Fluorescence microscopy methods for the determination of somatic cell count in raw cow's milk

Abstract

The direct fluorescence microscopy method with ethidium bromide staining can be used for somatic cell counting in raw cow's milk. However, this method has some limitations that may influence the results of the analysis. We therefore aimed at improving the procedure of somatic cell nuclei staining. We tested the hypothesis that ethidium bromide can better penetrate into the DNA of cells with degraded somatic cell walls or into dead cells. Therefore, we increased the temperature of the sample to 100 °C in order to disrupt the somatic cell wall membrane and to improve ethidium bromide penetration to somatic cell nuclei. In all, 90 samples of raw cow's milk were analysed in this experiment. Three parallel measurements were performed using each of the microscopic methods and the routine flow cytometry method. In all, 810 microscopic smears were analysed. The somatic cells were counted using fluorescence microscopic methods and flow cytometry. The increased temperature during the sample preparation improved (P < 0.005) the penetration of ethidium bromide into the somatic cell nuclei. It is concluded that the direct fluorescence microscopy method is suitable for precise laboratory analysis of somatic cell in raw cow's milk.