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

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 ...

Research

JoVE Journal

Biology

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

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

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

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

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

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...