Electron microscope imaging was conducted at the Electron Microscopy Unit of the Weizmann Institute of Science, supported in part by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. This research was also supported by the ISF I-CORE grant 152/1, Mr. and Mrs. Dan Kane, Ms. Lois Rosen, by a Yeda-Sela research grant, by the Larson Charitable Foundation, by Ruth and Herman Albert Scholars Program for New Scientists, by the Ilse Katz Institute for Materials Sciences and Magnetic Resonance Research grant, by the Ministry of Health grant for alternative research methods, and by the France-Israel Cooperation - Maimonide-Israel Research Program. IKG is a recipient of the Rowland and Sylvia Career Development Chair.

1.评估小分子抑制剂的影响薄皮生物膜集落形成<ol><li>制备定义生物膜诱导MSgg介质25的2倍的溶液未经氯化钙和氯化铁（III）六水合物。除菌过滤后，加入氯化钙。介质是准备直接使用，或者它可以被存储在4℃在黑暗中。 </li><li>准备在实验当天的1倍MSgg稀释。 <ol><li>稀释2倍MSgg介质用无菌蒸馏水（药膜）或无菌的3％热（80℃）琼脂（生物膜）为1x和添加铁（III）六水合氯化至50微米（药膜）或250μM的终浓度（生物膜）。添加抗生素或小分子抑制剂至所需浓度，搅拌均匀。例如，为了获得0.5mM的D-亮氨酸的终浓度在30毫升建立药膜或生物膜，添加196.6微升76.3毫米（10毫克/毫升）D-亮氨酸原液。 没有TE:最终1倍MSgg组合物在表1中描述相比于原来的配方25中 ，培养基中含有50微克/毫升的苏氨酸和铁浓度为固体MSgg培养基上生长的生物膜的菌落增加到2.5倍，以优化皱菌落形态。 </li></ol></li><li>琼脂凝固后，干燥的生物罩固体MSgg板为前接种30-45分钟。 </li><li>以选择与薄膜形成（ 图1）的机制干扰特异性抑制剂，排除使用的浓度影响浮游和静态的增长。 <ol><li>由每一小时测量的光密度在600nm直到静止生长期确定在一个简单的生长曲线浮游生长（超过在液体培养时间增加光密度）。 </li><li>以确认测得的培养的浊度表示活细胞计数，测定菌落形成单位数（CFU）细胞从振荡培养浮游增长阶段之后的几个时间点。 </li><li>要很好，在相同条件下接种在部分1.7-1.9描述的24孔细胞培养评估在23℃静态薄膜生长，收获细胞在3天的培养结束小分子抑制剂的作用并确定CFU。对于该控制，使用防护膜缺损株缺乏对细胞外基质成分编码操纵子（ 即 ， 枯草杆菌 ΔepsH，Δ 塔萨 ）。 注意:此菌株能够在静态条件下生长的，但在对比一个薄膜形成用野生型，它是在浮到液体-空气界面，在那里生长由于氧含量26增加青睐的能力不足。因此，该细胞外基质 - 和防护膜缺损株是建议的参考菌株，以评估在静态条件下的生长。 注:对于特定的前充足非规范D-氨基酸D-亮氨酸的以下描述，对浮游和静态生长的效果，其浓度与薄膜的形成被排除12,17干涉。确定浮游和静态生长方法详细17进行说明。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54612/54612fig1.jpg" /> 图1.一个强大的实验装置的识别概念性概述评估生物膜形成特异性抑制。对于小分子抑制剂，表明与生物膜形成不会对浮游增长显着的影响干扰特定选择标准。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54612/54612fig1large.jpg" target="_blank">请点击此处查看大图这一数字。</a> <ol start="5"><li>连胜了B.从-80℃股票杆菌 （LB文化前进在20％甘油冷冻重10 9细胞/ ml），以用无菌尖端或涂药棒上的LB-1.5％琼脂平板上分离单菌落。 </li><li>在30℃下生长过夜。 </li><li> 关键步骤:对于由非规范D-氨基酸如D-亮氨酸健壮防护薄膜组件的抑制，成长单菌落从LB-1.5％琼脂平板上挑取3毫升LB培养基在37℃下4小时的振荡培养箱（振荡速度200rpm）下。通过在6000 XG离心1.5毫升首发培养4分钟，小心除去上清液和再悬浮在1.5ml MSgg介质颗粒在接种前更换LB培养基生物膜诱导MSgg网上平台。培养的剩余部分可以被丢弃。 重要的是:为了保证系统的鲁棒性，在洗涤起子培养物在600nm（OD 600）的光密度应为0.6和1之间。 </li><li>在发酵剂培养物生长，准备12孔细胞培养板multidish containin克3毫升MSgg培养基没有或小分子抑制剂（ 例如 ，0.3，0.5，1mM的D-亮氨酸17）的浓度范围。为了排除边缘效应，横跨multidish板分配不同浓度的位置。另外，使用含有1.5毫升MSgg介质中的24孔细胞培养multidish板。 </li><li>接种12孔细胞培养multidish板的孔用3微升洗涤起子培养（1:1000稀释）。 注:较低的稀释比， 即 ，1:500都可以使用。这降低了药膜的开发时间。 </li><li>生长的菌膜在23℃的静态条件下三天。在此期间不要移动菌膜，因为它会影响薄膜的最终表面形貌。 </li><li>获得闪电的双眼和均匀曝光的照片。另外，取药膜的图片用高清晰度摄像机。以避免由于inconsis假象帐篷灯角度和阴影，采取自上而下的照片与固定在三脚架拍摄，并在双方的45°，用柔软的大光源。 注:另一种方法来研究B.枯草多细胞是固体，生物膜诱导MSgg介质上的生物膜菌落检测。像菌膜，此法可以让时空过程的研究。一旦小分子抑制剂的活性范围是确定的，其上生物膜集落形成的影响进行研究。 </li><li>生长生物膜菌落，对称地发现1.5微升未洗预培养上与模板的帮助下干燥MSgg 1.5％琼脂平板上（步骤1.7） - 每8.5厘米直径的培养皿4滴。让液滴吸附到板移动之前。 注意:模板有助于获得区域内的生物膜集落，其中细胞生长的相等分布。为了制备模板，绘制在原始尺度生长的总面积，将其划分为等于节ORS和纪念中心。 8.5厘米口径的圆形培养皿中，这种分配14厘米2一个生物膜殖民地。 </li><li>孵育在30℃下将板三天。在此期间，生物膜集落发展形成一个三维的，皱结构。 </li><li>拍照如步骤1.11。 </li></ol> 2.乙醇抗性测试<ol><li>如步骤1.1-1.7和1.12-1.14描述生长生物膜。 </li><li>在30℃下68小时生长后，切生物膜菌落成两个相等的部分用剃刀刀片和模板的帮助。 </li></ol><img alt="图2" src="/files/ftp_upload/54612/54612fig2.jpg" /> 图2.示例实验设计，生物膜集落细胞性评估消毒剂。用于生物膜菌落整个培养皿中的平等分配和切割（A）模板。 （B）未处理的野生型生物膜的自上而下的图像中生长，在30℃下为固体，限定生物膜诱导MSgg培养基上68小时。扩大了如何生物膜殖民地在相等的两半被削减。 （C）两个相等生物膜半部同样处理（对照，PBS）或PBS或灭菌剂和所述方法处理。比例尺:1厘米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54612/54612fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>小心提起生物膜集落的每一半由用小铲琼脂板，并将其移至含有500微升磷酸盐缓冲盐水（PBS）的1.5ml微量离心管中。如果需要的话，从板刮去剩余的细胞，并将其转移到微离心管，以及。 注:生物膜集落的后半部分被差分处理，这取决于它是否是控制或进行测试以免缝的阻力LIZING代理商。 </li><li>对于对照，孵育于500μlPBS中的生物膜集落的后半部分如在步骤2.3。为了评估耐灭菌剂，生物膜集落的后半部分转移到500μl的50％（体积/体积）乙醇。 可被采用的灭菌剂如次氯酸钠:注。对于所使用的所有消毒剂，确定了初步的实验中活性浓度和培养时间。 </li><li>孵育生物膜菌落在室温下的台式10分钟。 </li><li>离心5分钟，生物膜集落18,000 xg离心， 小心地用移液管去除上清。加入300微升的PBS。 </li><li>超声处理用超声波仪的微尖的细胞轻度（振幅10％，脉冲5秒）。 注:超声能量必须足以分离生物膜聚集体。但是，过于苛刻超声可裂解细胞。请事先确认减光镜的超声能量使用不溶解细胞，并且所有的聚集体溶解。 </li><li>加入700μl的PBS以1毫升的最终体积。在PBS中进行系列稀释（到10 -7），传播100微升3稀释使用无菌玻璃珠的LB-1.5％琼脂平板上。 注:待镀的最佳稀释度应在一个初步实验来确定，因为这取决于在感兴趣，细胞的存活率生物膜集落细胞的量响应于灭菌剂。 </li><li>孵育板在30℃过夜，算上CFU并确定CFU /毫升。从最终的CFU / ml的每个半生物膜集落，计算存活的百分比。 注意:当进行分析如所描述的，控制生物膜集落的两个半部和未处理相对于未处理的生物膜集落应该产生在活菌数在10％以下的差异的乙醇处理的一半，验证对称性或集落的电阻，respe沉着应对。或者，所述结果可以在总CFU表示。控制和未处理的生物膜集落的细胞计数应该保持在相同的数量级顺序。相反，小分子处理的生物膜集落的乙醇处理的一半的细胞计数，预计由最小两个数量级的权利要求用于向灭菌剂的敏感性增加下降。 </li></ol> 3.香港生物膜样品制备扫描电镜<ol><li>如步骤1.1-1.7 1.12-1.14和成长描述生物膜殖民地。 </li><li>制备一批新的2％（V / V）戊二醛，3％在100mM二甲胂酸钠（体积/体积）低聚甲醛溶液，5mM的氯化钙缓冲液，pH 7.3。准备5毫升固定液8.5厘米直径的每一个培养皿。 注意:戊二醛和多聚甲醛是危险的。用化学罩内的安全设备处理它们。丢弃的解决方案和污染材料的HAZardous浪费。 </li><li>小心地将固定剂添加到生物膜殖民地，而不是直接在生物膜顶部配药。 注:由于生物膜集落的疏水特性，菌落慢慢从琼脂分离并开始浮动。 </li><li>仔细密封板用封口膜的条带。孵育在旋转摇床上在室温下2小时，并随后将板转移到4℃过夜。 </li><li>第二天，小心与连接到真空泵的巴斯德玻璃吸管除去液体。 </li><li>小心加入10 mL的100mM二甲胂酸钠，5mM的氯化钙缓冲洗生物膜并孵育5分钟。轻轻从板的角上的巴斯德玻璃吸管除去液体，以避免损坏生物膜并通过温和移液添加新鲜的洗涤溶液。重复此步骤一次。 </li><li>对于生物膜殖民地的脱水，继续进行下面的步骤:在DDH 2 O 2个5分钟;在3 2X 20分钟0％的乙醇; 2×20分钟以50％的乙醇; 2×20分钟以70％的乙醇; 2×20分钟以96％的乙醇; 2×100％乙醇30分钟。 <ol><li>加入15mL液体8.5厘米直径的在每一步每一个培养皿中，每个孵化后，小心地取出液体。 </li></ol></li><li>使用两种不同的方法之一用于从乙醇干燥的样品。 <ol><li>对于乙醇空气干燥: <ol><li>切宿舍纤维素滤纸（9厘米口径）。简而言之淹没四分之一在100％的乙醇，然后一个浮动生物膜菌落仔细转移到它。放湿滤纸的陪替氏培养皿用滤纸衬里。盖培养皿，让殖民地生物膜在化学罩干通宵。 </li></ol></li><li>对于使用二氧化碳（CO 2）作为过渡流体-drying临界点（CP）: <ol><li>填充临界点干燥机械室用100％乙醇的75％。转移样品到一个保持器，每个采样到自身的Chamber。如果需要的话，用剪刀剪生物膜成更小的碎片。所有的处理过程中留在乙醇浸没样品。然后，保持器传送到腔室并紧紧关闭腔室。 </li><li>冷却室7°C，并开始搅拌。用液态二氧化碳完全填充室。期间7分钟温育时间，让与CO 2的乙醇混合。然后，排出该溶液的25％。 注意:不要空下样品的水平室。 </li><li>重复步骤3.8.2.2四次。 </li><li>重复步骤3.8.2.2仅为5分钟的温育时间五倍。最后，乙醇应完全由二氧化碳代替。 </li><li>在最后一轮中，腔室的仅5空％。关掉搅拌和冷却。启动腔室加热至42℃。在31.1℃的温度和73.9巴的压力，液体的 CO 2达到其临界点时，该状态下的气态ph值酶具有相同的密度作为溶剂27的液相中。一旦温度达到42℃，孵育10分钟。在42℃下，在腔室中的CO 2的形式存在的超临界气体。 注意:经常检查室的压力。压力应不超过42°C 120条。 </li><li>开始慢慢地持续加热释放气体。这样可以使中的CO 2气相试样，并防止样品形态的变形通过液体表面张力。通过微调将流量计5升/小时计量阀控制流量计。等到在腔室中的所有的压力被释放。现在打开室，并从支架小心地取出样品。 </li></ol></li></ol></li><li>涂层的电子显微镜存根与碳胶带。用镊子的帮助下，小心地转移生物膜菌落到存根。通过增加从汽车薄桥连接各殖民地存根盂兰盆胶带，这是在电子束下消除电荷至关重要。在这个阶段，小心处理生物膜菌落，它们是很脆弱的。存储样品在干燥器中至少24小时，或直到检查。 </li><li>用扫描电子显微镜检查的那天，溅射涂层2分钟生物膜集落中在金 - 钯溅射镀膜机60°角。重复此步骤两次，并通过120°之间旋转样品。最后，溅射涂层样品一次从顶部3分钟。金 - 钯的20纳米薄层提高了导电性和提高了样品的用于在SEM成像对比度。 </li><li>存储样品在干燥器中避免样品28的再水化之前，用扫描型电子显微镜29,30成像。 </li></ol>

The authors have nothing to disclose.

枯草杆菌形式健壮和高度结构化的生物膜无论在液体（药膜）和固体培养基上（集落）。因此，它可作为一种理想的模式生物表征特定生物膜抑制剂的作用方式。在固体培养基中，细胞形成具有独特的功能，不在药膜明显，像皱纹从中心到边缘的辐射的多细胞结构。因此，菌膜和菌落互补系统学习B.枯草多细胞。 这项研究的目标是开发一个B.生物膜（药膜和殖民地）抑制枯草模型系统。有几个步骤是生物膜干扰文章的重复性是至关重要的。第一关键步骤是，以确认对生物膜形成的影响是不是由于一般的毒性在浮游生活方式生长的细菌。这可以通过比较作为给定浓度的影响被认为上的相对生物膜抑制到该分子上浮游生长的效果商场分子抑制剂。生物膜发展的一个显着更强的效果可以保证的是生物膜的发展特别重要的目标机制的鉴定。此外，本文中所描述的研究表明的预培养条件药膜的发展及其与小分子灵敏度的重要性。表征小分子，可再现的和强大的实验条件（ 例如 ，薄膜生长温度和缺乏媒体组合物的敏感性的）来测试它们的效果的效果前应进行定义。找到这样的条件下，在预培养条件的介质，预培养，预培养介质取出，生长温度和生长媒体的生长阶段，应考虑。 一个主要的挑战，一旦生物膜形成的特异性抑制剂被发现，是寻找定量和表征生物膜发展和抵抗这些小分子抑制剂的效果的定性方法。这里，两个重要的方法是在可用于评估这样的抑制剂的效果详细描述。首先，我们介绍，可以重复地探测的处理与未经处理的细胞集落的生物膜以杀菌剂例如乙醇的电阻的简单定量分析方法。代表性的结果示于D-亮氨酸处理的生物膜菌落至50％的乙醇的灵敏度。然而，该文章可以容易地通过使用不同的灭菌剂或抗生素修改。第二，用SEM方法进行详细，允许在几个补充水平所引起的小分子抑制剂，生物膜集落的变化高分辨率检查描述:整体结构，组织和所述EPS矩阵的丰度和其组件，以及单细胞形态在整个生物膜。我们注意到dehydrati的这两种方法上（从使用二氧化碳作为过渡流体100％的乙醇或CP干燥空气干燥）可以同等生物膜菌落的SEM样品制备过程中使用。重要的是，生物膜样品的成功固定在很大程度上依赖于生物膜集落的疏水性。此疏水性为B.的固有特性枯草生物膜由于疏水表面层10,34。 有几种方法以前用来研究生物膜组装和发展B.枯草芽孢杆菌作为模式生物35。一些独立的研究证明媒体组成生物膜发展的36-38的影响。到目前为止然而，介质组成对生物膜抑制效果的系统的评价是，据我们所知，缺乏。这项研究表明，虽然B的薄膜抑制枯草由小分子是预培养条件敏感，它是UNA由温度和介质组成，从而为小分子抑制剂的系统的屏幕有用的效果ffected。此外，我们建立了一种简单的，可再现的和定量的方法来评估未处理的或小分子抑制剂治疗的生物膜菌落抗菌剂的抗性，目前缺乏的生物膜模型生物体B.枯草 。 与荧光报告组合枯草杆菌生物膜可以采取进一步寻找靶向的生物膜形成任一特定的发育步骤或生物膜内的细胞的特定亚群的小分子抑制剂。在本文描述的方法不生物膜内的基因表达方面提供单细胞分辨率。对于内B. EPS基质基因表达分析方法在单细胞水平杆菌生物膜成功39建立。 细菌生物膜是极其关键的中在农业，工业和临床设置升意义。在农业方面，以形成生物膜的能力增加许多植物的植物宿主定植病原体40，并在临床方面，生物膜是抗微生物剂21都具有抗性，并且在许多持久性和慢性细菌感染的核心。此外，它现在承认，生物膜具有巨大的成本影响到行业，因为它们非常难以去除和控制41。因此，制定一个实验性的框架微生物生物膜抑制剂的研究将提供显著农业42,43，临床21,44和技术进步45-47。 目前的方法被限制为B.枯草 。我们鼓励下面所描述的定量和定性的概念，研究特定生物膜小分子抑制剂在其他物种中也是如此。此外<html>本研究涉及用于通过D-亮氨酸生物膜抑制的具体例子，出若干特定生物膜抑制剂的先前17发布。重要的是，D-亮氨酸已表征为在植物病原体黄单胞菌柑桔 48的抗生物分子，呈现植物定植和生物膜形成之间的可能的相似性。生物膜形成（薄膜和皱纹的集落形成）也是常见的人类病原体（ 例如， 铜绿假单胞菌 49-51和尿路致病性大肠埃希氏菌 52,53）。所述方法可以进一步发展以寻找抑制生物膜形成和减少在临床研究的抗微生物剂生物膜介导的抗性特异性药物。总体而言，这里描述的方法可以被用作基础来开发的定量和定性概念研究特定生物膜小分子抑制剂在其它物种中也是如此。 NT"&gt;综上所述，我们提供了一个简单而有用的工具盒，演示了潜在的优势和缺陷在使用枯草芽孢杆菌 ，研究生物膜抑制剂。

多细胞细菌群落的自然和人为环境中发挥显著作用，可以是有益或有害的高度。这些多细胞集落被称为生物膜，其中所述个体的细胞包埋在自产生的细胞外聚合物（EPS）基质中。 EPS的强烈坚持的细胞，他们殖民表面。它们作为对机械和化学力盾，并创建相邻小区之间的紧密联系，促进蜂窝通信1。生物膜可以被看作是一个分化的社会，在那里细胞利用高度管制，编排流程在社区内协调其活动，以及跨品种2-5。从生长的浮游，自由生活模式到生物膜状态的转变通常与发育过程相关联。一个很好的例子是革兰氏阳性土壤细菌枯草芽孢杆菌 ，因此一个undomesticated应变作为一个强大的模式生物研究发展阶段，导致生物膜的形成。在这种细菌，游动细胞组织起来成执行特殊任务4显眼的多细胞结构。细胞的一组中，基质生产者分泌胞外多糖6中 ，淀粉样蛋白塔萨7,8，和表面疏水性蛋白质BSLA 9,10;所有这些参加EPS 11-13的组装。 考虑到自然和人为壁龛丰富生物膜和公认的致命伤害，他们可能会导致，有迫切需要找到办法，以防止它们的形成。小分子抑制剂可以在新的监管途径，酶和参与生物膜形成的结构蛋白的发现帮助，从而促进多社区装配的复杂过程的见解。作为B.枯草芽孢杆菌是一个生物充分研究模型成膜14,15，它可以被用来评估各种生物膜抑制剂的效果。这项研究铲球是评估生物膜的小分子抑制剂的反应键四种基本方法。首先，以确保这些抑制剂具有特定生物膜目标，从上生物膜形成的影响上浮游生长的作用的分离是至关重要的。大多数抗菌药物靶向的浮游生长期细胞，但该目标生物膜的生活方式分子是罕见的。另外，如不影响浮游生长的分子是没有毒性，它们能减少选择压力利于抗生素抗性突变体16。例如，当生物膜与D-氨基酸或某些其它细胞壁干扰分子治疗，他们要么干扰或拆卸，但这些抑制剂仅轻度影响浮游生长12,17。与此相反，许多抗生素大大削弱浮游生长，与L-ittle或生物膜形成17没有影响。 第二，建立一致的和健壮的试验框架研究小分子的作用是至关重要的。我们观察到的小分子抑制剂的活性浓度范围是对预培养条件，并用于研究这些小分子抑制剂的效果的实验装置的敏感。各种报告，特别是那些学习B.枯草芽孢杆菌，揭示了在浓度范围内的变化在该D-氨基酸抑制药膜的形成-浮细菌生物膜12,17-19。此处呈现的结果表明，下列因素考虑在活性浓度范围不同:预培养条件（对数12,17与晚平稳20生长阶段），在该预培养条件中使用的生长培养基（丰富，未定义[卢里亚肉汤，LB]对定义[味精- 甘油，MSgg]），接种率，特别是在接种前除去预培养基的。静态防护膜生长的温度显示出在小分子抑制剂D-亮氨酸，在这项研究中使用的代表性的D-氨基酸的活性范围不太重要的作用。 最后，一旦生物膜与特定的生物膜抑制剂，需要坚固的信息的方法来表征生物膜健身这些抑制剂的效果处理。 （1）的影响生物膜集落及其与抗菌剂抗性内对单个细胞:在这里，两种方法来独立地表征小分子抑制剂的效果进行详细说明。相对于自由生活的细菌21-23时生物膜细胞通常对抗生素更耐。虽然这种现象是多因素的EPS，以减少抗生素的渗透能力通常被认为是一个有吸引力的解释24 </suP&gt;。此方法评估预建立生物膜细胞暴露于抗菌物质后的存活。 （2）向小规模上生物膜集落结构的影响，从大。生物膜菌落可以通过三维结构和所述EPS的存在表征。利用扫描电子显微镜，在细胞形态，生物膜集落结构和EPS的结构和数量的变化可以从大（毫米）被可视化的小规模（微米）。

<table border="0" cellpadding="0" cellspacing="0" width="897">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Luria Broth, Lennox</td>
			<td style="width:305px;">Difco</td>
			<td style="width:127px;">240230</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate monobasic&#160;</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate dibasic&#160;</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher Scientific, 209.27 g/mol</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnesium chloride hexahydrate&#160;</td>
			<td>Merck, 203.30 g/mol&#160;</td>
			<td>1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride anhydrous</td>
			<td>J.T. Baker, 110.98 g/mol</td>
			<td>1311-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">manganese(II) chloride tetrahydrate</td>
			<td>Sigma, 197.91 g/mol</td>
			<td>31422-250G-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iron(III) chloride hexahydrate&#160;</td>
			<td>Sigma, 270.30 g/mo)</td>
			<td>F2877-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zinc chloride anhydrous&#160;</td>
			<td>Acros Organics, 136.29 g/mol</td>
			<td>424592500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thiamine hydrochloride</td>
			<td>Sigma, 337.27 g/mol</td>
			<td>T1270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-tryptophan</td>
			<td>Fisher Scientific, 204.1 g/mol</td>
			<td>BP395-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-phenylalanine</td>
			<td>Sigma, 165.19 g/mol</td>
			<td>P5482-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-threonine</td>
			<td>Sigma, 119.12 g/mol</td>
			<td>T8625-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol anhydrous</td>
			<td>Bio-Lab Itd</td>
			<td>712022300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamic acid monosodium salts hydrate&#160;</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>G1626-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-leucine</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>855448-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol anhydrous</td>
			<td>Gadot</td>
			<td>830000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">razor blade</td>
			<td>Eddison</td>
			<td>NA</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">circular cellulose filter papers</td>
			<td>Whatman, 90 mm</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glutaraldehyde</td>
			<td>EMS (Electron Micoscopy Science), 25% in water</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde&#160;</td>
			<td>EMS, 16% in water</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium cacodylate</td>
			<td>Merck, 214.05 g/mol&#160;</td>
			<td>8.2067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride 2-hydrate</td>
			<td>Merck, 147.02 g/mol&#160;</td>
			<td>1172113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stub-aluminium mount</td>
			<td>EMS, sloted head</td>
			<td>75230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">carbon adhesive tape</td>
			<td>EMS</td>
			<td>77825-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker 37&#176;C</td>
			<td>New Brunswick Scientific Innowa42</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf table top centrifuge 5424</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:301px;">Digital Sonifier, Model 250, used with Double Step Microtip</td>
			<td>Branson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 30 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 23 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter System, 500 ml, polystyrene</td>
			<td>Cornig Incorporated</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotary Shaker - Orbitron Rotatory II</td>
			<td>Boekel</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S150 Sputter Coater&#160;</td>
			<td>Edwards</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPD 030 Critical Point Dryer</td>
			<td>BAL-TEC</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Environmental Scanning Electron Microscope</td>
			<td>XL30 ESEM FEG Philips (FEI)</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

该薄膜法是一种方法来研究B的严格监管和动态过程枯草多细胞。除此之外，薄膜法适合于测试的范围内任一起动机前置条件或小分子的浓度在单细胞培养multidish板在一项实验。然而，B.枯草薄膜的形成是在培养前的条件是敏感的（ 例如 ，预培养的生长培养基和其生长阶段），接种比率并在除去预培养基的。因此，我们首先筛选了一个实验装置，使我们能够再现由D-亮氨酸薄膜抑制。我们的结果表明，D-亮氨酸再现的防护膜抑制可以如果细胞处于未定义，富LB培养基至中期对数生长期（单菌落在3ml LB预培养，在37℃4小时振荡得到），离心，以1重悬浮于定义MSgg介质之前:1,000 inoculat离子（ 图3A）。当细胞在MSgg培养基中生长到中期对数生长期（单菌落在1ml LB 2小时，再以1:100稀释至3ml MSgg，并生长5小时，在37℃下以200振荡转），除去预培养基的没有对D-亮氨酸活性的效果。细胞生长至稳定生长期（单菌落在1ml LB 2小时，再以1:100稀释至3ml MSgg，并在在室温下在滚筒生长长达20小时），并在中MSgg培养基洗涤接种是较不敏感。然而，这增加了向D-亮氨酸的防护膜抑制作用抗性可能是由于在预培养的更高的细胞密度，从而增加了接种比率。预培养，以在MSgg介质静态生长， 即接种比率，1:500，或1:1000，影响D-亮氨酸的活性范围和薄膜的发展（ 图3B）的时间。通过D-亮氨酸抑制薄膜发生在不同的生长温度（23℃和30℃， 图3C）。重要的是，当细胞对D-亮氨酸的敏感性依赖于预培养条件下，这种现象在不同温度下的再现性表明，由小分子D-亮氨酸防护膜抑制具有强大的功能。 <img alt="图3" src="/files/ftp_upload/54612/54612fig3.jpg" /> 图3.实施例的结果，显示对D-亮氨酸防护膜抑制各种预培养条件的影响的若干参数进行评估，以获得一个强大的实验装置，包括:（A）去除污染物从培养预增介质（未洗涤与洗涤），预培养的生长培养基（丰富，未定义LB培养基或定义生物膜诱导MSgg介质），和预培养物的生长状态（对数与静止生长期），（预培养的500）到最终薄膜生长介质，以及（C）生长温度（23℃与30℃）B.:B）的接种比率（1:1000对1。 枯草 NCIB 3610细胞中，在37℃或在摇动室温过夜（O / N）4小时指示（LB或MSgg）的培养基中生长，并预培养介质用MSgg取代，如果指明（洗涤或不洗）。最后，将起子培养物接种1:1000，如果没有另外说明并且药膜是静止的条件下生长，在23℃三天。自上而下的照片是用照相机获取的。井径为22毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54612/54612fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 文化先决条件的鉴定和实验装置，允许为D的可重复的活动范围后，- 亮氨酸上薄膜形成（在LB生长到中期对数生长期，在MSgg洗涤预培养，稀释1:1000，在23℃下生长三天），效果是为其鲁棒性的测试。为了这个目的，在薄膜形成D-亮氨酸的活性在不同的改性MSgg媒体（ 图4）进行了评估。在防护薄膜组件抑制D-亮氨酸的活性范围是在测试的五个媒体一致，并表明，D-亮氨酸的上薄膜形成的影响是鲁棒的。用D-酪氨酸，其中在浓缩防护薄膜组件抑制范围高达2微米在几个改性MSgg媒体（低级铁浓度并用氯化铵或与葡萄糖的碳源，数据未更换氮源一致观察到类似的趋势示出）。 <img alt="图4" src="/files/ftp_upload/54612/54612fig4.jpg" /> 图4.敏感性D-亮氨酸发生在各种生长介质。显示的是B的自上而下的照片枯草 NCIB 3610药膜，所定义，生物膜诱导MSgg介质或在不同的改性MSgg介质（碳源0.5％葡萄糖静态条件下在23℃生长;氮源0.5％的（NH 4）2 SO 4;高铁250μM的FeCl 3;低甘油0.125％甘油）三天，除了替代氮源培养基（其中药膜中生长5天）或无，或在指定浓度D-亮氨酸。起始培养中生长4小时，在37℃，在未定义的，丰富的LB培养基中振荡。在接种前，将细胞通过离心洗涤并且重新悬浮于相应的防护膜生长培养基中。起动培养物稀释1:1000，该井直径为22毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54612/54612fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </a&gt; 一种替代方法，研究B.枯草多细胞扎实MSgg介质上的生物膜菌落检测。像菌膜，此法可以让时空过程的研究。一旦小分子抑制剂的活性范围在防护薄膜组件系统被确定，它们对生物膜集落形成的影响进行研究。在这项研究中，生物膜集落被用于评估D-亮氨酸的生物膜集落的耐灭菌剂的效果，因为生物膜菌落不太易碎并且在固体背景，它们更容易操纵。当与D-亮氨酸处理的生物膜菌落暴露于50％乙醇10分钟，存活细胞的百分数急剧下降比未处理部分（ 图5）。这种方法可以进一步发展，以评估其他小分子抑制剂的作用和它们对于杀菌剂抗性效果（与不孕定义药物或抗生素）。 <img alt="图5" src="/files/ftp_upload/54612/54612fig5.jpg" /> 图5.未处理或处理的生物膜集落性的实施例的结果对灭菌剂。B的生物膜集落枯草 NCIB 3610在30℃生长固体，确定的培养基上68小时后，有或没有0.5毫D-亮氨酸。切割相等的两半的菌落后，一半用PBS（内部对照），而另一半用50％乙醇或PBS（对照）处理。温和超声处理，连续稀释，电镀和集落形成单位的数量之后，计算相对于内部对照的幸存者的百分比。显示的是用两种（A）或三个（B，C）复制和标准偏差的平均值三个独立实验的结果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54612/54612fig5large.jpg" target="_blank">请点击此处Ť</a>Ø查看此图的放大版本。 扫描电子显微镜（SEM）是一种强大的工具，可用于集中于生物膜集落皱纹，丰度和EPS的本地化和单细胞形态31的空间架构。扫描电镜成像需要，可以在高真空下被成像的样品。调查在高真空条件下的样品，所有本体水分子已被删除，因此，扫描电镜成像需要成像之前样品的脱水。或者，样品可以在低真空条件下，在环境扫描电子显微镜（ESEM），其中所述水合的样品轻轻地在显微镜室干燥湿模式成像。评价对生物膜集落架构，所述EPS组织和细胞形态的小分子抑制剂的效果，三种不同类型的样品脱水的进行了比较:干燥在根据湿莫显微镜德条件（ESEM），空气干燥从溶剂或临界点（CP），使用二氧化碳作为过渡流体-dried。在未经处理的和D-亮氨酸处理B.根据所用的方法， 枯草生物膜菌落不同水合阶段可以观察到。由ESEM湿模低真空条件下成像保留了生物膜殖民地的很自然的状态。然而，关于单细胞形态和车丰度和体系结构的信息是匮乏，作为对EPS仍完全水合，将细胞包埋在EPS（数据未显示）。在脱水方面，CP-干燥是最严格的程序。它除去大部分结构结合和本体水分子从组织，占30-70％的体积损失，这取决于组织32。与此相反，空气干燥保留了结合水分子。例如胚胎组织从挥发性溶剂乙醇丢失风干后其体积的18％和后59％的CP-干燥32 </SUP&gt;。由于EPS结合水，并且非常水合，建议考虑水在CP-干燥方法的完全丧失。当本研究的样品中的乙醇和空气干燥进行脱水，生物膜集落的三维结构被保留下来，单细胞出现了，在EPS被压扁，但它们仍连接并覆盖所述单电池（ 图6A） 。该脱水乙醇和生物膜菌落CP-干燥也仍保持其三维结构，并且在EPS出现像蜘蛛网（ 图6C）。在空气中干燥和CP-干燥的样品，在生物膜集落架构的小分子抑制剂D-亮氨酸的作用，单细胞形态和车结构是明显的（ 图6B和D）。在D-亮氨酸的存在下生长的生物膜的菌落较小，皱纹不太明显。的D-亮氨酸处理的细胞被拉长，表型是OBSERVED与细胞壁靶向分子33处理的细胞。此外，将细胞明显地少覆盖有EPS和经由中所见未处理生物膜集落中的EPS不紧紧连接到它们的相邻小区。所呈现的结果表明，生物膜集落脱水两种方法中，空气干燥，用乙醇或用二氧化碳CP-干燥作为过渡流体给予小分子上的单个细胞形态的效果有价值的见解，以及对所述EPS丰和体系结构。 <img alt="图6" src="/files/ftp_upload/54612/54612fig6.jpg" /> 图6.小分子可以改变大，小规模的生物膜殖民地建筑。B.生物膜菌落枯草 NCIB在不存在3610生长（A和D）或（B和D）在72小时0.5mM的D-亮氨酸的存在下在30℃下固体MSgg介质上如以下作为在协议中所述并从乙醇干燥中迷恋:（A和B）空气干燥，（C和D）的CP-干燥使用二氧化碳作为过渡流体。示出的是自上而下生物膜集落的双目图像（左，上图），自上而下干燥生物膜集落的照片安装在电子显微镜存根之前金 - 钯涂层（左，下图），低和高放大倍数通过SEM获得的图像，以显示生物膜集落皱纹或单细胞形态/ EPS组织的三维结构。比例尺从左至右依次为:5毫米，100微米，2微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54612/54612fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1.925毫米</td><td>锅assium磷酸二氢</td></tr><tr><td> 3.075毫米</td><td>磷酸氢二钾</td></tr><tr><td> 100毫米</td><td> 3-（N-吗啉代）丙磺酸，pH值7.1 </td></tr><tr><td> 2毫米</td><td>氯化镁六水合物</td></tr><tr><td> 700μM </td><td>无水氯化钙</td></tr><tr><td> 50μM的一个 </td><td rowspan="2">铁（III）六水氯化</td></tr><tr><td> 125μMb </td></tr><tr><td> 1μM </td><td>氯化锌无水</td></tr><tr><td> 2μM </td><td>盐酸硫胺</td></tr><tr><td> 50微克/毫升</td><td>色氨酸</td></tr><tr><td> 50微克/毫升</td><td>苯丙氨酸</td></tr><tr><td> 50微克/毫升</td><td>苏氨酸</td></tr><tr><td> 0.5％（V / V） </td><td>甘油无水</tD&gt; </tr><tr><td> 0.5％（重量/体积） </td><td> L-谷氨酸一钠盐水合物</td></tr><tr><td colspan="2"> 一个用于薄膜检测;生物膜法b </td></tr></tbody></table> 表1.用于本研究的最后1个MSgg组合物。

Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

methodologies-for-studying-b-subtilis-biofilms-as-model-for

Nanyang Technological University

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JoVE Journal

Immunology and Infection

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

15.0K 次观看.  Weizmann Institute of Science. This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

视频: Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...