The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

这里进行的实验方案在下面描述的顺序。微细加工的协议，用于创建微流体平台在步骤讨论1。步骤2中记载的细菌培养协议（ 图2），和第三步涉及到的实验配置（ 图3）的装配。最后，实际的实验步骤是在步骤4中描述。 1，芯片制造过程注意:适当的安全程序必须遵循下面描述的过程。咨询机构安全官员的详细资料。 <ol><li>设计与适当的软件（ 例如 ，L-EDIT）面具。该通道的设计由一个主微通道宽度625微米。的信道的中心区域包含的微柱体的阵列50微米的直径，间距25μm的间隔（参见参考文件）。 </li><li>打印当前设计上的玻璃（5"×5"的钠钙玻璃），它的厚度为0.09"，并且由约70纳米厚的一层铬（Cr）的涂布，使用以制备光掩模掩蔽。使用AZ400K显影剂，持续1分钟，然后，蚀刻使用铬蚀刻剂Cr层约1分钟用丙酮剥离光刻胶，用冷食人鱼溶液清洗。（H 2 SO 4和H 2 O 2的比例为3:1）。 </li><li>光刻<ol><li>化学清洁标准4"硅片20分钟食人鱼的解决方案。 </li><li>用去离子水清洗晶圆，并擦干。 </li><li>加热晶片的热板上（200℃，15分钟）。 </li><li>大衣的硅片光致抗蚀剂。这里，在正性光刻胶AZ4620旋涂在硅晶片上以2000rpm，持续25秒，得到12.5微米厚的层。 </li><li>由浮动的晶片90秒的在100℃氮气流下除去通过在热板上的晶片的软烘烤所有的溶剂。然后，保持在真空中在t他相同的温度，持续60秒。 </li><li>将晶圆在黑暗中24小时进行脱水。 </li><li>暴露晶片于UV光以所设计的图案转移到光致抗蚀剂。 </li><li>沉浸在光致抗蚀剂显影剂溶液（AZ400K）为240秒的晶片。然后，用异丙醇冲洗晶片并通过放置在氮气流中干燥。 </li></ol></li><li> ICP-DRIE（电感耦合等离子体 - 深反应离子蚀刻）过程<ol><li>应用DRIE蚀刻。根据所需的设备最终深度（50微米在本次调查）选择适当的蚀刻深度。光致抗蚀剂作为在此过程中的掩模层。 </li><li>除去丙酮的残留光致抗蚀剂和清洗晶片。 </li></ol></li><li> PDMS（聚二甲基硅氧烷）铸造<ol><li>对于硅烷化硅模具师傅使用三氯甲基硅烷（TCMS）。倒入2〜3滴三氯甲基硅烷的小瓶，并将其放置在旁边的干燥器硅模具师傅。允许2-3小时的硅烷化过程完成。 </li><li>在一个单独的容器中，混合的Sylgard 184聚硅氧烷基与固化剂以重量比10:1，制备PDMS。通过将其在真空条件下（约2小时）脱气PDMS。 </li><li>把硅母模的持有人。然后，倒在硅原型模具的PDMS形成PDMS压模。确保气泡不会在PDMS在此过程中形成。 </li><li>固化的PDMS 2小时，在80℃。 </li><li>剥去从母模的PDMS印章。然后，切PDMS压模成单独的芯片。最后，使用切割核心钻洞的入口（S）和出口（S）。 </li></ol></li><li>硅橡胶玻璃的粘接<ol><li>暴露盖玻片和PDMS压模，以氧等离子体进行30秒。债券的PDMS印章的盖玻片。 </li><li>实现PDMS压模和盖玻片之间的适当密封，通过将其在烘箱中在70℃下进行10分钟退火设备。 </LI&gt; </ol></li></ol> 2，细菌培养注意:正确的生物安全协议必须遵循的步骤2-4。咨询机构安全官员的详细资料。 <ol><li>准备LB琼脂平板<ol><li>添加20克的Luria-Bertani（LB）琼脂（米勒）粉末和500ml超纯水至1升烧瓶中。搅拌使之溶解的粉末。 </li><li>通过高压灭菌在15磅，121℃下进行15分钟杀菌。 </li><li>让烧瓶冷却至50-55℃的长椅上，或在水浴中的生物安全罩。 </li><li>添加抗生素四环素达到50微克/毫升的最终浓度。纷飞拌匀。 </li><li>将该混合物倒入平板上。填写每块板，直到1月2日 - 2月3日已满。 </li><li>火焰气泡简单地弹出他们，如果他们形成。凝固气泡难以普及细菌培养过。 </li><li>将平板在室温下冷却过夜。 </li><li>当他们冷静，放盘放回他们的袖子，封袋，标签（抗生素及日期），并且在4℃商店。 注:盖板用锡箔的股票，轻停用许多抗生素。 </li></ol></li><li> LB培养基的制备<ol><li>添加20克的Luria-Bertani（LB）肉汤（米勒）粉末和1升超纯水至烧瓶中。搅拌使之溶解的粉末。 </li><li>通过高压灭菌在15磅，121℃下进行15分钟杀菌。 </li><li>让烧瓶冷却至50-55℃的长椅上，或在水浴中的生物安全罩。 </li><li>添加抗生素四环素达到50微克/毫升的最终浓度。纷飞拌匀。 </li><li>当冷，可将标记的瓶子在4℃。 注:盖上瓶锡箔，轻停用许多抗生素。 </li></ol></li><li>在LB琼脂平板上培养细菌（该协议使用荧光假单胞菌 ） <ol><li>以细菌股价从冷冻（-80℃℃），并放置在冰上。 </li><li>放置在-80℃的细菌股票和生物安全罩内的LB琼脂平板上。 </li><li>条纹的细菌菌株上的LB琼脂平板上以Z字形图案。覆盖琼脂平板上，并培育其在30℃过夜。最后，存储盘在冰箱4℃。 </li></ol></li><li>准备细菌溶液（S1） <ol><li>倒入50毫升LB培养基介质高压灭菌瓶中。生物安全罩内执行此操作。 </li><li>从LB琼脂板烧瓶传输​​单个菌落。此操作也应生物安全罩内进行。 </li><li>将烧瓶在摇床培养箱中在30℃和150rpm下进行足够时间（4小时）。 </li></ol></li><li>制备的稀细菌溶液（S2）的<ol><li>倒入5ml的LB肉汤培养基经消毒的塑料管。 </li><li>稀释S1用的LB肉汤培养基混合。接着，旋涡振荡溶液。稀的溶液达到所需的光学人的密度值（OD测定在600nm = 0.1）。 注:生物膜的实验通常采用在这附近的OD值。 </li></ol></li></ol> 3，准备好实验装置<ol><li>使用镊子连接柔性塑料管（0.20"ID）到微芯片的入口（S）和（多个）出口的入口和出口进行了预先钻入微芯片（步骤1.5.5）的PDMS部，在该调查，微芯片包括两个入口和一个出口。 </li><li>填充注射器（S）与细菌溶液（S2解决方案），并删除所有气泡注射器（S）。 </li><li>连接注射器头（S）（30克0.5"钝针）到入口管（S），然后连接出口管（S）废容器。 </li></ol> 4，运行实验<ol><li>连接的注射器（S）到注射器尖端（次）。 </li><li>地方和固定注射器（S）到注射泵。然后将光学显微镜的合乎需求的物镜下的微芯片编倍率（ 例如 40倍）。盖上一个活细胞腔室装置，以保持恒定的温度环境细菌生长（30℃， 荧光假单胞菌 ）的微芯片。 </li><li>设置泵以所需流速水平（例如10微升/小时），并启动流体泵送。 </li><li>一旦细菌被引入到室中，生物膜的形成也被启动。生物膜的形成和成熟，通常会发生在一段几小时甚至几天。观察并通过显微镜利用生物膜生长的图像。 </li></ol>

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The authors have nothing to disclose.

我们证明了模拟多孔介质的生物膜研究发展的生境复杂的简单的微流体装置。它有决定了实验的结果，几个关键步骤。它们包括设备的几何形状。而后期的几何形状可以改变，充分的孔隙空间幡形成是必要的。此外，Valiei 等 。1表明，流光的形成只发生在一定的流量范围。在流率低于阈值的情况下，变形的生物膜成缆可能不会被观察到。但是，超过一定的阈另一个流量值，生物膜骨折可以支配，不允许形成幡。可困扰着这些实验的另一个问题是气泡，可以成为被困在微柱阵列中。通常这些泡沫必须通过增加流速最初，然后逐渐减小它为所需的值被删除。 微流控平台如这些提供了一些优势和一些限制。的平台使我们有小培养体积的工作，并具有结合用户定义的功能的灵活性。例如，不同的多孔结构可通过改变微柱阵列的几何参数进行模拟。即使是模仿实际多孔介质的随机结构结构可以制作在微流体平台18。此外，一些这样的信道可在单个设备允许收集显著相关数据进行准确的统计分析来实现。然而，微流体系统通常模仿多孔介质的二维结构。设备，可以模拟多孔介质的立体性通常是相当具有挑战性的制作。 幡的形成和演化都不能很好地理解的是，进一步的研究需要在这个方向。了解飘带是如何形成的，并导致格式成熟生物膜结构的离子将是相关的，以各种各样的方案，包括生物医学装置，如心脏支架，在土壤中的生物膜，和过滤系统的堵塞。我们的微流体平台是朝这个方向迈出的一步。

最近，我们展示了模仿多孔介质1的微流体器件细菌生物膜形成动态。细菌生物膜基本上是由胞外聚合物（EPS）2-4包裹表面聚集的细菌菌落。这些薄膜的细菌可以形成在几乎每一个可以想象小生从光滑表面的更复杂的生境的多孔介质。 Valiei 等人 1所用的微流体装置与微柱阵列来模拟多孔介质结构和研究生物膜形成在该装置的流体流速的函数。他们发现，在一定的水流流态，被称为幡丝状生物膜开始不同柱子之间出现。缆可在同一被拴或两端固体表面上，但是该结构的其余部分被悬浮在液体中。流光形成通常开始后生物膜的初始层已经形成，它的格式离子可支配生物膜的长期演变在这样复杂的栖息地。最近，一些研究人员调查了流光形成的过程中。亚兹迪等 5表明，幡可以在旋涡流从一个振荡的泡沫起源形成。在另一个实验中，RUSCONI 等 6研究了拖缆的形成信道的曲率和通道几何形状的影响。他们发现，该拖缆可在微通道的弯曲部分构成，并流光形态涉及蠕动。最近的研究已经表明，拖缆可以有各种天然的和人造的场景广泛影响，因为它们可以作为前体到成熟的结构中的多孔界面的形成，导致迅速和灾难性的生物膜增殖的生物医学系统，并且还造成实质性的流量 - 结构相互作用等 1,7-9。 生物膜幡常常表格IN复合栖息地，如多孔介质。理解生物膜生长在多孔介质中的环境相关的几个环境和工业过程，如污水生物处理10，保持在情况井筒的完整性，如CO 2的捕获11和在土壤中12堵孔。观察在如此复杂的栖息地生物被膜的形成往往是具有挑战性的，由于多孔介质的透明度。在这种情况下，基于微流体的多孔介质的平台可以证明非常有利的，因为它们允许实时和原位监测。微流体的另一个优点是，建立多个生物反应器在一个单一的生物微流体平台，同时允许在线监测和/或传感器的结合的能力。为执行在一个设备和收集显著相关数据进行准确的统计分析的能力，多个实验室实验的灵活性是重要的副词antage微流体系统13,14。 在上述讨论的范围内，在多孔介质环境理解流光形成动力学是将多个应用程序是有益的。在这项研究中，我们开发的协议，用于在设备模拟多孔介质调查流光形成。微流体平台的制造中，描述了用于细胞培养和实验所需的步骤。在我们的实验中， 荧光假单胞菌的野生型细菌菌株被使用。P.荧光假单胞菌，在土壤中自然存在，对维持土壤生态15了关键作用。该菌株采用已被基因工程改造以表达绿色荧光蛋白（GFP）组成。

<table><tbody><tr><td>Flourescent Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>LB agar</td><td>Fisher</td><td>BP1425-500</td><td>suspend 40 g in 1 L of purified water</td></tr><tr><td>LB broth</td><td>Fisher</td><td>BP1427-500</td><td>suspend 20 g in 1 L of purified water</td></tr><tr><td>Biosafety hood</td><td>Microzone corporation</td><td></td><td></td></tr><tr><td>Petri dish</td><td>Fisher</td><td>875712</td><td>sterile 100 mm x 15 mm polystyrene Petri dish</td></tr><tr><td>Incubator shaker</td><td>New Brunswick Scientific</td><td></td><td>Excella E24 incubator shaker series</td></tr><tr><td>50 ml sterilized centrifuge tube</td><td>Corning</td><td>430828</td><td>Polypropylene RNase-/DNase-free</td></tr><tr><td>Tetracycline free base</td><td>MP Biomedicals</td><td>103012</td><td>50 &amp;#956;g/ml</td></tr><tr><td>SYLGARD 184 silicone</td><td>Dow Corning Corporation</td><td>68037-59-2</td><td>Elastomer Base and curing agent</td></tr><tr><td>Positive photoresist (AZ4620)</td><td></td><td></td><td></td></tr><tr><td>Plastic tube</td><td>Cole-Parmer</td><td></td><td></td></tr></tbody></table>

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

几个细菌种类具有附着到表面并殖它们在薄膜中称为生物膜的形式的能力。生长在多孔介质生物膜有关的几个工业和环境过程，如污水处理和CO 2封存。我们使用荧光假单胞菌 ，革兰氏阴性好氧性细菌，调查生物膜形成中的微流体装置，模仿多孔介质。微流体装置由微型职位的阵列，它是用软光刻技术制造的。接着，生物膜形成中这些设备具有流量进行了调查，我们证明被称为我们的设备幡丝状生物膜的形成。此处提供的制作与微流体器件组装的详细协议以及细菌培养协议。是为实验与微流体器件的详细过程还伴随着代表介绍结果。

使用上述微细加工协议，PDMS微流体器件构成。 图1示出了扫描型电子显微镜（SEM）的PDMS的图像设备。 图1a示出了该装置的入口部分。甲叉状的入口被创建，以均衡压头跨器件。进一步SEM成像还表明，支柱壁几乎是垂直的（ 图1b）。将培养的细菌溶液（ 图2）中的稀释和其光密度调节至0.1的值。我们研究了生物膜的形成在微流体装置的输入流率的函数。当P。荧光假单胞菌注入设备在低流率的0.8微升/小时，细菌附着和生物膜的形成发生在该装置的壁上。即使经过较长时间（&gt; 20小时），比面拥抱生物膜其它没有其他细菌的结构我们再观察。接着，将同样的实验重复的8微升/小时的流速。在这种情况下，生物膜形成输液稀释的细菌培养物在几分钟后再次启动。然而，几个小时后，出现微柱之间延伸的丝状结构的附近观察到的装置（ 图4）的中间部分。这些丝状结构可以通过不动细菌的存在可视化。这些结构被称为拖缆和它们丝状生物膜那些只拴在一个或两个端部到表面上。该结构的其余部分通常是悬浮在液体介质（如在这种情况下）， 图4示出了生物膜流光结构随时间的演化。缆通常形成由于在粘弹性生物膜的流体剪切的效果， 图5示出了流线和速度轮廓为流过一系列支柱。仿真结果表明，该横幅的在我们的微流体系统的形式本质上是沿着流体流动的流线对齐。流动的结构和形成生物膜缆之间的相关性还没有得到很好的理解。然而，Das和库马尔16最近提出，这些幡形成的固有的粘弹性生物膜的高粘度液体状态。他们根据自己的猜想上的时间尺度生物膜流光形成通常远远超过生物膜的粘弹性松弛时间尺度的观察。生物膜是已知表现为粘弹性体的液体，因此在时间尺度比粘弹性松弛时间尺度要大得多，所以它们基本上表现为高粘性液体17。根据该配方，拖缆可以预计到起源于高剪切应力的位置， 图5示出了高的速度在通道中的位置，并且这些位置用的高剪切应力的位置重合。在最初的PHA<html>生长本身，拖缆附近观察到这些位置（ 图4）来发起。 <img alt="图1" src="/files/ftp_upload/51732/51732fig1highres.jpg" /> 图1的扫描电子显微镜（SEM）图像，所述微流体通道（顶视图）的。 A）入口段，含微柱B）地区。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/51732/51732fig2highres.jpg" /> 图涉及细菌培养2连续的步骤。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> NT"FO:保together.within页="总是"&gt; <img alt="图3" src="/files/ftp_upload/51732/51732fig3highres.jpg" /> 图3设置为微流体实验。1光学显微镜（倒置），2注射器泵，3 -图像及数据采集，4注射器含有染料（可选），5 -注射器含有细菌，6 -废物储存器。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/51732/51732fig4highres.jpg" /> 幡进化图4定时聚焦成像，图像平面对应于Z =设备25微米，即中间。虚线椭圆证明生物膜的飘带。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig4large.jpg" target="_blank">请点击</a>这里查看这个数字的放大版本。 <img alt="图5" src="/files/ftp_upload/51732/51732fig5highres.jpg" /> 图5计算流体力学模拟出流过微柱的流线和速度轮廓。流体流动是由上到下，速度规模为米/秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

protocol-for-biofilm-streamer-formation-microfluidic-device-with

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.1K 次观看.  University of Alberta. Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. 

视频: Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Fabrication and Visualization of Capillary Bridges in Slit Pore Geometry

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and functionalized to render their top surfaces hydrophilic. The combination of a physical feature (the pillar) with a chemical boundary (the hydrophilic film on the top of the pillar) provides both a physical and chemical heterogeneity that pins the triple contact line, a necessary feature to create stable long but narrow capillary bridges. The substrates with the pillars are attached to glass slides and secured into custom holders. The holders are then mounted onto four axis microstages and positioned such that the pillars are parallel and facing each other. The capillary bridges are formed by introducing a fluid in the gap between the two substrates once the separation between the facing pillars has been reduced to a few hundred micrometers. The custom microstage is then employed to vary the height of the capillary bridge. A CCD camera is positioned to image either the length or the width of the capillary bridge to characterize the morphology of the fluid interface. Pillars with widths down to 250 &#181;m and lengths up to 70 mm were fabricated with this method, leading to capillary bridges with aspect ratios (length/width) of over 1001.

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. The creation of capillary bridges relies on the formation of pillars to provide a directional physical and chemical heterogeneity to pin the fluid. Capillary bridges are formed and manipulated using microstages and visualized using a CCD camera.

狭缝孔几何中毛细金香桥的制造和可视化

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and ...

科研

工程学

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

生物工程

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

环境科学

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

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.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

研究多孔介质中生物堵塞的微流体平台

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

生物学

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...