1. Prepare Solutions for NGT-3D and NGB-3D
2. Prepare Bacteria Culture for NGT-3D and NGB-3D
3. Making NGT-3D and NGB-3D (200 ml)
4. Measure Fitness of Worm Population on NGT-3D (Relative Brood Size Assay)
5. Image and Record Worm Behavior on NGB-3D
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 C. elegans hermaphrodites in NGT-3D reproduce just as well as hermaphrodites in the standard 2D NGM plates when bacterial colonies are plentiful with at least 60 colonies (Figure 1B). Furthermore, larval development in NGT-3D also proceeds normally when there are plenty of bacterial colonies with the majority of worms in the adult state after 96 hours (Figure 1C). However, if bacterial colonies are sparse in the 3D matrix, both relative brood size (Figure 1B, left bar) and larval development (Figure 1C, left bar) are negatively impacted.
To test whether habitation in 3D has any effect on the long-term physiology of the worm, we conducted a lifespan assay comparing NGT-3D and NGM plates. The average lifespan for worms on NGT-3D was 15.6 ± 3.6 days compared to 14.8 ± 3.1 days for standard NGM plates. The lifespan curves for worms living on NGT-3D and NGM were nearly identical. Thus, it seems that worms survive equally well in 3D and 2D conditions.
Manufacturing the NGB-3D is also not difficult and should result in a clear, agar-filled culture bottle like that seen in Figure 2A. To easily view the bacterial colonies, we have expressed deoxyviolacein, a dark purple bacterial metabolite11, in the OP50 strain E. coli (Figures 2A, 2B; strain available on request). Live worms can be imaged under a transmission stereo-dissecting microscope (Figure 2B, Supplemental Movie 1).
Storage of NGT-3D and NGB-3D at room temperature is sufficient to maintain both of these culture chambers for up to 1 month. Desiccation of the agar is not a concern for either NGT-3D or NGB-3D if a plug-type or screw-type cap that fits the tube or bottle is applied.
Figure 1: Development, fertility and lifespan of C. elegans cultivated in NGT-3D is comparable with that of the standard 2D NGM. (A) Images of NGT-3D at several dilutions of bacteria. Left and right contains a 5 x 10-7 dilution, and the middle is a 1 x 10-7 dilution. (B) Relative brood size of wild-type worms in NGT-3D to fewer than 60 OP50 colonies (n = 24), greater or equal to 60 colonies (n = 14), or on 2D NGM plates (n = 29). Error bars indicate standard error. (C) Percent of F1 generation worms in the L3, L4 or adult developmental stage in NGT-3D with fewer than 60 OP50 colonies, greater than or equal to 60 colonies, or on 2D NGM plates. (D) Survival curve of wild-type C. elegans in NGT-3D or NGM plates. N.S. indicates not significantly different calculated by log-rank test. This figure has been modified from Lee9. Please click here to view a larger version of this figure.
Figure 2: Imaging C. elegans in 3D using NGB-3D. (A,B) Images of NGB-3D; (C) Images of adult C. elegans near a colony of OP50 strain E. coli expressing the dark purple pigment deoxyviolacein. Scale bar is 500 µm. Please click here to view a larger version of this figure.
Supplementary Movie 1: Adult C. elegans approach an E. coli OP50-violace in colony in NGB-3D. Please click here to download this file.
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 cm^2 |
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 |
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.
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.
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.