我们感谢马内尔·玻利瓦尔，他在野外工作的协助。我们感谢"自然公园德尔德蒙特格里，莱巴利阿里玛代I EL的Baix泰尔"他们对我们的研究和抽样权限的支持。水下机器人是由Ayelet Dadon-Pilosof设计和Pilosof先生制造。这项工作是由西班牙政府项目CSI-珊瑚[授权号CGL2013-43106-R RC来和MR]，然后由"部:EDUCACION，文化宫ÿDeporte（MECD）"，以TM一个FPU奖学金支持。这是从海洋生物地球化学贡献与全球变化研究小组由加泰罗尼亚政府[授权编号2014SGR1029]和ISF授权十三分之一千二百八十〇和BSF补助2012089到G Yahel资助。

1.预备步骤和清洁程序<ol><li> 清洗液 <ol><li>穿戴护具，白大褂和手套在任何时候。开展无烟尘一个干净的空间，这些准备步骤。 </li><li>使用新鲜的，高品质，双蒸水5-10％的盐酸（HCl）解决方案。 </li><li>制备5％的阴离子和非离子表面活性剂溶液的高度可溶基本混合物（见材料清单）用新鲜的，高品质，双蒸水。 </li><li>存放在干净的所有解决方案，酸洗容器。 </li></ol></li><li> 预备步骤和清洗程序（在实验室） 注意:如果磷化合物不感兴趣，盐酸洗可以通过高质量的磷酸（H 3 PO 4）洗涤（8％ 的 H 3 PO 4的最终浓度）来代替。 <ol><li>穿戴护具，白大褂和手套在任何时候。 </li><li>洗取样装置（不包括在行不锈钢Swinney过滤器支架）与高纯度的水足量。留在5-10％的HCl溶液中浸泡过夜装置。以高品质的双蒸水足量再次冲洗设备。 </li><li>用的高纯度的水足量清洗直列不锈钢Swinney滤波器持有者。离开过滤器托架，在5％浸泡的阴离子和非离子表面活性剂溶液高度可溶性碱性混合物过夜。充足的高品质双蒸水再次冲洗。 </li><li>干所有的取样装置，包裹在铝箔中并保持在清洁箱，直到使用。 </li></ol></li><li> 吸率控制 <ol><li>通过调整长度，并根据计划的工作深度和水温的吸入管的内径控制采样速率。使用下面的等式（与用于充分DEVEL的泊肃叶定律导出OPED层管流），作为指导: <img alt="式（1）" src="/files/ftp_upload/54221/54221eq1.jpg" />其中，F =流速（厘米3分钟-1），ΔP=差压（巴）中，r =进口管内部半径（厘米），K = 2.417×10 -9（秒-2）中，L =管的长度（厘米），V =水的粘度（G -1秒-1）。请参阅表1的更多细节。 </li><li>保持低于所研究的动物的泵送速率的1％，采样率。 注意:使用撤离容器，有时有未知的真空造成额​​外的并发症。因此，强烈建议现场试验。以10米的深度和〜22℃的海水（40 PSU），具有254微米的内径在50cm入口管提供26微升秒-1（1.56毫升分钟-1）的〜平均吸速率。 </li></ol></li><li> 采样船 <ol><li>对于小体积样品（3-20毫升， 例如，超浮游生物流量CYTometry）使用预先抽真空的无菌塑料管中。 注意:预抽真空的无菌塑料试管常规用于人类标准血液测试;确保无添加剂使用无菌试管中。这些抽真空的无菌塑料试管最好使用无菌，单次使用的管保持架的偏离中心的Luer采样。虽然这是最安全和最有效的采样装置，它有一个稍大死体积相比简单的针头。 </li><li>对于较大的水样，如营养物和溶解的有机物，使用符合挥发性有机分析环境保护署（EPA）的标准40或60毫升的玻璃小瓶中。这些小瓶包括聚四氟乙烯，硅面临隔膜的聚丙烯盖。 </li><li>对于更大的体积，使用带有橡皮塞或保温瓶瓶青霉素。 </li><li>使用高密度聚乙烯瓶（HDPE瓶）的二氧化硅样品。 </li><li>为了增加样本容量和减小塞子撞出的风险在上升过程中，用真空泵在潜水前撤离（真空）项目1.4.2-1.4.4。真空手动使用手真空泵或者甚至通过用注射器抽吸空气。然而，为了获得最佳效果，建议了良好的真空泵。标准冻干机提供高真空。 注意:使用大抽真空烧瓶时，确保了更快的初始吸率将不会污染呼出水样要特别注意。 </li></ol></li><li> 容器清洗程序 <ol><li>对于溶解的有机物和NH 4 +的分析，采用新的预清洁的EPA瓶。 </li><li>漂洗等营养成分的分析小瓶（玻璃和HDPE）如下: <ol><li>冲洗小瓶（玻璃和HDPE）和高品质的双蒸水的聚丙烯盖。安装新的硅隔膜。 </li><li>浸泡小瓶（玻璃和HDPE）在10％的HCl至少3天，并拥有充足的高品质双蒸水冲洗。 </li><li>燃烧该玻璃小瓶在450℃下进行4小时，并允许在炉中冷却。安装帽，并在铝箔包裹直到使用。 </li></ol></li></ol></li><li> 过滤器 <ol><li>使用无粘合剂的玻璃纤维过滤器对所有溶解的有机样品过滤（ 例如 ，DOC，DON），并为粒状有机物的集合（ 例如 ，POC，PON）。包在一个单独的铝箔信封中的每个玻璃过滤器。燃烧在400℃2小时以挥发在干净和干燥的容器中的有机残基和储存，直到使用。 </li><li>使用一个无粘合剂的玻璃纤维过滤器如上述，或0.2微米的聚碳酸酯膜的用于进行采样的无机营养物（如 PO 4 3-，NO x的- NH 4 +）。清洁一旦安装过滤网架下方（1.7.3）作为解释后者。 </li><li>使用0.2微米的石英采样聚碳酸酯膜过滤器。清洁它们一旦installed。在过滤网架下方（1.7.3）的解释。 </li></ol></li><li> 过滤组件的研制 <ol><li>使用它们来清洁过滤组件之前过滤通过0.2微米的过滤器阴离子和非离子表面活性剂溶液和高品质的双蒸水的高度可溶性基本组合。 </li><li>对营养和二氧化硅以外溶解性有机物过滤组件: <ol><li>将清洁后在线不锈钢Swinney过滤器支架内燃烧无粘合剂的玻璃纤维过滤器。 </li><li>使用酸清洗注射器穿过整个组件来运行百毫升阴离子和非离子表面活性剂溶液的5％的高度可溶性碱性混合物，然后加入100毫升的高品质的双蒸水。 </li></ol></li><li>一氧化硅4过滤组件: <ol><li>将清洁后的聚碳酸酯过滤器固定架（PC过滤器支架）内的聚碳酸酯过滤器。 </li><li>使用酸清洗注射器于r未30毫升5％盐酸，并通过整个组件30毫升高品质的双蒸水。 </li></ol></li></ol></li><li> 系统组装 <ol><li>组装系统使用PEEK（聚醚醚酮）油管为1.6毫米的外径（OD）和254微米或177微米的内径（ID）的水下工作。 </li><li>用锋利的刀或PEEK切割器管切割成所需的长度。 </li><li>在其远端（样品容器侧），适合每个管连接到注射器针一凸形Luer连接头。请务必遵循制造商的指示，并拧紧螺母绿色对准前用管的末端蓝色兰套圈的侧扁。 </li><li>通过使用绝缘胶带连接PEEK管到三脚架"武器"或定制的机械手。 </li><li>附加一次性注射器针头注射器阳连接器。保持针头的保护盖，以防受伤。 </li&gt; <li>清楚标示采样齿轮和色码所有吸入呼出组件（ 例如 ，绿色= IN，红色=例）。 </li><li>同样颜色代码组成对采样管的采样管按顺序编号。 </li></ol></li></ol> 2.工作水下<ol><li> 工作现场准备 <ol><li> 初步调查和标本的选择 注:由于水下取样协议的复杂性，费尽准备必要的时间将保证一个有效的采样跳水。 <ol><li>调查工作网站，并提前必要的准备。 </li><li>选择和标记可相对容易地访问合适的靶生物。由于不是所有的生物体可能一定是活跃在采样潜水的时候，准备多个工作站比您预期品尝。 </li></ol></li><li> 基座支架的安装 <ol><li> WHEN个工作水平的基板上: <ol><li>安装在1公斤潜水加权柔性三角架的原始快速释放夹子和简单地旁目标动物定位。 </li></ol></li><li>当垂直的墙壁上工作: <ol><li>对于VacuSIP系统安装底座支撑板，挂钩附件齿轮和衣架在工作现场准备阶段（2.1.1）携带托盘收集容器。 </li><li>当使用柔性的便携式三脚架，使用螺栓或双组分环氧树脂固定旁边的每个目标动物10×10厘米的PVC板。每个板需要有一个孔，以连接所述柔性便携式三角架的快速释放的剪辑。 </li><li>一旦树脂固化和底座牢固地贴在墙壁上，拧在快速释放剪辑，作为一个坚定执着点为VacuSIP系统灵活便携三脚架。 </li></ol></li></ol></li></ol></li><li> 安装弗吉尼亚州CUSIP <ol><li>检查样品是否通过释放旁边的吸入孔过滤荧光素染料泵送和确认该染料是新兴通过呼出孔口如Yahel 等人所述（2005）16。 </li><li>安装VacuSIP设备，并将吸入（IN）采样管吸入剂孔内或只是在旁边（内约5毫米）。确保该吸入管是不是在另一个exhalant孔的附近。 </li><li>精心指导exhalant（EX）采样管向吸盘/ exhalant虹吸非常温和地将其插入，直到它被定位在吸盘/ exhalant虹吸（ 见图1和补充视频1）内1-5毫米。请特别注意不要使接触或以其他方式干扰采样的有机体。 </li><li>之前和期间的采样双重检查两个管的位置。 </li><li>取样检查后是否将标本仍然抽为DEScribed上述（2.2.1）。 注:由于三脚架的一只手臂的运动操纵其他可能发生的时候，一定要首先放在吸入采样管二来exhalant管，这就需要更精确的操控。按照此顺序，即使exhalant采样管的操作可能会导致吸入管的运动，也不会影响到采样。 </li></ol></li><li> 模块化水下取样程序 注:所研究的问题之前，下面的每个实验步骤可以作为一个独立的实验进行。下面描述的完整代谢分布采样协议是一个漫长的过程，需要高达每个试样8小时（对于概述参见图2和表2）。潜水条件和规定采样点，地区和机构之间的不同，潜水计划不包括在此协议。然而，投入非常谨慎和METIculous规划的潜水计划。要特别小心，以避免饱和度和YOYO潜水资料。如果可能，最好是进行这些实验在深度浅（&lt;10米）。闭路循环呼吸器可以这样长时间的抽样方案非常方便。 <ol><li>实际采样开始之前，确保残留荧光没有明显的痕迹依然和悬浮物已经解决或已经飘荡了。 </li><li> 超浮游生物，（没有过滤器安装在该步骤！） <ol><li>用针头刺破IN（吸入的）和EX（呼出）真空无菌塑料管隔。验证水计划的速度滴水，并收集2-6毫升的水样。 </li><li>检索时，保持样品中冰冷箱。在实验室中，保持用1％多聚甲醛+ 0.05％的EM级戊二醛（最终浓度），或0.2％的EM等级戊二醛。冷冻在液氮中并储存冷冻管在-80＃176; C，直到分析。 </li></ol></li><li> 硅酸盐采样和存储 <ol><li>安装含有针和在该管的远端的鲁尔阳连接器之间的0.2微米的聚碳酸酯膜的预清洗直列不锈钢的PC过滤器保持器。 </li><li>皮尔斯的预清洗的高密度聚乙烯瓶（HDPE瓶）的隔片盖开始采样。验证两个采样器滴落并收集15ml水中的每个小瓶中。 </li><li>保持冷藏样品（4℃），直到分析。如果分析不能半个月，卖场内开始在-20°C。为了分析，确保将样品在50℃下解冻，至少50分钟，以溶解硅石凝胶。 注:根据需要，膜可以保存镜检或DNA分析。 </li></ol></li><li> 溶解无机物（PO 4 3-，NO x的- ，NH 4 +） <ol><li>替代T.他的PC过滤器组件与包含一个燃烧预玻璃过滤器的预清洁直列不锈钢过滤器保持器。 </li><li>采样之前，确保至少20毫升海水样品均通过使用EPA，高密度聚乙烯小瓶或其他真空容器以开始抽吸通过整个过滤系统通过。测量所收集的水的体积被丢弃之前。 </li><li>皮尔斯适当的EPA玻璃小瓶的隔膜帽开始采样，验证两个采样器滴，收集25-30毫升硝酸盐和磷酸盐的分析。 </li><li>切换到新的EPA玻璃瓶中氨的分析，验证两个采样器滴，收集20毫升每个小瓶。 </li><li>保持在冰上并存储一个冷箱样品在-20℃，直到分析。 注意:如果只滤液是所关注的，一次性注射器过滤器，可用于步骤2.3.3和2.3.4。 </li></ol></li><li> 溶解的有机物（DOC + DON）采样和STO形圈 注意:保持的样品一样直立地处理整个使水样不来与硅隔膜接触。 <ol><li>继续使用不锈钢过滤器组件和收集20毫升海水样品进入新的EPA玻璃小瓶，如上所述。 </li><li>经检索，保留样品中冰冷箱。在实验室中，使用预燃烧玻璃巴斯德吸管，修正正磷酸的样品（添加25％的痕量金属级酸5-6滴入20ml的样品，终浓度为0.04％）或盐酸（加2滴痕量金属的级浓酸成20 ml样品，最终浓度0.1％），并保持冷藏。 </li><li>保持冷藏样品（4℃），直到分析。如果样品没有在-20°C，直到分析收集，存储的一个星期内进行分析。 </li></ol></li><li> 颗粒有机物（POC，PON，POP） <ol><li>继续使用STainless钢过滤器组件和过滤器的至少500毫升海水进入抽空250毫升真空烧瓶中。如有必要，更换瓶。 </li><li>在检索，使用空气充式注射器从过滤器支架撤出所有剩余的海水，用铝箔纸包起来，然后在冰上冷箱存放。在实验室中，从过滤器托架去除的过滤器，并在-20℃下直到分析存储。 </li></ol></li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54221/54221fig1.jpg" /> 图1. VacuSIP的正确安装的一个例子:用绿色吸入和黄色呼出的水样（照片取样使用带有色码定制的机械手的海鞘桐子mytiligera（亚喀巴湾，红海）（A）由汤姆Shelizenger和尤瓦Yacobi）; （ 二 ）抽样海绵Agelas oroides（NW地中海海）为3毫米的排水孔的宽度，使用VacuSIP设备。使用的颜色代码是黄色为吸入和红色呼出的水样。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54221/54221fig1large.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/54221/54221fig2.jpg" /> 图2.概述协议部分中描述的VacuSIP技术。该实验室的工作是在黄色方框表示，在蓝盒子实地调查。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54221/54221fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54221/54221table1.jpg" /> 表1.整体平均采样率（毫升分钟-1），不同的容器获得用于水集合和不同的真空度:烧瓶没有真空（无）; EPA玻璃小瓶和HDPE小瓶抽真空的容积（半体积）的一半;无菌塑料试管已经由生产抽真空。在5-8米深度，18-22℃的水温合作，利用79厘米长度25微米内径的PEEK管。 <img alt="表2" src="/files/ftp_upload/54221/54221table2.jpg" /> 表2取样容器中，固定剂，在线过滤器组件中，存储的概述和在协议部分中描述的分析方法。所分析的化合物是:超浮游生物丰度（浮游生物&lt;10微米），硅酸盐（氧化硅4），磷酸盐（PO 4 3-），亚硝酸盐+硝酸盐（NO 2 - + NO 3 - ），溶解有机物质（DOM），铵（NH + 4）和颗粒有机物质（POM）。所有取样容器有硅隔膜帽和取样之前抽真空。所述固定剂是:多聚甲醛+戊二醛（戊二醛+ Parafor），正磷酸（H 3 PO 4）和盐酸（HCl）。所使用的在线过滤器组件是:聚碳酸酯过滤器固定器和聚碳酸酯膜0.2μm的滤膜（PC过滤器保持器+ PC膜）和不锈钢过滤器固定器和无粘合剂的玻璃纤维过滤器GFF。

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The authors have nothing to disclose.

准备步骤 收集瓶的DOM和营养分析 因为集电极船只可与溶解微成分相互作用并采样壁可以是用于生长30-34，对DOM和养分收集不同小瓶测试细菌的底物。不建议二氧化硅定量33,35硼硅酸盐，因为玻璃瓶最多可将两折增加二氧化硅的初始浓度如果样品不能迅速冻结30。我们的结果表明，使用预清洁的EPA小...

底栖食悬浮在海洋生态系统1的功能发挥重要作用。通过筛选大量的水2,3，它们捞出分泌颗粒（浮游生物和碎屑）和溶解的化合物1（和参考文献），并底栖中上层耦合4,5和养分循环6,7的重要代理人。精确测量的微粒和溶解的化合物除去由底栖悬浮馈线（如海绵，海鞘，多毛，和双壳类）排泄是基本了解他们的生理，代谢和摄食生态。连同泵送率测量，这也使得能够通过这些生物体和水质以及对生态系统规模的过程及其生态影响介导的养分通量的定量。 选择测量颗粒去除和生产率和溶解融为一体适当的方法英镑由悬挂滤食性是获得关于它们的觅食活动8可靠的数据至关重要。如通过Riisgård和其他不适当方法偏压结果指出，扭曲的实验条件下，产生摄取和某些物质排泄的不正确的估计，并能导致由这些生物处理的养分通量的错误的定量。 两个最频繁使用的方​​法来测量滤食微粒和溶解养分通量涉及要么孵化（间接技术）或环境的同时收集和呼出的水（直接技术）。孵化技术基于测量的变化率中的颗粒在温育水的浓度和溶解的营养物，并估计生产或除去速率相比充分控制8。然而，在孵育室包围生物体可以改变其还精g且泵送行为由于自然流动状态的变化，是由于在氧和/或在食物浓度，或由于在孵化水中7,9-排泄化合物（和其中的参考文献）的积累减少。除了 ​​分娩和改性水供给的影响，温育技术的重大偏差，从重新过滤效果（参见例如10）的茎。虽然其中一些方法问题已经通过使用合适的体积和形状的孵化容器11的或在原位 12引入再循环钟罩系统克服，这种技术经常低估去除和生产率。量化溶解化合物如溶于有机氮（DON）和碳（DOC）或无机营养素的代谢，已被证明是特别容易引起孵化技术13偏见。 在60年代末和70年代初，亨利·Reiswig9,14,15开创了直接技术的应用巨加勒比海海绵，量化通过单独采样，原位生物体吸入呼出的水粒子去除。由于施加在较小悬浮馈线和在更具挑战性的水下条件Reiswig的技术难度，研究在本领域的大部分仅限于实验室（体外 ）使用大多间接孵化技术16。 Yahel和同事改装Reiswig的直接原位技术，规模较小的条件下工作。他们的方法，被称为INEX 16，是基于原状生物吸入（In）和呼出（出）水的同时水下采样。不同浓度的一对样品（INEX）之间的物质（ 例如 ，细菌）的提供由动物的该物质的保持率（或生产）的量度。该INEX技术采用开放式的管依赖于所研究的生物体的泵送活动产生被动替换环境中的水收集管中的出水管喷射。而Yahel和同事在15种以上不同的悬浮液的研究已经成功地应用这种技术进料器类群（ 例如 ，17），该方法是由所需的高级别实践经验的制约，一些出水管孔的微小的尺寸，并通过海况。 为了克服这些障碍，我们开发的基础上，通过细管的水样的吸控制（外径&lt;1.6毫米）的替代技术。我们的目标是创造一种简单，可靠，廉价的设备，允许清洁和从一个非常具体的点， 在原位水采样控制如底栖悬浮馈线的出水管孔口。是有效的，该方法具有以非侵入以免影响周围流动状态或修改将b所研究的生物ehavior。这里介绍的设备被称为VacuSIP。它是由Yahel 等人开发的SIP系统的简化。 （2007年）18在深海ROV基于点采样。该VacuSIP是比原来的SIP相当便宜，它已被改编为根据SCUBA工作。该系统是根据用于实验室环境提出并通过Wright和斯蒂芬（1978）19和Møhlenberg和Riisgård（1978）20测试原理设计。 虽然VacuSIP系统是在海底悬浮馈线的代谢的原位研究设计用于，它也可用于实验室研究和任何需要控制和清洁，点源的水样。需要在延长的时间段（最小-小时） 或原位抽滤集成系统时是特别有用的。该VacuSIP已在2011年以来Yahel实验室成功地使用，并且还在最近的两个由加勒比和地中海海绵21种营养介导通量研究被使用（莫尔甘蒂等人提交）。 使用特定的采样器，该延长取样持续时间，和现场条件下，在被施加VacuSIP，而承受从标准海洋学协议一些偏差，收集，过滤，并存储样本敏感的分析物。由VacuSIP系统或水样的变形例的由收集后细菌活性的风险降低污染的风险，我们测试原位过滤和存储程序的各种。不同的过滤器，收集容器，并存储程序，以实现溶解无机（PO 4 3-，NO x的- ，NH + 4的SiO 4）的分析中最适合的技术进行了检查，有机（DOC +，DON）化合物，和超浮游生物（&lt;1081 M）和颗粒有机（POC + PON）采样。为了进一步减少污染的风险，尤其是在野外条件下，处理步骤的数量减少到最低限度。其中提出的方法的可视格式被定向，以促进再现性并减少以有效地应用该技术所需的时间。 系统总览 在从具有小到2毫米exhalant孔悬浮液供料器原位泵送水样，每个试样的泵送活性首先通过释放过滤荧光染色海水可视的吸入口（S）和观察从出水管孔16其流动旁（也参见图2B中18）。在研究样品（incurrent和出水管）吸入和呼出的水，然后同时通过使用一对安装在定制操纵分钟管的或在两个所述"芳的采样毫秒"倒置柔性便携式三角架的（ 图1和补充视频1）。在研究有机体吸入的水通过一管的近端仔细定位的内部或附近的研究生物的吸入孔收集。一个恒然后管被定位在出水管孔口内。此操作需要良好小心，以避免接触或动 ​​物的干扰， 例如 ，通过沉淀再悬浮。为了开始采样，潜水员刺穿在收集容器与连接到注射针的隔膜每个管的远端，允许外部水压力迫使采样水进入通过采样管的容器中。抽吸通过先前在小瓶中产生的真空和通过外部水和抽空样品容器之间的压力差启动。 为确保呼出的水干净收集和避免AMBI意外吸耳鼻喉科水16时，水的采样速率需要被保持在比所述出水管流速的显著降低率（&lt;10％）。抽吸速率由管的长度和它的内直径（ID）的控制。小内径也保证了可以忽略不计的死体积（&lt;每管200米微升）。采样过长时间的（几分钟到几小时）使得有可能感兴趣的大多数物质的固有斑块集成。以确保样品充分保留在延长的水下采样会话以及用于运输到实验室， 在原位过滤推荐用于敏感的分析物。取样容器，过滤组件和管道的选择是通过研究生物和具体的研究问题所决定的。下面描述的协议假定一个完整代谢分布是感兴趣（查看概述参见图2）。然而，协议的模块化特性允许˚F否则容易修改，以适应更简单，甚至非常不同的抽样方案。对于一个完整的代谢分布，采样协议应该包括以下步骤:（1）流量可视化; （2）取样超浮游生物馈送（浮游生物&lt;10微米）; （3）采样无机养分的吸收和排泄（使用在线过滤器）; （4）采样溶解有机的吸收和排泄（使用在线过滤器）; （5）颗粒喂养和排泄（使用在线过滤器）; （6）重复步骤2（超浮游生物喂养的质量检查）; （7）流量的可视化。 当后勤可行的，则建议的代谢分布测量用泵送速率组合（ 例如，染料前端速度的方法，在16）以及与呼吸测量。这些测量是在取样会话的开始和结束时最好服用。为呼吸测量，水下光极或微电极是优选的。

<table><tbody><tr><td>GorillaPod, Original</td><td>Joby</td><td>GP000001</td><td>flexible portable tripod&amp;nbsp;</td></tr><tr><td>Flangeless Ferrule</td><td>IDEX Health &amp;amp; Science&amp;nbsp;</td><td>P-200X</td><td>1/16&quot; in Blue/pk</td></tr><tr><td>Male Nut</td><td>IDEX Health &amp;amp; Science</td><td>P-205X&amp;nbsp;</td><td>1/16&quot; in Green/10&amp;nbsp;pk</td></tr><tr><td>Female to Female Luer</td><td>IDEX Health &amp;amp; Science&amp;nbsp;</td><td>P-658</td><td></td></tr><tr><td>Female-Male Luer</td><td>IDEX Health &amp;amp; Science&amp;nbsp;</td><td>P-655</td><td></td></tr><tr><td>Peek Tubing (254&amp;nbsp;&amp;micro;m ID)</td><td>IDEX Health &amp;amp; Science&amp;nbsp;</td><td>1531</td><td>1/16&quot; OD x 0.01&amp;nbsp;in ID x 5&amp;nbsp;ft length. Alternative ID can be used.</td></tr><tr><td>Two component resin epoxy</td><td>IVEGOR</td><td>9257</td><td>Mix well the two component resin before use</td></tr><tr><td>(TOC) EPA Vials</td><td>Cole -Parmer</td><td>03756-20</td><td>40 ml glass vials. Manufactured also by Thomas Scientific (ref. number 9711F09).</td></tr><tr><td>HDPE Vials</td><td>Wheaton</td><td>986701 (E78620)</td><td>20 ml high-density polyethylene vials</td></tr><tr><td>Vacuette Z no additive</td><td>Greiner bio-one</td><td>455001</td><td>pre-vacuum by the manufacturer&amp;nbsp;</td></tr><tr><td>Septum Sample Bottles</td><td>Thomas Scientific</td><td>1755C01</td><td>250 ml glass bottles&amp;nbsp;</td></tr><tr><td>Septum Cap 1</td><td>Wheaton</td><td>W240844SP (E7865R)</td><td>22-400 for HDPE vials&amp;nbsp;</td></tr><tr><td>Septum Cap 2</td><td>Wheaton</td><td>W240846 (1078-5553)</td><td>24-400 for glass vials and bottles. Also manufactured by Thermo Scientific National (ref. 03-377-42).</td></tr><tr><td>In-line stainless steel Swinney Filter holders</td><td>Pall</td><td>516-9067</td><td>13 mm of diameter</td></tr><tr><td>PTFE Seal Washer</td><td>Pall&amp;nbsp;</td><td>516-8064</td><td>ring for stainless steel filter holders</td></tr><tr><td>TCLP Glass Filters</td><td>Pall&amp;nbsp;</td><td>516-9126</td><td>binder-free glass fiber filters, 13&amp;nbsp;mm of diameter, pore size 0.7&amp;nbsp;&amp;micro;m</td></tr><tr><td>Polycarbonate Filter Holders</td><td>Cole -Parmer</td><td>17295</td><td>13 mm of diameter</td></tr><tr><td>Isopore Membrane Filters</td><td>Millipore</td><td>GTTP01300</td><td>13&amp;nbsp;mm of diameter, pore size 0.2&amp;nbsp;&amp;micro;m</td></tr><tr><td>Contrad 70 Solution&amp;nbsp;</td><td>Decon Labs</td><td>1002</td><td>highly soluble basic mix of anionic and non-ionic surfactant solution&amp;nbsp;</td></tr><tr><td>Sterile Syringe Filters</td><td>VWR International Eurolab S.L.</td><td>514-0061P</td><td>25&amp;nbsp;mm of diameter , pore size 0.2&amp;nbsp;&amp;micro;m</td></tr><tr><td>Fluorescein</td><td>Sigma-Aldrich</td><td>(old ref.28802) 46955-100G</td><td>100&amp;nbsp;g</td></tr><tr><td>Holdex, disposable,sterile</td><td>Greiner bio-one</td><td>450263</td><td>sterile, single-use tube holder with off-center luer for Vacuette</td></tr><tr><td>Sterile Needles</td><td>IcoGammaPlus</td><td>5160</td><td>0.7&amp;nbsp;mm x 30&amp;nbsp;mm</td></tr><tr><td>Cryovials Nalgene</td><td>Nalgene</td><td>V5007(Cat. No.5000-0020)</td><td>2&amp;nbsp;ml</td></tr><tr><td>Cryobox carton&amp;nbsp;</td><td>Rubilabor</td><td>M-600</td><td>145x145x55&amp;nbsp;mm p/microtube 1.5&amp;nbsp;ml</td></tr><tr><td>Orthophosphoric Acid</td><td>Sigma</td><td>79617 or Alternatively use Ultra-Pure Hydrochloric acid, final concertation 0.1%</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>500&amp;nbsp;g</td></tr><tr><td>Glutaraldehyde</td><td>Merck</td><td>8,206,031,000</td><td>25%, 1 L</td></tr><tr><td>Hand Vacuum Pump&amp;nbsp;</td><td>B&amp;uuml;rkle&amp;nbsp;</td><td>5620-2181 or Alternatively use standard laboratory vacuum pump</td><td></td></tr></tbody></table>

vacusip, an improved inex method for in situ measurement of particulate and dissolved compounds processed by active suspension feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

海水收集方法优化 收集瓶和清洗程序的选择 VacuSIP兼容收集容器应具有一个隔膜，允许采样到通过用注射器针头刺入发起。他们应该承受较高压力的水下（2-3条在工作深度潜水典型），并应维持真空。许多（但不是所有品牌）的挥发性有机物的分?...

Watch this Scientific Journal Video about VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders at JoVE.com

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

vacusip-an-improved-inex-method-for-situ-measurement-particulate

Universidad San Sebastian

Research

JoVE Journal

Environment

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

10.8K 次观看.  Centre d’Estudis Avançats de Blanes (CEAB-CSIC). We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

视频: VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.
We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.

定量测量原位流动，使用一个自包含的水下测速仪（SCUVA）

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

科研

生物工程

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

清洁取样和痕量金属研究河和河口水域分析

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

环境科学

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

农业流域营养盐和泥沙的连续河道监测

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

在排水过程中捕获农用运河中的流动加权水和悬浮颗粒

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality.

春季池塘连续水文水质监测

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

海洋浮游生物和悬浮颗粒的超洁净多层采集装置

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

海上双壳贝类悬浮饲料定量装置的设计与应用

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

在西图化学分析中，以检验水生生态系统中的微生物行为

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip (LOC) system. The simultaneous excitation of the fundamental frequency and its third harmonic, which is termed as dual-frequency excitation, to a pair of interdigital transducers (IDTs) could generate a new type of standing acoustic waves in a microfluidic channel. Varying the power and the phase in the dual-frequency excitation signals results in a reconfigurable field of the acoustic radiation force applied to the microparticles across the microchannel (e.g., the number and location of the pressure nodes and the microparticle concentrations at the corresponding pressure nodes). This article demonstrates that the motion time of the microparticle to only one pressure node can be reduced ~2-fold at the power ratio of the fundamental frequency greater than ~90%. In contrast, there are three pressure nodes in the microchannel if less than this threshold. Furthermore, adjusting the initial phase between the fundamental frequency and the third harmonic results in different motion rates of the three SSAW pressure nodes, as well as in the percentage of microparticles at each pressure node in the microchannel. There is a good agreement between the experimental observation and the numerical predictions. This novel excitation method can easily and non-invasively integrate into the LOC system, with a wide tenability and only a few changes to the experimental set-up.

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

双频激励下驻面声波的微粒操纵

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip ...

工程学

Dissolved Solute Sampling Across an Oxic-Anoxic Soil-Water Interface Using Microdialysis Profilers

Biogeochemical processes shift rapidly in both spatial (millimeter scale) and temporal (hour scale to day scale) dimensions at the oxic-anoxic interface in response to disturbances. Deciphering the rapid biogeochemical changes requires in situ, minimally invasive tools with high spatial and temporal sampling resolution. However, the available passive sampling devices are not very useful in many cases either due to their disposable nature or the complexity and extensive workload for sample preparation.
To address this problem, a microdialysis profiler with 33 individual polyethersulfone nanomembrane tubes (semipermeable, &#60;20 nm pore size) integrated into the one-dimensional skeleton (60 mm) was established to iteratively sample the dissolved compounds in porewater across the soil-water interface at a high resolution of 1.8 mm (outer diameter plus one spacing, i.e., 0.1 mm between probes). The sampling mechanism is based on the principle of concentration gradient diffusion. The automatic loading of degassed water allows minimal disturbance to the chemical species across the oxic-anoxic interface.
This paper describes the procedures of device setup and continuous porewater sampling across the soil-water interface on a daily basis. Concentration-depth profiles were selectively measured before (on Day 6) and after (on Day 7) disturbances induced by irrigation. The results showed that concentration-depth profiles were undergoing rapid changes, especially for redox-sensitive elements (i.e., iron and arsenic). These protocols can help investigate the biogeochemical responses across the soil-water interface under various disturbances caused by physical, chemical, and biological factors. The paper thoroughly discusses the advantages and disadvantages of this method for potential use in the environmental sciences.

A microdialysis profiler is described to sample dissolved porewater solutes across an oxic-anoxic soil-water interface in situ with minimal disturbance. This device is designed to capture rapid changes in concentration-depth profiles induced by disturbances at the soil-water interface and beyond.

使用微透析剖面仪在氧化-缺氧土壤-水界面上进行溶解溶质采样

Biogeochemical processes shift rapidly in both spatial (millimeter scale) and temporal (hour scale to day scale) dimensions at the oxic-anoxic interface ...

VacuSIP，对改进的方法INEX

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