这项研究得到了捷克科学基金会的支持，分别向K.和D.S.颁发了13-00243S和19-16554S的研究补助金。本文还得到由欧洲区域发展基金资助的"生物操作作为改善水坝水库水质的工具"项目（无CZ.02.1.01/0.0/0.0/0.0/16_025/0007417）项目的支持，该项目由欧洲区域发展基金资助，用于业务方案研究、开发和教育。

1. 样本收集
<ol><li>
水库水样收集：第一个案例研究（Exp I;低天然原位捕食者和猎物丰度系统）<ol>
<li>以适当的深度从所需位置收集水样。在运送到实验室过程中，将样品保存在温度控制的冷却器中，在原位温度下填充（避免温度冲击;应注意，原生动物的吸收率取决于温度）。 注：我们的取样是在中富营养峡谷形的切莫夫水库（南波希米亚，体积34.5 x 106 m3，最大深度43米，平均保留时间100天，二分）。取样地点位于30米深的大坝附近。2-l Friedinger 取样器采集了深度为 0.5 m 的混合样品。</li>
		<li>尽快继续第 1.2 节。</li>
	</ol>
</li>
	<li>
从食肉性尿反射植物的陷阱中收集液体：第二个案例研究（Exp II;具有高捕食者和猎物丰度、极小样本体积的系统）<ol>
<li>轻轻摇动水下的植物，从栽培容器中取出，放在吸水材料上吸收多余的水。根据植物的坚固性和陷阱的大小，选择8-10个植物个体。</li>
		<li>通过计算承载陷印器官的叶节点，将每个拍摄划分为大致相等的部分。每个拍摄部分将起到收集代表年轻、中年和旧陷阱的混合样本。</li>
		<li>将薄玻璃毛细管和 Eppendorf 小瓶连接到蠕动泵，用于收集样品。继续将毛细管尖端插入陷阱开口。使用真空泵，从每个疏水阀中吸出所有液体，直到为每个疏水阀年龄类别收集 900 ± 100 μL 的疏水阀液。</li>
		<li>使用池陷井液的200 μL的三联体子样品进行原石放牧实验。立即处理这些，详见第 3 节。保留样品的剩余 ±300 μL，用于对流体中微生物成分进行所有其他分析，详情如下（第 2 节）。</li>
		<li>立即转到第 2 节。</li>
	</ol>
</li>
</ol>2. 采集的样品的固定
<ol><li>Exp I 和 II：将水/陷阱液子样本固定在细菌枚举（第 4 节;分别为约 20 mL 和 0.3 mL）中，使用甲醛至少 1 小时，以获得 2% 的最终体积：每个样品的体积浓度。 注意：在烟罩中专门处理甲醛，并在操作样品时随时戴上手套。</li>
</ol>3. 样品过滤
<ol><li>样品稀释（Exp I）：储层水样无需稀释。（Exp II）：用无颗粒MQ水稀释疏水液样品10x-100x，在通过荧光显微镜计数之前，在过滤表面实现目标微生物的适当分布。</li>
	<li>使用过滤漏斗（直径 25 mm）过滤储液罐水的 1-2 mL （Exp I） 或疏水阀液子样品的 10-30 μL （Exp II），以便细菌计数到黑色 0.2 μm 孔径过滤器上。</li>
	<li>用DAPI（4'，6-二酰胺-2'-苯林多尔二盐，0.2%最终浓度）染色过滤器4分钟。 注意：避免皮肤和工作表面污染，并戴上手套。</li>
	<li>将装有浓缩微生物的过滤器放在显微镜幻灯片上的一滴浸入油（用于荧光显微镜）。将另一个油滴放在滤清器的中心，并盖上盖玻片，确保机油均匀分布。</li>
	<li>此时，最好立即处理样品，或者将其储存在冷冻室（-20°C）中数周至数月，直至进一步分析。</li>
</ol>4. 在过滤器上统计细菌数量
<ol><li>将幻灯片放在荧光显微镜下（过滤器设置与荧光DAPI相对应）。将 10 x 10 计数网格放入其中一个眼部。将幻灯片移动到随机位置。</li>
	<li>量化计数网格区域（放大倍数 1000x 下的细菌细胞（蓝色荧光）。在计数中，包括跨越计数网格左边缘和上边缘的单元格，同时排除位于右边缘和下边缘的单元格。</li>
	<li>移动到另一个随机位置，并在至少 10-15 个计数网格上重复枚举，总计 500 个单元格计数。</li>
	<li>根据对一个网格面积与过滤器总有效过滤面积的比率的了解，为给定显微镜和放大倍率建立转换系数。然后，将计数的细胞总数除以计数的网格数，每个网格产生平均细菌数。</li>
	<li>将后一个参数乘以建立的转换系数，并标准化样品每 mL 的结果数（取决于过滤的样本体积），以获得每 mL 的总细菌丰度。</li>
</ol>5. 确定原丰度
<ol><li>用谷醛（最终浓度为 1%，更适合含有叶绿素的颗粒在固定后数天到数周）固定水样（Exp I）或疏水液 （Exp II） 子样品，或使用在下面指定的二硫盐脱色技术。 注：这两种保存技术都防止从原药学家的食物中摄取的物分8。</li>
	<li>关于脱色剂脱色技术，将100μL/1 μL的Lugol溶液加入20mL/200 μL的水/陷阱流体亚样品（分别为Exp I/Exp II）。</li>
	<li>紧接着加入0.5 mL/50 μL的硼酸盐缓冲形式素，然后加入20μL/2μL的3%硫磺酸钠（分别为Exp I/Exp II）。 注：硫磺酸钠使Lugol的黄色变色，从而能够在荧光显微镜8下观察细胞。</li>
	<li>将已知样品体积（取决于目标原体数量）过滤到 1 μm 孔径黑色聚碳酸酯过滤器上。</li>
	<li>估计孕激素物种的数量是通过使用计数网格在放大倍数 600 倍下计算至少 200 个细胞（见上文）。</li>
	<li>将过滤漏斗中真空下的样品体积降低至约 2 mL。然后，释放压力下，加入DAPI氟铬（4'，6-二酰胺-2-苯二氯盐13，0.2%最终浓度）2分钟。</li>
</ol>6. 确定浮游生物样本中西利亚特动物的社区结构
注：淡水栖息地的西栖菌群落高度多样化，14、15、16、18，其微观测定具有挑战性。将纤毛组分为功能公会10、14、16、17，可以更详细地分析作为中上层细菌的不同纤细组。
<ol><li>通过结合以下两方面来评估纤毛社区结构：<ol>
<li>荧光显微镜中DAPI染色的样品（用不同尺寸和形态的宏观和微核的明亮荧光对纤细细胞进行局部化）与荧光标记细菌的吸收相结合（FLB8;详情，见下文），跟踪西利特以细菌为食的能力。</li>
		<li>在选定情况下进行现场抽样观察17，19。有关用于将纤毛分类分组到不同分类类别中的上述方法和标准的更多详细信息，请参阅以前的出版物16、17。 注：引用的研究表明，在硅酸中，来自锡科特里奇亚（属哈尔特里亚和佩拉戈哈尔特里亚）和奥利戈特里西亚（即里莫斯特龙比姆spp.）的杂食物种是在绝大多数淡水栖息地的浮游生物10，17，18。</li>
	</ol>
</li>
</ol>7. 估计西利特放牧率
<ol><li>根据示踪剂平均数量的变化（即每只毛草与孵育时间（5-15分钟）相关的FLB8]和添加的FLB的示踪量计算菌的取药率，最多占细菌总数的5%-15%。</li>
	<li>为了比较不同物种的接受率，将摄入率标准化为每小时每毛质动物的细菌数量，并根据实际孵育时间和添加的示踪剂FLB的比例进行计算。 注：FLB方法应用的一般方案，用于估计自然样本中细胞或物种特定和散装细菌率，如图1所示。</li>
	<li>从本地细菌菌株制备FLB到淡水环境8<ol>
<li>选择适当的大小（平均细胞体积 + MCV）和细菌的形态，以便它们有效地模仿被调查的水生系统中浮游杆菌/细菌细胞的典型大小。 注：对于Exp I，使用了从林诺比坦和多核细胞属的研究位置分离的菌株混合物（即，湖泊和池塘中典型的、高度丰富的浮游杆菌）20 。有关菌株形态和大小的详细信息，请参阅以前的出版物3、17、18、21。</li>
		<li>在早期固定阶段，通过离心（5，000 x g）从培养中收获细菌细胞15分钟，并在与所选位置的典型MCV细菌对应的混合物中以数值比例混合它们， 产生MCV + SD。</li>
		<li>将颗粒悬浮在10 mL的磷酸盐缓冲盐水中（PBS;pH = 9）。</li>
		<li>在磷酸盐盐缓冲液中向细胞悬浮液中加入2mg的黄绿色荧光染料5-（4，6-二氯三聚二苯-2-yl）氨基氟辛（DTAF，与蛋白质结合），并在60°C水浴中孵育2小时。</li>
		<li>孵育后，将细胞向下离心机，分解DTAF溶液，用PBS清洗和离心机3x。</li>
		<li>最后洗涤后，在PPi-盐碱缓冲液的20 mL中重新悬浮细胞。</li>
		<li>将FLB悬浮液和移液器1.5 mL等分液涡到2 mL低温瓶中，然后在PPi-盐碱缓冲液中保持冷冻（在-20°C下），直到使用。</li>
		<li>通过 0.2 μm 聚碳酸酯过滤器预过滤 PPi-盐碱缓冲液，用于下一步。</li>
		<li>要确定FLB浓度，将一个小等分（通常为20-40μL）转移到2 mL的无颗粒PPi-盐碱缓冲液，在30W下声波几次2s爆发，并过滤到0.2μm聚碳酸酯黑色过滤器上，通过荧光进行枚举在 DTAF （448 nm/520-540 nm） 的光学滤镜设置下进行显微镜（1，000 倍放大率）。</li>
	</ol></li>
	<li>用于估计西利茨细菌的跟踪器技术<ol>
<li>在天然木板栖息地进行放牧实验时，将300 mL样品放入经过良好冲洗的1 L瓶中，并在原位温度下孵育15分钟（让原生体从处理冲击中恢复）。</li>
		<li>添加FLB示踪剂，以占细菌总数的5%-15%，根据季节和水温增加量。 注： 在纤毛酸盐物种特定摄取率中，有非常广泛的、依赖于季节的光谱，跨越几个数量级（即，从 101-104细菌西利酸盐-1每小时）10、16 17，18，22，23，24。</li>
		<li>在具有高接受率的西利酸盐发生期间（通常在夏季），还运行平行孵育，FLB添加量非常低，仅占细菌总数的2%-4%，以避免由示踪剂FLB过度加载西利酸盐（参见图2中的示例 。</li>
		<li>用FLB孵育西利酸盐/浮游生物样品5-15分钟。</li>
		<li>有两种机会的样品固定，以防止从食物的食样剂的摄入材料的吸收8。通过添加 1% 谷醛（最终浓度更适合含叶绿素颗粒的样品，如藻类）来终止孵育。或者，将100μL/10 μL的Lugol溶液放入20 mL/200 μL的水/陷阱流体亚样品中，然后立即加入0.5 mL/10 μL的硼酸盐缓冲形式素，然后加入200 μL/2 μL的3%硫硫酸钠（Exp I/Exp II）。</li>
	</ol></li>
	<li>加入固定剂后，让样品在黑暗中在4°C下至少休息1小时，以确保完全保存纤毛细胞。</li>
	<li>从4-30 mL/10-30 μL（Exp I/Exp II;体积取决于西柳石丰度）和DAPI染色（最终浓度0.2%wt/vol;详情见上文步骤3.2）采集天然浮游生物子样本。</li>
	<li>通过 1 μm 黑色过滤器，并通过荧光显微镜检查，以计数纤毛（600 倍放大率），并列举摄念的FLB示踪剂数量（大部分以 1000 倍放大率），如前几篇出版物2、17 中详述的.保存后7天内检查样品。</li>
	<li>要估计原生动物/物种特异性放牧的总数量，请将所有西毛动物的平均接受率，或仅按原地丰度检测到的西利酸盐物种进行繁殖。</li>
	<li>从切莫夫水库的原位数据计算每单元的取水率的例子如下：<ol>
<li>假设细菌浓度为 3.55 x 106细菌/mL，添加示踪剂 FLB 为 0.25 x 106 FLB/mL，得出总细菌颗粒（天然细菌 + FLB = 100% 的猎物颗粒）的总和 3.8 x 106细菌/mL可用于天然样品中的噬菌体原生。 注：因此添加的FLB示踪剂代表细菌颗粒总量的6.58%（项目为0.25/0.038）。在5分钟的孵育中，每个哈尔特里亚sp的平均FLB为6.2FLB。</li>
		<li>要使每小时的摄入正常化，请使用以下计算：（6.2 x 12）/（6.58/100） = 每哈尔特里亚细胞/小时 1131 细菌。 注：有关在可变水温下检测到的Halteria sp.的单个摄取率分布的更多示例、添加的示踪剂FLB的量（百分比）以及FLB的不同孵育时间，请参见图3。</li>
	</ol></li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+DAPI+for+identifying+and+counting+aquatic+microflora.">The use of DAPI for identifying and counting aquatic microflora.</a> Limnology and Oceanography. 25, 943-948 (1980).</li><li>Foissner, W., Berger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user-friendly+guide+to+the+ciliates+(Protozoa,+Ciliophora)+commonly+used+by+hydrobiologists+as+bioindicators+in+rivers,+lakes,+and+waste+waters,+with+notes+on+their+ecology.">A user-friendly guide to the ciliates (Protozoa, Ciliophora) commonly used by hydrobiologists as bioindicators in rivers, lakes, and waste waters, with notes on their ecology.</a> Freshwater Biology. 35, 375-482 (1996).</li><li>Foissner, W., Berger, H., Schaumburg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+ecology+of+limnetic+plankton+ciliates.">Identification and ecology of limnetic plankton ciliates.</a> Informationsberichte des Bayer Landesamtes f&#252;r Wasserwirtschaft Heft. , 3-99 (1999).</li><li>&#352;imek, K., 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=Ciliate+grazing+on+picoplankton+in+a+eutrophic+reservoir+during+the+summer+phytoplankton+maximum:+a+study+at+the+species+and+community+level.">Ciliate grazing on picoplankton in a eutrophic reservoir during the summer phytoplankton maximum: a study at the species and community level.</a> Limnology &amp; Oceanography. 40, 1077-1090 (1995).</li><li>Skibbe, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+quantitative+protargol+stain+for+ciliates+and+other+planktonic+protists.">An improved quantitative protargol stain for ciliates and other planktonic protists.</a> Archiv f&#252;r. Hydrobiolgie. 130, 339-347 (1994).</li><li>Macek, M., 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=Growth+rates+of+dominant+planktonic+ciliates+in+two+freshwater+bodies+of+different+trophic+degree.">Growth rates of dominant planktonic ciliates in two freshwater bodies of different trophic degree.</a> Journal of Plankton Research. 18, 463-481 (1996).</li><li>Šimek, K., 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=Microbial+food+webs+in+hypertrophic+fishponds:+omnivorous+ciliate+taxa+are+major+protistan+bacterivores.">Microbial food webs in hypertrophic fishponds: omnivorous ciliate taxa are major protistan bacterivores.</a> Limnology &amp; Oceanography. , (2019).</li><li>Jezbera, J., 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=Major+freshwater+bacterioplankton+groups:+Contrasting+trends+in+distribution+of+Limnohabitans+and+Polynucleobacter+lineages+along+a+pH+gradient+of+72+habitats.">Major freshwater bacterioplankton groups: Contrasting trends in distribution of Limnohabitans and Polynucleobacter lineages along a pH gradient of 72 habitats.</a> FEMS Microbiology Ecology. 81, 467-479 (2012).</li><li>Kasalick&#253;, V., 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=The+diversity+of+the+Limnohabitans+genus,+an+important+group+of+freshwater+bacterioplankton,+by+characterization+of+35+isolated+strains.">The diversity of the Limnohabitans genus, an important group of freshwater bacterioplankton, by characterization of 35 isolated strains.</a> PLoS One. 8, 58209 (2013).</li><li>Stabell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliate+bacterivory+in+epilimnetic+waters.">Ciliate bacterivory in epilimnetic waters.</a> Aquatic Microbial Ecology. 10, 265-272 (1996).</li><li>Zingel, P., 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=Ciliates+are+the+dominant+grazers+on+pico-+and+nanoplankton+in+a+shallow,+naturally+highly+eutrophic+lake.">Ciliates are the dominant grazers on pico- and nanoplankton in a shallow, naturally highly eutrophic lake.</a> Microbial Ecology. 53, 134-142 (2007).</li><li>Bickel, S. L., Tang, K. W., Grossart, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliate+epibionts+associated+with+crustacean+zooplankton+in+german+lakes:+distribution,+motility,+and+bacterivory.">Ciliate epibionts associated with crustacean zooplankton in german lakes: distribution, motility, and bacterivory.</a> Frontiers in Microbiology. 3 (243), (2012).</li><li>Sirov&#225;, D., 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=Hunters+or+gardeners?+Linking+community+structure+and+function+of+trap-associated+microbes+to+the+nutrient+acquisition+strategy+of+a+carnivorous+plant.">Hunters or gardeners? Linking community structure and function of trap-associated microbes to the nutrient acquisition strategy of a carnivorous plant.</a> Microbiome. 6, 225 (2018).</li><li>&#352;imek, K., 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=Ecological+traits+of+a+zoochlorellae-bearing+Tetrahymena+sp.+(Ciliophora)+living+in+traps+of+the+carnivorous+aquatic+plant+Utricularia+reflexa.">Ecological traits of a zoochlorellae-bearing Tetrahymena sp. (Ciliophora) living in traps of the carnivorous aquatic plant Utricularia reflexa.</a> Journal of Eukaryotic Microbiology. 64, 336-348 (2017).</li><li>Pitsch, G., 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=The+green+Tetrahymena+utriculariae+n.+sp.+(Ciliophora,+Oligohymenophorea)+with+its+endosymbiotic+algae+(Micractinium+sp.),+living+in+the+feeding+traps+of+a+carnivorous+aquatic+plant.">The green Tetrahymena utriculariae n. sp. (Ciliophora, Oligohymenophorea) with its endosymbiotic algae (Micractinium sp.), living in the feeding traps of a carnivorous aquatic plant.</a> Journal of Eukaryotic Microbiology. 64, 322-335 (2017).</li><li>Nielsen, J. M., Clare, E. L., Hayden, B., Brett, M. T., Kratina, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+tracing+in+ecology:+Method+comparison+and+selection.">Diet tracing in ecology: Method comparison and selection.</a> Methods in Ecology and Evaluation. 9, 278-291 (2018).</li><li>Beisner, B. E., Grossart, H. P., Gasol, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+methods+for+estimating+phago-mixotrophy+in+nanophytoplankton.">A guide to methods for estimating phago-mixotrophy in nanophytoplankton.</a> Journal of Plankton Research. , 1-13 (2019).</li><li>Dolan, J. D., &#352;imek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+ingested+matter+in+Strombidium+sulcatum,+a+marine+ciliate+(Oligotrichida).">Processing of ingested matter in Strombidium sulcatum, a marine ciliate (Oligotrichida).</a> Limnology and Oceanography. 42, 393-397 (1997).</li><li>Massana, R., 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=Grazing+rates+and+functional+diversity+of+uncultured+heterotrophic+flagellates.">Grazing rates and functional diversity of uncultured heterotrophic flagellates.</a> The ISME Journal. 3, 588-596 (2009).</li><li>Gruj&#269;i&#263;, V., 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=Cryptophyta+as+major+freshwater+bacterivores+in+experiments+with+manipulated+bacterial+prey.">Cryptophyta as major freshwater bacterivores in experiments with manipulated bacterial prey.</a> The ISME Journal. 12, 1668-1681 (2018).</li></ol>

作者没有什么可透露的。

在水生系统中破译营养相互作用总是具有挑战性的28，特别是在纳米浮游生物秤涉及原发动物和他们的猎物，细菌。在营养摄入途径和定量方面，由于生物相互作用的复杂性高，在高营养水平下成功使用的方法的应用的可能性较小。例如，这些方法包括稳定的同位素标记方法。该协议显示了使用荧光显微镜和荧光标记细菌作为示踪剂来跟踪和半定量/估计碳流动的优点（细菌猎物?...

异养原核生物是水生系统中的关键生物成分，在浮游生物生物量1、2、3中占很大比例。控制其丰度、多样性和活性的因素对于理解其在生物地球化学循环中的作用（即有机碳和其他营养物质的命运以及从原核生物到更高的营养水平的能量流动）至关重要。原生动物放牧是这些重要因素之一。异营养纳米旗和纤毛的细菌对原核生物丰度、群落功能、结构、多样性，甚至细胞形态和特定细菌群的生长速率施加了强大的自上而下的控制。 5，6.在一些系统中，原发学家是细菌死亡的主要原因6，7。
用于评估原生动物细菌的标准方法，已经使用了一段时间，涉及使用荧光标记细菌（FLB）作为猎物类似物和荧光显微镜。细胞特异性的吸收率可以通过在选定的时间程8中量化前列腺食物中标记的猎物颗粒的数量来确定。这种方法有几个优点。追踪器被添加到自然样品与自然捕食者和猎物组合。孵育前有最少的样品操作，添加的FLB示踪剂对样品的改变最小，孵育时间较短，以确保在接近原位条件下获得良好结果。或者，在细菌原虫或浮游动物数量少的环境中（例如近海海洋系统），通过流动细胞测定，可长期（12-24小时）在样品中添加少量FLB的消失率（2%-3%示踪剂）孵化实验。然后，在起点和终点的FLB数量（集成所有细菌的影响）通过流动细胞测定法进行量化（详情请参阅以前的出版物9）。然而，这种参数只代表不能直接归因于任何特定的前列腺动物和浮游动物食草动物群体或物种的总细菌率。
总体而言，准确量化水生环境中的古生物物种或形态特定细菌死亡率可能具有挑战性。一些原生动物是选择性的放牧者，添加的FLB示踪剂的大小和细胞形状可能会扭曲猎物摄入的自然速率10，11。此外，前列腺活性和代谢高度温敏12;因此，需要针对每种样品类型仔细操纵添加的FLB示踪剂量（不仅基于细菌的自然丰度、大小和形态以及细菌的流行类型，还基于温度）。大多数研究侧重于散装牧草放牧活动;然而，特定原物种的细菌通常具有较高的信息价值，并且可能更可取。在这种情况下，需要对样本中存在的原科物种的分类学知识，并了解它们的行为。因此，需要大量的时间和劳动力，才能获得针对特定物种的细菌感染率的健全结果，该比率可归因于特定的前列腺组或物种。
尽管存在这些困难，这种方法仍然是目前可用于在自然环境中评估前列腺细菌的最合适工具。这里介绍了一种在水生微生物生态学研究中使用FLB作为示踪剂的综合性、易于遵循的方法。该方法的所有问题方面都得到了说明，并描述了改进的工作流程，其中有两个来自对比环境的实验以及对比的西利特物种作为示例。
第一个案例研究是在捷克共和国的中生代什莫夫水库的表观环境中进行的，结果表明，与大多数地表淡水体相媲美的食草和细菌丰度（参见第5、7）。第二个案例研究是在水生食肉植物Utricularia反射的陷阱内高度特殊的环境中进行的，这种反射中含有极高数量的两种放牧混合营养性纤酸（四海梅纳眼）和细菌细胞。显示了这两种样本中细胞特异性放牧率和细菌常备库的计算。然后讨论了对结果的一系列生态解释，最后提出了可能的后续研究实例。

<table border="0" cellpadding="0" cellspacing="0" style="width:1612px;" width="1611">
	<tbody>
		<tr height="19">
			<td height="19" style="height:20px;width:360px;">0.2-&#181;m pore-size filters&#160;</td>
			<td style="width:462px;">SPI supplies, https://www.2spi.com/</td>
			<td style="width:137px;">B0225-MB</td>
			<td style="width:654px;">Black, polycarbonate track etch membrane filters, diameter approprite for filtering apparatus used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)</td>
			<td>Any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Automatic pipettes with adjustable volumes&#160;</td>
			<td>Any brand, various sizes</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Centrifuge</td>
			<td>22 000 x g</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Cryovials</td>
			<td>Any brand, 2 mL size</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DAPI (4&#180;,6-Diamidino-2&#180;-phenylindole dihydrochloride)</td>
			<td>Any brand&#160;</td>
			<td></td>
			<td>1 mg ml-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Epiflorescence microscope</td>
			<td>Magnification from 400 x up to 1000 x</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;"></td>
			<td>Filters appropriate for viewing in the DAPI and DTAF range</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;"></td>
			<td>Counting grid in one of the oculars</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Filtering apparatus</td>
			<td>Usually with a diameter of 25 mm&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Formaldehyde</td>
			<td>A brand for microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Glutaraldehyde</td>
			<td>A brand for microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Immersion oil for microscopy</td>
			<td>Specific oil with low fluorescence</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Lugol&#180;s solution</td>
			<td>Any brand or see comment</td>
			<td></td>
			<td style="width:654px;">Make an alkaline Lugol&#39; solution as follows: Solution 1 - dissolve&#160; 10 g of potassium iodide in 20 ml in MQ water, then add 5 g of iodine. Solution 2 - add 5 g of sodium acetate&#160; to 50 ml of MQ water. Add the solution 2 to the solution 1 and thoroughly mix</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Methanol stabilized formalin</td>
			<td>Any brand available for microscopy purposes</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Microscope slides and cover slips</td>
			<td>Any brand produced for microscopy purposes&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">MQ water for diluting samples</td>
			<td style="width:462px;">Any brand 
			&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS; pH = 9)</td>
			<td>Any brand</td>
			<td></td>
			<td>0.05 M Na2HPO4-NaCl solution, adjusted to pH 9</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">PPi-saline buffer</td>
			<td>Any brand</td>
			<td></td>
			<td style="width:654px;">0.02 M Na4P2O7-NaCl solution. Add 0.53 g Na4P2O7 to 100 ml of MQ water plus 0.85 g NaCl&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Sampling device&#160;</td>
			<td>Appropriate for obtaining representative sample&#160;</td>
			<td></td>
			<td>e.g. Friedinger sampler for lake plankton</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Sodium thiosulfate solution</td>
			<td>Any brand</td>
			<td></td>
			<td>3% solution is used in the protocol</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Sonicator</td>
			<td>Any brand</td>
			<td></td>
			<td>30 W</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Vortex</td>
			<td>Any brand allowing&#160; thorough mixing of the solutes and samples</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Water bath</td>
			<td>Any brand allowing temperature to be maintained at 60 &#176;C</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescently labeled bacteria as a tracer to reveal novel pathways of organic carbon flow in aquatic ecosystems

阐明营养相互作用，如掠夺及其影响，是生态学许多研究者经常的任务。微生物群落的研究有许多局限性，确定捕食者、猎物和捕食率通常很困难。这里介绍的是一种基于添加荧光标记的猎物作为示踪剂的优化方法，它允许可靠地定量水生食肉真核生物的放牧率，并估计营养转移到更高的营养水平。

例如，实验是在切莫夫水库（南波希米亚，CZ），这是一个自然地点，较低的自然原位捕食者和猎物丰度。报告具有代表性的数据为杂食性香菜物种哈尔特里亚格兰尼拉，这是一个丰富和高效的肉食虫颗粒（<2 μm）颗粒10，16，17，18 ， 22.图...

Watch this Scientific Journal Video about Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems at JoVE.com

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

这里介绍的是一种基于单细胞的表氟显微镜技术的协议，用于以高精度和分类分辨率量化水生捕食真核生物的放牧率。

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

这种方法意义重大，因为它使我们能够以非常高的分辨率可视化和估计从原核生物到微生物食物网中更高营养水平的碳和能量流动。这是目前唯一可用于确定哪些原生物种原位使用细菌的技术，同时在单细胞水平上量化这个过程的速率。由于细菌是唯一重要的微生物，将溶解的有机碳改变为颗粒有机碳，因此，参与者的知识和发生这种物质的比率构成了了解水生生态系统的基础。我们强烈建议研究人员在进行放牧实验之前熟悉他们的研究系统。这涉及到光和荧光的显微镜检查样品，并了解主要分类存在和他们的季节性动态。演示程序将是吉特卡 · 耶兹贝罗夫

fluorescently-labeled-bacteria-as-tracer-to-reveal-novel-pathways

Research

JoVE Journal

Environment

7.0K 次观看.  Biology Centre CAS. 这种方法意义重大，因为它使我们能够以非常高的分辨率可视化和估计从原核生物到微生物食物网中更高营养水平的碳和能量流动。这是目前唯一可用于确定哪些原生物种原位使用细菌的技术，同时在单细胞水平上量化这个过程的速率。由于细菌是唯一重要的微生物，将溶解的有机碳改变为颗粒有机碳，因此，参与者的知识和发生这种物质的比率构成了了解水生生态系统的?...

视频: Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Alternative Method of Removing Otoliths from Sturgeon

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a transverse vertical incision on the top of the skull with a hand or electrical saw may damage the otolith if not performed correctly. Sturgeons (Acipenseridae) are one family in particular that have a very large and thick skull. A new laboratory method entering the brain cavity from the ventral side of the fish to expose the otoliths was easier than other otolith extraction methods found in the literature. Methods reviewed in the literature are designed for the field and are more efficient at processing large quantities of fish quickly. However, this new technique was designed to be more suited for a laboratory setting when time is not pressing and successful extraction from each specimen is critical. The success of finding and removing otoliths using this technique is very high and does not compromise the structure in any manner. This alternative technique is applicable to other similar fish species for extracting the otoliths.

The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses.

从鲟鱼去除耳石替代方法

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a ...

科研

环境科学

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

活性有机碳评估土壤中连续熏蒸孵化程序

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

了解溶解有机物生物地球化学通过<em&gt;原位</em&gt;在河流生态系统营养手法

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

对水生生命值评价悬浮泥沙影响的流通系统曝光

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the production of synthetic PM for laboratory studies have become a widespread necessity. Herein, experimental protocols demonstrate approaches to produce aerosolized organic PM by &#945;-pinene ozonolysis in a flow tube reactor. Methods are described for measuring the size distributions and morphology of the aerosol particles. The video demonstrates basic operations of the flow tube reactor and related instrumentation. The first part of the video shows the procedure for preparing gas-phase reactants, ozonolysis, and production of organic PM. The second part of the video shows the procedures for determining the properties of the produced particle population. The particle number-diameter distributions show different stages of particle growth, namely condensation, coagulation, or a combination of both, depending on reaction conditions. The particle morphology is characterized by an aerosol particle mass analyzer (APM) and a scanning electron microscope (SEM). The results confirm the existence of non-spherical particles that have grown from coagulation for specific reaction conditions. The experimental results also indicate that the flow tube reactor can be used to study the physical and chemical properties of organic PM for relatively high concentrations and short time frames.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

流管反应器中有机颗粒物的生产与测定

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it regulates the gravity-driven soil movement induced by runoff and tillage activities. The recent application of Light Detection and Ranging (LiDAR) data holds promise for generating high spatial resolution topographic metrics that can be used to investigate soil property variability. In this study, fifteen topographic metrics derived from LiDAR data were used to investigate topographic impacts on redistribution of soil and spatial distribution of soil organic carbon (SOC). Specifically, we explored the use of topographic principal components (TPCs) for characterizing topography metrics and stepwise principal component regression (SPCR) to develop topography-based soil erosion and SOC models at site and watershed scales. Performance of SPCR models was evaluated against stepwise ordinary least square regression (SOLSR) models. Results showed that SPCR models outperformed SOLSR models in predicting soil redistribution rates and SOC density at different spatial scales. Use of TPCs removes potential collinearity between individual input variables, and dimensionality reduction by principal component analysis (PCA) diminishes the risk of overfitting the prediction models. This study proposes a new approach for modeling soil redistribution across various spatial scales. For one application, access to private lands is often limited, and the need to extrapolate findings from representative study sites to larger settings that include private lands can be important.

Landscape processes are critical components of soil formation and play important roles in determining soil properties and spatial structure in landscapes. We propose a new approach using stepwise principal component regression to predict soil redistribution and soil organic carbon across various spatial scales.

利用主成分扩展地形模型绘制土壤再分配和土壤有机碳

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

热光学有机/元素碳测量的分割点分析与不确定性定量

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...