Development of a Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Severe and Moderate Clinical Mastitis

A rapid method for demonstration of Gram-positive and Gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8–3.0), which causes decolorization of Gram-negative bacteria, whereas Gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with severe and moderate clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92%, and 100% for Gram-positive, and Gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples from cows with severe and moderate clinical mastitis. The detection limit was 5 × 10⁶ CFU/ml for Staphylococcus aureus and 1 × 10⁶ CFU/ml for Escherichia coli in spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.