Name: utilization of capsules for negative staining of viral samples within biocontainment
Uploaded: 2017-07-19T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment at JoVE.com

我们要感谢John Carra博士和Rowena Schokman博士提供纯化的埃博拉纳维VLP，Rajini Mudhasani博士提供基孔肯雅病毒，以及Charles（Jason）Shoemaker博士提供表达埃博拉病毒糖蛋白的小鼠白血病VLP。我们还要感谢MAJ Carl Soffler为夏季实习计划（SIP）和科学与工程学徒计划（SEAP）和凯瑟琳·威廉森博士提供实验室安全培训。

1.在使用病毒样本之前，在BSL-2环境中进行实验准备<ol><li>准备或购买Formvar和碳涂层的TEM铜网格，通常为200-400目。 </li><li> 将涂层的TEM网格插入胶囊。 <ol><li>使用放大镜可以使此过程容易执行。一个或两个网格可以插入每个胶囊。如果需要，可以购买预装胶囊以消除此步骤。 </li></ol></li><li>将带有TEM涂层网格的胶囊与其他用品和试剂一起转移到生物体内，其中病毒将被染色。 </li></ol> 2.使用水性戊二醛和1％四氧化锇蒸气失活的生物达标中的阴性染色的胶囊方法<ol><li>在生物驯化BSC内，将40μL病毒悬浮液吸入附着在移液管上的胶囊。 注意:移液器仍然连接到e胶囊，直到过程完成。病毒准备是根据我们以前的出版物14 。 </li><li>将移液管放在其侧面10分钟，网格水平定向。这是为了促进病毒颗粒在涂层网格上的均匀分布。 </li><li> 在Biocontainment BSC内的胶囊内灭活病毒。 <ol><li>拿起移液管并按压柱塞将病毒溶液分配到废物容器中。 </li><li>将40μL2％戊二醛固定液吸入胶囊。 注意:戊二醛是一种危险化学品，需要适当的保护。戊二醛可以在正常BSC中短暂使用，但延长的开放试剂需要在管道BSC或化学通风橱中工作。 </li><li>将移液器放在其侧面20分钟。这是为了确保样品固定好。 </li><li>推出固定剂并将40μL的dI水吸入胶囊中灰掉了固定剂。重复此洗涤步骤总共3次漂洗循环。 </li></ol></li><li>吸入40μL的1％乙酸铀酰（UA）或1％磷酸钨酸（PTA）到胶囊中，并使其静置30秒。 注意:基于病毒样本，染色时间可能在10秒至1分钟之间变化。 注意:UA是α发射体，是一种累积的毒素。处理适当的保护。 </li><li>从移液管中取出胶囊，然后通过在网格保留在胶囊内的同时将一块滤纸接触网格边缘，来吸干网格。 </li><li> 氧化锇蒸汽灭活程序。 <ol><li>将胶囊盖子打开放入含有浸泡在1％四氧化锇溶液中的滤纸的50mL离心管中。 注意:四氧化锇在极低的蒸气压下毒性极大。必须在管道BSC或化学通风橱中使用。处理适当的保护。发布警告在工作区域的信息。 </li><li>将50 mL离心管密封1 h，以使四氧化锇蒸气完全渗透。然后，随后对BSL-2 EM设施进行净化和转移出生物处理。 </li></ol></li><li> 从胶囊中取出EM网格。 <ol><li>在BSL-2 EM设备中，从离心管中取出胶囊并将其放在移液管上。 </li><li>将40μL的dI水吸入胶囊中，并将水分配到废物容器中三次。 </li><li>从移液器上取下胶囊，用滤纸吸干网格以接触网格边缘。 </li><li>空气干燥后，存放电网用于随后的TEM成像。 </li></ol></li></ol> 3.用2％戊二醛生物达标灭活的胶囊方法，随后在BSL-2实验室中进行阴性染色<ol><li> 病毒灭活程序。 <ol><li&gt;在生物驯化BSC内，将病毒悬浮液与相同体积的4％戊二醛混合，达到终浓度为2％的戊二醛。 注意:戊二醛是一种危险化学品，需要适当的保护。戊二醛可以在正常BSC中短暂使用，但延长的开放试剂需要在管道BSC或化学通风橱中工作。 </li><li>在包装，去污和将其转移到BSL-2 EM设施之前，先将固定剂灭活至少24小时。 </li></ol></li><li>在BSL-2 EM设备中，将病毒和固定剂混合物吸入胶囊，包含两个连接到移液管上的TEM网格。 </li><li>将移液器水平放置10分钟，网格水平定向。 注意:这是为了促进病毒颗粒均匀分布到TEM网格上。 </li><li>拿起移液管并按压柱塞将病毒排出到废物容器。吸出40μL的dI水进入胶囊并将其排入废物容器中3次漂洗循环。 </li><li>将40μL的1％UA或1％PTA吸入胶囊中30 s。 注意:基于病毒样本，染色时间从10秒变化到1分钟。 注意:UA是α发射体，是一种累积的毒素。处理适当的保护。 </li><li>从移液器上取下胶囊，并通过将网格边缘接触到一张滤纸来吸干网格。空气干燥网格并存放它们用于随后的TEM成像。 </li></ol>

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文章中提出的意见，解释，结论和建议是作者的意见，并不一定得到美国陆军或国防部的认可。

负染是用于评估和筛选病毒，蛋白质复合物和纳米颗粒的有价值的TEM技术。通过手动将网格从试剂转移到阴性污渍，这些样品的液滴制备已经是半个多世纪以来的典型方案。这是一个简单的过程，但需要通过培训获得成功完成的专业知识。优秀的阴性染色仍被认为是最先进的技术，并且在许多TEM实验室中非常期望。胶囊方法相对于手动液滴方法具有几个明显的优点，特别是在生物实验室中。首先，手动液滴方法需要大量的培训和经验才可以成功执行，而使用简单的胶囊方法可以通过简单的移液技术进入入门级技术人员。手动液滴法最大的挑战是使用BSL-3和BSL-4 PPE中的镊子成功处理TEM网格o大大降低了触觉和灵巧度。脆性涂层TEM网格容易被镊子损伤或刺穿。涂层TEM网格的损伤通常不会透露，直到用TEM观察。胶囊的第二个优点是TEM格栅只有当它们插入胶囊中并且当它们被移除以插入电子显微镜时才被直接处理。无论是在生物实验室还是BSL-2 EM设施工作，都不会直接进行电网处理。胶囊方法的第三个优点是网格的两侧被病毒和污渍覆盖。当样品浓度低时，这是有用的;然而，如果浓度高，可能导致过多的样品，或者在使用前导致样品稀释。 使用手动液滴技术的阴性染色多个网格是耗时的，因为每个样品需要单独制备和染色。这导致了困难ning一致，可重复染色。此外，跟踪许多个别网格总是一个挑战。每个胶囊容纳两个网格，并且多个胶囊可以连接到多通道移液管，从而简化了该过程。因此，胶囊确保在一致的样品条件下和同时使用非常相似的方法对网格进行染色。胶囊可以标记以帮助组织，从而减少网格混合或错误识别的可能性。使用手动液滴方法时面临的另一个常见问题是网格污染。当网格留在露天太久或掉落时，可能会发生这种情况。胶囊可保护TEM格栅免受露天影响，并将其牢牢固定，使其不会掉落。当制备和阴性染色时，胶囊提供更大的实验控制和重复性，因为胶囊是更受控制的环境。 胶囊可以预购，如果de首创。将网格手动装入胶囊需要一些做法才能确保网格不被损坏。胶囊方法还需要比手动液滴方法稍微更多的样品和污点。与手动液滴法相比，胶囊方法需要至少40μL的样品，可以用少至8μL进行。 病毒灭活是BSL-3和BSL-4阴性染色的重要组成部分，因为它允许灭活的病毒从生物实验室中取出，用于EM设施中成像。使用固定剂灭活具有修复病毒的附加益处，防止降解。灭活病毒样本有两种不同的方法，这两种方法都在本研究中进行了研究。首先将病毒粘附到涂覆的TEM网格上，将病毒在2％戊二醛溶液中固定20分钟，然后将栅格暴露于四氧化锇蒸气1小时（ 图1C ）。这个灭活离子法是有利的，因为通过使样品在1小时20分钟后能够从生物实验室中取出来节省时间。它也可以防止病毒稀释到涂覆的TEM网格之前，因此可能需要怀疑接近TEM检测限的样品。然而，1％的四氧化锇蒸气会降低TEM图像的质量，因为锇可以进一步污染样品（ 图3 ）。以前的报告显示，四氧化锇蒸气暴露5分钟，当阴性染色时可以获得很好的效果;然而，绝对病毒灭活所需的更长的暴露产生正和负染色17的混合物。第二种灭活方法在2％戊二醛溶液中至少需要24小时，不需要四氧化锇蒸气处理（ 图1D ）。第二种方法需要更长时间才能完成，因为样品没有从生物修复实验室中移除但是可以有利于在BSL-2环境中进行阴性染色，而不需要有害的四氧化锇。我们的研究结果表明，当用四氧化锇蒸气处理样品时，24小时的戊二醛灭活产生更高质量的TEM图像（ 图3 ）。 在本实验中使用了两种阴性污染物:UA和PTA。两种污渍均用作1％溶液。 UA，铀的醋酸盐，作为一个负面的污点，因为浓铀原子散射电子5 。 PTA，一种杂多酸，类似地作为由于致密的钨原子而产生的负污染5 。 PTA有时比UA优选，因为它毒性低得多，如果吸入或接触只有轻微的刺激物。当阴性染色未固定样品时，必须考虑UA的较低pH，因为它可能导致损伤o样品。在该实验中使用的病毒需要固定以进行灭活，因此UA和PTA都实现了类似的结果，并且对于两种染色都没有检测到明显的优点。两种污渍易于使用，并且它们都产生类似质量的TEM图像（ 图4 ）。 总之，胶囊方法在生物环境中更容易使用，可用作手动液滴法的替代方法。使用胶囊可以减轻样品操作缺陷，并产生良好质量的TEM图像。使用胶囊方法产生的图像与使用手动液滴方法产生的图像相当。用四氧化锇进行短病毒灭活可提高速度，但只有降低图像质量可接受才能使用，否则疑似稀释病毒会使其低于手术的TEM检测限。 UA和PTA都与本研究中使用的固定病毒样品提供了类似的结果;浩wever，当染色未固定病毒时，结果可能不同。

透射电子显微镜（TEM）可以观察生物样本太小的形态和超微结构的有效工具与传统的光学显微镜1，2，3，4中看到的。 TEM通过非常薄的样品射出电子，产生更高分辨率的图像，因为电子的波长比光短得多。弯曲或阻挡电子的样品的区域看起来很暗，而电子透明的区域看起来是白色的。 缺少电子致密物质使得病毒难以在TEM下观察，因为它们不能散射电子。阴性染色是用于通过TEM创建对比度和观察病毒的最常用方法。 Brenner和Horne在1959年提出了第一个负染色程序，基于Hall（1955）和Huxley（1957）观察到的实验当浸入电子密度物质5时，反向对比生物结构的外观。阴离子染色过程在过去半个世纪几乎没有改变。阴性染色涉及在TEM网格上简单地将重金属盐溶液施用于样品，以试图用致密材料包围病毒而不渗透病毒6 。这产生了一个黑色的边框，并显示了颗粒的形状5 。本研究使用两种阴性染色试剂，乙酸双氧铀（UA）和磷钨酸钾（PTA）。这两个污渍通常用于小的生物样品，如病毒，蛋白质复合物和纳米颗粒7，8，9负染色。 常规的阴性染色技术是手动液滴阴性染色技术nique 7 。该方法需要用镊子精确处理小而脆弱的TEM网格，以应用少量病毒样品，染色和漂洗。典型的制备方法包括将一滴样品悬浮液施加到膜涂布的TEM网格的表面上（ 图1A ）。在将样品连接到膜表面之后，漂洗网格以除去非粘附病毒，并且根据样品的类型用UA或PTA染色数秒至一分钟。通过将一块滤纸接触到网格的边缘，将过量的液体从网格中除去。 手动液滴方法要求每个格子单独制作。如果不小心处理，涂层的TEM网格容易被刺穿，弯曲或污染。处理多个样品可能导致跟踪网格的困难，并确保每个样品的染色一致。本手册染色程序更多d由于这些环境所需的个人防护装备（PPE），在生物安全水平（BSL）-3和-4生物实验室进行培训时很困难。 PPE是繁琐的，与常规实验室相比，生物环境的变化更为动荡。在BSL-3生物实验室工作的人员必须佩戴2双手套，并在生物安全柜（BSC）工作。这双手套可以降低触感，并限制电动机运动。保护用户并有助于防止样品污染的BSC的气流可导致样品和污渍太快地干燥，从而影响染色质量。 BSC中强烈的湍流气流也可以快速吹走不太牢固的电网。在BSL-4生物实验室中，还有其他安全要求。人员需要穿正压服，这进一步限制身体运动和清晰看见和操纵的能力网格。在BSL-4工作的技术人员还穿着至少2双手套，外套是厚厚的手套，大大降低了灵巧和触感。最后，用于处理TEM网格的镊子是锋利的，因此由于其穿刺手套的能力而对技术人员造成风险。使用含有网格的胶囊，镊子不是必需的，因此提供了一种安全的无镊子的替代方案，用于操纵生物体的网格。最后，胶囊还提供了在加工过程中储存网格的有效方式，锇蒸气去污和储存期间;从而保持网格的组织和安全免受损害。 在本报告中，我们介绍负染色TEM网格在生物防护实验室利用mPrep / g的胶囊，网格处理和染色10，11，12基于胶囊的装置的新方法。胶囊accom改变两个TEM网格，最大限度地减少直接处理，并减少电网损坏的可能性。胶囊以与移液管尖端相同的方式直接附接到单通道或多通道移液管，允许将各种液体施加到包含在其中的网格上。这使得能够同时准备具有重复格栅的多个样品（ 图1B ）。用胶囊阴性染色，将病毒样品吸入胶囊中并保持10分钟，使病毒吸附在网格表面上。随后用去离子水（dI）洗涤带有吸附病毒的网格，并用UA或PTA染色数秒至1分钟。该方法使用与手动液滴法相同的方案步骤和试剂;不同之处在于，所有的工作都发生在胶囊内，没有物理处理网格。 （ 图1C ，1D ）。 本研究的目的是评估胶囊一种用于生物环境中病毒样品阴性染色的新方法。本研究还检测了由两种不同病毒灭活程序产生的TEM图像的质量:1）快速灭活，1％四氧化锇蒸气，2）用2％戊二醛灭活24小时。这些都是使用胶囊进行的。最后，我们评估了两种常用的阴性污染物UA和PTA，用于胶囊。 13

<table><tbody><tr><td>Formvar/carbon coated TEM grids</td><td>SPI</td><td>3420C-MB</td><td>200 mesh Cu Pk/100</td></tr><tr><td>mPrep/g capsules</td><td>EMS</td><td>85010-01</td><td>box</td></tr><tr><td>mPrep/f couplers</td><td>EMS</td><td>85010-11</td><td>standard 16/Pk</td></tr><tr><td>glutaraldehdyde</td><td>EMS</td><td>16320</td><td>50% solution, EM grade</td></tr><tr><td>Osmium Tetroxide</td><td>EMS</td><td>19190</td><td>4% aqueous solution</td></tr><tr><td>Uranyl Acetate</td><td>EMS</td><td>22400</td><td>powder</td></tr><tr><td>Potassium phosphotungstic acid</td><td>EMS</td><td>19500</td><td>powder</td></tr><tr><td>filter paper</td><td>Whatman</td><td>1450-090</td><td>size 50</td></tr><tr><td>Tranmission Electron Microscope</td><td>JEOL</td><td>JEM-1011</td><td>TEM</td></tr></tbody></table>

utilization of capsules for negative staining of viral samples within biocontainment

透射电子显微镜（TEM）用于观察病毒和其他微生物病原体超微结构的纳米分辨率。大多数生物材料不含能够散射电子的致密元件以产生图像;因此，需要在样品周围放置稠密的重金属盐的负污渍。为了在TEM下观察悬浮液中的病毒，它们必须应用于涂覆有仅纳米厚的透明表面的小网格。由于其尺寸小巧易碎，这些网格难以处理，容易被气流移动。薄表面容易损坏，使样品难以或不可能成像。传染病毒必须在生物安全柜（BSC）中进行处理，有些需要生物化学实验室环境。生物安全水平（BSL）-3和-4中的染色病毒特别具有挑战性，因为这些环境更为动荡，技术人员需要to佩戴个人防护装备（PPE），降低灵活性。 在这项研究中，我们评估了一种新的装置，以帮助负染色的病毒生物获得。该设备是一种胶囊，可用作专用移液器吸头。一旦将网格装入胶囊中，用户只需将试剂吸入胶囊中，将病毒和污渍传递到封装的网格上，从而消除用户对网格的处理。虽然这种技术专门设计用于BSL-3或-4生物测定，但它可以通过实现病毒容易的阴性染色来缓解任何实验室环境中的样品制备。同样的方法也可用于制备纳米颗粒，大分子和类似标本的负染色TEM样品。

胶囊方法产生优质的阴性染色用于TEM成像: 首先，我们通过使用手动液滴法和用于阴性染色扎伊尔埃博拉病毒的胶囊方法来评估产生的图像的质量。伏博病毒属于Filoviridae家族，还有Marburg病毒。埃博拉病毒的直径通常为80至100纳米，长度可超过1000纳米。埃博病毒必须在BSL-4生物环境中处理。 图2示出了使用手动液滴法和用于阴性染色的胶囊方法产生的图像。 图2A （手动液滴法）和图2B （胶囊法）显示在病毒粒子中心具有明确定义细胞核结构的微卫星病毒样品，可见的埃博拉病毒糖蛋白在表面上。因此，胶囊和液滴方法都具有产生类似质量的TEM图像的能力。 使用胶囊法，用戊二醛水溶液和1％四氧化锇蒸气快速灭活，并用2％含水戊二醛进行24小时灭活，比较和评估EM图像质量: 我们评估了使用基孔肯雅病毒从两种不同的样品灭活方法产生的TEM图像的质量。基孔肯雅病毒是披膜 病毒科中的甲病毒属的成员。它是直径为60-70nm的球形。病毒粒子含有丰富的糖蛋白的信封，其在病毒表面上形成三聚体峰。基孔肯雅病毒必须在BSL-3中处理。使用2％戊二醛20分钟后快速灭活，然后1小时暴露于1％四氧化锇蒸气，整个阴离子在染色过程中发生在BSC内的胶囊中的生物实验室（ 图1C ）。然而，当使用2％戊二醛24小时来灭活病毒时，灭活发生在生物环境中，但是在BSL-2实验室中使用胶囊法进行1％UA阴性染色程序（ 图1D ）。这些灭活程序不产生相同质量的图像（ 图3 ）。从图3可以看出，戊二醛中没有四氧化锇存在下的固定（ 图3A ，3B ）显示出比用戊二醛和四氧化锇制备的样品更多的超微结构细节（ 图3C ，3D ）。 胶囊法使用醋酸铀（UA）和磷酸盐戊酸（PTA）作为醛固定样品的阴性污渍。 图4中示出使用胶囊方法的UA和PTA阴性染色的实例在醛固定病毒样颗粒（VLP）上。 VLP是组装成病毒样结构的蛋白质，但不含有任何病毒遗传物质。它们通常用于疫苗开发和基础病毒研究。负染色是评估VLP装配和形态的有价值的工具。我们使用UA和PTA用胶囊法对VLP进行阴性染色。两种污渍都显示出高质量的结果，糖蛋白可见和明确界定埃博拉纳维VLP 15 （ 图4A ，4B ）和鼠白血病VLPs 16 （ 图4C ，4D ）。 图1:负染方法概述。 （ A ）通过将TEM网格从液滴逐步移动到样品液滴，冲洗和污渍来进行手动液滴法。 （ B ）用网格设置胶囊方法并应用样品悬浮液。 （ C ）使用胶囊方法在1％四氧化锇蒸气短时间灭活的典型程序。 （ D ）使用胶囊法在2％戊二醛长期灭活的生物培养中的推荐方法。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56122/56122fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2:1％PTA阴性埃博病毒颗粒的比较。 （ A ）用手动液滴法进行阴性染色。 （ B ）使用胶囊法进行阴性染色。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56122/56122fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/56122/56122fig3.jpg" /> 图3:使用不同灭活程序的基孔肯雅病毒胶囊方法1％UA阴性染色。 （ A ， B ）仅用2％戊二醛灭活至少24小时。 （ C ，D）用2％戊二醛和1％四氧化锇蒸气快速灭活。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56122/56122fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56122/56122fig4.jpg" /> 图4:使用胶囊方法的磷钨酸（PTA）和乙酸铀酯（UA）阴性病毒样颗粒（VLP）的实例。 （ A ）1％PTA染色的埃博拉纳维VLP的低放大概率。 （ B ）显示PTA染色的埃博拉纳米VLP的结构细节的高倍放射TEM图像。 （ C ）1％UA染色的小鼠白血病VLP的低放大概率。 （ D ）高倍放大TEM图像显示结构detUA染色的小鼠白血病VLP在其表面具有禽流感病毒糖蛋白。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56122/56122fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

该协议为负染色病毒样本提供了指导，可以很容易地用于BSL-2，-3或-4实验室。它包括使用创新的加工胶囊，其保护透射电子显微镜网格并且在生物达到的更湍流环境中为使用者提供更容易的处理。

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

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JoVE Journal

Immunology and Infection

10.1K 次观看.  United States Army Medical Research Institute of Infectious Diseases (USAMRIID). 该协议为负染色病毒样本提供了指导，可以很容易地用于BSL-2，-3或-4实验室。它包括使用创新的加工胶囊，其保护透射电子显微镜网格并且在生物达到的更湍流环境中为使用者提供更容易的处理。 

视频: Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

科研

免疫与感染

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

用不连续密度梯度用胶囊分离细菌

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

遗传学

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

生物化学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

自动测量隐球菌种多糖胶囊和细胞体

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

尺寸事项:新生隐球菌中胶囊直径的测定

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

生物学

Microscopy and Staining: Gram, Capsule, and Endospore Staining

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1 
1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
Bacteria are diverse microorganisms found nearly everywhere on Earth. Many properties help distinguish them from each other, including but not limited to Gram-staining type, shape and arrangement, production of capsule, and formation of spores. To observe these properties, one can use light microscopy; however, some bacterial characteristics (for example size, lack of coloration, and refractive properties) make it hard to distinguish bacteria solely with a light microscope (1, 2). Staining bacteria is necessary when distinguishing bacterial types with light microscopy. The two main types of light microscopes are simple and compound. The main difference between them is the number of lenses used to magnify the object. Simple microscopes (for example a magnifying glass) have only one lens to magnify an object, while compound microscopes have several lenses to enhance magnification (Figure 1). Compound microscopes have an objective lens close to the object which collects light to create an image of the object. This is then magnified by the eyepiece (ocular lens) which enlarges the image. Combining the objective lens and eyepiece allows for higher magnification than using a single lens alone. Typically, compound microscopes have multiple objective lenses of varying powers to allow for different magnification (1, 2). Here, we will discuss visualizing bacteria with Gram stains, Capsule stains, and Endospore stains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10513/10513fig1.jpg" /> 
Figure 1: A typical compound microscope. The most important parts of the microscope are labeled.
The Gram stain, developed in 1884 by the Danish bacteriologist Hans Christian Gram (1), differentiates bacteria based on the composition of the cell wall (1, 2, 3, 4). Briefly, a bacterial smear is placed on a microscope slide and then heat-fixed to adhere the cells to the slide and make them more readily accepting of stains (1). The heat-fixed sample is stained with Crystal Violet, turning the cells purple. The slide is flushed with an Iodine solution, which fixes the Crystal Violet to the cell wall, followed by a decolorizer (an alcohol) to wash away any non-fixed Crystal Violet. In the final step, a counterstain, Safranin, is added to color cells red (Figure 2). Gram-positive bacteria stain purple due to the thick peptidoglycan layer which is not easily penetrated by the decolorizer; Gram-negative bacteria, with their thinner peptidoglycan layer and higher lipid content, destain with the decolorizer and are counterstained red when Safranin is added (Figure 3). Gram staining is used to differentiate cells into two types (Gram-positive and Gram-negative) and is also useful to distinguish cell shape (spheres or cocci, rods, curved rods, and spirals) and arrangement (single cells, pairs, chains, groups, and clusters) (1, 3).

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10513/10513fig2.jpg" /> 
Figure 2: Schematic of the Gram Staining Protocol. The left column shows how Gram-negative bacteria react at each step of the protocol. The right column shows how Gram-positive bacteria react. Also, shown are two typical bacterial cell shapes: the bacilli (or rods) and the cocci (or spheres).

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10513/10513fig3.jpg" /> 
Figure 3: Gram Staining Results. A Gram stain of a mixture of Staphylococcus aureus (Gram-positive purple cocci) and Escherichia coli (Gram-negative red rods).
Some bacteria produce an extracellular viscous outer layer called a capsule (3, 5). Capsules are protective structures with various functions, including but not limited to adherence to surfaces and other bacteria, protection from desiccation, and protection from phagocytosis. Capsules are typically composed of polysaccharides containing more than 95% water, but some may contain polyalcohols and polyamines (5). Due to their mostly non-ionic composition and tendency to repel stains, simple staining methods do not work with capsule; instead, capsule staining uses a negative staining technique which stains the cells and the background, leaving the capsule as a clear halo around the cells (1, 3) (Figure 4). Capsule staining involves smearing a bacterial sample into an acidic stain on a microscope slide. Unlike Gram staining, the bacterial smear is not heat-fixed during a Capsule Stain. Heat-fixing can disrupt or dehydrate the capsule, leading to false negatives (5). Furthermore, heat-fixing can shrink cells resulting in a clearing around the cell which can be mistaken as a capsule, leading to false positives (3). The acidic stain colors the slide background; while follow up with a basic stain, Crystal Violet, colors the bacterial cells themselves, leaving the capsule unstained and appearing as a clear halo between the cells and the slide background (Figure 5). Traditionally, India ink has been used as the acidic stain because these particles cannot penetrate the capsule. Therefore, neither the capsule nor the cell is stained by India ink; instead, the background is stained. Congo Red, Nigrosin, or Eosin can be used in place of India ink. Capsule staining can help doctors diagnose bacterial infections when looking at cultures from patient samples and guide appropriate patient treatment. Common diseases caused by encapsulated bacteria include pneumonia, meningitis, and salmonellosis.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10513/10513fig4.jpg" /> 
Figure 4: Schematic of the Capsule Staining Protocol. The top panel shows the slide smear prior to any stain application. The middle panel shows how the slide and bacteria look after the primary stain, Congo Red. The final panel shows how the slide and bacteria look after the counterstain, Crystal Violet.

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/10513/10513fig5.jpg" /> 
Figure 5: Capsule Staining Results. Capsule staining of encapsulated Acinetobacter baumannii (denoted with black arrows) and non-encapsulated Escherichia coli (denoted with white arrows). Notice the background is dark and A. baumannii cells are stained purple. The capsule around A. baumannii cells is evident as a halo, while E. coli has no halo.
In adverse conditions (for example nutrient limitation, extreme temperatures, or dehydration), some bacteria produce endospores, metabolically inactive structures that are resistant to physical and chemical damage (1, 2, 8, 9). Endospores allow the bacterium to survive harsh conditions by protecting the genetic material of the cells; once conditions are favorable for growth, spores germinate, and bacterial growth continues. Endospores are difficult to stain with standard staining techniques because they are impermeable to dyes typically used for staining (1, 9). The technique routinely used to stain endospores is the Schaeffer-Fulton Method (Figure 6), which uses the primary stain Malachite Green, a water soluble stain that binds relatively weakly to cellular material, and heat, to allow the stain to break through the cortex of the spore (Figure 7). These steps color the growing cells (termed vegetative cells in the context of endospore biology), as well as endospores and any free spores (those no longer within the former cell envelope). Vegetative cells are washed with water to remove Malachite Green; endospores retain the stain due to heating the Malachite Green within the spore. Finally, the vegetative cells are counterstained with Safranin to visualize (Figure 8). Staining for endospores helps differentiate bacteria into spore formers and non-spore formers, as well as determines whether spores are present in a sample which, if present, could lead to bacterial contamination upon germination.

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/10513/10513fig6.jpg" /> 
Figure 6: Schematic of the Endospore Staining Protocol. The left column shows how spore forming bacteria react at each step of the protocol. The right column shows how non-spore forming bacteria react.

<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/10513/10513fig7.jpg" /> 
Figure 7: Diagram of Endospore Structure. Bacterial cell containing an endospore with the various spore structures labeled.

<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/10513/10513fig8.jpg" /> 
Figure 8: Endospore Staining Results. A typical staining of endospores of Bacillus subtilis. The vegetative cells (denoted with the white arrows) are stained red, while the endospores (denoted with the black arrows) are stained green.

Microscopy and Staining Techniques in Bacteria

显微镜和染色:革兰克、胶囊和内孔染色

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

教育

JoVE Science Education

高级生物学

微生物学

Capsule-Based Negative Staining of Virus Samples on TEM Grids: A Heavy Metal Staining Technique to Visualize the Ultrastructure of Enveloped Viruses

Source: Blancett, C. D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56122">Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the negative staining of enveloped virus samples using a processing capsule within the biocontainment environment. The negatively stained viruses can be visualized using transmission electron microscopy to investigate viral structure and morphology with nanometer resolution.

TEM 网格上病毒样品的基于胶囊的阴性染色:一种可视化包膜病毒超微结构的重金属染色技术

Source: Blancett, C. D., et al. Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment. J. Vis. Exp. (2017)
In this video, ...