This high throughput assay can be used to observe robust gravitactic behavior in caenorhabditis dauer larvae. Worm behavior is observed over large distances compared with assays performed on a petri dish. This enhances the sensitivity of the assay and allows for detailed data analysis.
Studying gravitaxis behavior can provide insight into how gravity sensation occurs in caenorhabditis, which has relevance for understanding the mammalian vestibular system. Creating the gravitaxis assay chambers, isolating dauers, and injecting the worms into the chambers takes practice. You can save time by isolating the dauers and creating the chambers a day in advance.
Begin by setting up a Bunsen burner, one to two razor blades, pliers, tweezers, and a plastic cutting surface, and a fume hood. To make a chamber, gather two 5 milliliter serological pipettes, and with tweezers, remove the cotton plug from one pipette. Hold a razor blade using pliers over the Bunsen burner until hot.
Use the heated blade to slice off the tapered end of the second pipette so that the entire pipette has a uniform diameter. Now, quickly bring the two modified pipette ends close to the flame and melt them slightly. Join these ends by pressing them together firmly, ensuring that the walls of both pipettes are continuous.
If any gaps are visible after joining, break them apart and repeat this step. Prepare NGM with 4%agar according to standard worm procedures and fill each chamber by attaching it to a serological pipetter while the agar is still molten. To minimize any variations in the consistency of the agar due to uneven cooling, hold the pipetter parallel to the bench top while drawing up the agar.
Then slowly draw the solution and seal the tip with parafilm before removing the chamber from the pipetter. Lay the chamber flat to cool and allow the agar to harden before moving. Once cooled, heat a three-millimeter hex key over a Bunsen burner and create a small plastic opening by firmly pressing it into the wall of each chamber about five millimeters to one side of the midline.
Next, use a heated blade to remove the cotton and tapered ends of the chamber and seal the ends with paraffin film. 10 to 15 days before the experiment, chunk each strain onto two to three large NGM plates with OP50 bacteria and wrap the paraffin film. Collect worms by rinsing the lids and plates with M9 Buffer and pipetting the solution into 15-milliliter centrifuge tubes.
Pellet the worms by spinning at 1600 x G for 30 to 60 seconds at room temperature and aspirate most of the M9 Buffer with a pipette or vacuum aspirator. Add seven milliliters of 1%sodium dodecyl sulfate solution to each worm pellet and leave the worms in it for 30 minutes. To allow aeration, keep rotating the tubes continuously during this time.
Then, remove the detergent by rinsing it three to five times with M9.After adding five milliliters M9, add five milliliters of cold filtered 60%sucrose solution. Mix thoroughly and create a separation gradient by centrifuging at 1600 x G for five minutes at room temperature. Fill new 15-milliliter centrifuge tubes with two milliliters of M9 Buffer.
Now crush the ends of glass Pasteur pipettes to widen the bore. And use these pipettes to transfer the top layer of the solution from the sucrose gradient to the new tubes. Rinse the dauers three to five times with M9 Buffer.
Centrifuge dauers at 1600 x G for five minutes at room temperature and aspirate most of the M9 solution with a pipette or vacuum aspirator. Next, estimate the worm density by manually counting the number of worms in three separate 1-microliter droplets under a dissecting microscope, and use the average of these counts to approximate the number of worms per microliter. Now widen the tip bore by cutting the end of 10, 20, or 200-microliter pipette tips.
Set the micropipette to a slightly larger volume than the intended aspiration volume. Aspirate approximately 1 to 2 microliters of the concentrated worm solution and allow a small amount of air to enter the tip. Gently push the pipette tip into the agar while depressing the micro pipette.
This will create an injection site without clogging the tip. Release the worms into the agar and use paraffin film to seal the opening. To eliminate as many variables as possible once the chambers are placed within a dark Faraday cage at room temperature, label each chamber and hang it vertically within the Faraday cage.
Test horizontal controls concurrently by laying some chambers flat within the same environmental conditions. Then use the vertically-oriented N2 worms as a positive control and horizontally-oriented chambers containing N2 dauers as an additional negative control against the experimental strains. After a few hours, the dauer worms begin to disperse from the start site and take several hours to reach either end of the chamber.
Leave the chambers undisturbed during this time and score within 12 to 24 hours following injection. Next, remove the chambers one at a time and score gravitaxis by looking for live dauers under a dissecting microscope and marking their locations with ink. Do not score worms within 2.5 centimeters to either side of the injection site as these worms are not likely to be demonstrating a directional preference.
Also, avoid scoring worms that appear dead or are trapped in the liquid and discard any chambers containing more than 50%dead or swimming worms. To quantify the results, use a marker to divide each half of the chamber into seven 3.5 centimeters, starting 2.5 centimeters away from the injection site. Use a manual tally counter to tally the number of worms observed in each section.
In caenorhabditis briggsae, a large percentage of dauer worms showed migration toward the top of the chambers. However, the horizontal controls showed distribution around the center of the chambers, indicating negative gravitactic behavior. Also, the vertical distributions of caenorhabditis briggsae and caenorhabditis elegans did not show any difference suggesting that caenorhabditis briggsae dauers show a negative gravitaxis behavior similar to caenorhabditis elegans dauers.
Paralytic agents such as sodium azide are not used in this protocol. Therefore, it is important to score the gravitaxis chambers as soon as they're removed from the Faraday cage. This assay led to the discovery of negative gravitaxis behavior in c.elegans.
Through testing several mutant strains, it also revealed genetic and neural requirements for gravitaxis in worms.
