这项工作已经由美国国家科学基金会资助的IOB＃0615996。

第1部分:准备线虫的生长介质（NGM）的Petri板实验线虫， 如线虫和 P. pacificus通常在6厘米的Petri板含有线虫的生长介质（NGM）1实验室培养。蠕虫是保持在20 ° C，但可以根据线虫的物种和实验设计，在一定温度范围内（4 ° C - 25 ° C之间）孵育。为了准备与NGM板，标准的无菌技术已被用来防止真菌和细菌的污染。以下是协议准备NGM板: <ol><li>称取蛋白胨2克，琼脂15克，2.4克氯化钠2.72克KH 2 PO 4 2升锥形瓶中，使体积至1 L与水，高压灭菌器，并允许它冷却到60 ° C水浴中。 </li><li>添加0.8毫升的1 M 氯化钙 ，胆固醇（5毫克/毫升乙醇中），1米硫酸镁 。为了防止真菌和细菌的污染，1 ml链霉素（100毫克/毫升）和1毫升制霉菌素（10毫克/毫升），可以被添加到每公升介质。 </li><li>使用Unispense机和保持无菌条件下，倒入所需的板媒体。板应装到约2 / 3 路 。一个标准的直径为60毫米的钢板，需要约10毫升的媒体。不要板块移动，直到琼脂完全gelified，否则会有琼脂表面凹凸不平，使其难以集中的体视显微镜上的蠕虫。 </li><li>一旦干燥，板材可立即接种或储存在4℃，直至与细菌播种彗星。 </li></ol>第2部分:准备OP50细菌和播种NGM的Petri板 P. pacificus是在实验室培养大肠杆菌 OP50应变喂养。 OP50上NGM板的有限增长，从而形成一个薄的草坪，方便的可视化的蠕虫1。要成长的OP50文化，使用LB -肉汤: <ol><li>添加5克，2.5克酵母提取物和2.5 g氯化钠bactotryptone螺丝帽瓶500毫升，用蒸馏水和高压灭菌容至500 ml。 </li><li>同一个OP50单菌落接种肉汤的无菌条件下培养板，让它成长一夜之间在37 ° C。 </li><li> LB - OP50是随时可以使用，可以储存在4 ° C几个月，如果保持无菌。每当种子板块需要无菌一个小烧杯，倒入少量的肉汤，这将有助于防止污染的股票文化。 </li><li>种子板，分配到60毫米直径的NGM的Petri板和漩涡传播的OP50 100-120μL。 </li><li>孵育这些种子草坪生长板在室温下过夜。这些种子的板，现在可以存储长达3个星期在4 ° C。它们存储在一个倒置的位置，有助于防止快速干燥。 </li></ol>第3部分:一挑在本节中，我们介绍了如何挑一个蠕虫。挑用于蠕虫从一个位置转移到另一个。挑30计90％的铂金10％铱丝，虽然一些研究人员可能更喜欢稍有不同的金属成分: <ol><li>剪下约3-4厘米的导线，巴斯德吸管（其尖端被打破到一个较小的长度）的提示，然​​后0.5厘米内入玻璃密封在一个本生灯。从玻璃突起线的长度大约是3-3.5厘米，但可以根据个人喜好而有所不同。 </li><li>拼合线是凸出使用锤子或钳子的结束。然后夷为平地的部分向上弯曲，形成了瓢。 </li><li>用沙纸，以防止破坏性的蠕虫或琼脂，理顺拿起锋利的边缘。 </li></ol>第4部分:采摘蠕虫<ol><li>挑蠕虫，火焰消毒的铂丝和拖细菌草坪扁平尖挑大衣与稠粘细菌的一角，小心不要穿刺或损坏的琼脂表面。 </li><li>使用立体显微镜，很轻轻一刷直到蠕虫被拾起棍子上挑的细菌的蠕虫病毒/蠕虫对粘提示。 </li><li>一旦蠕虫的挑尖，转移它立即到新盘，通过触摸或滑动的挑尖对新盘的细菌草坪和蠕虫抓取。必须小心不损坏板面的琼脂，其他的蠕虫爬进孔，使其难以检索。如果蠕虫停留挑太久，它可能变干。 </li></ol>第5部分:EMS诱变乙基甲烷磺酸盐（EMS）诱导突变在基因组中的2的广谱。突变体可以识别的缺陷，如产蛋，肌肉缺损S，性别决定的dauer形成，行为，和性腺形成。为EMS诱变的协议是类似的 C描述线虫 1。 <ol><li>准备500毫升1N氢氧化钠（或更多）。 </li><li>需4-5，直径6厘米的Petri板的蠕虫病毒，其中包含几百蠕虫幼虫少年第三阶段（J3），洗掉的蠕虫使用2-3毫升无菌每盘M9。 </li><li>在无菌的，一次性的15毫升离心管收集的所有的蠕虫。离心5-7分钟，或在1500 XG直到蠕虫形成一个颗粒。 </li><li>吸出液体，并用2毫升M9的重新暂停蠕虫。 M9的一个试管中2毫升添加20μL的环境管理体系和动摇解散。然后，添加这种蠕虫悬挂和漩涡轻轻2毫升EMS解决方案。 EMS的终浓度为47毫米。注意:EMS是一个非常强烈的诱变剂，并应在通风橱中处理。在这个诱变剂接触的所有材料（手套，滴管，提示），应被视为与1N氢氧化钠不活跃的EMS。双层手套穿在整个EMS诱变过程。根据您的机构的指导方针，处置危险废物的。 </li><li>将离心管中，并用封口膜覆盖的上限;水平放置在通风柜，让孵育3.5小时的蠕虫。管也可以放在低速摇臂。 </li><li>打开管，并用封口膜覆盖的上限;蠕虫下沉的垂直位置，直到孵化的地方。 </li><li>吸出上清液，并丢弃到一个烧杯/烧瓶中含有1N氢氧化钠。这将可灭活该环境管理体系。 </li><li>加入5毫升M9的蠕虫，倒置管的25-30倍，洗5-7分钟，或蠕虫，离心机在1500 XG直到蠕虫形成一个颗粒。吸出上清液，并丢弃到1N氢氧化钠烧杯。 </li><li>重复至少3次洗涤步骤。 </li><li>最后一次洗涤后，重新暂停少量的M9（〜500μL）的蠕虫病毒颗粒，并滴入含有OP50细菌草坪的NGM板出来。在这一点上，你不再需要工作在化学罩。 </li><li>让液体干燥几分钟，然后挑选健康的J4的幼虫转移到新鲜的种子板块。根据您所在机构的危险废物准则丢弃原板。 </li><li>让诱变蠕虫自我受精。使用立体显微镜下，屏幕上的F2代表型为隐性突变体的利益。 </li></ol>第6部分:补骨脂素诱变在本节中，我们描述了长波长紫外线（UV）一起使用TMP的突变过程。 TMP /紫外线诱变的方法被广泛用于诱导缺失突变在基因组 3 。 <ol><li> 4-5，直径6厘米的Petri板收集到15 mL离心管前面介绍的步骤中的蠕虫病毒。 </li><li>添加10毫升M9的反转20次，在1500 XG洗5分钟的蠕虫和离心机。弃上清，重复洗涤，共3次。吸出液体和重新暂停在10倍左右，M9的体积含有30微克/毫升的TMP的蠕虫。吸取20μL到2毫升M9的TMP（3毫克/毫升）。环境管理体系一样，TMP是一种强力致癌物质，必须采取适当的安全措施来处理。为了防止在紫外线照射下正确的眼睛穿的潜在风险，应使用。必须在适当的生物危害袋被丢弃的一次性。 </li><li>在黑暗中孵育15分钟的低转速的摇杆（40芬欧）在室温下管。设置覆盖的管垂直为10分钟，让所有的蠕虫下沉。 </li><li>从巴斯德吸管管的底部取出蠕虫和它们转移到一个非种子选手NGM板。丢弃以下机构的危险化学品的指引上清。 </li><li>保持在暗盘，直到多余的川芎嗪板吸收。 </li><li>使用紫外线强度计，设置之间的长波紫外线源的距离和钢板等，对钢板的强度是500微瓦 / 平方厘米4。删除铝盖，盖，盖，板，含有50秒川芎嗪治疗蠕虫的长波紫外线辐射暴露。 </li><li>盖，在室温下5小时，在黑暗中的蠕虫。 </li><li>挑选和健康的J4的幼虫转移到新鲜的种子板块。丢弃以下机构的危险化学品的指引的原板。 </li><li>使用立体显微镜，屏幕F2代利益的突变表型。 </li></ol>第7部分:代表结果一些在体育pacificus（可以通过EMS或TMP /紫外线）诱变产生的突变表型，如图1所示。每个突变体具有特征性的的表型，野生型动物，很容易分辨。从蠕虫被处理的诱变剂，30 J3/J4从亲代（P0）的幼虫被放置在单独的板块，并让其产卵，这是F1代。从板块表现出健康的F1子代，共1000 F1S被转移到个别板块和F2代表型的筛选。矮胖5,6，不协调6和变压器7表型的动物被发现。 <img src="/files/ftp_upload/2293/2293fig1.jpg" alt="图1" /> 图1。Pristionchus pacificus野生型和突变型的动物。 A. WT雌雄同体B. WT男三DPY突变D。 UNC突变E. TRA突变。前两名面板显示野生型雌雄同体（左）和男（右）的动物，在余下的面板不同的突变体进行比较。

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Cell Biol. 48, 31-31 (1995).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1381-1381 (1994).</li><li>Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. , 1-1 (2005).</li><li>Kenning, C., Kipping, I., Sommer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Genesis. 40 (3), 176-176 (2004).</li><li>Sommer, R. J., Carta, L. K., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental and Applied Nematology. 19 (6), 511-511 (1996).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genes &#38; development. 18 (10), 1198-1198 (2004).</li><li>Wei, A., Yuan, A., Fawcett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 30 (20), 110-110 (2002).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Rev Genet. 3 (5), 356-356 (2002).</li></ol>

突变是一个广泛采用的技术，在各种实验模式生物基因研究。通常用于突变实验，以确定一个基因的2的功能。这里所描述的突变程序可以使用，不仅为体育pacificus，但也为C。线虫和其他相关线虫物种。在这里，我们介绍两种方法，环境管理体系和TMP /紫外线诱变。 EMS是一种化学物质，诱导点突变或整个基因组的3,8小缺失。补骨脂突变（TMP / UV）...

an introduction to worm lab: from culturing worms to mutagenesis

这个协议描述的程序，以保持线虫在实验室，以及如何mutagenize他们使用两种可供选择的的方法:甲基磺酸乙酯（EMS）和4，5"，8 trimethylpsoralen结合紫外线（TMP /紫外线）。线虫是因为其简单的身体计划和交配系统，这是自我施肥雌雄同体和男性可以产生数以百计的每动物后代组成强大的生物遗传学的研究系统。线虫是保持中含有的细菌草坪的琼脂板，可以很容易地从一个板块转移到另一台使用一挑。 EMS是常用的一种烷化剂诱发点突变和小缺失，而TMP /紫外线主要是诱导删除。根据正在使用的线虫的物种，环境管理体系和TMP的含量进行优化。为了隔离隐性突变线虫Pristionchus pacificus，F2代的动物视觉筛选表型。为了说明这些方法，我们诱变蠕虫和不协调（UNC），矮胖（DPY）和变压器（TRA）突变体。

Watch this Scientific Journal Video about An Introduction to Worm Lab: from Culturing Worms to Mutagenesis at JoVE.com

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

与表型的缺陷突变体的筛选是一个简单的方法识别基因功能，在一个给定的生物过程。在这篇文章中我们将介绍如何对文化自由的生活的蠕虫（如，<em&gt; Pristionchus pacificus</em&gt;）在实验室中，并显示两个不同的诱变方法，环境管理体系和TMP /紫外线。

蠕虫病毒实验室介绍:从培养蠕虫突变

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

an-introduction-to-worm-lab-from-culturing-worms-to-mutagenesis

Research

JoVE Journal

Biology

34.3K 次观看.  University of Texas at Arlington. 与表型的缺陷突变体的筛选是一个简单的方法识别基因功能，在一个给定的生物过程。在这篇文章中我们将介绍如何对文化自由的生活的蠕虫（如， Pristionchus pacificus）在实验室中，并显示两个不同的诱变方法，环境管理体系和TMP /紫外线。

视频: An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

遗传学

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

环境科学

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

生物学

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Source: Prior, H., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57518/a-rapid-facile-pipeline-for-generating-genomic-point-mutants-c">A Rapid and Facile Pipeline for Generating Genomic Point Mutants in&#160;C. elegans&#160;Using CRISPR/Cas9 Ribonucleoproteins.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes a technique to rapidly screen a single nematode for a genetic marker by PCR screening. In the example protocol, we screen for CRISPR-based genome editing.

蠕虫病毒实验室介绍:从培养蠕虫突变

摘要

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此视频中的章节

生殖系化学毒性的利用线虫综合评估

光遗传学随机突变用组蛋白miniSOG在

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

培养

可筛选的

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

单蠕虫 PCR:一种提取和扩增基因组 DNA 的方法

蠕虫病毒实验室介绍:从培养蠕虫突变

摘要

探索更多视频

此视频中的章节

生殖系化学毒性的利用线虫综合评估

光遗传学随机突变用组蛋白miniSOG在

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

培养

可筛选的

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

单蠕虫 PCR:一种提取和扩增基因组 DNA 的方法

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预