我们感谢金钗实验室的所有成员对生物膜测定进行有益的讨论。这项研究得到了国立卫生研究院 (NIH) 授予 R21 AI125801 (C.J.N.) 的支持。D.L.R. 得到了加利福尼亚大学墨西哥和美国研究所 (UC-MEXUS) 和理事会国家 Ciencia y Technologia (委员会) 的博士奖学金的支持。

1. 真菌细胞培养制剂
注意: 在生物安全柜内进行细胞培养工作 (即打开低温储存管、细胞培养管和烧瓶)。在工作前打开机箱的紫外线 (uv) 杀菌灯至少1小时, 并在机柜中积极工作时关闭紫外线灯。在实验开始前, 戴上手套、安全眼镜和适当的个人防护设备, 清除工作台表面的污管和70% 乙醇。建议使用无菌过滤器提示和熟悉基本无菌微生物技术。
<ol>
	<li>条纹白念珠菌菌株 (野生类型, bcr1 δ/δ, 和efg1 δ/δ) 酵母提取蛋白胨葡萄糖 (YPD) 培养基 (1% 酵母提取物, 2% 蛋白胨, 2% 葡萄糖; pH 6.8) 琼脂板。在30° c 下孵育 48 h, 遵循标准微生物操作19。</li>
	<li>选择一个单一的孤立的群体从板块接种到4毫升的 YPD 液体培养基 (1% 酵母提取物, 2% 蛋白胨, 2% 葡萄糖; pH 6.8) 和孵化在30° c 的震动在225分钟的标准震动孵化器 12-15 h, 遵循标准的微生物学实践tices19。</li>
	<li>按照标准微生物做法19, 通过测量试管 (1 毫升, 1 厘米路径长度) 600 nm 的光学密度 (OD) 来确定培养细胞的密度。</li>
	<li>将细胞培养物稀释到最终 OD600 0.5, 相当于 2 x 107单元格/mL, 在罗斯威尔公园纪念研究所 (RPMI)-1640 介质 (含 165 mM 3-morpholinopropane-1-磺酸 (拖把), l-谷氨酰胺, 无钠碳酸氢盐, ph 值 7.0) 或蜘蛛培养基 (1% 营养液, 1% 甘露醇, 0.4% K2HPO4, ph 7.2)。 注: 可供选择的介质可用于支持特定的增长菌株的利益。 注意: 不要让细胞稀释太远。建议在3.3 步 (如下所述) 后开始稀释, 以防止在观察通道中植入细胞之前形成菌丝。</li>
</ol>
2. 微流控板微流控通道的制备
注: 请参阅微流控系统用户手册 (见材料表), 了解有关板和仪器设置的信息。
<ol>
	<li>在运行微流控实验之前, 在37° c 的恒温箱中预热20毫升的 RPMI-1640 介质或蜘蛛介质。根据步骤2.8 中的计算, 可能需要额外的媒体卷。在实验中, 预热介质防止气泡形成。</li>
	<li>打开微系统, 其中包括控制器、两个电源单元、显微镜、照相机和连接到系统的计算机。从计算机上的程序菜单打开控制系统的软件 (请参阅材料表)。打开程序后将出现两个窗口。</li>
	<li>找到使用的车牌号码, 并在软件提示时输入号码。使用两个单独的计算机屏幕查看两个窗口中的软件。一个窗口 (控制模块) 包含的控制板和第二个窗口 (蒙太奇, 成像模块) 包含的控制显微镜和相机。</li>
	<li>打开控制器上的加热器电源按钮, 并使用温度控制器上的箭头按钮将微系统温度设置为37° c。</li>
	<li>使用无菌水清洁接口板 (图 1), 如下所示。用不育的水喷洒在盘子的底部, 用透镜纸去除灰尘。将界面板放在干净的镜片纸上, 晾干。使用70% 异丙醇和透镜纸清洁界面板的顶部。 注: 接口板将微流体系统连接到含有电池和介质的板上。确保没有液体残留, 所有的纤维和污垢都被去除。不正确的清洁可能导致低质量的图像和视频。</li>
	<li>从接口板上的管子上除去冷凝。<ol>
		<li>将盘面放在镜片纸上。在控制模块窗口单击手动模式的板。在 "剪切控制" 菜单部分中, 单击 1-4 和 5-8 列的流体选择, 然后从下拉菜单中选择 "37 ° c"。在剪切控制部分, 将流模式设置为常量, 并将最大切变设置为2达因/cm2 (0.2 Pa) (图 2A)。</li>
		<li>点击入口口井, 启动无菌气流通过界面板管除去冷凝。5分钟后, 单击 "井板控制" 部分 (图 2A) 中的 "停止" 按钮。观察接口板中的管子, 确认所有冷凝已被清除。如果水滴仍然可见, 重复上述的无菌空气流动, 再5分钟。 注: 进口管连接到0.22 微米的过滤器, 以确保只有无菌空气被引入到微系统。该流可由 "控制模块" 窗口中屏幕上的事件日志监视。</li>
	</ol>
	</li>
	<li>在显微镜台上放置48井0-20 达因微流控板。</li>
</ol>
3.白色念珠菌微流控系统中的生物膜形成
<ol>
	<li>要记录生物膜的形成, 增加600µL 的 5ml RPMI-1640 介质或蜘蛛介质到入口井 (见图 1的板布局)。每个实验条件都有两个复制。<ol>
		<li>为了检测生物膜形成的野生型和突变株, 将600µL 的蜘蛛培养基添加到实验板的入口井中. </li>
		<li>用于在存在抗真菌药物 (或其他感兴趣的化合物) 的情况下检测生物膜的形成, 将600µL RPMI-1640 培养基添加到两口井 (负控制) 和600µL RPMI-1640 培养基中, 辅以两性霉素 B (16 µg/毫升) (或所需药物选择浓度) 到另外两个入口井。 注: 对于 12 h 生物膜的形成实验, 增加600µL 的5ml 介质到入口井。为更长的实验增加另外的媒介 (50 µL/小时) 到入口井。不要超过井的最大容积 (1500 µL)。</li>
	</ol>
	</li>
	<li>慢慢降低48井微流控板上的清洗界面板。将界面板稍稍向左滑动, 直到相间板上的管子位于入口和出水口的顶端。</li></ol>将接口板上的拉杆从左向右转动, 以锁定接口板, 确保实验是密闭的。 注: 从接口板上的管子中流出的无菌空气会将液体推入进风口或出水口 (取决于在计算机上使用软件所选择的方向), 使液体流经入口和出口之间的查看通道井在自定义的方向和速度由用户口授。
	<li>在剪切控制部分, 将流模式设置为常量, 并将最大切变设置为1达因/cm2 (0.1 Pa)。点击入口井与介质, 开始从入口到出口井 (图 2A) 的媒体流量, 以总理的渠道。5分钟后, 点击停止按钮在井板控制部分。取下接口板, 观察微流控板井。在启动通道后, 出口井中只有一小滴介质可见。如果下降是不可见的, 以2分钟为增量的渠道, 直到在出口井中的小滴媒体可见。 注: 需要启动时, 从查看通道中取出空气, 确保通道充满所需的介质。</li>
	<li>从微流控板上取下接口板, 并将其放在不育的表面上。添加50µL 的细胞培养 (在步骤1.5 中描述) 到每个出口井。<ol>
		<li>用于测试C. 白念珠菌野生型和突变株, 添加50µL 野生型 (SC5314) 细胞培养在蜘蛛媒体到两个出口井, bcr1 δ/δ在蜘蛛介质到两个出口井, 和efg1 δ/δ在蜘蛛介质中的两个出水口。将接口板放回微流控板上并锁定盖子 (请参阅图 1进行版面布局)。 注: 如步骤3.1.1 所述, 所有对应的输入井均含有蜘蛛介质。</li>
		<li>为了在两性霉素 B 或其他复利的存在下测试白色念珠菌生物膜的形成, 在 RPMI-1640 培养基和四口井中添加50µL 的野生型 (SC5314) 细胞培养物。将接口板放回微流控板上并锁定盖子 (请参阅图 1进行版面布局)。 注: 如步骤3.1.2 所述, 所有对应的输入井均含有与两性霉素 B 或其他复利的 RPMI-1640 介质。</li>
	</ol>
	</li>
	<li>在剪切控制部分, 将流模式设置为常量, 并将最大切变设置为2达因/cm2 (0.2 Pa)。点击出口井与细胞开始流介质从入口到出口井 (图 2A) 开始播种的C. 白色念珠菌。3秒后, 单击井板控制部分的 "停止" 按钮, 防止单元格进入输入井。 注: 细胞从出水口向入口井移动;在细胞到达入口井之前, 必须停止流动。这确保了入口井不会受到细胞培养的污染, 并且在实验期间, 入口井中的介质保持不育。所需的剪切力和时间是基于介质的粘度和细胞的大小。</li>
	<li>允许细胞保持静止不动 (0 达因/厘米2), 在观察通道中进行 10-20 分钟的初始细胞粘附。在这 10-20 分钟坚持步骤, 设置计算机捕捉相机阶段的位置, 并开始获取 pre-flush 图像 (详细说明在 4-6)。</li>

4. 设置微流控实验的舞台位置
注: 在开始试验前, 应设置工作台位置和板的标定。此设置允许计算机存储每个查看通道的位置, 以便在实验期间捕获图像。在设置舞台位置后, 不应干扰微流控板。如果板块被移动, 舞台位置将不得不在实验开始前复位。
<ol>
	<li>使用可视化分析软件 (见材料表) 设置舞台位置;每个查看通道都是一个阶段 (1-24) (图 1D), 每个阶段都有三亚 (1-3), 用于在查看通道 (图 1C) 的不同位置捕获图像。在 "图像处理模块" 窗口中, 通过单击 mda 选项卡 (图 2B) 打开 "多维获取" (mda) 模块。</li>
	<li>在 MDA 模块中, 单击 left-hand 端菜单上的 "舞台" 选项卡 (图 2B)。在舞台部分, 加载48井 0-20 达因微流控板的主阶段列表。每个查看通道都应该有三阶段的图像采集。</li>
	<li>通过单击 "多点" 选项卡和 "映射" 选项卡 (图 2B), 打开 "示例重新加载调整" (多点) 和 "移动阶段到绝对位置" (map) 模块。在该模块中, 按 "实时" 按钮以查看带有十字线的图像预览。</li>
	<li>使用操纵杆, 定位井板, 使箭头的尖端 (物理上位于板上) 毗邻查看通道4与该软件的十字线对准。在 "映射" 菜单中, 按 "设置原点" 按钮 (图 2B)。当前位置现在应在 X、Y 和 Z 中读取0。</li>
	<li>在该模块中, 单击 left-hand 菜单中的 "初始参考点" 部分 (图 2B)。单击 "加载设置" 并加载48井 0-20 达因微流控板的文件。</li>
	<li>在 MDA 模块的阶段部分, 双击 "舞台位置 222"。此位置表示查看通道编号22的中间 (sub-stage 2)。这将带来显微镜观看阶段和相机的焦点接近箭头标记通过查看通道22。使用操纵杆, 定位井板, 使箭头的尖端 (物理上位于板块) 毗邻查看通道22与十字线对准。</li>
	<li>将 MDA 模块中的 y 坐标复制到多对多模块 (图 2B) 的 "更新参考点" 选项卡中的点2的 y 位置。</li>
	<li>在 "更新阶段位置" 选项卡中, 单击 "应用于 MDA 阶段列表" (图 2B)。 注: 这将更新板 (1-24) 中的所有观察阶段位置, 以校准到显微镜阶段的特定微流控板位置。</li></ol>该板块现在已准备好进行图像采集, 并将使用校准阶段位置移动, 同时捕捉所有24查看通道中的三位置的图像。

5. 在微流体实验中建立图像捕获的获取
<ol>
	<li>在 "图像处理模块" 窗口中, 使用 MDA 模块并单击 left-hand 菜单中的 "保存" 选项卡 (图 2B)。选择实验的文件名和文件夹以将获取的图像存储在计算机上。</li>
	<li>在 MDA 模块中, 单击 left-hand 菜单中的 "时差" 选项卡, 并将其设置为获取145总图像;将图像之间的时差设置为5分钟。这将导致1图像每5分钟的 12 h 生物膜的开发实验。 注意: 图像的时间间隔和图像的总数量可以根据实验要求进行调整。如果成像超过12小时, 在步骤3.1 的入口井中添加额外的介质, 以确保油井不会干涸。为更长的实验增加另外的媒介, 根据需要 (50 µL/小时) 到入口井。</li>
	<li>在 MDA 模块中, 单击 left-hand 菜单中的 "波长" 选项卡 (图 2B), 并将实验的波长数设置为1。设置波长以捕获50% 明和50% 相机, 曝光时间为 12-20 ms, 0.6 增益和 20 MHz 数字化仪。 注: 可以使用额外的波长, 包括波长的荧光成像 (例如, 我们已经成功地可视化 mCherry 和 GFP 标记的C. 白念珠菌菌株使用此微流控装置)。根据实验要求, 也可以改变采集参数。例如, 建议为初学者设置第二个波长, 选择 "自动对焦在每个 Timepoint" 和第三波长, 并选择 "Autoexposure 在每个 Timepoint", 以最大限度地提高图像获取的机会, 是重点。</li>
	<li>在 MDA 模块中, 选择 left-hand 菜单中的阶段列表选项卡。11阶段–243将显示。依次单击每个阶段, 并使用细微的焦点设置手动将焦点放在每个阶段的查看通道中的单元格上。一旦视图的字段处于焦点, 请单击舞台列表旁边的黑色箭头以更新和保存每个阶段的设置。计算机将使用这些手动定义的位置设置和焦点设置来获取每个时间点的图像。使用 "删除" 箭头从列表中删除所有未使用的阶段。 注: MDA 模块和程序可交替地将查看通道视为阶段, 软件使用相同的术语。</li>
</ol>
6. 运行微流体实验
<ol>
	<li>在 "图像处理模块" 窗口中, 使用 MDA 模块, 选择 "获取" 开始捕获所有亚 (图 2B) 的粘附单元格的 pre-flush 图像。 注意: "图像处理模块" 窗口和照相机控件现在将显示正在捕获的图像, 而映射、多模式和 MDA 模块将不再可见。捕获的图像还会在侧面显示一个计时器, 以指示捕获的图像数和在下一个图像捕获周期开始之前的时间间隔。等待一轮图像被捕获, 然后再继续下一步。 注意: 不要在 "图像处理模块" 窗口屏幕上使用 "暂停" 或 "标记事件" 按钮。在实验过程中, 将鼠标放在第二个计算机屏幕上的 "控制模块" 窗口 (图 2A)。这对于避免干扰成像模块窗口很重要。</li>
	<li>在控制模块窗口停留在手动方式为板材。在剪切控制部分, 将流模式设置为常量, 并将最大切变设置为1达因/cm2 (0.1 Pa)。单击出口水井 O1、O7、O13 和 O19, 开始从入口到出口井 (图 2A) 的介质流, 以删除 non-adhered 电池。5分钟后, 点击停止按钮在井板控制部分。允许获得第二轮图像。图像可以可视化, 并且可以在图像模块窗口的第二个屏幕上监视时间推移。</li>
	<li>在剪切控制部分, 通过单击最大切变列中的数字并使用键盘进入 0.5, 将最大切变流调整为0.5 达因/cm2 (0.5 Pa)。按键盘上的 enter 键, 并确认 "控制模块" 窗口 (图 2A) 中事件日志中的流率变化。让实验在黑暗中不受干扰地保持12小时。 注意: 暴露在光和运动/振动可能会严重降低在整个实验中获得的图像的质量。在微流控实验结束时, 成像模块窗口将不会显示任何图像, 并还原为显示多目标、MAP 和 MDA 模块。成像模块软件可以用来查看图像, 组装视频, 并量化的生物膜形成 (描述如下)。</li>
</ol>
7. 分析结果
<ol>
	<li>在 "图像处理模块" 窗口中, 选择 "分析工具" 选项卡, 然后从下拉菜单中单击 "查看多维数据" (RMD)。在 RMD 模块中, 单击 "选择基本文件"。这将打开 "多维数据集实用程序" 窗口。</li>
	<li>单击 "选择目录" 按钮, 然后选择用于在步骤5.1 中保存实验的文件夹。在 "数据集" 列表中, 选择实验文件日志 (. nd 扩展名)。加载日志后, 单击 "查看" 按钮。</li>
	<li>通过单击 left-hand "波长" 菜单中的选项选择波长, 然后从下拉菜单中选择要查看的舞台位置。通过右键单击图像编号, 从模块顶部的数字中选择要加载的图像。选定后, 单击 "加载图像" 按钮以加载图像。您可以一次查看一个图像或所有图像。</li>
	<li>选择所有图像, 使时间推移视频。选定的图像将在新窗口中作为视频打开。单击 "图像处理模块" 窗口中的 "文件" 选项卡, 然后从下拉菜单中选择 "另存为"。将生成的视频保存为默认文件 (TIFF/MetaSeries)。</li>
	<li>使用 ImageJ 软件打开并加载保存的视频文件。在 ImageJ 中, 单击 "文件" 选项卡, 然后单击 "另存为"。从下拉菜单中选择 ". avi", 并将文件转换为. avi 文件, 使用 JPEG 转换设置为10帧/秒。 注意: 视频的速度可以通过改变帧/秒来调整。</li>
	<li>要量化生物膜, 重复步骤7.2、7.3 和7.4。</li></ol>选择最后一个图像 (数字 144), 然后单击 "加载图像" 按钮以加载图像。图像将在新窗口中打开。单击 "阈值图像" 按钮并选择 "包含" 阈值。移动光标, 直到生物膜细胞呈红色, 没有细胞的区域不会被着色。
	<li>单击 "打开日志" 按钮进行日志测量 (该按钮将只在最初显示 "打开日志"; 一旦日志处于活动状态, 该按钮将被重命名为 "F9: 日志数据")。在 "打开数据日志" 窗口中选择 "动态数据交换", 然后单击 "确定"。</li>
	<li>在 "图像处理模块" 窗口中, 选择 "分析工具" 选项卡, 然后单击 "显示区域统计信息"。这将打开一个新窗口, 显示图像的度量值。在 "测量" 选项中选择 "整个图像", 然后单击 "F9: 日志数据"。将显示一个具有所有度量值的 excel 文件。找到 "区域" 和 "值区域" 的值, 并将后者除以前者, 以获取被生物膜占据的区域的数值。 注: 在实验中获得的每个图像的 "面积" 是相同的, 因此 "值面积" 测量可以用来评估野生型菌株与突变菌株之间生物膜形成的差异, 或评估对生物膜形成的药物进行了测试。</li>

<ol>
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E., Marchillo, K., Spiegel, C. A., Andes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+in+vivo+Candida+albicans+biofilm+denture+model.">Development and validation of an in vivo Candida albicans biofilm denture model.</a> Infect Immun. 78 (9), 3650-3659 (2010).</li><li>Nett, J. E., 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=Rat+indwelling+urinary+catheter+model+of+Candida+albicans+biofilm+infection.">Rat indwelling urinary catheter model of Candida albicans biofilm infection.</a> Infect Immun. 82 (12), 4931-4940 (2014).</li><li>Krom, B. P., Willems, 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=In+Vitro+Models+for+Candida+Biofilm+Development.">In Vitro Models for Candida Biofilm Development.</a> Methods Mol Biol. 1356, 95-105 (2016).</li><li>Hawser, S. P., Douglas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+Candida+species+on+the+surface+of+catheter+materials+in+vitro.">Biofilm formation by Candida species on the surface of catheter materials in vitro.</a> Infect Immun. 62 (3), 915-921 (1994).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. 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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=Global+Identification+of+Biofilm-Specific+Proteolysis+in+Candida+albicans.">Global Identification of Biofilm-Specific Proteolysis in Candida albicans.</a> mBio. 7 (5), (2016).</li><li>Nobile, C. J., 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+recently+evolved+transcriptional+network+controls+biofilm+development+in+Candida+albicans.">A recently evolved transcriptional network controls biofilm development in Candida albicans.</a> Cell. 148 (1-2), 126-138 (2012).</li><li>Fox, E. 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=An+expanded+regulatory+network+temporally+controls+Candida+albicans+biofilm+formation.">An expanded regulatory network temporally controls Candida albicans biofilm formation.</a> Mol Microbiol. 96 (6), 1226-1239 (2015).</li><li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell-surface+genes+and+biofilm+formation+by+the+C.+albicans+transcription+factor+Bcr1p.">Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p.</a> Curr Biol. 15 (12), 1150-1155 (2005).</li><li>Baker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the bench: A laboratory navigator. 27, (2005).</li></ol>

金钗是 BioSynesis 的创始人, Inc. 是一家开发抑制剂和诊断白色念珠菌生物膜的公司。

本文所描述的可定制微流控生物膜法可以在一个固定速率的层流和恒定温度下, 在单个细胞水平上 real-time 生物膜形成的可视化。它为研究野生型和突变菌株的生物膜的发展提供了有力的手段, 以及在模拟临床环境中观察到的生理条件下抗菌剂处理对生物膜的影响。不同于大多数的体外生物膜测定法, 这种方法允许检查在 real-time 形成的生物膜的发展。
这种方法可用于高?...

白色念珠菌是人类微生物的一个共生成员, 但它也是一种机会病原体, 能够引起表面和严重的真菌感染1,2。一个主要的毒力性状的白色白念珠菌是它的能力形成弹性和抗药性生物膜, 细胞群落附着在一个表面和封闭在一个细胞外基质材料1,3。白色念珠菌生物膜是高度结构化的, 包含多个细胞类型的多层 (圆芽酵母形态细胞, 椭圆形菌丝细胞, 和管状菌丝细胞)4。C. 白色念珠菌生物膜的发育始于圆的酵母形态细胞附着在表面 (播种生物膜), 其次是这些细胞在表面的增殖, 然后将未成熟的生物膜结构成熟为由胞外基质材料包围的完全形成的生物膜4。成熟的生物膜主要由形成致密和互连网络的细长菌丝细胞组成, 为生物膜4提供了结构稳定性。在整个生物膜的生命周期中, 圆芽酵母细胞从成熟的生物膜中分散开来, 并可能前往其他部位引起播散性感染或种子新的生物膜在其他站点4,5。白色念珠菌"可以在生物表面形成生物膜, 如粘膜表面和整个寄主组织, 以及在非生化表面, 如导管、心脏起搏器、假牙和假肢接头。由于生物膜的顽固性质, 它们非常难以根除, 而且在许多情况下, 唯一有效的治疗策略是去除感染的设备4。因此, 在类似于临床环境中观察到的条件下, 研究生物膜的形成至关重要。
有几个关键的体内动物模型用于研究C. 白色念珠菌生物膜形成6,7,8;然而, 这些研究可能是昂贵的, 耗时, 并受到限制的菌株和抗菌剂的数量, 可以在给定的时间进行测试。体外生物膜测定, 另一方面, 允许快速, 高通量的抗真菌化合物和突变株的评估, 并更 cost-effective 和伦理比在动物模型中进行的生物膜测定9, 10,11,12,13,14。在这里, 我们描述了一个体外分析, 我们开发和优化, 以观察在层流下的生物膜形成使用可定制的微流控设备14,15。该方法可以对生物膜形成的各个阶段进行可视化, 包括初始黏附步骤、细胞增殖、生物膜成熟和细胞分散。该分析也有助于可视化细胞形态学的变化, 在整个发展的生物膜。
孔板, 通常用于体外生物膜测定, 而高吞吐量, 不允许受控流条件。传统的层流细胞系统允许在受控流条件下对生物膜的形成进行连续的评估, 但通常这些都是建立起来的, 并且往往有有限的动态范围控制和吞吐量。这里使用的微流控装置克服了这些限制, 将高吞吐量板 (含48口井) 与内置层流室结合起来, 具有高度的重现性、通用性和可定制性。
在这里, 我们描述了一个使用商业上可用的自动微流控装置的协议来评估野生型的C. 白念珠菌菌株的生物膜形成, 已知的抗真菌剂对生物膜的发育和生物膜的影响在两个突变株的形成 (bcr1δ/δ和efg1 δ/δ), 以前报告有生物膜缺陷和在体内16,17, 18。所述的协议可用于测试抗菌剂在生物膜形成过程中抑制生物膜生成的功效, 并通过筛选突变文库来识别正常生物膜发育所需的基因。

<table>
	<tbody>
		<tr>
			<td>BioFlux 1000z</td>
			<td>Fluxion</td>
			<td></td>
			<td>Automated microfluidic device for live cell analysis</td>
		</tr>
		<tr>
			<td>48-well plate 0-20 dyne</td>
			<td>Fluxion</td>
			<td>910-0047</td>
			<td>Microfluidic plate</td>
		</tr>
		<tr>
			<td>Montage Software</td>
			<td>Fluxion</td>
			<td>Version 7.8.4.0</td>
			<td>Visualization analysis software</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>NIH</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Criterion</td>
			<td>C7341</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (D-Glucose)</td>
			<td>Fisher Scientific</td>
			<td>D163</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma-Aldrich</td>
			<td>R6504</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Broth</td>
			<td>Criterion</td>
			<td>C6471</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco D-Mannitol</td>
			<td>BD Biosciences</td>
			<td>217020</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Criterion</td>
			<td>C5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Corning</td>
			<td>30-003-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Inoculating Loops</td>
			<td>VWR</td>
			<td>30002-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14-955-127</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Paper</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate and Cuvette Spectrophotometer</td>
			<td>BioTek</td>
			<td>EPOCH2TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>Eppendorf</td>
			<td>M12820004</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of biofilm formation in candida albicans using an automated microfluidic device

白色念珠菌是人类最常见的真菌病原体, 导致大约15% 的医院感染败血症病例。一个主要的毒力属性的白色白念珠菌是它的能力, 形成生物膜, 结构化群落的细胞附着在生物和非生物学表面。白色念珠菌生物膜可以在宿主组织 (如粘膜层) 和医疗设备 (如导管、心脏起搏器、假牙和关节修复体) 上形成。生物膜具有重大的临床挑战, 因为它们对物理和化学的扰动具有很强的抵抗力, 并且可以作为水库种子传播感染。各种体外检测方法已被用于研究白色念珠菌生物膜的形成, 如孔板测定、干重测量、细胞活力分析和共聚焦扫描激光显微镜。所有这些化验都是单一的 end-point 化验, 其中生物膜的形成是评估在特定的时间点。在这里, 我们描述了一个在层流条件下使用自动微流控装置研究 real-time 生物膜形成的协议。这种方法允许观察生物膜的形成随着时间的推移, 生物膜的发展, 使用可定制的条件, 模仿宿主, 如那些在血管导管中遇到的。该协议可用于评估遗传突变体的生物膜缺陷以及抗菌剂对 real-time 生物膜发育的抑制作用。

我们在两种介质条件 (RPMI-1640 和蜘蛛介质) 下使用野生型的C. 白念珠菌菌株进行微流控生物膜测定, 这种菌株存在于已知的抗真菌药物两性霉素 B (16 µg/毫升) 的 RPMI,和两个变种菌株以前报告的生物膜形成缺陷 (bcr1δ/δ和efg1 δ/δ) 在蜘蛛媒体。
视频1显示了野生型生物膜在 RPMI-1640 培养基中的发展, 以及抗真菌?...

Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

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

全自动微流控装置在白色念珠菌生物膜形成中的可视化

本协议描述了在宿主生理条件下, 使用可定制的自动微流控装置来可视化白色念珠菌中的生物膜形成。

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-biofilm-formation-candida-albicans-using-an-automated

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

10.5K 次观看.  University of California, Merced. 本协议描述了在宿主生理条件下, 使用可定制的自动微流控装置来可视化白色念珠菌中的生物膜形成。

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

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. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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

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

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

可视化痰生物膜发展影响使用腔室盖玻片型号

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

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

红光光疗法调节白色念珠菌生物膜生长

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...