Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1720, USA.
J Ind Microbiol Biotechnol. 2010 Oct;37(10):1081-9. doi: 10.1007/s10295-010-0756-z. Epub 2010 Jun 15.
To better engineer and analyze beneficial biofilms as well as to develop strategies to control detrimental biofilms (e.g., biomedical device-based infections), it is critical to quantify bacterial species compositions within biofilms. A non-invasive method is described here that determines local and overall bacterial concentrations within a biofilm, using optical microscopy and digital image analysis techniques. The method is based upon a calibration of cell fluorescence to known cell number concentrations and is verified by direct cell counts of destructive samples of cultivated biofilms. Two GFP mutants, each with unique emission colors were used with both epi-fluorescent microscopy and one-photon confocal microscopy to determine local spatial biofilm cell concentrations in pure and mixed-strain biofilms. Our microbial system comprises Pseudomonas putida containing either green fluorescent protein (GFP) or containing the red fluorescent protein (DsRed). Strains expressing a green or red fluorescent protein were detected by two different microscopy methods: epi-fluorescence and single-photon confocal laser scanning microscopy. Overall biofilm cell concentrations determined directly from destructive samples were in good agreement with non-invasive measurements of adherent cell concentrations calculated from the measured "integrated fluorescent density" minus any background fluorescence. Results show the areal cell concentration (cell number/area) determined from non-destructive direct counts in a pure culture or binary-strain biofilm varied with the biofilm depth. Use of this method to estimate local dynamic plasmid segregational loss and plasmid conjugation transfer kinetics will be reported in a subsequent manuscript.
为了更好地设计和分析有益的生物膜，并开发控制有害生物膜的策略（例如，基于生物医学设备的感染），定量生物膜内细菌种类组成至关重要。这里描述了一种非侵入性方法，该方法使用光学显微镜和数字图像分析技术来确定生物膜内的局部和整体细菌浓度。该方法基于细胞荧光与已知细胞数浓度的校准，并通过破坏性培养生物膜样本的直接细胞计数进行验证。使用两种 GFP 突变体，每种都具有独特的发射颜色，结合 epi 荧光显微镜和单光子共聚焦显微镜，用于确定纯和混合菌株生物膜中局部空间生物膜细胞浓度。我们的微生物系统包括含有绿色荧光蛋白（GFP）或红色荧光蛋白（DsRed）的假单胞菌。表达绿色或红色荧光蛋白的菌株通过两种不同的显微镜方法检测： epi 荧光和单光子共焦激光扫描显微镜。直接从破坏性样品中确定的总生物膜细胞浓度与从测量的“积分荧光密度”减去任何背景荧光计算得出的粘附细胞浓度的非侵入性测量值非常吻合。结果表明，从纯培养物或二元生物膜中的非破坏性直接计数确定的面积细胞浓度（细胞数/面积）随生物膜深度而变化。将在随后的一篇论文中报告使用该方法估计局部动态质粒分离损失和质粒共轭转移动力学的结果。
