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...

<ol>
	<li>Schulenburg, H., F&#233;lix, M. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+biotic+environment+of+Caenorhabditis+elegans.">The natural biotic environment of Caenorhabditis elegans.</a> Genetics. 206 (1), 55-86 (2017).</li><li>Petersen, C., Dirksen, P., Prahl, S., Strathmann, E. A., Schulenburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+Caenorhabditis+elegans+across+1.5+years+in+selected+North+German+locations:+the+importance+of+substrate+type,+abiotic+parameters,+and+Caenorhabditis+competitors.">The prevalence of Caenorhabditis elegans across 1.5 years in selected North German locations: the importance of substrate type, abiotic parameters, and Caenorhabditis competitors.</a> BMC Ecology. 14 (1), 4 (2014).</li><li>Petersen, C., 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=Travelling+at+a+slug's+pace:+possible+invertebrate+vectors+of+Caenorhabditis+nematodes.">Travelling at a slug's pace: possible invertebrate vectors of Caenorhabditis nematodes.</a> BMC Ecology. 15 (1), 19 (2015).</li><li>Berg, M., 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=Assembly+of+the+Caenorhabditis+elegans+gut+microbiota+from+diverse+soil+microbial+environments.">Assembly of the Caenorhabditis elegans gut microbiota from diverse soil microbial environments.</a> The ISME Journal. 10 (8), 1998-2009 (2016).</li><li>Samuel, B. S., Rowedder, H., Braendle, C., F&#233;lix, M. -. A., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+responses+to+bacteria+from+its+natural+habitats.">Caenorhabditis elegans responses to bacteria from its natural habitats.</a> Proceedings of the National Academy of Sciences. 113 (27), 3941-3949 (2016).</li><li>Dirksen, P., 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=The+native+microbiome+of+the+nematode+Caenorhabditis+elegans:+gateway+to+a+new+host-microbiome+model.">The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model.</a> BMC Biology. 14 (1), 38 (2016).</li><li>Johnke, J., Dirksen, P., Schulenburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+assembly+of+the+native+C.+elegans+microbiome+is+influenced+by+time,+substrate+and+individual+bacterial+taxa.">Community assembly of the native C. elegans microbiome is influenced by time, substrate and individual bacterial taxa.</a> Environmental Microbiology. 22 (4), 1265-1279 (2020).</li><li>Zhang, F., 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=Caenorhabditis+elegans+as+a+model+for+microbiome+research.">Caenorhabditis elegans as a model for microbiome research.</a> Frontiers in Microbiology. 8, 485 (2017).</li><li>Dirksen, P., 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=CeMbio+-+The+Caenorhabditis+elegans+microbiome+resource.">CeMbio - The Caenorhabditis elegans microbiome resource.</a> G3 Genes|Genomes|Genetics. 10 (9), 3025-3039 (2020).</li><li>Zimmermann, J., 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=The+functional+repertoire+contained+within+the+native+microbiota+of+the+model+nematode+Caenorhabditis+elegans.">The functional repertoire contained within the native microbiota of the model nematode Caenorhabditis elegans.</a> The ISME Journal. 14 (1), 26-38 (2019).</li><li>Kissoyan, K. 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L., 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=A+multi-parent+recombinant+inbred+line+population+of+C.+elegans+allows+identification+of+novel+QTLs+for+complex+life+history+traits.">A multi-parent recombinant inbred line population of C. elegans allows identification of novel QTLs for complex life history traits.</a> BMC Biology. 17 (1), 24 (2019).</li><li>Avery, L., Shtonda, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+transport+in+the+C.+elegans+pharynx.">Food transport in the C. elegans pharynx.</a> Journal of Experimental Biology. 206 (14), 2441-2457 (2003).</li><li>Zhang, J., 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=A+delicate+balance+between+bacterial+iron+and+reactive+oxygen+species+supports+optimal+C.+elegans+development.">A delicate balance between bacterial iron and reactive oxygen species supports optimal C. elegans development.</a> Cell Host &amp; Microbe. 26 (3), 400-411 (2019).</li><li>Petersen, C., 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=Ten+years+of+life+in+compost:+temporal+and+spatial+variation+of+North+German+Caenorhabditis+elegans+populations.">Ten years of life in compost: temporal and spatial variation of North German Caenorhabditis elegans populations.</a> Ecology and Evolution. 5 (16), 3250-3263 (2015).</li><li>F&#233;lix, M. -. 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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. 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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL</td>
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			<td>multiphysics simulation software</td>
		</tr>
	</tbody>
</table>

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....

Watch this Scientific Journal Video about Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans at JoVE.com

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 ...

This protocol allows new insights into the natural microbiota of C.elegans. Simultaneously, the isolated microbes can be used in controlled laboratory experiments to characterize microbiota-host interactions and C.elegans biology. This protocol focuses on isolating microbes associated with C.elegans in nature, which are therefore key for understanding the biology of this important model host in a more natural context.It will be helpful to be familiar with the appearance of Caenorhabditis compared to other nematodes. Pipetting under the dissecting scope can initially be challenging, but practice will be helpful. Begin by collecting the environmental samples like compost or rotten fruits in separate plastic bags, tubes, or clean containers.Evenly spread the pieces of the collected environmental samples in a sterile nine centimeter empty Petri dish and add approximately 20 milliliters of sterile viscous medium to cover the sample with the medium. Under the dissecting microscope, search for Caenorhabditis nematodes following the guidelines on isolating Caenorhabditis elegans and related nematodes. Using a 20 to 100 microliter pipette, collect the smallest possible amount of liquid containing the Caenorhabditis nematodes from the plate and transfer it to the three centimeter Petri dish containing one to three milliliters of sterile M9T.To establish a worm population, the individual worms can be transferred onto a plate with a nematode growth medium or NGM. To wash the nematodes and remove the microbes present outside the nematodes, incubate them in M9T for 10 to 15 minutes. Pipette out the smallest possible amount of liquid containing the nematodes and transfer it to a new sterile three centimeter Petri dish containing one to three milliliters of fresh M9T.For unbiased identification of nematode-associated microbes, prepare a 96 well-plate with approximately three sterile beads of one millimeter diameter and add a mixture of 19.5 microliters of PCR buffer and 0.5 microliters of proteinase K per well. Transfer a single washed nematode to each well with as little liquid as possible. Break up the transferred nematodes using a bead homogenizer for three minutes at 30 hertz and centrifuge the plates or tubes at 8, 000 G for 10 seconds at room temperature to get the liquid to the bottom.To identify C.elegans, heat the samples in a PCR cycler for one hour at 50 degrees Celsius and 15 minutes at 95 degrees Celsius to isolate DNA of individual nematodes, or use commercial kits to isolate DNA of worm populations. Freeze the isolated DNA at minus 20 degrees Celsius for long-term storage. Use the DNA and the primer pair NLP 30 forward and NLP 30 reverse and produce a 154 base pair PCR product.To isolate the bacteria, prepare a 96-well plate with approximately three sterile one millimeter beads, 20 microliters of M9 buffer per well, and pipette a single washed nematode to each well with as little liquid as possible. Break up the nematodes as demonstrated before using a bead homogenizer, followed by brief centrifugation of the plate to get the liquid to the bottom. Once done, collect the suspension and serially dilute it at one to 10 proportion.Plate up to 100 microliters of the diluted suspension onto nine centimeter agar plates. Then incubate the plates at 15 to 20 degrees Celsius for 24 to 48 hours. To obtain the pure bacterial culture, pick a single colony from the incubated plate using a sterile loop or toothpick and streak it onto a new agar plate containing the same agar medium used during the purification.Ensure to only use 1/3 of the plate. Then sterilize a reusable loop or use a new sterile loop and drag it through the first streak to create a second streak on another 1/3 part of the sample plate. Repeat it by dragging a loop through the second streak and creating the third streak to achieve the single colony growth.Incubate the plate under the same growth conditions used for isolation, and if required, repeat the purification step. Grow the pure colonies in a liquid medium using the same temperature and growth conditions. Next, characterize the bacteria by extracting the bacterial DNA from pure liquid cultures amplifying the 16S ribosomal RNA using 27 forward and 1495 reverse primers and performing the PCR as described in the text.When the collected fruit and compost samples were distributed in Petri dishes and submerged in a liquid or viscous medium, it resulted in the worms leaving the substrate material and swimming to the medium's surface. The microbes were isolated as pure single colonies and the purification played an essential role as some C.elegans associated bacteria can form biofilms or easily aggregate, resulting in multi-species cultures. The PCR using the C.elegans specific primers, namely NLP 30 forward and NLP 30 reverse, resulted in the amplification of a 154 base pair product for C.elegans.The DNA isolated from single worms resulted in weak PCR bands, whereas DNA extraction from the worm populations consisting of at least 50 worms yielded a larger amount and clearer bands. The success of C.elegans specific PCR was confirmed by running the DNA of a laboratory strain namely N2 as a positive control simultaneously with the DNA of the isolated nematodes Microbes are everywhere. Hence, it is essential to work under sterile conditions to ensure that those microbes that are isolated are truly associated with the worm in nature.The isolated microbes can be used to study host-microbiota interactions or the role of microbes on host biology, for example, aging, development, or immunity in controlled laboratory experiments. Microbes isolated with this technique are currently used by the C.elegans community to improve the understanding of microbiota diversity on host fitness or the role of microbes on immunity.

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2.8K Views.  University of Kiel. This protocol allows new insights into the natural microbiota of C.elegans. Simultaneously, the isolated microbes can be used in controlled laboratory experiments to characterize microbiota-host interactions and C.elegans biology. This protocol focuses on isolating microbes associated with C.elegans in nature, which are therefore key for understanding the biology of this important model host in a more natural context.It will be helpful to be familiar with the appearance of Caenorhabdit...