Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

The nematode Caenorhabditis elegans is easy to cultivate, to maintain, to manipulate, and to be used in experiments for biological science teaching, and also for students to carry out their projects. This protocol brings two easy and low-cost assays that are based on the transparency of the nematode, and also on the use of colored dyes. The first protocol allows the investigation of metabolic chains following different diets, and the second protocol allows the observation of the aging process in a short period of time.

These both assays are interesting for educational purposes under limited resources. The first assay takes an advantage of the worm's transparency, and the fact that it produce and it stores glycogen. Using Lugol iodine solution, we can dye the worms and verify whether different diets or other factors can modify its levels.

Aging is a natural process, but some factors, such as ultraviolet, food, and chemical exposure can accelerate this process. The intestinal barrier is modified with the agent, and epithelium loses integrity, causing leakage of the substance the worm's intake, such as We call this assay Smurf Test because the worms get totally blue when there is a reduction of intestinal integrity. 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 seeded 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 1, 400G 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 1, 000 worms per microliter.

Transfer 500 to 1, 000 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 micro tube 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 resuspending 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. To begin, transfer 500 to 1, 000 L1 stage synchronized Caenorhabditis elegans worms on NGM agar plate and incubate at 20 degrees Celsius until the day of the test.

Design the assay schedule for two experimental groups. Using M9 buffer, collect L4 worms and adulthood worms on the respective day. Transfer the collected worms to 1.5 milliliter micro tubes and wait for worms to sediment by gravity.

Wash the worms thrice with one milliliter of M9 buffer, leaving approximately 500 microliters in the tube after the last wash. Using an automatic pipette, add 10 microliters of the worm suspension to a microscope slide, and calculate the volume required to obtain 100 worms per microliter. In a new micro tube, add the volume required for 100 worms and 100 microliters of 25%erioglaucine disodium salt solution.

Then, add 200 microliters of E.coli OP50 culture and increase the volume to 500 microliters using an M9 buffer. Incubate the tube for three hours with agitation in a mixer, protecting it from light. Upon incubation, allow the worms to sediment by gravity before washing them with one milliliter of M9 buffer until the staining solution is completely removed.

After the last wash, resuspend the worms in 250 microliters of M9 buffer and transfer approximately 50 microliters onto the slide. Place the cover slip, and incubate the slide at minus 20 degrees Celsius for 10 minutes to paralyze the worms. Using a stereo microscope under bright field mode, count the total number of worms and the total dyed worms.

Intestinal permeability assay displayed a strong intestinal barrier in young worms, preventing dye leakage. In contrast, aged worms exhibited dye extravasation throughout their bodies, indicating intestinal membrane damage.