FM4-64和Hoechst染料在活细菌胞膜和拟核标记定位中的条件优化与应用

Optimization of labeling and localizing bacterial membrane and nucleus with FM4-64 and Hoechst dyes

摘要:

摘要:【目的】为了观察细菌细胞膜和拟核的形态结构，准确对亚细胞进行定位。【方法】本文利用FM4-64和Hoechst两种染料，分别采用不同浓度和不同时间对大肠杆菌活细胞进行染色和荧光共聚焦显微成像，并对7种细菌的活细胞(金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、鼠疫耶尔森菌、军团菌、霍乱弧菌和炭疽芽孢杆菌)进行染色观察。【结果】相同观察条件下，不同染料浓度和不同的染色时间影响了细胞膜、拟核的荧光强度;确定了FM4-64染色通用条件(浓度20 μg/mL 染色1 min)和Hoechst的最佳条件(浓度20 μg/mL染色20min)。上述条件下，Hoechst对8种细菌染色效果均较理想，然而FM4-64对细菌染色效果不同，革兰氏阴性菌(大肠杆菌、肺炎克雷伯菌、鼠疫耶尔森菌、霍乱弧菌、铜绿假单胞菌和军团菌)表现较好的细胞膜轮廓，革兰氏阳性菌(炭疽芽孢杆菌和金黄色葡萄球菌)效果较差。【结论】FM4-64和Hoechst两种染料共染8种细菌，对细胞膜和拟核的染色观察，可为原核细胞结构染色提供借鉴，并为大分子的亚细胞定位研究奠定基础。

Abstract:

Abstract:［Objective］To observe cell membrane and nucleus in bacteria for subcellular localization.［Methods］FM4-64 and Hoechst were dyed that can label cell membrane and nucleus，respectively． Both dyes were used to co-stain the membranes and nucleus of eight bacterial strains ( Escherichia coli，Staphylococcus aureus，Pseudomonas aeruginosa，Klebsiella pneumoniae，Yersinia pestis，Legionella pneumonia，Vibrio cholerae and Bacillus anthracis)． E.coli was dyed with different dye concentrations and times and then observed by confocal fluorescence microscopic imaging.［Results］Fluorescence intensity of cell membrane and nucleus is affected by dye concentrations and times． The optimal conditions were determined as follows: staining cell membrane with 20 μg/mL FM4-64 for 1 min and cell nucleus with 20 μg/mL Hoechst for 20 min．Gram-negative bacteria were dyed better than gram-positive bacteria with FM4-64dye．［Conclusion］FM4-64 and Hoechst can be used to stain membrane and nucleus in different types of bacteria．Co-staining bacterial membrane and nucleus provides the reference to observe cell structure in prokaryotes for studying subcellular localization．