Pharyngeal Pumping Assay

Using an eyelash pick, pick 10 x L4s of each worm strain to be assayed onto 2 x 90 mm plates (pre-seeded with OP50) and incubate at 20°C

Prepare and pour the following number of NGM plates per worm strain to be studied:

• 5 x 60 mm (15mL agar per plate)
• 2 x 90 mm (35mL agar per plate)

Therefore make 150mLNGM per strain

To ensure accuracy when weighing out media components it is recommended that you do not prepare agar in volumes less than 250 mL at a time!

Prepare NGM agar and autoclave

Protocol for preparing NGM: dx.doi.org/10.17504/protocols.io.bvh7n39n

In the laminar flow hood, pour 5 x 60 mm and 2 x 90 mm plates per worm strain

Plate Pouring

Once plates have dried, store agar-side up in an airtight container in the cold room

Dry 2 x 90 mm plates per worm strain in the cabinet dryer (setting 2) for 1.5 hours

Seed the 90 mm plates with OP50 and leave to dry overnight at room temperature

Bleach synchronise worms (picked in Step 1) and store eggs/larvae in a 15 mL falcon tube on the rotator that is constantly spinning at 20°C until feeding

Bleach synchronisation of C. elegans

Refeed bleach synchronised L1s

At 16:50, spin L1s bleached in step 4 using centrifuge program 1 (2500 rpm, 2 mins)

Using a plastic pipette remove the supernatent, taking care to not disturb the worm pellet, and leave ~1 mL of M9 in the tube

Gently flick the falcon tube to resuspend the L1s

Using a glass pipette, drop 1 small droplet around the edges (off food) of the pre-seeded 90 mm plates (prepared in Step 3)

It is recommended that you check the density of worms in the droplet down a microscope and add another if necessary

Place plates agar-side down in the 20°C incubator to allow droplets to dry (~5-10 mins), then invert plates and allow to grow for 2.5 days

In the morning, take the 60 mm plates from the cold room, and weigh three random plates without their lids

Dry plates in in cabinet dryer (setting 2) with lids off until they have lost 3-5% of their weight (approx 2 hours)

Aseptically prepare 1:10 dilution of OP50 in M9 in a 15 mL falcon tube:

1mL OP50

9mL M9

Working by a flame, dispense 30µL of the dilute OP50 into the centre of each 60 mm plate and allow droplets to dry at room temp

Ensure that you do not touch the agar with the pipette tip and that plates are kept on a flat surface so you get a nice, even circle of food once dried

Once the food has dried, leave plates agar-side up at room temp overnight

Label 5 x 60 mm plates seeded with 30µL OP50 (around the edge, so not to obscure the bacterial lawn) per worm strain

At 09:00, use an eyelash-pick to transfer 10 x young adult worms (prepared in Step 5) onto the edge of each 60 mm plate (away from food)

This yields a total of 50 worms per strain

Incubate plates, agar-side up, at 20°C for 1 hour

This allows worms to crawl to the bacterial lawn and start foraging again

Working one plate at a time, place a 60 mm plate upon a dissecting stereo microscope at the highest magnification possible and bring the worms/bacterial lawn to focus

Working on a single worm at a time, identify the grinder (in the terminal bulb of the head) of each worm and ensure this is clearly in focus

See: http://www.wormbook.org/chapters/www_measurepharyngeal/measurepharyngeal.html for detailed images/diagrams of pharynx and terminal bulb

Use a tally counter to count the grinder movements of a worm over a 20 second period

Repeat this 3 times per worm and record the result as an average of the three counts

Go clockwise around the bacterial lawn, and repeat the steps above for each worm on the 60 mm plate. Then repeat for each 60 mm plate remaining in the incubator

Calculate pharyngeal pumps per minute of each strain:

pumps per minute = pumps per 20 seconds * 3