We acknowledge financial support from the German Science Foundation (projects A1.1 and A1.2 of the Collaborative Research Center 1182 on the Origin and Function of Metaorganisms). We thank the members of the Schulenburg lab for their advice and support.

1. Preparation of buffers and media
<ol>
	<li>Prepare S-buffer by adding 5.85 g of NaCl, 1.123 g of K2HPO4, 5.926 g of KH2PO4, and 1 L of deionized H2O to a flask and autoclave for 20 min at 121 &#176;C.</li>
	<li>Prepare a viscous medium by adding S-buffer containing 1.2% (w/v) hydroxymethylcellulose (the substance causing viscosity of the medium), 5 mg/mL cholesterol, 1 mM MgSO4, 1 mM CaCl2, and 0.1% (v/v) acetone. Autoclave and stir the viscous medium until it is completely homogeneous. 
	NOTE: This can take multiple hours. Also, S-buffer can be directly prepared and added to the viscous medium without prior sterilization.</li>
	<li>Prepare M9-buffer by adding 3 g of KH2PO4, 6 g of NA2HPO4&#183;2 H2O, 5 g of NaCl, and 1 L of deionized H2O to a 1 L flask. Autoclave the solution, and after cooling, add 1 mL of 1 M MgSO4 (123.24&#160;g of MgSO4&#183;7H2O in 500 mL of deionized H2O, filter sterilized).</li>
	<li>Prepare 10% (v/v) Triton X-100 stock solution by mixing 5 mL of Triton X-100 with 45 mL of M9-buffer. Filter-sterilize the solution using a 0.2 &#181;m filter.</li>
	<li>Prepare M9-buffer with Triton X-100 (M9-T) by adding 2.5 mL of the 10% (v/v) Triton X-100 stock solution to 1 L of M9-buffer after autoclaving to obtain 0.025% (v/v) M9-T.</li>
	<li>Prepare 30% (v/v) glycerol in S-buffer by mixing 15 mL of sterile 100% glycerol and 35 mL of sterile S-buffer in a 50 mL tube.</li>
</ol>
2. Preparation of environmental samples (Figure 1)
<ol>
	<li>Collect environmental samples like compost or rotten fruits and place each sample in an individual plastic bag, tube, or other clean container. 
	NOTE: In order to attract nematodes, apples can be placed on compost several weeks prior to the sampling.</li>
	<li>Spread pieces of the environmental sample evenly in an empty, sterile, 9 cm Petri dish. 
	NOTE: Samples with higher levels of decay are more likely to contain C. elegans. Optionally, a Petri dish filled with peptone-free agar medium (PFM) can be used to increase contrast.</li>
	<li>Cover the sample carefully with approximately 20 mL of sterile viscous medium. 
	NOTE: Nematodes float to the surface within 1-2 h. The viscous medium slows down the movement of the worms and makes it easier to sample them. Sterile M9-buffer can be used as an alternative, however, worm movement will be faster.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64249/64249fig01.jpg" /> 
Figure 1: Isolation of nematodes from substrates. Substrate samples are placed in empty Petri dishes and covered with viscous medium to flush out nematodes. Nematodes are transferred to M9-T and repeatedly washed to remove bacteria from the outside. Individual nematodes can be used for DNA isolation, isolation of associated bacteria, or placed on agar plates to culture worm populations. Figure created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64249/64249fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Isolation of Caenorhabditis nematodes (Figure 1)
<ol>
	<li>Search for Caenorhabditis nematodes using a dissecting microscope, following the guidelines presented in the WormBook chapter on the isolation of C. elegans and related nematodes by Barri&#232;re and F&#233;lix27. 
	NOTE: Caenorhabditis hermaphrodites/females have a gut of light brown color (under transmitted illumination). Moreover, the gut cells have large cell nuclei, which are visible as white dots. By contrast, other nematode species often have a dark brown gut and may show anteroposterior asymmetry in pigment intensity and usually no visible nuclei. The vulva of adult Caenorhabditis hermaphrodites/females is found in the middle of the animal. This vulva localization is not always shown by other nematode taxa. Caenorhabditis nematodes possess two pharyngeal bulbs, which are both visible with sufficient magnification and transmitted illumination and contrast with many other nematode taxa. Caenorhabditis nematodes have a pointed tail, whereas some other nematodes have a round tail.</li>
	<li>Collect the Caenorhabditis nematodes under a dissecting microscope in as little liquid as possible using a 20-100 &#181;L pipette. Transfer the collected nematodes directly to 1-3 mL of sterile M9-T in a sterile 3 cm Petri dish for washing the worms to remove non-attached microbes (step 3.3) or, alternatively, transfer individual worms onto a plate with nematode growth medium (NGM) to establish a worm population (step 3.4).</li>
	<li>Wash the nematodes to remove microbes from the outside of the nematode. 
	NOTE: This protocol enriches for gut bacteria but does not completely eliminate bacteria sticking to the worm cuticle.
	<ol>
		<li>Incubate the nematodes for 10-15 min in M9-T.</li>
		<li>Pipette the nematodes in as little liquid as possible into 1-3 mL of fresh M9-T in a new sterile 3 cm Petri dish.</li>
		<li>Repeat the incubation and transfer the nematodes to fresh M9-T two more times. 
		NOTE: The following steps can be done either with individual worms or with worm populations. The worms can now be used for the characterization of C. elegans and associated microbes, as well as the isolation of bacteria (steps 4 and 5). Alternatively, they can be transferred individually to NGM plates to establish a worm population (step 3.4).</li>
	</ol>
	</li>
	<li>To obtain a worm population, pipette an individual nematode to a NGM plate. 
	NOTE: Only hermaphroditic nematodes such as C. elegans or C. briggsae are able to produce offspring from single worms. However, single worms of other species may still produce offspring if they were already mated.
	<ol>
		<li>Naturally isolated nematodes usually contain bacteria in their gut, which they shed onto the NGM plates, where these bacteria grow and are available as food for C. elegans. Do not add separate food organisms like the standard laboratory food organism, E. coli strain OP50. 
		NOTE: Rearing worms on plates influences the composition of the associated bacterial community, yet the composition is still comparable to that of the natural C. elegans isolates6,7.</li>
		<li>Allow the nematodes to proliferate for up to 10 days at the appropriate temperature (e.g., 15-20 &#176;C for temperate locations). Freeze these nematodes (step 3.5) or use them for the characterization of C. elegans and associated microbes as well as the&#160;isolation of microbes (steps 4 and 5).</li>
	</ol>
	</li>
	<li>Freezing nematodes for long-term storage
	<ol>
		<li>Leave the nematodes on plates until the food bacteria have disappeared, and there are mainly small larval stages on the plates. Wash the worms off the plates in 1.5 mL of S-buffer.</li>
		<li>Mix 500 &#181;L of worm-containing S-buffer thoroughly with 500 &#181;L of 30% (v/v) glycerol in S-buffer in a sterile 2 mL tube. Freeze the tubes promptly at -80 &#176;C for long-term storage, otherwise the glycerol may harm the nematodes.</li>
	</ol>
	</li>
</ol>
4. Preparation of the worms for molecular identification of C. elegans and microbes
<ol>
	<li>For unbiased identification of nematode-associated microbes, prepare a 96-well plate with three sterile 1 mm beads, 19.5 &#181;L of PCR-buffer, and 0.5 &#181;L of Proteinase K (20 mg/mL) per well. Pipette an individual, washed nematode to each well, transferring as little liquid as possible.</li>
	<li>Worm populations can also be used. For this, wash the worms from the plates with M9-buffer and transfer ~300 &#181;L of worm-containing M9-buffer to 2 mL tubes with 10-15 beads.</li>
	<li>Break up the nematodes using a bead homogenizer (e.g., bead-beating for 3 min at 30 Hz). Centrifuge the plate or tubes briefly to get the liquid to the bottom (e.g., for 10 s at 8000 x g at room temperature [RT]).</li>
	<li>Identification of C. elegans
	<ol>
		<li>Isolate the DNA of individual nematodes by heating the samples in a PCR cycler for 1 h at 50 &#176;C and 15 min at 95 &#176;C. Isolate the DNA of worm populations using any isolation method of choice (example protocols of different isolation methods using commercial kits7,9). Freeze the DNA at -20 &#176;C for long-term storage.</li>
		<li>For the identification of C. elegans, use the DNA and the primer pair nlp30-F (Table 1, 5&#39;-ACACATACAACTGATCACTCA-3&#39;) and nlp30-R (Table 1, 5&#39;-TACTTTCCCCATCCGTATC-3&#39;) in a PCR, following the instructions of a Taq supplier of choice.</li>
		<li>Use the following PCR conditions: initial denaturation step at 95 &#176;C for 2 min, followed by 35 cycles of 95 &#176;C for 45 s, 55 &#176;C for 30 s, 72 &#176;C for 1 min, and a final elongation step at 72 &#176;C for 5 min. C. elegans produces a 154 bp PCR product.</li>
	</ol>
	</li>
	<li>Characterize the nematode-associated bacteria via 16S amplicon sequencing of the V3-V4 region, using the isolated DNA.
	<ol>
		<li>Prepare a 16S library with the 16S primers of choice and follow the library preparation kit protocol. One option is to use the primers 341F (Table 1, 5&#39;-CCTACGGGNGGCWGCAG-3&#39;) and 806R (Table 1, 5&#39;-GACTACHVGGGTATCTAATCC-3&#39;) covering the V3-V4 region of the 16S rRNA gene, which results in sequences that can be classified with standard databases with good resolution7. 
		NOTE: The amount of input DNA is critical in this step. DNA acquired from single worms is going to be much less than that obtained from worm populations. For single worms, it might be necessary to increase the amount of input DNA or to increase the amount of PCR cycles7.</li>
		<li>Libraries can be sequenced on a sequencing platform using a suitable sequencing kit. 
		NOTE: Here, the MiSeq platform is used with a suitable MiSeq Reagent Kit. The reagents are improved constantly and should be chosen to the newest standards.</li>
	</ol>
	</li>
</ol>
5. Isolation and cultivation of nematode-associated bacteria (Figure 2)
<ol>
	<li>To isolate bacteria, prepare a 96-well plate with three sterile 1 mm beads, 20 &#181;L of M9-buffer per well, and pipette an individual, washed nematode to each well, transferring as little liquid as possible. 
	NOTE: Alternatively, worm populations can be washed from the plates with M9-buffer and ~300 &#181;L of worm-containing M9-buffer can be transferred to 2 mL tubes with 10-15 beads. In all cases, the worms should come from a culture that was identified to be C. elegans using steps 4.1-4.4.</li>
	<li>Break up the nematodes using a bead homogenizer (e.g., bead-beating for 3 min at 30 Hz). Centrifuge the plate or tubes briefly to get the liquid to the bottom (e.g., for 10 s at 8000 x g at RT). 
	NOTE: The usage of this method led to the isolation of bacterial taxa similar to those revealed by 16S rDNA amplicon sequencing7, suggesting that the described bead-beating leaves most bacterial cells intact.</li>
	<li>Collect the supernatant, serially dilute it at 1:10, and plate up to 100 &#181;L onto 9 cm agar plates.
	<ol>
		<li>To ensure that most bacteria can be cultivated, use a variety of alternative media with different nutrient compositions, including diluted trypticase soy agar (TSA, 1:10 dilution), MacConkey agar, Sabouraud glucose agar, potato dextrose agar, or yeast peptone dextrose agar.</li>
	</ol>
	</li>
	<li>Incubate the plates at the average temperature conditions of the sampling location (e.g., temperatures between 15-20 &#176;C for temperate locations) for 24-48 h.</li>
	<li>Use the standard three-streak technique and a sterile loop to obtain pure bacterial cultures (Figure 2).
	<ol>
		<li>Pick a single colony from a plate using a sterile loop or toothpick and streak it out onto a new agar plate containing the same agar medium as used for purification. Make sure to use only roughly 1/3 of the plate.</li>
		<li>Either use a new sterile loop or sterilize a reusable loop and drag it through the first streak to create a second streak on another 1/3 of the same plate.</li>
		<li>Repeat this step by dragging a sterile loop through the second streak.</li>
		<li>Incubate the plate at the same growth conditions used for isolation. This technique must result in single colonies growing in the area of the third streak. 
		NOTE: It might be necessary to repeat the purification step multiple times as natural isolates tend to form biofilms and/or aggregates.</li>
	</ol>
	</li>
	<li>Grow the pure colonies in a liquid medium (of the same type as the agar medium) using the same temperature and growth time as above (step 5.4)</li>
	<li>Prepare bacteria stocks by adding 300 &#181;L of the bacterial culture to 200 &#181;L of 86% (v/v) glycerol (in the respective growth medium, e.g., TSB) and pipette up and down to mix properly. Alternatively, prepare DMSO stocks by mixing 50 &#181;L of bacterial culture with 50 &#181;L of 7% (v/v) DMSO. Freeze at -80 &#176;C for long-term storage.</li>
	<li>Characterizing bacteria using sequencing of the full 16S ribosomal RNA gene
	<ol>
		<li>Extract the bacterial DNA from pure liquid cultures using a suitable technique (e.g., a DNA extraction kit; from experience, a CTAB-based extraction protocol works very well22).</li>
		<li>Amplify the 16S rRNA gene using the primers 27F (Table 1, 5&#39;-GAGAGTTTGATCCTGGCTCAG-3&#39;) and 1495R (Table 1, 5&#39;-CTACGGCTACCTTGTTACGA -3&#39;)28 and the following PCR conditions: 95 &#176;C, 2 min, 22x (95 &#176;C, 30 s; 55 &#176;C, 30 s; 72 &#176;C, 100 s), and a final extension period at 72 &#176;C, 5 min.</li>
		<li>In order to acquire the full sequences, additionally use two internal sequencing primers, such as 701F (Table 1, 5&#39;-GTGTAGCGGTGAAATGCG-3&#39;) and 785R (Table 1, 5&#39;-GGATTAGATACCCTGGTAGTCC-3&#39;)6.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64249/64249fig2v2.jpg" /> 
Figure 2: Species identification and isolation of individual bacteria. Individual nematodes are broken up using a bead homogenizer, and DNA is isolated for species determination via PCR or sequencing. Alternatively, the broken-up nematode material is serially diluted and plated onto growth medium plates. Plates are incubated until bacterial colonies appear, and single colonies are streaked to new plates to obtain pure cultures. Single colonies of the pure cultures are used to grow liquid bacterial cultures for the preparation of bacterial stocks for long-term storage at -80 &#176;C. Figure created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64249/64249fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+C.+elegans+and+related+nematodes.">Isolation of C. elegans and related nematodes.</a> WormBook. , 1-19 (2014).</li><li>Weisburg, W. G., Barns, S. M., Pelletier, D. A., Lane, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=16S+ribosomal+DNA+amplification+for+phylogenetic+study.">16S ribosomal DNA amplification for phylogenetic study.</a> Journal of Bacteriology. 173 (2), 697-703 (1991).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Crombie, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+adaptation+and+spatiotemporal+patterns+of+genetic+diversity+revealed+by+repeated+sampling+of+Caenorhabditis+elegans+across+the+Hawaiian+Islands.">Local adaptation and spatiotemporal patterns of genetic diversity revealed by repeated sampling of Caenorhabditis elegans across the Hawaiian Islands.</a> Molecular Ecology. 31 (8), 2327-2347 (2022).</li><li>Haber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+history+of+Caenorhabditis+elegans+inferred+from+microsatellites:+Evidence+for+spatial+and+temporal+genetic+differentiation+and+the+occurrence+of+outbreeding.">Evolutionary history of Caenorhabditis elegans inferred from microsatellites: Evidence for spatial and temporal genetic differentiation and the occurrence of outbreeding.</a> Molecular Biology and Evolution. 22 (1), 160-173 (2004).</li><li>Watson, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+network+rewiring+of+propionate+flux+compensates+vitamin+B12+deficiency+in+C.+elegans.">Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans.</a> eLife. 5, 17670 (2016).</li></ol>

We declare that we have no conflict of interest.

The nematode Caenorhabditis elegans is one of the most intensively studied model organisms in biological research. It was introduced by Sydney Brenner in the 1960s, originally for understanding the development and the function of the nervous system29. Since then, C. elegans has become a powerful model for studying fundamental processes across all biological disciplines, including behavioral biology, neurobiology, aging, evolutionary biology, cell biology, developmental biology, a...

In nature, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps1. It is also associated with certain invertebrate hosts such as slugs and woodlice2,3. These habitats are rich in microbes, which not only serve as food for the worm, but may also form stable associations with it. Information on the diversity of naturally associated microorganisms was only published in 20164,5,6. Since then, these and only a few more recent studies have revealed that C. elegans is associated with a variety of bacteria and fungi, most commonly including bacteria of the genus Pseudomonas, Enterobacter, Ochrobactrum, Erwinia, Comamonas, Gluconobacter, and several others6,7,8. Several associated bacteria can stably colonize the worm gut, although not all6,9,10,11,12. They are likely to be of key importance for our understanding of C. elegans biology because they can provide nutrition, protect against pathogens and possibly other stressors, and affect central life-history traits such as reproductive rate, development, or behavioral responses.
As an example, naturally associated isolates of the genera Pseudomonas, Ochrobactrum, and also Enterobacter or Gluconobacter can protect the worm from pathogen infection and killing in distinct ways5,6,11,13,14. A specific isolate of the genus Comamonas influences nematode dietary response, development, lifespan, and fertility15,16,17. Providencia bacteria produce the neuromodulator tyramine and thereby modulate host nervous system activity and resulting behavioral responses18. A set of different naturally associated bacteria were demonstrated to affect population growth rate, fertility, and behavioral responses5,6,9,11,19.
To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations are not fully understood, and further associations between the worm and microbes from its environment remain to be uncovered. Several previous studies used bacterial strains isolated from some soil environment, natural C. elegans habitats, or from mesocosm experiments (i.e., lab-based environments that recreate natural habitats) with C. elegans laboratory strains4,5,20. Even though these studies obtained new insights into the influence of microbes on specific nematode traits (e.g., nematode metabolism21), the relevance of these interactions for C. elegans biology in nature is unclear. Therefore, this manuscript describes the methods to directly isolate C. elegans from nature and to isolate and subsequently characterize the naturally associated microbes from both single worms and groups of worms. The described methods are an updated and improved version of the procedures used previously for the isolation and characterization of natural C. elegans and its native microbiota2,6,7. Considering that C. elegans is widely found in decomposing plant matter across the globe (especially in rotting fruits, temperate regions, and in autumn)1,2,22,23,24,25, this protocol can be applied by any lab whenever there is interest in relating C. elegans traits to naturally associated microbes and thus a more naturally relevant context. The latter is pivotal for a full understanding of the nematode&#39;s biology because it is known from a diversity of other host systems that the associated microbiota can affect diverse life history characteristics26, an aspect which is currently largely neglected in the multitude of C. elegans studies across almost all life science disciplines.

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isolation and characterization of the natural microbiota of the model nematode caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

The nematode C. elegans is frequently found in decomposing fruits, such as apples, and also compost samples. In Northern Germany, C. elegans as well as congeneric species (particularly C. remanei but also C. briggsae) are mainly found from September until November2. The nematodes are most commonly found in decomposing plant matter, especially rotting fruits such as apples or pears, and also compost, particularly material that shows a high grade of decomposition....

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Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

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2.9K Views.  University of Kiel. Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

Video: Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions 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.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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.

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 ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

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Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory