Induced and Natural Inhibitory Behavior of Milk and Significance to Antibiotic Disc Assay Testing

Abstract

Summary The nature, incidence, and inducement of natural inhibitory zoning by Grade A raw milks against the test organism Bacillus subtilis , using three types of disc assays, were studied for 2 yr. Natural inhibitory zoning reactions by fresh, normal pH milks predominated on whey agar disc assays and were almost totally absent on Penassay agar and reverse-phase, nonnutrient agar plates. In 707 fresh, raw, Grade A milks, 81 gave small zones by whey agar, occurring mostly during the summer months, whereas only eight milks produced zones on the two other assay methods combined. All natural inhibitory zones disappeared when positive-zoning, normal pH, fresh milks were heated to 180 F—5min. Prior heat treatment of milk to avoid false positives has merit in whey agar assaying, but with Pennassay agar or reverse-phase agar assays such heating may not be quite as necessary. Incubation of Grade A milk at 33C, with resulting low pH and high bacterial numbers, gave to many milks, not hitherto endowed, the ability to produce inhibitory zones against B. subtilis on all three disc assays. Milks reduced to pH 6.0-5.5 showed up to 68% of their numbers with inhibitory zoning ability, while those reduced to pH 5.5-4.6 showed up to 87%. Many low pH milks heated to 180F for 5min retained their zoning ability, even though heat coagulation occurred. Bacterial antibiotic development in low pH milks, resulting from normal milk flora fermentation, apparently explains this newly acquired ability to form inhibitory zones against B. subtilis .