应用绿色荧光蛋白基因标记细菌进行生物膜结构定量化新方法

绿色荧光蛋白基因pEGFP经CaCl₂转化法标记E.coli JM109菌株,获得的标记菌株作为模式细菌接种含50μg/mL氨苄的LB培养基,在摇瓶中与火山岩颗粒共混培养挂膜(37℃,120r/min,16h).用激光共聚焦扫描显微镜摄取获得火山岩填料生物膜250μm×250μm区域不同层面的图片堆,所获图片堆经COMSTAT程序处理可以获得相关的定量化参数,如16h生物膜平均厚度为0.120844μm,生物膜最大厚度为10.5μm,生物膜体积为0.136986μm³/μm²,生物膜表面积21338.1μm²,生物膜比表面积为3.36854μm²/μm³.该方法也可以扩展至其他绿色荧光蛋白基因标记细菌的生物膜结构定量化.

New method for spatial structure quantification of biofilm with GFP tagged bacteria

Green fluorescent protein (GFP) gene was applied to mark E. coli JM109 by the method of CaCl2 transformation. The GFP-tagged cells was inoculated into the LB medium containing 50 microg/mL of Ampicillin to develop biofilm on the surface of lava by mix-culture with lava particles in flask under 37 degrees C, 120 r/min for 16 h. The fluorescent images stack of different layer at specific area (250 microm x 250 microm) on lava covered with biofilm was obtained by confocal scanning laser microscopy. The quantifying parameters of biofilm spatial structure could be acquired after the image information was calculated by COMSTAT program, i. e., the average thickness of specific area of biofilm after 16 h was 0.120 844 microm, the maximum thickness was 10.5 microm, the volume was 0.136 986 microm3 /microm2, the surface area was 21 338.1 microm2, and the surface area per volume biofilm was 3.36 854 microm2 /microm3. This method might be expanded to reflect the spatial structure of biofilm with any other GFP tagged bacteria.