激光共聚焦显微镜观察阴道来源乳杆菌生物膜的形成

目的:采用激光共聚焦显微镜技术动态观察一株女性阴道来源卷曲乳杆菌生物膜的形成过程. 方法:使用体外盖玻片生物膜培养法,培养阴道来源卷曲乳杆菌的生物膜,在培养2、4、8、12、16、20、24、48、72、96、120 h后取出盖玻片,用异硫氰酸荧光素标记的刀豆蛋白A( fluorescein isothiocyanate-conjugatedconcanavalin A,FITC-conA)和碘化丙啶( andpropidium,PI)双重免疫荧光染色,激光共聚焦显微镜( confocal laser scanning microscopy, CLSM)观察卷曲乳杆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,观察到卷曲乳杆菌在4 h即开始有散在的细菌黏附于盖玻片上,为可逆吸附期;8~20 h细菌黏附量逐渐增加,进入不可逆吸附期,20 h细菌聚集成团,生物膜初步形成;24~48 h形成大片的生物膜菌落,细菌镶嵌在大量多糖基质中,结构紧密,形态稳定,生物膜成熟;72 h后生物膜菌落开始解聚. 培养20 h乳杆菌生物膜密度为42. 7 × 10 -3 ± 6. 83 × 10 -3 ,24 h上升为102.5 ×10 -3 ±23.14 ×10 -3,两者相比差异具有统计学意义(P<0.05),表明培养24 h乳杆菌生物膜形成. 结论:该株阴道来源卷曲乳杆菌在体外可形成生物膜,24 h可形成成熟的生物膜,72 h生物膜开始解聚再定植.

Confocal laser scanning electron microscopy for assessment of vaginal Lactobacillus crispatus biofilm

Objective:To investigate the female vaginal Lactobacillus crispatus biofilm by using confocal laser scanning microscopy (CLSM),thus revealing the formation of biofilm. Methods:The cover slide biofilm culture approach in vitro was employed for induction of the vaginal Lactobacillus crispatus biofilm formation. Following the culture for 2, 4, 8, 12, 16, 20, 24, 48, 72, 96 and 120 hours, the cover slide was removed for subsequent staining with the fluoresce in isothiocyanate-conjugated concanavalin A ( FITC-ConA) and propidium ( PI) . This was followed by determination of the formation and characteris-tics of the vaginal Lactobacillus crispatus biofilm by using CLSM. Results:The CLSM images of biofilm formation at different time points were captured, suggesting that the vaginal Lactobacillus crispatus adhe-sion occurred at h 4, which was in reversible attachment, then more and more Lactobacillus crispatus ag-gregated at h 8 to h 20, which was in irreversible attachment. Lactobacillus crispatus clustered at h 20, with early development of biofilm architecture. Then the biofilm with extracellular matrix around the bacte-ria was set up at h 24,with gradual matureation at h 24 to h 48. The biofilm dispersed at h 72. The bio-film density of cultivating for 20 hours was 42 . 7 × 10 -3 ± 6 . 8 × 10 -3 , and for 24 hours increased to 102. 5 × 10 -3 ± 23. 1 × 10 -3 , suggesting a significant difference, P<0. 05. This meant that mature bio-film was formed at h 24 . Conclusion:The vaginal Lactobacillus crispatus is able to form typical biofilm with distinct developmental phases and architecture characteristics. Mature biofilm is formed at h 24 to h 48, then the biofilm begins to disperse.