Detection of Penicillin, Cephapirin, and Cloxacillin in Commingled Raw Milk by the Spot Test

The objective of this study was to compare Spot Test results with the results of the Bacillus stearothermophilus disc assay. Commingled raw milk samples were subdivided and spiked with penicillin, cephapirin, or cloxacillin. All subsamples, including unspiked subsamples, were analyzed by the Spot Test (3 or 9 replicates) and disc assay (9 replicates). Mean zone diameter for every subsample was calculated; subsamples having a mean zone diameter greater than or equal to 16 mm were considered positive. At penicillin concentrations less than or equal to 3.7 ng/ml, agreement between the Spot Test and disc assay was 83.4%, and false positive and negative percentages were 14.4 and 2.2%, respectively. Above 3.7 ng/ml, agreement was 100%. At cephapirin concentrations less than or equal to 12 ng/ml, agreement between the two tests was 67.9%, and false positive and negative percentages were 28.8 and 3.3%, respectively. Above 15 ng/ml, agreement was 99.3% with .7% false negatives. At cloxacillin concentrations less than or equal to 50 mg/ml agreement between the two tests was 54.5 with 45.5% false positives. At cloxacillin concentrations greater than or equal to 62.5 ng/ml, agreement between the two tests was 87.2% and false positive and negative percentages were 12.6 and .2%, respectively. In a field trial consisting of 823 bulk samples, Spot Test and disc assay agreement was 100%. The Spot Test is a rapid and reliable method for detecting penicillin, cephapirin, and cloxacillin residues in raw milk at concentrations that will produce a 16-mm zone.