Simple disk technique for detection of nitrate reduction by anaerobic bacteria

The laboratory and clinical evaluation of a potassium nitrate-saturated disk for the rapid detection of nitrate reductase production in anaerobes was investigated. The optimal disk concentration and incubation time were determined by utilizing triplicate sets of quadrant plates prepared with supplemented brucella (Difco) blood agar and swabbed with a 24-h broth (BBL; 135 C thioglycolate) suspension of the test organism. Each set of plates received one control disk and three disks of varying concentrations of potassium nitrate (1 to 8 mg) with 0.1% sodium molybdate. All sets were incubated in GasPak jars for 24, 48, or 72 h, and subsequently sulfanilic acid and 1,6-Cleve's acid were added to each disk. A pink or red color change was indicative of nitrate reductase production. Eighty-eight stock isolates, 23 American Type Culture Collection strains, and 214 fresh clinical isolates were evaluated and compared with results obtained with tubes of preduced indole-nitrite medium (BBL) incubated for 7 to 10 days. The 6-mg disk incubated for 48 h yielded an overall agreement of 89% with the conventional tube technique, and fresh clinical isolates demonstrated better disk-tube agreement (93%) than previously frozen stock strains. The simplicity and ease of this disk test suggest its value as a preliminary screening procedure for nitrate reductase production. There were no false positives. Negative results by disk should be rechecked by tube.