Yazdankhah S P, Sørum H, Larsen H J, Gogstad G
Department of Pharmacology, Microbiology, and Food Hygiene, Norwegian School of Veterinary Science, N-0033 Oslo, Norway.
J Clin Microbiol. 2001 Sep;39(9):3228-33. doi: 10.1128/JCM.39.9.3228-3233.2001.
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 to 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 moderate or severe 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. The detection limit was 5 x 10(6) CFU/ml for Staphylococcus aureus and 1 x 10(6) 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.
本文描述了一种用于检测牛奶中革兰氏阳性菌和革兰氏阴性菌的快速方法。该技术基于将样品在培养基中稀释，然后通过具有一定孔径的多孔聚砜膜进行过滤，从而截留并浓缩样品中的细菌。浓缩在膜表面的细菌用一种阳离子染料（甲苯胺蓝）染色，这种染料肉眼可见。染色后，将膜用乙醇 - 乙酸（pH 2.8至3.0）处理，这会使革兰氏阴性菌脱色，而革兰氏阳性菌则保留染色。该方法不需要热固定、电力、显微镜检查或经过专门培训的人员。进行该测试所需的时间约为5分钟。该技术应用于人工感染的牛奶以及患有中度或重度临床乳腺炎奶牛的牛奶，以检测和区分细菌。与牛奶样品的传统细菌培养相比，过滤法对革兰氏阳性菌和革兰氏阴性菌的灵敏度分别为92%和100%。在加标牛奶样品中，金黄色葡萄球菌的检测限为5×10⁶CFU/ml，大肠杆菌的检测限为1×10⁶CFU/ml。该方法的总体特异性为86%。这种诊断方法可为兽医在乳腺炎治疗中优化抗生素的使用提供现场指导。
