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 less than or equal to 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 less than or equal to 25 mm, for proper interpretation. Samples with zone diameters greater than or equal to 16 mm and less than or equal to 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 greater than or equal to 16 mm. This assay cannot, however, separate ceftiofur from cefazolin. The modified B. stearothermophilus disc assay provides an inexpensive and simple method to detect antibiotics in milk and to differentiate parent ceftiofur from most other beta-lactam and non-beta-lactam antibiotics.
