方法表征的共同发展生物膜和生境异质性
Summary
Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.
Abstract
生物膜是具有复杂的结构，并产生显著的空间异质性表面附着的微生物群落。生物膜发展被周围流动和营养环境强烈调节。生物膜生长也通过产生复杂的流场和溶质运输模式增加了局部微环境的不均一性。为了研究异质性的生物膜及其局部微栖息地之间的生物膜和互动发展，我们长大绿脓杆菌的单种生物膜和P.双种生物膜根据营养梯度的微流体流动池假单胞菌和大肠杆菌 。我们对流动单元内创建养分梯度和用于生长和在这些条件下可视化生物膜发展提供详细协议。我们还进行了一系列的光学方法本协议来量化空间模式在生物膜结构，流DISTRIbutions在周围生物膜，和大众交通和生物膜殖民地之内。这些方法支持联合开发的生物膜和生境异质性的全面调查。
Introduction
微生物附着于表面，并形成生物膜-封闭在细胞外聚合物基质1细胞集合体。生物膜表现非常不同，从单个微生物细胞，由于生物膜具有戏剧性的空间异质性，从 内部溶质运移限制和空间变化的细胞代谢2,3的组合产生。氧气和营养物浓度急剧降低，在生物膜和周围的流体并获得内生物膜2进一步贫化之间的接口。在生物膜呼吸和蛋白质合成的空间变化，也可能发生由于局部氧和养分有效性2的响应。
在水体和土壤环境中，大部分细菌住在生物膜。天然生物膜进行重要的生物地球化学过程，包括骑自行车的碳和氮，减少金属4,5。在临床上，生物膜的形成是responsIBLE长时间肺部和泌尿系统感染6。生物膜相关的感染是很成问题，因为细胞在生物膜得抗菌素极高电阻相比，其浮游对口6。由于生物膜是在不同的设置很重要，研究了大量一直专注于了解控制生物膜活动和空间异质性的生物膜和周围的微环境中的环境因素。
先前的研究已经发现，生物膜发展强烈地受到许多环境因素的调节：生物膜发展的各种流动状态下不同的形态;氧气和养分供应影响生物膜形态;和流体剪切应力影响浮游细胞的附着于表面和细胞从生物膜7-9脱离。此外，外部流动状况影响的基板INT交付O和生物膜10中。生物膜的增长也改变了周围的物理和化学条件。例如，生物膜生长导致氧气和营养2的局部耗竭;生物膜积聚来自周围环境11的无机和有机化合物;和生物膜集群转移流动，增加表面摩擦力12,13。因为生物膜与其周围环境中非常复杂的方式相互作用，这是至关重要的，以同时获取对生物膜特性和环境条件的信息，和多学科的方法需要使用全面表征生物膜与环境的相互作用。
在这里，我们提出了一系列的综合方法，以在单种和双种生物膜中的微生物生长空间格局特征下强加的营养梯度，并观察所产生的局部化学和流体微环境的改变。我们杉ST描述了使用最近开发的双入口的微流体流动池的观察下明确定义的化学梯度生物膜生长。然后，我们说明如何使用该微流体流动池的观察下的范围内的营养条件2种细菌， 绿脓杆菌和大肠杆菌 ，在生物膜的生长。我们展示了如何在荧光示踪剂传播到生物膜集落原位可视可以用于定量评估生物膜溶质运输模式。最后，我们显示如何微尺度粒子跟踪测速，在共聚焦显微镜进行的，可以被用于获得周围生长生物膜局部流场。
Protocol
Representative Results
Discussion
我们表现出了一套方法来描述三个重要的生物膜与环境的相互作用：生物膜反应化学梯度，生物膜生长在周围的微环境流量，和生物膜的异质性，从内部运输的局限性造成的影响。
我们首先显示了使用一种新颖的微流体流动池，以施加用于生物膜发展的一个明确定义的化学梯度。以产生流动单元内的明确定义的化学梯度，以维持相同的流速为两个入口是重要的。确保该?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢马特Parsek在华盛顿大学（西雅图）提供P.铜绿假单胞菌和E.大肠杆菌菌株和罗杰·诺克斯在坎特伯雷（新西兰）的大学提供访问流软件。这项工作是由卫生部国家过敏研究所全国学院和传染病资助R01AI081983支持。在西北大学的生物成像设备（BIF）进行聚焦成像。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Peristaltic Pump | Gilson | Miniplus 3 | Flow cell setup and inoculation |
| PUMP TUBING 0.50MM OVC, Orange/Yellow | Gilson | F117934 | Flow cell setup and inoculation |
| Three-way Stopcock w/ Swivel male Luer lock | Smiths Medical | MX9311L | Flow cell setup and inoculation |
| Sylgard 184 Solar Cell Encapsulation for Making Solar Panels | ML Solar LLC | Flow cell setup and inoculation | |
| Pyrex Medium Bottle, 1L, GL45 | VWR | 16157-191 | Flow cell setup and inoculation |
| C-FLEX Tubing | Cole-Parmer | 06422-02 | Flow cell setup and inoculation |
| 1 mL TB Syringe | BD | 309659 | Flow cell setup and inoculation |
| Polymer Tubing | IDEX | 1520G | Flow cell setup and inoculation |
| Sterile Intramedic Luer Stub Adapter | Clay Adams | 427564 | Flow cell setup and inoculation |
| PrecisionGlide Needle | BD | 305195 | Flow cell setup and inoculation |
| Spectrophotometer | HACH | Flow cell setup and inoculation | |
| Syringe filters- sterile (0.2 μm) | Fisherbrand | 09-719A | Flow cell setup and inoculation |
| MAXQ Shaker | Thermo Scientific | Flow cell setup and inoculation | |
| Ammonium sulfate | Sigma Aldrich | A4418 | Growth media |
| Sodium phosphate dibasic anhydrous | Sigma Aldrich | RES20908-A7 | Growth media |
| Monobasic potassium phosphate | Sigma Aldrich | P5655 | Growth media |
| Sodium chloride | Sigma Aldrich | S7653 | Growth media |
| Magnisium chloride | Sigma Aldrich | M8266 | Growth media |
| Calcium chloride | Sigma Aldrich | C5670 | Growth media |
| Calcium sulfate dihydrate | Sigma Aldrich | C3771 | Growth media |
| Iron(II) sulfate heptahydrate | Sigma Aldrich | 215422 | Growth media |
| Manganese(II) sulfate monohydrate | Sigma Aldrich | M7634 | Growth media |
| Copper(II) sulfate | Sigma Aldrich | 451657 | Growth media |
| Zinc sulfate heptahydrate | Sigma Aldrich | Z0251 | Growth media |
| Cobalt(II) sulfate heptahydrate | Sigma Aldrich | C6768 | Growth media |
| Sodium molybdate | Sigma Aldrich | 243655 | Growth media |
| Boric acid | Sigma Aldrich | B6768 | Growth media |
| Dextrose | Sigma Aldrich | D9434 | Growth media |
| Luria Bertani Broth | Sigma Aldrich | L3022 | Growth media |
| TCS SP2 Confocal Microscopy | Leica | Fluorescent imaging | |
| SYTO 62 | Life Technology | S11344 | Fluorescent imaging |
| Cy5 | GE Healthcare Life Sciences | PA15100 | Fluorescent imaging |
| Red Fluorescent (580/605) FluoSphere | Life Technology | F-8801 | Fluorescent imaging |
| BioSPA | Packman Lab | Image Processing | |
| ImageJ | NIH | Image Processing | |
| Volocity | PerkinElmer | Image Processing | |
| Streams 2.02 | University of Cantebury | Image Processing |
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