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。

1.流通池设置和接种注意：使用在Song 等人所述的双入口的微流体流动池，2014 14生长生物膜。这个流动池是能够创造良好定义的平滑化学梯度。流动池设计示于图1和流动池制造以前在乐曲等人的，2014年14。在这里，我们详细我们的方法通过使用P。铜绿假单胞菌和E.大肠杆菌以形成生物膜，但其他物种可能是合适的了。我们使用P.绿脓杆菌 PAO1- GFP，其中组成性表达绿色荧光蛋白（GFP），作为模式生物的生物膜发展。此外，我们使用E.大肠杆菌 DH5α形成混种生物膜与P.铜绿假单胞菌。P.铜绿假单胞菌和E.大肠杆菌菌株生长在LB琼脂平板上。 <ol><li>准备使用聚二甲基硅氧烷流动池（PDMS）绑定到一个通过氧等离子处理的玻璃盖玻片如14所述。流动池室的尺寸是23毫米×13毫米×0.24毫米（长×宽×深）。 </li><li>制备改性FAB生长培养基15。观察下的流动单元内的营养梯度生物膜生长，引入改性FAB用0.6μM的葡萄糖培养基15在一个进口和没有通过另一入口任何碳源引入FAB培养基。过滤器 - 杀菌（孔径= 0.2微米）加入到该FAB介质之前葡萄糖储液（60毫摩尔）。在使用之前，还高压灭菌FAB介质循环1（121°C，17磅的液体，15分钟）。 </li><li>消毒流动系统。在接种前，高压釜用周期1中，除了位于所述流动池上游的塑料三通阀（一次性的预灭菌的）整个流路（介质瓶，管，气泡陷阱，流动池）。 （三途径阀用于注射小区culture，荧光示踪剂和微球）。要避免污染装配过程中，覆盖所有的管路和接头开口用铝箔或高压反应釜前袋。 </li><li>连接流系统。仔细装配流动池系统的组件（参见流动系统组件的视频），并通过一个蠕动泵精确地控制流量递送生长培养基到流动池。 </li><li>通过将P.的殖民地准备的细胞培养接种绿脓杆菌或E.从大肠杆菌 LB板至3ml LB肉汤，动摇文化O / N在225转，37℃。稀释在1ml无菌水中至最终OD 600的O / N的细胞培养物= 0.01作为接种物。 （文化和稀的细菌在层流罩，以避免污染。） </li><li>对于具有混合物种的生物膜的实验中，稀释2细菌培养物为1：1的比例在1ml用等效的OD 600 = 0.01对于每个细菌。 </li><li>接种流动池。注射1毫升接种物从三通阀的流动池入口。注射后，暂停所述流1小时，以允许细菌细胞附着到玻璃盖。 （请确保流动池放置盖玻片的一面朝下，以使悬浮细胞定居到盖玻片）。 </li><li> 1小时后，恢复的流量和泵的生长培养基到流动池以0.03毫升/分的每个入口3天的恒定速率。 </li></ol> 2.表征生物膜发展应对营养梯度利用共聚焦显微镜注：P.假单胞菌可以与组成型表达的GFP进行成像，但大肠杆菌大肠杆菌在混合物种的生物膜必须由复染进行成像。 <ol><li>观察使用共焦显微镜的3天的生物膜。对于代表性的结果，观察使用共焦显微镜用63X的目标生物膜。此前成像，标记双入口流动细胞上的玻璃盖侧的网格。 （此网格的目的是允许实验者流动池室中定位在成像区域）。 </li><li>为了对，稀释10微升的细胞穿透物的红色荧光核酸染料，例如绿色荧光核酸染料，如STYO 62，在990微升原液（1毫米）的灭菌水，然后缓慢注入用注射器向稀释污点流动池室从上游三通阀。保持流动池停滞和在黑暗中30分钟，然后恢复流洗出未结合的染色15分钟。 </li><li>执行共聚焦成像。激活488nm的氩激光激发和发射收集渠道的GFP（500-535纳米）和核酸染色（绿色荧光核酸染料，650-750纳米）。调节增益和偏移的两个通道有光洁的图像。选择XYZ和同步成像模式。使用Z-控制旋钮IDENtify在外地生物膜的底部和顶部。设置的Z轴步骤至0.5微米，用于获取三维图像栈。 （为了保证高的图像质量，使用线路平均四个用于图像采集。） </li><li>映射生物膜发展的流动单元内的空间模式，图像生物膜在三个或更多的纵向（x）的沿所述流动池入口的距离，和在三个横向（y）的位置相对于所施加的营养梯度， 如图1 。 </li></ol> 3.内部表征溶质运移保守荧光示踪剂的注射注：保守的荧光示踪物，如Cy5标记，可用于生物膜内表征溶质运输模式。 <ol><li>溶于水的Cy5（单活性NHS酯），以10毫克/毫升作为原液的最终浓度。存储Cy5的原液在-20℃的冰箱中。 注：由于Cy5的是光SENSITIVE，存储，稀释并在黑暗中注入Cy5的解决方案。 </li><li>在此之前的荧光示踪剂的注射剂，取3天的生物膜用共焦显微镜的三维图像栈（具有63X物镜）。 （生物膜菌落宽区域是理想的染料传输的时间序列的观察）。另外选择的z平面与用于成像以及分离的菌落（ 如图5A）。 </li><li>对于每个Cy5的注入实验中，稀释2微升Cy5的原液在998微升灭菌水，得到具有20微克/ ml的Cy5的浓度的注射溶液。 </li><li>停止泵暂停流并注入所述Cy5的溶液成可经由三通阀使用注射器流动池的上游。 （重要的是要防止气泡进入流路，同时注入是非常重要的。气泡陷阱应保持开放的背面注射期间）。 </li><li>注入Cy5的解决方案后，关闭泡沫陷阱，调整三通阀，并重新启动泵提供所注入的Cy5的溶液注入流动池。 </li><li>开关共焦成像模式XYT。激活下同时成像模式的Cy5和GFP渠道。调整Cy5的强度共焦设置（激光强度，增益和偏移），以避免饱和的信号，这对于量化的关键。 （高时间分辨率是优选的可视化染料渗透，然而，快速扫描降低图像的质量。）选择适当的扫描速度和线平均平衡成像的时间分辨率和图像质量。此协议使用一条线平均4和0.15赫兹帧频。 </li><li>恢复流来提供Cy5的入流通池（仍然以0.03毫升/分钟的流速）。同时启动的时间序列共聚焦成像。 </li><li>通过径向平均不到2-D循环生物膜殖民地Cy5的强度量化Cy5的渗透。进行平均，首先确定一个生物膜簇的边缘，然后生成一个距离的地图为集群的2-D部，最后的Cy5强度的平均径向图案，以产生渗透曲线（参见图6）。 <ol><li>为了计算实现步骤3.8，使用BioSPA（生物膜空间格局分析）软件包（未出版，软件和手动可应要求提供）或其他图像分析程序。 </li><li>要使用BioSPA，先导入设置成BioSPA图像。然后选择合适的阈值，GFP和Cy5渠道进行二值图像。在分析/计算菜单中，选择高级分析，然后扩散分析。使用GFP通道以限定一集群的边缘，并使用多边形工具选择一个簇作为感兴趣区域（ROI）的区域。 </li><li>双击选定的生物膜集群进行扩散分析。保存扩散分析结果和使用校准曲线校准Cy5的强度，以Cy5的浓度。 <ol><li>以产生Cy5的浓度的校准曲线，过的Cy5 conce测量Cy5的强度根据共焦显微镜ntration梯度。 （Cy5的强度和浓度具有线性关系。） </li></ol></li></ol></li></ol> 4.定性周围流场跟踪荧光颗粒注：荧光颗粒，如荧光微珠，可用来围绕生物膜流场的特征。生物膜周围流场可以使用粒子跟踪测速软件颗粒归仓的时间序列的观测来计算。此处显示的结果与流2.02软件包16（软件下载，并在可用的用户指南获得<a href="http://www.civil.canterbury.ac.nz/streams.shtml">http://www.civil.canterbury.ac.nz/streams.shtml</a> ）。 <ol><li>稀释的荧光微珠（直径约1微米）在无菌水中，以0.2％的最终固体浓度为0.5ml的最终体积。涡流以确保微珠被良好地分散。 </li><li>注入和交付稀释荧光微球进入流通池下面的步骤，从3.3到3.5的过程。以允许粒子路径的精确跟踪，可使用相对低的流率（0.01毫升/小时的粒子追踪结果在本文中）。 </li><li>切换共焦设置XYT模式在同一z轴切片来跟踪颗粒运动，并采取时间序列图像。 （1赫兹的帧速率被用于在本文中示出的代表性结果。）重复粒子注射和图像在不同的z轴位置，以产生三维流场。 </li><li>预过程的时间序列的共焦图象中减去使用（可在软件下载和手动ImageJ的背景荧光信号（在图像组的第一获取的图像信号） <a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a> ）或其他程序。 </li><li>在ImageJ的，打开一个图像集的第一张图像，然后导入图像集。在过程/图像钙lculator，从图像集合中减去第一图像。保存处理前的图像集。 </li><li>要计算流动载体，导入时间序列共聚焦图像流2.02。在“开立流程视图”中，选择并执行“确定粒子”。使用适当的门槛，以确定粒子。用0.5和5微米的最小和最大直径阈值1微米的颗粒。 </li><li>然后在“打开进程查看”，选择并执行“PTV分析管道”产生的粒子的路径。检查粒子的路径，看看他们是否代表粒子的运动。最后，选择并执行“创建速度场”，以产生流动矢量地图。 </li></ol>

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The authors have nothing to disclose.

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?...

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。

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:89px;">Company</td>
			<td style="width:104px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Peristaltic Pump</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">Miniplus 3</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PUMP TUBING 0.50MM OVC, Orange/Yellow</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">F117934</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Three-way Stopcock w/ Swivel male Luer lock</td>
			<td style="width:89px;">Smiths Medical&#160;</td>
			<td style="width:104px;">MX9311L</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Sylgard 184 Solar Cell Encapsulation for Making Solar Panels</td>
			<td style="width:89px;">ML Solar LLC</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Pyrex Medium Bottle, 1L, GL45</td>
			<td style="width:89px;">VWR</td>
			<td>16157-191</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">C-FLEX Tubing</td>
			<td style="width:89px;">Cole-Parmer</td>
			<td style="width:104px;">06422-02</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">1 mL TB Syringe</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">309659</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Polymer Tubing</td>
			<td style="width:89px;">IDEX</td>
			<td style="width:104px;">1520G</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Sterile Intramedic Luer Stub Adapter</td>
			<td style="width:89px;">Clay Adams</td>
			<td style="width:104px;">427564</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PrecisionGlide Needle</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">305195</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Spectrophotometer</td>
			<td style="width:89px;">HACH</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Syringe filters- sterile (0.2 &#956;m)</td>
			<td style="width:89px;">Fisherbrand</td>
			<td style="width:104px;">09-719A</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">MAXQ Shaker</td>
			<td style="width:89px;">Thermo Scientific</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Ammonium sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">A4418</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium phosphate dibasic anhydrous</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">RES20908-A7</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Monobasic potassium phosphate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">P5655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">S7653</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Magnisium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M8266</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C5670</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium sulfate dihydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C3771</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Iron(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">215422</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Manganese(II) sulfate monohydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M7634</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Copper(II) sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">451657</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Zinc sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">Z0251</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Cobalt(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium molybdate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">243655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Boric acid</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">B6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Dextrose</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">D9434</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Luria Bertani Broth</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">L3022</td>
			<td>Growth media</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">TCS SP2 Confocal Microscopy</td>
			<td style="width:89px;">Leica</td>
			<td style="width:104px;"></td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:227px;">SYTO 62</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">S11344</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:227px;">Cy5</td>
			<td style="width:89px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">PA15100</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Red Fluorescent (580/605) FluoSphere</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">F-8801</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">BioSPA</td>
			<td style="width:89px;">Packman Lab</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">ImageJ</td>
			<td style="width:89px;">NIH</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">Volocity</td>
			<td style="width:89px;">PerkinElmer</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Streams 2.02</td>
			<td style="width:89px;">University of Cantebury</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
	</tbody>
</table>

methods for characterizing the co-development of biofilm and habitat heterogeneity

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。

双入口的微流体流动池允许观察生物膜生长的下由两种溶液的流动室中混合形成的明确定义的化学梯度。由此产生的化学梯度的前身是由染料注射观察其特征详细宋等人。14。形成平滑的浓度梯度在横向方向上， 如图1中的浓度分布是陡峭的邻近入口和下游得到放宽由于扩散（ 图1）。观察下一个营养梯度生物膜发展，我们使用一个定义的最小生长培养基（F...

Watch this Scientific Journal Video about Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity at JoVE.com

Methods for Characterizing 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 ...

methods-for-characterizing-co-development-biofilm-habitat

Research

JoVE Journal

Bioengineering

9.8K 次观看.  Northwestern University. 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. 

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

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

方法Nanoformulated的抗逆转录病毒药物的血源性巨噬细胞运输的发展

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

科研

生物工程

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

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

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 porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

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 ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...