This work was supported by NIH R35GM133588 to G.L.S., a United States National Academy of Medicine Catalyst Award to G.L.S., the State of Arizona Technology and Research Initiative Fund administered by the Arizona Board of Regents, and the Ellison Medical Foundation.

<ol>
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The authors state that they do not have any conflicts of interest to disclose.

The WorMotel system is a powerful tool for gathering individualized data for hundreds of isolated C. elegans over time. Following the earlier studies using multi-well devices for applications in developmental quiescence, locomotory behavior, and aging, the goal of this work was to optimize the preparation of multi-well devices for the long-term monitoring of activity, health, and lifespan in a higher-throughput manner. This work provides a detailed protocol for preparing multi-well devices that optimizes many of...

Caenorhabditis elegans are commonly used in aging studies because of their short generation time (approximately 3 days), short lifespan (approximately 3 weeks), ease of cultivation in the laboratory, high degree of evolutionary conservation of molecular processes and pathways with mammals, and wide availability of genetic manipulation techniques. In the context of aging studies, C. elegans allow for the rapid generation of longevity data and aged populations for the analysis of late-life phenotypes in live animals. The typical approach for conducting worm aging studies involves manually measuring the lifespan of a population of worms maintained in groups of 20 to 70 animals on solid agar nematode growth media (NGM) in 6 cm Petri plates1. Using age-synchronized populations allows the measurement of lifespan or cross-sectional phenotypes in individual animals across the population, but this method precludes monitoring the characteristics of individual animals over time. This approach is also labor-intensive, thus restricting the size of the population that can be tested.
There are a limited number of culture methods that allow for the longitudinal monitoring of individual C. elegans throughout their lifespan, and each has a distinct set of advantages and disadvantages. Microfluidics devices, including WormFarm2, NemaLife3, and the &#34;behavior&#34; chip4, among others5,6,7, allow the monitoring of individual animals over time. Culturing worms in liquid culture using multi-well plates similarly allows the monitoring of either individual animals or small populations of C. elegans over time8,9. The liquid environment represents a distinct environmental context from the common culture environment on solid media in Petri plates, which can alter aspects of animal physiology that are relevant to aging, including fat content and the expression of stress-response genes10,11. The ability to directly compare these studies to the majority of data collected on aging C. elegans is limited by differences in potentially important environmental variables. The Worm Corral12 is one approach developed to house individual animals in an environment that more closely replicates typical solid media culture. The Worm Corral contains a sealed chamber for each animal on a microscope slide using hydrogel, allowing the longitudinal monitoring of isolated animals. This method uses standard brightfield imaging to record morphological data, such as body size and activity. However, animals are placed in the hydrogel environment as embryos, where they remain undisturbed throughout their lifespan. This requires the use of conditionally sterile mutant or transgenic genetic backgrounds, which limits both the capacity for genetic screening, as each novel mutation or transgene needs to be crossed into a background with conditional sterility, and the capacity for drug screening, as treatments can only be applied once to the animals as embryos.
An alternative method developed by the Fang-Yen lab allows the cultivation of worms on solid media in individual wells of a microfabricated polydimethylsiloxane (PDMS) device called a WorMotel13,14. Each device is placed into a single-well tray (i.e., with the same dimensions as a 96-well plate) and has 240 wells separated by a moat filled with an aversive solution to prevent the worms from traveling between wells. Each well can house a single worm for the duration of its lifespan. The device is surrounded by water-absorbing polyacrylamide gel pellets (referred to as &#34;water crystals&#34;), and the tray is sealed with Parafilm laboratory film to maintain the humidity and minimize the desiccation of the media. This system allows healthspan and lifespan data to be gathered for individual animals, while the use of solid media better recapitulates the environment experienced by animals in the vast majority of published C. elegans lifespan studies, thus allowing more direct comparisons. Recently, a similar technique has been developed using polystyrene microtrays that were originally used for microcytotoxicity assays15 in place of the PDMS device16. The microtray method allows for the collection of individualized data for worms cultured on solid media and has improved capacity for containing worms under conditions that would typically cause fleeing (e.g., stressors or dietary restriction), with the trade-off being that each microtray can only contain 96 animals16, whereas the multi-well device utilized here can contain up to 240 animals.
Presented here is a detailed protocol for preparing multi-well devices that is optimized for plate-to-plate consistency and the preparation of multiple devices in parallel. This protocol was adapted from the original protocol from the Fang-Yen laboratory13. Specifically, there are descriptions for techniques to minimize contamination, optimize the consistent drying of both the solid media and the bacterial food source, and deliver RNAi and drugs. This system can be used to track individual healthspan, lifespan, and other phenotypes, such as body size and shape. These multi-well devices are compatible with existing high-throughput systems to measure lifespan, which can remove much of the manual labor involved in traditional lifespan experiments and provide the opportunity for automated, direct longevity measurement and health tracking in individual C. elegans at scale.

<table><tbody><tr><td>2.5 lb weight</td><td>CAP Barbell</td><td>RP-002.5</td><td></td></tr><tr><td>Acrylic sheets (6 in x 4 in x 3/8 in)</td><td>Falken Design</td><td>ACRYLIC-CL-3-8/1224</td><td>Large sheet cut to smaller sizes&amp;nbsp;</td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich</td><td>A9518</td><td></td></tr><tr><td>Autoclavable squeeze bottle</td><td>Nalgene</td><td>2405-0500</td><td></td></tr><tr><td>Bacto agar</td><td>BD Difco</td><td>214030</td><td></td></tr><tr><td>Bacto peptone</td><td>Thermo Scientific</td><td>211677</td><td></td></tr><tr><td>Basin, 25 mL</td><td>VWR</td><td>89094-664</td><td>Disposable pipette basin&amp;nbsp;</td></tr><tr><td>Cabinet style vacuum desiccator&amp;nbsp;</td><td>SP Bel-Art</td><td>F42400-4001</td><td>Do not need to use dessicant, only using as a vacuum chamber.&amp;nbsp;</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Acros Organics</td><td>349615000</td><td></td></tr><tr><td>&lt;em&gt;Caenorhabditis elegans&lt;/em&gt; N2</td><td>Caenorhabditis Genetics Center (CGC)</td><td>N2</td><td>Wildtype strain</td></tr><tr><td>Carbenicillin&amp;nbsp;</td><td>GoldBio</td><td>C-103-25</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>360902</td><td></td></tr><tr><td>Cholesterol</td><td>ICN Biomedicals Inc</td><td>101380</td><td></td></tr><tr><td>Compressed oxygen tank</td><td>Airgas</td><td>UN1072</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemical</td><td>C493-500</td><td></td></tr><tr><td>Dry bead bath incubator</td><td>Fisher Scientific</td><td>11-718-2</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; OP50&amp;nbsp;</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>Floxuridine</td><td>Research Products International</td><td>F10705-1.0</td><td></td></tr><tr><td>Hybridization oven</td><td>Techne</td><td>731-0177</td><td>Used to cure PDMS mixture, any similar oven will suffice</td></tr><tr><td>Incubators</td><td>Shel Lab</td><td>2020</td><td>20 &amp;deg;C incubator for maintaining worm strains and 37 &amp;deg;C incubator to grow bacteria&amp;nbsp;</td></tr><tr><td>Isopropyl &amp;szlig;-D-1-thiogalactopyranoside (IPTG)</td><td>GoldBio</td><td>I2481C100</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemical</td><td>P288-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemical</td><td>P286-1</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>Low melt agarose</td><td>Research Products International</td><td>A20070-250.0</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemical</td><td>M-8900</td><td></td></tr><tr><td>Microwave&amp;nbsp;</td><td>Sharp</td><td>R-530DK</td><td></td></tr><tr><td>Multichannel repeat pipette, 20&amp;ndash;200 &amp;micro;L LTS EDP3</td><td>Rainin</td><td>17013800</td><td>The exact model used is no longer sold, a similar model&#039;s catalog number has been provided</td></tr><tr><td>NaCl</td><td>Fisher Bioreagents</td><td>BP358-1</td><td></td></tr><tr><td>Nunc OmniTray</td><td>Thermo Scientific</td><td>264728</td><td>Clear polystyrene trays</td></tr><tr><td>Parafilm M</td><td>Fisher Scientific</td><td>13-374-10</td><td>Double-wide (4 in)</td></tr><tr><td>Petri plate, 100 mM&amp;nbsp;</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>Petri plate, 60 mM&amp;nbsp;</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Plasma cleaner</td><td>Plasma Etch, Inc.</td><td>PE-50</td><td></td></tr><tr><td>PLATINUM vacuum pump</td><td>JB Industries</td><td>DV-142N&amp;nbsp;</td><td></td></tr><tr><td>PolyJet 3D printer</td><td>Stratasys&amp;nbsp;</td><td>Objet500 Connex3</td><td>PolyJet 3D printing services provided by ProtoCAM (Matrial: Vero Rigid; Finish: Matte; Color: Gloss; Resolution: X-axis: 600 dpi, Y-axis: 600 dpi, Z-axis: 1600 dpi)</td></tr><tr><td>Shaking incubator</td><td>Lab-Line</td><td>3526CC</td><td></td></tr><tr><td>smartSpatula</td><td>LevGo, Inc.</td><td>17211</td><td>Disposable spatula</td></tr><tr><td>Superabsorbent polymer (AgSAP Type S)</td><td>M2 Polymer Technologies</td><td>Type S</td><td>Referred to in main text as &quot;water crystals&quot;</td></tr><tr><td>SYLGARD 184 Silicone Elastomer base</td><td>The Dow Chemical Company</td><td>2065622</td><td></td></tr><tr><td>SYLGARD 184 Silicone Elastomer curing agent</td><td>The Dow Chemical Company</td><td>2085925</td><td></td></tr><tr><td>Syringe filter (0.22 &amp;micro;m)</td><td>Nest Scientific USA Inc.&amp;nbsp;</td><td>380111</td><td></td></tr><tr><td>Syringe, 10 mL&amp;nbsp;</td><td>Fisher Scientific</td><td>14955453</td><td></td></tr><tr><td>TWEEN 20</td><td>Thermo Scientific</td><td>J20605-AP</td><td>Detergent</td></tr><tr><td>Vacuum pump oil</td><td>VWR</td><td>54996-082</td><td></td></tr><tr><td>VeroBlackPlus</td><td>Stratasys&amp;nbsp;</td><td>RGD875</td><td>Rigid 3D printing filament</td></tr><tr><td>Weigh boat</td><td>Thermo Scientific</td><td>WB30304</td><td>Large enough for PDMS mixture volume</td></tr></tbody></table>

long-term culture and monitoring of isolated caenorhabditis elegans on solid media in multi-well devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

The WorMotel culture system can be used to gather a variety of data, including regarding lifespan, healthspan, and activity. Published studies have utilized multi-well devices to study lifespan and healthspan13,14, quiescence and sleep22,23,24, and behavior25. Lifespan can be scored manually or through a collection of images and downstream imaging...

Watch this Scientific Journal Video about Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices at JoVE.com

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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Research

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Biology

1.6K Views.  University of Arizona. Wanneer we veroudering en zee-elegans bestuderen, volgen we meestal een populatie dieren in de loop van de tijd. Dit geeft ons een glimp van wat dat proces dat je bestudeert doet in de populatie op elk tijdstip, maar het vertelt je niet hoe het proces in de loop van de tijd verandert bij individuele dieren. Deze omgevingen met één diercultuur stellen ons in staat om dynamische veranderingen in moleculaire processen en fysiologische processen in individuele dieren in de loop van de tijd te v...

Video: Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

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 ...

Genetics

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Biochemistry

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.

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.

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

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Bioengineering

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

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.

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.

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 ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

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