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目的实时观察铜绿假单胞菌生物膜形成并测量其厚度。方法将绿色荧光蛋白表达质粒转化铜绿假单胞菌,再采用激光共聚焦显微镜对活的生物膜进行水平逐层扫描,观察生物膜形成过程与形态,根据三维坐标系Z轴距离计算生物膜厚度。结果铜绿假单胞菌生物膜形成过程分3个阶段:0~6 h为黏附阶段;6~24 h为聚集阶段,24~72 h为成熟阶段;6,24,48和72 h的生物膜厚度分别为(6.1±2.8),(29.2±2.3),(61.4±1.4),(61.8±1.1)μm。统计学分析表明,生物膜厚度6 h<24 h<48 h,差异有统计学意义(均P<0.01),48与72 h的生物膜厚度差异无统计学意义(P>0.05)。结论激光共聚焦实时观察法能够用于铜绿假单胞菌生物膜形成检测以及生物膜厚度测量。
Objective To observe the biofilm formation and measure the thickness of Pseudomonas aeruginosa in real time. Methods The green fluorescent protein expression plasmid was transformed into Pseudomonas aeruginosa. The living biofilm was scanned by laser scanning confocal microscope and the biofilm formation process and morphology were observed. The biofilm thickness was calculated according to the Z axis distance in the three-dimensional coordinate system . Results The biofilm formation of Pseudomonas aeruginosa was divided into three stages: 0 ~ 6 h for the adhesion stage, 6 ~ 24 h for the aggregation stage and 24 ~ 72 h for the mature stage. The biofilms for 6, 24, 48 and 72 h The thicknesses were (6.1 ± 2.8), (29.2 ± 2.3), (61.4 ± 1.4) and (61.8 ± 1.1) μm, respectively. Statistical analysis showed that the biofilm thickness of 6 h <24 h <48 h, the difference was statistically significant (P <0.01), 48 and 72 h biofilm thickness difference was not statistically significant (P> 0.05). Conclusion Confocal real-time laser scanning can be used to detect the biofilm formation and biofilm thickness of Pseudomonas aeruginosa.