To begin, select a nematode growth medium or NGM plate occupied by starved L1 stage Caenorhabditis elegans larvae. Using one milliliter of sterile water, wash the worms off the plate gently. Aliquot approximately 300 microliters of the worm population onto NGM plates seeded with concentrated OP50.
Allow the plates to dry using a plate dryer or a Bunsen burner, and then incubate at 20 degree Celsius until a significant population becomes gravid adults. To collect the worms, wash them off the plate with up to 10 milliliters of sterile water and transfer a warm water mixture into a 15-milliliter conical tube. Allow the worms to settle by gravity at the bottom of the tube for approximately four minutes.
Using a small pipette tip, carefully aspirate and discard the supernatant. Then add up to 15 milliliters of sterile water. After the final wash, remove the supernatant and add five milliliters of a freshly prepared solution of bleach and sodium hydroxide.
Incubate the worms for five minutes at room temperature while vortexing every minute for at least 10 seconds. Monitor the progress using a dissecting microscope. Once all the adults break open or dissolve, add M9 buffer to neutralize the reaction, bringing the final volume to 15 milliliters.
Then centrifuge the tube for two minutes at 1, 300 G.Wash the eggs two more times with 13 milliliters of M9 buffer via centrifugation followed by a single wash with 15 milliliters of S complete buffer. Then centrifuge the eggs for two minutes at 1, 300 G.After centrifugation, aspirate the supernatant and add 10 milliliters of S complete buffer. Rotate the tube gently at room temperature overnight using a mutator or a similar device.
Using a dissecting microscope, assess if the worms have hatched. Count the worms in 10-microliter drops of S buffer under the microscope. Prepare a liquid worm mixture with 60 worms per milliliter in S complete medium containing carbenicillin, amphotericin B, and OP50.
Add 120 microliters of medium with worms to rows A to G and no worms medium to row H.Then seal the plate with a tape sealer to prevent contamination and evaporation. Incubate the sealed plates for approximately 65 hours at 20 degrees Celsius until the animals become L4 worms. Add 30 microliters of a 0.6-millimolar fluorodeoxyuridine stock solution to each well to sterilize the animals at the L4 stage.
Reseal the plate using tape sealers and agitate it for 20 minutes at 800 RPM on a microtiter plate shaker. Return the plates to the 20-degree-Celsius incubator. The following day, add the drug of interest to the day one culture.
Reseal the plates with tape sealer and shake for 20 minutes at 800 RPM on a plate shaker. Then after removing the lid and seal, measure the OD600 in a plate reader for the day one measurement. Reseal and return the plates to a 20-degree-Celsius incubator.
Using an inverted microscope, preferably with a two times objective, count the worm population in each well and record the numbers in a spreadsheet. After counting, return the plates to the 20-degree-Celsius incubator. The dose response curve's a full change in food intake as a function of serotonin concentration showed that the N2 strain can overeat in a dose-dependent manner.
The daf-16 mu86 mutant exhibits higher basal feeding than N2.However, it cannot respond to serotonin in the same dose-dependent manner as the N2 strain. A significant difference was observed in the food intake of worms treated with loxapine but fed with bacteria killed by either x-rays, gamma rays, or paraformaldehyde. Comparison of the food intake of a series of genetic strains showed that exc-4 and cgr-1 mutants eat less while srp-6 mutants eat more.
