利用Fura-2/AM荧光探针法和LCSM法研究受磁场处理S.aureus细胞Ca2+的跨膜行为

研究受脉冲磁场处理金黄色葡萄球菌(S.aureus)细胞Ca2+的跨膜行为,为此研究了表征S.aureus胞内Ca2+浓度变化Fura-2/AM荧光探针检测法,并检测了不同脉冲数下经脉冲磁场处理后S.aureus细胞荧光强度的变化,采用激光共聚焦扫描显微镜(LCSM)观察了经脉冲磁场处理前后S.aureus胞内Ca2+浓度的变化。研究结果表明,Fura-2/AM可成功的负载于S.aureus中,可以应用于S.aureus胞内游离钙离子浓度变化的测定。经脉冲磁场处理后,S.aureus胞内钙离子浓度显著上升,且与活菌数的减少高度相关,相关度达到-0.989 15;胞内光点显著增多,光点荧光强度明显增大,说明大量胞外钙离子跨膜进入胞内。因此,可以判断微生物细胞膜通透性的改变和胞内Ca2+浓度的上升是高强度脉冲磁场具有杀菌作用的重要原因。

Study on Ca2+ transmembrane behaviors of magnetic-treated S. aureus with Fura-2/AM fluorescence probe and LCSM

The purpose of the present paper is to study Ca2+ transmembrane behaviors of the S. aureus treated with high intensity pulsed magnetic field. For this purpose, the method of Fura-2/AM fluorescence probe was investigated to determine the change in intracellular Ca2+ concentration in S. aureus, fluorescence intensities of S. aureus cells treated by pulsed magnetic field under different pulse number were determined, and the change in intracellular Ca2+ concentration was observed by laser confocal scanning microscope (LCSM). Research results showed that Fura-2/AM can load in S. aureus successfuuly, and can be used to determine the change in intracellular free Ca2+ concentration. After being treated with high intensity pulsed magnetic field, intra cellular free Ca2+ concentration of S. aureus went up significantly, and the regulation of Ca2+ increase was close consistent with the decrease in living bacterium number, and relatedness reached to -0.989 15; the number and fluorescence intensity of intracellular lightspot increased significantly, meaning that large amount of exocellular calcium ions across cell membrane enter into inside of cells. So, it was judged that the change in microbial membrane permeability and increase in intracellular free Ca2+ concentration are important reasones why high intensity pulsed magnetic field has sterilization effect.