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.
The previously reported sodium polyanethol sulfonate disk test for the identification of Peptostreptococcus anaerobius (Graves et al., 1974) was evaluated, with modifications. Three bands of brucella agar, three inoculum sizes, and two inoculum sources were compared. Nine stock cultures of P. anaerobius (eight normal flora isolates and ATCC 27337) and 16 fresh clinical isolates were used. All cultures of P. anaerobius showed inhibition zones of 12 to 30 mm in diameter, regardless of test conditions. Out of 103 clinical isolates of other species of anaerobic gram-positive cocci tested, only two had an inhibition zone size in this range (one P. micros of 11 studied had a zone of 12 mm and one P. prevotii of 14 studied had a zone of 16). The test had an overall accuracy of 98% in the identification of P. anaerobius from clinical specimens. Since P. anaerobius accounts for one-fifth to one-third of all anaerobic gram-positive cocci encountered in clinical specimens, this simple and rapid technique can be very useful for presumptive identification.
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