激光共聚焦显微镜观察铜绿假单胞菌生物膜的形成

Confocal laser scanning microscopy for assessment of pseudomonas aeruginosa biofilm formation

目的采用激光共聚焦显微镜技术观察铜绿假单胞菌形成生物膜,进一步揭示了铜绿假单胞菌生物膜形成过程。方法使用盖玻片生物膜培养法,培养铜绿假单胞菌的生物膜,在培养2、4、8、12、16、24、48和72h后取出盖玻片,用异硫氰酸荧光素(FITC)标记的刀豆蛋白A(FITC-ConA)和碘化吡啶(PI)双重免疫荧光技术染色,用激光共聚焦显微镜(CLSM)观察铜绿假单胞菌生物膜形成过程与特点。结果获得生物膜形成过程不同时间点的CLSM图像,观察到铜绿假单胞菌一般在24h后开始逐渐形成生物膜,在72h形成稳定的生物膜。结论双重免疫荧光染色技术和CLSM是观察细菌生物膜形成过程的有效手段。

Objective To investigate the Pseudomonas aeruginosa biofilm by using confocal laser scanning microscopy （CLSM）, thus revealing the formation of biofilm. Methods The cover slide biofilm culture approach was employed for induction of Pseudomonas aeruginosa biofilm formation. Following the culture for 2, 4, 8, 12, 16, 24, 48 and 72 hours, the cover slide was removed for subsequent staining with the fluoreseein isothiocyanate-labeled concanavalin A （FITC-ConA） and iodidepyridine （PI）. This was followed by determination of the formation and characteristics of Pseudomonas aeruginosa biofilm by using CLSM. Results The CLSM images of biofilm formation at different time points were captured, suggesting that the biofilm formed at hour 24 and grew robustly at hour 72. Conclusion The double immunofluoreseence staining and CLSM are effective means for assessment of Pseudomonas aeruginosa biofilm formation.