Name: visualization of biofilm formation in candida albicans using an automated microfluidic device
Uploaded: 2017-12-14T15:00:00.000+00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

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>

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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。
这种方法可用于高通量的方式, 其中多达24样本可以同时运行, 以评估生物膜的形成。该方法还可用于对突变库进行遗传筛选, 以确定正常生物膜发育所需的基因, 并可用于评估复合型图书馆筛网中抗菌化合物的生物膜的有效性。我们预计, 该微流体设备的未来应用将包括这样的屏幕。然而, 我们注意到, 某些药物治疗测试将受到限制的生物膜的厚度使用这种微流控装置作为生物膜生长 #62; 16 h 倾向于堵塞查看通道。因此, 药物测试实验将需要进行和 #60; 16 小时。
总的来说, 这个设备是高度可定制的和多才多艺的, 并且可以被调整评估不同的微生物种类横跨王国、流速、温度、潜伏期和媒介。所述方法提供了关于生物膜形成的若干不同方面的信息, 包括初始细胞黏附、生物膜成熟和细胞散布。虽然我们只报告物种生物膜形成的结果, 这一议定书也可以适应研究双和混合物种生物膜的形成。
两个步骤对于成功执行此协议至关重要。首先, 必须通过在实验温度下预热介质来避免系统中的气泡。一个常见的失误是在没有预热的培养基中稀释细胞。在用于进水井的介质中, 应将电池稀释。其次, 必须仔细监测从出口到入口井的细胞流动情况;如果细胞流动太远, 入口介质将被污染, 实验不会产生解释的结果。另一方面, 如果不允许单元格一直移动到查看通道中, 则在实验过程中, 通道中的单元格将不可见, 从而导致空白图像。另外的要点要记住的是, 微流控板中的井之间的体积和移动是连接在一起的每一个栏目, 这是重要的保持尽可能多的介质一致性 (具有相似的粘度, 细胞密度,成分) 和类似大小的细胞 (如果可能), 因为不同的媒体和不同的细胞大小将有不同的流速在该列。在实验中, 使用显微镜对细胞的回流进行监测;但是, 一次只能查看两个通道 (使用10X 目标)。因此, 强烈建议进行模拟实验, 以测量从出口到入口井的时间, 使用计划在实际实验中使用的电池和介质。
总的来说, 我们强烈建议使用这种微流控生物膜法作为一种体外检测方法, 用于在动物实验之前的体内。然而, 该化验仍然是一个体外化验, 结果将需要在相关的动物模型中得到验证。在我们的经验中, 这种微流控生物膜法的结果对体内的生物膜测定具有最好的预测价值, 与其他的体外检测方法相比, 我们已经评估了14。

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, 以及抗真菌药物, 两性霉素 B 对生物膜形成的影响。在野生型条件下, 几个细胞强烈黏附在通道上, 细胞随着时间的推移形成菌丝, 插菌丝和酵母细胞可以观察到厚的生物膜。在微流控实验结束时, 野生型生物膜完全填满了观察通道。然而, 两性霉素 B 的存在对减少生物膜有明显的影响。具体来说, 细胞的黏附作用被减少, 并且, 与未处理的条件不同, 在两性霉素 B 存在的情况下, 几个细胞可以被看见流动与媒介的切变流动。随着时间的推移, 细胞不能形成菌丝, 生物膜也不会形成。这些差异也显示在图 3中, 它描述了0小时、2 h、6 h 和 12 h 的检测中的4时点。
视频2显示了一个野生型生物膜和两个以前报告的生物膜缺陷突变体 (bcr1 δ/δ和efg1 δ/δ) 在蜘蛛介质中的发展。与基因野生型菌株相比, bcr1 δ/δ和efg1 δ/δ菌株都显示出严重的生物膜形成。对于efg1 δ/δ应变, 我们观察到黏附的细胞不形成菌丝, 由此产生的生物膜是严重缺陷。对于bcr1 δ/δ应变, 我们观察到, 不仅是在生物膜形成中有缺陷的bcr1 δ/δ应变, 而且它还显示了一个清晰的粘附缺陷, 其中的细胞在剪切流方向漂移, 因为它们不能坚持查看频道。这些差异也显示在图 4中, 它描述了在0小时、2 h、6 h 和 12 h 的检测中的4时间点。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56743/56743fig1.jpg"> 
图 1: 本实验中使用的微流体仪器.面板A显示了所用的微流体仪器, 面板B显示了接口板, 面板C显示了一个单一的查看通道 (阶段) 和三亚在实验中捕获的示意图轮廓, 并面板D显示了48井微流控板的示意图, 其中使用了出口和入口井的示意图和查看窗口。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56743/56743fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56743/56743fig2.jpg"> 
图 2: 选择图像 modulesoftware 和微流控板控制系统的屏幕截图.面板A显示控制模块, 其中包含微流控板的控件, 面板B显示了 "图像处理模块" 窗口, "移动阶段到绝对位置" (MAP) 模块, "多维获取" (MDA) 模块,和 "样本重装调整" 模块。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56743/56743fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56743/56743fig3.jpg"> 
图 3: 暴露于两性霉素 B 会导致生物膜形成缺陷.RPMI-1640 培养基中生物膜形成的时间依赖性可视化 (1 栏) 和 RPMI-1640 培养基在微流控系统的2° c 下, 在动态流 (0.5 达因/cm37) 下, 辅以16µg/mL 两性霉素 B (列 2)。代表 0 h (post-adherence 和初始洗涤), 2 h, 6 h, 和 12 h 图像 (顶部到底部) 显示的基因野生型应变 SN250 (列1和 2)。每个面板中的刻度线为20µm。在视频 1中提供了生物膜形成的相应时间推移视频。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56743/56743fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56743/56743fig4.jpg"> 
图 4: 删除BCR1或EFG1会导致生物膜形成缺陷。在微流控系统中, 在37° c 下的动态流 (0.5 达因/cm2) 下, 在 post-adherence 中的生物膜形成的时间依赖性可视化 (1-3 列)。代表性 0 h (post-adherence 和初步洗涤), 2 h, 6 h, 和 12 h 图像 (上到下) 显示为基因野生型应变 SN250 (列 1), bcr1δ/δ应变 (列 2), 和efg1 δ/δ应变 (列 3)。每个面板中的刻度线为20µm。在视频 2中提供了生物膜形成的相应时间推移视频。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56743/56743fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
视频 1:<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56743/Video-1-WT_and_WT_plus_AmpB.mp4">请单击此处下载此文件.</a>
视频 2:<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56743/Video-2-WT_bcr1_efg1_Mutants.mp4">请单击此处下载此文件.</a>

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 San Sebastian

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

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

生物工程

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

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 ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

医学

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

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 ...

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

白假丝酵母体内小鼠模型中的生物膜形成

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

全自动微流控装置在白色念珠菌生物膜形成中的可视化

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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

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

协议生物膜形成的流光在微流体器件与微柱

使用高通量的

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

用两相循环流系统实时成像和定量真菌生物膜的研究

澄清和成像念珠菌生物膜

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

一种基于四唑的可溶性还原测定，用于评估抗体对热带念珠菌生物膜的影响

白假丝酵母体内小鼠模型中的生物膜形成

全自动微流控装置在白色念珠菌生物膜形成中的可视化

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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

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