我们感谢迈克尔 Jahn 和黄玉庭提供了纯文化和活性污泥群落的程序和数据集。我们进一步感谢 Katrin Mörters 对沼气群落样品的分析。这项工作由 Fachagentur Nachwachsende Rohstoffe e (FNR, 项目沼气-指纹 Nr) 资助. 22008313) 代表德国联邦粮食和农业部 (BMEL), 联邦部中小企业的中央创新方案 (星)经济事务和能源 (BMWi) (INAR-ABOS, 16KN043222), 德意志 Bundesstiftung Umwelt (DBU, 项目按需 Produktion 冯 Phosphatdünger 澳大利亚的 Reststoffen 和 Brauerei 33960/01-32), 德国联邦教育部和研究 (BMBF) (FZK 03XP0041G) 和亥姆霍兹协会的面向项目的资金 (光纤 III R31 主题3生物能源)。

<ol>
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作者没有什么可透露的。

对微生物种群和群落的成功分析需要经过良好调整的 cytometers, 适当的细胞参数选择, 以及从取样和固定到测量和数据评估的可靠工作流程。所选单元格参数必须与可用的激发波长匹配。我们使用和推荐 DAPI, 这是非常敏感的低浓度;但需要被紫外线激光激发, 这通常不包括在标准的细胞仪设置。其他染料, 如 SYBR 绿 I, 也玷污了整个人口或社区, 但一般提供劣质决议。我们不建议在微生物群落中使用鱼类程序或生存能力测试。这些方法无法量化、验证和控制, 因为它们对群落中个别物种的影响是不确定的。只要有相当一部分典型的社区仍然不能作为纯文化存在, 就无法对它们进行可靠的测试。
该协议的关键步骤包括取样和固定程序以及细胞分类。由于某些纯净的文化和微生物群落的倾向聚集到絮凝体或附着在样品基质中的微粒, 取样可能会变得复杂。在引入流细胞分析以保证可靠的结果之前, 必须在样品中消散这些聚集物并将细胞分离出来。对所提出的制备方案进行了优化, 以实现单细胞分析。最后50µm 过滤是在测量之前执行, 以去除任何残余骨料和防止70µm 喷嘴的细胞分拣器和流试管的分析仪从堵塞。污水处理厂的细胞絮凝体尤其丰富、健壮, 对系统的功能至关重要。对某大型污水处理厂不同储罐中的浮游污泥基微生物群落进行了研究, 对所建立的协议进行了测试。污泥形成细胞团聚体明显消散, 群落组成保持稳定。此外, 浮游和污泥为基础的群落在所有三取样罐中表现出显著的相似性 (图 8, 补充文件 1-S10)。这些结果通过比较 16S rDNA 扩增子测序的新鲜, 甲醛处理-, 甲醛和乙醇固定-, 和排序样本10验证。然而, 极具挑战性的环境样品, 如强的生物膜或在紧密相连的链条中生长的细菌, 可能无法消散。土壤样品可能是特别疑难的, 因为无处不在的微粒出现在直方图中, 并且可以通过纯粹的丰度掩盖细胞。在这种情况下, 需要采用传统的测序方法。
在设计实验前, 必须对每套新样品集的固定稳定性进行测试, 以保证结果的有效性。良好的固定稳定性也能在一天内使多样本时间点的汇集染色和测量。在实验结束和最后评价后, 它还能使复制测量和回溯细胞分类成为决定性的时间点。对纯培养物的固定稳定性进行了28天的验证 (图 9)。对于包括样品运输在内的现场试验, 应在较长时期内对固定稳定性进行试验 (在这种情况下, 活性污泥群落为60天, 沼气群落195天, 补充文件 1-S9)。
染色通常不是问题, 但需要控制与生物标准, 以允许应用的生物信息学评估工具。我们使用了模拟应变大肠杆菌BL21 (DE3), 这是根据 ASC 协议和储存, 以用于每一个染色批次。
除了 DAPI, SYBR 绿我已经成功地应用于解决微生物群落14 年,23,24,25,26。SYBR 绿色我可以用在线工作中的活细胞来解决社区变成高和低核酸子集 (海航和放大器)。然而, DAPI 在对 DNA 的结合上更具体, 并且在一个样本集合中能够区分超过50子社区, 但需要在染色前采取固定步骤。
细胞分类是这种方法的一个突出特点, 如果某些子社区有进一步的兴趣, 就可以应用。需要强调的是, 无论是粉煤灰 (仅执行30分钟), 还是乙醇处理或 DAPI 染色10都对随后的16S 扩增子测序产生负面影响。固定和染色程序对子社区 metagenome 分析的潜在影响仍需检验。
在涉及生物信息学工具的情况下, 许多关于社区功能和趋势动态的信息可以独立于分类方法中, 并通过细胞层次来解决虚拟细胞的社区特征, 并将这些特征参数。
最近, 一些新的生物信息学评估工具, 所有有用的 SYBR 绿色 I 和 DAPI 方法, 已开发解决细胞社区模式。这些是 FlowFP27, 这项研究中使用的包 (flowCHIC20, flowCyBar28), 一个与淡水社区29建立的反褶积模型和一个工具, 用于区分菌株根据其生理特征30。此外, 细胞多样性可以确定25 , 甚至微生物群落的稳定性特性现在可以遵循在线工具10。
因此, 在快速评价微生物群落和可能的非生物参数相关性方面, 流细胞分析具有巨大的优势。但是, 该方法仍然存在一些限制。由于有经验的基于门设置的程序, 它不能真正地在网上使用 flowCyBar 工具。此外, 研制定制、使用方便的流 cytometers 将极大地促进流细胞菌群分析方法的扩散。在这个方向上的第一步是进行28。
微生物流式细胞术的未来应用可以在微生物生态学中得到展望, 因为它允许在细菌产生时间范围内进行高频监测, 并表明生态学范式适用于细胞数据 5. ,10。该方法对在瑞士31的强制性饮水控制等自然环境的常规筛查非常有用。它也可以是一个宝贵的工具, 医疗应用, 如人类15或动物32微生物筛查。此外, 生物信息学的最新发展可能使微生物流式细胞术成为管理微生物系统过程控制中的一个整体传感器。微生物群落细胞术也为细胞分类提供了一个筛选工具, 它允许对特定群落子集进行更高分辨率的基因组调查。

微生物在人类生活的许多方面起着至关重要的作用。它们是行星碳、氮、磷和硫循环1的主要生物驱动因素, 其作用是降解2、3以及在各种工业 (如废水处理) 中合成4用于生物催化剂。5或生物技术6。它们甚至构成了人类的微生物菌群, 直接影响到人类的健康和新陈代谢7,8。因此, 如果我们的目标是充分理解和操纵这些系统, 那么关于微生物的结构、功能和动态行为的信息, 以响应它们的直接环境是必要的。下一代测序技术是解决菌群结构和功能9的既定方法。但是, 对内部数据的分析和评估不能产生量化信息, 而且成本高昂, 耗时, 而且在性能走廊内也没有准备好提供现场结果。有些细菌在1小时以下显示世代时间并且导致经常改变的社区结构在某些环境10。在这样的动态下, 使用排序方法将超过大多数科学实验室的财务和人力能力。
相比之式细胞术可以提供类似于数据的社区指纹 , 同时保留更高的时间、劳动和成本效率。在这里, 我们提出的流动细胞技术能够遵循微生物群落动力学在网上的单一细胞水平。与本研究不同, 流式细胞术并不提供功能基因的系统进化隶属关系或信息, 而是提供定量的细胞数量。利用流式细胞仪, 可以将微生物群落分解为子社区, 具有明显的光散射和荧光特性 (前向散射 (FSC) 和侧散射 (SSC), 分别提供细胞大小和粒度的指示)。所提出的方法主要利用细胞大小和与某些细胞类型和生理 (生长) 状态相关的 DNA 数量相关信息。dna 含量的量化使用紫外兴奋 4 ', 6-diamidino-2 '-苯基吲哚 (DAPI) 染料, 它绑定到一个 T 丰富的地区的 DNA, 甚至可以解决不同的染色体水平。通过结合 DAPI 荧光和 FSC 超过50子社区可以区分和监测, 以改变丰度随着时间的推移11。子社区丰度变化可与微环境环境的变化相关, 如 pH 值和产品效价12, 具有全球参数, 如天气13,14, 或肠道或唾液的情况下微生物与某些医疗治疗15。这些相关性揭示了关键子社区负责在整个社区的新陈代谢网络中的特定功能。然后, 通过改变周围的微环境, 或者为随后的测序15或蛋白质组调查16进行排序, 可以特别促进或抑制关键子社区。
然而, 微生物群落流式细胞术还没有得到广泛的建立, 因为大量的流动细胞装置能够正确地解决微生物种群或群落。此外, 缺乏经验、社区分析管道和有效的数据评估可能会给面临微生物分析挑战的研究人员带来进入障碍。我们已经建立了全面的工作流程来解决这些问题。为了说明它们的一般适用性, 我们将在三示范样本集 (补充文件 1-S1) 上展示它们, 即 i) 一种纯文化 (PC), 用于在明确的培养基中生长的生物技术应用 (单胞菌恶臭KT 2440 上葡萄糖), ii) 在透明介质 (合成废水活性污泥群落 (ASC)) 和 iii) 的一个复杂的实验室社区, 从一个稠密基质 (BC) 的天然环境中的一个复杂的社区 (玉米青贮)。
几个因素影响了每个样本集的协议选择。深冻放弃使用有毒化学品, 但主要限于实验室环境, 因为使用液态氮气的冲击霜。甲醛稳定和随后的乙醇固定允许散装取样, 不需要整除准备, 并已证明是稳定的长期。然而, 富含蛋白质的样品如人唾液15可能是问题, 因为蛋白质絮凝体, denaturized 由甲醛, 可能导致不赞同的信号到噪声比率。样品干燥将花费多数时间 (大约 1 h 在一共) 过程在这个比较, 但可以在现场执行, 不用毒物化学制品。它在管子中产生稳定的颗粒, 可以很容易地运送而不冷却或额外的危险预防措施。此外, 还提出了许多替代固定方法11。在引入新的样本集时, 我们强烈建议测试不同协议的分辨率和固定稳定性。
我们使用了两个不同的流 cytometers 来分析集合: i) 一个高度复杂和昂贵的, 研究中心的细胞分拣器和 ii) 一个应用重点的台顶分析仪, 似乎更适合现场应用5。BC 用工作台顶部分析仪测量, 而 PC 和 ASC 用细胞分拣机测量。分析三示范样本集需要不同的程序, 以优化重现性, 样品稳定性和工作流程的方便。下面的协议将指定三不同系统的各部分。

<table border="0" cellpadding="0" cellspacing="0" style="width:985px;" width="985">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Milli-Q system Synthesis A10</td>
			<td style="width:335px;">Merck, Darmstadt, (GER)</td>
			<td style="width:149px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioPak Ultrafiltration Cartridge</td>
			<td style="width:335px;">Merck, Darmstadt, (GER)</td>
			<td>CDUF BI0 01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MoFlo Legacy cell sorter</td>
			<td style="width:335px;">Beckman-Coulter, Brea, California (USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Innova 90C</td>
			<td style="width:335px;">Coherent, Inc , Santa Clara (USA)</td>
			<td style="width:149px;"></td>
			<td>334 nm - 364 nm, 100 mW</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Innova 70C&#160;</td>
			<td style="width:335px;">Coherent, Inc , Santa Clara (USA)</td>
			<td style="width:149px;"></td>
			<td>488 nm, 400 mW</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genesis MX488-500 STM OPS</td>
			<td style="width:335px;">Coherent, Inc , Santa Clara (USA)</td>
			<td style="width:149px;"></td>
			<td>488 nm, 400 mW</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xcyte CY-355-150</td>
			<td style="width:335px;">Lumentum, Milpitas, California, USA</td>
			<td style="width:149px;"></td>
			<td>355 nm, 150 mW</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photomultiplier tubes R928 and R3896</td>
			<td style="width:335px;">Hamamatsu Photonics, Hamamatsu City (Japan)</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Summit 4.3 software</td>
			<td style="width:335px;">Beckman-Coulter, Brea, California (USA)</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 &#181;m&#160; FluoSpheres</td>
			<td style="width:335px;">Molecular Probes, Eugene, Oregon (USA)</td>
			<td style="width:149px;">F8815</td>
			<td>350/440 blue fluorescent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 &#956;m YG FluoSpheres</td>
			<td style="width:335px;">Molecular Probes, Eugene, Oregon (USA)</td>
			<td style="width:149px;">F-8827</td>
			<td>505/515 yellow-green fluorescent beads&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">0.5 &#956;m Fluoresbrite BB Carboxylate microspheres</td>
			<td style="width:335px;">PolyScience, Niles, Illinois (USA)</td>
			<td>18339</td>
			<td>360/407</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 &#956;m Fluoresbrite BB Carboxylate microspheres</td>
			<td style="width:335px;">PolyScience, Niles, Illinois (USA)</td>
			<td>17458</td>
			<td>360/407 blue fluorescent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 &#181;mYG FluoSpheres</td>
			<td style="width:335px;">Molecular Probes, Eugene, Oregon (USA)</td>
			<td>F-13081</td>
			<td>505/515 yellow-green fluorescent beads&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">KH2PO4</td>
			<td>Sigma-Aldrich, St. Louis (USA)</td>
			<td style="width:149px;">CAS no. 7778-77-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">KCl</td>
			<td>Sigma-Aldrich, St. Louis (USA)</td>
			<td style="width:149px;">CAS no. 7447-40-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyflow Space&#160;</td>
			<td>Sysmex Corporation, Kobe (Japan)</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Laser Genesis CX,</td>
			<td style="width:335px;">Coherent, Inc , Santa Clara (USA)</td>
			<td style="width:149px;"></td>
			<td>355 nm, 150 mW</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">H6779-32 series PMTs</td>
			<td style="width:335px;">Hamamatsu Photonics, Hamamatsu City (Japan)</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">FloMax software</td>
			<td style="width:335px;">Sysmex Partec GmbH, G&#246;rlitz, (GER)</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">all optical filters</td>
			<td>Carl Zeiss, Jena (GER)</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">KH2PO4</td>
			<td>Sigma-Aldrich, St. Louis (USA)</td>
			<td>CAS no. 7778-77-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">KCl</td>
			<td style="width:335px;">Carl Roth, Karlsruhe (GER)</td>
			<td align="right">6781.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo 10.0.8r1</td>
			<td>FlowJo, LLC, Ashland, Oregon (USA)</td>
			<td style="width:149px;"></td>
			<td>Build number: 42398</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">R-software v.3.4.3</td>
			<td>R Core Team</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">flowCHIC</td>
			<td colspan="2"><a href="http://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html">http://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">flowCyBar</td>
			<td colspan="2"><a href="http://www.bioconductor.org/packages/devel/bioc/html/flowCyBar.html">http://www.bioconductor.org/packages/devel/bioc/html/flowCyBar.html</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing microbiome dynamics &#8211; flow cytometry based workflows from pure cultures to natural communities

对纯文化的调查和微生物群落动态监测对于了解和控制微生物驱动的自然生态系统和技术应用至关重要。下一代测序方法被广泛地用于解决微生物, 但它们通常是资源和时间密集型的, 提供的主要是质量信息。流细胞菌群分析不受这些缺点的影响, 可以提供相对子社区丰度和绝对细胞数的在线。虽然它不提供直接的系统系统的信息, 它可以提高分析的深度和解决的排序方法。流式细胞术在研究和常规设置中与医学应用形成鲜明对比, 目前仍未广泛应用于微生物菌群分析。缺少样本准备和数据分析管道的信息可能会为面临微生物分析挑战的研究人员带来进入障碍, 这通常是教科书流式细胞术的应用。在这里, 我们分别为纯文化、复杂社区在具有挑战性的矩阵中的清晰介质和复杂社区提供三个综合工作流。我们描述各自的抽样和固定程序和优化染色协议的各个样本集。本文以复杂的研究为中心, 以应用为重点的平台顶部设备, 描述了细胞分类程序, 并提出了数据分析包, 细胞分析。此外, 我们还提出了重要的实验控制, 并将所提出的工作流应用于相应的样本集。

以下代表的结果强调了在三样本集上复制和举例说明不同的数据可视化和分析技术的必要性。他们展示了可能的数据评估管道的结果适合回答标准研究问题关于各自集合。然而, 所提出的技术并不排斥它们所呈现的样本集。流式细胞仪原始数据是公开提供的 FlowRepository ID FR-FCM-ZY46。以前上载的 ASC 数据在 ID FR-FCM-ZYD7 下可用。
根据已发表的11号议定书, 进行了生物和技术复制, 编写和分析。从个人计算机和 ASC 的生物复制从三个平行的烧瓶被采取了。BC 的生物复制被采取了作为三后继抽样在一个地点在一个试验性规模植物。三整除数的样品进行固定、染色和分析, 以确保技术重现性。根据先前公布的程序11, 对重现性进行了评估。一个样本集的所有样本 (补充文件 1-S6) 的门模板用于计算每门的标准偏差 (%)。
对于 PC, 相对丰度的最大标准偏差为 3.44%, 平均标准偏差为 2.13% (图 1)。对于 ASC 和 BC, 相对丰度的最大标准偏差为1.27% 和 0.88%, 而标准偏差的平均值为2.13% 和 0.21% (补充文件 1-S8)。
一个标准的程序, 当调查纯应变培养, 是准备一个增长曲线, 以分析滞后阶段, 产生时间和细胞密度在特定条件下。我们将此过程与流程细胞分析工作流相结合, 以实现对系统的更深入的了解 (图 2)。FSC 与 DAPI 荧光图揭示了间歇培养的不同点的细胞周期状态。建立了主门模板 (补充文件 1-S6), 以量化细胞的比例与一个 (c1n), 两个 (c2n) 和多染色体 (航空公司,图 2B)。接种细胞的主要比例只有一条染色体 (93.7% 在 0 h)。这在指数增长期间发生了剧烈的变化, 几乎所有的细胞包含不止一个 (99.6%, 4 小时) 和超过一半的人口 (53.1%) 甚至包含两个以上的染色体。这说明P. 恶臭的染色体复制速度比它的生成时间快。
流细胞分析已被证明是非常有用的监测微生物群落的演变。它可以跟随社区动力学比更多资源密集的分子方法5,10更加接近。我们在一部电影中强调了这些动态, 并获得了随着时间的推移, 活性污泥群落变化的概述。每1/2 帧显示一个 FSC 与 DAPI 荧光图的样本点 (补充文件 2)。在0天和4天之间的一个非常明显的转变之后, 在7天之后建立了一个核心社区。另外的子社区在21天出现了。我们建立了一个主门模板 (补充文件 1-S6), 使评估微生物动力学的子社区水平。实验的不同阶段的显性子社区使用 CyBar 工具进行了清晰的识别 (图 3)。将它与相对子社区丰度的频率分布结合起来, 有助于选择有趣的门 (在关键时间点触发的大量的显著变化) 和可行 (超过5% 相对丰度) 进行排序和进一步分析22。
工业规模的生物反应器应用可以面对潜在的空间异质性由于搅拌限制。它们也经常接触到变化的操作参数, 如非合成基底质量的改变。BC 代表这样一个系统并且从不同的现场取样在一个动态地被驾驶的塞流反应器。FSC 与 DAPI 荧光地块 (图 4) 的模范抽样点显示只有很少的空间, 但明显的时间异质性。BC 是进一步调查使用, 快速自动化别致和更深入的主门模板基于 CyBar 方法。
它的自动化, 快速和无偏见的性质, 使别致特别有趣的现场控制发酵在一个工业环境。它比较了所有可用样本的原始. fcs 数据。其中的两个比较显示在图 5中。由此产生的不同值证实了以前关于空间和时间异质性的观察。NMDS 地块生成的别致工具不同矩阵 (图 6) 进一步证实这一结果, 并据此形象化。
此外, 我们计算了公元前23子社区的相对丰度, 使用主浇口模板 (补充文件 1-S6)。对48例一年期的样品进行相关分析, 了解微生物群落中的功能关系。这可以帮助确定感兴趣的门进一步调查 (4 细胞分类)。强正或负相关的非生物参数, 如产品的效价可以帮助了解和优化生态系统和生物技术的过程。这一相关性中包含的有机负载率 (图 7) 是这种非生物参数的例证。G5、G4 和 G10 具有较强的正相关性, 可能含有发酵类, 而 G8 中的负相关可能暗示甲烷古菌。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58033/58033fig1.jpg"> 图 1: 纯文化细胞分析的重现性.与控制珠 (A, B) 和条形图的3子社区丰度 [%] 的 DAPI 荧光地块, 其中误差条代表了一个标准偏差 (C, D)。相对丰度的确定与门模板显示在补充文件 1-S6。三生物复制 A, B 和 C 被采取了增长曲线在 4 h 和三技术复制 P1, P2, P3 从样品 A 和三次被测量了, 分别: M1, M2, M3, 并且给以他们各自的标准偏差。5万事件被记录在细胞门。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig1large.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/58033/58033fig2.jpg"> 图 2: 在 12 h (a) 的生长曲线实验过程中所采取的纯文化的细胞周期分析.FSC 与 DAPI 荧光图的双小时抽样在指数生长阶段表现出 DNA 含量的变化。这个测量系列不包括珠子 (参见补充文件 1-S3)。(B).基于光密度的生长曲线, 误差条代表了子社区的一个标准偏差和相对丰度。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig2large.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/58033/58033fig3.jpg"> 图 3: 活性污泥群落结构演化24天以上.(A) 在 boxplots 中, 根据趋势的相似性、相应的颜色键 (B) 以及在 NMDS 图 < 0.001 中的相似性分析, flowCyBar 与叠加频率分布可视化的, 分别为:C). 基础地块显示在补充文件2的电影序列中。使用辅助文件 1-S6 中的栅极模板确定了相对丰度。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig3large.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/58033/58033fig4.jpg"> 图 4: 工业级插头流沼气反应器中微生物群落结构的时空异质性.(A) 在223天, 对四个取样口的微生物群落结构进行比较。(B) 将0天至384日的时间依赖性社区结构变化与第二港口的比较。噪音已被切断, 以提高可视化。25万事件被测量了在细胞门 (补充文件 1-S6)。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58033/58033fig5.jpg"> 图 5: 细胞直方图图像比较-别致.通过比较两个表示空间和时间异质性的样本, 说明了别致算法的基本原理。(一) 在选择两个要显示的参数后, 从. fcs 文件创建别致图像 (FSC 与 DAPI 荧光)。灰度 (0–255) 在像素中对事件计数进行编码。(ii) 在指定包含单元格的区域后生成别致子集 (这里: 200 < x < 4000, 200 < y < 2200)。(iii) 通过比较子集之间的像素来创建 XOR 组合图像。没有区别等于 0, 并显示为黑色。最大差等于 200, 显示为白色。通过在 xor 图像中添加所有像素的灰度值来计算相应的 XOR 值。(iv) 叠加组合图像是通过添加子集像素的灰度值创建的。对应的叠加值是通过计算不等于零的叠加图像的像素并因此包含信息来计算的。(v) 不同值的计算方法是除以 XOR 和叠加值。这些是图 6中 NMDS 图的基础。不同的价值和 NMDS 的情节显示了一个微不足道的空间和明显的世俗群落异质性。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58033/58033fig6.jpg"> 图 6: 基于别致的沼气群落样本相似性分析.0天的样品被排除在这个情节由于它的极端不同的社区结构歪曲分析 (补充文件 1-S11)。NMDS 剧情证实了使用别致的工具做的低空间和更高的世俗异质性的假设并且解释了图 5。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58033/58033fig7.jpg"> 图 7: 沼气群落的相关分析.用长矛的秩阶系数计算矩阵, 并以辅助文件 1-S6 中主浇口模板确定的23子社区的相对丰度为基础。在一年的时间内, 它们与48份样品的有机负荷率 (OLR) 相关。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58033/58033fig8.jpg"> 图 8: 废水中浮游 (P) 和污泥基 (S) 群落的相似性分析.样品取自一个全尺寸污水处理厂的主澄清池 (PCL)、活性污泥池 (AS) 和消化池 (DT) (补充文件 1-S10 中的点地块)。使用辅助文件 1-S6 中显示的门模板确定了相对丰度。这些子社区丰度还与 flowCyBar 工具进行了分析, 以创建一个 CyBar (补充文件 1-S10) 和 NMDS 情节 (R < 0.01)。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58033/58033fig9.jpg"> 图 9: 纯文化的固定稳定性.在0小时的生长曲线实验样品中, 28 天内储存了15% 甘油的-80 摄氏度。对控制珠的 DAPI 荧光图进行了对比。测量系列不包括珠子 (补充文件 1-S3)。5万事件被记录在细胞门 (补充文件 1-S6)。<a href="https://www.jove.com/files/ftp_upload/58033/58033fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充文件 1:<a href="https://www.jove.com/files/ftp_upload/58033/Supplementary-File-1.pdf" target="_blank">请点击这里下载此文件.</a>
补充文件 2:<a href="https://www.jove.com/files/ftp_upload/58033/Supplementary_File_2.gif" target="_blank">请点击这里下载此文件.</a>

Watch this Scientific Journal Video about Characterizing Microbiome Dynamics Ã¢â‚¬â€œ Flow Cytometry Based Workflows from Pure Cultures to Natural Communities at JoVE.com

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

流细胞分析已证明对研究纯文化和监测微生物群落动力学有价值。我们提供了三个全面的工作流程, 从抽样到数据分析, 到纯文化和复杂社区, 在清晰的介质中, 以及在挑战性的矩阵, 分别。

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

characterizing-microbiome-dynamics-flow-cytometry-based-workflows

Research

JoVE Journal

Environment

11.8K 次观看.  Helmholtz Centre for Environmental Research - UFZ. 流细胞分析已证明对研究纯文化和监测微生物群落动力学有价值。我们提供了三个全面的工作流程, 从抽样到数据分析, 到纯文化和复杂社区, 在清晰的介质中, 以及在挑战性的矩阵, 分别。

视频: Characterizing Microbiome Dynamics – Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), &#34;thorny-headed worms&#34; (acanthocephalans), &#34;flukes&#34; (trematodes), and &#34;tapeworms&#34; (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 &#956;m sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris&#39; hematoxylin or Semichon&#39;s carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.

Wild animals are commonly parasitized by a wide range of helminths.&#160; The four major types of helminths are &#8220;roundworms&#8221; (nematodes), &#8220;thorny-headed worms&#8221; (acanthocephalans), &#8220;flukes&#8221; (trematodes), and &#8220;tapeworms&#8221; (cestodes).&#160; Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.&#160;

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), ...

科研

环境科学

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

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 ...

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread breeding distribution, that has been recently listed as endangered in some habitats of its range. Waterbird monitoring programs exist to track populations through time; however, some of the more intensive approaches require entering colonies and can be disruptive to nesting populations. This paper describes a protocol that utilizes a minimally invasive surveillance system to continuously monitor common tern nesting behavior in typical ground-nesting colonies. The video monitoring system utilizes wireless cameras focused on individual nests as well as over the colony as a whole, and allows for observation without entering the colony. The video system is powered with several 12 V car batteries that are continuously recharged using solar panels. Footage is recorded using a digital video recorder (DVR) connected to a hard drive, which can be replaced when full. The DVR may be placed outside of the colony to reduce disturbance. In this study, 3,624 h of footage recorded over 63 days in weather conditions ranging from 12.8 &#176;C to 35.0 &#176;C produced 3,006 h (83%) of usable behavioral data. The types of data retrieved from the recorded video can vary; we used it to detect external disturbances and measure nesting behavior during incubation. Although the protocol detailed here was designed for ground-nesting waterbirds, the principal system could easily be modified to accommodate alternative scenarios, such as colonial arboreal nesting species, making it widely applicable to a variety of research needs.

A Video Surveillance System to Monitor Breeding Colonies of Common Terns Sterna Hirundo

This paper describes a protocol that uses a remote video monitoring surveillance system to continuously monitor breeding colonies of ground-nesting waterbirds. The system includes five cameras monitoring individual nests and one camera monitoring the colony as a whole, and is powered by car batteries that are recharged via solar panels.

监测常见燕鸥繁殖菌落的视频监控系统 (燕鸥家燕)

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread ...

Necropsy-based Wild Fish Health Assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

基于尸检的野生鱼类健康评估

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

A Guide to Concentration Alternating Frequency Response Analysis of Fuel Cells

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency response analysis (cFRA) on proton-exchange membrane (PEM) fuel cells. During cFRA experiments, the modulated concentration feed was sent to the cathode of the cell at different frequencies. The electric response, which can be cell potential or current depending on the control applied on the cell, was registered in order to formulate a frequency response transfer function. Unlike traditional electrochemical impedance spectroscopy (EIS), the novel cFRA methodology makes it possible to separate the contribution of different mass transport phenomena from the kinetic charge transfer processes in the frequency response spectra of the cell. Moreover, cFRA is able to differentiate between varying humidification states of the cathode. In this protocol, the focus is on the detailed description of the procedure to perform cFRA experiments. The most critical steps of the measurements and future improvements to the technique are discussed.

We present a protocol for concentration alternating frequency response analysis of fuel cells, a promising new method of studying fuel cell dynamics.

燃料电池浓度交替频率响应分析指南

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency ...