作者想感谢弗朗西斯克里克研究所的 BRF 游泳队的技术帮助和关键投入。这项工作得到了弗朗西斯克里克研究所的支持, 它获得了癌症研究英国 (FC001999)、英国医学研究理事会 (FC001999) 和惠康信托基金 (FC001999) 的核心资助。

1. 在循环系统中暴露预过滤哨兵
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设置一个清洁的8升坦克从一个循环系统。用坑的水来填满它。从系统中添加2个生物介质的陶瓷珠或海绵立方体 (图 1)。添加 1-2鱼/L (即, 12 条鱼)。使用野生型鱼类的优势遗传背景的设施, 例如 AB。
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<li>选择至少6条鱼如下: 至少一个女性和一个男性在6月的年龄以下, 一女性和一男性在6和18月之间, 并且一女性和一男性在18月年龄之上。</li>
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每天喂一次。改变饮食 (如: 如, 干性饮食, 盐水虾), 以确保哨兵是暴露在所有的饮食使用的斑马鱼设施。更改水在星期一, 星期三, 星期五,即, 每周三次, 为期4月。
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<li>进行水的变化, 转移哨兵和生物媒体到一个临时的坦克。清空完全的哨兵坦克和清洁它。只用水池水重新填充哨兵舱。把哨兵和生物媒体放回去</li>
	</ol>
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	<li>把哨兵4月的时间暴露在水池里安乐通过一种经批准的方法, 如浸泡在 2-乙醇 (3 毫升/升) 过量溶液中的鱼。</li>
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确认死亡, 在停止鳃运动后等待10分钟。
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<li>用钳子抓住尸体, 将其完全冻结在-80 ° c 的容器中。这将用于 PCR12,15。</li>
		<li>另外, 切下 tailat 的尾梗, 尼克腹壁, 并设置在4% 福尔马林。 注意: 使用手套和通风柜进行组织病理学。标记示例容器。</li>
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</ol>2. 水池拭子
<ol><li>使用塑料轴的无菌干拭子。戴上手套找到要擦拭的表面 (在水面上的水池壁), 并移除任何防止容易接触到表面的物品。选择低流量的水槽表面。</li>
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拔出通过除去外包装, 将无菌棉签暴露在空气中。避免接触拭子的交叉污染, 注意不要触摸未经测试的表面。
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<li>用拭子在 5-10 厘米的水池壁上吸收水和生物膜在水池水面水平。</li>
		<li>将拭子套回或将笔尖折断成无菌离心管。给样品贴上标签, 在-80 ° c 时进行 PCR 检测或冷冻。</li>
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</li>
</ol>3. 在罐底检测毛白杨卵
<ol><li>
污泥的显微分析
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<li>使用60毫升注射器9来吸入污水坑底部的污泥或任何包括哨兵在内的鱼罐。将样品分成15毫升的管子。用螺丝顶盖上管子, 并将管子贴上标签。</li>
		<li>通过在177毫升的热水中掺入227克砂糖, 用磁性搅拌器14来制备糖饱和溶液 (比重 = 1.27)。</li>
		<li>离心机的15毫升管在 175-250 x g 为10分钟的离心与摆动桶。醒酒管, 并保持在他们的管沉淀物。</li>
		<li>用糖饱和溶液将管子填满一半。关闭管与他们的螺丝顶部和彻底混合的沉淀物与解决方案。</li>
		<li>把管子放在离心机的摆动桶里, 用糖饱和溶液填满它们的顶端。在每根管子的顶端轻轻地设置一个盖子玻璃, 并与糖饱和溶液接触。</li>
		<li>离心机在 175-250 x g 10 分钟注意到, 一些盖玻璃可能在离心过程中脱落和断裂, 因此每个60毫升样品有4管。提起盖子玻璃并将其放在玻璃滑梯上。用铅笔或记号笔标记幻灯片。<ol>
<li>以防太多的盖子玻璃破损发生, 填补了大部分的管与糖饱和溶液, 离心机在 175-250 x g 10 分钟, 填补了与糖饱和溶液的顶部, 然后轻轻地设置盖玻璃。等待30分钟。</li>
		</ol>
</li>
		<li>使用显微镜 13 (图 2和视频 1) 查找毛的毛鸡蛋。在放大倍数 400X6中识别双极性插头。 注: 卵的大小为 57-78 µm 长和 27-39 µm 直径16,17。请注意, 一个积极的幻灯片是足够的60毫升样本被宣布为正数。</li>
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</li>
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在检疫中筛选进口动物为毛白杨卵
	<ol>
<li>在坦克中设置男性和女性的D. 鱼。添加到坦克通常用于收获和保存产卵的设备, 但在这里使用它收集粪便 (图 3)。 注: 例如, 一个完整的1升养殖罐 (外罐和带磨碎底部的内胆) 完全浸没在一个13升的水箱中, 允许鱼自由进出繁殖装置。</li>
		<li>在一周后取出繁殖装置, 并在3.1 步 "显微镜下的污泥分析" 中所描述的繁殖装置中收获收集的污泥。</li>
	</ol>
</li>
</ol>4. 污泥沉积物的 PCR
<ol><li>用60毫升注射器9将容器底部的污泥吸吸, 并将样品转移到60毫升管。关闭管与它的螺丝顶部和标签的管。把注射器处理掉</li>
	<li>摇动60毫升管和转移15毫升到15毫升管。关闭管与它的螺丝顶部和标签的管。</li>
	<li>离心机的15毫升管在 175-250 x g 为10分钟的离心与摆动桶。醒酒管, 并保持在管道中的沉淀物。</li>
	<li>
拔出用棉签除去外包装, 将无菌的棉签暴露在空气中。避免接触拭子的交叉污染, 注意不要触摸未经测试的表面。
	<ol>
<li>在试管中拭去十五年代的沉淀物。</li>
		<li>将拭子套回或将笔尖折断成无菌离心管。标签的样本, 冻结在-80 ° c, 并发送 PCR 测试。 注:45 毫升保持在60毫升管。如步骤3.1 所述, 可以通过显微镜下的污泥分析来检测出毛白杨卵, 以确认 PCR 结果。在步骤3.2 之后, 可以尝试在污泥中筛选出进口动物的 PCR 方法。</li>
	</ol>
</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Assessment+of+the+Efficacy+of+Five+Veterinary+Broad-Spectrum+Anthelmintics+to+Control+the+Intestinal+Capillariasis+in+Zebrafish+(Danio+rerio).">Experimental Assessment of the Efficacy of Five Veterinary Broad-Spectrum Anthelmintics to Control the Intestinal Capillariasis in Zebrafish (Danio rerio).</a> Zebrafish. 12 (3), 255-267 (2015).</li><li>Collymore, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+Efficacy+of+Emamectin+Benzoate+and+Ivermectin+for+the+Treatment+of+Pseudocapillaria+tomentosa+in+Laboratory+Zebrafish+(Danio+rerio).">Tolerance and Efficacy of Emamectin Benzoate and Ivermectin for the Treatment of Pseudocapillaria tomentosa in Laboratory Zebrafish (Danio rerio).</a> Zebrafish. 11 (5), 490-497 (2014).</li><li>Chang, C. T., Whipps, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Antibiotics+against+Mycobacterium+Species+Commonly+Found+in+Laboratory+Zebrafish.">Activity of Antibiotics against Mycobacterium Species Commonly Found in Laboratory Zebrafish.</a> Journal of Aquatic Animal Health. 27 (2), 88-95 (2015).</li><li>Chang, C. T., Doerr, K. M., Whipps, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+treatment+of+zebrafish+mycobacteriosis:+tolerance+and+efficacy+of+treatments+with+tigecycline+and+clarithromycin.">Antibiotic treatment of zebrafish mycobacteriosis: tolerance and efficacy of treatments with tigecycline and clarithromycin.</a> J Fish Dis. , (2017).</li><li>Peterson, T. S., Ferguson, J. A., Watral, V. G., Mutoji, K. N., Ennis, D. G., Kent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramecium+caudatum+enhances+transmission+and+infectivity+of+Mycobacterium+marinum+and+M.+chelonae+in+zebrafish+Danio+rerio.">Paramecium caudatum enhances transmission and infectivity of Mycobacterium marinum and M. chelonae in zebrafish Danio rerio.</a> Dis Aquat Organ. 106 (3), 229-239 (2013).</li><li>Watts, S. A., Lawrence, C., Powell, M., D'Abramo, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vital+Relationship+Between+Nutrition+and+Health+in+Zebrafish.">The Vital Relationship Between Nutrition and Health in Zebrafish.</a> Zebrafish. 13, 72-76 (2016).</li></ol>

作者没有什么可透露的。

技术、关键步骤和疑难解答的限制:
哨兵的年龄、性别、应变和暴露时间不规范。这在表 1中显示。在6月龄以下的鱼类或老鱼的筛查很少。可能有一些病原体影响幼鱼, 因为有一些病原体是较常见的在老年人口10,18,19,20。同样, 在某些哨兵组的选择中也没有考虑到性别问题, 尽管有些报告说某些病原体存在性别偏见21。建议的技术试图解决这些问题, 虽然应变的选择可以根据特定的病原体进行监测。例如, TU 可以帮助检测分枝杆菌,12,22, 但有一个风险, 哨兵将作为一个水库或显示临床症状。关于曝光时间的长短, 斑马鱼国际资源中心的做法10增加了检测病原体的几率, 而这些病菌在污染期内可能会被遗漏。长期暴露的需要意味着哨兵不容易获得。添加的环境样品允许一些灵活性和增殖的筛选事件。例如, 取样可以每隔一个月进行一次, 每次筛选方法间隔4月。这可能会减少在新引入的病原体被检测之前的时间的流逝。
环境筛选技术依赖于对环境中病原体的检测。病原体由鱼流下, 因此在系统水中稀释。没有探讨通过水过滤来捕获病原体的可能性23 。我们所描述的方法只有当病原体有足够的时间在鱼和生物膜上繁殖以达到允许检测的污染阈值时才有效。通过对取样点的关键选择, 将这种技术的限制降到最低限度: 对罐内的污泥进行取样, 而不是在废油坑污泥中取样, 而水和生物膜则是在污水坑的表面采样, 而不是在罐内或后过滤。尽管如此, 同一系统中的所有样本都不可能提供相同的结果。使用另一种方法 (组织病理学、PCR 或污泥分析) 可以证实对毛白杨的阳性结果。杆菌 PCR 阳性结果可以通过培养或其他诊断实验室确认。然而, 在建立健康状况时, 建议进一步的样本, 以确认任何环境筛选技术的负面结果。
技术在现有/替代方法方面的重要性:
分枝杆菌在环境中是常见的, 它们在污水坑中的存在并不能预测其致病性12。马可9表明, 监测死亡率是调查健康问题发展的关键。动物标本的使用对任何兽医调查都是必不可少的。健康监测意味着在一个设施中检测所有流行的病原体, 而这不能仅仅通过环境筛选技术来实现。然而, 缺乏灵敏度的诊断工具可以延缓或防止准确描述健康状况。虽然哨兵的使用减少了检测在人群中流行的微生物所需的鱼的数量, 但是缺乏敏感性增加了使用多种方法的重量, 包括环境筛选5,23。事实上, 特定的病原体自由状态通常被定义为在设施中缺少一个物种, 这样环境和动物样本必须测试负的24,25。
该水池拭子的结果, 以确定分枝杆菌, 表明, 依赖于鱼类样本可能会导致一个虚假的负面健康状况。6试验过的杆菌种被描述为斑马鱼的致病或潜在致病性15 , 有些不会被鸡蛋表面消毒, 氯26在检疫中例行执行。因此, 伪负可能会对导入行的协作者产生一定的影响。例如, m 杆菌在鱼样本上被 pcr 遗漏, 但超过一半的废油坑棉签 pcr 检测到它。考虑到这些分枝杆菌对氯的耐药性比其他人多, 而且它们在水系统中的生长能力27, 这是不受污染的进口设施的风险。为了允许导入行, 经理需要信任和比较出口设施的健康报告与他们的。委员会性能评估程序28是啮齿类动物过程中的关键。RESAMA 网络报告法语D. 鱼11中的m gordonae和m mucogenicum的检测。这些分枝杆菌不是在我们使用的商业实验室的面板中提出的。这将是有用的扩展委员会程序和统一的诊断化验以及致病物种名单29。
水气也是一种病原体, 在导入动物时有可能被引入, 尽管它对氯气的敏感度30使其在常规的蛋表面消毒过程中更容易消除。水池, 棉签和污泥的结果表明, 环境筛选可以用来检测这种病原体。其他细菌如分枝杆菌在污泥中被检测到 PCR23。这种类型的样品是特别相关的, 因为它可以检测棚病原体。例如, 另一个新的应用是污泥分析筛检进口鱼在检疫为P. 毛白杨。寄生虫是威胁动物的健康13和肿瘤模型16。此外, 在常规斑马鱼蛋表面消毒中使用的氯浓度不是有效的31。因此, 以一周的营业额和没有任何鱼类安乐死来筛选进口动物的能力似乎很有吸引力。这种技术可以通过允许进口的分流来影响检疫和生物安全规则。然后根据出口设施中流行的病原体、进口鱼样本中检测到的病原体以及危害进口设施的健康状况的风险, 设计了一个决策过程10。
掌握这些技术后的未来应用或方向:
即使是常规的检疫治疗是选择的选择, 这种药物的功效32,33,34,35,36可以用育种装置的污泥分析评估。更普遍地, 环境筛选可以被用于测试化合物反对细菌和寄生虫根除, 包括在鱼群落生境。环境筛选的另一个利基应用是监测活饲料中的病原体的数量37,38。虽然这些技术的主要应用是作为一个宝贵的除了诊断工具箱的健康监测在斑马鱼设施。由于更准确, 成本和时间的有效定义的健康状况, 废油坑棉签和污泥分析是互补的哨兵监测和例行检疫的做法。事实上, 这些技术的未来将成为任何水生实验室健康报告的例行部分。

为了保护研究和动物福利1,2, 在动物设施内监测病原体的存在。在斑马鱼, 健康监测3,4,5,6,7,8,9,10,11通常依赖于分析的动物尸检后的经病理组织学, 细菌培养, 或分子方法。仅检测菌落动物不是推荐的方法, 因为鱼的数量和相关的费用将被要求检测低发病率的病原体。因此, 首选的方法是将一小群动物暴露在更高的污染物负荷中。这些鱼被称为预过滤哨兵。这种暴露持续了几个月, 它涉及增加动物看护人的工作量和/或一些专门建造的工程解决方案。另一项挑战是, 在检疫中筛选出可育的动物要保持活力的进口线, 这与对胴体的常规化验不相容。
我们在这里描述了一些方法来检测某些斑马鱼病原体 (水气, 分枝杆菌, 和毛白杨) 通过筛选水生系统的环境。目的是减少用于健康监测的鱼的数量, 并优化检测的营业额、成本和灵敏度。这种方法是一种替代动物的使用, 一些技术可以用于甄别进口的检疫。例如, 马可9能够通过在水池拭子上进行 PCR 而不是斑马鱼 (包括哨兵) 来识别更多的致病杆菌物种, 这是以较少的样本获得的。在同一研究中, 利用浮选和显微显微镜对坦克污泥进行筛选, 而不是通过 PCR 和组织病理学检测鱼, 来检测出更敏感的毛白杨卵。
表 1概述了哨兵程序的各种特征345,6789用于许多斑马鱼设施。后过滤哨兵接收水的方式与任何殖民地的鱼一样, 而预过滤哨兵接收水一旦它已经通过殖民地的鱼缸首先。例如, 可以在循环系统上设置预过滤哨兵, 不断接收污水坑水。这可能不是一个选项, 当有许多独立的系统在一个房间。在这种情况下, 一罐预过滤哨兵可以用来屏蔽整个房间。哨兵是在一个静态的坦克, 出循环系统, 他们的水是定期改变, 只使用预过滤水即, 从所有的系统在房间的污水。该技术被描述为与环境筛选效果比较的基线。该建议的设置是为了控制水质问题, 如降低 pH 值或氮污染。
细菌环境筛选的概念依赖于这样的假说, 即细菌在生物膜中被发现, 如在水表面的污水坑壁上或在罐底的污泥中找到。在循环养殖系统中, 水池似乎是一个理想的取样点, 因为它从所有的罐前过滤器收集废物 (水、粪便、饲料和其他有机物)。水池的表面经常容易地是可到达的, 抽汲是快速的, 并且它可以执行菌避免样品的交叉污秽 (从手套例如)。该概念用于鉴别斑马鱼系统中常见的致病性分枝杆菌9,12。下面介绍了该技术, 我们还报告了斑马鱼水槽表面拭子和污泥中的水气检测。
对寄生虫卵进行环境筛选是基于默里et al.的检测13和浮选技术是常规用于寄生虫和粪便中寄生虫卵的显微筛选14。马可9提出了一种替代取样过程的方法, 并表明该技术可用于检测鱼类群落生境的其他种类。感染的D. 鱼通过P. 毛白杨将它们的粪便和虫卵留在池底的污泥中。它们可以在那里收集, 因为它们的密度大于水。鸡蛋的密度也用来处理环境样品。第一次浮选与离心分离水和轻的残骸从重的物质。第二次离心依赖于饱和糖溶液 (密度大于密度的毛白杨卵), 允许寄生虫卵出现在试管的表面。
通过对第一次离心后获得的污泥样品进行 PCR, 对生物膜中的细菌和罐底的毛白杨进行筛选。这优化了取样时间。该方法如下所述。我们还建议在隔离环境中使用这些技术。为了屏蔽进口的成年斑马鱼 , 需要保持活着 , 一个育种装置入到检疫坦克。一周后, 在育种装置中的粪便和其他废弃物的收集和筛选通过显微镜或 PCR。下面介绍了该技术, 并在这方面显微镜检测到一些毛白杨卵。

<table border="0" cellpadding="0" cellspacing="0" width="683">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Aqua-Sed 250 mL</td>
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			<td style="width:191px;">2-phenoxyethanol</td>
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			<td height="21" style="height:21px;width:229px;">Tubed Sterile Dryswab Tip</td>
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			<td style="width:124px;">MW100</td>
			<td>Sump surface</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">BD Plastipak Disposable Syringe 50mL Eccentric</td>
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			<td height="21" style="height:21px;width:229px;">Centrifuge tube 15 mL Corning</td>
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			<td style="width:124px;">430766</td>
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			<td height="63" style="height:63px;width:229px;">Centaur 2 benchtop centrifuge with 4 x 200 mL Swing&#8211;Out Rotor (unsealed)</td>
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			<td style="width:124px;">MSB020.CX1.5</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Cover Glass 22 mm x22 mm</td>
			<td style="width:140px;">Menzel-Glaser</td>
			<td style="width:124px;">MNJ-350-020H</td>
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		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:229px;">Plain Swab Sterile Plastic Applicator Rayon Tipped White Cap</td>
			<td style="width:140px;">Sterilin Ltd Thermo Fisher Scientific</td>
			<td style="width:124px;">F155CA</td>
			<td>Swab sediment from sludge</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">50 mL Self-Standing Centrifuge Tube CentriStar Cap&#160;</td>
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			<td style="width:124px;">430921</td>
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			<td height="42" style="height:42px;width:229px;">In-Tank Spawning Tray Set&#160;</td>
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			<td></td>
		</tr>
	</tbody>
</table>

environmental screening of aeromonas hydrophila, mycobacterium spp., and pseudocapillaria tomentosa in zebrafish systems

健康监测系统是开发和使用在斑马鱼的研究设施, 因为病原体的斑马鱼, 如单胞菌水气, 分枝杆菌, 和Pseudocapillaria 毛白杨有潜力损害动物福利和研究。鱼通常被分析为检测微生物的宰后。使用哨兵是一个建议的方式, 以提高灵敏度的监测和减少动物数量的样本。介绍了一种用于循环系统的预过滤哨兵箱的设置。该技术的发展是为了防止水污染, 并通过仔细选择年龄、性别和菌株来代表鱼类种群。为了使用最小的动物数量, 屏幕环境的技术也详细。聚合酶链反应 (PCR) 在表面废油坑拭子是用来显着改善检测一些流行和致病杆菌物种, 如分枝杆菌杆菌, 分枝杆菌 haemophilum, 和龟分枝杆菌。另一种环境方法是处理在贮水池底部的污泥, 以寻找毛白杨卵。这是一种廉价和快速的技术, 可以应用在检疫的饲养设备被淹没在进口动物的控股罐。最后, 将 PCR 技术应用于污泥样品, 并在底池和表面检测到水气。一般来说, 这些特定的病原体的环境筛选技术, 比起前过滤哨兵的测试, 更敏感。

在图 4中的结果支持与鱼样相比, 废油坑拭子的优势, 以确定流行的分枝杆菌。在测试的115条鱼中, 分别在5% 和3% 中检测到了m 龟和m haemophilum 。没有发现其他致病的杆菌种。从同一系统中, 49 个水池拭子发现有5杆菌的物种存在。赔率比是计算的假设, m 龟和m. 杆菌是检测更频繁的 PCR 在表面水池拭子比在鱼样本。这是统计学上的显著性与各自的赔率比率 11 (95% CI: 4 至 29;p < 0.0001) 和 306 (95% CI:18 至 5208;p = 0.0001)。结果表明, 表面底壳拭子技术是一个有价值的替代方法的唯一使用哨兵筛选斑马鱼设施的分枝杆菌。环境示例还用于筛选水气。这些细菌是在鱼、污泥和表面样品中检测到的 (图 4)。这也支持建议的技术的能力筛选鱼群落生境。
关于污泥分析检测到的毛白杨卵, 马可9通过 PCR 和组织病理学检测了27% 条鱼样本中的寄生虫, 而在93% 的同一系统中检测出的污泥中发现了虫卵。在这里, 该技术被挑战, 以重现检疫检查。在这种情况下, 不能对进口的动物进行取样, 及时评估其健康状况有助于生物安全管理。从P. 毛白杨阳性装置中的未知健康状态的鱼在8罐中设置了繁殖装置: 最大 16 fish/13 L 罐, 7 转基因和1野生型线, 混合性别, 年龄 4-24 月。一周后从装置中收获污泥, 用显微镜进行分析。在 7 (88%) 样品中观察到了毛的卵. 最后, 对该寄生虫的 PCR 检测试用在底泥和污泥沉积物上。4 6 污泥样品中均为 PCR 阳性, 表面拭子均为阴性。这并不奇怪, 因为污泥筛选依赖于鸡蛋的能力下降到底部的坦克。这表明, 污泥分析技术可用于筛选鱼水族馆的毛白杨害虫, 这些方法可用于筛选进口动物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55306/55306fig1.jpg"> 图 1: 从循环系统中取出预过滤哨兵箱.一个8升坦克充满了水和生物介质从系统的坑的屏幕。两个白色陶瓷生物媒体珠坐在底部的坦克 (在图片中间)。根据年龄、品系和性别选择12条鱼, 并将其添加到哨兵池中。<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55306/55306fig2.jpg"> 图 2: 在污泥分析过程中通过显微镜检测到的毛. 使用的放大倍数是400X。箭头表示双极性插头。缩放栏 = 50 µm.<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55306/55306fig3.jpg"> 图 3: 在鱼缸中浸泡的养殖装置收集污泥进行分析.这个坦克是设置在一个长凳上的图片的目的;它在循环系统否则被设置。<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55306/55306fig4.jpg"> 图 4: 在鱼、表面水池拭子和废油坑污泥中, 用 PCR 方法对分枝杆菌、水气和毛白杨进行鉴定的百分比.百分比是通过将每种病原体的阳性结果除以被试样品的数量来获得的。阳性结果的百分比由样本类型作为鱼、面或污泥, 并在细菌的名称后表示。用 PCR 法检测了115条鱼和49种表面底池拭子, 鉴定了分枝杆菌。数据是用马可的9结果编译的, 因为它是本研究的扩展。根据协议1条, 鱼类主要是预过滤哨兵。很少, 当哨兵不是可利用的, 逃脱者和老殖民地鱼 (> 18 月) 被抽样了。对分枝杆菌的所有测试系统都在鱼和油槽拭子上进行了测试。赔率比是计算的假设, m 龟和m. 杆菌是检测更频繁的 PCR 在表面水池拭子比在鱼样本。这是统计学上的显著性与各自的赔率比率 11 (95% CI: 4 至 29;p < 0.0001) 和 306 (95% CI:18 至 5208;p = 0.0001)。marinum 分枝杆菌PCR 是阴性的所有样品, 因此它被认为没有从这些设施和不包括在分析。在水气的存在下, 用 PCR 方法检测了12条鱼、14表面水池拭子和 6个废油坑污泥。对6坑在表面和污泥中的存在进行了测试. 聚合酶链反应。CI = 置信区间。* 和 ** 表示统计学意义;其他比较没有统计学意义。<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Video 1" src="/files/ftp_upload/55306/55306vid1.jpg"> 视频 1: 扫描一个鸡蛋的毛白杨与显微镜.滑块是从污泥分析中获得的, 如步骤3.1 所述。将扫描该字段以检测P. 毛白杨的卵。一旦结构被识别, 它将被放大以在更高的分辨率下确认识别。<a href="http://cloudflare.jove.com/files/ftp_upload/55306/video_scan_for_egg.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>作者</td>
			<td>接触开始时的年龄</td>
			<td>曝光长度</td>
			<td>取样年龄</td>
			<td>前或后 过滤</td>
			<td>性别</td>
			<td>应变 (s)</td>
		</tr>
<tr>
<td>巴顿et al.3
</td>
			<td>4月</td>
			<td>6月</td>
			<td>10月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>博尔et al.4
</td>
			<td>6月</td>
			<td>6月</td>
			<td>12月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>AB 野生型</td>
		</tr>
<tr>
<td>弗兰克·科利莫尔et al.5
</td>
			<td>尽可能年轻</td>
			<td>3月</td>
			<td>< 6 月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>格斯勒et al.6
</td>
			<td>4月</td>
			<td>4月</td>
			<td>8月</td>
			<td>前和后 过滤</td>
			<td>N/A</td>
			<td>AB 野生型</td>
		</tr>
<tr>
<td>刘et al.7
</td>
			<td>3月</td>
			<td>1-6 月</td>
			<td>4-9 月</td>
			<td>前和后 过滤</td>
			<td>N/A</td>
			<td>野生型</td>
		</tr>
<tr>
<td>马丁斯et al.8
</td>
			<td>3月</td>
			<td>3月</td>
			<td>6月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>野生型</td>
		</tr>
<tr>
<td>马可9
</td>
			<td>< 6 月, 6-12 月, > 18 月</td>
			<td>4月</td>
			<td>7-24 月</td>
			<td>预过滤</td>
			<td>1女性和 1男性 每个年龄组</td>
			<td>AB 野生型</td>
		</tr>
<tr>
<td>默里et al.10
</td>
			<td>3-4 月</td>
			<td>3月, 6 月, 1 年</td>
			<td>7、10和16 月</td>
			<td>前和后 过滤</td>
			<td>N/A</td>
			<td>AB 野生型</td>
		</tr>
</tbody></table>表 1: 斑马鱼设施中哨兵设置的比较.哨兵鱼可根据年龄、性别或应变选择。他们被暴露在一个明确的时期, 他们接收预先或后过滤系统的水。这些数据是从2016的斑马鱼杂志健康特刊上编译的3,4,5,678910。

Watch this Scientific Journal Video about Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems at JoVE.com

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

本议定书描述了使用水槽拭子和对斑马鱼系统的污泥分析, 这导致检测比唯一使用哨兵检测病原体, 如单胞菌水气, 分枝杆菌, 和Pseudocapillaria 毛白杨。此外, 还提出了一种在检疫中监测毛白杨卵的系统。

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

environmental-screening-aeromonas-hydrophila-mycobacterium-spp

Research

JoVE Journal

Biology

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

9.7K 次观看.  The Francis Crick Institute. 本议定书描述了使用水槽拭子和对斑马鱼系统的污泥分析, 这导致检测比唯一使用哨兵检测病原体, 如单胞菌水气, 分枝杆菌, 和Pseudocapillaria 毛白杨。此外, 还提出了一种在检疫中监测毛白杨卵的系统。

视频: Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

用于高内涵筛选和分析应用的大规模生产球体的稳健方法

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

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