Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

My research focuses on understanding the transcriptional and metabolic pathways underlying drug-induced changes in feeding behavior. I am working to identify drugs that lower food intake and extend lifespan, indicative of activating healthy fasting pathways. Using our bacterial clearance assay, we identified two distinct pathways that modulate serotonin-induced food intake and antipsychotic-induced food intake.

I am now studying how these pathways relate to feeding and long-lived mutants. This protocol allows researchers to study the relationship between feeding and longevity. The feeding rate is usually characterized by pharyngeal pumping.

However, the pumping rate in C.Elegans is not necessarily proportional to the amount of food being eaten. When added to those previous assays, this protocol gives a quantifiable amount of food intake per worm population. Our assay allows us to quantitatively modulate feeding using small molecules and to determine how the induced change alter lifespan simultaneously.

To begin, inoculate a single OP50 colony in five-milliliters of LB with ampicillin and amphotericin B, and incubate for approximately six hours at 37 degrees Celsius in a bacterial shaker. Once the culture becomes cloudy, dilute the pre-inoculate culture of OP50 by 1:2, 000 in 250 milliliters of TB with ampicillin and glycerol. Then transfer the OP50 liquid culture into a sterile centrifugation tube.

Centrifuge the tube for 15 minutes at 3, 100 g in a tabletop centrifuge at four degrees Celsius. Discard the supernatant, and resuspend the OP50 pellet in sterile water before recentrifugation. After the second wash, discard the supernatant, and resuspend the OP50 pellet in sterile water to a volume of 50 milliliters.

Transfer it to a pre-weighed 50-milliliter conical tube Centrifuge tube for 20 minutes to pellet the OP50. At the end of the centrifugation, carefully remove all remaining supernatant, ensuring no water remains in the tube. Calculate the weight of the pellet by subtracting the weight of the empty centrifugation tube from the weight of the centrifugation tube with the pellet.

Thoroughly resuspend the OP50 pellet in S-Complete Buffer to achieve a concentration of 100 milligrams per milliliter. Test the OP50 suspension on an agar plate to check for any contamination. Store the OP50 suspension in S-Complete Buffer at four degrees Celsius.

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 a 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 the worm 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 Fluoro-deoxyuridine 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 degrees Celsius incubator. The following day, add the drug of interest to the Day 1 worm 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 1 measurement. Reseal, and return the plates to a 20 degree Celsius incubator. Using an inverted microscope, preferably with a 2x objective, count the worm population in each well, and record the numbers in a spreadsheet.

After counting, return the plates to the 20 degrees Celsius incubator. The dose response curves of fold 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 show that exc-4 and cgr-1 mutants eat less, while srp-6 mutants eat more.