聚维酮碘溶液对金黄色葡萄球菌细菌生物膜的体外杀菌效果

发布时间：2018-06-21 00:25

本文选题：金黄色葡萄球菌 + 钛板　；参考：《福建医科大学》2015年硕士论文

【摘要】：目的通过在钛板表面建立金黄色葡萄球菌细菌生物膜模型,在激光扫描共聚焦显微镜(CLSM)下观察细菌生物膜(BBF)的结构、中层及底层的活菌率;研究0.5%、0.25%浓度的聚维酮碘溶液对金黄色葡萄球菌BBF的体外杀菌效果。方法选取斜面保种的金黄色葡萄球菌(SA),接种于预先配制的胰蛋白胨大豆肉汤(TSB)中,37℃(5%CO2)的恒温培养箱中培养16小时获得菌液,通过比浊仪将菌液浓度调成1.0×106 CFU/ml备用。将15枚清洁、钝化、消毒过的钛板随机分装至3个25cm2细胞培养瓶,每个细胞培养瓶内5枚钛板、1ml上述调配好的菌液、20ml TSB,置37℃(5%CO2)的恒温培养箱中培养24小时形成成熟BBF。从以上3个细胞培养瓶中随机取出10枚钛板在避光环境下由SYTO9/PI组成的染色剂染色20分钟,在CLSM下观察BBF的结构、活菌率,另外5枚在经过漩涡振荡器、超声降解、离心机离心处理后接种至血平板培养基做细菌培养。根据上述建模方法,在45枚钛板表面建立金黄色葡萄球菌BBF模型,取出后用PBS冲洗3次,将45枚钛板随机分为3组,每组15枚,其中两组分别置于0.5%和0.25%的聚维酮碘溶液中作用5分钟,通过由硫代硫酸钠、吐温80、卵磷脂组成的中和剂将钛板表面的碘残留去除,这两组中的每组其中10枚钛板通过上述染色方法染色20分钟后在CLSM下观察BBF的结构、活菌率的变化,另外5枚在经过漩涡振荡器、超声清洗、离心机离心处理后接种至血平板培养基做细菌培养。另外一组其中10枚钛板作为对照组,剩余5枚在经过漩涡振荡器、超声清洗、离心机离心、稀释处理后接种至血平板培养基做细菌培养。结果①BBF在CLSM下呈高度组织化的群体结构。BBF在CLSM下的图像由橙色或橘黄色荧光、绿色荧光、红色荧光、暗性区域组成。其中橙色或橘黄色荧光由活菌和死菌在同一位置叠加而成,绿色荧光代表活菌,红色荧光代表死菌。②CLSM下的BBF是团块状的多聚物。活菌大多被包裹在生物膜的中间,死菌大多在BBF同一层面的外围。③培养24小时后成熟BBF中层活菌率为(67.35±1.06)%,底层活菌率为(39.42±2.23)%。中层和底层的BBF活菌率具有明显的统计学意义(P0.01)。④0.5%聚维酮碘溶液作用10分钟后的BBF的中层、底层活菌率分别为(66.65±1.37)%,(38.22±3.15)%。⑤0.25%维酮碘溶液作用10分钟后的BBF的中层、底层活菌率分别为(66.53±1.00)%,(39.13±2.41)%。⑥0.5%聚维酮碘溶液作用10分钟后的BBF的中层、底层活菌率与0.25%聚维酮碘溶液作用10分钟后的BBF的中层、底层活菌率和空白对照组相比差异都无统计学意义(P0.05)。⑦模型组、0.5%和.25%聚维酮碘组做细菌培养的血平板培养基内均可见菌落形成,经鉴定均为SA。结论①本实验所采用的建立金黄色葡萄球菌BBF模型方法相对简单、可重复性强,可用于研究同种细菌不同异物表面细菌生物膜的形成能力。②在BBF内,中层活菌率较底层活菌率高,死菌大多出现在活菌同一层面的外围。③0.5%、0.25%浓度的聚维酮碘溶液对钛板表面金黄色葡萄球菌BBF无明显杀菌效果。
[Abstract]:Objective to establish the bacterial biofilm model of Staphylococcus aureus on the surface of titanium plate, the structure of bacterial biofilm (BBF), the rate of living bacteria in the middle layer and the bottom were observed under the laser scanning confocal microscope (CLSM), and the bactericidal effect of 0.5%, 0.25% concentration of polyvidone iodine solution on Staphylococcus aureus BBF in vitro was studied. Staphylococcus aureus (SA) was inoculated in the pre prepared tryptone soybean broth (TSB), and cultured for 16 hours in a constant temperature incubator of 37 C (5%CO2), the bacterial liquid concentration was adjusted to 1 x 106 CFU/ml by turbidimetry. 15 clean, passivated and sterilized titanium plates were randomly assigned to 3 25cm2 cell culture bottles and each cell culture was cultured. 5 titanium plates in the bottle were raised in the bottle. 1ml, 20ml TSB, and 37 C (5%CO2) incubator was cultured for 24 hours to form mature BBF., and 10 titanium plates were randomly removed from the 3 cell culture bottles for 20 minutes under the light environment. The structure of BBF, the living bacteria rate, and the other 5 were observed under CLSM. According to the above modeling method, the BBF model of Staphylococcus aureus BBF was established on the surface of 45 titanium plates, and 3 times were washed with PBS, and 45 titanium plates were randomly divided into 3 groups, each group was 15, of which two groups were placed in 0.5% and 0.25% of the polyvidone iodine respectively. In the solution for 5 minutes, the iodine residue on the surface of the titanium plate was removed by the neutralizer composed of sodium thiosulfate, Twain 80 and lecithin, and 10 titanium plates of each of the two groups were stained by the above staining method for 20 minutes to observe the structure of BBF under CLSM, the change of the living bacteria rate, and the other 5 in the vortex oscillator, ultrasonic cleaning, centrifugation. The machine was centrifuged and inoculated to the blood plate culture medium for bacterial culture. The other 10 titanium plates were used as the control group, the remaining 5 were passed through the vortex oscillator, ultrasonic cleaning, centrifuge centrifuge, and then inoculated to the blood plate culture medium for bacterial culture. Results (1) BBF under CLSM, the highly organized group structure.BBF was under CLSM. The images are composed of orange or orange fluorescent, green fluorescence, red fluorescence and dark area. The orange or orange fluorescence is superimposed on the same position by living bacteria and dead bacteria. The green fluorescence represents the living bacteria and the red fluorescence represents the dead bacteria. (2) the BBF under CLSM is a lump polymer. The live bacteria are mostly wrapped in the middle of the biofilm and dead bacteria Mostly at the periphery of the same level of BBF, after 24 hours of culture, the rate of mature BBF medium living bacteria was (67.35 + 1.06)%, and the living bacteria rate at the bottom was (39.42 + 2.23)%. The rate of BBF living bacteria in the middle and bottom layers had significant statistical significance (P0.01). (4) the rate of living bacteria in the middle layer of BBF after 10 minutes' action of 0.5% polyvidone iodine solution was (66.65 + 1.37)%, respectively, 38.2 2 + 3.15)% of the middle layer of BBF in the middle layer after 10 minutes' action of 0.25% dimensional ketone iodine solution (66.53 + 1)% and (39.13 + 2.41)% respectively. 6. 0.5% polyvidone iodine solution in the middle layer of BBF after 10 minutes' action, the bottom living bacteria rate and the middle layer of BBF after the action of 0.25% polyvidone iodine solution for 10 minutes, the difference of the living bacteria rate of the bottom layer and the blank control group There was no statistical significance (P0.05). (7) model group, 0.5% and.25% povidone iodine group had bacterial colony formation in the blood plate culture medium of bacterial culture. The identification was SA. conclusion (1) the method of establishing Staphylococcus aureus BBF model used in this experiment is relatively simple and reproducible, and can be used to study the surface bacteria of different foreign bodies of the same bacteria. The ability to form biofilm. In BBF, the middle lower rate of viable bacteria rate, dead bacteria occurs mostly in the periphery of live bacteria of the same level. In 0.5%, the concentration of 0.25% povidone iodine solution without obvious bactericidal effect on Staphylococcus aureus BBF titanium plate surface.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R515

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