这项工作是由德意志研究联合会（DFG）资助的资助KO4741 / 3-1。此外，Á.TK实验室是由玛丽·居里罗多夫斯卡集成事业补助金（PheHetBacBiofilm），并授予来自DFG KO4741 / 2-1的支持。 TH，AD，RG-M，和EM被国际马克斯·普朗克研究学院分别，亚历山大·冯·洪堡基金会，理事会全国国立西恩西亚ŸTECNOLOGIA，德意志学术交流中心（国家科学技术委员会-DAAD）和JSMC奖学金支持。

1.培养基，半固态琼脂和生物膜板，预培养物的制备<ol><li>中准备蜂拥及滑动<ol><li>溶解2克伦诺克斯肉汤（LB）和0.7g在100ml离子交换水中琼脂和高压釜的为在120℃20分钟。使用小体积（50-200毫升），以提高实验之间的重现性。 </li><li>立即灭菌后，关闭介质瓶盖以减少蒸发和放置在55℃的培养箱中至少2小时。 </li><li>介质的温度已经回火到55℃后，倒入20毫升琼脂LB培养基到实验室的无菌罩下的90mm直径的聚苯乙烯培养皿。时间推移实验，倒？35毫米直径的聚苯乙烯培养皿5毫升琼脂LB培养基。 </li><li>关闭培养皿浇注后立即，堆叠在彼此的顶部不超过4板和让琼脂培养基固化至少1小时。 </li></ol></li><li> 2xSG介质P赔偿殖民地生物膜<ol><li>溶解1.6克营养肉汤的将0.2g的KCl，0.05g的MgSO 4上的7H 2 O，和1.5g在100ml离子交换水中琼脂和高压釜的为在120℃20分钟。使用小体积（50-200毫升），以提高实验之间的重现性。 </li><li>灭菌后，立即关闭介质瓶盖以减少蒸发，并将瓶在55℃培养箱中至少2小时。 </li><li>介质的温度具有自调节到55℃后，添加0.1毫升过滤灭菌1M的Ca（NO 3）2溶液中加入0.1ml过滤除菌的100mM的MnCl 2溶液中加入0.1ml过滤除菌1mM的硫酸亚铁溶液，和0.5ml无菌的20％葡萄糖溶液。 </li><li>在实验室无菌罩，每倒直径90mm的聚苯乙烯培养皿20毫升琼脂2倍SG网上平台。时间推移实验，倒？35毫米直径的聚苯乙烯培养皿5毫升琼脂LB培养基。 </li><li>克洛斯e本培养皿浇注后立即，堆叠在彼此顶部的板，但不超过4板，并让琼脂培养基固化至少1小时。 </li></ol></li><li>发酵剂的制备 注:B.芽孢杆菌 168，在下面组成中所述的方法中使用NCIB 3610衍生菌株产生绿光或红荧光蛋白和8,27之前进行了描述。菌株常规储存在-80℃冰箱中。 <ol><li>接种-80℃储存起始培养在3ml LB培养基，并在37℃下具有水平摇动（225转）孵育过夜（16-18小时）。不要孵化文化长于18小时野生B.分离株枯草大多容易发生聚集并形成在试管的生物膜。 </li></ol></li></ol>将荧光标记的菌株2.联合接种面撒<ol><li>半固态琼脂平板的蜜蜂分群和干燥B.滑动枯草 。 <ol><li>干琼脂平板蜂拥和接种前滑动20分钟。在层流罩露出干板（ 见图1）。 注:细菌蜂拥和滑动依赖于半固体琼脂培养基的干。干燥不足使得导致鞭毛介导的游泳琼脂培养基上积水。延长干燥时间导致缺乏蜂拥的。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54752/54752fig1.jpg" /> 图1:实验工作流程中的常用的方法是在图中所示，包括准备培养介质，干燥板，接种和荧光显微镜检测（从左至右） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/54752fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <ol start="2"><li>共接种对于蜂拥及滑动细菌培养<ol><li>确定隔夜发酵剂培养物的光密度在600 nm和混合B.密度归绿光和红色荧光蛋白生产菌株枯草 NCIB 3610或在1.5ml反应管其ΔHAG衍生物。例如，对于应变2.轻度涡流（最大速度3秒）的微升（[菌株2的过夜培养物的OD 600] / 100 * [应变1的过夜培养物的OD 600]）为混合100μl的应变1的均匀分布。 注:B.枯草 NCIB 3610菌株接种到观察蜂拥，而它们的Δ 女巫衍生物用于滑动。 </li><li>斑点2微升上的预干燥板的中间混合培养的（ 见图1），并进一步干燥所述板为接种后的10分钟。 </li><li>在37℃下孵育板直立，以允许多余的水分凝结在盖而不是在琼脂表面。&lt;BR /&gt;注:孵育时间为B.枯草蜂拥一般是8-16小时之间。一般地，群的边缘到达在8小时90毫米的培养皿的一侧。滑动是一个缓慢的过程，需要培养至少16至42小时。 36小时后，将滑动前达到90毫米的培养皿的一侧。 </li><li>时间推移实验中，将35毫米直径的培养皿在预热阶段孵育室设置在37℃。确保在培养皿的盖保持在整个实验期间除去。舞台培养箱的盖设定为40℃至规避培养箱的顶部水分形成。 </li></ol></li></ol>将荧光标记的菌株3.联合接种不同初始细胞密度<ol><li>琼脂平板B的殖民地生物膜形成的干燥枯草 。 <ol><li>干燥菌落生物膜发展板无盖在层流罩接种前15分钟。 注:增加湿度和游泳或蜂拥不足的干燥效果是可能的29。在干燥生物膜小殖民地太久的结果。 </li></ol></li><li> 10倍稀释发酵剂培养菌落生物膜准备<ol><li>混合100微升绿光和红色荧光蛋白的生产B.过夜起始培养芽孢杆菌 168在1.5ml反应管中并温和涡旋均匀分布。准备在LB培养基中10倍稀释系列。 </li><li>的未稀释或10 1，10 2斑点2微升，10 3，在含有生物膜诱导培养基平板10 4稀释混合培养。 注:6至9生物膜集落可以在单个90毫米培养皿考虑到的菌落以相等距离彼此分开启动。 </li><li>在30℃下孵育板直立，以允许多余的水分凝结在盖和不是在琼脂表面。 注:孵育时间B.枯草芽孢杆菌生物膜是1天3之间。一般情况下，B的殖民地生物膜枯草达到在2天后的平均尺寸和复杂的结构。 </li><li>时间推移的实验中，将单种菌在35毫米直径的培养皿的中央，并将培养皿在预热阶段孵育室设定在30℃。确保在培养皿的顶部保持在整个实验期间除去。舞台培养箱的盖设置为35℃至规避培养箱的顶部水分形成。 </li></ol></li></ol>标记的菌株4.荧光显微镜检测<ol><li>设备描述成像。 <ol><li>为了检测表面殖民化和荧光信号，使用配有0.5X PlanApo目的电动荧光立体变倍显微镜（见材料表清单），两个LED冷光源（一个用于荧光检测，一个用于可见光），过滤器组的GFP（激发在四十零分之四百七十零nm，发射在五十零分之五百二十五纳米）和MRFP（激发在二十五分之五百七十二nm，发射在629 / 62纳米），和高分辨率单色相机。 </li><li>与可用于立体变倍显微镜，包括多通道和时间推移模块相应的软件进行图像采集和处理。对于时间流逝的实验中，使用安装在与适配器立体声变焦显微镜标准加热阶段的孵化器。 </li></ol></li><li>和蜜蜂分群滑动扩展的影像<ol><li>用最低的放大倍率拍摄90毫米的钢板最大可能的区域。设置接种的原点（90毫米的培养皿的中间）至可见领域的监测径向细菌膨胀和荧光角球。 </li><li>取决于荧光信号的强度调整最佳曝光时间。 注:对于组成expressed荧光基因B.枯草芽孢杆菌 ，绿光和红荧光为1.5和3秒的曝光时间，可以使用分别。此外，10毫秒的曝光时间是适当的可见光。 </li></ol></li><li>使用允许整个生物膜集落的检测放大率和视场的中间调整菌落。 注:对于蜂拥和滑动扩展，最佳曝光时间，以检测在所述生物膜集落的荧光信号依赖于荧光蛋白编码基因的表达水平。对于下面的代表性结果，使用1和3秒的曝光时间间隔，分别检测绿光和红光荧光。 </li><li>对于时间推移成像，获取使用恒定的曝光时间以一定的间隔图像。 </li><li>保存的文件格式的立体荧光图像的记录是由ImageJ的软件定量数据分析的认可。 </li></ol> 5.逸一个分析<ol><li>分析由每个不同地标记的荧光应变占据的区域中，打开与一个BioVoxxel插件扩展中的ImageJ软件感兴趣的文件。 <ol><li>当一个名为"生物格式导入选项"窗口出现，其中仅选择"打开全系列"的选择和"自动定标"，单击"确定"打开该文件。 注:文件显示为三个图像，一个用于用来记录在显微镜（绿光，红光荧光和明视场图像）的图像的每个信道的堆叠。 </li><li> "栈" - - 选择"图像"分开堆叠成单独的通道图像"堆栈图像"在ImageJ的控制面板。 注:图像出现，编号为1/3（绿色通道），2/3（红色通道）和3/3（明场）。这里，亮场图像被从分析中排除。 </li></ol></li><li>来分析图像，通过选择变换每个为8位的图像"图像" - "型" - "8位"。 </li><li> "设置比例" -确定像素2的占用面积，使用"分析"重置图像的规模。当一个窗口，不同规模的选项弹出，选择"单击以清除水垢"复位规模。选中选项"全球"，以去除水垢对所有打开的图像。 </li><li>要删除的背景下，利用画在ImageJ的控制面板上的"椭圆"工具荧光区域外的椭圆形区域（感兴趣区域，ROI）。 <ol><li>以确保该背景椭圆的大小是所有分析的图像是相同的，通过键盘的[T]字符它添加到的ROI经理。一个ROI管理器窗口出现在背景椭圆ROI可以通过"更多"保存 - "保存"选项。 </li><li>如果背景椭圆的ROI是在图像上可见的，测量的区域的强度通过选择"分析" - "测量"。 注:结果其中，除其他的平均荧光强度显示在标有"平均"的列中显示的窗口。 </li><li> "数学" - - "减"和插入测量值被取消选择背景椭圆ROI，单击"进程"中减去从图像的平均背景荧光强度值。 </li></ol></li><li> "调整" - - "阈值"选项通过"图像"应用​​到图像的阈值。选择方法大津和黑白（B＆W）。检查"黑暗背景"选项，并单击"应用"应用的门槛。 注:其中阈值以上的区域中的白色和被示于下面的黑色示出了阈值的图像改变为二进制图像。 </li><li>通过选择"分析"一切超出阈值 - "分析粒子"的选项。在与设置的窗口，保持默认选项，并保持了"显示结果"和"Summarize"选项选中，单击"确定"，显示在结果窗口中的总结和展示在标柱被占领区"总面积"。 </li></ol>

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</ol>

作者宣称，他们没有竞争的经济利益。

<html>细菌的荧光工具箱的可用性促进不仅异质基因表达30,31和蛋白定位32的研究，但还菌株的空间分布的群体8内的分析。具有足够不同激发和发射波长的荧光标记物允许清楚本地化两株混合时，否则是无法区分彼此。所描述的协议可以被用于观察菌株或物种间在混合培养物中的人口动态， 例如竞争实验或协同作用。确定相对丰度的荧光标记的菌株在混合群体的能力并不限于表面附蜂拥，滑动，或者生物膜集落，但是也可以用于其他多细胞生物膜系统，包括淹没，流通池或空气介质接口生物膜27,33-35。 _content"&gt;虽然提出的技术是，以检测应变和设计竞争实验的空间分布的有力工具，它也允许以下扩大菌落基因表达的异质性。这里所描述的培养条件适用于枯草芽胞杆菌和对扩张的确切参数琼脂介质可能，需要为其它物种或菌株20的优化。在孵育室放置样品而成像允许实验者跟随人口动态时刻虽然应当重视的孵育期间在腔室中的湿度水平。 此处所描述的技术还需要检测细菌菌株的遗传修饰，使得该菌株表达可以从彼此区分的荧光标记。此外，除了具有不同激发和发射光谱时，建议这两个选择的荧光标记物也有类似的定量微米的产率和在一个水平相当表示（ 即发射吸收的光子的比率）。此外，在时间的相对强度的变化可被测量并归一化到一个实验的早期时间点。的相对增加或减少可以与不同的量子效率不同的荧光团之间可以进行比较。对于所提出的实验系统，不同的绿光和红色荧光蛋白先前测试36,37选择为可以在B中检测出的最优化的荧光对枯草 。最佳曝光时间应为每个荧光蛋白和样品来确定。可能需要一定的细胞密度或细胞的多个层的人口内有效地检测该信号。某些荧光蛋白可能具有在细菌细胞由于低效的表达和/或蛋白质的翻译，因此低量子产率低的强度。这种低效率的荧光标记物可以ř得出该系统的灵敏度和扩大检测细菌菌株可能导致细胞毒性由激发光所需要的时间。的荧光强度可以通过改变用于表达荧光报道编码基因的启动子作相应的修改。的表达水平太高可能导致荧光蛋白导致对细菌有害健身成本的不必要的生产过剩。当执行竞争实验，应该考虑特定荧光蛋白生产中的单元的成本。对照实验，其中荧光标记的竞争菌株或在两个基因株只有在他们的荧光标记的不同会相互竞争之间交换，总是需要来确定对一个标记任何偏见。在细胞内的荧光蛋白的寿命也可能会影响测量的强度。此外，某些细菌物种的自发荧光S可能会发生需要使用比这里所描述的其它不同的荧光标记物。 精确地确定不同细菌菌株的空间分布与丰度，从第一荧光蛋白始发，同时使用用于第二荧光标记物，反之亦然荧光滤波器背景信号应单独对单一样品进行测试（含有产生仅一个标记的细菌）。这允许重叠的荧光信号强度的减法。重要的是，作为立体显微镜从膨胀菌落上述记录荧光信号，所提出的协议是方便的确定在两个维度的空间排列。膨胀细菌群体的体系结构，可能会导致不同的荧光水平（ 即皱纹状结构可能包含更多的细胞显示更高的局部荧光强度）。因此，图像的描述分析ðetermines的空间分布，而不是大量的应变的一定位置内。上一页协议描述的样品制备用于细菌菌落38蜂拥人口动态20或荧光成像，但我们的协议结合了这些技术。其它显微技术允许人口结构的三维分辨率（ 例如共焦激光扫描显微镜39,40或结构照明显微镜41）的观察可以应用于具有增加结构的复杂性的样品。这些附加技术也支持菌株31用立体显微镜不可用的单细胞基于检测。

在过去的几十年，微生物已被确认为与各种生态系统在地球上1,2有关的社会团体。相反在一般实验室实践中使用的浮游培养物，在环境中的微生物显示根据对生态环境的空间社区结构的一个不同的范围。简单微生物系统可以用来了解空间结构的社会互动3,4的进化的结果。同时使用真核生物和原核生物模型系统在过去的2 - 3年的出版物强调空间结构对合作的稳定性微生物种群内5-8的影响。此外，预留的相互作用中的微生物， 如代谢跨喂养，也可能改变互动的合作伙伴9-11的空间分布。空间结构的这些研究的影响是使用表面附着固着细胞居住在这样大多审查-called生物膜或在菌落生长的琼脂培养基的表面上。导致高空间分类遗传漂在微生物菌落观察，其中在细胞分裂介导的扩张导致一系列的遗传瓶颈，导致某些克隆linages 12高的局部固定概率边缘养分枯竭。遗传漂变可以使用，因此，研究空间隔离的微生物菌落的作用。 在环境中，生物膜是由自产聚合物基质13包围多种类的社区。生物膜的结构，功能和稳定性取决于社会交往的复杂网络，其中的细菌进行信号交换，基质成分和资源，或争夺空间和使用的毒素和抗生素的营养物质。 枯草芽孢杆菌是一种土壤住宅和根定殖细菌，开发高度组织上生物膜社区14。类似于社会昆虫，B.芽孢杆菌细胞雇佣劳动力战略的一个部门，开发细胞外基质的生产者和食人族，运动细胞，休眠孢子和其他细胞类型15,16亚群。分化过程是动态的，并且可以通过环境条件17,18被改变。 由细菌表面定植策略可以在实验室条件下在生长培养基修改琼脂浓度容易操纵。在低琼脂水平（0.2-0.3％），窝藏细菌鞭毛活跃能游泳，而半固体琼脂（0.7-1％琼脂）有利于带动鞭毛社区传播，称为蜂拥19-21。在没有鞭毛的，某些细菌菌株能够通过滑动， 即依赖于生长的人口膨胀由多糖基质和其他分泌疏水蛋白的化合物22-24便利，以便移到半固体介质。最后，细菌这是capabl硬琼脂培养基（1.2-2％）14,17,25生物膜发展形式架构复杂的菌落如虽然这些性状是通过精确调整的条件，在自然栖息地在实验室中检测了这些表面传播的策略可能逐渐从一个过渡到另一个取决于环境条件26。而单一的基于细胞运动是在革兰氏阳性和阴性菌27，B的复杂菌落生物膜的空气-液体界面生物膜发展的开始期间临界枯草不受鞭毛运动28的删除。然而，B的发育过程中的空间组织枯草菌落生物膜取决于用于发起生物膜8的细菌接种物的密度。 在这里，我们使用B.枯草表明，表面殖民化过程中空间隔离取决于种群水平motili机制TY（ 即蜂拥或滑动），和菌落生物膜发展取决于创始人的细胞密度。我们提出了可以适用于连续监测微生物生物膜生长，表面定植和品种在宏观尺度的荧光显微镜工具。另外，提出了一种定量方法来确定相对应变丰度的人口。

<table><tbody><tr><td>Lennox Broth (LB)</td><td>Carl Roth GmbH</td><td>X964</td><td></td></tr><tr><td>Agar-agar, Kobe I</td><td>Carl Roth GmbH</td><td>5210</td><td></td></tr><tr><td>Petri dish (90 mm diameter)</td><td>any</td><td>NA</td><td>Use Petri dishes without ventillation cams</td></tr><tr><td>Petri dish (35 mm diameter)</td><td>any</td><td>NA</td><td>Use Petri dishes without ventillation cams</td></tr><tr><td>Difco Nutrient Broth</td><td>BD Europe</td><td>234000</td><td></td></tr><tr><td>KCl</td><td>any</td><td>NA</td><td></td></tr><tr><td>MgSO,&lt;sub&gt;4&lt;/sub&gt; 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>any</td><td>NA</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2 &lt;/sub&gt;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>any</td><td>NA</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>any</td><td>NA</td><td></td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;</td><td>any</td><td>NA</td><td></td></tr><tr><td>D-Glucose</td><td>any</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Fluorescence AxioZoom V16 time-lapse microscope&lt;/strong&gt;</td><td>&lt;strong&gt;Carl Zeiss Microscopy GmbH&lt;/strong&gt;</td><td></td><td>&lt;strong&gt;see bellow detailed description&lt;/strong&gt;</td></tr><tr><td>AxioZoom V16 Microscope body</td><td>Carl Zeiss Microscopy GmbH</td><td>435080 9030 000</td><td></td></tr><tr><td>Phototube Z 100:0 for Axio Zoom V16</td><td>Carl Zeiss Microscopy GmbH</td><td>435180 9020 000</td><td>without eyepieces</td></tr><tr><td>Fluar Illuminator Z mot Fluorescence intermediate tube for Axio Zoom.V16</td><td>Carl Zeiss Microscopy GmbH</td><td>435180 9060 000</td><td></td></tr><tr><td>Controller EMS 3</td><td>Carl Zeiss Microscopy GmbH</td><td>435610 9010 000</td><td></td></tr><tr><td>System Control Panel SYCOP 3</td><td>Carl Zeiss Microscopy GmbH</td><td>435611 9010 000</td><td></td></tr><tr><td>Reflector module Z</td><td>Carl Zeiss Microscopy GmbH</td><td>435180 9160 000</td><td>For Fluar Illuminator Z mot on Axio Zoom.V16 and SYCOP 3</td></tr><tr><td>Filter set 38 HE eGFP shift free (E)</td><td>Carl Zeiss Microscopy GmbH</td><td>489038 9901 000</td><td>EX BP 470/40, BS FT 495, EM BP 525/50</td></tr><tr><td>Filter set 63 HE mRFP shift free (E)</td><td>Carl Zeiss Microscopy GmbH</td><td>489063 0000 000</td><td>EX BP 572/25, BS FT 590, EM BP 629/62</td></tr><tr><td>Mount S</td><td>Carl Zeiss Microscopy GmbH</td><td>435402 0000 000</td><td></td></tr><tr><td>Objektive PlanApo Z 0,5x/0,125 FWD 114 mm</td><td>Carl Zeiss Microscopy GmbH</td><td>435280 9050 000</td><td>164 mm parfocal length; M62x0.75 thread at front</td></tr><tr><td>Coarse/fine drive with profile column</td><td>Carl Zeiss Microscopy GmbH</td><td>435400 0000 000</td><td>490 mm, 10 kg load capacity, compatible with stand bases 300/450</td></tr><tr><td>Stand base 450</td><td>Carl Zeiss Microscopy GmbH</td><td>435430 9902 000</td><td></td></tr><tr><td>Cold-light source Zeiss CL 9000 LED CAN</td><td>Carl Zeiss Microscopy GmbH</td><td>435700 9000 000</td><td></td></tr><tr><td>CAN-bus cable</td><td>Carl Zeiss Microscopy GmbH</td><td>457411 9011 000</td><td>2.5 m length</td></tr><tr><td>Slit-ring illuminator</td><td>Carl Zeiss Microscopy GmbH</td><td>417075 9010 000</td><td>d = 66 mm</td></tr><tr><td>Flexible light guide 1500</td><td>Carl Zeiss Microscopy GmbH</td><td>417063 9901 000</td><td>8/1,000 mm&amp;nbsp;</td></tr><tr><td>Illumination Adapter for light guide</td><td>Carl Zeiss Microscopy GmbH</td><td>000000 1370 927</td><td></td></tr><tr><td>Lightguide HXP with liquid fill</td><td>Carl Zeiss Microscopy GmbH</td><td>000000 0482 760</td><td>&amp;oslash;3 mm x 2,000 mm</td></tr><tr><td>Camera Adapter 60N-C&amp;nbsp;</td><td>Carl Zeiss Microscopy GmbH</td><td>426113 0000 000</td><td>2/3&quot; 0.63X</td></tr><tr><td>High Resolution Microscopy Camera AxioCam MRm Rev. 3 FireWire</td><td>Carl Zeiss Microscopy GmbH</td><td>426509 9901 000</td><td></td></tr><tr><td>AxioCam FireWire Trigger Cable Set</td><td>Carl Zeiss Microscopy GmbH</td><td>426506 0002 000</td><td>for direct shutter synchronization</td></tr><tr><td>ZEN pro 2012</td><td>Carl Zeiss Microscopy GmbH</td><td>410135 1002 120</td><td>Blue edition, requires min. Windows 7 64-bit</td></tr><tr><td>ZEN Module Time Lapse</td><td>Carl Zeiss Microscopy GmbH</td><td>410136 1031 110</td><td></td></tr><tr><td>Standard Heating Stage Top Incubator</td><td>Tokai Hit</td><td>INUL-MS1-F1</td><td></td></tr><tr><td>Zeiss Stereo Microscope Base Adapter</td><td>Tokai Hit</td><td>MS-V12</td><td></td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health, Bethesda, MD, USA</td><td>v 1.49m</td><td></td></tr><tr><td>BioVoxxel plugin</td><td>BioVoxxel</td><td></td><td>http://www.biovoxxel.de/development/</td></tr></tbody></table>

monitoring spatial segregation in surface colonizing microbial populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

<html>细菌种群的实验室系统提供一个有吸引力的方式，探索生态或进化的问题。这里，三B的表面定植模式枯草被用来研究人口品种的出现， 即基因相同的分离，但荧光标记不同的菌株。一窝蜂，这是B的鞭毛依赖集体的地面活动枯草芽孢杆菌 ，结果在高度混合群体。在这些蜂拥殖民地，绿光和红色荧光的细菌定植地区被重叠（ 见图2A）。快速曲面定植可以遵循的时间（ 视频图1）。在枯草芽孢杆菌的蜂拥，细胞的薄层从孵育几个小时后接种中心扩展（参见图2B）。 4752 / 54752fig2.jpg"/&gt; 图2:B.蜂拥扩张枯草芽孢杆菌蜂拥殖民地包含涨跌互现1绿光和红色荧光菌:接种前1。 （A）（分别的GFP和RFP，）15小时，在绿光和红光荧光后用适当的荧光过滤器进行检测。 （B）蜂拥B.薄层图像枯草芽孢杆菌是从视频图1.比例尺=5毫米提取选定的时间点显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/54752fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 然而，当二枯草杆菌菌株，即缺乏官能鞭毛但能与产生外多糖，疏水和枯草的帮助下进行传播，被发现半固态琼脂培养基上，在不同标记的菌株在某些DEF分离INED扇区（ 见图3A）。滑动菌落的发展可以被记录在时间（参见图3B或视频图2）。 <img alt="图3" src="/files/ftp_upload/54752/54752fig3.jpg" /> 图3:滑动B.菌落枯草芽孢杆菌菌落包含涨跌互现1绿光和红色荧光菌:接种前1。 （A）后（分别为GFP和RFP，）24小时，在绿光和红色荧光适当的荧光过滤器进行检测。 （B）中的B的图像滑动盘枯草正在从视频图2.比例尺=5毫米提取选定的时间点显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/54752fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 虽然蜂拥和SLI的分类级别鼎扩大殖民地无法修改，在殖民地生物膜不同标记的荧光菌的空间隔离可以由起始细胞密度的影响。当B的殖民地生物膜枯草用混合种群的高细胞密度启动，绿光和红光的荧光菌株显示轻微或没有空间分类（ 见图4）。与此相反，当细胞密度以引发biolfilm低，明确绿光和红光荧光扇区可以通过荧光显微镜进行检测。该品种水平显然取决于生物膜发起人口的稀释水平。 视频图3和4展示用于接种菌株的最低和最高稀释菌落扩大。 <img alt="图4" src="/files/ftp_upload/54752/54752fig4.jpg" /> 图4:在B中的集落的生物膜拼盘水平在枯草（:分别非稀释至稀释起始培养物10 的5倍，从上方到下方）不同初始细胞密度绿光和红光荧光菌株的菌落生物膜后第2天，用不同的初始细胞密度接种被示出。比例尺= 5毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/54752fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 绿光和红色荧光菌株的比率可以使用ImageJ软件，允许用于该实验的菌株群体结构和竞争力的定量表征可以进一步量化。 <img alt="视频1" src="/files/ftp_upload/54752/54752video1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/Video_Fig1.mov" target="_blank">视频图1:时间流逝IMAG</a>B.蜂拥的ES 枯草 1开始:1混合绿光和红色荧光的菌株（右击下载）视频显示10小时的时间过程。比例尺= 7毫米。 <img alt="视频2" src="/files/ftp_upload/54752/54752video2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/Video_Fig2.mov" target="_blank">视频图2:侧滑B.时间推移图像枯草 1开始:1混合绿光和红色荧光的菌株 （右击下载）</a>视频显示24小时的时间过程。比例尺= 5毫米。 <img alt="视频3" src="/files/ftp_upload/54752/54752video3.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/Video_Fig3.mov" target="_blank">视频图3:B.时间推移图像绿色1混合: 枯草殖民地生物膜与1开始</a>-在高细胞密度红色荧光菌（点击下载）。视频显示48小时的时间过程。比例尺= 5毫米。 <img alt="视频4" src="/files/ftp_upload/54752/54752video4.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/Video_Fig4.mov" target="_blank">视频图4:B.时间推移图像在低细胞密度1混合绿光和红色荧光的菌株: 枯草殖民地生物膜与1开始。</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54752/Video_Fig4.mov" target="_blank">（点击下载）。</a>视频显示48小时的时间过程。比例尺= 5毫米。

Watch this Scientific Journal Video about Monitoring Spatial Segregation in Surface Colonizing Microbial Populations at JoVE.com

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

在进化的场景分类（空间隔离）的作用，可以在允许控制的空间分布调整实验室使用简单微生物系统进行检查。通过修改创始人细胞密度时，各品种水平可在枯草芽孢杆菌的菌落生物膜使用荧光标记的细菌菌株进行可视化。

在表面定植微生物种群监测空间分异

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

monitoring-spatial-segregation-surface-colonizing-microbial

Nanyang Technological University

Research

JoVE Journal

Genetics

11.0K 次观看.  Friedrich Schiller University, Jena. 在进化的场景分类（空间隔离）的作用，可以在允许控制的空间分布调整实验室使用简单微生物系统进行检查。通过修改创始人细胞密度时，各品种水平可在枯草芽孢杆菌的菌落生物膜使用荧光标记的细菌菌株进行可视化。 

视频: Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

科研

环境科学

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

实时检测和捕获共培养物中的侵袭细胞亚群

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

癌症研究

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

发育生物学

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

量化诱导剂梯度板上细菌表面聚集运动

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

生物化学

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

生物工程

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

在浮动水培系统中监测阿拉伯菌的殖民化与维护

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

免疫与感染

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

生物学

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

微殖器的高通量实时成像，测量生长和基因表达中的异质性

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

在表面定植微生物种群监测空间分异

摘要

探索更多视频

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

实时检测和捕获共培养物中的侵袭细胞亚群

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

量化诱导剂梯度板上细菌表面聚集运动

微孔阵列平台中微生物群落发展的组装与跟踪

在浮动水培系统中监测阿拉伯菌的殖民化与维护

采用共培养，以检测化学介导的种间相互作用

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

微殖器的高通量实时成像，测量生长和基因表达中的异质性

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

在表面定植微生物种群监测空间分异

摘要

探索更多视频

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

实时检测和捕获共培养物中的侵袭细胞亚群

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

量化诱导剂梯度板上细菌表面聚集运动

微孔阵列平台中微生物群落发展的组装与跟踪

在浮动水培系统中监测阿拉伯菌的殖民化与维护

采用共培养，以检测化学介导的种间相互作用

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

微殖器的高通量实时成像，测量生长和基因表达中的异质性

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用