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

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