Name: bile salt-induced biofilm formation in enteric pathogens: techniques for identification and quantification
Uploaded: 2018-05-06T14:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification at JoVE.com

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</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</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</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F1635-500</td><td></td></tr><tr><td>Gluteraldehyde</td><td>Sigma-Aldrich</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</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&amp;deg;C Shaking Incubator</td><td>New Brunswick Scientific Excella E25</td><td></td><td></td></tr><tr><td>37&amp;deg;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

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

科研

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

分析结肠上皮细胞中 志贺氏菌 感染的方案

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

作者聚焦:了解和对抗 志贺氏菌 感染的进展

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

志贺氏菌上皮细胞感染分析

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

生物学

不动杆菌生物膜形成的评估和生物膜的表征

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

不动杆菌生物膜的定量、活力评估和可视化策略

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

摘要

探索更多视频

此视频中的章节

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

细胞和生物膜细胞外成分的定量并发

方法学研究

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

志贺氏菌上皮细胞感染分析

志贺氏菌上皮细胞感染分析

志贺氏菌上皮细胞感染分析

Measuring the Migration and Biofilm Formation of Various Bacteria

不动杆菌生物膜的定量、活力评估和可视化策略