这项工作是由健保局以R01授予GM59690授予索尼KLV支持。

1。初步表征和注意事项<ol><li>生物膜的形成是受细胞密度和增长率。因此，有必要确定增长率（光密度值（OD值）随着时间的推移增加）利益应变（S）和产量（最后的手机号码），通过执​​行简单的生长曲线和细胞电镀检测。在增长，或OD和细胞数量之间缺乏相关的缺陷，分析发现实验结果时，必须考虑到。 </li><li>评估时形成皱纹的殖民地，作为小板到板的变化可能会影响生物膜的发展，包括在同一板块上的适当的阳性和阴性对照。 </li><li>确定发现的最好的条件，即那些揭示之间的控制和突变（S）的利益，最明显的差异。生长在不同条件下，如不同的媒体或温度，液体培养菌株，不同阶段增长（指数或固定相）事先向斑点。文化点10μL，在适当的介质上的各种细胞密度，在所需的温度下，孵化直至菌落形态显现。 </li><li>端点检测涉及评估皱纹的集落形成斑点（即48小时）后，在预定的时间点。这项评估是有益的菌株，展览（或拟参展）在生物膜形成的严重缺陷。 </li><li>时间课程实验评估过了一段时间（即每隔一小时）后发现，集落形成皱纹。此法允许的生物膜形成的半定量评估，允许皱纹殖民地的形成和发展格局，过了一段时间开始的决心。对于一个给定的时间当然采集点的数量和实验的持续时间应确定在初步实验。 </li><li>可以轻松地保持跟踪时间后发现，设置在IMER以计数。 </li></ol> 2。 五皱菌落形态的微观评估鲵 <ol><li>接种五。到5毫升LB盐（LBS）的培养基13（1％W / V]蛋白胨，0.5％W / V]酵母提取物，2％W / V氯化钠，50毫米的Tris-HCl [ 鲵细胞pH值7.5），其中包含任何必要的抗生素和孵育，摇晃，一夜之间在28°C。在早晨，用1:100稀释的细胞传代到5毫升的新鲜磅中型和在同等条件下孵育，直到细胞已达到所需的600外径（例如，外径600 = 0.2或0.5）。 </li><li>吸取1毫升到离心管和离心机最大速度为一分钟的离心集文化。删除愿望上清。洗细胞重新悬浮颗粒在1毫升70％的无菌人工海水（ASW）的（35毫米硫酸镁4 7H 2 O 7毫米氯化钙，以消除残余媒体和外组件2-2H 2，210毫米氯化钠，氯化钾7毫米），重复离心。水洗颗粒悬浮在1毫升70％反潜。 </li><li>确保每个样品中含有相同数量的细胞，在600 nm的OD估计。额外的70％，屈臣氏集团更浓缩的样品稀释与任何必要的调整。在初步实验中，当场文化在不同的初始OD值，以确定一个最佳的起点外径为给定株或条件。得到最好的结果为五鲵从文化与外径约0.2点。 </li><li>发现10μL洗细胞上的LBS板包含任何必要的抗生素。当察觉到文化的一个板块，确保稳定的正上方的琼脂表面移液器（用手指）。发现垂直，而不是在一个角度，慢慢弹出的光斑均匀分布的液体。通常每一株被发现一次，每盘（每盘长达6点），机智h动板（2-3），每个实验。 </li><li>以确保均匀分布的斑点文化遗存，允许当场晾干后方可移动板的孵化器。反转板，培养他们在28°C。 </li><li>在接种后12-15小时监测的增长点每小时开始的形态。我们用相机附件（PROGRES C10的平台 ）和PROGRES CapturePro和ImageJ的软件程序，观察和记录菌落形态和评估皱集落形成的开始和进展解剖范围（蔡司STEMI 2000-C的）。 </li><li>在步骤2.5中列出的具体设置，从下面照射点与CL 1500 EC冷光源通过透明的玻璃舞台，而从上面捕获的图像。此设置是最佳成像皱纹殖民地发展，因为五鲵殖民地（点）是半透明的。 </li><li>在ST中列出的特定设备的情况下2.5 EP，显示器皱纹菌落形态，使用任何解剖显微镜，允许全殖民地观看和有可调光源，最好，一个附加的相机。如果有必要，也可以利用数码相机在没有附加的相机，但是这是不是最佳的。 </li><li>到最好的可视化皱纹殖民地的发展，这是必要的调整等，就可以看出端倪的三维形态的发展生物膜的细菌菌落下方的照明强度和反射角。确定最佳的照明条件下提供之间的强烈对比发现菌落周围的琼脂背景，这样的殖民地架构（皱纹）清晰可辨。 </li><li>斑点殖民地色彩，在某些情况下，还必须考虑到，作为殖民地色彩的改变生物膜的形成过程中可能出现。调整光源的强度和角度揭示这些微妙的色彩变化。一旦达到相应的设置，在实验期间，他们保持。 </li><li>多少时间的流逝，从接种时间的皱纹菌落形成启动每一株或条件时注意。定义为图案和三维结构的形成（即条纹形成的外缘或&#39;涟漪&#39;发生在中心）是第一个明显的时间点开始形成皱纹殖民地。突变的细胞可能会出现下降或增加皱纹的集落形成的起始时间（例如， 图2）。 </li><li>通过捕捉适当的数字图像文件开始皱纹的集落形成和发展。收集整个实验过程中的图像时，重要的是要使用相同的放大倍率。切换从显微镜目镜的电脑屏幕上，使用位于相机背面的杠杆。当开关之间的目镜和电脑屏幕上观看，调整意见，并相应地对焦。 </li><li>前成像斑文化，培养皿的盖子通常删除，以提供清晰的图像。然而，这是一个可选的步骤，可以通过使用设置步骤2.5和2.6中列出的盖子成像斑点文化。 </li><li>皱纹的集落形成的开始，在每个时间点，请注意皱纹殖民地发展的格局。架构可能由内而外的发展，或外面。这个评估提供了一种机制来区分不同品系或不同条件下形成的生物膜。 </li><li>在每个时间点测量发展菌落直径。这可以手工完成，或数字使用相关的软件程序。如果实现数字化，使用的PROGRES CapturePro的软件程序，并首次在实验中使用的具体倍率校准的标尺。包括在每个拍摄的图像的比例尺。 </li><li>使用ImageJ的软件程序来计算菌落直径，打开每个图像文件。规范比例尺如下：选择“直线”选项“从工具栏。覆盖具有相同的长度和宽度的直线嵌入的比例尺。选择标记的标签“设置量表”，“分析”。在“已知的距离”框中，插入相应的长度（从原来的比例尺确定，也就是说，2毫米），并选择“OK”。 </li><li>使用“直线”选项插入一个水平线上，整个现场。分析“选项卡下，选择”办法“。在新成果的窗口，将提供计算长度。通过插入垂直线跨当场获得第二次测量。重新计算直径。记录两次测量的平均值。图中每个点的平均直径在每个时间点使用的软件程序，如Excel。 </li><li>实验结束时，无论是在指定的时间点，或当没有进一步生物膜的发展。一旦最终达成，合并成一个数字（S）的可视化发展的格局，超过时间使用软件程序，如PowerPoint的图像。 </li></ol> 3。代表结果在这些实验中，我们用五鲵作为一种模式生物，研究生物膜的形成，通过评估固体琼脂表面上的皱纹殖民地的发展。 五生物膜生产菌株具有广泛的三维建筑鲵形式的殖民地，40小时内（ 图1），8,9,14。当过一段时间课程研究，很明显的，通过控制应变的皱纹殖民地形成启动早约12小时后接种（上的具体情况而定）（ 图2）9。相比之下，有代表性的突变形成生物膜延迟约4小时，直到约16小时后接种9启动。 repea这些实验的TS建议的时机是比较一致的，这一评估半定量9。第二个半定量生物膜形成的措施，随着时间的推移发展殖民地的直径变化。正如图所示。 3A，具有代表性的的生物膜主管应变形成的殖民地，在相对 ​​有代表性的菌株，并不构成生物膜的复杂性和直径增加。仔细测量发现，超过时间，这两个菌株形成的菌落直径的的生物膜主管殖民地的大小，在一个更​​大的比生物膜负的殖民地率增加，结束时间点在两个不同近2 - 倍（ 图3B）。因此，虽然后期有代表性的时间或“终点”的菌落形态的图像经常在文学，另外，半定量的数据可以被收集，将更好地了解允许生物膜缺陷的。<img alt="图1" src="/files/ftp_upload/4035/4035fig1.jpg" /> 图1。终点检测。这个数字是一个终点检测，使用非生物膜形成（左）和生物膜形成（右）代表五株鲵 。在40小时后，发现这些图像采集。这些图像生成与野生型五鲵含矢量控制（非生物膜的形成）或RSCS过度质粒（生物膜的形成），并为Morris 等人，2011年收集的数据的一部分。 <img alt="图2" src="/files/ftp_upload/4035/4035fig2.jpg" /> 图2。时间课程实验。上游面板包含由生物膜主管的控制应变生物膜形成的代表图像五鲵在选定的时间过程。在12小时启动生物膜的形成是显而易见的。较低的面板包含代表我法师的一个五，突变株鲵 ，展品中的集落形成的皱纹随着时间的推移开始延误（4小时），在接种后16小时开始形成生物膜，。请注意，在40个小时的菌株看起来类似的强度和生物膜形成的图案，而这些菌株之间的细微差别仅在较早的时间点观察。这些图像生成与RSCS过度野生型和突变sypE五的鲵细胞和数据集收集Morris 等人。，2011。 <img alt="图3" src="/files/ftp_upload/4035/4035fig3.jpg" /> 图3。菌落直径为生物膜形成的半定量分析。 （一）代表生物膜形成（上图）和非生物膜的形成（下） 五株生物膜形成的时间过程鲵 。需要注意的是生物膜形成的应变表现出更大的增长随着时间的推移，相对非生物膜形成应变的直径。这些图像生成与野生型五鲵含有RSCS过度质粒（生物膜形成）或矢量控制（非生物膜形成）和部分数据设置为Morris 等人，2011年收集的。 （B）面板A.使用ImageJ的Excel软件生成这些数据在协议中所述的两个菌株在菌落直径随着时间的推移增加的图形表示。

<ol>
	<li>Donlan, R. M., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+survival+mechanisms+of+clinically+relevant+microorganisms.">Biofilms: survival mechanisms of clinically relevant microorganisms.</a> Clin. Microbiol. Rev. 15, 167-167 (2002).</li><li>Stoodley, P., Sauer, K., Davies, D. G., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+as+complex+differentiated+communities.">Biofilms as complex differentiated communities.</a> Annu. Rev. Microbiol. 56, 187 (2002).</li><li>Branda, S. S., Vik, S., Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+the+matrix+revisited.">Biofilms: the matrix revisited.</a> Trends Microbiol. 13, 20 (2005).</li><li>Branda, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruiting+body+formation+by+Bacillus+subtilis.">Fruiting body formation by Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11621 (2001).</li><li>Beyhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+rugosity+and+biofilm+formation+in+Vibrio+cholerae:+comparison+of+VpsT+and+VpsR+regulons+and+epistasis+analysis+of+vpsT,+vpsR,+and+hapR.">Regulation of rugosity and biofilm formation in Vibrio cholerae: comparison of VpsT and VpsR regulons and epistasis analysis of vpsT, vpsR, and hapR.</a> J. Bacteriol. 189, 388 (2007).</li><li>Enos-Berlage, J. L., Guvener, Z. T., Keenan, C. E., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+determinants+of+biofilm+development+of+opaque+and+translucent+Vibrio+parahaemolyticus.">Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus.</a> Mol. Microbiol. 55, 1160 (2005).</li><li>Merritt, J. H., Brothers, K. M., Kuchma, S. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SadC+reciprocally+influences+biofilm+formation+and+swarming+motility+via+modulation+of+exopolysaccharide+production+and+flagellar+function.">SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function.</a> J. Bacteriol. 189, 8154-8154 (2007).</li><li>Yip, E. S., Geszvain, K., DeLoney-Marino, C. R., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+symbiosis+regulator+rscS+controls+the+syp+gene+locus,+biofilm+formation+and+symbiotic+aggregation+by+Vibrio+fischeri.">The symbiosis regulator rscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri.</a> Mol. Microbiol. 62, 1586-1586 (2006).</li><li>Morris, A. R., Darnell, C. L., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+a+novel+response+regulator+is+necessary+for+biofilm+formation+and+host+colonization+by+Vibrio+fischeri.">Inactivation of a novel response regulator is necessary for biofilm formation and host colonization by Vibrio fischeri.</a> Mol. Microbiol. 82, 114 (2011).</li><li>Nyholm, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Vibrio+fischeri+and+nonsymbiotic+bacteria+in+the+dynamics+of+mucus+secretion+during+symbiont+colonization+of+the+Euprymna+scolopes+light+organ.">Roles of Vibrio fischeri and nonsymbiotic bacteria in the dynamics of mucus secretion during symbiont colonization of the Euprymna scolopes light organ.</a> Appl. Environ. Microbiol. 68, 5113 (2002).</li><li>Yip, E. S., Grublesky, B. T., Hussa, E. A., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+conserved+cluster+of+genes+promotes+symbiotic+colonization+and+Ï&#x0192;54-dependent+biofilm+formation+by+Vibrio+fischeri.">A novel, conserved cluster of genes promotes symbiotic colonization and Ï&#x0192;54-dependent biofilm formation by Vibrio fischeri.</a> Mol. Microbiol. 57, 1485 (2005).</li><li>Mandel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+regulatory+gene+is+sufficient+to+alter+bacterial+host+range.">A single regulatory gene is sufficient to alter bacterial host range.</a> Nature. 458, 215 (2009).</li><li>Graf, J., Dunlap, P. V., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+transposon-induced+motility+mutations+on+colonization+of+the+host+light+organ+by+Vibrio+fischeri.">Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.</a> J. Bacteriol. 176, 6986 (1994).</li><li>Geszvain, K., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hybrid+sensor+kinase+RscS+integrates+positive+and+negative+signals+to+modulate+biofilm+formation+in+Vibrio+fischeri.">The hybrid sensor kinase RscS integrates positive and negative signals to modulate biofilm formation in Vibrio fischeri.</a> J. Bacteriol. 190, 4437 (2008).</li><li>Krasteva, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+cholerae+VpsT+regulates+matrix+production+and+motility+by+directly+sensing+cyclic+di-GMP.">Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP.</a> Science. 327, 866 (2010).</li><li>Romling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+rdar+morphotype,+a+multicellular+behaviour+in+Enterobacteriaceae.">Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae.</a> Cell Mol. Life. Sci. 62, 1234 (2005).</li><li>Berleman, J. E., Chumley, T., Cheung, P., Kirby, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rippling+is+a+predatory+behavior+in+Myxococcus+xanthus.">Rippling is a predatory behavior in Myxococcus xanthus.</a> J. Bacteriol. 188, 5888 (2006).</li><li>Lee, K., Veeranagouda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicrocells+form+by+reductive+division+in+macroscopic+Pseudomonas+aerial+structures.">Ultramicrocells form by reductive division in macroscopic Pseudomonas aerial structures.</a> Environ. Microbiol. 11, 1117 (2009).</li></ol>

没有利益冲突的声明。

在这项工作中，我们描述了一个半定量的方法来评估生物膜形成使用V。鲵作为一种模式生物。具体来说，我们利用解剖显微镜用相机附件监视随着时间的推移皱纹殖民地形成了坚实的琼脂表面生物膜的形成和发展。在这个协议中，我们勾勒出两种方法，我们通常使用评估皱的集落形成的特定类型。首先是终点检测，这使我们能够在选定的“最终”的时间点观察最终的，整体的3D架构，图案，和花斑文化直径。这种方法是最有用的突变株或条件，导致生物膜形成中的戏剧性缺陷评估。然而，这种方法不区分更细微的差别在选定的终点前发生的时间点。为了更密切监察皱纹的集落形成，我们使用一个时间过程的检测，这使我们能够识别的W开始rinkled集落形成，随着时间的推移，看其发展。由于这种方法的结果，更细微的差别，集落形成皱纹，3D架构和模式的时机可以鉴别。我们利用这个时间当然法生成两个半定量检测生物膜的形成。首先，应变开始开发3D建筑的时间可以比对照菌株。我们发现，在生物膜形成一个特定的突变，在同等条件下的延迟是相当一致。例如，在图中显示的数据。 2，突变的一贯表现，约4小时延时启动生物膜的形成。半定量生物膜形成的第二个措施是皱的殖民地（点）的直径大小的变化。我们发现，皱纹菌落直径逐步不同于非生物膜殖民地的是，达到约2倍的差异，在结束时间点（ 图3 </&gt;）（莫里斯和Visick，未发表资料）。到今天为止，我们没有观察到一个没有其他的（即没有形成菌落直径增加，生物膜）（未发表资料）型，虽然它仍然是有可能的，有些突变会表现不同。事实上，它已经为五霍乱一些皱纹菌落菌落直径在大幅增加的结果，而有的则没有15。仍然直径的变化，随着时间的推移评估可以帮助表征潜在的生物膜突变和/或提供额外的突变体的定量措施，延误发展。生物膜形成的两项措施（时间和直径），确定皱纹的集落形成的开始，是较为敏感，但也更主观，确定点的直径比。即便如此，这两项措施提供了一个表型的半定量的评估，这是非常有用的生物膜的研究人员，但不容易服从到quantificatioN。 当进行培养实验发现，重要的是要考虑在其中发现的菌株培养的环境条件。皱纹殖民地的形成往往受各种环境条件，包括养分供应，温度和湿度。为了减少实验之间的变异性，它是有助于规范这些条件尽可能多的可能（即规范平板一套体积和培养发现菌株在温度控制）。要进一步控制斑点实验之间的变异性，是重要的，包括在每一组实验中适当的控制株。最后，在解释这些实验数据，它是要执行任何一个实验多次（3 +），尤其是当评估在皱纹的集落形成的细微差别（例如，在生物膜形成延迟或图案的差异）。本协议的一些限制：1）确定ING细胞是否有增长的缺陷，可能很难：液体培养细胞的生长，可能无法准确反映固体培养基上，增长率和增长生物膜形成的细胞，这可能会粘在一起，可能无法准确测定; 2）菌株的生长缺陷问题的分析; 3）它可能无法区分菌株的直径与微妙的生物膜表型的差异;同心环在不长的4株），它可能无法准确地测量在直径变化; 5），而图案可以观察生物膜形成过程中，有没有办法量化，导致生物膜的图案; 6）有没有办法来衡量这个实验设置了生物膜的Z维。尽管有这些限制，但这个协议提供了一种手段获得的数值数据，以帮助评估皱落形成。 在这个协议中，我们利用一个特定的成像系统（即，蔡司解剖显微镜和PROGRES CapturePro成像软件）来观察和评估皱纹的集落形成。这里描述的是成像系统强大：检测皱纹的集落形成的开始，因此，同一个时间过程的方法评估发展的能力，通过解剖显微镜的使用大大增强。然而，如果这项技术是不可用的，该协议可以适用于其他设备的使用，包括一个简单的数码相机变焦焦点。虽然这个协议上的皱纹殖民地发展评估的重点，它也可以被修改，以评估薄膜的形成，生物膜的形式，发展当细胞生长在液体培养静态。该协议也可能被证明有用的评估生物膜形成的其他指标，包括纳入特定染料的生物膜发展过程中的斑​​点殖民地。这些措施包括，但不仅限于，染料，如刚果红和calcoflu的或可以绑定到纤维素的细菌生物膜的共同组成部分16。此外，该协议中，虽然使用与五鲵 ，并不仅限于这种微生物，但可以是广义的研究在许多不同的生物，如枯草芽孢杆菌 ， 霍乱弧菌 ， 副溶血性弧菌 6， 绿脓杆菌 7，所有展品皱集落形成，生物膜形成。最后，它也可以适应研究有一个发展模式，如可能， 绿脓杆菌 18增长17 黏细菌和天线结构的发展过程中出现的图案，其他菌落形态。因此，这个协议是微生物和生物膜的研究人员一般使用。

<html><table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>蔡司STEMI 2000-C的解剖显微镜</td><td>蔡司</td><td> 455053000亿</td><td> （通过卢卡斯显微镜获得的） </td></tr><tr><td> PROGRES C10的PLUS </td><td> JENOPTIK光学系统有限公司</td><td> 017953-602-26 </td><td>相机附件（通过卢卡斯显微镜获得） </td></tr><tr><td> CL 1500欧共体冷光源</td><td>蔡司</td><td> 43554000亿</td><td> （通过卢卡斯显微镜获得的） </td></tr><tr><td> PROGRES CapturePro </td><td> JENOPTIK光学系统有限公司</td><td>免费注册相机软件</td><td>图像采集的计算机程序</td></tr><tr><td>图像J </td><td>国立卫生研究院</td><td>免费软件：IJ / download.html“目标=”_blank“&gt; http://rsb.info.nih.gov/ij/download.html </td><td>直径测量的计算机程序</td></tr></tbody></table>

a semi-quantitative approach to assess biofilm formation using wrinkled colony development

生物膜，或封装在细胞外基质的细胞表面连接社区，代表了许多细菌的共同生活方式。细菌细胞内生物膜，往往表现出改变的生理，包括增强抗病能力，抗生素和其它环境压力1。此外，生物膜可以在宿主 - 微生物相互作用中发挥重要作用。生物膜细菌过渡时从个别发展，浮游细胞形成复杂的多细胞的社区2。在实验室，生物膜研究评估具体生物膜表型的发展。一种常见的生物膜表型涉及的皱纹或皱纹固体琼脂培养基菌落形成。皱落形成提供了一个特别简单的和有用的手段，识别和描述参展改变生物膜表型的菌株，并探讨影响生物膜形成的环境条件。 WRInkled集落形成作为生物膜形成的各种细菌，包括革兰氏阳性菌，如枯草芽孢杆菌和革兰氏阴性菌，如霍乱弧菌 ， 副溶血性弧菌 ， 绿脓杆菌 7 6 5，指示灯， 弧菌鲵 8。 海洋细菌五鲵已成为一个模型对生物膜形成由于主机殖民化过程中生物膜的关键作用：生物膜产生五。鲵促进其殖民夏威夷短尾的鱿鱼Euprymna scolopes 8-10。重要的是， 在体外观察到的生物膜表型相关的五能力鲵细胞有效殖民宿主动物，：障株在体外生物膜形成具有殖民缺陷9,11，而株参展增加生物膜表型增强殖民8,12。 五。鲵 ，因此提供了一个简单的模型系统评估机制，细菌调节生物膜的形成和生物膜影响主机殖民。 在这份报告中，我们描述了一个半定量的方法来评估生物膜形成使用五鲵作为一个模型系统。这种方法涉及小心细菌培养，发现在规定的浓度和固体琼脂培养基上卷;花斑文化是一个单一的细菌菌落的代名词。可以利用这个&#39;斑疹文化技术比较单一的，特定的时间点（终点法）总额的生物膜表型，或通过识别和表征生物膜发展和菌落直径测量过程检测微妙的生物膜表型，这是由生物膜形成的影响。因此，这种技术提供了一个生物膜形成的半定量分析，每mitting评价的时机和皱纹殖民地的发展和发展结构，超出了简单的整体形态特征的相对大小的图案。

Watch this Scientific Journal Video about A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development at JoVE.com

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

我们提供一个简单的，半定量的方法来研究生物膜的形成<em&gt;在体外</em&gt;。这种方法需要利用蔡司STEMI 2000-C的解剖显微镜（相机附件）监测的时间和生物膜形成的格局，发展由皱殖民地的评估。

一个半定量的方法来评估生物膜形成皱殖民地的发展

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

a-semi-quantitative-approach-to-assess-biofilm-formation-using

Research

JoVE Journal

Biology

23.1K 次观看.  Loyola University Medical Center. 我们提供一个简单的，半定量的方法来研究生物膜的形成在体外。这种方法需要利用蔡司STEMI 2000-C的解剖显微镜（相机附件）监测的时间和生物膜形成的格局，发展由皱殖民地的评估。

视频: A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

科研

生物学

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

软琼脂集落形成分析

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots located in Xinjiang Province, northwest China. To prevent high correlations among observations from the same sampling unit, we developed the model using a linear mixed-effects approach with random plot effect to account for stochastic variability. Various tree- and stand-level variables, such as indices for tree size, competition, and site condition, were included as fixed effects to explain the residual variability. In addition, heteroscedasticity and autocorrelation were described by introducing variance functions and autocorrelation structures. The optimal linear mixed-effects model was determined by several fit statistics: Akaike&#8217;s information criterion, Bayesian information criterion, logarithm likelihood, and a likelihood ratio test. The results indicated that significant variables of individual-tree basal area increment were the inverse transformation of diameter at breast height, the basal area of trees larger than the subject tree, the number of trees per hectare, and elevation. Furthermore, errors in variance structure were most successfully modeled by the exponential function, and the autocorrelation was significantly corrected by first-order autoregressive structure (AR(1)). The performance of the linear mixed-effects model was significantly improved relative to the model using ordinary least squares regression.

Mixed-effects models are flexible and useful tools for analyzing data with a hierarchical stochastic structure in forestry and could also be used to significantly improve the performance of forest growth models. Here, a protocol is presented that synthesizes information relating to linear mixed-effects models.

使用线性混合效应方法开发单树基数增量模型

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots ...

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

收获和分解：生物膜方法研究中被忽视的步骤

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...

不动杆菌生物膜形成的评估和生物膜的表征

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

不动杆菌生物膜的定量、活力评估和可视化策略

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...