Staudt C, Horn H, Hempel D C, Neu T R
Department of River Ecology, UFZ Centre for Environmental Research Leipzig-Halle, D-39114 Magdeburg, Germany.
Biotechnol Bioeng. 2004 Dec 5;88(5):585-92. doi: 10.1002/bit.20241.
In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.
在本研究中，使用从活性污泥中培养出的富集培养物来研究完全水合的多物种生物膜的结构。利用共聚焦激光扫描显微镜（CLSM）对生物膜的结构和体积进行评估。用核酸特异性染料SYTO 60确定细菌细胞分布，而用金耳Alexa-488标记的凝集素对细胞外聚合物（EPS）的糖缀合物进行染色。采用数字图像分析对三维CLSM数据集进行可视化和定量分析。对聚合物和细胞生物膜成分的比容进行了定量。此外，通过重量法测量来确定生物膜的干质量和厚度。然后将CLSM技术记录的数据与重量法数据进行比较。结果表明，对于生长缓慢的异养和化学自养生物膜，两种方法测定的生物膜厚度吻合良好。此外，对于生长缓慢的生物膜，由CLSM计算得到的体积和质量与重量法测量得到的生物量也具有可比性。对于在高葡萄糖浓度下培养的快速生长的异养生物膜，数据集的拟合度较低，但仍呈现相同的总体趋势。与传统的重量法测量相比，CLSM能够对多个生物膜参数进行差异记录，并随后进行三维可视化和定量分析。CLSM记录的定量三维结果是理解、控制、利用和模拟生物膜的重要基础。
