天然菌斑生物被膜中变形链球菌、远缘链球菌和血链球菌的荧光原位检测

目的 检测菌斑生物被膜初期形成过程中的变形链球菌(Streptpcoccus mutans)、远缘链球菌(Streptpcoccus sobrinus)和血链球菌(Streptpcoccus sanguis).方法 从植入釉质磨片表面获得完整的菌斑生物被膜标本,应用激光共聚焦扫描显微镜和荧光原位杂交技术相结合的方法,对天然菌斑生物被膜形成初期的变形链球菌、远缘链球菌和血链球菌进行原位的、实时的动态观察,通过连续断层扫描及三维重建,观察这3种菌在天然菌斑生物被膜中的空间分布,测量3种细菌的扫描厚度.实验中的扫描厚度结果采用方差分析方法进行统计处理,SPSS11.5统计软件辅助完成,α设在0.05.结果 在激光共聚焦扫描显微镜下观察生物被膜呈三维立体结构,形态多样,生物被膜内层和外层细菌较稀疏,中间层较密集,细菌之间可见许多黑色空隙,贯穿整个生物被膜.菌斑生物被膜变形链球菌、远缘链球菌和血链球菌在菌斑生物被膜形成初期的平均扫描厚度随时间延长而增加,1 h时平均扫描厚度分别为20.43、11.50、14.76 μm,到24 h时平均厚度最大,分别为70.25、75.40、79.98 μm.结论 应用荧光原位杂交技术结合激光共聚焦扫描显微镜,可以快速、灵敏地检测出菌斑生物被膜变形链球菌、远缘链球菌和血链球菌.

Application of rapid identification for Streptpcoccus mutans, Streptpcoccus sobrinus and Streptpcoccus sanguis in the native dental plaque biofilm by fluorescence in situ hybridization

Objective To examine Streptpcoccus mutans,Streptpcoccus sobrinus and Streptpcoccus sanguis in the early formation of native dental plaque biofilm. Methods An experimental dental plaque biofilm model in the oral cavity was established using enamel slabs. The spatial distribution of S. mutans, S.sobrinus and S. sanguis in the early colonization of dental plaque biofilms on the enamel surface was observed bv in situ, real-time and dynamic observations and optical sections utilizing confocal laser scanning microscopy(CLSM) and fluorescence in situ hybridization(FISH). The experiment data were analyzed with One-Way AVOVA, α=0.05 using SPSS11.5. Results Dental biofilm had a certain degree of thickness and various forms in three-dimensioned structure. The bacteria in the structure were sparse at the inner layers and the outer layers. In the middle layers the bacteria were closely compacted. There were many voids traversing from the outside of the biofilm to the enamel surface. At the initial stage of dental biofilm formation, the scanned average thickness of S. mutans,S. sobrinus and S. sanguis increased with time elapsing,the mean thicknesses of 1 h biofilms were 20.43 μm,11.50 μm and 14.76 μm,respectively,and those of 24 h were the thickest in terms of average level,the mean values were 70.25 μm,75.40 μm and 79.98 μm,respectively. Conclusion The fluorescence in situ hybridization combined with CLSM are thought to be convenient and sensitive to detect S. mutans, S. sobrinus and S. sanguis in the dental plaque biofilms.