Abstract: To quantify the sequential development of Cronobacter sakazakii biofilm structure in biofilm process, with hyphened technique of the program confocal laser scanning microscopy (CLSM) and COMSTAT, C. sakazakii biofilm model in vitro was constructed on glass slice in cell culture plate and biofilm development was monitored at different time intervals (6 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h).  Meanwhile, the total biomass of biofilms was analyzed using the crystal violet (CV) staining method. The change of A570 value ascended before 60 h and then descended. With the elongation of culture time, total biomass, average thickness and average diffusion distance increased first and decreased afterwards, and reached the peak at 60 h, whereas live/dead ratio, roughness coefficient and surface to volume ratio rose after the first drop, and the minimum values occurred at 60 h, 48 h and 60 h, respectively. The biofilm extracellular polysaccharide (EPS) increased first and then decreased with the extension of culture time. The formation of C. sakazakii biofilm was divided into four steps: adhesion (0~12 h), development (12~48 h), maturation (48~60 h) and dispersal (>60 h). CLSM-COMSTAT method provides not only direct qualitative observation of biofilm spatial structure but also the respective quantitative structural parameters, and furthermore the change of total biomass measured by CLSM-COMSTAT method was in accordance with the CV staining method. CLSM-COMSTAT method can be used as a regular method for quantitative analysis of C. sakazakii biofilm structure, which is beneficial to study the formation mechanism and prevention techniques of C. sakazakii biofilm.

Key words: Cronobacter sakazakii, biofilm, confocal laser scanning microscope, COMSTAT, quantitative analysis

摘要： 为了建立阪崎克罗诺杆菌的孔板置片成膜模型，将不同培养时间(6 h、12 h、24 h、36 h、48 h、60 h和72 h)的生物膜经荧光染料标记后，运用激光共聚焦扫描显微镜(confocal laser scanning microscope, CLSM)联合COMSTAT定量分析生物膜形成过程的结构变化，同时用结晶紫(crystal violet, CV)染色法分析生物量。结果显示：随着培养时间延长，A570先升后降，在60 h有最大值；总生物量、平均厚度及平均扩散距离也先升后降，在60 h有最高值，活死比例、粗糙系数及表面积与生物量比值先降后升，最低值分别在60 h、48 h与60 h；胞外多糖先增后减。阪崎克罗诺杆菌生物膜形成包括：粘附阶段(0~12 h)、发展阶段(12~48 h)、成熟阶段(48~60 h)及解聚再定植阶段(>60 h)。CLSM-COMSTAT法在定性观察生物膜结构的同时，还可定量分析结构参数，且该法得到的总生物量变化趋势与CV染色法一致，说明CLSM-COMSTAT法可作为分析阪崎克罗诺杆菌生物膜的常规方法。

关键词: 阪崎克罗诺杆菌, 生物膜, 激光共聚焦扫描显微镜, COMSTAT, 定量分析