Thank you to Dr. Christian Braendle and the Centre Nationale de la Recherche Scientifique (CNRS) Nouragues Field Station.

1. Inducing dauer formation for wild nematodes on NGM plates
<ol>
	<li>Grow the wild Caenorhabditis strain on standard NGM plates with OP50-1 E. coli as a food source after obtaining the worms with an unculturable microbe of interest. Incubate the nematodes&#160;at 20 &#176;C. 
	NOTE: The standard temperature to grow Caenorhabditis nematodes is between 15-25 &#176;C, but should be determined empirically to prevent loss of the microbe of interest.</li>
	<li>Starve the plate of animals at 20 &#176;C for 4-5 days until all the OP50-1 is consumed and the majority have reached the dauer stage. 
	&#8203;NOTE: Dauers are long-lived larvae that develop due to the absence of nutrition and have protective cuticles.</li>
</ol>
2. Washing the nematodes
<ol>
	<li>Add 5 mL of M9 minimal salts media (42 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 19 mM NH4Cl) to a 6 cm plate of starved worms.</li>
	<li>Using a sterile glass pipette and bulb, pipette up the M9 and worms from the plate and transfer them to a sterile 15 mL centrifuge tube.</li>
	<li>Using a clinical centrifuge, spin down the worms in the tube at 1000 x g for 30 s at room temperature.</li>
	<li>Using a sterile 15 mL pipette, remove and discard the M9 supernatant above the pellet of worms. Do not disturb the live worms at the bottom of the tube by leaving approximately 50 &#181;L of M9 above the worms.</li>
	<li>Add 10 mL of M9 + 0.05% of Triton X-100 to the same centrifuge tube and adequately tighten the tube&#39;s cap.</li>
	<li>Incubate the tube on a nutator for 20 min at room temperature to remove the external microbes. Remove the tube from the nutator and spin down the worms at 1000 x g for 30 s.</li>
	<li>Using a sterile pipette, remove and discard the M9 supernatant above the pellet of worms. Do not disturb the live worms at the bottom of the tube by leaving approximately 50 &#181;L of M9 above the worms.</li>
	<li>Follow and repeat steps 2.5-2.8 three more times.</li>
</ol>
3. Disinfecting the nematodes
<ol>
	<li>Prepare an antibiotic and SDS solution in 10 mL of M9 buffer by adding 0.25% SDS (250 &#181;L of 10% SDS), 50 &#181;g/mL of carbenicillin, 25 &#181;g/mL of kanamycin, 12.5 &#181;g/mL of tetracycline, 100 &#181;g/mL of gentamycin, 50 &#181;g/mL of streptomycin, 37.5 &#181;g/mL of chloramphenicol, and 200 &#181;g/mL of cefotaxime (see Table of Materials).</li>
	<li>Incubate the tube containing nematodes in the antibiotic and SDS solution on a nutator at room temperature overnight. 
	&#8203;NOTE: All non-dauer worms and embryos will die, but many of the dauer worms will survive this cleaning process.</li>
</ol>
4. Removing the antibiotic and SDS solution
<ol>
	<li>Spin down the worms in the tube at 1000 x g for 1 min at room temperature. Using a sterile pipette, remove the supernatant from the centrifuge tube without disturbing the worms at the bottom of the tube.</li>
	<li>Add 10 mL of M9 + 0.05% Triton X-100 and adequately tighten the cap of the tube. Incubate the tube on a nutator for 20 min at room temperature.</li>
	<li>Remove the tube from the nutator and spin down the worms at 1000 x g for 1 min. Follow and repeat steps 4.2-4.3 three times.</li>
	<li>After the last wash, leave the worm pellet undisturbed at the bottom in 100 &#181;L of the solution and discard the rest.</li>
</ol>
5. Propagate a clean nematode strain
<ol>
	<li>Using a sterile glass pipette, transfer 100 &#181;L of the supernatant and the pellet to the center of a 10 cm NGM plate seeded with OP50-1. Allow the plate to dry without disturbing the liquid at the center.</li>
	<li>Allow the dauers to crawl out of the center and through the OP50-1 lawn for 5-10 min.</li>
	<li>Carefully pick up one single dauer and plate it onto a 6 cm NGM seeded plate. In total, pick at least 10 dauers and plate them in individual 6 cm NGM plates seeded with OP50-1 (10 plates total).</li>
	<li>Incubate the plates for 4-5 days at 20 &#176;C until dauers have grown and the following generation (F1) has reached the adult stage. Visually check for contamination on all the plates, seen as non-OP50-1 microbial growth.</li>
	<li>For each clean plate, verify the propagation of the microbe of interest via Normarski or fluorescent microscopy, such as fluorescent in situ hybridization (FISH)12, depending on the phenotype of interest.</li>
</ol>
6. Intestinal dissection and PCR identification of microbial species
<ol>
	<li>Grow animals at 20 &#176;C for 3-4 days to allow them to starve and reduce the amount of OP50-1 bacteria. Add 5 mL of M9 media to the plate of starved worms.</li>
	<li>Using a sterile glass pipette and bulb, pipette up the M9 + worms from the plate and transfer them to a sterile 15 mL centrifuge tube.</li>
	<li>Using a clinical centrifuge, spin down the worms in the tube at 1000 x g for 30 s at room temperature.</li>
	<li>Using a sterile 15 mL pipette, remove the supernatant from the centrifuge tube without disturbing the live worms at the bottom of the tube.</li>
	<li>Add 10 mL of M9 + 0.05% Triton X-100 and incubate the tube on a nutator for 20 min to remove external microbes. Repeat four times to wash the worms.</li>
	<li>After the last wash, using a sterile pipette tip, remove the supernatant without disturbing the pellet at the bottom in 100 &#181;L of the solution.</li>
	<li>Using a sterile pipette tip, transfer M9 and the worms to an unseeded NGM plate and let the plate dry while the worms crawl around for 20 min to help remove OP50-1 from the cuticle and the intestine.</li>
	<li>Add 250 &#181;L of M9 to the dried plate and use a glass pipette to transfer M9 + worms to a new unseeded NGM plate. Again, let that plate dry and allow the worms to crawl around for 20 min.</li>
	<li>Add 250 &#181;L of M9 to the plate and transfer 100 &#181;L of the M9 + worms to a clean watch glass.</li>
	<li>Using two sterile 26 G syringe needles, decapitate the nematodes by holding the worm down with one needle and using the other needle to cut the worm. After decapitation, the intestine (granular) and the gonad (transparent) from the body will naturally come out of the body.</li>
	<li>Cut off a piece of the exposed intestine by holding down the intestine with one needle and cutting it with the other. 
	NOTE: If possible, verify that the microbe of interest is still present in everted intestines using Normarski microscopy, FISH, or immunohistochemistry23.</li>
	<li>Transfer a single dissected intestine into a 0.5 mL PCR tube containing 10 &#181;L of sterile water. Repeat for a total of at least 5 PCR tubes containing intestines from different animals.</li>
	<li>Freeze the PCR tubes at -80 &#176;C for a minimum of 5 min. Remove the PCR tubes from the freezer and thaw out the samples.</li>
	<li>Pipette the liquid up and down a few times to separate the bacteria from the intestine.</li>
	<li>Conduct PCR using universal primer pairs against the DNA sequence of the small ribosomal subunit of bacteria, yeast, or microsporidia, depending on the suspected type of microbe. 
	NOTE: For example, a sample protocol using universal bacterial 16S primers is presented in Table 1.</li>
	<li>Purify the amplicon using a filter-based DNA cleaning and concentration kit (see Table of Materials) and perform Sanger sequencing.</li>
</ol>

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The authors declare no conflicts of interest.

This protocol describes the isolation and identification of microbes from wild-isolated Caenorhabditis nematodes using a series of cleaning procedures. Numerous microbes are associated with wild-isolated nematodes, and some of them have exciting phenotypes that can be used for future studies in host-microbe interactions and innate immunity. Many culturable microbiome and pathogenic bacteria have been isolated from wild Caenorhabditis nematodes using standard techniques for in vitro bacterial growth25,26. However, not all microbes can be cultured in vitro, and it becomes necessary to enrich them in wild nematodes. Some microbes have a resistant spore stage, such as microsporidia, and high concentrations of SDS can be used to kill most bacteria and fungi, allowing for specific enrichment of spores12. This protocol presents a method to enrich unculturable intestinal microbes that are not resistant to SDS and antibiotic treatment.
The technique presented here takes advantage of the environmental resistance seen in dauer animals due to physiological changes such as strengthening of the cuticle, suppressing pharyngeal pumping, and covering the mouth with a buccal plug27. A critical step in this protocol is the overnight incubation with various antibiotics and 0.25% SDS. This step is used to kill all external microbes while leaving internal microbes intact. While C. elegans dauers have been demonstrated to survive SDS concentrations as high at 10% for 30 min27, this protocol uses a moderate but prolonged incubation to not only kill microbes but further expose bacteria to antibiotics. Furthermore, a moderate concentration of SDS can help ensure that dauers from other Caenorhabditis species survive, as exposure of C. tropicalis to 1% SDS overnight resulted in the death of all dauer animals. If all of the dauers die, then the concentration of SDS and/or the length of exposure to SDS should be reduced. Conversely, if the F1 generation plates still have visible contamination after cleaning, the SDS concentration and incubation time should be increased.
Another critical step is the isolation of single dauer animals after cleaning. This step is crucial as not all animals are clean after SDS and antibiotic treatment. Therefore, the animals are placed in the center of a 10 cm NGM plate with OP50-1 and allowed to crawl radially outward. Often it is best to pick more distal animals, as extended crawling through OP50-1 appears to help remove any potential surviving microbes attached to the cuticle. However, this leads to a limitation of the protocol, as it will be more challenging to enrich for a microbe of interest if it is not present in the population at a high frequency. Here, the adhering Alphaproteobacteria was present in 90%-95% of the population; therefore, most clean plates had the microbiome bacterium. However, if a microbe of interest is present at a much lower frequency in the population, it may be necessary to screen many more F1 plates.
This protocol could likely be used to isolate any number of non-culturable microbes of interest found in wild nematodes. However, the microbe must be in a tissue protected by the dauer cuticle, capable of surviving in dauer animals, and have an observable phenotype in the host. As such, this technique can be used to enrich other microbiome bacteria in the intestinal lumen besides the Alphaproteobacteria species described here, including bacteria that do not adhere. Also, the protocol was used to enrich for a facultative intracellular bacterium, Bordetella atropi, which infects the nematode Oscheius tipulae28. After enrichment, B. atropi was found to form colonies on NGM plates, showing that a microbe of interest may be discovered to be culturable in vitro once faster-growing contaminants are removed. This technique would likely work for microsproidians and viruses, including the Orsay virus, given this capacity to enrich an intracellular bacterium. However, these microbes must be capable of surviving the transition into and out of dauer.
It is important to remember that while this protocol can be performed in a Biosafety Level 1 laboratory, a sterile technique must be maintained throughout to prevent further microbial contamination. The protocol can be changed according to the researcher&#39;s needs, including the types/concentrations of antibiotics, the percentage of SDS, and/or the addition of antifungals such as nystatin. Often, the number of contaminating microbes found in a wild-isolated nematode can vary dramatically. Here, the apparent loss of non-OP50-1 E. coli growth on NGM plates was used as a readout for a clean nematode strain. But, there may be non-culturable populations of contaminating microbes present, so it is essential to conduct a metagenomics method such as 16S rRNA amplicon sequencing to see the extent of contamination26. Once the worm strain is cleaned, it can be frozen and stored away for future studies. Overall, this protocol allows researchers to enrich unculturable microbes in wild nematodes, allowing them to study effects on host fitness, characterize phenotypes of colonization or infection, and take advantage of genetic tools to understand the mechanisms underlying host-microbe interactions.

The genetic model organism C. elegans is an excellent in vivo system to study host-microbe interactions1,2. They have relatively simple physiology compared to other animals, yet, much of their cell biology is fundamentally similar to mammals making them a good model for biological research1,3,4. Additionally, they are microscopic, easy to maintain, and remain transparent throughout their short lifespan. These properties enable rapid studies into the mechanisms governing host-microbe interactions and visualization of in vivo infection and colonization of the genetically pliable hosts5,6. Finally, C. elegans rapidly&#160;responds to bacterial, fungal, and viral infections, making them an excellent model to study host-microbe interactions and the gut microbiome7,8,9.
An increase in the sampling of wild C. elegans and other nematodes has allowed for research into the ecology of free-living nematodes and natural genetic variation10,11. Concurrently, sampling has also increased the discovery of naturally occurring biological pathogens and microbes that interact with C. elegans12,13,14,15, leading to the establishment of many host-microbe model systems that study interactions with&#160;viruses, bacteria, microsporidia, oomycetes, or fungi16,17,18,19,20. Typically, wild C. elegans is found in rotting stems and fruits, often in more temperate climates, and mostly they are self-reproducing21. When these samples are brought into the lab, wild nematodes are isolated into clonal populations, carrying an array of associated microbiota. When discovering new microbes of interest in Caenorhabditis nematodes, animals are often directly screened for infection or colonization by microscopy using easily visualized phenotypes. For example, viral infection can be visualized as a disintegration of intestinal structures, and microsporidian stages can be seen inside host cells as spores or meronts14,22. When a microbe of interest is discovered for future investigation, it must be separated from the other contaminating microbes found in the wild nematodes so that it can be studied in isolation. In many cases, the microbe of interest cannot be cultured in vitro, making it essential to enrich the microbe in the host nematode.
For example, this protocol describes a wild isolate of C. tropicalis containing a bacterium that colonizes within the intestinal lumen of nematodes, adhering to the intestinal epithelial cells in a directional manner. Phenotypically, the bacterium grows perpendicular along the intestinal lumen&#39;s inner sides, giving it a bristle-like appearance, visualized on a standard Normarski microscope in all stages of the animal, including the dauer stage. The nematode growth medium (NGM) plate on which this wild C. tropicalis strain was grown contained visible contamination with other microbes. This protocol was developed to reduce additional contaminating microbial growth on the plates for studying this unknown adhering bacterium. The nematodes were forced into the dauer stage to protect the bacteria in the lumen, and then cleaned using a series of washes. Afterward, the unknown bacterial species was identified by dissection of the intestines and PCR amplification of the 16S ribosomal DNA for sequencing.
Overall, this protocol can potentially enrich any microbe of interest associated with a wild-caught nematode. Afterward, researchers will identify the target microbe, visualize in vivo infection or colonization phenotypes via microscopy, and study effects on host fitness or other aspects of host-microbe interactions. The isolation and investigation of novel microbial species that interact with Caenorhabditis nematodes can uncover the genetic mechanisms of host immunity and novel paradigms of host-microbe interactions relevant to microbial pathogenesis and microbiome studies.

<table><tbody><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP1356</td><td></td></tr><tr><td>10% SDS</td><td>Invitrogen</td><td>AM9822</td><td></td></tr><tr><td>BD PrecisionGlide Needle - 26 G</td><td>Fisher Scientific</td><td>305115</td><td></td></tr><tr><td>Carbenicillin</td><td>Millipore-Sigma</td><td>C1389-1G</td><td></td></tr><tr><td>Cefotaxime</td><td>Millipore-Sigma</td><td>C7039-500mg</td><td></td></tr><tr><td>Chloramphenicol</td><td>Millipore-Sigma</td><td>C0378-25G</td><td></td></tr><tr><td>DNA Clean and Concentrator Kit</td><td>Zymo Research</td><td>11-303C</td><td></td></tr><tr><td>DreamTaq Polymerase</td><td>Fisher Scientific</td><td>EP0711</td><td></td></tr><tr><td>Gentamycin</td><td>Millipore-Sigma</td><td>G1264-250mg</td><td></td></tr><tr><td>Kanamycin</td><td>Millipore-Sigma</td><td>K1876-1G</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P-286</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S-671</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific</td><td>A-661</td><td></td></tr><tr><td>Streptomycin</td><td>Millipore-Sigma</td><td>S6501-50G</td><td></td></tr><tr><td>Tetracyclin</td><td>Millipore-Sigma</td><td>T7660-5G</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP-151</td><td></td></tr><tr><td>Watch glasses</td><td>VWR</td><td>470144-850</td><td></td></tr></tbody></table>

selective cleaning of wild caenorhabditis nematodes to enrich for intestinal microbiome bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

A wild C. tropicalis strain (JU1848) was isolated from rotten palm tree fruits in the Nouragues Forest of French Guiana (Figure 1A)24. This strain was found to have thin microbes that colonize the lumen of the intestine in a directional manner (Figure 1B). This microbe was easily transferred to C. elegans strain N2 via co-culture with strain JU1848, where it colonized the lumen of the intestine similarly.
Propagation of JU1848 on standard NGM plates seeded with E. coli OP50-1 over multiple generations continually resulted in visible contamination, seen as various dark, mucoid colonies on and off the OP50-1 lawn (Figure 2A). A plate of wild JU1848 was starved to force animals into dauer and cleaned as described. Single dauer animals that survived cleaning were plated onto individual 6 cm NGM plates seeded with OP50-1 and allowed to grow for 4 days at 20 &#176;C. Multiple plates of F1 progeny were observed without visible microbial contamination (Figure 2B). The F1 progeny were verified to still&#160;contain adhering bacteria in the lumen of the intestine (see below).
Clean JU1848 animals were washed and decapitated to isolate intestinal pieces as described in the protocol (steps 6.1-6.12). Adhering bacteria in the lumen of the dissected intestine was verified via Nomarski microscopy (Figure 3). The microbe in the lumen of JU1848 was suspected to be a bacterium, so the dissected intestines were used as a template for PCR using universal bacterial 16S primers, 27F, and 1492R. From a total of six individual dissected intestines, the PCR products were sequenced via Sanger, and clean chromatographs showed that all the six sequences were identical. Based on these sequences, this bacterium was identified as a new species in the class Alphaproteobacteria but could not be classified into a known order or genus (Supplementary File 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62937/62937fig01.jpg" /> 
Figure 1: Adhering bacteria colonizing the lumen of a wild C. tropicalis. (A) Field sample image of rotten Euterpe sp. (Family: Arecaceae) palm tree fruits in the Nouragues forest of French Guiana.&#160;(B) Nomarski image of strain JU1848 seen with thousands of long, thin bacteria that form a brush-like appearance in the lumen (lu) of the host intestine (in). The bacterial (ba) layers coating the intestine are indicated with brackets ([). <a href="https://www.jove.com/files/ftp_upload/62937/62937fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62937/62937fig02.jpg" /> 
Figure 2: Contaminating microbial growth is lost after nematode cleaning. (A) Wild strain JU1848 propagates noticeable microbial growth on standard 6 cm NGM plates seeded with E. coli OP50-1 bacteria. (B) After cleaning, a plate of F1 progeny from a single dauer shows no visible microbial contamination after 4 days of incubation at 20 &#176;C. <a href="https://www.jove.com/files/ftp_upload/62937/62937fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62937/62937fig03.jpg" /> 
Figure 3: Adhering bacteria are seen in the lumen of the dissected intestine. Nomarski image of a clean JU1848 animal that was decapitated so that the intestines spill out. The colonizing bacteria (ba) are indicated with a bracket ([) and are seen in the lumen (lu) of a piece of the intestine (in) that is outside of the nematode body (nb). <a href="https://www.jove.com/files/ftp_upload/62937/62937fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Concentration</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>27F primer&#160; (5&#8217;-AGAGTTTGATCMTGGCTCAG-3&#8217;)</td>
			<td>20 mM</td>
			<td>2.5 &#181;L</td>
		</tr>
		<tr>
			<td>1492R primer (5&#8217;-GGTTACCTTGTTACGACTT-3&#8217;)</td>
			<td>20 mM</td>
			<td>2.5 &#181;L</td>
		</tr>
		<tr>
			<td>Dissected intestine in water</td>
			<td>N/A</td>
			<td>3 &#181;L</td>
		</tr>
		<tr>
			<td>10x PCR buffer</td>
			<td>10x</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>10 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>Taq Polymerase</td>
			<td>5 U/&#181;L</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>N/A</td>
			<td>35.5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Sample PCR protocol using universal bacterial primers and dissected intestine.
Supplementary File 1: MUSCLE alignment of bacterial 16S rDNA sequences derived from PCR of six dissected JU1848 intestines. <a href="https://www.jove.com/files/ftp_upload/62937/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria at JoVE.com

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

selective-cleaning-wild-caenorhabditis-nematodes-to-enrich-for

Research

JoVE Journal

Immunology and Infection

2.7K Views.  San Diego State University. Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Video: Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode research community, there is a need for an affordable and effective way to maintain large, age-matched populations of C. elegans. Here, we present a methodology for mechanically sorting and cleaning C. elegans. Our aim is to provide a cost-effective, efficient, fast, and simple process to obtain animals of uniform sizes and life stages for their use in experiments. This tool, the Caenorhabditis Sieve, uses a custom-built lid system that threads onto common conical lab tubes and sorts C. elegans based on body size. We also demonstrate that the Caenorhabditis Sieve effectively transfers animals from one culture plate to another allowing for a rapid sorting, synchronizing, and cleaning without impacting markers of health, including motility and stress-inducible gene reporters. This accessible and innovative tool is a fast, efficient, and non-stressful option for maintaining C. elegans populations.

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

The current protocol includes a methodology for the sorting and cleaning of age-matched populations of Caenorhabditis elegans. It uses a simple, inexpensive, and efficient custom-made tool to obtain a large experimental population of nematodes for research.

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode ...

Developmental Biology

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Biology

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

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.

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.

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

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Summary

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Chapters in this video

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Rapid Isolation of Wild Nematodes by Baermann Funnel

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

Hand Dissection of Caenorhabditis elegans Intestines

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

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans