Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide
Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide
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DOI：
10.1002/jbm.a.30077
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发表时间：
2004-08-01
影响因子：
4.9
通讯作者：
Matsuda, T
中科院分区：
文献类型：
--
作者：
Maeyama, R;Mizunoe, Y;Matsuda, T
我们开发了一种使用表达绿色荧光蛋白的大肠杆菌和胞外多糖的红色荧光染色来评估合成材料上生物膜结构的新方法。共焦激光扫描显微镜观察揭示了聚氨酯薄膜上生物膜原位三维结构特征变化的时间过程，而没有结构破坏：最初粘附的细胞生长形成细胞聚集体并分泌胞外多糖。这些细胞在早期孵化时间点状分布在表面上，但随着孵化时间的推移融合形成垂直生长的生物膜。荧光强度是细胞数量的测量，使用荧光计测定，并且通过共焦激光扫描显微镜垂直图像测定生物膜厚度，发现对于量化生物膜的时间依赖性生长是有效的。产生 Curli（与纤连蛋白和层粘连蛋白特异性结合的表面纤维）的大肠杆菌菌株 YMel 在纤连蛋白包被的聚氨酯上显着增殖，而 Curli 缺陷的同基因突变体 YMel-1 却没有。对分子和形态事件中生物膜结构的理解以及新的荧光显微技术可能有助于具有高抗菌潜力的生物材料的逻辑表面设计。 (C) 2004 年 Wiley 期刊公司。
We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein-expressing Escherichia coli and red fluorescence staining of exopolysaccharides. Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three-dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides. These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time. Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time-dependent growth of biofilms. The curli (surface-located fibers specifically binding to fibronectin and laminin)-producing Escherichia coli strain, YMel, significantly proliferated on fibronectin-coated polyurethane, whereas the curli-deficient isogenic mutant, YMel-1, did not. The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential. (C) 2004 Wiley Periodicals, Inc.