identify samples capable of reducing nitrates.