Delatolla Robert, Tufenkji Nathalie, Comeau Yves, Lamarre Daniel, Gadbois Alain, Berk Dimitrios
Department of Chemical Engineering, McGill University, Montreal, Quebec H3A 2B2, Canada.
Water Res. 2009 Apr;43(6):1775-87. doi: 10.1016/j.watres.2009.01.009. Epub 2009 Jan 27.
Methods for characterizing nitrifying bacteria within biofilms are of key importance to understand and optimize the nitrification kinetics of attached growth treatment facilities. In this work, we propose an analytical protocol based upon environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CSLM) in combination with fluorescent in situ hybridization (FISH) to characterize the structure of nitrifying biofilm as it remains attached to the original reactor substratum. This protocol minimizes the loss of mass and distortion of in situ perspective commonly associated with traditionally applied microscopic techniques and thereby enables a more accurate estimation of the nitrifying biomass within biofilm attached to the substratum. The use of ESEM eliminates the destructive preparatory procedures associated with traditional scanning electron microscopy and thus the loss of mass and shrinking of the samples. ESEM is used in this study to evaluate the percent coverage of the substratum with biofilm and the biofilm thickness. CLSM-FISH is used to determine cell counts in the biofilm and to characterize the undisturbed substratum/biofilm interface. By hybridizing and analyzing the nitrifying biofilm using CLSM as it remains attached to the substratum, the loss of material and distortion of in situ perspective associated with the biofilm detachment process is minimized. Moreover, by conducting the CLSM analysis directly on the nitrifying biofilm as it remains attached to the substratum it is shown that cell counts at the substratum/biofilm interface differ significantly from that located above the interface.
表征生物膜中硝化细菌的方法对于理解和优化附着生长处理设施的硝化动力学至关重要。在这项工作中，我们提出了一种基于环境扫描电子显微镜（ESEM）和共聚焦激光扫描显微镜（CSLM）并结合荧光原位杂交（FISH）的分析方案，以表征附着在原始反应器基质上的硝化生物膜的结构。该方案将通常与传统应用的显微镜技术相关的质量损失和原位视角失真降至最低，从而能够更准确地估计附着在基质上的生物膜中的硝化生物量。ESEM的使用消除了与传统扫描电子显微镜相关的破坏性制备程序，从而避免了样品的质量损失和收缩。本研究中使用ESEM评估生物膜对基质的覆盖百分比和生物膜厚度。CLSM-FISH用于确定生物膜中的细胞数量，并表征未受干扰的基质/生物膜界面。通过在附着于基质的硝化生物膜上使用CLSM进行杂交和分析，与生物膜分离过程相关的材料损失和原位视角失真被降至最低。此外，通过直接在附着于基质的硝化生物膜上进行CLSM分析表明，基质/生物膜界面处的细胞数量与界面上方的细胞数量有显著差异。
