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>
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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. 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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 ...

culture-assay-large-scale-mixed-stage-caenorhabditis-elegans

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4.9K Views.  University of Georgia. 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.

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

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 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

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

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

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

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

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

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

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 is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

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

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

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

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 contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

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

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

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

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

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

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. 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)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through factors such as non-coding RNAs and chromatin modifications. The phenomenon of RNA interference (RNAi) inheritance in the nematode Caenorhabditis elegans is an effective model to investigate TEI that takes advantage of this model organism's short life cycle, self-propagation, and transparency. In RNAi inheritance, exposure of animals to RNAi leads to gene silencing and altered chromatin signatures at the target locus that persist for multiple generations in the absence of the initial trigger. This protocol describes the analysis of RNAi inheritance in C. elegans using a germline-expressed nuclear green fluorescent protein (GFP) reporter. Reporter silencing is initiated by feeding the animals bacteria expressing double-stranded RNA targeting GFP. At each generation, animals are passaged to maintain synchronized development, and reporter gene silencing is determined by microscopy. At select generations, populations are collected and processed for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) to measure histone modification enrichment at the GFP reporter locus. This protocol for studying RNAi inheritance can be easily modified and combined with other analyses to further investigate TEI factors in small RNA and chromatin pathways.

Author Spotlight: RNAi Inheritance and ChIP in C. elegans 

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

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