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.

C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria⁶. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter^7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory.

To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short⁹. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates¹⁰. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ.

In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

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 autoclave. Sterilize the cholesterol solution using a 50 ml syringe with a 0.45 µm syringe filter. This will also remove any undissolved cholesterol.
4. (Optional) Prepare 10 ml of a 150 mM of 2'-deoxy-5-fluorouridine (FUdR) stock. In a 15 ml conical tube, mix 0.3693 g of FUdR and 10 ml of distilled water. Shake well.

2. Prepare Bacteria Culture for NGT-3D and NGB-3D

1. Inoculate 10 ml LB broth with bacteria. The standard bacteria used for C. elegans feeding is E. coli strain OP50.
2. Culture the bacterial inoculation at 37 °C in a shaking incubator overnight.
3. In preparation for a serial dilution, aliquot 9 ml of the NaCl solution into sterile 15 ml conical tubes. For example, aliquot 9 ml into a total of 7 tubes to make a 10^-7 dilution of OP50 strain E. coli.
4. Using a sterile 1,000 µl pipette, pipet 1 ml of the bacterial culture or diluted bacterial culture into the sterile new tube with 9 ml of NaCl solution and vortex the 10 ml mixture well. Repeat until the desired bacterial dilution is reached.
   NOTE: The desired bacterial dilution depends on the type and condition of bacteria as well as the exact experimental conditions. For example, a 10^-6 - 10^-8 dilution of OP50 strain E. coli was sufficient to produce from 1 - 200 bacterial colonies per 6.5 ml NGT-3D tube. Worms reliably developed and reproduced normally when the number of colonies was over 60, which occurred at a dilution of 10^-6 - 10^-7. For NGB-3D, use a dilution of 10^-8.

3. Making NGT-3D and NGB-3D (200 ml)

1. After preparing the bacterial culture, mix 0.6 g NaCl, 1 g granulated agar, and 0.5 g peptone in a 500 ml flask. Insert a magnetic stir bar into the flask.
2. Add 195 ml distilled water and cover mouth of the flask with aluminum foil.
3. Autoclave 121 °C for 15 min.
4. Place the hot autoclaved flask onto a stir plate, and stir at a moderate speed for at least 2 hr. Cool the flask to 40 °C. Be sure to cool sufficiently as continuing from here at high temperatures may lead to clouding of the finished agar. However, lower temperatures may result in premature hardening of the agar.
   1. (Optional) To speed up the cooling process, place the flask in a 40 °C water bath 15 min before placing on a stir plate.
5. When the temperature of the agar media reaches 40 °C, add 200 µl 1 M CaCl₂, 200 µl of 5 mg/ml cholesterol solution, 200 µl 1 M MgSO₄ and 5 ml 1 M KPO₄ buffer as the solution continues to stir to final concentrations of 1 mM CaCl₂, 5 µg/ml cholesterol, 1 mM MgSO₄, 1 mM KPO₄.
   1. (Optional) For NGT-3D lifespan assay add 80 µl of 150 mM FUdR to a final concentration of 120 µM¹².
6. Add 6 ml of the 10 ml diluted bacterial culture from step 2.4 into the flask directly.
   1. (Optional) For NGT-3D, remove 6 ml of agar media before step 3.6 and keep it warm separately. This media will be used for the bacteria-free top layer.
7. Using sterile procedures, dispense the media into a sterile culture chamber. Make sure the cover of the chamber closes tightly.
   1. (Optional) To prevent bacterial colonies from forming at the top surface of the agar, make a layer of bacteria-free agar media from step 3.6.1 on the top before the media from step 3.7 completely hardens. This will create a bacteria-free zone at the top of the NGT-3D.
   2. For NGT-3D, pour 6.5 ml media into the 8 ml clear plastic test tubes to make a bacteria agar layer, and carefully dispense 200 µl of the bacteria-free media on top of the semi-hardened 3D media to make a thin bacteria-free layer on top.
   3. For NGB-3D, pour 65 ml of media into the 25 cm² clear plastic cell culture bottle. This amount should fill the body of the 25 cm² cell culture bottle.
8. Leave chambers vertically at room temperature for one week to allow bacterial colonies to grow to a considerable size of at least 1 mm diameter.
   1. (Optional) For lifespan assay in NGT-3D, cover chambers with aluminum foil to prevent light degradation of FUdR.

4. Measure Fitness of Worm Population on NGT-3D (Relative Brood Size Assay)

1. Pick an L4 stage worm using a platinum wire pick and transfer on a bacteria-free NGM plate. Allow worm to freely move around for a few minutes to remove bacteria attached to its body.
2. Repeat step 4.1 and make sure that the worm is free from bacteria. Generally, two repeats of 4.1 are enough.
3. Carefully place the clean worm on the surface of the 3D media with a platinum wire pick. The worm should eventually enter the agar into the 3D agar matrix.
4. Close the cover loosely allowing some air to get into the tube, but preventing drying of the agar media.
5. Incubate for 96 hr at 20 °C.
6. After 4 days, close the lids tightly and place the NGT-3D culture chamber into an 88 °C water bath to melt the agar. The heat kills worms but their bodies remain intact.
   NOTE: Using 8 ml tubes for NGT-3D, 20 - 30 min incubation is usually sufficient.
7. Using a glass pipette, transfer the melted media onto a 9 cm plastic petri dish. Plastic pipettes are not advised, as worms can often stick to them.
8. Using a transmission stereo dissecting microscope, count the number of worms at the L3, L4, and adult stages. Do not count worms that are L2 stage and younger, as the F1 and F2 generations can be confused here. Thus, this assay is a relative brood size assay rather than a total brood size assay.

5. Image and Record Worm Behavior on NGB-3D

1. Pick an L4 stage worm using a platinum wire pick and remove any bacteria stuck to the surface by transferring to a bacteria-free NGM plate and allowing it to freely move for a few min.
2. Repeat step 5.1 to make sure the worm is free from bacteria.
3. Carefully place the clean worm onto the center of the agar surface of the NGB-3D near the neck of the bottle with a platinum wire pick. The worm should eventually enter the agar into the 3D agar matrix.
4. Close the cover loosely allowing some air to get into the tube, while preventing drying of the agar media.
5. Image or record the worm under a transmission stereo dissecting microscope. Adjust the focus as the worm moves through the 3D matrix.

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

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