Name: culture and assay of large-scale mixed-stage caenorhabditis elegans populations
Uploaded: 2021-05-05T14:00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations at JoVE.com

We thank members of the Edison Lab for helpful discussions and feedback on this manuscript; in particular, B.M. Garcia. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and CeNDR, which is funded by NSF Living Collections CSBR 1930382. This work was supported by a grant from the NIH (U2CES030167).

<ol>
	<li>F&#233;lix, 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> Current Biology. 20 (22), 965-969 (2010).</li><li>Corsi, A. K. <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> WormBook. , 1-31 (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+behaviour.">The genetics of behaviour.</a> British Medical Bulletin. 29 (3), 269-271 (1973).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: The Online Review of C. elegans Biology. , (2006).</li><li>Xiong, H., Pears, C., Woollard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+C.+elegans+based+platform+for+toxicity+assessment.">An enhanced C. elegans based platform for toxicity assessment.</a> Scientific Reports. 7 (1), 9839 (2017).</li><li>Loer, C. 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=Cuticle+integrity+and+biogenic+amine+synthesis+in+Caenorhabditis+elegans+require+the+cofactor+tetrahydrobiopterin+(BH4).">Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4).</a> Genetics. 200 (1), 237-253 (2015).</li><li>Li, Y., Paik, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+role+for+fatty+acid+biosynthesis+genes+during+molting+and+cuticle+formation+in+Caenorhabditis+elegans.">A potential role for fatty acid biosynthesis genes during molting and cuticle formation in Caenorhabditis elegans.</a> BMB Reports. 44 (4), 285-290 (2011).</li><li>Meli, V. S., Osuna, B., Ruvkun, G., Frand, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MLT-10+Defines+a+Family+of+DUF644+and+Proline-rich+Repeat+Proteins+Involved+in+the+Molting+Cycle+of+Caenorhabditis+elegans.">MLT-10 Defines a Family of DUF644 and Proline-rich Repeat Proteins Involved in the Molting Cycle of Caenorhabditis elegans.</a> Molecular Biology of the Cell. 21 (10), 1648-1661 (2010).</li><li>Fritz, J. A., Behm, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CUTI-1:+A+novel+tetraspan+protein+involved+in+C.+elegans+CUTicle+formation+and+epithelial+integrity.">CUTI-1: A novel tetraspan protein involved in C. elegans CUTicle formation and epithelial integrity.</a> PloS One. 4 (4), 5117 (2009).</li><li>Golden, J., Riddle, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pheromone+influences+larval+development+in+the+nematode+Caenorhabditis+elegans.">A pheromone influences larval development in the nematode Caenorhabditis elegans.</a> Science. 218 (4572), 578-580 (1982).</li><li>Jeong, P. Y., 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=Chemical+structure+and+biological+activity+of+the+Caenorhabditis+elegans+dauer-inducing+pheromone.">Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone.</a> Nature. 433 (7025), 541-545 (2005).</li><li>Srinivasan, J., 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+blend+of+small+molecules+regulates+both+mating+and+development+in+Caenorhabditis+elegans.">A blend of small molecules regulates both mating and development in Caenorhabditis elegans.</a> Nature. 454 (7208), 1115-1118 (2008).</li><li>Kaplan, F., 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=Ascaroside+Expression+in+Caenorhabditis+elegans+Is+Strongly+Dependent+on+Diet+and+Developmental+Stage.">Ascaroside Expression in Caenorhabditis elegans Is Strongly Dependent on Diet and Developmental Stage.</a> PLoS One. 6 (3), 17804 (2011).</li><li>Golden, J., Riddle, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pheromone+influences+larval+development+in+the+nematode+Caenorhabditis+elegans.">A pheromone influences larval development in the nematode Caenorhabditis elegans.</a> Science. 218 (4572), 578-580 (1982).</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>Andersen, E. C., Bloom, J. S., Gerke, J. P., Kruglyak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Variant+in+the+Neuropeptide+Receptor+npr-1+is+a+Major+Determinant+of+Caenorhabditis+elegans+Growth+and+Physiology.">A Variant in the Neuropeptide Receptor npr-1 is a Major Determinant of Caenorhabditis elegans Growth and Physiology.</a> PLoS Genetics. 10 (2), 1004156 (2014).</li><li>Rosenblatt, 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+central+limit+theorem+and+a+strong+mixing+condition.">A central limit theorem and a strong mixing condition.</a> Proceedings of the National Academy of Sciences. 42 (1), 43-47 (1956).</li><li>Boyd, W. A., Smith, M. V., Freedman, 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=Caenorhabditis+elegans+as+a+model+in+developmental+toxicology.">Caenorhabditis elegans as a model in developmental toxicology.</a> Methods in Molecular Biology. 889, 15-24 (2012).</li><li>Nika, L., Gibson, T., Konkus, R., Karp, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Beads+Are+a+Versatile+Tool+for+Staging+Caenorhabditis+elegans+in+Different+Life+Histories.">Fluorescent Beads Are a Versatile Tool for Staging Caenorhabditis elegans in Different Life Histories.</a> Genes, Genomes, Genetics. 6 (7), 1923-1933 (2016).</li><li>Denver, D. R., Morris, K., Streelman, J. T., Kim, S. K., Lynch, M., Thomas, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+consequences+of+mutation+and+natural+selection+in+Caenorhabditis+elegans.">The transcriptional consequences of mutation and natural selection in Caenorhabditis elegans.</a> Nature Genetics. 37 (5), 544-548 (2005).</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>Kevin, S., Pulak, R., Strange, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+Analysis,+Sorting,+and+Dispensing+of+C.+elegans+on+the+COPAS+Flow-Sorting+System.">Techniques for Analysis, Sorting, and Dispensing of C. elegans on the COPAS Flow-Sorting System.</a> C. elegans. Methods in Molecular Biology. 351, 275-286 (2006).</li><li>Lee, D., 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=Selection+and+gene+flow+shape+niche-associated+variation+in+pheromone+response.">Selection and gene flow shape niche-associated variation in pheromone response.</a> Nature Ecology &amp; Evolution. 3 (10), 1455-1463 (2019).</li><li>Cook, D. E., Zdraljevic, S., Roberts, J. P., Andersen, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CeNDR,+the+Caenorhabditis+elegans+natural+diversity+resource.">CeNDR, the Caenorhabditis elegans natural diversity resource.</a> Nucleic Acids Research. 45, 650-657 (2016).</li><li>Nika, L., Gibson, T., Konkus, R., Karp, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Beads+Are+a+Versatile+Tool+for+Staging+Caenorhabditis+elegans+in+Different+Life+Histories.">Fluorescent Beads Are a Versatile Tool for Staging Caenorhabditis elegans in Different Life Histories.</a> Genes, Genomes, Genetics. 6 (7), 1923-1933 (2016).</li></ol>

The authors have nothing to disclose.

A variety of vessels can be used as a LSCP. In this protocol, a standard glass baking dish was used. The LSPCs in use had outer dimensions of 35.56 x 20.32 cm, inner dimensions of 27.94 x 17.78 cm, and approximately 4.45 cm deep and came with a fitted lid. Thus, the amount of bacteria used here has been optimized for a LSCP with the above dimensions to yield a large population of mixed-stage worms. Bacterial volume and concentration can be adjusted to fit the experimental needs.
Contamination by mold, fungi, or other bacterial sources can occur at any step in the LSCP method, so handle samples with care. Prior to starting any step in the protocol, ensure that the working space is cleaned with 70% ethanol and 10% bleach. If available, treat used areas with UV light for 30 min and turn on a HEPA air-filter 30 min prior to starting each step.
By growing the LSCP in a controlled setting (i.e., in a CT room set at 20 &#176;C), the user can more easily track the growth of the sample and document potential contamination. If the surface of the LSCP becomes contaminated, either cut out the contamination when possible and let the sample continue to grow or discard the sample if the contamination is not possible to control. It is imperative to address contamination quickly to reduce unwanted growth and to ensure it is not outcompeting worms for resources.
This method is meant for those who want to grow large-scale mixed-population cultures of C. elegans. Although it may be possible to grow synchronized populations of worms on the LSCP as done on commercially available Petri dishes and in liquid culture, the authors have not tested this option. Additionally, if users wish to grow more than approximately 2.4 million worms on average in a given sample, a different method is recommended4. Growth success is dependent on the strain being processed in the pipeline. The authors were able to successfully grow populations of approximately 2.4 million worms in at least five biological replicates of 15 C. elegans strains, indicating that the method is robust.
Prior to starting the experiment note that the age and health of a given worm can influence fecundity and subsequent population growth time. Ensure that worms are maintained in healthy conditions with minimal stress prior to being used in this pipeline. It is assumed that stock samples have been created, frozen, and kept at -80 &#176;C to reduce genetic drift over time.
Depending on the needs of a given experiment, the number of starting gravid adults on a LSCP can be changed. Altering the number of starting gravid adults on the LSCP will change the growth rate and thus time to harvest. Five gravid adults are used to seed each LSCP for the following reasons: (1) A simple, fast, and efficient way to seed many C. elegans strains onto LSCPs at one time was needed and (2) to reduce the age differences amongst the gravid adults picked that could lead to the growth heterogeneity.
This method allows the user to harvest large populations of worms with all life cycle stages present. With current methods available, collecting large-scale samples of C. elegans requires bleach synchronization to obtain the number of worms desired for downstream work. Given this approach, one can now grow as many worms as previously possible in fermenters or large-scale liquid cultures without the difficulties associated with bleach synchronizing and multiple handling steps. Our protocol allows one to target strains of interest efficiently, use minimal handling time in growing the sample itself, and isolate stages of worms or the population as needed in downstream pipelines.
A LPFC was utilized as a tool to document the population distribution and size in a given LSCP. The LPFC used is a continuous flow system that analyzes, sorts, and dispenses worms based on their size (TOF) and optical density. As a given worm passes through the flow cell, the axial light-loss detector captures the amount of signal light blocked by a 488 nm-solid state laser for the length of time it takes a worm to pass through, giving the user the TOF and optical density of the worm. Fluorescence collection optics and detectors can also be utilized to maximize fluorescence sensitivity and collection on each sample. LPFC collection parameters will vary based on instrument. Users can employ a variety of platforms to capture worm size and are not limited to using this protocol if a LPFC is not available.
The authors are using samples grown in the method described here to identify unknown metabolites in various strains of C. elegans via Liquid Chromatography &#8211; Mass Spectrometry, NMR spectroscopy, and RNA sequencing. The authors plan to continue to use this method for growth of samples in this pipeline with a variety of C. elegans strains as new strains of interest can be easily processed using this pipeline.

C. elegans is a small free-living nematode that is found throughout the world in a variety of natural habitats1. Its relative ease of growth, fast generation time, reproduction system, and transparent body make it a powerful model organism that has been widely studied in developmental biology, aging, neurobiology, and genetics2,3. The copious work on C. elegans makes it a prime candidate to use in -omics studies to comprehensively link phenotypes with complex biological components and their relationships in a given organism.
To use C. elegans in collaborative -omics research, a method is needed to generate large mixed-stage populations of animals where a single sample can be split and used across diverse platforms and instruments for comparative analyses. Creating a pipeline to generate such a sample requires keen awareness of diet, environment, stress, population structure, and sample handling and collection. Therefore, it is crucial to have standard and reproducible culturing conditions integrated into large-scale pipelines. In C. elegans research, two traditional methods are used to culture worms - agar Petri dishes and liquid culture4.
Historically, when large quantities of C. elegans are needed, they are grown in liquid culture4. The steps involved in generating a large population of worms in liquid culture require multiple handling steps that often include bleach synchronization to rupture gravid adult cuticles, releasing embryos to achieve the desired population size. However, when bleach synchronization is used, population growth is dependent on starting census size and, therefore, effects subsequent growth and population numbers. In addition, C. elegans strains vary in their cuticle sensitivity, exposure time, and stress response to bleach synchronization making it difficult to assay many strains at a time5,6,7,8,9.
Additionally, worm growth in liquid culture requires a couple of transfer steps as it is often recommended to grow just one generation of worms before harvesting because overcrowding can easily occur if grown for multiple generations and lead to dauer formation despite the presence of food10. Dauer formation occurs through small signaling molecules such as ascarosides, often referred to as &#8220;dauer pheromones&#8221;11,12,13,14, are released into liquid media and effect the growth of the population. Furthermore, growing large worm populations in liquid culture leads to excess bacteria accumulation in the culture, creating difficulties when a clean sample is needed for downstream phenotypic assays. Lastly, when a liquid culture becomes contaminated, it is more difficult to maintain as fungal spores or bacterial cells are easily dispersed throughout the media15.
The other traditional method of growing C. elegans is on agar Petri dishes. Commercially available Petri dishes allow one to easily grow multiple generations of mixed-stage worms without the rapid effects of overcrowding and high dauer formation as seen in liquid cultures. However, a disadvantage to worm growth on traditional agar Petri dishes is that the largest commercially available Petri dish does not yield large worm populations for an -omics study without adding in a bleach synchronization step. In summary, culturing mixed-stage populations of C. elegans on agar Petri dishes is more suitable for collecting -omics data, but we required a method to generate very large population sizes without liquid culturing.
Here, we present a method to culture and collect large mixed-stage C. elegans populations on large scale culture plates (LSCP). Collecting samples through this pipeline yields enough sample to gather phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation of the animals, less user prep time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility.

<table>
	<tbody>
		<tr>
			<td>10 mL Sterile Serological Pipettes</td>
			<td>VWR</td>
			<td>89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ul pipette tips</td>
			<td>VWR</td>
			<td>89079-438</td>
			<td></td>
		</tr>
		<tr>
			<td>100 ul pipette tips</td>
			<td>VWR</td>
			<td>89079-442</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Graduated Cylinder</td>
			<td>VWR</td>
			<td>10124-380</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 ul pipette tips</td>
			<td>VWR</td>
			<td>89079-488</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR</td>
			<td>89039-668</td>
			<td></td>
		</tr>
		<tr>
			<td>190 Proof Ethanol</td>
			<td>VWR</td>
			<td>89125-166</td>
			<td></td>
		</tr>
		<tr>
			<td>2 L Wide Neck Erlenmeyer Flask</td>
			<td>VWR</td>
			<td>75804-654</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>VWR</td>
			<td>75874-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>05040-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BVC Control G Fluid Aspiration System</td>
			<td>Vacuubrand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>449709-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C3045-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorox Bleach</td>
			<td>VWR</td>
			<td>89414-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Conviron Control Temperature Room</td>
			<td>Conviron</td>
			<td></td>
			<td>https://www.conviron.com/environmental-rooms</td>
		</tr>
		<tr>
			<td>Corning Low Volume 384 Well Black with Clear Flat Bottom Polystyrene TC-Treated Microplate</td>
			<td>VWR</td>
			<td>89089-866</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Scientific Accuspin 3R</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-Bottom 24-Well Plate</td>
			<td>VWR</td>
			<td>29443-952</td>
			<td></td>
		</tr>
		<tr>
			<td>Honeywell True HEPA Purifier 465 sq ft.</td>
			<td>Home Depot</td>
			<td>204390560</td>
			<td></td>
		</tr>
		<tr>
			<td>HT115 E. coli (DE3)</td>
			<td>CGC</td>
			<td>HT115(DE3)</td>
			<td>https://cgc.umn.edu/strain/HT115(DE3)</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>VWR</td>
			<td>470224-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Scale Culture Plate (LSCP)</td>
			<td>Pyrex</td>
			<td>1090948</td>
			<td>Pyrex 2-quart Glass Baking Dish with Red Lid</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>C86677-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>VWR</td>
			<td>97062-998</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Plain Slides</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Filter</td>
			<td>Millipore</td>
			<td>1.11727.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Devices ImageXpress</td>
			<td>Molecular Devices</td>
			<td>Model Number:IXMConfocal</td>
			<td>https://www.moleculardevices.com/products/cellular-imaging-systems/high-content-imaging/imagexpress-micro-confocal#gref , Authors used MetaXpress Software Version 6.5.4.532</td>
		</tr>
		<tr>
			<td>Nystatin (10mg/mL)</td>
			<td>Sigma</td>
			<td>N6261-25MU</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Sigma</td>
			<td>P7750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes (6 cm)</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Controller</td>
			<td>VWR</td>
			<td>613-4180</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher</td>
			<td>P217-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>VWR</td>
			<td>0781-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasssium Hydroxide</td>
			<td>Fisher</td>
			<td>P250-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Fluroscent Microspheres</td>
			<td>Polysciences</td>
			<td>19507-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>746398-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher</td>
			<td>111357</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Fisher</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Gilson Pipette Set</td>
			<td>Gilson</td>
			<td>FA10002M, FA10004M, FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin (100mg/mL)</td>
			<td>Sigma</td>
			<td>S6501-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Union Biometrica COPAS BioSorter</td>
			<td>Union Biometrica</td>
			<td></td>
			<td>https://www.unionbio.com/biosorter/ , authors used: Flow Pilot software version 1.6.1.3.</td>
		</tr>
	</tbody>
</table>

culture and assay of large-scale mixed-stage caenorhabditis elegans populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Growth of C. elegans using the LSCP method yields an average of approximately 2.4 million mixed-stage worms per sample over 12.2 days. Growth of C. elegans using the LSCP method enables users to generate large mixed-stage populations of C. elegans with little handling and manipulation of the animals, which is ideal for large-scale -omics studies (Figure 1). Once a LSCP has become full of adult worms, reached a large population size, and has minimal bacteria left, users can harvest and estimate the population size. This point can also serve as a quality control by evaluating whether the population is sufficient to use in an -omics pipeline (Figure 2). Population dynamics are dependent on the strain itself, behavior of the strain (i.e., burrowing strains tended to have lower worm recovery), and growth success (i.e., contamination). The LSCP method was tested on 15 strains of C. elegans containing a mixture of Caenorhabditis Genetics Center (CGC) mutants and Caenorhabditis elegans Natural Diversity Resource (CeNDR) wild strains25. Strain genotypes are described in Supplementary Table 3.
The LSCP method yielded population sizes from approximately 94,500 to 9,290,000. The mean population size within the reference strain, PD1074, and across strains was approximately 2.4 million worms (Figure 3). No significant differences were found in estimated population sizes between C. elegans strains over the course of an average of 12.2 LSCP growth days (Figure 4). PD1074 LSCPs took between 10 &#8211; 14 days to grow to a full mixed-stage population. The mean growth time across PD1074 was 10 days. The slowest growing strain grew for a maximum of 20 days, and the fastest growing strain grew for a minimum of 10 days (Figure 4).
Therefore, using this LSCP method, users can easily integrate new strains of interest into a study with little knowledge of developmental timing and background expertise. Note that strains and phenotypes that have to be maintained by picking, have fecundity defects, are heterozygous, or have growth defects may not work well in this pipeline.
Large particle flow cytometry and imaging of samples allows users to document population distribution. A wide variety of platforms can be used to measure successful population growth.
For reproducible -omics measurements, it is important to grow consistent cultures. The metrics of culture reproducibility are number of worms and a consistent size distribution for a given strain. We show the sample distribution for the reference strain, PD1074 &#8211; a variant of the original N2 Bristol strain, using the LPFC23,26 and micro confocal microscope images as proxies for growth success. As worms were measured from the L1 stage through gravid adult on the LPFC distribution (Figure 5), subsequent imaging (Figure 6), and the variation in the population distribution across samples (Figure 7), we can see that this pipeline generated a mixed-stage population of C. elegans.
To take a closer look at the population distribution of our mixed-stage samples, we looked at the distribution of 35 PD1074 LSCPs by looking at the percent of worms that fall within each region across the entire Time of Flight (TOF) (i.e., body length) distribution (Figure 7A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61453/61453fig01.jpg" /> 
Figure 1: Overview of the LSCP worm growth pipeline.&#160;(A) Once received in the lab, all strains were prepared and frozen for long-term storage at -80 &#176;C2. (B) A &#8220;master chunk&#8221; plate was prepared from a frozen worm stock and stored at 15 &#176;C to be used for no longer than one month. (C) Each sample went through four successive chunking steps to reduce generational stress prior to growing on the LSCP. (D) 5 individual gravid adults were picked from the &#8220;chunk 4&#8221; 6 cm plate in Step (D) and spot bleached on five given areas of the LSCP. (E) The LSCP was placed in a Controlled Temperature room and grown at 20 &#176;C until the LSCP was full of adult worms, reached a large population size, and had minimal bacteria left. (F) The worm population was harvested and collected for downstream steps. (G) Aliquots were created from the LSCP and were flash frozen for downstream desired applications. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61453/61453fig02.jpg" /> 
Figure 2: Overview of LSCP harvesting and estimating population size.&#160;(A) 50 mL of M9 were used to wash worms off the NGMA surface. Worm suspension was pipetted into a 50 mL conical tube. Step (A) was repeated twice. (B) 15 mL of worm suspension was poured into a new 15 mL conical tube. Worms were pelleted by centrifuging. M9 + debris were aspirated off without disturbing worm pellet. Step (B) was repeated until all 150 mL of worm suspension were collected. (C) The worm pellet was washed and centrifuged three times with M9 to eliminate remaining debris. Once the sample was clean, the worm pellet was resuspended in 10 mL of ddH2O. (D) A serial dilution of the sample was created to estimate worm population size. The dilution factor(s) that allowed worms to be counted accurately were used. The dilution factor(s) used changed depending on the population size of the LSCP. (E) Once the dilution factor(s) were chosen, all worms from all three aliquot replicates of that dilution were pipetted onto a clean slide and worms were counted under a dissecting microscope. (F) Sample was split into appropriate-sized aliquots. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61453/61453fig03.jpg" /> 
Figure 3: LSCP method generated on an average a population of 2.4 million mixed-stage worms.&#160;The LSCP yields population sizes in the smallest population growths at around 94,500 and at the biggest population growths at around 9,290,000. The mean population size across all strains was 2.4 million worms. Bars underneath C. elegans strain names indicate whether a strain is a CGC mutant or CeNDR natural isolate. LSCP sample size is displayed for each strain. Comparisons for all pairs using Tukey&#8217;s HSD Test were performed. No significant differences were observed between estimated population sizes across C. elegans strains (F(14,108) = 0.7, p = 0.77). Colored bars indicate standard color displays for respective C. elegans strain representation. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61453/61453fig04.jpg" /> 
Figure 4: LSCP method generated large mixed-stage populations of worms in 10 &#8211; 20 days.&#160;A given C. elegans LSCP grew until the sample was full of adult worms, reached a large population size, and had minimal bacterial lawn left. LSCPs took between 10 &#8211; 20 days to grow to a full mixed-stage population, depending on the strain. The mean growth time across the strains was 12.2 days. LSCP sample size is displayed for each strain. Each error bar was constructed using 1 standard deviation from the mean. Levels not connected by same letter are significantly different. Comparisons for all pairs using Tukey&#8217;s HSD Test. A significant difference was found in the amount of growth time on LSCP needed across C. elegans strains (F(14,108) = 8.8, p &#60; 0.0001*). Colored bars indicate standard color displays for respective C. elegans strain representation. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61453/61453fig5v2.jpg" /> 
Figure 5: Mixed population and growth measurement of the wild-type reference strain, PD1074.&#160;A representative LPFC distribution of one LSCP growth of the wild-type reference strain, a variant of the original N2 Bristol strain, (PD1074) documents the size distribution and event counts of a mixed-stage population. The x-axis displays the length (Time of Flight, TOF) of the worms sorted. The y-axis displays the optical density (optical extinction, EXT) of the worms sorted. Each data point is a worm that was documented in the sample. Each TOF region that was used for image analysis is displayed in a different color. Twenty TOF regions were created (R2 &#8211; R21) ranging from a TOF of 50 to 2050. Details on each TOF region can be found in Supplementary Table 1. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61453/61453fig06.jpg" /> 
Figure 6: Images of worms sorted from TOF regions ranging from R2 &#8211; R12 show the PD1074 LPFC distribution.&#160;In region R2, L1 worms can be identified and in region R9 predominately gravid adults are identified, spanning the two developmental larval extremes giving us approximate regions within the flow cytometer distribution of where stages are expected in the distribution. Scale bar represents 1 mm. Representative images were taken from the LPFC distribution displayed in Figure 5, and the colored boxes correspond to regions from Figure 5. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61453/61453fig07.jpg" /> 
Figure 7: Population distribution across time of flight (TOF) regions in the wild-type reference strain, PD1074.&#160;Distribution of worms across the entire TOF region showing&#160;the regions where worms were found. Each PD1074 LSCP is represented as an individual color. (A) The x-axis shows the twenty TOF regions (R2 &#8211; R21) observed and counted for the LSCP, displaying the entire size distribution. The y-axis shows the percent of worms from a given LSCP that had a body size that fell into a given TOF region. (B) As a smaller fraction of the worm population falls between the R7-R21 regions, the log of the percent of worms that fell within each region was taken to display the population distribution. The x-axis displays the R7-R21 TOF regions. The y-axis displays the log of the percent of worms from a given LSCP that had a body size that fell into a given TOF region. <a href="https://www.jove.com/files/ftp_upload/61453/61453fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Mean daily temperature (&#176;C) of growth conditions under which the LSCP was grown and handled. Reported temperatures of the Controlled Temperature (CT) room were documented and collected throughout the six-month span of sample growth and collection. The average daily temperature is reported here. No significant differences were observed between the temperature in which the LCSP grew during the duration of the project (F(5,24) = 2.59, p = 0.0524). The entire temperature difference spanned no greater than 0.003 &#176;C throughout the six-month duration of sample growth and generation. <a href="https://cloudflare.jove.com/files/ftp_upload/61453/61453supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Table 1: TOF gated regions used to sort worms into 384-well plates for imaging. Binned regions were created to span a TOF of 100 across the entire TOF distribution from 50 &#8211; 2050. Gated regions can be changed and optimized to suit your needs. Each TOF region that was used for image analysis is displayed in a different color. <a href="https://cloudflare.jove.com/files/ftp_upload/61453/Supplementary_Table1_FINAL.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 2: 384-well plate template of TOF regions and replicate layout. Every sample was sorted into a 384-well plate for imaging. Four replicates were created for each region selected for sorting. Gated regions can be changed and optimized to suit your needs. See Supplementary Table 1 for specific gated regions created and used in this protocol. Each TOF region that was used for image analysis is displayed in a different color. <a href="https://cloudflare.jove.com/files/ftp_upload/61453/Supplementary_Table2_FINAL.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 3: C. elegans strains used in this protocol contain a mixture of CGC and CeNDR strains. The strain, genotype, strain source, and details are described in this table. <a href="https://cloudflare.jove.com/files/ftp_upload/61453/Supplementary_Table3_FINAL.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations at JoVE.com

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Research

JoVE Journal

Genetics

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Manipulation of Ploidy in Caenorhabditis elegans

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried ...

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

Epigenetic Inheritance and ChIP in Caenorhabditis elegans

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

RNAi Inheritance and ChIP in C. elegans (Video) | JoVE

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...