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目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P<0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P<0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P<0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P<0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。
Objective To establish a model of biofilm (Staphylococcus epidermidis) biofilm (BF) in vitro and observe and quantitatively analyze the dynamic formation of Staphylococcus epidermidis BF. Methods The BF model of Staphylococcus epidermidis (RP62A) was established and the in vitro BF model was established by plate method. The tetrazolium salt (XTT) was used to reduce the change of bacterial activity during BF formation. The confocal laser scanning-ning microscopy (CLSM) combined with image structure analyer (ISA) to analyze the structural parameters of BF formation process. Scanning electron microscope (SEM) was used to observe the morphological structure during BF formation. Results At 12, 24 and 48 h, the values of XTT reduction A450 were 2.39 ± 0.48, 3.41 ± 0.18 and 3.92 ± 0.27, respectively, P <0.05; the quantitative analysis of ISA software showed that the values of APA were 0.84 ± 0.08,0.68 ± 0.01 and 0.59 ± 0.13, P <0.05. The values of average diffusion distance (ADD) were 1.34 ± 0.24, 1.49 ± 0.09 and 1.89 ± 0.39, respectively, P <0.05. The values of structural entropy (TE) 4.71 ± 0.82, 8.69 ± 0.68 and 8.94 ± 0.28, 24 h, 48 h compared with 12 h, P <0.05. Conclusion The formation of Staphylococcus epidermidis BF is a dynamic process. BF formed at 24 h, and the structure of BF at 48 h was more complicated. XTT reduction method, CLSM combined with ISA software, SEM combined use of three methods is to observe and quantitative analysis of BF in vitro model is the ideal method.