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.
