Name: isolation and characterization of the natural microbiota of the model nematode caenorhabditis elegans
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我们感谢德国科学基金会的财政支持（关于超生物起源和功能的1182合作研究中心的项目A1.1和A1.2）。我们感谢舒伦堡实验室成员的建议和支持。

1. 缓冲液和培养基的制备
<ol><li>通过将5.85gNaCl，1.123gK 2 HPO 4，5.926gKH 2 PO4和1L去离子H2O加入烧瓶中并在121°C高压灭菌20分钟来制备S缓冲液。</li>
	<li>通过添加含有 1.2% （w/v） 羟甲基纤维素（引起培养基粘度的物质）、5 mg/mL 胆固醇、1 mM MgSO4、1 mM CaCl2 和 0.1% （v/v） 丙酮的 S 缓冲液来制备粘性培养基。高压灭菌并搅拌粘性介质，...

<ol>
	<li>Schulenburg, H., F&#233;lix, M. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+biotic+environment+of+Caenorhabditis+elegans.">The natural biotic environment of Caenorhabditis elegans.</a> Genetics. 206 (1), 55-86 (2017).</li><li>Petersen, C., Dirksen, P., Prahl, S., Strathmann, E. 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S., Rowedder, H., Braendle, C., F&#233;lix, M. -. 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线虫秀丽隐杆线虫是生物学研究中研究最深入的模式生物之一。它是由悉尼·布伦纳（Sydney Brenner）在1960年代引入的，最初是为了了解神经系统的发展和功能29。从那时起，秀丽隐杆线虫已成为研究所有生物学科基本过程的强大模型，包括行为生物学、神经生物学、衰老、进化生物学、细胞生物学、发育生物学和免疫学。它的成功基于优势特性的独特组合，包括大约...

在自然界中，秀丽隐杆线虫常见于腐烂的植物物质中，尤其是苹果等腐烂的水果或堆肥堆1。它还与某些无脊椎动物宿主有关，例如蛞蝓和木虱2，3。这些栖息地富含微生物，它们不仅可以作为蠕虫的食物，还可以与蠕虫形成稳定的联系。关于天然伴生微生物多样性的信息仅在2016年发布4，5，6。从那时起，这些和最近的一些研究表明，秀丽隐杆线虫与多种细菌和真菌有关，最常见的包括假单胞菌属、肠杆菌、赭石杆菌、欧文氏菌、Comamonas、葡萄糖酸杆菌和其他几种细菌6，7，8。几种相关的细菌可以稳定地定植在蠕虫肠道中，尽管不是全部6，9，10，11，12。它们可能对我们对秀丽隐杆线虫生物学的理解至关重要，因为它们可以提供营养，防止病原体和可能的其他压力源，并影响中心生活史特征，如繁殖率、发育或行为反应。
例如，假单胞菌属、赭石属以及肠杆菌或葡萄糖酸杆菌属的天然相关分离株可以以不同的方式保护蠕虫免受病原体感染和杀戮5，6，11，13，14。Comamonas属的特定分离株影响线虫的饮食反应，发育，寿命和生育能力15，16，17。普罗维登西亚细菌产生神经调节剂酪胺，从而调节宿主神经系统活动和由此产生的行为反应18。一组不同的天然相关细菌被证明会影响人口增长率，生育率和行为反应5，6，9，11，19。
迄今为止，原生秀丽隐杆线虫微生物群在栖息地和地理位置上的确切多样性和一致性尚未完全了解，蠕虫与其环境中的微生物之间的进一步关联仍有待发现。之前的几项研究使用了从某些土壤环境、秀丽隐杆线虫天然栖息地或中生虫实验（即重建自然栖息地的实验室环境）中分离的细菌菌株，秀丽隐杆线虫实验室菌株4，5，20。尽管这些研究对微生物对特定线虫性状（例如线虫代谢21）的影响有了新的见解，但这些相互作用与自然界秀丽隐杆线虫生物学的相关性尚不清楚。因此，本手稿描述了从自然界中直接分离秀丽隐杆线虫的方法，以及从单个蠕虫和蠕虫群中分离并随后表征天然相关微生物的方法。所描述的方法是以前用于分离和表征天然秀丽隐杆线虫及其天然微生物群的程序的更新和改进版本2，6，7。考虑到秀丽隐杆线虫广泛存在于全球分解植物物质中（特别是在腐烂的水果、温带地区和秋季）1，2，22，23，24，25，只要有兴趣与秀丽隐杆线虫相关，任何实验室都可以应用该协议 自然相关微生物的特征，因此具有更自然的相关背景。后者对于充分了解线虫的生物学至关重要，因为从其他宿主系统的多样性中得知，相关的微生物群可以影响不同的生活史特征26，这一方面目前在几乎所有生命科学学科的众多秀丽隐杆线虫研究中在很大程度上被忽视。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL</td>
			<td></td>
			<td>multiphysics simulation software</td>
		</tr>
	</tbody>
</table>

isolation and characterization of the natural microbiota of the model nematode caenorhabditis elegans

线虫秀 丽隐杆线虫 与自然界中各种各样的微生物相互作用。一般来说， 秀丽隐杆线虫 常见于腐烂的植物物质中，尤其是苹果或堆肥堆等腐烂的水果。它还与某些无脊椎动物宿主有关，如蛞蝓和木虱。这些栖息地富含微生物，这些微生物是 秀丽隐杆 线虫的食物，也可以持续地定植线虫肠道。迄今为止，原生 秀丽隐杆线虫 微生物群在栖息地和地理位置上的确切多样性和一致性尚不完全清楚。在这里，我们描述了一种从自然界中分离秀 丽隐杆线 虫并表征蠕虫微生物群的合适方法。线虫可以很容易地从堆肥材料、腐烂的苹果、蛞蝓中分离出来，或者通过将苹果放在堆肥堆上来吸引线虫。在北半球发现 秀丽隐杆线虫 的黄金时间是 9 月至 11 月。通过将底物浸入缓冲溶液中，可以将蠕虫从收集的底物材料中洗掉，然后收集线虫并将其转移到线虫生长培养基或PCR缓冲液上以进行后续分析。我们进一步说明了如何使用样品来分离和纯化蠕虫相关微生物，并处理蠕虫以进行微生物群落组成的16S核糖体RNA分析。总体而言，所描述的方法可能会激发对跨栖息地和地理位置的秀 丽隐杆 线虫微生物群特征的新研究，从而有助于全面了解线虫微生物群的多样性和稳定性，作为未来功能研究的基础。

线虫秀丽隐杆线虫经常出现在分解的水果中，例如苹果，以及堆肥样本。在德国北部，秀丽隐杆线虫以及同类物种（特别是C. remanei，但也包括C. briggsae）主要在9月至11月2日发现。线虫最常见于分解植物物质，尤其是苹果或梨等腐烂的水果，以及堆肥，特别是显示出高分解等级的材料。水果和堆肥样品可以带入实验室（图3A，<strong...

Watch this Scientific Journal Video about Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans at JoVE.com

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

秀丽隐杆线虫 是生物学中的主要模式物种之一，但几乎所有的研究都是在没有其天然相关微生物的情况下进行的。这里描述的方法将有助于提高我们对相关微生物多样性的理解，作为未来 功能性秀丽隐杆线虫 研究的基础。

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

该协议允许对秀丽隐杆线虫的天然微生物群有新的见解。同时，分离的微生物可用于受控实验室实验，以表征微生物群 - 宿主相互作用和秀丽隐杆线虫生物学。该协议侧重于分离自然界中与秀丽隐杆线虫相关的微生物，因此这是在更自然的背景下理解这一重要模型宿主的生物学的关键。与其他线虫相比，熟悉盲肠炎的外观会有所帮助。在解剖范围内移液最初可能具有挑战性，但实践会有所帮助。首先将堆肥或腐烂的水果等环境样本收集在单独的塑料袋、管子或干净的容器中。将收集的环境样品碎片均匀地铺在无菌的9厘米空培养皿中，并加入约20毫升无菌粘性培养基以用培养基覆盖样品。在解剖显微镜下，按照分离秀丽隐杆线虫和相关线虫的指南搜索线虫。使用20至100微升移液管，从平板中收集尽可能少量的含有Caenorhabditis线虫的液体，并将其转移到含有一至三毫升无菌M9T的三厘米培养皿中。为了建立蠕虫种群，可以将单个蠕虫转移到带有线虫生长培养基或NGM的平板上。要清洗线虫并去除线虫外部存在的微生物，请将它们在M9T中孵育10至15分钟。移出含有线虫的尽可能少量的液体，并将其转移到含有一到三毫升新鲜M9T的新无菌三厘米培养皿中。为了无偏地鉴定线虫相关微生物，准备一个 96 孔板，其中包含大约三个直径为 1 毫米的无菌磁珠，每孔加入 19.5 微升 PCR 缓冲液和 0.5 微升蛋白酶 K 的混合物。用尽可能少的液体将单个洗涤的线虫转移到每个孔中。使用珠子均质器在30赫兹下分解转移的线虫三分钟，并在室温下以8， 000 G离心板或管10秒钟以使液体到达底部。要鉴定秀丽隐杆线虫，请在PCR循环仪中将样品在50摄氏度下加热1小时，在95摄氏度下加热15分钟，以分离单个线虫的DNA，或使用商业试剂盒分离蠕虫种群的DNA。将分离的DNA冷冻在零下20摄氏度下长期储存。使用 DNA 和引物对 NLP 30 正向和 NLP 30 反向，产生 154 碱基对 PCR 产物。为了分离细菌，准备一个96孔板，其中包含大约三个无菌的一毫米珠子，每孔20微升M9缓冲液，并用尽可能少的液体将单个洗涤的线虫移液到每个孔中。如使用珠均质器之前所示分解线虫，然后对板进行短暂离心以使液体到达底部。完成后，收集悬浮液并以 1 到 10 的比例连续稀释。将多达 100 微升的稀释悬浮液接种到 9 厘米琼脂平板上。然后将板在 15 至 20 摄氏度下孵育 24 至 48 小时。为了获得纯细菌培养物，使用无菌环或牙签从孵育板中挑选单个菌落，并将其划线到含有纯化过程中使用的相同琼脂培养基的新琼脂平板上。确保只使用板的 1/3。然后对可重复使用的环进行灭菌或使用新的无菌环并将其拖过第一条条纹，以在样品板的另一 1/3 部分上形成第二条条纹。通过拖动穿过第二条条纹的循环并创建第三条条纹来实现单个菌落生长来重复它。在用于分离的相同生长条件下孵育板，如果需要，重复纯化步骤。使用相同的温度和生长条件在液体培养基中培养纯菌落。接下来，通过使用 16 个正向和 1495 个反向引物扩增 27S 核糖体 RNA 并从纯液体培养物中提取细菌 DNA 并按照文本中所述进行 PCR，来表征细菌。当收集的水果和堆肥样品分布在培养皿中并浸没在液体或粘性介质中时，导致蠕虫离开基质材料并游到培养基表面。微生物被分离为纯单菌落，纯化起着至关重要的作用，因为一些秀丽隐杆线虫相关细菌可以形成生物膜或容易聚集，从而产生多物种培养物。使用秀丽隐杆线虫特异性引物（即NLP 30正向和NLP 30反向）的PCR导致秀丽隐杆线虫的154个碱基对产物扩增。从单个蠕虫中分离的DNA导致PCR条带较弱，而从至少50个蠕虫组成的蠕虫种群中提取DNA产生更多和更清晰的条带。秀丽隐杆线虫特异性PCR的成功是通过将实验室菌株（即N2）的DNA与分离的线虫的DNA同时运行作为阳性对照来证实的。因此，必须在无菌条件下工作，以确保那些被分离的微生物真正与自然界中的蠕虫相关。分离的微生物可用于研究宿主-微生物群相互作用或微生物对宿主生物学的作用，例如，受控实验室实验中的衰老、发育或免疫。用这种技术分离的微生物目前被秀丽隐杆线虫群落用于提高对宿主适应性微生物群多样性或微生物对免疫作用的理解。

Research

JoVE Journal

Biology

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

科研

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

从环境中自然产生的趋磁细菌的采集，分离和富集

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...