Name: long-term culture of individual caenorhabditis elegans on solid media for longitudinal fluorescence monitoring and aversive interventions
Uploaded: 2022-12-02T14:30:00
Description: Watch this Scientific Journal Video about Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions at JoVE.com

This work was supported by NIH R35GM133588 to G.L.S.,&#160;an NIHT32GM008659 training grant to L.E., a United States National Academy of Medicine Catalyst Award to G.L.S., and the State of Arizona Technology and Research Initiative Fund administered by the Arizona Board of Regents.

1. Recipes
NOTE: Prepare stock solutions before starting microtray plate preparation.
<ol>
	<li>Stock solutions for low-melt agarose nematode growth media (lmNGM)
	<ol>
		<li>Prepare 1 M K2HPO4 by dissolving 174.18 g of K2HPO4 in 1 L of sterile deionized water in a 1 L bottle. Autoclave the solution at 121 &#176;C, 15 psi, for 30 min and store it at room temperature (RT).</li>
		<li>Prepare 1 M KPi (pH 6.0) by dissolving 136.09 g o...

<ol>
	<li>Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Cell+Biology.">Methods in Cell Biology.</a> WormBook: The Online Review of C. elegans Biology. , (2006).</li><li>Boulin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+genes+are+required+for+functional+reconstitution+of+the+Caenorhabditis+elegans+levamisole-sensitive+acetylcholine+receptor.">Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor.</a> Proceedings of the National Academy of Sciences. 105 (47), 18590-18595 (2008).</li><li>Massie, M. R., Lapoczka, E. M., Boggs, K. D., Stine, K. E., White, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+the+metabolic+inhibitor+sodium+azide+induces+stress+protein+expression+and+thermotolerance+in+the+nematode+Caenorhabditis+elegans.">Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans.</a> Cell Stress &amp; Chaperones. 8 (1), 1-7 (2003).</li><li>Xian, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WormFarm:+a+quantitative+control+and+measurement+device+toward+automated+Caenorhabditis+elegans+aging+analysis.">WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis.</a> Aging Cell. 12 (3), 398-409 (2013).</li><li>Rahman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NemaLife+chip:+a+micropillar-based+microfluidic+culture+device+optimized+for+aging+studies+in+crawling+C.+elegans.">NemaLife chip: a micropillar-based microfluidic culture device optimized for aging studies in crawling C. elegans.</a> Scientific Reports. 10 (1), 16190 (2020).</li><li>Chronis, N., Zimmer, M., Bargmann, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+approaches+for+Caenorhabditis+elegans+research.">Microfluidic approaches for Caenorhabditis elegans research.</a> Animal Cells and Systems. 24 (6), 311-320 (2020).</li><li>Atakan, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+platform+for+long-term+culture+and+high-content+phenotyping+of+single+C.+elegans+worms.">Automated platform for long-term culture and high-content phenotyping of single C. elegans worms.</a> Scientific Reports. 9 (1), 14340 (2019).</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> Journal of Visualized Experiments. (49), e2496 (2011).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+biosensing+with+fluorescent+C.+elegans+strains.">High-throughput screening and biosensing with fluorescent C. elegans strains.</a> Journal of Visualized Experiments. (51), e2745 (2011).</li><li>Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+swim+sessions+in+C.+elegans+induce+key+features+of+mammalian+exercise.">Single swim sessions in C. elegans induce key features of mammalian exercise.</a> BMC Biology. 15 (1), 30 (2017).</li><li>&#199;elen, &. #. 3. 0. 4. ;., Doh, J. H., Sabanayagam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+liquid+cultivation+on+gene+expression+and+phenotype+of+C.+elegans.">Effects of liquid cultivation on gene expression and phenotype of C. elegans.</a> BMC Genomics. 19 (1), 562 (2018).</li><li>Churgin, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+imaging+of+Caenorhabditis+elegans+in+a+microfabricated+device+reveals+variation+in+behavioral+decline+during+aging.">Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging.</a> eLife. 6, 26652 (2017).</li><li>Turner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+and+monitoring+of+isolated+nematodes+on+solid+media+in+WorMotels.">Long-term culture and monitoring of isolated nematodes on solid media in WorMotels.</a> Journal of Visualized Experiments. , (2022).</li><li>Pittman, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+apparatus+for+individual+C.+elegans+culture.">A simple apparatus for individual C. elegans culture.</a> Methods in Molecular Biology. 2144, 29-45 (2020).</li><li>Breimann, L., Preusser, F., Preibisch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-microscopy+methods+in+C.+elegans+research.">Light-microscopy methods in C. elegans research.</a> Current Opinion in Systems Biology. 13, 82-92 (2019).</li><li>Mittal, K. K., Mickey, M. R., Singal, D. P., Terasaki, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotyping+for+homotransplantation.+18.+Refinement+of+microdroplet+lymphocyte+cytotoxicity+test.">Serotyping for homotransplantation. 18. Refinement of microdroplet lymphocyte cytotoxicity test.</a> Transplantation. 6 (8), 913-927 (1968).</li><li>. Worm paparazzi-a high throughput lifespan and healthspan analysis platform for individual Caenorhabditis elegans Available from: <a target="_blank" href="https://repository.arizona.edu/handle/10150/661628">https://repository.arizona.edu/handle/10150/661628</a> (2021)</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal of Visualized Experiments. (64), e4019 (2012).</li><li>Sutphin, G. L., Kaeberlein, 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+on+solid+media.">Measuring Caenorhabditis elegans life span on solid media.</a> Journal of Visualized Experiments. (27), e1152 (2009).</li><li>Moore, B. T., Jordan, J. M., Baugh, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WormSizer:+high-throughput+analysis+of+nematode+size+and+shape.">WormSizer: high-throughput analysis of nematode size and shape.</a> PloS One. 8 (2), 57142 (2013).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook: The Online Review of C. Elegans Biology. , 1-17 (2013).</li><li>Roussel, N., Sprenger, J., Tappan, S. J., Glaser, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+tracking+and+quantification+of+C.+elegans+body+shape+and+locomotion+through+coiling,+entanglement,+and+omega+bends.">Robust tracking and quantification of C. elegans body shape and locomotion through coiling, entanglement, and omega bends.</a> Worm. 3 (4), 982437 (2014).</li><li>Raizen, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethargus+is+a+Caenorhabditis+elegans+sleep-like+state.">Lethargus is a Caenorhabditis elegans sleep-like state.</a> Nature. 451 (7178), 569-572 (2008).</li><li>Peters, M. 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Here, we describe a novel culture system that adapts microtrays, originally developed for human leukocyte antigen tissue typing assays, to allow the isolation and characterization of single C. elegans over time in a solid media environment that is contextually similar to the agar-based NGM that is the standard in C. elegans research. This system is compatible with a variety of interventions, including dietary restriction, exogenous drug treatment, a challenge with chemical or environmental stressors, an...

C. elegans are a powerful model organism for research in genetics, cellular biology, and molecular biology, because they are easily cultured in the laboratory, have a short generation time and lifespan, share a high degree of protein homology with mammals, and have a transparent body structure that allows in vivo visualization of fluorescent proteins and dyes1. As a result of the long-standing use of C. elegans as a major model system in a range of fields, including developmental biology and aging, their growth and development are well-understood, their genome has been fully sequenced, and a host of powerful genetic tools have been created, including genome-wide RNAi feeding libraries and thousands of mutant and transgenic strains. Historically, C. elegans are cultivated as populations on solid agar nematode growth media (NGM), and phenotypes are manually evaluated either by direct observation or by imaging and downstream analysis. Fluorescent microscopy is used to capture a variety of molecular phenotypes using dyes or transgenically expressed fluorescent tags in individual C. elegans. Fluorescent imaging typically involves fixing or paralyzing an animal on slides containing thin agarose pads, which is invasive and often lethal. It also involves the use of chemicals, such as levamisole or sodium azide, which can potentially interfere with the molecular process of interest2,3. Together, these approaches allow cross-sectional, population-level data to be collected across a broad range of phenotypes, but do not allow the tracking of individual animals over time.
In recent years, several approaches have emerged to cultivate isolated C. elegans, allowing researchers to capture dynamic changes in physiological and molecular phenotypes of animals over time utilizing new imaging technologies. One category of C. elegans culture approach is microfluidics devices, including WormFarm4, the Nemalife chip5, and the &#39;behavior&#39; chip by Chronis et al.6, among various others7,8,9. Related to these are liquid-based culture methods, that use multi-well plates to characterize individual worms or small populations over time10,11. Microfluidics and microplate systems provide excellent quantitative measurements of phenotypic responses in C. elegans down to a single animal, but the culture environment presents a key limitation. The vast majority of past research in C. elegans, particularly in the field of aging, has been completed on solid agar-based media. Liquid culture causes C. elegans to swim continuously and represents a distinct environmental context that can alter the underlying biology. For example, animals cultured in liquid media have drastically altered fat content and gene expression &#8212; particularly for genes involved in the stress response &#8212;&#160;relative to animals cultured on agar-based solid NGM12,13. An alternative category of single-animal imaging methods involves polydimethylsiloxane (PDMS) devices that isolate individual animals on solid media, in an effort to more closely mimic the standard environment experienced by worms cultured on solid NGM in group culture on Petri plates. The WorMotel is a 240-well PDMS device designed to culture individual animals on solid media. Each well is filled with a modified NGM using low-melt agarose in place of agar and seeded with bacterial food, creating a solid media environment similar to the most common culture system using Petri plates. The well walls are round, allowing each animal to be imaged regardless of location in the well (avoiding the visual obscuring caused by an animal near a wall in a multi-well plate). Copper sulfate in a narrow moat surrounding each well is used as a deterrent to keep animals in their wells14,15. A limitation of this approach is that the copper sulfate is ineffective at preventing worms from fleeing when aversive environmental conditions are present, including dietary restriction, pathogenic bacteria, or chemicals that induce cellular stress (e.g., paraquat).
A second system that uses solid media is the Worm Corral, which employs a hydrogel to create a small sealed environment for each worm on a slide, allowing long-term monitoring of individually isolated animals16. A key limitation is that animals must be sealed into the environment as eggs, requiring the use of sterile animals to prevent reproduction, and limiting drug treatments to a single application. Multi-dose drug trials can be accomplished in the WorMotel either by conducting multiple rounds of exposure prior to transferring worms to the device or by topically adding additional drugs to the wells during the experiment; however, in the latter case, the actual exposure dose after adding an additional drug to an existing well is difficult to precisely quantify and depends on how rapidly the drug degrades. Both the WorMotel and the Worm Corral are excellent for brightfield or darkfield imaging to capture information related to activity and animal physiology (e.g., growth and development). While these systems can be used to monitor fluorescence, in our experience, the PDMS used to create the other single-worm imaging technologies is prone to forming microbubbles, capturing particulate, and other small abnormalities that generate irregular fluorescent artifacts that interfere with consistent fluorescence visualization and quantification, especially in the emission range for GFP, the most common fluorophore used in C. elegans research. To date, live fluorescence imaging of C. elegans individual animals in a longitudinal manner primarily relies on microfluidics devices17.
Here, we describe a novel method for culturing individual C. elegans on solid media that is compatible with both aversive interventions and direct fluorescent imaging. This approach is similar in concept to other single-worm imaging technologies, except that the custom-molded PDMS chip is replaced with commercially available polystyrene microtrays originally developed for micro cytotoxicity assays (also commonly called Terasaki trays)18. These microtrays feature wells that can be filled with solid media and seeded with bacterial food, closely mimicking the environment experienced by animals under standard solid NGM culture methodology. Each well is surrounded by an aversive barrier of palmitic acid rather than copper sulfate. Palmitic acid is commonly used to prevent worms from fleeing solid media, using standard group culture on Petri plates in experiments where worms are challenged with an aversive environment like dietary restriction or exposure to a chemical stressor. The microtrays also produce minimal and consistent fluorescent background, allowing fluorescent imaging of animals directly in their culture environment. This new single-animal solid agar-based culture system not only allows for tracking individual animals throughout life and monitoring growth, development, activity, and lifespan, but is also compatible with direct fluorescent microscopy. Because the worms can be imaged without paralysis or fixation, in vivo fluorescence biomarkers can be quantified longitudinally in individual animals remaining on their culture media, allowing the observation of dynamic changes over the lifetime of each animal. This culture system is also compatible with current generation automated systems for tracking lifespan and other health metrics14,19. We provide a detailed protocol for culturing individual C. elegans in this microtray-based system, discuss potential pitfalls and troubleshooting, and discuss the advantages and limitations relative to other systems, and in particular, an updated and optimized WorMotel protocol15.
Each single-worm culture environment consists of a microtray mounted inside a standard single-well tray using a custom 3D-printed adapter (Figure 1A). The wells are filled with low-melt agarose nematode growth media (lmNGM), seeded with concentrated bacteria as a food source, and surrounded by a palmitic acid coating to prevent worms from fleeing (Figure 1B). The space between the microtray and the walls of the single-well plate is filled with saturated water crystals to maintain humidity (Figure 1B). A detergent coating is applied to the tray lid to prevent condensation. A single worm is added to each well, and the single-well tray is sealed with Parafilm to maintain moisture and allow oxygen exchange. Up to six microtrays can reasonably be prepared in parallel by a single practiced researcher.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D-printed terasaki inserts</td>
			<td>Custom printing company</td>
			<td>Robot_Terasaki_tray_insert_10-20 
			-2021.STL</td>
			<td>FDM printing, nozzle size 0.6 mm using standard PLA plus filament</td>
		</tr>
		<tr>
			<td>AirClean systems AC624LF vertical laminar flow fume hood</td>
			<td>Fisher Scientific</td>
			<td>36-100-4376</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>Thermo Scientific</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Acros organics</td>
			<td>349615000</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans N2</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>N2</td>
			<td>Wildtype strain</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Goldbio</td>
			<td>C-103-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>ICN Biomedicals Inc</td>
			<td>101380</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50&#160;</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>OP50</td>
			<td>Standard labratory food for C. elegans</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore</td>
			<td>ex0276-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Vortex Genie 2</td>
			<td>Fisher Scientific</td>
			<td>G-560</td>
			<td></td>
		</tr>
		<tr>
			<td>FUdR&#160;</td>
			<td>Research Products International</td>
			<td>F10705-1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrating water crystals&#160;</td>
			<td>M2 Polymer Technologies</td>
			<td>Type S</td>
			<td>Type S super absorbent polymer</td>
		</tr>
		<tr>
			<td>Isopropyl &#223;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>GoldBio</td>
			<td>I2481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher Chemical</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>KimTech</td>
			<td>34155</td>
			<td>Task wipes</td>
		</tr>
		<tr>
			<td>LB Broth, Lennox</td>
			<td>BD Difco</td>
			<td>240230</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica K5 sCMOS monochrome camera</td>
			<td>Leica Microsystems</td>
			<td>11547112</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica M205 FCA Fluorescent Stereo Microscope</td>
			<td>Leica Microsystems</td>
			<td>10450826</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melt agarose</td>
			<td>Research Products International</td>
			<td>A20070-250.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#160;</td>
			<td>Fisher Chemical</td>
			<td>M-8900</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher bioreagents</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc OmniTray Single-Well Plate</td>
			<td>Thermo Scientific</td>
			<td>264728</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Sigma</td>
			<td>N1538</td>
			<td></td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>Acros organics</td>
			<td>129700010</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Coastwide Professional</td>
			<td>365374</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Parafilm</td>
			<td>16-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stratagene UV Stratalinker 2400</td>
			<td>Stratagene</td>
			<td>400075</td>
			<td>UV crosslinker</td>
		</tr>
		<tr>
			<td>Terasaki trays (Lambda)</td>
			<td>One Lambda</td>
			<td>151431</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolyne Dri-bath</td>
			<td>Thermolyne</td>
			<td>DB28125</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween&#160;</td>
			<td>Thermo Scientific</td>
			<td>J20605-AP</td>
			<td></td>
		</tr>
	</tbody>
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long-term culture of individual caenorhabditis elegans on solid media for longitudinal fluorescence monitoring and aversive interventions

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

The microtray-based single-worm culture environment described here can be used to monitor a variety of phenotypes, including lifespan and health span, activity and movement, body shape and crawling geometry, and the expression of transgenically expressed fluorescent biomarkers in individual animals over time. The microtray culture system is compatible with lifespan analysis through either manual scoring or image collection and downstream imaging analysis. As with standard culture on Petri plates21...

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

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

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

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

While other solid culture methods exist, this is the first solid culture system that allows automated longitudinal tracking of individual animal life and health span and longitudinal imaging of fluorescent biomarkers in one system. This protocol provides comparable results to standard C.elegans experiments on solid NGM is compatible with both aversive interventions and direct live fluorescence imaging. This system will be invaluable for aging research, especially regarding long-term exposure to environmental stressors and observing gene and protein dynamics over extended periods.Avoiding contamination and getting consistent drying across the wells are both challenging. We highlighted key points in the written protocol to minimize these issues. To begin, grind 500 milligrams of solid palmitic acid in a mortar and pestle.Combine it with 15 milliliters of TWEEN 20 in a 50 milliliter conical vial, and mix by vortexing to dissolve the crystals. Add 17.5 milliliters of 100%ethanol, and vortex the solution to dissolve as much palmitic acid as possible. Then add 17.5 milliliters of ultrapure water, and vortex vigorously.Gently heat the solution in a bead bath at 80 degrees Celsius until the palmitic acid dissolves completely. Reheat the bead bath to 80 degrees Celsius. Immediately before use, aliquot one milliliter of the palmitic acid working solution per Microtray into a separate sterile container, and add four microliters of nystatin per one milliliter of palmitic acid solution before heating.Now spray inside and outside the Microtray with ethanol to saturation, and shake off the excess ethanol. To sterilize the Microtray, place the plate and lid separately in the UV crosslinker. After 20 minutes, flip the plate and lid.Next, place the 15 milliliter conical vial with the palmitic acid working solution in the bead bath, and allow any visible crystals to dissolve completely before use. Warm up the Microtray by placing it on the ledge surrounding the bead bath with the lids on for two minutes. Remove the Microtray lid, and slowly add 200 microliters of palmitic acid working solution across the back wall of the Microtray.Replace the lids, and let the Microtray sit on the bead bath for two to three minutes to allow the palmitic acid solution to settle across the bottom of the Microtray. Rotate the Microtray 180 degrees, and add the palmitic acid working solution as demonstrated earlier. Prepare lmNGM by adding the components described in the manuscript.Then pipette 20 microliters of lmNGM into each Microtray placed on the bench. Be sure to cover the Microtray when refilling the pipette to minimize contamination. To seed the Microtray wells with bacterial food, centrifuge a culture grown overnight at 3, 500 G for 20 minutes.Remove the supernatant, and resuspend the bacteria culture at a tenfold concentration with 85 millimolar sodium chloride solution. Then carefully pipet five microliters of tenfold concentrated bacterial culture onto the surface of the lmNGM in each well. Avoid allowing the bacteria culture to run over the side of the well and into the palmitic acid, as this will attract worms to leave the well.Place the sterile 3D printed insert into a sterile single-well plate. Place the Microtray into the 3D printed adapter in the single-well plate. Liberally dispense water crystals on top of the 3D printed insert between the outer well of the Microtray and the inner wall of the single-well plate.If worms are not being added immediately, seal the Microtray to use the next day. Close the tray lid, and use a single piece of Parafilm to wrap all four sides to seal the Microtray. Repeat the sealing step with Parafilm two more times for three layers.Add one worm per well once they've reach the desired life stage. Add 40 microliters of detergent solution to the lid of a single-well plate, and use a task wipe to coat the lid evenly. Use additional task wipes to spread the detergent solution until vision through the lid is not distorted.Stretch the first piece of Parafilm slightly to cover two sides of the tray. Wipe the top of the tray with a task wipe, and damp it with 70%ethanol to remove any fingerprints. Measure the lifespan by gently tapping the side or top of the plate, or shining a bright blue light on the plate, and observing each animal.Score an animal dead if it does not move within a few seconds. Repeat this task every one to three days until all worms have died. Perform fluorescent imaging on single wells.Exposure to copper sulfate and dithiothreitol significantly reduced worm lifespan and health span. In contrast, copper sulfate decreased the proportion of life spent in good health, while dithiothreitol did not. Copper sulfate reduced average activity across individuals within the population to a greater extent than dithiothreitol at all ages.The population average of the individual animal area under the lifetime activity curve is substantially impaired by copper sulfate or dithiothreitol. Cumulative activity for individual animals up to day 10 correlates with their lifespan for dithiothreitol challenged animals, but not for untreated controls or copper sulfate challenged animals. A drastic increase in KYNU-1 expression is observed between days three and eight of adulthood, and there is a progressive decline with age after that.Cumulative KYNU-1 mScarlet expression through day 14 of adulthood correlates with animal lifespan. Since KYNU-1 expression is dynamic throughout a lifespan, we compared KYNU-1 expression at different ages to overall survival across individual animals. At no single time point was KYNU-1 expression significantly correlated with survival.When attempting this procedure, the most important steps are addition of the palmitic acid, pipetting the agar, and introducing the worms. This technique allows longitudinal tracking of lifespan and health span, as well as fluorescent biomarkers in individual worms in a way that is directly comparable to NGM assays.

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