TITLE: Measure feeding during large population swarming with bioluminescent bacteria
AUTHORS: Serena Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol uses bioluminescently labelled bacteria (Gregor et al, 2008, Addgene Plasmid #107879, </span><a href="https://www.pnas.org/content/115/5/962" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.pnas.org/content/115/5/962</span></a><span style = "font-weight:bold;">) </span><span>to measure population feeding during large-scale C. elegans swarming.</span></div></div>

[STEPS]
?. [Grow bacteria overnight]
Day -2 Streak out bioluminescent bacteria frozen stock onto an LB plate containing 50 μg/mL ampicillin and incubate overnight at .
37 °C
This step does not need to be performed every time. A streaked plate can be re-used within 1 month, so long as single colonies are still available. Otherwise re-streak onto a new LB/ampicillin plate for single colonies.
?. [Seed bacteria for imaging (Day 0 ~2.30pm)]
Take imaging plates (two each of 90mm and 35 mm Petri dish containing 35 mL and 3.5 mL low peptone agar, respectively) out of the  cold room, let them incubate at  for 30 minutes to warm up.
4 °C
0 Room temperature
?. [Seed bacteria for imaging (Day 0 ~2.30pm)]
Seed 500 μL and 20 μL of the bacteria culture onto the centre of each 90 mm and 35 mm imaging plate, respectively. Dry the plate in a laminar flow hood (setting II) for 2 hours and 30 minutes, respectively.
?. [Initialise the IVIS imaging system (Day 0 ~4.30pm)]
Initialise the IVIS system via the Living Image software.
?. [Seed bacteria for imaging (Day 0 ~2.30pm)]
Give the flask containing the bacteria overnight culture a swirl to resuspend the bacteria cells that may have settled during the day.
?. [Seed bacteria for imaging (Day 0 ~2.30pm)]
Measure bacteria culture optical density with a spectrophotometer for the record.
?. [Inoculate worms for experiment (Day 0 ~5.30pm)]
Retrieve imaging plates from the flow hood. Select one each of 90 mm and 35 mm plates for the actual experiment, and label them with sample name.
?. [Initialise the IVIS imaging system (Day 0 ~4.30pm)]
Set up imaging parameters within the Living Image software interface. Set stage temperature to  , designate auto-saving directory for acquired images, and set up acquisition sequence parameters.
20 °C
Bioluminescence acquisition parameters: subject height: 0.5 cm; field of view: C; expososure: 1 second; frame interval: 1 minute (for N2 worms) and 2 minutes (for DA609 worms); excitation: block; emission: open; luminescence binning: 4; photograph binning: 4; luminescence F/stop: 1; photograph F/stop: 1; batch sequence: true; sequence 1 length: 99; sequence 2 length: 35.These imaging parameters have been determined in advance for the experiment and are saved as a .xsq configuration file, which can be loaded into the imaging software.
?. [Inoculate worms for experiment (Day 0 ~5.30pm)]
Harvest synchronised adult worms using M9 buffer and transfer them into a 15 mL Falcon tube, pooling between three large plates. Wash them twice with M9 buffer by topping up the tube with M9, spinning at 1500 rpm/210 rcf (ascending 9, descending 7), and then aspiring the supernatent leaving the loose pellet intact. After the last wash, leave the loose worm pellet.
This spinning programme separates worms (pellet) from bacteria (supernatant), so bacteria can be removed. Soft brake prevents the loose worm pellet from breaking up.
?. [Inoculate worms for experiment (Day 0 ~5.30pm)]
Remove as much supernatant as possible. Using a glass pipette with a medicine dropper bulb attached, transfer all worms to the 90 mm imaging plate.
Try to keep the droplet small so it does not spill onto the bacterial lawn.
?. [Signal extraction and export (Day 1)]
After image sequence acquisition, read signal across the acquisition sequence using the Living Image software by measuring radiance (photons/s) for a defined region of interest.
?. [Signal extraction and export (Day 1)]
Export signal as a tab-delimited text file for downstream analysis.
?. [Grow bacteria overnight]
Day -1 ~5pmTo a 300 mL volumetric flask, add 100 mL LB broth, 100 μL ampicillin stock solution (50mg/mL) , and a single colony of bioluminescent bacteria from the streaked plate. Place on  incubator shaking at 220 rpm overnight.
37 °C
?. [Grow bacteria overnight]
Day 0 ~10amTake the bacteria overnight culture off of the shaking incubator and leave at room temperature on the bench until use that afternoon.
Do not put bacteria culture at 4 °C  to save for later, as this will abolish the signal. Instead, a fresh overnight culture should be grown for each day of experiment.
?. [Image (Day 0)]
Place imaging sample on the 90 mm plate and the no-worm 35 mm control plate inside the IVIS chamber inside the laser grid guidelines. Close the chamber door, and start acquisition via the software immediately.
No need to wait for the liquid to be absorbed into the media first before acquisition, as this can take a while and can be monitored by brightfield images.
?. [Bleach synchronise worms (Day -7 to -4)]
Bleach synchronise animals from three 90 mm plates, making sure lots of gravid hermaphrodites are present. Leave on rotator at 20 °C until use.
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?. [Re-feed worms (Day -3)]
Re-feed worms onto three pre-seeded 90 mm plates using a glass pipette. Incubate at 20 °C.
Monitor these plates until use to ensure worms do not run out of food. Three full plates of synchronised young adults give approximately 10,000 animals.
?. [Inoculate worms for experiment (Day 0 ~5.30pm)]
Put the 15 mL Falcon tube with the worm pellet inside a styrofoam box, and take them to the IVIS imaging system.