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

Emily A. Gardea; Destiny DeNicola; Samuel Freitas; Will Peterson; Hope Dang; Karissa Shuck; Christopher Fang-Yen; George  L. Sutphin

doi:10.3791/64681

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Method Article

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

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DOI:

10.3791/64681

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December 9th, 2022

Emily A. Gardea¹ , Destiny DeNicola¹ , Samuel Freitas¹ , Will Peterson¹ , Hope Dang¹ , Karissa Shuck¹ , Christopher Fang-Yen² , George L. Sutphin¹

¹Department of Molecular & Cellular Biology, University of Arizona, ²Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania

Summary

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.

Abstract

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.

Introduction

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 gr....