Counting Caenorhabditis elegans: Protocol Optimization and Applications for Population Growth and Toxicity Studies in Liquid Medium

Leona D Scanlan,Steven P Lund,Elijah J Petersen,Patricia Lapasset,John T Elliott,Bryant C Nelson,Karina Brignoni,Shannon K Hanna,Monique E Johnson,Sanem Hosbas Coskun,Christopher M Sims

doi:10.1038/s41598-018-19187-3

Abstract

The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research. However, standardized methods for counting nematodes in liquid culture do not exist despite the wide use of nematodes and need for accurate measurements. Herein, we provide a simple and affordable counting protocol developed to maximize count accuracy and minimize variability in liquid nematode culture. Sources of variability in the counting process were identified and tested in 14 separate experiments. Three variables resulted in significant effects on nematode count: shaking of the culture, priming of pipette tips, and sampling location within a microcentrifuge tube. Between-operator variability did not have a statistically significant effect on counts, even among differently-skilled operators. The protocol was used to assess population growth rates of nematodes in two different but common liquid growth media: axenic modified Caenorhabditis elegans Habitation and Reproduction medium (mCeHR) and S-basal complete. In mCeHR, nematode populations doubled daily for 10 d. S-basal complete populations initially doubled every 12 h, but slowed within 7 d. We also detected a statistically significant difference between embryo-to-hatchling incubation period of 5 d in mCeHR compared to 4 d in S-basal complete. The developed counting method for Caenorhabditis elegans reduces variability and allows for rigorous and reliable experimentation.

Highlights

The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research
The nematode C. elegans is an important model organism widely used in diverse fields of study
Nematodes in liquid culture are convenient for different types of experimental procedures and are highly amenable for high-throughput studies

Summary

Introduction

The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research. The developed counting method for Caenorhabditis elegans reduces variability and allows for rigorous and reliable experimentation Alternative model organisms such as the nematode Caenorhabditis elegans are increasingly used to study the toxicity of chemicals[1,2,3]. In order to expand the use of C. elegans in toxicological testing, protocols and experimental methods that detail and enumerate specific aspects of nematode culturing, sampling and toxicant exposure are essential. Procedures describing how nematode counts were determined, or how the correct number of C. elegans were transferred from one system to another (e.g., from a culture vessel to an exposure vessel) are often not clearly stated This makes it challenging to verify or reproduce experiments, a current key topic in biological research[15,16]. COPAS instrumentation is very expensive, and is not widely available in many laboratories, which limits its use for toxicity testing with C. elegans[32]

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Results

Conclusion