The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. M.R.H., D.P., L.B., K.J. and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: S.M. as an employee of MRI Global; M.G.L. as an employee of Lovelace Respiratory Research Institute, Inc.; and J.H.K. as an employee of Tunnell Government Services, Inc.

1.实验室进入<ol><li>收集从BSL-2实验室所有用品进入BSL-4实验室在实验前（ 如电池，媒体和消耗品）。 </li><li>完成BSL-4进入过程（在参考文献7中详细描述的）。 </li></ol>在BSL-4实验室II级生物安全柜的2.准备工作<ol><li>一旦BSL-4实验室内，确保日常内部检查表（ 图1）已经完成。完成该清单如果清单先前尚未填写并指示哪些病毒将被使用。如果该清单已经完成，加名和病毒将用于该列表。 </li><li>清洗由BSC的整个内部（包括窗框），用5％的双季铵盐消毒剂铵溶液喷洒下来（ 如正烷基二甲基苄基氯化铵，正-烷基二甲基乙基苄基铵的II类BSC正在使用的氯或其他消毒剂适合剂）和擦拭用纸巾10,11。喷雾机柜和窗框内70％的乙醇溶液以除去粘性消毒液。 </li><li>如果坐着，在一个舒适的高度设置在II级BSC前面的椅子上，以保证机箱的背面可以达到和实验室专家的面位于前开口以上（ 图2）11。 </li><li>准备在生物安全柜是浪费容器。确保在废物容器双季铵盐消毒剂铵溶液中的最终浓度是不低于5％。相应的规划，使废物将被添加的10％溶液至其将稀释的消毒剂。此外，放置一个喷雾瓶与II类BSC内的5％的双季铵盐消毒剂铵溶液中和在测定完成后喷去除和戴手套的手前的任何物品。 </li> &lt;li&gt;将在II类BSC需要整个实验早在机壳尽可能避免反复材料引入到空气流11的II类BSC和破坏的合适的原料。 </li></ol> 3.举例:斑块检测<ol><li>检索含病毒样品，并用手从存储位置控制病毒，并在37℃的培养箱中解冻材料。 </li><li>标签根据规划的病毒稀释液中使用的6孔板的孔中。标记板的盖子和主体，以确保成功的匹配，如果盖子和本体分开。 </li><li>汇集来自步骤3.1-3.2材料进入II级BSC。地方没有或一侧与病毒接触不会来（"干净侧"）和废物在另一边（"脏侧"）的所有项目。如果可能的话，保持"干净项目"30cm以上除了"肮脏的物品"中气溶胶的活动11。 </li><li>作为内线中锋的设计是为了保护自己的11最有效的位置工作，尽量靠近II类BSC尽可能的内线中锋。 </li><li>除去50μl的病毒样品和对照病毒的并放入450微升的Dulbecco改进的Eagle培养基中，用2％的胎牛血清。与制作系列稀释尽量根据需要进行（ 图3A）进行。 </li><li>在稀释液，缓慢并小心地用移液管尖端混合病毒样本的至少5倍，而试图在样品中气泡产生最小化。每次添加下一个稀释以及之后更改枪头。 </li><li>在最后稀释，样品5次混合后，弃去50微升病毒样品放入废物容器，以确保稀释液等体积。 </li><li>当提示处置，冲洗与废液桶消毒剂溶液各前尖expell以净化尖端的内外ING尖到废液桶。 </li><li>一旦稀释液制成，除了设置稀释孔，并开始从细胞培养板的孔中吸移媒体，留下500微升的媒体在一个6孔板的每个孔中。 </li><li>当使用手动，音量可调的移液管尖，吸消毒剂从废物桶到吸管的顶端溶液完成，按钮移液器，以保证移液管的内部的适当的去污。离开尖端插入废物桶，直到实验结束。 </li><li>一旦媒体已经从板中除去，一式两份加入100微升正确样品到适当的孔中的预标记的板，改变用于每个样品的每个提示。 </li><li>经噬斑分析接种后，喷洒过戴手套的手的消毒液，并使用用消毒剂溶液浸湿的纸巾用手放置板放回孵化器之前擦拭所有板的外部。回覆直到所有板块均进入孵化器泥炭这个过程。 </li><li>岩石中的图8的运动的板，以确保样品的适当的分散在细胞上，每15分钟1小时。 </li><li>在摆动板的完成时，返回板进入II类BSC与和1.6％西黄蓍胶2×Eagle氏最低必需培养基的混合物，半固体覆盖比琼脂糖容易操纵，用于噬菌斑测定的覆盖沿。 </li><li>在用于覆盖的均匀分布在整个的井的表面的图8的运动2毫升覆盖混合物再次添加到各孔中一个6孔板和岩石。直到每个使用以及与覆盖混合叠加重复此过程。 </li><li>确保血清吸管尖端不接触任何液体中的井进行叠加的过程，以避免交叉污染。 </li></ol> 4.废物处置仪器和生物安全柜的清洁和 &lt;OL&gt; <li>完全淹没在废物桶为至少10分钟的接触时间的5％消毒剂溶液的所有废料。消毒枪头，血清移液管，并与上述其他废物。另外，喷雾的废物桶（内部和外部），用5％消毒剂溶液让该溶液保持与废物桶10分钟的接触时间的接触。 </li><li>在等待的接触时间结束的浪费，浸泡，用5％消毒剂溶液纸巾，擦拭仪器，如微量，和从BSC移除。把戴手套的手出了II级BSC之前，可以先喷戴手套的手用5％消毒液。 <ol><li>从本BSC的微量后，擦拭用70％的乙醇溶液独立的纸巾，以避免对仪器粘堆积。喷雾不能有效地擦拭具有5％消毒剂任何仪器，并与在BSC的溶液10分钟接触离开。 </li></ol></li><li>经过足够的接触时间，请从BSC中的所有项目。取放物品，包括含有废料的废物桶，向下沉。清洗可重复使用的去除残留的消毒剂项目。返回的所有项目的存储位置。 </li><li>清洁II类BSC的工作表面，机壳侧面和背面，玻璃的内部，窗框1，用5％消毒剂溶液，然后用70％的乙醇溶液。 </li><li>拉出沥干从废物桶和地方表面消毒的血清移液管到单独的吸管盘的血清移液管高压灭菌（血清移液管可以提出一个锐器的危险，并可能通过垃圾袋撕破。之前将这些吸管的硬边容器高压灭菌）。 </li><li>倒入废物桶的内容到放置在水槽底部的过滤器。 </li><li>提起这衬以红色生物危害袋的水槽的生物危险废物的容器和转储straine的内容r记录到红色生物危害袋。不要将手伸入过滤去除微量的提示，可能会粘到过滤器内部。打滤网沿着垃圾容器内，直到所有的技巧都去掉，或者使用镊子从过滤器中删除的提示。 </li><li>冲洗出来的废桶和地方晾晒旁边的水槽机架上。 </li></ol> 5.高压灭菌废物<ol><li>在上车的高压釜托盘中取出来自生物危险废物的容器和地点的生物危害袋。 </li><li>离开生物危害袋打开，将一块釜胶带在袋外，连接高压釜盘的两侧，以保持固定在托盘袋。 </li><li>放置在血清枪头托盘高压釜胶带的两件，并与一个人的声母和日期进行标记。广场上与高压锅托盘车的血清吸管托盘。 </li><li>打开高压釜。连接提供釜装载/卸载CART到高压釜，并带出了可伸缩的平台釜上休息的车。 </li><li>从高压釜中取出金属棒并打开顶部。放置装有土芽孢杆菌属的嗜热脂肪芽孢到金属杆的生物指示剂小瓶，检查高压灭菌周期成功完成。 </li><li>金属棒放入废物袋在高压釜托盘的中心，但确保了金属杆仍然很方便。 </li><li>充满垃圾和血清吸管盘釜托盘放置到伸缩釜平台，并把它推回高压釜中。 </li><li>从分离高压釜釜装货/卸货车并关闭车门釜。 </li><li>启动高压釜中。 </li><li>不要离开高压釜，直到周期已经开始。将高压釜的操作屏幕将显示剩余时间该运行。 </li><li>高压釜运行完成后，取出生物INDicator和评价在指定培养箱加热增长。如果在生物指示剂中检测的生长，重新运行高压釜中的垃圾，并评估重新指示器。如果没有检测到增长，从设施去除垃圾。 </li></ol> 6.示例:修复及斑块检测的染色<ol><li>相应的天数（这是取决于所使用的病毒）已经过去之后，返回回实验室，并再次执行从第1和第2步骤。 </li><li>小心地从孵化器在步骤3.1.2完成清除牙菌斑实验板，并放入II级BSC手。 </li><li>移液器关闭所有的媒体和覆盖材料，并分配到消毒液容器中，用10％中性缓冲福尔马林和0.8％结晶紫12,13的混合物代替。让该混合物保持在板30分钟以失活在板上的病毒。 </li><li>灭活后，小心地取出中性在一个单独的废物容器福尔马林/结晶紫混合和地点之前，处置中和。 </li><li>喷戴手套的手消毒剂和传递出来的II类BSC如上所述之前擦拭板的外部。 </li><li>继续进行到接收器，冲洗所述板以除去过量的染色，然后将所述板在一个购物晾干。 </li><li>一旦板完全干燥后，用灯箱来算斑块。记录所有的计数和从标准方程式（ 图3B）计算的病毒滴度。 </li></ol> 7.从BSL-4实验室取出样品<ol><li>灭活，将BSL-2实验室条件下进行操作的任何样品。遵循的，用10％中性缓冲的福尔马林（NBF）或Trizol试剂的LS（苯酚，异硫氰酸胍，硫氰酸铵，醋酸钠，甘油）-1,14-在IRF-弗雷德里克批准的内部生物安全办公室两种方法之一。传输SAmples到BSC的外包装之前从BSL-4实验室去除一个新的清洁的管或板。 </li><li>热封灭活样品中含有足够的5％消毒剂或固定剂溶液来消毒袋的内部和接受转出的BSL-4实验室的样品管的外部的热密封袋管或板。把这个袋子到另一个袋子下面同样的程序。 </li><li>密封第二袋并放置在热密封袋入含有5％消毒剂溶液至少10分钟的扣篮罐消毒热密封袋的外侧。 </li><li>通过划定在管的数量和管的大小，数量填写实验室内扣篮坦克记录簿，代理使用，灭活方法使用，室内到的样本将被转移。 </li><li>与在BSL-4实验室的外侧的同事坐标检索来自扣篮罐的袋和取样的BSL-2实验室。 </li></ol>

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The authors have nothing to disclose.

在BSL-4实验室工作，需要大量的时间和更多的对细节的关注。在这种环境中的任何类型的工作，需要训练有素，深入，认真的人。标准的病毒斑块检测提供了一个共同的过程的精确模型与在BSL-4实验室后果严重病原体的工作，作为测定包括在实验室工作人员必须经过培训，几个主要的概念。 第一个主要概念是在II类BSC，其功能为高病原后果一级容器的正确使用和安全操作的应用程序。的II类BSC功能将如何决定实践，极大地限制暴露风险的个人理解。从清洁区的工作流程（"干净方"）向污染区（"脏侧"），在整个作业区在第二类BSC还有助于避免交叉污染11。清洁被污染的材料和增刊IES应该被隔离以限制污染的物品在清洁的物品的移动。 第二个主要的概念是物理和生物废弃物管理。在处理这两种类型的废物适当的措施是在确保实验室的专家保持安全和环境不被污染至关重要。在实验中步骤旨在灭活和摧毁病原体样本带出BSL-4实验室前。这样的关键步骤的例子包括:吸移消毒剂进入每个尖端，允许至少一个与消毒剂，灭菌废物可能受到污染的材料的10分钟的接触时间，并在高压釜周期验证无菌。这些步骤被设计成冗余以确保高后果病原体的破坏。

A correction was made to the authors section in: Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices
One of the authors names was corrected from:
Nicole Joselyn
to:
Nicole Josleyn

A correction was made to the authors section in: Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Erratum: Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

由于实验室人员处理高后果的病原体的安全性（无感染预防就也没有治疗方案存在）是最重要的，健康和人类服务的美国部门已建立了设施建设和工作的安全行为准则的最佳实践，在生物医学病原体和从生物安全的角度来看1临床实验室。通过立法和监管，许多做法和程序已成为强制性要求必须遵守与这些病原体的工作。在美国，这很容易从人与人之间传播的病原体，导致高病死率，和/或具有重大公共卫生影响和生物恐怖主义的潜力，被归类为过敏和传染病的健康/国立研究所病（NIH / NIAID）排序的一种病原体和或疾病控制中心和预防中心（CDC）生物恐怖A类代理商2。此外，H如果这些病原体是潜在的生物恐怖制剂，有大量人员伤亡和毁灭性影响到经济，重要基础设施，或公众信心3 IGH潜力，后果的病原体被列为一级代理商选择。 BSL-4行动，包括访问院所BSL-4实验室，更高度控制比BSL-2/3的操作。例如，它是基本上更难以获得由于大量西装培训要求，广泛指导要求，以及额外的医疗生物安全的先决条件访问BSL-4实验室相比，BSL-2或BSL-3实验室。此外，还有典型的一个BSL-4设施与一个BSL-2或BSL-3设施4-6多个物理安全屏障。正如我们在第一篇文章中概述了BSL-4出入境手续，实验室工作人员进行广泛的培训和心理筛查资格进入的BSL-4实验室7。 W¯¯ithin的BSL-4实验室，感染和错误的风险是可以避免的，或者按照既定程序缓解。研究必须细心谨慎进行，以最小的多任务处理或分心。在正压力服弯腰是困难的，并且面罩可能限制方法如显微术。笨重的手套阻碍了精细动作的任务，如搬运小件物品或标记管的性能。为了最大限度地减少在BSL-4的实验室所花费的时间，实验室专家应该检查的工作程序，以确定可以提前在BSL-2实验室中进行的步骤，然后传输这些材料进BSL-4实验室的任务（多个）的完成。在BSL-2实验室中除去以进行进一步处理的材料时，材料是固定的，从在密封次级容器的BSL-4实验室除去。可能需要被删除的样本的实例包括:将通过酶联immunosorbe被分析的固定板或感染材料的管核苷酸测定（ELISA），免疫荧光测定（IFA）或聚合酶链反应（PCR）。 除了通过在BSL-4实验室所需的个人防护设备施加较大的物理限制相比，那些在BSL-2实验室中，为了在细胞培养板和废物处置高后果病原体灭活程序比所需更少致病病毒严格研究了一个BSL-2实验室。在最低限度，这些方法应符合CDC要求。例如，受污染的细胞培养板和其他材料可以与化学试剂，如中性缓冲的福尔马林灭活。处理过的细胞培养板或管是通过填充有液体消毒剂扣篮罐被放入含有福尔马林热封袋和除去从实验室。废物桶填充有消毒液和喷雾消毒剂用于暂时接收该实验期间和DISI产生的废物nfecting手套，清洁生物安全柜表面和仪器，分别为。在列出的浓度消毒剂季铵溶液被认为是所有美国BSL-4实验室（巴尔Ĵ，个人通信，2015年）的黄金标准。从废物桶固体废物灭菌，以消除污染的可能性。 在努力目视证明工作流和普通BSL-4程序的限制，我们使用了一个标准的病毒噬菌斑测定作为常用的病毒程序的一个例子。而病毒检测程序在一般描述中，我们强调用来保证在本协议中的实验室人员的安全生物安全程序。请参考以前的古典牌匾检测为可视化的斑块检测技术8,9更多的背景。 这里介绍的程序遵循疾病预防控制中心1中列出的BMBL规范。然而，所提出的协议是具体到IRF-弗雷德里克。每个BSL-4设备具有的BSL-4实验室内影响实验的执行标准不同操作程序（SOP）和操作方法。废物流管理和斑块检测的执行替代程序可能会有所不同基于这些实验室的管理和运作。尽管如此，BSL-4实验室的西装，为BSL-4环境中与表演类II工作程序橱柜的设置一个大致的了解将有助于考虑高风险的病原体研究时，科学家们理解的制约因素和安全隐患。增加的周围在BSL-4实验室工作中的困难外协作者的认识可导致调整的预期并在研究团体显影医疗对策更容易。

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			<td>21-402-581</td>
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			<td>Lonza</td>
			<td>12-604Q</td>
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			<td>Sigma</td>
			<td>F2442-500mL</td>
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			<td>Lonza</td>
			<td>17-602E</td>
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			<td>Quality Biological</td>
			<td>115-073-101</td>
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			<td>Drummond</td>
			<td>4-000-101</td>
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			<td height="21" style="height:21px;">1000-&#956;l Pipette&#160;</td>
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			<td>&#160;L-1000XLS+</td>
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			<td>&#160;L-200XLS+</td>
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			<td>Rainin</td>
			<td>L12-200XLS+</td>
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			<td height="21" style="height:21px;">8-Well, Multichannel 1000-&#956;l Pipettor</td>
			<td>Rainin</td>
			<td>LA8-1200XLS</td>
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			<td height="21" style="height:21px;">Attest</td>
			<td>Express Medical Supplies&#160;</td>
			<td>MMM12192</td>
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			<td>GEB 2404 AMB-2</td>
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		<tr height="21">
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			<td>Fisher</td>
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			<td>Fisher</td>
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			<td>Fisher</td>
			<td>WU-39321-00</td>
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			<td>Rubbermaid Commercial</td>
			<td>FG614500 RED</td>
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			<td height="21" style="height:21px;">Biohazard Bag</td>
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safety precautions and operating procedures in an (a)bsl-4 laboratory: 2. general practices

在生物安全4级（BSL-4）封闭实验室工作需要时间，非常注重细节。这是在BSL-2实验室与非高病原后果做了同样的工作将在BSL-4设置显著更长的时间。这增加的时间要求是由于多种因素的旨在保护从实验室获得性感染，从潜在污染的工作环境和高病原后果可能释放当地社区的研究员。实验室内，运动由于连接到强制全身安全服空气软管的限制。此外，需要是从II级生物安全柜（的BSC）中除去每个项目的消毒。实验室专家必须在BSL-4实验室的做法进行培训，必须表现出他们正在执行的技能熟练度高。本文的重点是勾勒出合适的程序和技术，以确保实验室biosafety和实验精度使用标准病毒噬菌斑测定，例如，过程。特别是，适当的技术，在BSL-4环境中安全工作的时候进行实验将在视觉上强调。这些技术包括:建立II级BSC的实验中，当完成工作的二类BSC的正确的清洁，废物管理和无害化处理的BSL-4实验室内产生的废物，并从里面BSL-去除灭活样本4实验室到BSL-2实验室。

在BSL-4实验室内继适当的程序，是确保试验的安全，有效的完成是至关重要的。参照每天完成内部检查表（ 图1），实验室工作人员保证设备完全正常运行。在BSC的中心适当的身体定位确保了实验最佳空气流动条件（ 图2）下进行的。该病毒样品被连续稀释，以获得具有每板30-300噬斑（ 图3A），并确定病毒滴度（ 图3B）板。许多因素会影响形成斑块，包括病毒趋性为宿主细胞系，接种技术，对病毒生长，适当稀释范围，并覆盖选择8的条件。在手术过程中在BSC产生的废弃物被正确由前高压灭菌从BSC并再次除去之前消毒离开BSL-4环境。通过遵循这些程序，没有实验室获得性感染已经在IRF-弗雷德里克BSL-4的研究过程中记录。 <img alt="图1" src="/files/ftp_upload/53600/53600fig1.jpg" /> 图1:样品每天的内部系统清单此清单的每日完成，确保实验室工作人员已开始工作前检查设备实验室（最重要的是BSC）之内。如果发现在BSC是外部的校准范围的，这BSC不能使用和维护应通知。所有的BSC必须正确校准和运行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53600/53600fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53600/53600fig2.jpg" /> 数字<html> 2:含黄色消毒液，并用吸管在II级生物安全柜实验室专家移液样品的背面和侧面视图（a）废物桶中的孔板（B）的权利，消毒剂喷雾瓶是废桶（A）的右侧。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53600/53600fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53600/53600fig3.jpg" /> 图3:样品的病毒滴度的计算病毒滴度表示为每毫升的噬斑形成单位（pfu）。计算病毒滴度，通过稀释因子（四）倍稀释的病毒的量加入到孔中（Ⅴ）的产物计算明确定义斑（PFU）和除法的数目。D / 53600 / 53600fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices at JoVE.com

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

安全注意事项和操作步骤在（A）BSL-4实验室:2.常规做法

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

17.8K 次观看.  National Institutes of Health (NIH). Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

视频: Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

艾滋病毒/艾滋病的综合成本有效的实验室监测:非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

鞭虫感染muris:在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

安全注意事项和操作步骤在（A）BSL-4实验室:1.生物安全4级实验室西服套房出入境手续

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

安全注意事项和操作步骤在（A）BSL-4实验室:4.医疗成像过程

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

安全注意事项和操作步骤在（A）BSL-4实验室:3，大气生物学

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...