Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Summary

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.

Abstract

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.

Introduction

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades¹. C. elegans was the first multicellular organism to have its genome sequenced in 1998², and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level^3-5.

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Protocol

1. Prepare Solutions for NGT-3D and NGB-3D

1. Prepare the following sterile solutions: 1 L of 0.1454 M NaCl solution, 1 L of 1 M CaCl₂, 1 L of 1 M MgSO₄, Lysogeny Broth (LB), 1 L of 1 M KPO₄ buffer (108.3 g of KH₂PO₄ and 35.6 g of K₂HPO₄, and fill H₂O to 1 L). Production of NGM using these solutions can be found in a previous protocol¹⁰.
2. Autoclave solutions at 121 °C, 15 min.
3. Prepare 50 ml of a sterile 5 mg/ml cholesterol solution. In a 50 ml conical tube, mix 0.25 g of cholesterol and 50 ml of 99.99% ethanol and mix well. Do not....

Results

The construction of NGT-3D is a simple and straightforward protocol that results in an agar-filled test tube with small bacterial colonies spaced throughout the agar (Figure 1A). Worms can freely move through the agar matrix, finding and consuming the bacterial colonies. To confirm whether C. elegans can reproduce and grow normally in NGT-3D, we compared fertility and larval development in 3D with standard 2D NGM plates. In the relative brood size assay, adult

Discussion

The laboratory cultivation of C. elegans using the classical nematode growth media plates was crucial to the hundreds of important discoveries that research in C. elegans has provided. Here, we present new methods to cultivate C. elegans in an environment that more accurately reflects their natural three-dimensional habitats. Although other methods have been used to observe C. elegans in 3D¹³, this is the first protocol that allows cultivation of worms in a solid 3D matrix. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB broth, Miller (Luria-Bertani)	Difco	224620
Sodium chloride	DAEJUNG	7548-4400	58.44 MW
Agar, Granulated	Difco	214530
Peptone	Bacto	211677
Calcium chloride, dihydrate	Bio Basic	CD0050	2*H2O; 147.02 MW
Cholesterol	Bio Basic	CD0122	386.67 MW
Ethyl alcohol	B&J	RP090-1	99.99%; 46.07 MW
Magnesium sulfate, anhydrous	Bio Basic	MN1988	120.37 MW
Potassium phosphate, monobasic, anhydrous	Bio Basic	PB0445	136.09 MW
2'-Deoxy-5-fluorouridine	Tokyo Chemical Industry	D2235	246.19 MW
Potassium phosphate, dibasic, anhydrous	Bio Basic	PB0447	174.18 MW
Multi-Purpose Test Tubes	Stockwell Scientific	ST.8570	8 ml
Test Tube Closures	Stockwell Scientific	ST.8575
Cell Culture Flask	SPL Lifescience	70125	25 cm2
Research Stereo Microscope	Nikon	SMZ18
High-Definition Color Camera Head	Nikon	DS-Fi2
PC-Based Control Unit	Nikon	DS-U3
NIS-Elements Basic Research, Microscope Imaging Software	Nikon	MQS32000