Name: visualizing the effects of sputum on biofilm development using a chambered coverglass model
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TB承认囊性纤维化加拿大研究奖学金。

需要注意的是研究伦理委员会（REB）来收集和痰标本从人类受试者的存储。这些研究是由病童医院REB＃1000019444批准。 1.准备CF痰液标本<ol><li>收集患者的痰液标本中的囊性纤维化诊疗常规访问，保持在冰上。 </li><li>在冰上运痰样品收集的第一个小时内，向研究实验室，经过处理。 </li></ol> 2.痰处理<ol><li>记录所得到的痰样品的体积。添加磷酸盐缓冲盐水（PBS）至2倍的样品（ 即，2份PBS中，1份样品）的体积。 </li><li>用移液管混合样品井。涡上1分钟后，完全混合最高设置的样本。 </li><li>等分试样将1ml上述混合物的入适当数量的1.5 ml离心管中并离心以5,000 xg离心在4℃下20分钟。 </li><li>离心后，除去上清液并弃沉淀。 </li><li>过滤通过0.22微米的过滤器消毒上清，在一个干净的离心管收集。 注意:滤液的无菌通过电镀在LB琼脂和接种液体培养基试验。 </li><li>在-80℃，以备将来使用存储痰上清。 注:痰从多个患者也可以下列过滤合并。 </li><li>在使用之前，稀释痰滤液1/10体积/体积中所需的介质（100微升痰，900微升的介质）。 注:在这里，使用标准LB培养基（LB）的媒体。 </li></ol> 3.生物膜形成腔室盖玻片方法<ol><li>在37℃生长在期望的媒体关注隔夜的细菌分离用颤抖的（200转）。 注意:使用了许多不同的细菌，包括临床分离株绿脓杆菌 ， 金黄色葡萄球菌 ，洋葱伯克霍尔德菌复杂和木糖氧化无色杆菌。的媒体选择取决于菌株和感兴趣的条件下，但是LB培养基可以用于最初的实验。 </li><li>从过夜培养，将40微升培养物分成4毫升的新鲜培养基中，并在37℃振荡（200rpm）下在大约0.5 600纳米（OD 600），以获得与光学密度的培养生长3-4小时-0.6。 </li><li>稀释步骤3.2文化在1/5所需的媒体有10％痰滤液或无痰滤液（作为对照）。痰滤液其他浓度可以进行测试（ 即，50％或100％）。 </li><li>用200微升稀释的种子的幻灯片室井。 </li><li>使细菌4小时，在37°C不晃动重视。 </li><li> 4小时后，取出媒体，轻轻地用1X新鲜培养基洗生物膜。用200微升新鲜培养基更换。 注:研究痰的效果上生物膜的FRES^ h媒体应该包含痰上清。 </li><li>允许生物膜到的时间所需量的在37℃不摇动生长，替换媒体每12小时，未经洗涤直到时间为显微镜。 注:研究生物膜药敏痰上清液的效果，抗生素加入到以下生物膜生长的24小时的媒体和被保持在媒体直至染色和生物膜的成像。 </li></ol> 4.染色生物膜和共聚焦显微镜<ol><li>继所需生长时间（24-48小时效果最好），从室移去培养基，并轻轻地用300微升无菌PBS洗涤各室的两倍。 </li><li>通过混合1微升每种染料（在可行性套件中提供）为所需的每个毫升溶液的准备生物膜染色的混合物。使水或媒体解决方案染料。 注:水是由制造商推荐的。 </li><li>加入200μl染料混合物到腔式covergla的每个孔SS和孵育在室温下，在黑暗中45分钟。 </li><li>从腔室中移除染色混合物并用300μl无菌PBS冲洗每个孔中。除去PBS并用清水或媒体取代。 </li><li>通过共聚焦显微镜生物膜的可视化进行。 </li></ol> 5.可视化生物膜与共焦显微镜<ol><li>染色（在1个小时）后，立即阅读室染色生物膜。通过一次染色八月一日至2日孔腔最小化在载玻片的可视化延迟。 </li><li>利用共聚焦显微镜用激光激发和过滤器集收购进行成像。 注:在这里，北极光光谱的激光旋转盘共聚焦系统被用于激励（绿色:561纳米:491nm，红色）。用于发射滤光片套四十零分之五百十五和四十零分之六百二十四的由生物膜生存能力套件形象化的污渍。 </li><li>以使用与相机共聚焦显微镜25X一水的目标图像。 </li><l我&gt;使用Z-堆栈生物膜模型。拍摄图像每隔0.5-1微米从第一焦点对准平面开始到生物膜的最后的聚焦框（通常跨越30-80微米48小时生物膜） </li><li>从每口井以3-5的图像。 注:因此，对于一个8孔腔玻璃罩，将产生24-40图像。 </li><li>保存图像进行分析。 注意:图片应保存为使用COMSTAT 18,19分析OME-TIFF文件。生物膜图像分析说明可以在http://www.comstat.dk/找到。一旦图像被导入，参数，如平均厚度，生物质和表面覆盖率为每个通道（红色和绿色）进行分析。 </li></ol>

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A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Biofilm+Formation+in+an+8-well+Chamber+Slide.">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481 (2011).</li></ol>

本文描述的方法允许在外源产物的存在下生长的细菌生物膜的可视化。不令人惊讶地，使用这种类型的系统时生产exoproducts的是重要的。例如，二硫苏糖醇（DTT），经常被用来对人痰样品来帮助液化样本。然而，DTT的单独的效果可降低生物膜发展和活力（数据未显示）。因此，对于所有条件适当的控制是必要的。此外，加入的人类痰产品由于这一事实，即每个病人的痰中有一个独特的微生物产生的实验固有可变性。要对此进行调整，我们已经使用共用的痰标本，以避免病人具体成果。此外，使用任何模型系统当相应的介质的选择是重要的。对于预期生物体的生物膜生长标准媒体推荐用于该系统的初始设置。如果该实验的目的是ident命令IFY如何外源性产品的影响生物膜的形成，提供了充足的生物膜形成一种营养丰富的媒体提出了建议。这将允许足够的营养生长，同时确定外生因素会如何影响生物膜。其他媒体，例如最小或确定的培养基，可用于更好地模拟某些条件。 （数据未显示）的其他媒体已被用于在该系统的良好的结果。生物膜的向腔玻璃罩附件是健壮的，但是大必须小心除去/添加媒体或在生物膜的洗涤步骤，以防止破坏生物膜时。 使用活死染色按照制造商的协议中已取得了所描述的系统良好的效果。许多因素可影响图像的荧光比率（包括细菌染率，生物膜和检测器增益设置相对密度），但是，这种方法应与什么在图像中观察到的秒。可以使用其它措施来代表死/活生物膜的相对量，如从死细胞中存在的生物膜的总生物量（从COMSTAT衍生）的比例的生物质。使用细菌计数，以确认在反复实验生物膜存活建议确认该模型的视觉观察。由于生物膜的固有非均匀性，从每个室和多个腔室的多个图像应该被用于在实验中每种条件。这增加了这些实验的成本和时间。 图像的获取是从这些实验数据的分析之前重要。必须小心确定显微镜安装时，应考虑到在不饱和的图像和。取决于所需的详细程度和可用的显微镜​​，许多不同的目标（10X，25X，40X和63X）可用于获取图像，尽管我们发现，25X物镜给出更好的图像。的厚度t他Z堆栈也可以影响整体图像质量和细节的水平。具有每0.5-1微米拍摄的图像似乎在25X物镜提供清晰的图像，同时保持在Z堆叠图像在可由COMSTAT在标准的计算机系统进行分析的尺寸。 这些实验都比较昂贵和耗时比其它连接测定法，并且不能以高通量方式进行。在此过程中，细菌附着于硼硅表面，这是不反光的体内条件，并可能影响生物膜的形成和发展。然而，它们提供额外的信息，并且可以生成用于向生物膜抗生素抗性和外源信号的生理反应相关的机制假说。如果该实验的目的是要了解生物膜响应如何变化到不同的因素，而不是确定使用例如高通量方法抗生物化合物中，附加信息用t获得他的技术使得该方法值得的。因此，当前的方法是在前面的有限附着测定中显著进步，如结晶紫测定法，这是最常见的用于研究生物膜。 关键步骤这个过程包括:1）确保痰标本，以避免退化的及时处理; 2）是温柔与生物膜媒体更改，以避免生物膜的破坏和3）做好生物膜成像的重复测量，以确保生物膜厚度的精确表示由于生物膜形成的异质性。 一旦系统建立为一个给定的细菌物种，可以测试多种不同的条件，包括在多个临床分离外来因素的影响。此系统已用于研究的抗生素的效果上生物膜暴露于人类痰16和比较高抗的中等抗性临床分离物。 17修改到腔玻璃罩，如涂布粘蛋白或胶原蛋白微支架（如 puracol）的底部，可以进一步扩展的可能实验的范围。它可能会适应这一系统来研究的直接相互作用，如上皮细胞和细菌，或之间不同的细菌物种之间。这是通过Jurcisek 等人描述的模型的扩展。 20的方法，使用类似的是，这里所描述和使用福尔马林到可视化之前修复生物膜滑动室的生长。另外，在这个方法中，我们可以形象的生物膜染色后立即不使用固色剂。此外，我们添加外源因子来测试抗生素对生物膜的影响。因此，这种模式有大大促进我们在人类疾病的细菌生物膜的理解的潜力。

细菌生物膜是微生物组附加到彼此并以自分泌的基质包裹。 1,2-传统上，它们代表细菌物理连接到流量的条件下形成的非生物或生物的表面上。生物膜也已经显示在静态条件（无流动）和远离表面，如在形成于试管热水池或药膜的空气 - 液体界面生长。这些生物膜早已在环境中识别和是主要损害工业过程，因为它们可以在蓄水池或管道形成，导致生物结垢，腐蚀和堵塞。 3,4 生物膜也是在医疗设置关键的，因为它们已被证明参与导管相关感染，囊性纤维化患者的肺感染，以及在许多其他感染。 5,6-一个生物膜感染的标志是德折痕细菌对抗生素的敏感性，并通过先天免疫系统受损的间隙。 7-9最充分研究，涉及基于生物膜感染临床相关情形发生在患者的囊性纤维化（CF），谁是慢性感染绿脓杆菌生物膜。 铜绿假单胞菌能建立慢性感染，使得它很难治疗的过程中发生了一些变化。 10,11生物膜可以激活差异的先天免疫和炎症驱动。 12-14由于这些感染导致CF患者发病率和死亡率，它是要明白，在这方面可以影响生物膜发展的因素是至关重要的。 最近的一项研究表明，宿主因素是在铜绿假单胞菌生物膜聚集体的形成是至关重要的。 15这些生物膜有助于减少对抗生素的敏感性和宿主防御机制。该prese的宿主衍生的因素，如嗜中性粒细胞弹性蛋白酶，以及来自本在CF肺部的微生物分泌的产品NCE，必须大大调制生物膜形成和发展的潜力。 16此外，生物膜与宿主相互作用来调节多种途径表达并开始发炎。而高通量的方法，例如标准的结晶紫测定法中，可以为用户提供关于生物膜过程中的一些信息，响应于这些因素的生物膜的可视化提供更深入的信息。 在这个手稿我们描述了使用来自CF患者的痰因素来研究生物膜的体外发育的方法。此方法允许暴露于使用商业生物膜存活包含痰宿主因素生物膜的快速可视化。这种技术可以用于可视地识别在exogeno存在生物膜生长过程中发生的变化我们的产品，并表示改进的方法来分析各种条件下的生物膜发展的变化。

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

生物膜是由以自分泌的基质包裹细菌的群体。它们在工业污染，以及在许多健康相关性感染的发展和维持中发挥重要作用。一种在人类疾病中最充分描述并研究生物膜发生在囊性纤维化患者的慢性肺部感染。当在主机的情况下学习生物膜，许多因素会影响生物膜的形成和发展。为了鉴定宿主因素如何影响生物膜的形成和发展，我们使用一个静态腔室盖玻片方法中的宿主衍生的因子在痰上清液的形式存在下生长生物膜。细菌接种到商会和暴露于痰滤液。继增长48小时，生物膜沾满共聚焦显微镜和分析之前的商业可行性生物膜套件。以下图像采集，生物膜特性可以使用不同的软件平台来评估。这个方法允许我们想象中不同物质，包括抗生素的存在生物膜生长的关键性质。

实验的整体设计在图1中表示。使用这种协议的提供了一个方便的方法来可视化在生长的不同时间（ 例如，24，48或72小时）的生物膜的变化。重要的是，外源信号，如痰的滤液，可以被添加到可视化生物膜发展的变化。如在图2中看到的那样，10％的痰的滤液的存在可以改变生物膜的结构（ 图2A，下图）。 16这些图像可以使用COMSTAT软件获得关键的生物膜矩阵，包括生物膜的平均厚度，总生物量和覆盖面进行分析。这反映在生物膜厚度的整体增加（ 图2B和3）。 16对在存在痰生物膜抗生素的影响示于图3。通过visuali<html>查懋生物膜发展的变化，就可以更好地理解不同的因素如何影响生物膜生长，以比传统的生物膜测定法，如结晶紫染色好得多的程度。 <img alt="图1" src="/files/ftp_upload/54819/54819fig1.jpg" /> 图1:实验的整体设计。流量基本协议的图。过夜（O / N）培养物稀释1 / 1,000，并使其生长至最终的OD 0.5 600。这进一步稀释1 / 1,000和200微升接种到腔室盖玻片的井，并使其为3-4小时连接。在此之后，除去培养基并用新鲜的培养基替换。生物膜允许生长所需的时间。媒体，外源产物或抗生素可以被添加到生物膜。以下生长，除去培养基，生物膜染色，并进行共聚焦成像。ge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54819/54819fig2.jpg" /> 图2: 暴露于痰滤液生物膜形成的代表图像。 （A）的洋葱伯克霍尔德菌复杂的代表性图像（BCC）以下在单独用（上图）介质腔室盖玻片生长48小时的临床分离株或在随后用生物膜存活试剂盒染色10％痰的滤液（下图）的存在。 1刻度单位代表19.68微米。 （B - C）株（B）和死的平均厚度:BCC的多个图像的活比率（C）的菌株生长在滑动室在不存在48小时（白色条）或存在（黑色条）的痰滤液。每个条代表45图像绘制的平均与平均值的标准误差。 ** P &lt;0.001相比，使用秩和检验测试控制（媒体单独）。图改编自Kennedy 等。 16 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54819/54819fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54819/54819fig3.jpg" /> 图3: 暴露于痰滤液生物膜抗生素治疗的代表性的图像。 （A）以下伯克霍尔德vietnamiensis临床分离图像生长48小时与单独的培养基（左）的腔室玻璃罩或在10％的痰的滤液（右）有或无妥布霉素1000微克/立方米的存在。生物膜在媒体单独生长24小时或介质补充有10％（V / V）痰的滤液。 24小时后，除去培养基并用中介取代含抗生素（+/-痰）。 1刻度单位代表19.68微米。 （B - C）株（B）和死的平均厚度多张图片的活比（C）（9）B. vietnamiensis的菌株生长在幻灯片室在没有或痰滤液存在48小时。每个条代表与平均值的标准误差作图45的图像的平均值。 ** P &lt;0.001相比，使用Kruskal-Wallis检验控制（0微克/毫升）。图改编自Kennedy 等。 16 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54819/54819fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

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

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 ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.3K 次观看.  Hospital for Sick Children. 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.

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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. ...

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 ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

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.

可视化痰生物膜发展影响使用腔室盖玻片型号

摘要

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此视频中的章节

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

铜绿假单胞菌和酿酒酵母流细胞生物膜

酶标菜生物膜形成实验

在体外生物膜的形成以及8商会幻灯片

使用高通量的

方法学研究

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

全自动微流控装置在白色念珠菌生物膜形成中的可视化

囊性纤维化患者痰液中铜绿假单胞菌的可视化

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响

可视化痰生物膜发展影响使用腔室盖玻片型号

摘要

探索更多视频

此视频中的章节

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

铜绿假单胞菌和酿酒酵母流细胞生物膜

酶标菜生物膜形成实验

在体外生物膜的形成以及8商会幻灯片

使用高通量的

方法学研究

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

全自动微流控装置在白色念珠菌生物膜形成中的可视化

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

囊性纤维化患者痰液中铜绿假单胞菌的可视化

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响