This work was supported by a New Investigator Grant [2014R1A1A1005553] from the National Research Foundation of Korea (NRF) to J.I.L; and a Yonsei University Future Leader Challenge Grant [2015-22-0133] to J.I.L.

1. Prepare Solutions for NGT-3D and NGB-3D
<ol>
	<li>Prepare the following sterile solutions: 1 L of 0.1454 M NaCl solution, 1 L of 1 M CaCl2, 1 L of 1 M MgSO4, Lysogeny Broth (LB), 1 L of 1 M KPO4 buffer (108.3 g of KH2PO4 and 35.6 g of K2HPO4, and fill H2O to 1 L). Production of NGM using these solutions can be found in a previous protocol10.</li>
	<li>Autoclave solutions at 121 &#176;C, 15 min.</li>
	<li>Prepare 50 ml ...

<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>

The laboratory cultivation of C. elegans using the classical nematode growth media plates was crucial to the hundreds of important discoveries that research in C. elegans has provided. Here, we present new methods to cultivate C. elegans in an environment that more accurately reflects their natural three-dimensional habitats. Although other methods have been used to observe C. elegans in 3D13, this is the first protocol that allows cultivation of worms in a solid 3D matrix. ...

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.

The construction of NGT-3D is a simple and straightforward protocol that results in an agar-filled test tube with small bacterial colonies spaced throughout the agar (Figure 1A). Worms can freely move through the agar matrix, finding and consuming the bacterial colonies. To confirm whether C. elegans can reproduce and grow normally in NGT-3D, we compared fertility and larval development in 3D with standard 2D NGM plates. In the relative brood size assay, adult <e...

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

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

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 ...

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.

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17.6K visualizações.  Yonsei University. 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 res...