Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

C. elegans are a powerful model organism for research in genetics, cellular biology, and molecular biology, because they are easily cultured in the laboratory, have a short generation time and lifespan, share a high degree of protein homology with mammals, and have a transparent body structure that allows in vivo visualization of fluorescent proteins and dyes1. As a result of the long-standing use of C. elegans as a major model system in a range of fields, including developmental biology and aging, their growth and development are well-understood, their genome has been fully sequenced, and a host of powerful genetic t....

Protocol

1. Recipes

NOTE: Prepare stock solutions before starting microtray plate preparation.

  1. Stock solutions for low-melt agarose nematode growth media (lmNGM)
    1. Prepare 1 M K2HPO4 by dissolving 174.18 g of K2HPO4 in 1 L of sterile deionized water in a 1 L bottle. Autoclave the solution at 121 °C, 15 psi, for 30 min and store it at room temperature (RT).
    2. Prepare 1 M KPi (pH 6.0) by dissolving 136.09 g o.......

Representative Results

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

Discussion

Here, we describe a novel culture system that adapts microtrays, originally developed for human leukocyte antigen tissue typing assays, to allow the isolation and characterization of single C. elegans over time in a solid media environment that is contextually similar to the agar-based NGM that is the standard in C. elegans research. This system is compatible with a variety of interventions, including dietary restriction, exogenous drug treatment, a challenge with chemical or environmental stressors, an.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
3D-printed terasaki insertsCustom printing companyRobot_Terasaki_tray_insert_10-20
-2021.STL
FDM printing, nozzle size 0.6 mm using standard PLA plus filament
AirClean systems AC624LF vertical laminar flow fume hoodFisher Scientific36-100-4376
Bacto peptoneThermo Scientific211677
CaCl2Acros organics349615000
Caenorhabditis elegans N2Caenorhabditis Genetics Center (CGC)N2Wildtype strain
CarbenicillinGoldbioC-103-25
CholesterolICN Biomedicals Inc101380
Escherichia coli OP50 Caenorhabditis Genetics Center (CGC)OP50Standard labratory food for C. elegans
EthanolMilliporeex0276-4
Fisher Vortex Genie 2Fisher ScientificG-560
FUdR Research Products InternationalF10705-1.0
Hydrating water crystals M2 Polymer TechnologiesType SType S super absorbent polymer
Isopropyl ß-D-1-thiogalactopyranoside (IPTG)GoldBioI2481C100
K2HPO4Fisher ChemicalP288-500
KimwipesKimTech34155Task wipes
LB Broth, LennoxBD Difco240230
Leica K5 sCMOS monochrome cameraLeica Microsystems11547112
Leica M205 FCA Fluorescent Stereo MicroscopeLeica Microsystems10450826
Low-melt agaroseResearch Products InternationalA20070-250.0
MgSO4 Fisher ChemicalM-8900
NaClFisher bioreagentsBP358-1
Nunc OmniTray Single-Well PlateThermo Scientific264728
NystatinSigmaN1538
Palmitic acidAcros organics129700010
Paper towelsCoastwide Professional365374
Parafilm MParafilm16-101
Stratagene UV Stratalinker 2400Stratagene400075UV crosslinker
Terasaki trays (Lambda)One Lambda151431
Thermolyne Dri-bathThermolyneDB28125
Tween Thermo ScientificJ20605-AP

References

  1. Shaham, S. Methods in Cell Biology. WormBook: The Online Review of C. elegans Biology. , (2006).
  2. Boulin, T., et al. Eight genes are required for functional reconstitution of the Caenorhabditis elegans levami....

Explore More Articles

Caenorhabditis ElegansSolid MediaLongitudinal Fluorescence MonitoringAversive InterventionsAging ResearchEnvironmental StressorsGene And Protein DynamicsPalmitic AcidTWEEN 20EthanolNystatinUV CrosslinkerMicrotray

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved