我们感谢雷切尔 b. Chanin 和利亚诺斯的技术援助。我们感谢安东尼 t. Maurelli, 布莱恩 p. 赫尔利, 塞科法萨诺, 布雷特 e Swierczewski, 和鲍比 Cherayil 为本研究中使用的菌株。这项工作得到了国家过敏和传染性疾病研究所赠款 K22AI104755 (脑脊液) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

1. 试剂的制备
<ol><li>胆汁盐培养基: 制备含有0.4% 胆汁盐 (重量/容积) 的胰蛋白酶大豆汤 (并用重悬), 50 毫升蒸压的200毫克胆汁盐。使用0.22 µm 过滤器进行杀菌消毒。每周制作新鲜媒体。 注: 经常使用的胆汁盐是1:1 混合胆酸钠和脱氧钠, 从绵羊和牛胆囊分离。如前所示的4, 胆汁盐诱导的生物膜形成需要葡萄糖的存在。Luria-Bertani (磅) 汤增加了葡萄糖;因此, 在志贺菌和其他肠道病原体分析中, 足以诱发生物膜的形成。根据要分析的细菌, 可能需要不同的葡萄糖浓度或不同的糖要求。</li>
	<li>0.5% 瓦特/v 水晶紫在水: 溶化 2.5 g 水晶紫罗兰在500毫升蒸馏水。使用0.22 µm 过滤器进行杀菌消毒。</li>
	<li>刀豆蛋白 (错那县) 共轭异硫氰酸荧光素 (FITC): 重组 1x PBS 的股票。稀释10毫克集中的股票与 400 ul 1x PBS 的最终浓度25µg/毫升, 并保护免受光。</li>
	<li>pbs + 葡萄糖: 溶解0.2 克葡萄糖在10毫升 1x PBS (2% 瓦特/v 葡萄糖决赛)。使用0.22 µm 过滤器进行杀菌消毒。在使用的当天进行新鲜。</li>
	<li>pbs + 胆汁盐: 溶解40毫克在10毫升 1x PBS (0.4% 瓦特/v 胆汁盐决赛)。使用0.22 µm 过滤器进行杀菌消毒。在使用的当天进行新鲜。</li>
	<li>pbs + 葡萄糖和胆汁盐: 溶出40毫克胆汁盐和0.2 克葡萄糖10毫升 1x PBS (0.4% 瓦特/v 胆汁盐和2% 瓦特/v 葡萄糖决赛)。使用0.22 µm 过滤器进行杀菌消毒。在使用的当天进行新鲜。</li>
	<li>准备 LB 琼脂盘子。</li>
	<li>甲醛/戊二醛修复: 添加810µL 甲醛 (37% 库存溶液, 3% 最终浓度) 和125µL 戊二醛 (25% 股票溶液, 0.25% 最终浓度) 到14毫升 1x PBS。彻底混合, 并存储在4摄氏度。修复应该是冷的, 以便正确使用。 注意: 修复是有毒的, 需要危险的废物处理。</li>
	<li>Antifade mountant 解决方案: 使用含有 46-diamidino-2 苯基吲哚 (DAPI) 染色的 Antifade mountant 溶液, 抑制免疫荧光显微镜样品漂白, 同时荧光染色细菌的 DNA。</li>
</ol>2. 细菌的制备
<ol><li>在不育的试管中, 通过接种3毫升的一只独立的菌落, 在一夜之间培养细菌菌株。孵育在37°c 与震动在 225 rpm 为隔夜孵化 (16-24 h)。 注: 每2至4周, 应从冷冻库存中 restreaked 菌株, 并在盘子上保持不超过2周的时间。</li>
</ol>3. 固相粘附试验
注: 本试验采用96井板法对粘附细菌进行量化。细菌在平底板上静态生长。洗涤被执行去除不黏附的细菌和黏附的细菌被染用水晶紫罗兰色。水晶紫染色结合肽在细菌细胞壁和可可溶性使用乙醇。根据晶紫保留量确定粘附细菌的数量。
<ol><li>设置两个1.5 毫升管。标签用的是 a 和钠 + 胆汁盐 (BS)。</li>
	<li>在各自的管子上加1毫升的 a 和 b。</li>
	<li>接种试管与20µL 的隔夜文化 (在1:50 稀释)。</li>
	<li>在无菌、清、平底、组织培养处理的96井板上, 添加130µL/井适合控制介质, 以三口井作为空白控制。为每种媒体类型 (所用的) 测试设置三个控制井。</li>
	<li>添加130µL/井的接种培养成三口井, 并重复, 直到所有实验条件被镀三份。</li>
	<li>4-24 小时孵育在37摄氏度静态。</li>
	<li>使用板读取器, 记录 OD600。将控制井设置为 "空"。确认控制介质清晰, 无混浊的迹象。如果检测到浊度, 则丢弃实验。如果细菌菌株之间的生长速率有显著差异, 则可使用 OD600值对数据进行规范化。</li>
	<li>用真空线去除培养基, 轻轻倾斜板, 慢慢地将井下边缘的介质吸走。一定要收集所有的培养基, 而不会扰乱塑料表面的粘附细菌种群。如果 EPS 矩阵是在孵化时间产生的, 矩阵将被可视化为白色沉淀。请勿干扰 EPS 矩阵。</li>
	<li>用200µL 消毒 PBS 轻轻冲洗一次水井。使用真空线卸下 PBS 清洗。</li>
	<li>把盘子倒干。允许至少20分钟的干燥。 注: 由于生物膜必须在染色前彻底干燥, 所以该协议可以在这个步骤上暂停几个小时甚至一夜。添加的干燥时间不会改变染色过程, 而不完全干燥将影响定量的结果和重现性。</li>
	<li>添加150µL 0.5% 水晶紫对每个实验和控制好。</li>
	<li>室温下孵育5分钟 (RT)。</li>
	<li>用400µL 的蒸馏水冲洗一次水井。增加的体积有助于去除残余的晶体紫染色从两侧的水井。用真空线卸下清洗。</li>
	<li>用200µL 蒸馏水冲洗水井五次。用真空线卸下清洗。 注: 彻底清洗对量化很重要。当从蒸馏水中清除空白井, 不含任何残留的结晶紫染色时, 继续下一步。</li>
	<li>将盘子倒置, 使其免受光线的侵害。确保完全干燥的板材如上所述。</li>
	<li>用200µL 乙醇 Destain 井。在振动筛上孵出30分钟的盘子。为了避免蒸发, 特别是在更高的温度下, 执行这个步骤在4摄氏度。</li>
	<li>使用板读取器, 记录 OD540。 注: 晶体紫最大吸光度的波长接近 590 nm。文献报告一个范围从 od540 -od5954,13,14,15,16为水晶紫吸收;因此, 选择一个波长可利用的板阅读器。</li>
</ol>4. EPS 矩阵检测
注: 这些免费化验量化和可视化 EPS。在这两种情况下, EPS 被检测使用凝集素来绑定多糖。荧光共轭蛋白允许量化 (步骤 4.1) 或可视化 (步骤 4.2)。
<ol><li>EPS 的半定量检测<ol>
<li>设置两个1.5 毫升管。标签与公司或信托 + BS。</li>
		<li>在各自的管子上加1毫升的 a 和 b。</li>
		<li>接种试管与20µL 的隔夜文化 (1:50 稀释)。</li>
		<li>在无菌、黑色、平底、组织培养处理的96井板上, 添加130µL/井适合控制介质, 以三口井作为空白控制。为要测试的每种介质类型设置三控制井。添加130µL/井的接种培养到水井, 并重复, 直到所有实验条件被镀三份。</li>
		<li>4-24 小时孵育在37摄氏度静态。</li>
		<li>用吸管将培养基转化为清晰的96井板, 理想情况下是多通道吸管。小心收集所有的培养基, 而不破坏附着的人口和/或在塑料表面上的 EPS。留。</li>
		<li>在 1x PBS 中使用甲醛/戊二醛的200µL/井, 在 RT 上固定黑色板15分钟。</li>
		<li>当黏附的人口是固定的, 评估上清分数从步4.1.6。使用板读取器, 记录 OD600。将控制井设置为 "空"。确认控制介质清晰, 无混浊的迹象。如果检测到浊度, 则丢弃实验。<ol>
<li>或者, 整个化验可以在一个黑色清晰的底部板进行。在这种情况下, 可以记录 OD600值, 并且在继续执行步骤4.1.7 之前可以丢弃区域性媒体。可以使用 OD600值对数据进行规范化, 在这种情况下, 细菌菌株之间的增长率有显著差异。</li>
		</ol>
</li>
		<li>删除修复和处置危险废物。</li>
		<li>用200µL 的无菌 PBS 轻轻冲洗两次水井。使用真空线去除 PBS 清洗, 方法是轻轻地倾斜盘子, 慢慢地在井的下边缘吸洗。</li>
		<li>添加150µL/井25µg/毫升错那县 FITC, 并孵育15分钟在 RT。</li>
		<li>用200µL 的 PBS 轻轻地洗两次。</li>
		<li>在每个井中添加150µL 的 PBS。记录荧光在 488 nm。</li>
	</ol>
</li>
	<li>EPS 生产的共焦显微可视化及生物膜厚度的计算<ol>
<li>设置两个1.5 毫升管。标签与公司或信托 + BS。</li>
		<li>在各自的管子上加1毫升的 a 和 b。</li>
		<li>接种试管与20µL 的隔夜文化 (在1:50 稀释)。</li>
		<li>建立一个无菌的24井板与12毫米无菌圆形玻璃盖玻片。添加400µL/井适合控制介质到三井作为空白控制。为要测试的每种介质类型设置三控制井。</li>
		<li>将接种培养基的400µL, 再重复一次, 直到所有实验条件都被电镀。</li>
		<li>4-24 小时孵育在37摄氏度静态。</li>
		<li>可视化地确认在该条件下的生长, 生长和 EPS 沉淀 (白色), 在无菌控制井中没有生长和/或白色沉淀。删除上清液。</li>
		<li>在 1x PBS 中使用甲醛/戊二醛溶液的200µL/井, 在 RT 上固定15分钟。</li>
		<li>删除修复和处置危险废物。</li>
		<li>用200µL 的无菌 PBS 轻轻冲洗两次水井。使用真空线卸下 PBS 清洗。</li>
		<li>添加150µL/井25µg/毫升错那县 FITC 和孵化15分钟的 RT。</li>
		<li>轻轻洗两次与200µL/井的 PBS。</li>
		<li>安装与 DAPI antifade mountant 解决方案。 注意: 该解决方案是一个现成的, 甘油为基础的安装解决方案, 其中含有 DAPI 保存荧光标签, 同时染色的 DNA 在细菌细胞, 以可视化为 counterstain。在成像样品之前, 按照试剂盒的指示和建议进行潜伏期。在应用不含 DAPI 的 antifade mountant 解决方案之前, 可以执行 DAPI 染色过程。</li>
		<li>用共焦显微镜评价。在共聚焦显微镜下, 将激光器设置为 495 nm/519 nm FITC 可视化的激发/发射。设置第二个激光到 360 nm/460 nm 激发/发射可视化 DAPI。通过聚焦 DAPI 通道找到生物膜。使用 DAPI 和 FITC 通道来确定整个厚度并设置上、下成像边框。在将外围设置为 z 堆栈的顶部和底部后, 每0.25 µm 捕获图像, 记录全厚度的 z 堆栈图像。有关共聚焦显微学的更多信息, 请参阅围场et 等的出版物。17,18,19,20
</li>
		<li>重建 ImageJ (https://imagej.nih.gov/ij/) 中的3D 图像, 以可视化全生物膜形成, 并计算生物膜厚度。为福氏胆汁盐诱导的生物膜获得的平均厚度为14µm4。</li>
	</ol>
</li>
</ol>5. 色散测定法
注: 在本试验中, 检测到生物膜通过细菌分散而拆卸。在这里, 成熟的生物膜建立和随后 (通常第二天), 媒体被替换为 pbs 或补充 pbs。然后对上清部分进行评估, 以定量与生物膜分离的细菌数量。
<ol><li>设置两个1.5 毫升管。标签与公司或信托 + BS。</li>
	<li>在各自的管子上加1毫升的 a 和 b。</li>
	<li>接种试管与20µL 的隔夜文化 (在1:50 稀释)。</li>
	<li>在无菌、清、平底、组织培养处理的96井板上, 添加130µL/井适合控制介质, 以三口井作为空白控制。为要测试的每个介质类型设置三个控制井。</li>
	<li>添加130µL/井的接种培养到三口井, 并重复, 直到所有实验条件被镀三份。</li>
	<li>在37摄氏度静态地孵化 4-24 小时的板材。</li>
	<li>在继续之前, 温暖 pbs, pbs + 葡萄糖, pbs + 胆汁盐和 pbs + 胆汁盐 + 葡萄糖到37°c。每天新鲜地准备这些试剂。</li>
	<li>使用板读取器, 记录 OD600。将控制井设置为 "空"。确认介质清晰, 无浑浊的迹象。如果检测到浊度, 则丢弃实验。如果细菌菌株之间的生长速率有显著差异, 则可使用 OD600值对数据进行规范化。</li>
	<li>使用真空线去除培养基。一定要收集所有的培养基, 而不破坏塑料表面的附着填充。</li>
	<li>用200µL 的无菌 PBS 轻轻冲洗两次水井。使用真空线卸下 PBS 清洗。</li>
	<li>将以下130µL/井的洗涤更换为三口井: pbs、pbs + 葡萄糖、pbs + 胆汁盐、pbs + 胆汁盐 + 葡萄糖。这些试剂必须预先预热到37摄氏度。</li>
	<li>在37摄氏度孵育30分钟的盘子。</li>
	<li>小心地从37°c 的孵化器中取出盘子。把上清液转移到一个新鲜的, 不育的96井板。</li>
	<li>使用96井板或稀释块, 准备10倍 (1:10) 连续稀释的上清成无菌 PBS。 注: 稀释系列应从未稀释到 10-6不等, 具体取决于要测试的细菌应变。可以进行试验以确定最佳稀释范围。</li>
	<li>斑点板5µL 每稀释到 LB 琼脂使用多通道吸管。孵化在37°c 过夜。计数殖民地在第二天和解释稀释因素, 当确定恢复殖民地形成单位 (CFU)。要计算相对于 1x pbs 控件的百分比色散 (设置为 100%), 请从 1x pbs 控制示例中的恢复 CFU 划分每个处理条件的恢复 CFU。 注意: 常规培养基板对于细菌性菌株也可以使用。例如,志贺菌可以在刚果红板上电镀。确保板材干燥, 以适当的电镀工艺。</li>
</ol>

作者没有披露。

由于生物膜的动态性质以及菌株、材料、实验室和化验结果之间的变异性, 生物膜形成的分析具有挑战性。本文提出了在胆盐暴露后肠道病原体中生物膜形成的几种策略, 并对其进行了实验研究, 以促进重现性。还有其他的考虑, 以确保重现性。首先, 我们建议执行至少三个独立的实验, 每个与技术 triplicates, 以确认观察和统计意义, 由于可能发生的变化。第二, 使用积极和消极的控制是至关重要的, ?...

生物膜形成是恶劣环境条件下引起的一种重要的细菌生存策略。暴露在杀菌化合物如抗生素或营养或氧的变化, 导致细菌的压力状态, 可以通过生物膜形成缓解。生物膜的特点是细菌附着于表面或其他细菌, 并伴随着一个 EPS 矩阵的分泌, 主要由多糖1,2,3。生物膜形成是一个动态过程, 其中一连串事件的高潮形成一个成熟的黏附细菌社区1,2,3。细菌产生 adhesins, 以方便早期附着, 而转移黏附基因表达谱, 以加强附着在生物膜成熟。同时, EPS 生产发生在基质中覆盖细菌群落, 以保护细胞不受初始压力源的侵害。生物膜内含有的细菌生长缓慢;因此, 大多数抗生素都是无效的。此外, 缓慢的增长节省能源, 直到条件改变有利于细菌生长1,2,3。在严酷的条件已经过去了, 细菌分散生物膜和恢复浮游生活方式1,2,3。传统上, 生物膜被观察在表面上, 并代表了持续的临床挑战, 因为感染水库存在于导管和居住设备1,2,3。
最近对几种肠道病原体的生物膜形成进行了描述;感染小肠或结肠的细菌4。对于志贺菌物种,感染发生在人类结肠经胃肠道的大部分转运后。在通过小肠的通道中,志贺菌暴露于胆汁中;一种脂质降解的洗涤剂分泌到肠道, 以促进脂肪的消化, 同时杀死大多数细菌5。肠道病原体具有抵御胆汁6杀菌效果的独特能力。我们最近的分析利用了体内类似的葡萄糖和胆汁盐的组合, 以显示在S. 福氏以及其他种类的志贺菌,致病性大肠杆菌, 和 沙门氏菌4。此前,沙门氏菌 enterica钩伤寒被证明形成胆汁诱导生物膜, 由于在慢性感染期间胆囊的独特殖民化7,8,9, 10. 此外, 以前的研究与弧菌11和弯曲的12表明生物膜形成的反应胆汁。因此, 分析将胆汁诱导的生物膜形成观察扩展到其他病原体, 有助于建立一种保守的肠道病原体对胆汁的反应。不同于慢性生物膜, 其中细菌基因转录是有限的, 细胞衰老可能发生1,2,3, 我们建议, 肠道胆汁诱导生物膜是更短暂的性质。这种瞬态, 剧毒生物膜是虚幻快速拆卸 (如在弥散检测中看到) 和增强的毒力基因表达观察在生物膜的人口4,6。 
由于生物膜的形成是一个多方面的, 动态的过程和使用胆汁盐作为一个起始因素最近才被描述的大多数肠道病原体, 使用的工具和技术是独特的和创造性的应用传统方法。因此, 这里提出了三免费战略, 以量化的几个重要特征的胆汁盐诱导生物膜形成, 包括细菌黏附, 生产的 EPS 基质, 和分散的活菌从生物膜。这些技术主要用于研究与志贺菌;因此, 评估其他肠道病原体可能需要优化。然而, 来自所有三种化验的阳性数据都支持生物膜的鉴定, 并建立了胆汁盐诱导生物膜形成的可再生的协议。

<table>
	<tbody>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma</td>
			<td>C6158-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin-A FITC</td>
			<td>Sigma</td>
			<td>C7642-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bile Salts</td>
			<td>Sigma</td>
			<td>B8756-100G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma</td>
			<td>L7533-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL culture tubes, 17 x 100 mm, plastic, sterile</td>
			<td>Fisher</td>
			<td>14-959-11B</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield hard-set antifade with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F1635-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 96-well plates (clear)</td>
			<td>TPP</td>
			<td>92696</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 96-well plates (black)</td>
			<td>Greiner Bio-One&#160;</td>
			<td>655076</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 24-well plates (clear)</td>
			<td>TPP</td>
			<td>92424</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 12mm, round</td>
			<td>Fisher</td>
			<td>08-774-383</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate reader</td>
			<td>Spectramax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flourescent plate reader</td>
			<td>Biotek Synergy 2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal or Fluorescent Microscope</td>
			<td>Nikon A1 confocal microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Shaking Incubator</td>
			<td>New Brunswick Scientific Excella E25</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Plate Incubator</td>
			<td>Thermolyne Series 5000</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bile salt-induced biofilm formation in enteric pathogens: techniques for identification and quantification

生物膜形成是一种动态的、多级的过程, 在恶劣的环境条件下或受压力的时代发生在细菌中。对于肠道病原体, 在胃肠道转运和胆汁暴露过程中, 引起了明显的应力反应, 这是人类消化的正常成分。为了克服胆汁的杀菌作用, 许多肠道病原体形成生物膜假说, 允许在小肠中转时存活。在这里, 我们提出了定义生物膜形成的方法, 通过固相粘附测定, 以及胞外高分子物质 (EPS) 矩阵检测和可视化。此外, 还提出了生物膜色散评估法, 模拟了在感染过程中引发细菌释放的事件的分析。在高通量的96井板粘附试验中, 用结晶紫染色法检测粘附细菌。eps 生产评价由两种方法确定, 即 eps 基质的显微染色和荧光共轭多糖结合凝集素的半定量分析。最后通过菌落计数和电镀来测量生物膜的分散性。来自多种化验的阳性数据支持生物膜的特性, 可用于鉴别其他细菌菌株中的胆汁盐诱导生物膜的形成。

在图 1中, 在含有胆汁盐的培养基的生长过程中, 大多数六种肠道病原体均诱导生物膜形成。在几乎所有的菌株中都能观察到胆汁盐暴露后黏附细菌的显著增加。异常是 enteroaggregative大肠杆菌(EAEC);但是, 请注意对Δ aaf突变4的诱导观察。结果表明, 在没有聚集黏附放线菌菌毛 I (AAF)21 的情况下, EAEC ...

Watch this Scientific Journal Video about Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification at JoVE.com

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

该协议使读者能够分析胆汁盐诱导的肠道致病菌的生物膜形成, 采用多层面的方法来通过评估附着体、胞外高分子物质基质形成来捕获细菌生物膜的动态特性,和分散。

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, ...

bile-salt-induced-biofilm-formation-enteric-pathogens-techniques-for

Research

JoVE Journal

Immunology and Infection

12.3K 次观看.  Massachusetts General Hospital. 该协议使读者能够分析胆汁盐诱导的肠道致病菌的生物膜形成, 采用多层面的方法来通过评估附着体、胞外高分子物质基质形成来捕获细菌生物膜的动态特性,和分散。

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

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 ...

科研

免疫与感染

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. ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially 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 ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

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. ...

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. ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...