Non-invasive method to quantify local bacterial concentrations in a mixed culture biofilm.
Non-invasive method to quantify local bacterial concentrations in a mixed culture biofilm.
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DOI：
10.1007/s10295-010-0756-z
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发表时间：
2010-10
影响因子：
3.4
通讯作者：
Bryers, James D.
中科院分区：
文献类型：
--
作者：
Ma, Hongyan;Bryers, James D.
关键词：
为了更好地设计和分析有益的生物膜，以及制定控制有害生物膜的策略(例如，基于生物医学设备的感染)，量化生物膜内的细菌种类组成是至关重要的。这里描述了一种非侵入性的方法，它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对已知细胞数浓度的校准，并通过培养生物膜破坏性样品的直接细胞计数来验证。用表观荧光显微镜和单光子共聚焦显微镜研究了两个GFP突变体，每个突变体都具有独特的发射颜色，以确定纯生物膜和混合菌株生物膜中局部空间生物膜细胞的浓度。我们的微生物系统由恶臭假单胞菌组成，其中含有绿色荧光蛋白(GFP)或含有红色荧光蛋白(DsRed)。用两种不同的显微镜方法：EPI荧光法和单光子共聚焦激光扫描显微镜对表达绿色或红色荧光蛋白的菌株进行检测。直接从破坏性样品中测定的总生物膜细胞浓度与非侵入性测量的贴壁细胞浓度非常一致，该测量是通过测量的“综合荧光密度”减去任何背景荧光计算得出的。结果表明，在纯培养或双菌种生物膜中，由非破坏性直接计数确定的面积细胞浓度(细胞数/面积)随生物膜深度的不同而不同。使用这种方法估计局部动态的质粒分离损失和质粒接合转移动力学将在后续的手稿中报道。
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 nondestructive 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.
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影响因子：
3.8
作者：
HUANG, CT;PERETTI, SW;BRYERS, JD
通讯作者：
BRYERS, JD
影响因子：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S
影响因子：
3.5
作者：
Lewis, PJ;Marston, AL
通讯作者：
Marston, AL
影响因子：
56.9
作者：
CHALFIE, M;TU, Y;PRASHER, DC
通讯作者：
PRASHER, DC
影响因子：
4.4
作者：
Cowan, SE;Gilbert, E;Keasling, JD
通讯作者：
Keasling, JD