The quantitive analysis of sequential development of biofilm spatial structure of P.aeruginosa tagged with green fluorescence protein

Chen Boman,  Yu Jialin,  Liu Guanxin,  Hu Linyan,  Li Fang  and  Yang Hua


Objective  To quantify the sequential development of biofilm spatial structure in biofilm process with Image Structure Analyer (ISA) software, which will provide assistance to further research on the biological behavior of biofilm.  Methods  P.aeruginosa PAO1 biofilm model in vitro was constructed on glass slice and biofilm development was monitored in different time intervals (6 hours, 1 day, 3 days and 6 days). The fluorescence images stack of different layer in biofilm model were obtained by confocal laser scanning microscopy (CLSM), based on fluorophores from PAO1 with green fluorescent protein (GFP) genetically tagged. Quantitative parameters describing biofilm spatial structure could be acquired after the image information was calculated by ISA software.  Results  ①The PAO1 biofilm process was investigated successfully by CLSM after genetically tagged with GFP. ②The quantitative data from ISA software showed that the thickness of biofilm accumulated during biofilm growth, the rate was more obvious during the first 3 days than the last 3 days (19.6μm vs. 6.1μm); meanwhile the areal porosity (AP) decreased from 0.98±0.01 at 6h to 0.92±0.02 at 6d, the average diffusion distance (ADD) increased slightly from 1.00±0.009 at 6h to 1.06±0.027 at 6d; the textural entropy (TE) increased from 0.7±0.08 at 6h to 4.3±0.09 at 6d.  Conclusions  The ISA software could provide useful information of bacterial biofilm spatial structure in PAO1 biofilm process. Quantifying bacterial biofilm structure permitsed correlating biofilm development with biofilm performance.