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The effect of fresh whole milk without pasteurization or other pretreatment on in vitro antibacterial activity of selected compounds was determined in broth dilution. The milk was collected by hand directly from dairy goats, or by syringe or cannula from bovine quarters showing low bacterial counts. Antibacterial activity was determined in 50% (v/v) milk-broth medium against sensitive mastitis-etiologic strains of Streptococcus agalactiae and Staphylococcus aureus. The indicator salt 2,3,5-triphenyltetrazolium chloride was incorporated in the milk broth medium to determine inoculum growth. Contaminant interference was circumvented through early as well as late readings and comparisons with uninoculated control tubes, with and without the test compounds. Application of the method with more than 75 compounds, including nitrofurans, antibiotics, and other chemicals uncovered marked degrees of milk interference. The method warrants routine use among preliminary screens to relate in vitro with in vivo observations of antimicrobial activity. Similar procedures may be used with serum, skim milk, or mastitis-milk media for separating effects due to protein, lipid, or other elements in product evaluation.