The overall goal of the following experiment is to quantify the chemotactic response of C elegance using a simple and inexpensive assay. This is achieved by isolating staged worms and placing them at the center of a marked plate. Equidistant from all test and control sites deposited at each site is a compound of interest or a control compound, both combined with an anesthetic that will arrest the worms as they approach.
After 60 minutes, the final distribution of the worms on the plate is tallied, and the ratio of the worms locations is used to provide an assay index. Ultimately, quantifying the attraction of sea elegance to certain compounds can be used to evaluate the behavioral consequences of underlying experimental manipulations. The main advantage of this technique is that it includes many beneficial aspects of its predecessors, while allowing for a greater ease of use.
This assay provides insight into the behavior of sea elegance and is especially useful in combination with neurogenetic manipulations and other neurobiological assays. Begin with five centimeter plates loaded with chemotaxis agar or NGM. Mark the underside of the plates into four equal quadrants.
Prepare at least three plates for at least three trials per genetic strain being tested and per condition tested. Next, mark a circle with a 0.5 centimeter radius around the origin. Then mark points in the opposite quadrants with a T for test or a C for control, ensuring that the sites are equidistant from the origin and from each other.
The points must be at least two centimeters away from the origin for this assay. Have prepared young adult worms in synchronized development on five centimeter chemotaxis plates. Worms that are in different stages of development may respond differently to the analytes.
As a result, it is very important that all the worms employed in the assay are in the same developmental phase. Once the worms have cleared a lawn of OP 50 e coli, pipette two milliliters of S basal buffer onto the plate. Manipulate the plate to get all the worms into the buffer to wash the worms.
Pipette one milliliter of the worms in buffer into a micro centrifuge tube and centrifuge them for 10 seconds. Using a pico fuge at 6, 600 RPM, aspirate the as basil, leaving the pellet of worms undisturbed. Now wash these worms three more times using the same method.
Begin by adding the S basil buffer to the tube and inverting the tube to put the worms back in solution. After the final wash, centrifuge the worms for another 10 seconds and then aspirate the supernatant to a final volume of 100 microliters. Next, ensure a sufficient density of worms by pipetting two microliters of the washed worms from the pellet onto an NGM plate.
There must be at least 50 worms, but not much more than 250 worms. If there are too many worms, increase the volume of ESP basil solution. And if there are too few worms, spin them down and resus suspend them in a lower volume.
Set the worm tube in a fume hood. Once prepared, the worms are good to use in the chemo taxes assay for up to an hour. First, prepare the solutions.
Mix the test solution by combining equal volumes of the test compound with the anesthetic 0.5 molar azide. Next, mix a control solution of the solvent used to dilute the test compound with 0.5 molar sodium azide. Now prepare a test plate.
First pipette two microliters of worm solution from the pellet onto the origin of a test plate. Second, add the odorants pipette two microliters of the test solution onto the two T sites and pipette two microliters of the control solution onto the two T sites. Once the worms and odorant drops have been absorbed into the agar, replace the lids, invert the plates, and allow 60 minutes for the assay time.
For the sodium. ASI diffusion time to be standardized between trials. It is important that the worms are added immediately before the odorants.
Also, each plate should be fully prepared and that assay begun before preparing the next plate. After the 60 minute trial, place the worms in a four degree Celsius refrigerator. Later, remove the plate from the refrigerator when it can be counted.
Not counting them immediately and leaving the worms at room temperature may allow them to mobilize.Again. Record the location of worms that completely crossed out of the inner circle from the data. Calculate the chemotaxis index.
The value will range from negative one to positive one, representing maximum repulsion to maximum attraction. Repeat the process using the control solution in both the test and control quadrants. This will serve as the control plate.Three.
Such plates should be made and run for each experimental condition.Diacetyl. A known C elegance chemo attractant was used to compare wild type worms to mutants that lack the receptor for diacetyl. As expected, diacetyl elicited a significant chemo attractive response from wild type worms.
In contrast, the ODR 10 mutants were no more attracted to diacetyl as they were to the control compound ethanol Once mastered. This technique can be completed in less than 90 minutes if performed properly. After watching this video, you should have a good understanding of how to efficiently evaluate the chemotactic response of sea elegance to analyte solutions.
Take special care to ensure all tested worms earn a synchronized stage of development and that sufficient number of worms are employed. Each trial.
