Rapid method for detection of gram-positive and -negative bacteria in milkfrom cows with moderate or severe clinical mastitis

A rapid method for demonstration of gram-positive and gram-negative bacteri a in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane wit h a pore size retaining and concentrating bacteria from the sample. The bac teria concentrated on the surface of the membrane are stained with a cation ic dye (toluidine blue) that can be visualized by the naked eye. After stai ning, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), whi ch causes decolorization of gram-negative bacteria, whereas grain-positive bacteria retain the stain. The method does not require heat fixation, elect rical power, microscopic examination, or specially trained personnel. The t ime needed to perform the test is approximately 5 min. The technique was ap plied to artificially infected milk and milk from cows with moderate or sev ere clinical mastitis for detection and differentiation of bacteria. The se nsitivity of the filtration method was 92 and 100% for gram-positive and gr am-negative bacteria, respectively, compared with traditional bacteriologic al culture of milk samples. The detection limit was 5 x 10(6) CFU/ml for St aphylococcus aureus and 1 x 10(6) CFU/ml for Escherichia coli in spiked mil k samples. The overall specificity of the method was 86%. This diagnostic m ethod can provide on-site guidance to the veterinarian to optimize use of a ntibiotics in mastitis therapy.