作者感谢囊性纤维化基金会为这项研究提供资金。

1. 细菌分离物收集
<ol><li>从 SickKids（多伦多）接受吸入妥布霉素根除治疗的 CF 儿科患者队列中获取 铜绿假单胞菌 分离株。在-80°C的柠檬酸甘油中冷冻分离物，并在使用前进行至少三次传代培养。</li>
</ol>2. 体外 生物膜的形成
注：使用腔室盖玻片方法1 进行体外生物膜形成并进行修饰。该模型的整体工作流程如图 1 所示。
<ol><li>在用胰蛋白酶大豆琼脂和5%绵羊血制备的血琼脂上培养 铜绿假单胞菌 分离物过夜（参见 材料表）。</li>
	<li>将血琼脂中的 1-2 个细菌菌落接种到 4 mL 溶原肉汤 （LB） 中。在设置为225rpm的振荡器上在37°C下生长过夜。</li>
	<li>通过在 4 mL 新鲜 LB 中加入 40 μL 培养物来制备 1：100 稀释的过夜接种物。 在 37 °C 下在设置为 225 rpm 的振荡器上生长 3-4 小时，以在 600 nm （OD600） 处达到约 0.1 的光密度（早期对数期）。</li>
	<li>将 220 μL 接种物转移到 8 室盖玻片的每个孔中。在37°C下不受干扰地孵育24小时。</li>
	<li>从每个孔中缓慢取出培养基，以防止生物膜在基底脱落。 注意： 将载玻片向前倾斜 45° 角，并从每个腔室孔的底角吸出培养基，而不用移液器吸头接触底座。</li>
	<li>制备 100 μL 56 μg/mL 荧光标记单克隆抗体 （mAb）（参见 材料表）并缓慢加入到指定腔室孔的一侧。在室温 （RT） 下孵育 1 小时，以使抗体附着在细菌抗原表位上。 注：使用前，将荧光标记的（红色）mAb Psl0096 稀释在 LB 中，以获得 56 μg/mL 的终浓度。Psl0096 是一种抗 Psl mAb（Cam003 的优化亲和衍生物），它与 I 类 Psl 表位结合，Psl 表位是 铜绿假单胞菌 生物膜的关键 EPS 基质成分，参与初始细胞附着和结构完整性15。</li>
	<li>准备并缓慢加入 100 μL 的 1000 μg/mL 抗生素溶液（参见 材料表）到指定腔室孔的一侧。在37°C下不受干扰地孵育24小时。 注：使用前，在 LB 中稀释 50 mg/mL 妥布霉素抗生素储备液，以获得 1000 μg/mL 的终浓度。</li>
</ol>3. 生物膜荧光染色
<ol><li>制备 0.01 mM 活细胞染色荧光染料溶液。慢慢地从腔室孔中取出培养基，并向每个孔中加入 200 μL 染料混合物。在室温下在黑暗中孵育 45 分钟。 注：使用前，通过将 4 μL 的 5 mM 原液加入 2 mL LB 中来制备活细胞染色（绿色）荧光染料。</li>
	<li>从每个孔中缓慢取出培养基，并用 200 μL 新鲜 LB 洗涤 2 次。</li>
	<li>向每个孔中加入 200 μL 新鲜 LB，并通过共聚焦显微镜进行检查。</li>
</ol>4. 通过共聚焦显微镜进行图像采集
注：图像处理和 COMSTAT 分析程序如图 2 所示。在生物膜染色的同一天采集孔的图像。如果可视化延迟超过 1 小时，则将带腔室的盖玻片在黑暗中冷藏，直到进一步处理。
<ol><li>使用具有适当激光激发波长和滤光片组的共聚焦显微镜系统（参见 材料表）获取孔的图像。 注：此处，分别使用 561 nm 和 491 nm 激发波长激发荧光标记（红色）mAb 和活细胞（绿色）染料。</li>
	<li>使用 20-25 倍水浸镜头以 0.3 μm 的增量捕获分层 z 堆栈图像（从基质到每个生物膜段的顶部）。每孔至少采集 6 个图像堆栈。 注意：在这里，使用带有 25 倍水浸镜头的高分辨率相机可视化图像，并使用图像分析软件进行处理（参见 材料表）。在单次实验中保持所有采集的软件设置和数字成像参数（即亮度和灵敏度）不变。</li>
	<li>将图像另存为 OME-TIFF 以进行 COMSTAT 分析。 注意： 确保为每个通道（即红色和绿色）单独保存 OME-TIFF。此步骤因所使用的图像分析软件而异。</li>
	<li>重复步骤 2.1-4.3，从每个细菌分离株总共 3 个生物学重复（即 3 个独立实验）中捕获图像。</li>
</ol>5. COMSTAT分析
注意：使用免费提供的计算机程序 COMSTAT16,17 定量分析图像，该程序被重写为插件 （Comstat2） 到 ImageJ。阅读下载的软件包中分析生物膜图像堆栈的一般说明。本文提供了一个总结的方案，其中推荐了选定的 ImageJ 处理步骤和 COMSTAT 特征，用于量化抗菌剂对生物膜形成的影响。
<ol><li>从 http://www.comstat.dk/ 下载 Comstat2 包。在已安装的文件夹中，找到 ImageJ 并运行它。</li>
	<li>在桌面上创建一个源文件夹，并向该文件夹添加一个 OME-TIFF。</li>
	<li>从源文件夹中打开 OME-TIFF，并删除任何不包含生物质的空图层。这些层将是 z 堆栈的第一层或最后几层。 注意：显微镜定义的 z 堆栈边界有时会被用户高估。删除这些空层可为 COMSTAT 分析建立更精细的 z 堆栈边界。</li>
	<li>通过选择 “文件”|“ 在 ImageJ 中导入 OME-TIFF进口 |图像序列。找到源文件夹，高亮显示而不打开它，然后单击“ 选择”。将出现一个“序列选项”窗口。选择“ 确定”。 注意：要在 ImageJ 中导入其他图像，请先从源文件夹中删除之前的 OME-TIFF，然后将新的 OME-TIFF 添加到文件夹中，并重复步骤 5.3 和 5.4。</li>
	<li>通过选择 “图像”|“来翻转生物膜的方向 |转型 |翻转 Z 形，将基质定位为第一个（最顶部）堆栈。 注意：COMSTAT 算法从顶部（堆栈 1）到底部在 z 方向上读取生物膜。根据所使用的共聚焦显微镜系统，OME-TIFF输出可以反转。因此，通过将基质定位为图像堆栈 1 来颠倒切片的顺序非常重要，以防止输出数据产生缺陷。</li>
	<li>通过选择“ 图像”|”属性。将出现一个“源”窗口。<ol><li>将“长度单位”指定为“微米”。</li>
		<li>使用以下公式以数学方式确定“像素宽度”和“像素高度”： <img alt="Equation 1" src="/files/ftp_upload/61759/61759equ01.jpg"> (1) 这里，“像素宽度”和“像素高度”定义为0.427，其中电荷耦合器件（CCD）上像素的物理长度为16 μm;总放大倍率为25倍;放大镜位置为 1.5 倍。 注意：用于计算每个像素的单元大小的公式可能因显微镜相机制造商而异。或者，“像素宽度”和“像素高度”可以通过空间校准来定义（参见 https://imagej.net/Spatial_Calibration）。</li>
		<li>将“体素深度”定义为 0.3（即每个 z 堆栈层之间的增量空间）。在“源”窗口中选择 “确定 ”。</li>
	</ol></li>
	<li>通过选择“ 图像”|”调整 |阈值。将出现一个“阈值”窗口。 注意：图像中的对象将显示为红色，背景为灰度。或者，可以使用“阈值”窗口中的下拉菜单将“红色”更改为“黑白”，从而以黑白方式调整阈值。<ol><li>在图像窗口中，将滑块调整到最右侧（即生物膜的最顶层）。要消除背景噪声，请使用显示图像直方图的“阈值”窗口手动设置最大和最小阈值。首先，通过尽可能向右调整下滑块来设置最大阈值。其次，使用上部滑块调整最小阈值，该阈值将图像分割为两个独立的阶段：红色生物量和灰度背景（图 3）。 注：本文所述的体外生物膜形成和荧光显微镜程序在理想情况下生成 OME-TIFF，允许通过简单的直方图阈值方法将图像分割成两个不同的阶段。然而，在某些情况下，不同阶段之间的直方图区别并不那么清楚。这可能是由于存在广泛的背景噪声、不同的背景强度或生物量和背景之间的低强度对比。在这种情况下，用户应采用增强的分割程序18,19。<ol><li>或者，使用“阈值”窗口中设置为“默认”的左侧下拉菜单通过算法调整单个图像的阈值。此功能提供了 17 种不同的算法阈值选项供您选择（请参阅 https://imagej.net/Auto_Threshold）。选择最适用的选项，然后选择“自动”以设置阈值。</li>
		</ol></li>
		<li>调整阈值后，使用图像窗口中的滑块滚动浏览每一层，以确保在整个过程中充分消除背景噪声。</li>
		<li>在“阈值”窗口中选择“ 设置 ”以首先修复较低的阈值。将出现“设置阈值水平”窗口。选择“ 确定”。再次选择“ 设置 ”，然后重复此步骤以修复最大阈值。 注意： 每次选择“设置”时，下方滑块可能会自动调整。在这种情况下，请手动重新调整滑块（如果使用其中一个算法阈值，请选择“自动”）并重复步骤 5.6.2–5.6.3。主要思想是，每当滑块自动重新调整时，应在之后再选择两次“设置”，以确保上限和下限阈值都固定。</li>
		<li>选择“ 应用 ”，将出现“将堆栈转换为二进制”窗口。选择“ 确定”，然后退出“阈值”窗口。</li>
		<li>通过选择 Plugins |生物格式 |生物格式导出器|.输入新文件名并在源文件夹中另存为 OME-TIFF。将出现“生物格式导出器 - 多个文件”窗口。选择“ 确定”。将出现一个“生物格式导出器选项”窗口。选择“ 确定”。 注意：在继续进行 COMSTAT 分析之前，请确保仅将新调整的黑白 OME-TIFF 保存在源文件夹中。从文件夹中删除所有原始 OME-TIFF。</li>
	</ol></li>
	<li>通过选择 Plugins |Comstat2 |.将出现一个“关于”窗口。选择“ 确定”。将出现三个窗口。<ol><li>在“观察到的目录”窗口（右上角）中，选择 “添加”。找到源文件夹，高亮显示而不打开它，然后选择“选择”。将出现一个“目录中的图像”窗口（左上角），其中列出了要通过 COMSTAT 分析的 OME-TIFF。</li>
		<li>在“Comstat 2.1”窗口（右下角）上，取消选择“自动阈值（Otsu 方法）”，以确保软件使用之前为单个 OME-TIFF 设置的阈值。此外，取消选择 连接体积过滤器g （CVF），以确保分析中包括生物膜的非常薄的部分以及在生物膜结构空隙中发现的自由漂浮细胞或生物质。 注意：在这里，取消选择CVF，因为COMSTAT分析是在非常早期的生物膜（24小时初始生长）和抗菌处理后剩余的浮游细胞/菌落上进行的。对于成熟的生物膜，选择CVF以确保仅对连接到生物膜结构的生物量进行量化。</li>
		<li>在“Comstat 2.1”窗口（右下角）上，选择所需的定量分析特征。在这里，选择 生物质量、厚度分布 和 表面积。选择 “转到 ”以运行程序。在“日志”窗口（左下角）中，输出数据显示正在处理中，直到出现“完成选定的功能/图像！记录 COMSTAT 测量值。这些测量值也会自动保存为源文件夹中的 TXT 文件。</li>
	</ol></li>
</ol>

<ol>
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T., 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=Quantitating+the+cell:+turning+images+into+numbers+with+ImageJ.">Quantitating the cell: turning images into numbers with ImageJ.</a> Wiley Interdisciplinary Reviews: Developmental Biology. 6, 260 (2017).</li><li>Daims, H., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+uncultured+microorganisms+by+fluorescence+microscopy+and+digital+image+analysis.">Quantification of uncultured microorganisms by fluorescence microscopy and digital image analysis.</a> Applied Microbiology and Biotechnology. 75, 237-248 (2007).</li><li>Yerly, J., Hu, Y., Jones, S. M., Martinuzzi, R. 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+two-step+procedure+for+automatic+and+accurate+segmentation+of+volumetric+CLSM+biofilm+images.">A two-step procedure for automatic and accurate segmentation of volumetric CLSM biofilm images.</a> Journal of Microbiological Methods. 70, 424-433 (2007).</li><li>Lee, B., 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=Heterogeneity+of+biofilms+formed+by+nonmucoid+Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis.">Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis.</a> Journal of Clinical Microbiology. 43, 5247-5255 (2005).</li><li>Stapper, A. P., 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=Alginate+production+affects+Pseudomonas+aeruginosa+biofilm+development+and+architecture,+but+is+not+essential+for+biofilm+formation.">Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation.</a> Journal of Medical Microbiology. 53, 679-690 (2004).</li><li>Reichhardt, C., Parsek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+scanning+microscopy+for+analysis+of+Pseudomonas+aeruginosa+biofilm+architecture+and+matrix+localization.">Confocal laser scanning microscopy for analysis of Pseudomonas aeruginosa biofilm architecture and matrix localization.</a> Frontiers in Microbiology. 10, 677 (2019).</li><li>Yang, X., Beyenal, H., Harkin, G., 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=Quantifying+biofilm+structure+using+image+analysis.">Quantifying biofilm structure using image analysis.</a> Journal of Microbiological Methods. 39, 109-119 (2000).</li><li>Yang, X., Beyenal, H., Harkin, G., 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=Evaluation+of+biofilm+image+thresholding+methods.">Evaluation of biofilm image thresholding methods.</a> Water Research. 35, 1149-1158 (2001).</li><li>Ross, S. 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=Quantification+of+confocal+images+of+biofilms+grown+on+irregular+surfaces.">Quantification of confocal images of biofilms grown on irregular surfaces.</a> Journal of Microbiological Methods. 100, 111-120 (2014).</li><li>Ma, L., 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=Assembly+and+development+of+the+Pseudomonas+aeruginosa+biofilm+matrix.">Assembly and development of the Pseudomonas aeruginosa biofilm matrix.</a> PLoS Pathogens. 5, (2009).</li><li>Mah, T. F., 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+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426, 306-310 (2003).</li><li>Srinandan, C. S., Jadav, V., Cecilia, D., Nerurkar, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrients+determine+the+spatial+architecture+of+Paracoccus+sp.+biofilm.">Nutrients determine the spatial architecture of Paracoccus sp. biofilm.</a> Biofouling. 26, 449-459 (2010).</li><li>Ramos, I., Dietrich, L. E., Price-Whelan, A., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenazines+affect+biofilm+formation+by+Pseudomonas+aeruginosa+in+similar+ways+at+various+scales.">Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales.</a> Research in Microbiology. 161, 187-191 (2010).</li><li>Ma, L., Jackson, K. D., Landry, R. M., Parsek, M. R., Wozniak, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Pseudomonas+aeruginosa+conditional+psl+variants+reveals+roles+for+the+psl+polysaccharide+in+adhesion+and+maintaining+biofilm+structure+postattachment.">Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment.</a> Journal of Bacteriology. 188, 8213-8221 (2006).</li></ol>

没有规定的方法来定量比较 体外 生物膜结构的三维图像，并且由于操作者之间的可变性，在这种情况下描述的程序通常难以标准化20。因此，该协议为寻求量化不同抗菌条件下 体外 生物膜结构变化的COMSTAT应用提供了一个简单且可重复的框架。该技术的优势是使用 CF 铜绿假单胞菌 分离物建模的，该分离物以生物膜重复的形式生长，并暴露于妥布霉素和抗 Psl...

生物膜是微生物的聚集体，定向在自产生的细胞外聚合物物质 （EPS） 基质中。EPS 基质非常复杂，主要由细菌细胞、水、蛋白质、多糖、脂质和核酸1 组成，所有这些都使生物膜与自由生活的浮游细胞截然不同。生物膜EPS相互粘附在各种表面上。EPS 基质具有介导代谢物、遗传物质和用于细胞间信号转导和防御的化合物的细胞间交换的特性2。这些特性共同提供了生物膜结构完整性和抵御外部压力源的保护，有助于免疫逃避和抗菌素耐药性3.
铜绿假单胞菌是一种公认的医院病原体，已知它对抗菌药物采取逃避生物膜生长策略。一个典型的例子发生在患有隐性遗传疾病囊性纤维化 （CF） 的患者身上。生物膜在抗菌耐药性铜绿假单胞菌4 的发展中起着关键作用，并允许 CF 患者建立慢性肺部感染，导致肺功能加速下降和过早死亡5。因此，进行体外生物膜研究以测试抗生素和新型抗感染剂对从 CF 6,7 患者获得的铜绿假单胞菌分离株的疗效。生物膜形成后，将抗菌剂应用于结构外部，并使用共聚焦激光扫描显微镜 （CLSM） 生成生物膜段的高分辨率三维重建。通常的做法是使用计算机软件COMSTAT作为ImageJ的插件，以量化生物膜结构的变化8,9,10,11。
尽管COMSTAT可用于量化生物膜结构，但图像分析的可重复性和标准化问题较少得到解决。例如，在运行COMSTAT之前执行的图像处理程序是客观的，但在设置图像阈值12,13时包含主观性因素。以类似的方式，COMSTAT程序允许操作员选择从基本到高级的图像分割条件和参数，以及十种定量分析功能（例如，厚度分布、表面积、生物量、无量纲粗糙度系数）。众多的用户选项，再加上不同的操作人员专业知识水平，可能会导致对生物膜行为的误导性报告。
因此，该协议的目标是提出一种相对简单的方法，用于使用COMSTAT对体外生物膜结构进行定量比较。本文中，使用带腔盖玻片模型14 通过 CLSM 捕获来自铜绿假单胞菌分离物的生物膜片段的三维图像——这是一种用于执行可重复体外生物膜实验的成熟技术。利用 COMSTAT 作为 ImageJ 的插件，该方法使研究人员能够在不同条件下定量识别存在抗菌剂的情况下生物膜结构的变化。总体而言，该方法旨在消除与COMSTAT手动操作相关的主观差异，从而促进各中心协议的标准化。

<table>
	<tbody>
		<tr>
			<td>Anti-Psl mAb, Psl0096</td>
			<td>Medimmune</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar (TSA with 5 % Sheep Blood) Medium</td>
			<td>Fisher Scientific</td>
			<td>R01200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eight-well Chambered Coverglass w/ non-removable wells</td>
			<td>Thermo Fisher Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen SYTO 9 Green Fluorescent Nucleic Acid Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S34854</td>
			<td></td>
		</tr>
		<tr>
			<td>LB BROTH (LENNOX), Liquid Autoclave Sterilized</td>
			<td>BioShop Canada</td>
			<td>LBL666</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobramycin, 900 &#181;g/mg</td>
			<td>Alfa Aesar by Thermo Fisher Scientific</td>
			<td>J66040</td>
			<td>It is recommended to perform a minimal inhibitory concentration (MIC) test for every batch made to ensure quality control of antimicrobial potency</td>
		</tr>
		<tr>
			<td>Quorum Volocity 6.3</td>
			<td>Quorum Technologies</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

quantifying the effects of antimicrobials on in vitro biofilm architecture using comstat software

生物膜是微生物的聚集体，它们依赖于自产生的细胞外聚合物物质基质来保护和结构完整性。已知医院病原体 铜绿假单胞 菌采用生物膜生长模式，导致囊性纤维化 （CF） 患者发生慢性肺部感染。计算机程序COMSTAT是一种有用的工具，用于通过从三维共聚焦图像中提取数据来量化 铜绿假单胞菌 生物膜结构中抗菌诱导的变化。然而，软件的标准化操作较少得到解决，这对于生物膜行为的最佳报告和跨中心比较非常重要。因此，该协议的目的是提供一个简单且可重复的框架，用于通过COMSTAT量化不同抗菌条件下的 体外 生物膜结构。该技术使用铜 绿假单胞菌 分离物建模，以生物膜重复的形式生长，并暴露于妥布霉素和抗 Psl 单克隆抗体 Psl0096。循序渐进的方法旨在减少用户的歧义，并最大限度地减少忽视关键图像处理步骤的机会。具体而言，该协议强调消除与COMSTAT手动操作相关的主观变化，包括图像分割和选择适当的定量分析功能。尽管这种方法要求用户在运行COMSTAT之前花费额外的时间处理共聚焦图像，但它有助于最大限度地减少自动输出中错位的生物膜异质性。

从感染的CF患者中培养的 铜绿假单胞菌 分离物用于证明该方法在准确量化抗菌诱导 的体外 生物膜结构变化方面的优势。该模型的整体工作流程如图 1 所示。ImageJ 中的图像处理和 COMSTAT 分析过程如图 2 所示。 图 3 显示了 ImageJ 中用于图像分割的简单直方图阈值方法，该方法应用于 CLSM z 堆栈图像（另存为 OME-TIFF）。?...

Watch this Scientific Journal Video about Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software at JoVE.com

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

抗菌诱导的 铜绿假单胞 菌生物膜结构变化在从囊性纤维化和慢性肺部感染患者培养的临床分离株中有所不同。在共聚焦显微镜之后，COMSTAT软件可用于量化单个分离株的生物膜结构（例如，表面积、厚度、生物量）的变化，以评估抗感染剂的功效。

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 ...

quantifying-effects-antimicrobials-on-vitro-biofilm-architecture

Research

JoVE Journal

Immunology and Infection

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

3.1K 次观看.  Hospital for Sick Children. 抗菌诱导的 铜绿假单胞 菌生物膜结构变化在从囊性纤维化和慢性肺部感染患者培养的临床分离株中有所不同。在共聚焦显微镜之后，COMSTAT软件可用于量化单个分离株的生物膜结构（例如，表面积、厚度、生物量）的变化，以评估抗感染剂的功效。

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

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. ...

科研

免疫与感染

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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

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

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

可视化痰生物膜发展影响使用腔室盖玻片型号

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

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

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. ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响