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A collaborative study was conducted to determine the reliability of a Bacillus stearothermophilus disc assay method for differentiating various concentrations of penicillin in raw milk. Participating laboratories tested 10 different samples (including one negative) in blind duplicate. Triplicate standards were alternated with triplicate unknowns around the periphery of each of 5 different plates. Zone diameters were measured and the difference in zone size of pairs of adjacent standard and unknown samples were analyzed by a paired t-test. Penicillin concentrations 0.003 IU/mL different from the reference concentrations were consistently distinguishable at a 95% confidence level. Such discriminatory power was determined to be possible with as few as 3 plates (9 replicates) per unknown. The method has been adopted official first action.