Name: methods for characterizing the co-development of biofilm and habitat heterogeneity
Uploaded: 2015-03-11T14:00:00.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity at JoVE.com

我们感谢马特Parsek在华盛顿大学（西雅图）提供P.铜绿假单胞菌和E.大肠杆菌菌株和罗杰·诺克斯在坎特伯雷（新西兰）的大学提供访问流软件。这项工作是由卫生部国家过敏研究所全国学院和传染病资助R01AI081983支持。在西北大学的生物成像设备（BIF）进行聚焦成像。

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

<ol>
	<li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+From+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: From the natural environment to infectious diseases.</a> Nat Rev Microbiol. 2 (2), 95-108 (2004).</li><li>Stewart, P. S., Franklin, 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=Physiological+heterogeneity+in+biofilms.">Physiological heterogeneity in biofilms.</a> Nat Rev Microbiol. 6 (3), 199-210 (2008).</li><li>Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T., McFeters, 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=Spatial+physiological+heterogeneity+in+Pseudomonas+aeruginosa+biofilm+is+determined+by+oxygen+availability.">Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.</a> Appl Environ Microb. 64 (10), 4035-4039 (1998).</li><li>Costerton, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Biofilms+in+Nature+and+Disease.">Bacterial Biofilms in Nature and Disease.</a> Annu Rev Microbiol. 41, 435-464 (1987).</li><li>Battin, T. J., Kaplan, L. A., Newbold, J. D., Hansen, C. M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+microbial+biofilms+to+ecosystem+processes+in+stream+mesocosms.">Contributions of microbial biofilms to ecosystem processes in stream mesocosms.</a> Nature. 426 (6965), 439-442 (2003).</li><li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+A+common+cause+of+persistent+infections.">Bacterial biofilms: A common cause of persistent infections.</a> Science. 284 (5418), 1318-1322 (1999).</li><li>Stoodley, P., Dodds, I., Boyle, J. D., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+hydrodynamics+and+nutrients+on+biofilm+structure.">Influence of hydrodynamics and nutrients on biofilm structure.</a> J Appl Microbiol. 85, 19S-28S (1999).</li><li>Stoodley, P., Lewandowski, Z., Boyle, J. D., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+deformation+of+bacterial+biofilms+caused+by+short-term+fluctuations+in+fluid+shear:+An+in+situ+investigation+of+biofilm+rheology.">Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: An in situ investigation of biofilm rheology.</a> Biotechnol Bioeng. 65 (1), 83-92 (1999).</li><li>Wasche, S., Horn, H., Hempel, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+growth+conditions+on+biofilm+development+and+mass+transfer+at+the+bulk/biofilm+interface.">Influence of growth conditions on biofilm development and mass transfer at the bulk/biofilm interface.</a> Water Res. 36 (19), 4775-4784 (2002).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Convection+around+biofilms.">Mini-review: Convection around biofilms.</a> Biofouling: The Journal of Bioadhesion and Biofilm Research. 28 (2), 187-198 (2012).</li><li>Flemming, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorption+sites+in+biofilms.">Sorption sites in biofilms.</a> Water Sci Technol. 32 (8), 27-33 (1995).</li><li>Debeer, D., Stoodley, P., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Flow+in+Heterogeneous+Biofilms.">Liquid Flow in Heterogeneous Biofilms.</a> Biotechnol Bioeng. 44 (5), 636-641 (1994).</li><li>Schultz, M. P., Swain, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+biofilms+on+turbulent+boundary+layers.">The effect of biofilms on turbulent boundary layers.</a> J Fluid Eng-T Asme. 121 (1), 44-51 (1999).</li><li>Song, J. S. L., Au, K. H., Huynh, K. T., Packman, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+Responses+to+Smooth+Flow+Fields+and+Chemical+Gradients+in+Novel+Microfluidic+Flow+Cells.">Biofilm Responses to Smooth Flow Fields and Chemical Gradients in Novel Microfluidic Flow Cells.</a> Biotechnol Bioeng. 111 (3), 597-607 (2014).</li><li>Shrout, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+quorum+sensing+and+swarming+motility+on+Pseudomonas+aeruginosa+biofilm+formation+is+nutritionally+conditional.">The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional.</a> Mol Microbiol. 62 (5), 1264-1277 (2006).</li><li>Maxworthy, T., Nokes, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+gravity+currents+propagating+down+slopes.+Part+1.+The+release+of+a+fixed+volume+of+heavy+fluid+from+an+enclosed+lock+into+an+open+channel.">Experiments on gravity currents propagating down slopes. Part 1. The release of a fixed volume of heavy fluid from an enclosed lock into an open channel.</a> J Fluid Mech. 584, 433-453 (2007).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+experimental+measurements+of+effective+diffusive+permeabilities+and+effective+diffusion+coefficients+in+biofilms.">A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms.</a> Biotechnol Bioeng. 59 (3), 261-272 (1998).</li><li>Schramm, A., De Beer, D., Gieseke, A., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironments+and+distribution+of+nitrifying+bacteria+in+a+membrane-bound+biofilm.">Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm.</a> Environ Microbiol. 2 (6), 680-686 (2000).</li><li>Santegoeds, C. M., Schramm, A., de Beer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsensors+as+a+tool+to+determine+chemical+microgradients+and+bacterial+activity+in+wastewater+biofilms+and+flocs.">Microsensors as a tool to determine chemical microgradients and bacterial activity in wastewater biofilms and flocs.</a> Biodegradation. 9 (3-4), 159-168 (1998).</li><li>Debeer, D., Stoodley, P., Roe, F., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Biofilm+Structures+on+Oxygen+Distribution+and+Mass-Transport.">Effects of Biofilm Structures on Oxygen Distribution and Mass-Transport.</a> Biotechnol Bioeng. 43 (11), 1131-1138 (1994).</li><li>Liu, Y., Tay, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+role+of+hydrodynamic+shear+force+in+the+formation+of+biofilm+and+granular+sludge.">The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge.</a> Water Res. 36 (7), 1653-1665 (2002).</li><li>Zhang, W., et al. <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+Planar+Flow+Cell+for+Studies+of+Biofilm+Heterogeneity+and+Flow-Biofilm+Interactions.">A Novel Planar Flow Cell for Studies of Biofilm Heterogeneity and Flow-Biofilm Interactions.</a> Biotechnol Bioeng. 108 (11), 2571-2582 (2011).</li><li>Tseng, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+protects+Pseudomonas+aeruginosa+biofilms+by+limiting+the+penetration+of+tobramycin.">The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.</a> Environ Microbiol. 15 (10), 2865-2878 (2013).</li><li>Debeer, D., Srinivasan, R., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Chlorine+Penetration+into+Biofilms+during+Disinfection.">Direct Measurement of Chlorine Penetration into Biofilms during Disinfection.</a> Appl Environ Microb. 60 (12), 4339-4344 (1994).</li></ol>

The authors have nothing to disclose.

我们表现​​出了一套方法来描述三个重要的生物膜与环境的相互作用:生物膜反应化学梯度，生物膜生长在周围的微环境流量，和生物膜的异质性，从内部运输的局限性造成的影响。 我们首先显示了使用一种新颖的微流体流动池，以施加用于生物膜发展的一个明确定义的化学梯度。以产生流动单元内的明确定义的化学梯度，以维持相同的流速为两个入口是重要的。确保该蠕?...

微生物附着于表面，并形成生物膜-封闭在细胞外聚合物基质1细胞集合体。生物膜表现非常不同，从单个微生物细胞，由于生物膜具有戏剧性的空间异质性，从 ​​内部溶质运移限制和空间变化的细胞代谢2,3的组合产生。氧气和营养物浓度急剧降低，在生物膜和周围的流体并获得内生物膜2进一步贫化之间的接口。在生物膜呼吸和蛋白质合成的空间变化，也可能发生由于局部氧和养分有效性2的响应。 在水体和土壤环境中，大部分细菌住在生物膜。天然生物膜进行重要的生物地球化学过程，包括骑自行车的碳和氮，减少金属4,5。在临床上，生物膜的形成是responsIBLE长时间肺部和泌尿系统感染6。生物膜相关的感染是很成问题，因为细胞在生物膜得抗菌素极高电阻相比，其浮游对口6。由于生物膜是在不同的设置很重要，研究了大量一直专注于了解控制生物膜活动和空间异质性的生物膜和周围的微环境中的环境因素。 先前的研究已经发现，生物膜发展强烈地受到许多环境因素的调节:生物膜发展的各种流动状态下不同的形态;氧气和养分供应影响生物膜形态;和流体剪切应力影响浮游细胞的附着于表面和细胞从生物膜7-9脱离。此外，外部流动状况影响的基板INT交付O和生物膜10中。生物膜的增长也改变了周围的物理和化学条件。例如，生物膜生长导致氧气和营养2的局部耗竭;生物膜积聚来自周围环境11的无机和有机化合物;和生物膜集群转移流动，增加表面摩擦力12,13。因为生物膜与其周围环境中非常复杂的方式相互作用，这是至关重要的，以同时获取对生物膜特性和环​​境条件的信息，和多学科的方法需要使用全面表征生物膜与环境的相互作用。 在这里，我们提出了一系列的综合方法，以在单种和双种生物膜中的微生物生长空间格局特征下强加的营养梯度，并观察所产生的局部化学和流体微环境的改变。我们杉ST描述了使用最近开发的双入口的微流体流动池的观察下明确定义的化学梯度生物膜生长。然后，我们说明如何使用该微流体流动池的观察下的范围内的营养条件2种细菌， 绿脓杆菌和大肠杆菌 ，在生物膜的生长。我们展示了如何在荧光示踪剂传播到生物膜集落原位可视可以用于定量评估生物膜溶质运输模式。最后，我们显示如何微尺度粒子跟踪测速，在共聚焦显微镜进行的，可以被用于获得周围生长生物膜局部流场。

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

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

生物膜是具有复杂的结构，并产生显著的空间异质性表面附着的微生物群落。生物膜发展被周围流动和营养环境强烈调节。生物膜生长也通过产生复杂的流场和溶质运输模式增加了局部微环境的不均一性。为了研究异质性的生物膜及其局部微栖息地之间的生物膜和互动发展，我们长大绿脓杆菌的单种生物膜和P.双种生物膜根据营养梯度的微流体流动池假单胞菌和大肠杆菌 。我们对流动单元内创建养分梯度和用于生长和在这些条件下可视化生物膜发展提供详细协议。我们还进行了一系列的光学方法本协议来量化空间模式在生物膜结构，流DISTRIbutions在周围生物膜，和大众交通和生物膜殖民地之内。这些方法支持联合开发的生物膜和生境异质性的全面调查。

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

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

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

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.9K 次观看.  Northwestern University. Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies. 

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

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.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

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 ...

科研

遗传学

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

环境科学

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

免疫与感染

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

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

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

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

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

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

生物工程

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.

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 ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

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

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

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 ...

生物学

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

用于研究体外宿主-病原体相互作用的器官型组织模型系统

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under different growth conditions and after treatment with matrix modifying molecules. To better quantitate the material properties of 3D biofilms, an experimental and computational workflow was developed and applied to examine differences between Enterococcus faecalis, Salmonella enterica serotype Typhimurium and Escherichia coli biofilms as well as the role of the amyloid curli in confirming rigidity to Enterobacteriaceae biofilms. The spatio-temporal dynamics of 1 &#181;m carboxylate beads in biofilms were tracked in 20 &#181;m 3D biofilms over 20 minutes. The 4D image stacks were processed using the Mosaic plugin in ImageJ to produce 3D trajectory data of bead movement. This trajectory data was analyzed with a newly developed Bead Evaluator toolbox, where movement data, including trajectory lifespans, bead velocities, cell densities along trajectories, and bounding box information were computed and stored in csv-files. This paper presents the workflow from experimental setup and image recording to bead trajectory computation and analysis. The structure of curli-containing biofilms resulted in more stable bead interactions and less bead movement than in curli-mutant and Enterococcal biofilms. Bead movement did not appear strongly dependent on cell density when measuring the bead velocity and trajectory bounding box volume, supporting the hypothesis that other material properties of the biofilms control the bead dynamics. This technique is widely applicable to quantitating differences in biofilms of different matrix compositions as well as biofilms before and after matrix-modifying treatments.

We describe a method for analyzing and quantifying the movement pattern of 1 &#181;m carboxylate beads through heterogeneous bacterial biofilms. Comparison of the movement patterns can be used to quantitate differences in material properties of biofilms.

Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under ...

方法表征的共同发展生物膜和生境异质性

摘要

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此视频中的章节

在表面定植微生物种群监测空间分异

流式细胞仪对水生生物膜的表征

细胞和生物膜细胞外成分的定量并发

使用高通量的

方法学研究

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

一个半定量的方法来评估生物膜形成皱殖民地的发展

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响

用于研究体外宿主-病原体相互作用的器官型组织模型系统

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

方法表征的共同发展生物膜和生境异质性

摘要

探索更多视频

此视频中的章节

在表面定植微生物种群监测空间分异

流式细胞仪对水生生物膜的表征

细胞和生物膜细胞外成分的定量并发

使用高通量的

方法学研究

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

一个半定量的方法来评估生物膜形成皱殖民地的发展

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

用于研究体外宿主-病原体相互作用的器官型组织模型系统

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响