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 of a sterile 5 mg/ml cholesterol solution. In a 50 ml conical tube, mix 0.25 g of cholesterol and 50 ml of 99.99% ethanol and mix well. Do not autoclave. Sterilize the cholesterol solution using a 50 ml syringe with a 0.45 &#181;m syringe filter. This will also remove any undissolved cholesterol.</li>
	<li>(Optional) Prepare 10 ml of a 150 mM of 2&#39;-deoxy-5-fluorouridine (FUdR) stock. In a 15&#160;ml conical tube, mix 0.3693 g of FUdR and 10 ml of distilled water. Shake well.</li>
</ol>
2. Prepare Bacteria Culture for NGT-3D and NGB-3D
<ol>
	<li>Inoculate 10 ml LB broth with bacteria. The standard bacteria used for C. elegans feeding is E. coli strain OP50.</li>
	<li>Culture the bacterial inoculation at 37 &#176;C in a shaking incubator overnight.</li>
	<li>In preparation for a serial dilution, aliquot 9 ml of the NaCl solution into sterile 15 ml conical tubes. For example, aliquot 9 ml into a total of 7 tubes to make a 10-7 dilution of OP50 strain E. coli.</li>
	<li>Using a sterile 1,000 &#181;l pipette, pipet 1 ml of the bacterial culture or diluted bacterial culture into the sterile new tube with 9 ml of NaCl solution and vortex the 10 ml mixture well. Repeat until the desired bacterial dilution is reached. 
	NOTE: The desired bacterial dilution depends on the type and condition of bacteria as well as the exact experimental conditions. For example, a 10-6 - 10-8 dilution of OP50 strain E. coli was sufficient to produce from 1 - 200 bacterial colonies per 6.5 ml NGT-3D tube. Worms reliably developed and reproduced normally when the number of colonies was over 60, which occurred at a dilution of 10-6 - 10-7. For NGB-3D, use a dilution of 10-8.</li>
</ol>
3. Making NGT-3D and NGB-3D (200 ml)
<ol>
	<li>After preparing the bacterial culture, mix 0.6 g NaCl, 1 g granulated agar, and 0.5 g peptone in a 500 ml flask. Insert a magnetic stir bar into the flask.</li>
	<li>Add 195 ml distilled water and cover mouth of the flask with aluminum foil.</li>
	<li>Autoclave 121 &#176;C for 15 min.</li>
	<li>Place the hot autoclaved flask onto a stir plate, and stir at a moderate speed for at least 2 hr. Cool the flask to 40 &#176;C. Be sure to cool sufficiently as continuing from here at high temperatures may lead to clouding of the finished agar. However, lower temperatures may result in premature hardening of the agar.
	<ol>
		<li>(Optional) To speed up the cooling process, place the flask in a 40 &#176;C water bath 15 min before placing on a stir plate.</li>
	</ol>
	</li>
	<li>When the temperature of the agar media reaches 40 &#176;C, add 200 &#181;l 1 M CaCl2, 200 &#181;l of 5 mg/ml cholesterol solution, 200 &#181;l 1 M MgSO4 and 5 ml 1 M KPO4 buffer as the solution continues to stir to final concentrations of 1 mM CaCl2, 5 &#181;g/ml cholesterol, 1 mM MgSO4, 1 mM KPO4.
	<ol>
		<li>(Optional) For NGT-3D lifespan assay add 80 &#181;l of 150 mM FUdR to a final concentration of 120 &#181;M12.</li>
	</ol>
	</li>
	<li>Add 6 ml of the 10 ml diluted bacterial culture from step 2.4 into the flask directly.
	<ol>
		<li>(Optional) For NGT-3D, remove 6 ml of agar media before step 3.6 and keep it warm separately. This media will be used for the bacteria-free top layer.</li>
	</ol>
	</li>
	<li>Using sterile procedures, dispense the media into a sterile culture chamber. Make sure the cover of the chamber closes tightly.
	<ol>
		<li>(Optional) To prevent bacterial colonies from forming at the top surface of the agar, make a layer of bacteria-free agar media from step 3.6.1 on the top before the media from step 3.7 completely hardens. This will create a bacteria-free zone at the top of the NGT-3D.</li>
		<li>For NGT-3D, pour 6.5 ml media into the 8 ml clear plastic test tubes to make a bacteria agar layer, and carefully dispense 200 &#181;l of the bacteria-free media on top of the semi-hardened 3D media to make a thin bacteria-free layer on top.</li>
		<li>For NGB-3D, pour 65 ml of media into the 25 cm2 clear plastic cell culture bottle. This amount should fill the body of the 25 cm2 cell culture bottle.</li>
	</ol>
	</li>
	<li>Leave chambers vertically at room temperature for one week to allow bacterial colonies to grow to a considerable size of at least 1 mm diameter.
	<ol>
		<li>(Optional) For lifespan assay in NGT-3D, cover chambers with aluminum foil to prevent light degradation of FUdR.</li>
	</ol>
	</li>
</ol>
4. Measure Fitness of Worm Population on NGT-3D (Relative Brood Size Assay)
<ol>
	<li>Pick an L4 stage worm using a platinum wire pick and transfer on a bacteria-free NGM plate. Allow worm to freely move around for a few minutes to remove bacteria attached to its body.</li>
	<li>Repeat step 4.1 and make sure that the worm is free from bacteria. Generally, two repeats of 4.1 are enough.</li>
	<li>Carefully place the clean worm on the surface of the 3D media with a platinum wire pick. The worm should eventually enter the agar into the 3D agar matrix.</li>
	<li>Close the cover loosely allowing some air to get into the tube, but preventing drying of the agar media.</li>
	<li>Incubate for 96 hr at 20 &#176;C.</li>
	<li>After 4 days, close the lids tightly and place the NGT-3D culture chamber into an 88 &#176;C water bath to melt the agar. The heat kills worms but their bodies remain intact. 
	NOTE: Using 8 ml tubes for NGT-3D, 20 - 30 min incubation is usually sufficient.</li>
	<li>Using a glass pipette, transfer the melted media onto a 9 cm plastic petri dish. Plastic pipettes are not advised, as worms can often stick to them.</li>
	<li>Using a transmission stereo dissecting microscope, count the number of worms at the L3, L4, and adult stages. Do not count worms that are L2 stage and younger, as the F1 and F2 generations can be confused here. Thus, this assay is a relative brood size assay rather than a total brood size assay.</li>
</ol>
5. Image and Record Worm Behavior on NGB-3D
<ol>
	<li>Pick an L4 stage worm using a platinum wire pick and remove any bacteria stuck to the surface by transferring to a bacteria-free NGM plate and allowing it to freely move for a few min.</li>
	<li>Repeat step 5.1 to make sure the worm is free from bacteria.</li>
	<li>Carefully place the clean worm onto the center of the agar surface of the NGB-3D near the neck of the bottle with a platinum wire pick. The worm should eventually enter the agar into the 3D agar matrix.</li>
	<li>Close the cover loosely allowing some air to get into the tube, while preventing drying of the agar media.</li>
	<li>Image or record the worm under a transmission stereo dissecting microscope. Adjust the focus as the worm moves through the 3D matrix.</li>
</ol>

<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 authors have nothing to disclose.

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 ...

cultivation-caenorhabditis-elegans-three-dimensions

Research

JoVE Journal

Biology

17.7K Views.  Yonsei University. 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.

Video: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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 hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

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

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan 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

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode 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

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

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

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

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

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 function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

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

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

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 documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.
Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

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)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

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

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile 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

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

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