不同培养条件对口腔细菌生物膜生长的影响

目的：研究5种口腔细菌在体外形成生物膜的能力、形成过程及不同培养条件对生物膜形成的影响，并通过激光共聚焦扫描显微镜（CISM）观察细菌生物膜的形成及结构特征。方法：选择与龋病发生关系密切的5种口腔细菌，分别接种在含牛心脑浸液培养基（BHI）、人工唾液（BM5）以及人唾液培养基的标准96孔板中，在培养6h、8h、12h、24h、36h、48h和60h后取出相应的微孔板，11％Crystal Violet染色生物膜，95％乙醇复吸Crystal Violet，HTS7000PLUS多孔板高效分析仪测定各时段细菌生物膜的生长情况，并绘制生长曲线。CLSM观察细菌生物膜形成的结构变化。结果：在BHI及BM5中5种口腔细菌均能形成生物膜，在人唾液中没有形成生物膜。细菌生物膜形成表现为缓慢的非线性生长，出现了一个相对的生长停滞期，这个时期出现在36～48h之间，不同细菌略有差别。激光共聚焦扫描显微镜（asM）观察发现在培养6h后仅有少量细菌粘附形成散在微菌落，24h后出现生物膜的基本结构，48h形成成熟的生物膜。结论：研究发现在体外细菌形成生物膜的能力与培养基条件密切相关，细菌生物膜形成缓慢，其间有一个相对生长停滞期。细菌定植，粘附，共集聚是生物膜形成的重要步骤。

Study of five cariogenic bacteria biofilm formation in vitro

Objective:This research was to standardize a polyvinyl chloride(PVC) micro-titer plate assay to compare the ability of 5 cariogenic bacteria to form biofilms and to explore its characteristic,ultra structure,and influences by different medium.Method:A total of 5 cariogenic bacteria strains were grown in three different medium included BHI,BM5 and saliva medium in PVC micro-titer plate wells.Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance,using distaining solution.The biofilm growth curves of 5 laboratory strains were made.The ultrastructures of bioilm were observed under Co focal Laser Scanning Microscope(CLSM).Result:5 cariogenic bacteria strains formed biofilm structure in BHI and BM5 medium,but in saliva medium no biofilm were observed.The developmental step in biofilm formation include initial attachment to a surface,the formation of micro colonies and an mature biofilm structure.Conclusion:The ability of cariogenic bacteria to form biofilm was influenced by different strains and culture medium.The necessary steps of biofilm formation include initial attachment,the formation of micro colonies and co-aggregation.