中文摘要：

目的 探索混合细菌生物膜（biofilm,BF）培养的适宜条件,建立混合细菌BF体外模型,并为研究混合细菌BF的调控机制奠定基础.方法 分别培养铜绿假单胞菌（Pseudomonas aeruginosa）菌株PAO1、轻型链球菌（Streptococcus mitis）标准株ATCC49456.紫外分光光度计、CCK-8（cell counting kit-8）比色法比较2种细菌在不同pH值、培养基、混合比例、加入顺序和混合时间条件下的生长情况.建立单独及混合细菌BF后,荧光探针SYTO9/PI标记,激光共聚焦显微镜（confocal laserscanningmicroscopy,CLSM）观察BF内部活死菌比例.扫描电镜（scanning electron microscopy,SEM）观察BF结构.结果 pH值为7.5最适宜2种细菌生长,P＜0.01：2种细菌在脑心浸出液培养基中生长明显优于胰大豆蛋白胨肉汤培养基,P＜0.01;PAO1与Smitis以1：3混合,且PAO1优先加入,能形成较好的BF,P＜0.01;2种细菌混合的时机以PAO1培养后立即混合Smitis为优,P＜0.01.CLSM结果显示PAO1可形成成熟致密的BF;S.mitis单独培养不形成BF;混合细菌BF较单独PAO1组活菌比例增多,3组厚度分别为（19.02±1.298）μm,（2.250±0.25）μm和（28.76±3.472）μm,P＜0.05.SEM显示混合细菌BF内两种细菌共存,结构更加致密立体.结论 成功建立PAO1混合Smitis的BF体外模型,方法简便易行,结果可靠,重复性强.初步证实S.mitis可参与混合菌BF形成,并起促进作用.

英文摘要：

Objective To explore the appropriate conditions for cultivating mixed-biofilm and set up a model for further study on the regulation of muitispeeies bacterial biofilm. Methods Pseudornonas aeruginosa （P. aeruginosa） and Streptococcus mitis （S. mitis） were cultured independently. The density of bacteria was measured by ultraviolet speetrophotometer and cell counting kit-8 under different conditions, including different pH, culture medium, and mix proportion, sequence and timing. The mono-biofilm and mixed-biofilm were observed via confoeal laser scanning microscopy （CLSM）, stained by STYOg/PI and imaged by scanning electron microscopy（SEM）. Results Medium with pHT.5 was optimal for both the bacteria to grow （P〈0.01）. Both P. aeruginosa and S. mitis grew better in brain heart infusion broth medium than tryptone soy broth medium （P〈0.01）. When the ratio of P. aeruginosa and S. mitis was 1：3 and adding S. mitis after P. aeruginosa, the biofilm grew best among setting groups （P〈0.01）. Referring to timing, adding at the same time was optimal （P〈0.01）. The CLSM images showed that the S. mitis alone failed to form biofilm, while P aeruginosa itself formed mature and compact biofilm. However, the mixed bacterial biofilm was thicker and had higher ratio of live bacteria. The thickness of biofilm formed by P. aeruginosa alone, S. mitis alone and both bacteria was （19.02±1.298）μm, （2.250±0.25）μm and （28.76±3.472）μm, respectively （P〈0.05）. The images of SEM confirmed that the biofilm structure of multi-species was more solid. Conclusion The multi-species bacterial biofilm model in vitro was successfully established. The model is convenient and has good reliability and reproducibility. It is preliminarily proved that the S. mitis facilitates formation of the biofilm.