To begin, prepare six nematode growth media or NGM agar plates. The next day, add 200 microliters of Escherichia coli OP50 culture onto the plates, and incubate at room temperature for one day. After incubation, add 200 microliters of 0.025 molar D-glucose to four NGM agar plates, and allow them to dry at room temperature.
Transfer different staged three to four chunks of Caenorhabditis elegans Bristol N2 from a maintenance NGM agar plate to a new NGM plate seated with E.coli OP50. Incubate the C.elegans worms at 20 degrees Celsius with humidity of more than 95%for three days. Three days after, add distilled water onto the plate, and using a Pasteur pipette, collect the worms.
Transfer the collected worms to a 50 milliliter centrifuge tube, and allow them to sediment by gravity before removing the supernatant from the tube. Then, add distilled water into the tube. Once the volume of the tube is reduced to five milliliters, add 10 milliliters of bleaching solution and vigorously shake the tube for approximately six minutes.
Then, fill the tube to a 50 milliliter capacity with M9 buffer, and centrifuge at 1400 g for three minutes. Remove the supernatant up to five milliliters and add 45 milliliters of M9 buffer into the tube. After the last wash, remove the supernatant up to the 15 milliliter mark and transfer the remaining liquid to a plate.
Observe the plate under the microscope for the release of eggs, and place the plate at 20 degrees Celsius with humidity of more than 95%for 14 hours. After incubation, collect the worms into a 15 milliliter tube. Using an automatic pipette, add 10 microliters of the synchronized worms to a microscope slide.
Count the number of worms and calculate the volume required to obtain 500 to 1000 worms per microliter. Transfer 500 to 1000 larval stage one, or L1, synchronized worms to the NGM agar plates. Previously prepared and seeded with E.coli OP50 and glucose.
Incubate the plate at 20 degrees Celsius until the worms reach larval stage four, or L4.After 48 hours, add M9 buffer onto the plate, and using a Pasteur pipette, collect L4 larvae. Transfer the collected larvae to a 1.5 milliliter tube. Design the assay schedule for three experimental groups.
Transfer approximately 100 microliters of worm suspension from each group to the 1.5 milliliter microtube containing 400 microliters of 5%Lugol's Iodine Solution. Gently agitate the tube in a mixer for five minutes. After removing the tube from the mixer, wait for worms to sediment by gravity.
Wash the worms thrice with one milliliter of M9 buffer. After re-suspending the worms in 100 microliters of M9 buffer. Transfer approximately 50 microliters onto the slide.
Place the cover slip onto the slide. Next, on a stereo microscope, select the bright-field mode and observe the worms. Save the images containing a minimum of 10 worms per group as a jpeg file.
Finally, calculate the glycogen content based on the iodine staining of worms. Visual observations of glycogen content assay revealed that fasting worms displayed a weaker stain compared to those fed with E.coli OP50 and those with E.coli OP50 and D-glucose, which exhibited the most intense staining.
