TITLE: Pharyngeal Pumping Assay
AUTHORS: Thomas J J. O'Brien

[DESCRIPTION]
The pharynx is a is a neuromuscular pump found at the anterior end of the alimentary tract, consisting of 20 muscles and 20 neurons. A proper feeding rate in worms is coordinated by the precise timing of pharyngeal movements, with one complete cycle of synchronous contraction and relaxation of the corpus and terminal bulb termed a “pump”. A simple way to measure C. elegans feeding is to count how many times worms pump in a minute (pumps per minute). Movement of the grinder (in the terminal bulb) can easily be observed using a stereomicroscope, and because cycles of contraction/relaxation are synchronised along the pharynx, pumps per minute can be measured simply by counting grinder movements.

[STEPS]
SECTION: Pick L4 worms for bleaching (Day 1)
1. 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

SECTION: Pour 60 mm NGM agar plates (Day 2)
2. Prepare and pour the following number of NGM plates per worm strain to be studied:
5 x 60 mm (15 mL agar per plate)
2 x 90 mm (35 mL agar per plate)

Therefore make 150 mLNGM 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! 

SECTION: Pour 60 mm NGM agar plates (Day 2)
2.1. Prepare NGM agar and autoclave

Protocol for preparing NGM: dx.doi.org/10.17504/protocols.io.bvh7n39n
SECTION: Pour 60 mm NGM agar plates (Day 2)
2.2. In the laminar flow hood, pour 5 x 60 mm and 2 x 90 mm plates per worm strain
 

SECTION: Refeed L1s (Day 8)
5. Refeed bleach synchronised L1s
SECTION: Dry/Seed 90 mm plates and bleach worms (Day 5)
3. Dry 2 x 90 mm plates per worm strain in the cabinet dryer (setting 2) for 1.5 hours
SECTION: Refeed L1s (Day 8)
5.1. At 16:50, spin L1s bleached in step 4 using centrifuge program 1 (2500 rpm, 2 mins)
SECTION: Pour 60 mm NGM agar plates (Day 2)
2.3. Once plates have dried, store agar-side up in an airtight container in the cold room
SECTION: Dry/Seed 90 mm plates and bleach worms (Day 5)
3.1. Seed the 90 mm plates with OP50 and leave to dry overnight at room temperature
SECTION: Dry/Seed 90 mm plates and bleach worms (Day 5)
4. 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  
 


SECTION: Refeed L1s (Day 8)
5.2. Using a plastic pipette remove the supernatent, taking care to not disturb the worm pellet, and leave ~1 mL of M9 in the tube
SECTION: Refeed L1s (Day 8)
5.3. Gently flick the falcon tube to resuspend the L1s
SECTION: Refeed L1s (Day 8)
5.4. 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
SECTION: Refeed L1s (Day 8)
5.5. 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

SECTION: Dry and seed 60 mm plates (Day 10)
6. In the morning, take the 60 mm plates from the cold room, and weigh three random plates without their lids
SECTION: Dry and seed 60 mm plates (Day 10)
7. Dry plates in in cabinet dryer (setting 2) with lids off until they have lost 3-5% of their weight (approx 2 hours)
SECTION: Dry and seed 60 mm plates (Day 10)
8. Aseptically prepare 1:10 dilution of OP50 in M9 in a 15 mL falcon tube:
1 mL  OP50
9 mL  M9

SECTION: Dry and seed 60 mm plates (Day 10)
9. Working by a flame, dispense 30 µL 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

SECTION: Dry and seed 60 mm plates (Day 10)
10. Once the food has dried, leave plates agar-side up at room temp overnight
SECTION: Assaying rate of pharyngeal pumping (Day 11)
11. Label 5 x 60 mm plates seeded with 30 µL OP50 (around the edge, so not to obscure the bacterial lawn) per worm strain 

SECTION: Assaying rate of pharyngeal pumping (Day 11)
12. 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
SECTION: Assaying rate of pharyngeal pumping (Day 11)
13. Incubate plates, agar-side up, at 20 °C for 1 hour

This allows worms to crawl to the bacterial lawn and start foraging again
SECTION: Assaying rate of pharyngeal pumping (Day 11)
14. 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
SECTION: Assaying rate of pharyngeal pumping (Day 11)
14.1. 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
SECTION: Assaying rate of pharyngeal pumping (Day 11)
14.2. 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
SECTION: Assaying rate of pharyngeal pumping (Day 11)
14.3. 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
SECTION: Assaying rate of pharyngeal pumping (Day 11)
15. Calculate pharyngeal pumps per minute of each strain:

pumps per minute = pumps per 20 seconds * 3