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

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 KP_i (pH 6.0) by dissolving 136.09 g o.......

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.......

Materials

Name	Company	Catalog Number	Comments
3D-printed terasaki inserts	Custom printing company	Robot_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 hood	Fisher Scientific	36-100-4376
Bacto peptone	Thermo Scientific	211677
CaCl2	Acros organics	349615000
Caenorhabditis elegans N2	Caenorhabditis Genetics Center (CGC)	N2	Wildtype strain
Carbenicillin	Goldbio	C-103-25
Cholesterol	ICN Biomedicals Inc	101380
Escherichia coli OP50	Caenorhabditis Genetics Center (CGC)	OP50	Standard labratory food for C. elegans
Ethanol	Millipore	ex0276-4
Fisher Vortex Genie 2	Fisher Scientific	G-560
FUdR	Research Products International	F10705-1.0
Hydrating water crystals	M2 Polymer Technologies	Type S	Type S super absorbent polymer
Isopropyl ß-D-1-thiogalactopyranoside (IPTG)	GoldBio	I2481C100
K2HPO4	Fisher Chemical	P288-500
Kimwipes	KimTech	34155	Task wipes
LB Broth, Lennox	BD Difco	240230
Leica K5 sCMOS monochrome camera	Leica Microsystems	11547112
Leica M205 FCA Fluorescent Stereo Microscope	Leica Microsystems	10450826
Low-melt agarose	Research Products International	A20070-250.0
MgSO4	Fisher Chemical	M-8900
NaCl	Fisher bioreagents	BP358-1
Nunc OmniTray Single-Well Plate	Thermo Scientific	264728
Nystatin	Sigma	N1538
Palmitic acid	Acros organics	129700010
Paper towels	Coastwide Professional	365374
Parafilm M	Parafilm	16-101
Stratagene UV Stratalinker 2400	Stratagene	400075	UV crosslinker
Terasaki trays (Lambda)	One Lambda	151431
Thermolyne Dri-bath	Thermolyne	DB28125
Tween	Thermo Scientific	J20605-AP