四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型

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,P0.05;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,P0.05,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,P0.05,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,P0.05。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。

Observation and quantitative analysis of the formation of Staphylococcus epidermidis biofilm by using tetrazolium salt reduction assay and confocal laser scanning microscopy

Objective To establish the model of Staphylococcus epidermidis biofilm in vitro,observe and quantitatively analyze the dynamic procedure of the biofilm formation.Method Biofilm model of biofilm-producing strain Staphylococcus epidermidis RP62A was formed on plate.The metabolic activity of the bacteria inside biofilm was measured by tetrazolium salt（XTT） reduction assay.The parameters of BF structure were analyzed through pictures from Confocal Laser Scanning Microscopy（CLSM） with Image Structure Analyer（ISA） software.The structure of the biofilm was detected by scanning electron microscope（SEM）.Result The data of A450 by XTT reduction assay at different time intervals（12 h,24 h,48 h） were 2.392±0.481,3.410±0.177 and 3.917±0.274,respectively.There were significant difference between any two groups（P0.05）.The data from ISA software showed that the areal porosity（AP） were the 0.84±0.08, 0.68±0.01 and 0.59±0.13,respectively;the average diffusion distance（ADD） were 1.34±0.24,1.49±0.09 and 1.89±0.39,respectively;There were significant difference between any two groups（P0.05）.The textural entropy（TE） were 4.71±0.82,8.69±0.68 and 8.94±0.28,respectively.There were significant difference between the 12h group and the other two groups（P0.05）.Conclusion The formation of Staphylococcus epidermidis BF is a dynamic procedure.Mature biofilm was formed at the 24th hour.The structure became more complex at the 48th hour.XTT reduction assay,CLSM with ISA and SEM are the ideal methods to observe and quantitatively analyze the biofilm model in vitro.