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

Abstract

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.

Servicios tecnológicos de laboratorio para el sector agropecuario Colombiano: un análisis científico de los paisajes

“…Además de mejoras en las técnicas de laboratorio para el conteo de células somáticas en leche cruda, como la microscopia por fluorescencia o MIRS (Mid-infrared spectroscopy) para predecir los rasgos de calidad de la leche, la composición de FA (fatty acid -ácidos grasos), MCP (milk coagulation properties), contenido mineral, IBC (Individual Bacterial Cells -Células Bacterianas Individuales), CFU (Colony Forming Units -Unidades Formadoras de Colonias) entre otros. Esto permite vislumbrar otras características de la leche asociados con su valor nutricional, rendimiento de queso y componentes de suero, como glutatión, α-tocoferol y vitamina C (De Marchi, Toffanin, Cassandro, & Penasa, 2014;Tomáška et al, 2006;Zajac, Zubricka, Capla, & Zelenakova, 2016).…”

Rapid Milk Somatic Cell Counting Based on a Customized Multichamber Chip

“…The chamber height of the customized multichamber chip is a very important parameter, which determines whether the milk somatic cells after fluorescent staining can be accurately identified and the accuracy of the cell counting method. Through consulting a large number of milk somatic cell counting articles based on microchamber chips, it was found that the height size of almost all of the microchamber chips involved is 100 μm and below. ,,,, To better study the relationship between the microchamber chip layer height and milk somatic cell count, a batch of cell counting chips was customized by Nanning Matsui Tianlun Company (China), which consisted of a three-layer structure. The bottom layer is a normal glass slide, the middle part is a customized chamber wall structure, which is made of silicone and produced by a customized mold, and the top layer is a glass coverslip.…”

“…Previous studies have shown that highly accurate milk somatic cell counts can be obtained by staining milk somatic cells with fluorescent dyes, which also allows the differentiated analysis of milk somatic cells based on the unique staining characteristics of different milk somatic cell types . In the method of staining milk somatic cells with fluorescent stains, many fluorescent stains can stain the nucleus, such as propidium iodide (PI) stain, DAPI stain, CFDA stain, PYMG stain, PicoGreen stain, and EB stain. ,− Fluorescence detection is to use an excitation light source to excite the target substance to emit fluorescence at a specific wavelength. Only the target substance will absorb a large amount of excitation light energy and produce a fluorescence effect, thus eliminating the interference of other substances and being less affected by external influences.…”

Fabrication of spectroscopic microfluidic chips for mastitis detection in raw milk

“…The microdevice was attached to the optical fiber and connected using the mini-spectrometry method to set up the microchannel connection for the experiment. Structure 1 is the Somatic Cells Filter System, which filters out lymphocytes with a size of 7-18 µm and neutrophils with a size of 10-15 µm, accounting for 70-95% of total somatic cells in milk 26 . Structure 2 is the Mixing System, which mixes the somatic cells filtered from Structure 1 with AO dye.…”