Name: cultivation of caenorhabditis elegans in three dimensions in the laboratory
Uploaded: 2016-12-12T14:00:00
Description: Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

这项工作是由支持的新研究员格兰特[2014R1A1A1005553]韩国国家研究基金会（NRF）为JIL;和延世大学未来之星挑战格兰特[2015-22-0133]为JIL

1.准备NGT-3D和NGB-3D解决方案<ol><li>准备以下的无菌溶液：1升的0.1454摩尔NaCl溶液中，1升的1M的CaCl 2，1升的1M 硫酸镁的，LB培养基（LB），1升1M的KPO 4缓冲液（108.3克KH 2的PO 4和35.6克K 2的HPO 4，并填写H 2 O操作1升）。利用这些解决方案NGM的生产可以在先前的协议10被发现。 </li><li>在121℃，15分钟的高压釜的解决方案。 </li><li>准?...

<ol>
	<li>Corsi, A. K., Wightman, B., Chalfie, 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+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Consortium, C. E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282 (5396), 2012-2018 (1998).</li><li>Choi, J. I., Yoon, K. H., Kalichamy, S. S., Yoon, S. S., Lee, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+natural+odor+attraction+between+lactic+acid+bacteria+and+the+nematode+Caenorhabditis+elegans.">A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans.</a> ISME J. 10 (3), 558-567 (2016).</li><li>Gaertner, B. E., Phillips, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+platform+for+molecular+quantitative+genetics+and+the+systems+biology+of+natural+variation.">Caenorhabditis elegans as a platform for molecular quantitative genetics and the systems biology of natural variation.</a> Genet Res (Camb). 92 (5-6), 331-348 (2010).</li><li>Cutter, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+evolution+in+the+wild.">Caenorhabditis evolution in the wild.</a> Bioessays. 37 (9), 983-995 (2015).</li><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=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Felix, M. A., Braendle, C. <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+history+of+Caenorhabditis+elegans.">The natural history of Caenorhabditis elegans.</a> Curr Biol. 20 (58), R965-R969 (2010).</li><li>Barriere, A., Felix, 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=High+local+genetic+diversity+and+low+outcrossing+rate+in+Caenorhabditis+elegans+natural+populations.">High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations.</a> Curr Biol. 15 (13), 1176-1184 (2005).</li><li>Lee, T. Y., Yoon, K. H., Lee, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGT-3D:+a+simple+nematode+cultivation+system+to+study+Caenorhabditis+elegans+biology+in+3D.">NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.</a> Biol Open. 5 (4), 529-534 (2016).</li><li>Lewis, J. A., Flemming, J. T., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Culture+Methods.">Basic Culture Methods.</a> Caenorhabditis elegans.: Modern Biological Analysis of an Organism. 48, 4-27 (1995).</li><li>Choi, S. Y., Yoon, K. H., Lee, J. I., Mitchell, 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=Violacein:+Properties+and+Production+of+a+Versatile+Bacterial+Pigment.">Violacein: Properties and Production of a Versatile Bacterial Pigment.</a> Biomed Res Int. , 465056 (2015).</li><li>Solis, G. M., Petrascheck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+in+96+well+microtiter+plates.">Measuring Caenorhabditis elegans life span in 96 well microtiter plates.</a> J Vis Exp. (48), (2011).</li><li>Kwon, N., Pyo, J., Lee, S. J., Je, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+worm+tracker+for+freely+moving+C.+elegans.">3-D worm tracker for freely moving C. elegans.</a> PLoS One. 8 (2), e57484 (2013).</li><li>Pierce-Shimomura, J. T., Chen, B. L., Mun, J. J., Ho, R., Sarkis, R., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc Natl Acad Sci U S A. 105 (52), 20982-20987 (2008).</li><li>Kwon, N., Hwang, A. B., You, Y. J., SJ, V. L., Je, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+C.+elegans+behavioral+genetics+in+3-D+environments.">Dissection of C. elegans behavioral genetics in 3-D environments.</a> Sci Rep. 5, 9564 (2015).</li></ol>

采用经典的线虫的生长介质板线虫的实验室培养是至关重要的该研究线虫提供了数百个重要的发现。在这里，我们提出新的方法来培养线虫中更准确地反映其三维天然栖息地的环境。尽管其它方法已被用于在3D 13观察线虫 ，这是第一个协议，它允许蠕虫培养在固体三维基质。这里展示的两种方法，NGT-3D和NGB-3D，让科学家问3D养身健体，发展壮大的问题，也是图像...

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LB broth, Miller (Luria-Bertani)</td>
			<td>Difco</td>
			<td>224620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>DAEJUNG</td>
			<td>7548-4400</td>
			<td>58.44 MW</td>
		</tr>
		<tr>
			<td>Agar, Granulated</td>
			<td>Difco</td>
			<td>214530</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride, dihydrate</td>
			<td>Bio Basic</td>
			<td>CD0050</td>
			<td>2*H2O; 147.02 MW</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Bio Basic</td>
			<td>CD0122</td>
			<td>386.67 MW</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>B&#38;J</td>
			<td>RP090-1</td>
			<td>99.99%; 46.07 MW</td>
		</tr>
		<tr>
			<td>Magnesium sulfate, anhydrous</td>
			<td>Bio Basic</td>
			<td>MN1988</td>
			<td>120.37 MW</td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic, anhydrous</td>
			<td>Bio Basic</td>
			<td>PB0445</td>
			<td>136.09 MW</td>
		</tr>
		<tr>
			<td>2&#39;-Deoxy-5-fluorouridine</td>
			<td>Tokyo Chemical Industry</td>
			<td>D2235</td>
			<td>246.19 MW</td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic, anhydrous</td>
			<td>Bio Basic</td>
			<td>PB0447</td>
			<td>174.18 MW</td>
		</tr>
		<tr>
			<td>Multi-Purpose Test Tubes</td>
			<td>Stockwell Scientific</td>
			<td>ST.8570</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Test Tube Closures</td>
			<td>Stockwell Scientific</td>
			<td>ST.8575</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>SPL Lifescience</td>
			<td>70125</td>
			<td>25 cm^2</td>
		</tr>
		<tr>
			<td>Research Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Definition Color Camera Head</td>
			<td>Nikon</td>
			<td>DS-Fi2</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-Based Control Unit</td>
			<td>Nikon</td>
			<td>DS-U3</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Basic Research, Microscope Imaging Software</td>
			<td>Nikon</td>
			<td>MQS32000</td>
			<td></td>
		</tr>
	</tbody>
</table>

cultivation of caenorhabditis elegans in three dimensions in the laboratory

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 know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

NGT-3D的结构是一个简单的和直接的协议，其导致在整个琼脂（ 图1A）间隔开小的细菌菌落在琼脂填充试管。蠕虫可通过琼脂矩阵自由移动，寻找和消费的细菌菌落。为了证实线虫是否可以复制和NGT-3D正常生长，我们比较土壤肥力与3D幼虫发育与标准的2D NGM板。在育雏相对大小分析，成年线虫在NGT-3D雌雄同体只是重现，以及在标准的2D NGM板雌雄同?...

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

我们提出了一个简单的方法来构建三维线虫培养系统称为NGT-3D和NGB-3D。这些可以被用来研究在更类似于天然线虫栖息比标准2D实验室线虫培养板栖息线虫健身和行为。

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 ...

The overall goal of this procedure is to manufacture a three dimensional cultivation system for nematodes that may better simulate natural conditions. This method can answer key questions in the field of C elegans development, physiology and behavior. We first had the idea for this method when we were studying natural nematode behavior in rotting fruit which is a 3D environment.This technique is advantage compared to standard two dimensional cultivation techniques because nematodes reside in 3D habitats in nature. Though this method has been developed C elegans cultivation, it can also be applied to other nematodes. Demonstrating this procedure will be Tong Young Lee, a graduate student in my laboratory.To begin this procedure, Otto Clave the prepared solutions at 121 degrees Celsius for 15 minutes. Next, prepare 50 milliliters of the sterile cholesterol solution at 5 milligrams per milliliter. In a 50 milliliter conical tube, add 0.25 grams of cholesterol and 50 milliliters of 99.99%ethanol and mix well.Next, sterilize the cholesterol solution using a 50 milliliter syringe with a 0.45 vented syringe filter and remove any undeveloped cholesterol. Then, inoculate 10 milliliters of lysogeny broth with bacteria. Culture the bacterial inoculation at 37 degrees Celsius in a shaking incubator overnight.In preparation for a serial dilution, allocate nine milliliters of the Sodium Chloride solution into seven sterile 15 milliliter conical tubes. To make a tent of the seventh fold dilution of Opi 50 Strain E Coli. Afterward, pop out one milliliter of the bacterial culture or diluted bacterial culture to a new tube with nine milliliters of Sodium Chloride solution.And vortex the mixture well. After preparing the bacterial culture, add 0.6 grams of Sodium Chloride, one gram of granulated auger and 0.5 grams of peptone in a 500 milliliter flask. Place a magnetic stir bar into the flask.Then add 195 milliliters of distilled water and cover the flask with aluminum foil. Next, Otto Clave the sample at 121 degrees Celsius for 15 minutes. Subsequently, place the hot Otto Claved flask into a stir plate, stir at a moderate speed for at least two hours until it reaches 40 degrees Celsius.Alternatively, the flask can be cooled to 40 degrees Celsius by placing it in a 40 degrees Celsius water bath for 15 minutes before transferring it on a stir plate. Be sure to cool sufficiently as continuing from here. A high temperature may lead to clouding of the finished auger but temperature that is too low may result in premature hardening of the auger.When the temperature of auger media reaches 40 degrees Celsius, add Calcium Chloride, Cholesterol Solution, Magnesium Sulfate and Potassium Phosphate Buffer and continue to stir the solution. For NGT-3D lifespan assay, add 80 microliters of 150 millimeter FUDR to the media to result in a final concentration of 120 micromolar. Then, remove six milliliters of auger media into a sterile conical tube and keep it warm in an incubator.This media will be used for the bacteria free top layer. Next add six milliliters of the diluted bacteria culture into the flask directly on a sterilized bench. For NGT-3D, pour 6.5 milliliters of media into the eight milliliter clear plastic test tubes to form a bacteria auger layer.And carefully dispense 200 micro liters of the bacteria free media on top of the semi-hard 3D media to make a thin bacteria free layer on top. Then close the cover of the chamber tightly. For NGB-3D, pour 65 milliliters of media into a 25 centimeter squared clear plastic cell culture bottle.Then position the chamber vertically at room temperature for one week to allow bacterial colonies to grow to considerable size of at least one millimeter diameter. In this procedure, pick a L four stage worm and transfer it to a bacteria free NGM plate. Then allow the worm to freely move around for a few minutes in order to remove bacteria from it's body.And repeat the procedure two more times. Afterward, carefully place the clean worm on the surface of the 3D media. The worm should've actually entered the auger into the 3D auger matrix.Then close the cover loosely to incubate for 96 hours at 20 degrees Celsius. After four days, close the lid tightly and place the NGT-3D culture chamber into a water bath at 88 degrees Celsius to melt the auger. Using a glass pipette, transfer the melted media onto a nine centimeter plastic petri dish.Afterward, using a transmission stereo dissecting microscope, count the number of worms at the L3, L4 and adult stages. As shown here, the construction of NGT-3D is a simple and straightforward protocol that results in an auger filled test tube with small bacterial colonies spaced throughout the auger. To confirm whether C elegans can reduce and grow normally in NGT-3D, fertility and larva development are compared in 3D with the standard 2D NGM plates.In the Relative brood size assay, adult C elegans hermaphrodites in NGT-3D reproduced just as well as hermaphrodites in the standard 2D MGM plates when bacterial colonies are plentiful with at least 60 colonies. Furthermore, larva development in NGT-3D also proceeds normally when there are plenty of bacterial colonies with the majority of worms in the adult state after 96 hours. However, if bacterial colonies are sparse in the 3D matrix, both relative brood size and larva development are negatively impacted.Here is a survival curve of the wild type C elegans in NGT-3D or NGM plates. Once mastered, this technique can be done in three hours if performed properly. Following this procedure, other methods like life span assays and behavior analysis can be performed in order to answer questions about aging and behavior in 3D.After its development, this technique can pave the way for researchers in the fields of genetics to explore questions about ecology and evolution in C elegans. After watching this video, you should have a good understanding of how to cultivate nematodes in a 3D laboratory environment.

Research

JoVE Journal

Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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 ...

科研

生物学

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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 ...

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

三维声带振动的半喉设置研究

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...