慢性难愈创面细菌生物膜体外模型的建立及分析

发布时间：2017-12-28 06:37

本文关键词：慢性难愈创面细菌生物膜体外模型的建立及分析　出处：《内蒙古大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：建立慢性难愈创面临床常见细菌的生物膜体外模型,利用激光共聚焦显微镜观察细菌生物膜形成过程,探索细菌生物膜检测的有效手段。方法：(1)建立细菌生物膜体外模型,摸索生物膜体外模型的建立条件,首先将传统的平板培养法与引导片培养法进行比较,在此基础上,以引导片法体外培养生物膜,观察不同时间点和温度对生物膜形成的影响。(2)激光共聚焦显微镜观察各菌株生物膜形成过程。采用FITC-ConA和碘化丙啶(PI)分别标记多糖和细菌DNA,激光扫描共聚焦显微镜观察生物膜形成情况。结果：(1)传统的平板培养法最终测得的结果稳定性较差,引导片培养法最终所得的结果较稳定,试验重复性较好,37℃时生物膜的生长状态较22℃更好。(2)CLSM观察可见,培养24h时,各菌株均有散在的绿色荧光,不密集。48h时绿色荧光明显增多,部分与红色荧光重叠,形成视野中黄色荧光,金黄色葡萄球菌明显。在绿色荧光的强度和分布上,临床菌株明显多于标准株。培养72h时铜绿假单胞菌和其标准株的绿色荧光增多。培养5d时绿色荧光较分散。金黄色葡萄球菌在48h形成成熟的生物膜,铜绿假单胞菌则在72h。结论：(1)慢性创面临床常见的金黄色葡萄球菌和铜绿假单胞菌均能形成生物膜。(2)采用免疫荧光技术和激光扫描共聚焦显微镜研究生物膜形成过程是一种简便可行的方法。金黄色葡萄球菌、铜绿假单胞菌形成生物膜的时间、生物膜的形态与特点都有显著的差异。
[Abstract]:Objective: to establish a biofilm in vitro model of chronic refractory wounds clinical common bacteria, confocal microscopy observation of bacterial biofilm formation by laser, explore the effective means of bacterial biofilm detection. Methods: (1) establish the bacterial biofilm model in vitro, explore the conditions of establishment of biofilm model in vitro, the traditional plate culture method was compared with the guide piece culture method, on this basis, in order to guide plate biofilm method in vitro to observe the effect of different time and temperature on the biofilm formation. (2) the biofilm formation of each strain was observed by laser confocal microscope. FITC-ConA and propidium iodide (PI) were used to mark polysaccharide and bacterial DNA, and the formation of biofilm was observed by laser scanning confocal microscope. Results: (1) the results of the traditional plate culture method were not stable. The results obtained by the guided culture method were stable and the reproducibility was good. The growth state of biofilms was better than 22 degrees at 37 degrees. (2) CLSM observation showed that when the 24h was cultured, all the strains had the green fluorescence, which was not dense. At 48h, the green fluorescence increased obviously, partly overlapped with red fluorescence, forming yellow fluorescence in the field of vision, and the Staphylococcus aureus was obvious. In the intensity and distribution of green fluorescence, the clinical strains were obviously more than the standard strains. The green fluorescence of Pseudomonas aeruginosa and its standard strains were increased when 72h was cultured. When the 5D was cultured, the green fluorescence was more dispersed. Staphylococcus aureus forms a mature biofilm in 48h, and Pseudomonas aeruginosa is in 72h. Conclusions: (1) the common Staphylococcus aureus and Pseudomonas aeruginosa in the chronic wound surface can form the biofilm. (2) it is a simple and feasible method to study the process of biofilm formation by using immunofluorescence and laser scanning confocal microscopy. The time of the biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa, the morphological and characteristics of the biofilm are significantly different.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R641;R-332

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