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

Summary

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.

Abstract

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.

Introduction

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 animals^3,4. 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 used^5,6,7,8,9,10. 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 expressions^13,14,15. 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.

Protocol

1. Caenorhabditis Sieve Construction and Use

1. Construction protocol
   1. Acquire 2 lids from 50 mL conical tubes (Figure 1A).
   2. 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 injury.
   3. Clean and sand the cut edges and top the surface with a curved file or a rotary grinding tool (e.g., Dremel). See Figure 1C.
   4. Cut the circle of the monofilament mesh to the appropriate diameter (Figure 1D). For this, trace a lid onto a monofilament nylon mesh sheet and cut just inside the line drawn.
   5. Cut grooves/slashes into the top surface of the lids to enhance the adhesion of the two lids when glue is applied to the plastic (Figure 1E).
   6. Clean the lids with ethanol and let them dry.
   7. Apply cyanoacrylate glue to the top surface of both lids, keeping to the outer edge.
      NOTE: A little glue goes a long way.
   8. Lay the monofilament mesh, according to Table 1, on one glued lid (Figure 1F). Place the second lid inverted on top of the mesh; both lids must have their tops together. Press firmly together (Figure 1G). Ensure that the mesh is taut.
      NOTE: As a safety measure, use tweezers to place the mesh on the lids.
   9. Once the initial layer of glue has dried, apply a ring of cyanoacrylate glue around the outer gap between the lids. Be generous as this adds integrity and prevents any peeling apart or leaking.
   10. Label the filter with the mesh pore size of the monofilament mesh.
       NOTE: Here, we use two mesh pore sizes—20 µm and 50 µm.
2. Use protocol
   1. Pre-wet the sieve.
      1. Pipette a saline solution, such as M9 [5 g of NaCl, 6 g of Na₂HPO₄, 3 g of KH₂PO₄, 1 L of ultrapure H₂O, and 1 mL of MgSO₄ (1 M)]¹⁷, through the center of the sieve until a droplet forms, condenses, and drips off the center of the filter bottom (Figure 2A). Optionally, apply a lint-free wipe (e.g., Kimwipe) to the bottom to shape or spread a moisture droplet on the mesh.
      2. Place the sieve over a 50 mL conical tube. Label the tube as 'Waste tube' (Figure 2B).
   2. Wash a population of C. elegans off an agar plate.
      1. Wash the plate with the M9 buffer and place the worm-containing medium on the topside of the monofilament mesh 1 mL at a time (Figure 2C). Make sure to operate in the center of the mesh. Wash all worms from the plate.
         NOTE: Typically, 3 mL of M9 for a 60 mm plate is sufficient.
      2. To minimize the number of worms lost in pipetting, use a glass Pasteur pipette to move the worms off the plate and onto the mesh center (repeat as needed).
      3. Rinse the filter with the M9 buffer from the top. Again, make sure to operate in the center of the mesh and rinse all worms in that area. Wash as many times as necessary to ensure that all the bacteria and smaller worms have passed through the mesh.
   3. Harvest the size-matched animals from the sieve.
      1. Attach a new 50 mL conical tube to the top of the sieve, with the adult worms from step 1.2.2.3 facing the inside of the new collection tube (Figure 2D).
      2. Remove the first tube (waste tube) and quickly flip over the sieve and new tube to keep the droplet from migrating (Figure 2E).
         NOTE: If sterility is not required, use a lint-free wipe (e.g., Kimwipe) to wick fluid from bottom of the mesh prior to flipping.
      3. Rinse the mesh with M9 into the new 50 mL tube from the new topside (Figure 2F). Again, operate in the mesh center and maintain a droplet on the underside of the mesh.
      4. Allow the worms to settle or gently spin them down (e.g., <16 x g) for approximately 1 min (Figure 2G).
      5. Aspirate the buffer solution from the pellet of worms, ideally down to >0.5 mL, or remove as much fluid as possible without disturbing the pellet.
      6. Pipette the worms using a glass Pasteur pipette onto an NGM plate and let them dry. Space multiple droplets out on a new plate so they dry faster (Figure 2H).
   4. Clean the sieve.
      1. Rinse the sieve gently and thoroughly with ethanol and reverse-osmosis water. Let it dry.
      2. Store it in a clean container for indefinite future use.
      3. Discard it when the mesh develops a "sag" appearance.

2. Validation of Caenorhabditis Sieve Sorting Method

1. General maintenance
   1. For all experiments, culture worms on a standard Nematode Growth Media¹⁷ (1 L of NGM consists of 2.5 g of peptone, 17 g of agar, 3 g of NaCl, 975 mL of double-distilled water, 1 mL of 5 mg/mL cholesterol, 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 25 mL of 1 M KHPO₄, and 0.5 mL of 100 mg/mL streptomycin) at 25 °C.
2. Experimental treatment administration
   1. Compare the three treatment groups: pick, fluorodeoxyuridine (FUDR), and Caenorhabditis Sieve.
      1. For the FUDR treatment group, add 100 mg/mL FUDR to the NGM media to a final concentration of 100 μM to prevent any progeny production, and transfer the worms every other day to a fresh NGM plate to avoid food depletion.
      2. For the pick group, select and transfer the worms manually using a platinum loop.
      3. For the Caenorhabditis Sieve treatment group, follow step 1.2 and pipette the worms onto an NGM plate.
3. Caenorhabditis Sieve percentage yield
   1. To quantify how effective the sieve is at sorting C. elegans, grow age-synchronous N2 animals to day 1 of adulthood at 25 °C (i.e., 48 h after egg laying) and then transfer them by picking them to fresh NGM plates (totaling N = 50 or N = 100 animals per treatment group).
   2. After 24 h of recovery, transfer the population to new NGM plates with the Caenorhabditis Sieve following the above protocol (see step 1.2) and count the number of successfully transferred animals.
   3. Calculate the percentage yield as the ratio of the number of animals transferred in comparison to the starting number at the beginning of the transfer multiplied by 100 (%).
4. Healthspan assays
   NOTE: Score healthspan parameters of the motility class, the pharyngeal pump rate, and both the anterior and the posterior gentle touch response on days 2, 4, 6, and 8 of adulthood for age-synchronized N2 animals maintained at 25 °C.
   1. Motility tracking
      1. Assign motility scores based on a class-based system (classes A, B, and C) following the methods of Herndon et al.¹⁸. Compare the effects of the three experimental groups using an ordinal logistic statistics model in statistical analysis software.
         NOTE: Class A individuals move spontaneously in a normal, sinusoidal pattern. Class B individuals move in markedly non-sinusoidal movements and may require prodding to encourage movement. Class C individuals move their head and/or tail in response to prodding but are unable to move across the agar.
   2. Pharyngeal pump rate
      1. Count the grinder movement of the animal’s terminal pharyngeal bulb visually under a stereomicroscope at a 600X final magnification for 1 min.
      2. Conduct a statistical analysis with a one-way ANOVA with α = 0.05 and Bonferroni post-tests with α = 0.05.
   3. Touch response
      1. Record a gentle touch response and compare between the three treatment groups. Perform the assays based on the methods described by Calixto et al.¹⁹.
      2. Record the anterior and posterior touch response by gently stroking an eyelash pick perpendicularly across the tail or the head (5x each, alternating head and tail) of the animals.
      3. Score any movement in the opposite direction of the stroke as 1 point on a scale of 0 to 5 for both the anterior and the posterior response.
      4. Conduct statistical analysis with a one-way ANOVA with α = 0.05 and Bonferroni post-tests with α = 0.05.
5. Fecundity assay
   1. To determine the use of the Caenorhabditis Sieve's impact on reproduction, grow age-synchronous N2 animals to day 2 of adulthood at 25 °C.
   2. 60 h after egg lay, transfer animals to new NGM plates with either a platinum pick or the Caenorhabditis Sieve and give them 4 h to recover (dry the sieved plates for 20–30 min).
   3. After recovery, individually plate the animals via an eyelash pick to NGM plates, give them 24 h to lay eggs, and remove the animals. Allow the progeny on each plate to develop under normal conditions at 25 °C for another 24 h.
      NOTE: An eyelash pick is a human eyelash secured to the end of a Pasteur pipette with nail polish and sterilized with ethanol.
   4. Count the number of viable F1 generation individuals. Perform a statistical analysis using a T-test with α = 0.05.
      Note: Viable offspring are considered to be eggs that successfully hatch and begin their development through the regular larval cycles.
6. Fluorescent reporter stress response assays
   1. Perform three commonly used fluorescent reporter assays to detect potential markers of stress: a DAF-16::GFP translocation into the nuclei of cells in a strain [TJ356- zIs356 (pDAF-16::DAF-16-GFP;rol-6)]²⁰; an hsp-16.2 expression [TJ375- gpIs1 (hsp-16.2p::GFP)]²¹; and a sod-3 expression [CF1553-muIs84([pAD76]sod-3p::GFP+rol-6[su1006])]²².
   2. For each assay, culture age-synchronous animals from the three treatment groups at 20 °C and examine them at day 3 of adulthood: use a negative control (on a daily basis, transfer a group of animals manually with a platinum pick), a positive control (on a daily basis, transfer a group of animals manually with a platinum pick plus an established stressor), and the Caenorhabditis Sieve (pass the animals through the sieve and allow them to recover for 30 min on NGM just before imaging).
   3. In the DAF-16::GFP assay, heat shock the positive control animals at 37 °C for 30 min before imaging²⁰. For the hsp-16.2 assay, heat shock the positive control animals for 90 min, 20 h before imaging²¹. For the sod-3 assay, treat the positive control animals with 100 mM paraquat for 4 h before imaging²².
   4. Harvest the worms immediately with an eyelash pick and mount them to a coverslip with 1 μL of a 36% poloxamer 407 surfactant solution to immobilize the worms.
   5. Sandwich the mounted worms with another coverslip. Mount the two coverslips to a standard glass microscopy slide and image the worms using an 8X magnification on an inverted fluorescent microscope (for an overall magnification of 80X) and a constant exposure with a FITC filter.
   6. To detect any differences between the three groups in the DAF-16::GFP assay, categorize the animals based on the location of the DAF-16::GFP reporter (nuclear if the reporter translocated to nuclei, cytosolic if the reporter located in cytosol, and intermediate if the reporter located in both nuclei and cytosol).
   7. Compare the results using an ordinal logistics statistical model in statistical analysis software. For the hsp-16.2 and the sod-3 expression assays, use a one-way ANOVA with α = 0.05 and Tukey's post hoc tests with α = 0.05 to compare the total fluorescence of the head region.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Safety glasses	Uline	S-21076
Protective heat resistant glove	Grainger	Item # 3AT17 Mfr. Model # 3AT17 Catalog Page # 1703
50 mL conical tube	Falcon	14-432-22
Synthetic Nylon mesh	Dynamic Aqua-Supply Ltd	NTX20 and NTX50
Cyanoacrylate glue	Scotch Super Glue Liquid	SAD114
Pliers	Vampliers	VMPVT-001-8
Dremmel tool with circular file	Lowe's	Item # 525945 Model # 100-LG
FUDR	Sigma	F0503
M9 chemicals ( NaCl, Na2HPO4, KH2PO4, MgSO4)	Sigma	S7653, RES20908-A7, 1551139, M7506
NGM plate chemicals (Bactopeptone, Agar, KH2PO4, K2HPO4, CaCl2,Cholesterol, Streptomycin)	BD Biosciences (bactopeptone) , Lab express (agar), Sigma ( rest)	BD bioscience 211677, Lab Express 1001,  Sigma 1551139, 1551128, C1016, C8667, S6501
Pluronic F-127	Sigma	P2443
Paraquat dichloride hydrate	Sigma	36541
Inverted fluorescence microscope	Olympus	FSX100