Name: caenorhabditis sieve: a low-tech instrument and methodology for sorting small multicellular organisms
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The authors would like to thank Heather Currey for her initial contribution to the study design, and Dr. Swarup Mitra for his critical review of the manuscript. We would also like to thank Dr. Michael B. Harris for comments, refinements and assistance in producing the demonstration of this methodology. The strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). The research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990 and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 5P20GM103395-15. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.

1. Caenorhabditis Sieve Construction and Use
<ol>
	<li>Construction protocol
	<ol>
		<li>Acquire 2 lids from 50 mL conical tubes (Figure 1A).</li>
		<li>Remove the center area inside the inner lip of the lids (when viewed from the bottom, Figure 1B) using a Bunsen burner and a hot metal probe or a soldering iron or stepped drill bit. 
		NOTE: Using heat to cut the plastic lid is preferable to a blade because there is less risk of...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mechanisms of Ageing Development. 6 (6), 413-429 (1977).</li><li>Zhang, B., 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=Environmental+Temperature+Differentially+Modulates+C.+elegans+Longevity+through+a+Thermosensitive+TRP+Channel.">Environmental Temperature Differentially Modulates C. elegans Longevity through a Thermosensitive TRP Channel.</a> Cell Reports. 11 (9), 1414-1424 (2015).</li><li>Michaelson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans:+A+Practical+Approach.+Ian+A.+Hope+(ed.).+Oxford+University+Press,+Oxford.+1999.+Pp.+281.+ISBN+0+19+963738+5.">C. elegans: A Practical Approach. Ian A. Hope (ed.). Oxford University Press, Oxford. 1999. Pp. 281. ISBN 0 19 963738 5.</a> Heredity. 85 (1), 97-100 (2000).</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>Herndon, L. 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=Stochastic+and+genetic+factors+influence+tissue-specific+decline+in+ageing+C.+elegans.">Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.</a> Nature. 419 (6909), 808-814 (2002).</li><li>Calixto, A., Chelur, D., Topalidou, I., Chen, X., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+neuronal+RNAi+in+C.+elegans+using+SID-1.">Enhanced neuronal RNAi in C. elegans using SID-1.</a> Nature Methods. 7 (7), 554-559 (2010).</li><li>Henderson, S. T., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-16+integrates+developmental+and+environmental+inputs+to+mediate+aging+in+the+nematode+Caenorhabditis+elegans.">daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.</a> Current Biology. 11 (24), 1975-1980 (2001).</li><li>Rea, S. L., Wu, D., Cypser, J. R., Vaupel, J. W., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stress-sensitive+reporter+predicts+longevity+in+isogenic+populations+of+Caenorhabditis+elegans.">A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.</a> Nature Genetics. 37 (8), 894-898 (2005).</li><li>Libina, N., Berman, J. 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Herein, we introduced the design and use of the accessible, effective Caenorhabditis Sieve as a tool for sorting and maintaining C. elegans. This tool has several advantages to manually picking individual animals, washing populations, chemical treatments (e.g., FUDR), and more expensive methods of segregating animals. First, the Caenorhabditis Sieve efficiently and quickly (less than 20 min) sorts progeny from large mixed populations of animals (Table 2). Also, the use...

The nematode worm, Caenorhabditis elegans, is a premier model organism. In addition to the straightforward and controlled nature of their cultivation in the laboratory, their entire genome is sequenced1 and the developmental fate of each cell is known2. Due to these features, C. elegans is a widely used model organism for genetic studies. However, along with these beneficial characteristics come some challenges for researchers. Due to their rapid generation time, C. elegans populations can quickly run out of food and/or become mixed populations with multiple generations and developmental stages present at once. Thus, experiments performed on solid nematode growth media (NGM) require researchers to physically move animals to fresh plates before the bacterial food source depletes and new larvae develop. This can be tedious as a frequent transferring of the animals is required to prevent the experimental populations from becoming mixed with offspring generations. Still, some experiments require both large numbers of animals and extended time points (e.g., DNA or RNA extraction in adulthood). This compounds the challenges of accurately maintaining a synchronized population and transferring large numbers of animals.
Current methods of transferring C. elegans cultured on NGM are picking or washing the animals from plate to plate; chemically treating the animals (e.g., with the DNA replication inhibitor fluorodeoxyuridine or FUDR); or using flow cytometry to sort the animals in multi-well plates. Picking involves the use of a hand tool, made with either a thin platinum wire or an eyelash, to manually transfer individual or multiple animals3,4. This method is accurate but requires both skill and time and is a limitation for studies involving large numbers of animals. Picking may also be physically damaging and stressful to the animals by potentially subjecting individuals to unnatural and inconsistent amounts of disturbance and force. Washing involves rinsing a culture dish with a buffer solution and transferring the solution with the animals via glass Pasteur pipette to a new culture plate. This method is rapid and efficient but is not accurate as multiple generations and developmental stages of animals are transferred in bulk. Chemical treatments, such as FUDR, can be dissolved in the culturing media to prevent the production of offspring through blocking any DNA replication, and thus, the gamete production and egg development. While effective, this method must be applied after developmental maturation as to not disrupt the normal developmental processes, and this means that there is still a requirement to transfer the animals prior to its administration3. This method also influences multiple cellular signaling pathways, resulting in noticeable effects on the animals as they age (e.g., a lifespan extension or an altered proteostasis) depending on the strain of C. elegans used5,6,7,8,9,10. Flow cytometry methods automatically sort and transfer individual C. elegans from one multi-well plate to another11. While this method is very effective and efficient, flow cytometry equipment is prohibitively expensive and inaccessible to many researchers. An alternative to transferring animals is to use mutant models that are temperature sensitive, such as fer-15 and fem-1, which become sterile with temperature adjustment12. While using mutant animals is useful in some situations, these specific strains grow slower than wild-type animals and they rely on an altered genome, serving as poor representatives for aging or healthy worms. In addition, the reliance on a temperature shift to induce sterility also results in the absence of a static environment, and temperature changes have been readily shown to influence gene expressions13,14,15. Research groups have previously published techniques describing the use of a mesh to filter C. elegans by size16. However, we were unable to find previous work testing for any changes in the overall health outcomes that may be associated with the use of such filters.
There is, thus, a need within the C. elegans research community for an affordable, efficient, rapid, and accurate method for transferring large numbers of animals between culture plates. We have developed an improved, accessible piece of equipment (named the Caenorhabditis Sieve) and an associated protocol for its manufacture and operation that meets the needs of the C. elegans research community. Herein, we share the design of the Caenorhabditis Sieve and methods for its use, and we demonstrate that its use does not impact the common health or any stress markers when compared to standard manual picking and a treatment with the commonly-used, fertility-restricting chemical FUDR.

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Safety glasses</td>
			<td style="width:129px;">Uline</td>
			<td style="width:119px;">S-21076&#160;</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Protective heat resistant glove</td>
			<td style="width:129px;">Grainger</td>
			<td style="width:119px;">Item # 3AT17 Mfr. Model # 3AT17 Catalog Page # 1703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL conical tube</td>
			<td style="width:129px;">Falcon</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Synthetic Nylon mesh</td>
			<td style="width:129px;">Dynamic 
			Aqua-Supply Ltd</td>
			<td style="width:119px;">NTX20 and NTX50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cyanoacrylate glue</td>
			<td style="width:129px;">Scotch Super Glue Liquid</td>
			<td style="width:119px;">SAD114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pliers</td>
			<td style="width:129px;">Vampliers</td>
			<td style="width:119px;">VMPVT-001-8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dremmel tool with circular file</td>
			<td style="width:129px;">Lowe&#39;s</td>
			<td style="width:119px;">Item # 525945 Model # 100-LG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FUDR</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">F0503</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">M9 chemicals ( NaCl, Na2HPO4, KH2PO4, MgSO4)&#160;</td>
			<td style="width:129px;">Sigma&#160;</td>
			<td style="width:119px;">S7653, RES20908-A7, 1551139, M7506</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">NGM plate chemicals (Bactopeptone, Agar, KH2PO4, K2HPO4, CaCl2,Cholesterol, Streptomycin)</td>
			<td style="width:129px;">BD Biosciences (bactopeptone) , Lab express (agar), Sigma ( rest)</td>
			<td style="width:119px;">BD bioscience 211677, Lab Express 1001,&#160; Sigma 1551139, 1551128, C1016, C8667, S6501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluronic F-127</td>
			<td style="width:129px;">Sigma&#160;</td>
			<td style="width:119px;">P2443&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraquat dichloride hydrate</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">36541&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted fluorescence microscope</td>
			<td>Olympus&#160;</td>
			<td style="width:119px;">FSX100&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The Caenorhabditis Sieve consists of 2 screw caps, securing an area of woven nylon monofilament mesh smaller than the body diameter of the desired developmental age, used to extract live populations of organisms using a simple washing technique. It attaches to standard conical tubes and uses the mesh screen to mechanically sort animals by body diameter, leaving the desired animals in the tube ready for further maintenance and experimentation (e.g., transfer or genetic ha...

Watch this Scientific Journal Video about Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms at JoVE.com

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 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 ...

This method can help answer key questions in molecular biology, involving assays that require specific developmental stages of C.elegans. Like assays of gene and protein expression levels. The advantage of this technique is that it allows for accurate and efficient selection of large numbers of C.elegans by body size, this facilitates the use of assays that would normally be time and cost prohibitive.Generally, individuals new to this method will struggle because they can't perform the arching turnover step or they don't keep solution flow in the center of the mesh. Visual demonstration of this method is critical, as the washing and flipping steps are difficult to learn. Some may have trouble interpreting and visualizing specific motions in text.To begin, acquire two 50 mL conical tubes with lids, then use a stepped drill bit to remove the center from the inner lip of the lids. If necessary, use a file, sandpaper, or a rotary tool to clean and sand the edges and tops of the lids. Then cut a disc of nylon monofilament woven mesh, slightly smaller than the diameter of the lid.Texture the top surfaces of the lids, and clean them with ethanol. After allowing the lids to dry, apply cyanoacrylate glue to the top surfaces of both lids. Next, lay the monofilament mesh disc on one glued lid.Invert the second lid on top of the mesh, and press the lids firmly together. After the first layer of glue dries, apply a generous ring of cyanoacrylate glue around the outer gap between the lids. Next, label the filter with its respective mesh pore size.First, place the sieve over a labeled 50 mL conical tube. Then pipette saline solution through the center of the sieve until a droplet forms, coalesces, and drips off of the center of the filter. Using M9 buffer, wash a population of C.elegans off of an auger plate, then use a glass pasteur pipette to transfer the buffer containing the worms on the top side of the monofilament mesh.Operating in the center of the mesh, rinse the filter with M9 buffer, repeat this rinse step as needed to pass all bacteria and smaller worms through the mesh. Next, attach a new 50 mL conical tube to the top of the sieve, the previously collected worms larger than the pores in the mesh will be facing the inside of the new tube. Remove the waste tube and quickly flip the sieve.Then rinse the mesh with M9 buffer into the new collection tube. Allow the collected worms to settle for approximately one minute, then carefully aspirate the buffer solution away from the pellet of worms. Using a glass pasteur pipette, create droplets of worms on an NGM plate and allow them to dry.Finally, rinse the sieve with reverse-osmosis water and ethanol. After the sieve dries, store it in a clean container for future use. Discard the sieve when it develops a sagging appearance.In this protocol a sieve was constructed to sort and clean age-matched populations of C.elegans. Animals in the sieve treatment group exhibited normal and spontaneous movement pattern throughout adulthood. Additionally, the pick and sieve groups did not show any significant differences in pharyngeal pump rate.Animals in the sieve group also responded normally to anterior or posterior mechanical stimulus when compared to all of the treatment groups on any of the testing days. Finally, the fecundity of the treatment groups was compared;the use of the sieve did not significantly impact the number of progeny when compared to the pick treatment group. Once mastered, this technique can be done in less than ten minutes.While attempting this procedure, it's important to remember to operate in the center of the mesh area. After watching this video, you should have a good understanding of how to assemble and properly use a Caenorhabditis sieve to sort worms by body size.

Research

JoVE Journal

Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

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Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...