The Bacillus stearothermophilus disc assay is routinely used by the dairy industry to screen milk for antibiotic residues. Although the assay detects the presence of beta-lactam antibiotics, it does not distinguish cephalosporins from other beta-lactam antibiotics. In this study, the B. stearothermophilus disc assay was modified to allow it to distinguish parent ceftiofur from other antibiotics by incorporation of the enzymes penicillinase and cephalosporinase into the assay. The modified B. stearothermophilus disc assay involves determining the zone of inhibition of a sample on an agar plate after the plate was incubated at 65 degrees C for 2.5 to 3 h as well as determining the zone of inhibition after the sample was treated with penicillinase or cephalosporinase. Samples in which this zone diameter was > 19 mm and < or = 25 mm were interpreted using the data from the primary assay. Samples with zone diameters > 25 mm must be diluted 2- to 10-fold and reassayed to obtain a zone diameter > 19 and < or = 25 mm, for proper interpretation. Samples with zone diameters > or = 16 mm and < or = 19 mm must also be reassayed using dilute enzyme solutions for proper interpretation. When these modifications of the B. stearothermophilus disc assay are used, ceftiofur can be distinguished from ampicillin, amoxicillin, penicillin, cephapirin, cloxacillin, novobiocin, and pirlimycin for samples with zone diameters > or = 16 mm. This assay cannot, however, separate ceftiofur from cefazolin.(ABSTRACT TRUNCATED AT 250 WORDS)
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