激光扫描共聚焦显微镜观察细菌生物膜形成的方法学研究

目的:建立激光扫描共聚焦显微镜观察生物膜形成过程的方法,为进一步研究生物膜的形成机制奠定基础。方法:以临床分离金葡菌X428为研究对象,在盖玻片上形成生物膜,分别于接种后的4、8、12、16、24和48h取出玻片,采用免疫荧光技术标记多糖和细菌,激光扫描共聚焦显微镜(CLSM)观察生物膜形成情况。结果:取得了生物膜形成过程的不同时间点的CLSM图像,4h时细菌在盖玻片上粘附形成小菌落,8h和12h细菌聚集成簇,多糖基质产生并逐渐增多,至16h形成成熟生物膜结构;24h和48h生物膜已经播散,其结构变小。结论:应用免疫荧光技术和激光扫描共聚焦显微镜技术研究生物膜形成过程是一种简便可行的方法。

Study on Method for Observation of Bacterial Biofilm Formation under Confocal Laser Scanning Microscope

Objective:To establish the method of observation of bacterial biofilm formation under confocal laser scanning microscope(CLSM),so as to provide the basis for further study on the mechanism of biofilm formation.Methods:X428,a clinical isolation of Staphylococcus Aureus(SA),was selected to be investigated.Cover glasses were used to culture biofilms and collected at 4th,8th,12th,16th,24th and 48th hour of incubation.Polysaccharides and bacteria were labeled by immunofluorescent technique. The condition of SA biofilm formation was observed under confocal laser scanning microscope(CLSM).Results:A series of images of biofilm were collected by CLSM.Tiny colonies adhered on glasses at the 4th hour of incubation.Bacteria aggregated into dusters and polysaccharides were created at the 8th and 12th hour.Polysaccharides accumulated gradually until mature biofilms were formed at the 16th hour.Biofilms had dispersed at the 24th and 48th hour.Conclusions:It's a simple and feasible method to study the process of biofilm formation by using immunofluorescent technique and confocal laser scanning microscope.