TITLE: Measure 40 worm feeding with bioluminescent bacteria after drug treatment
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 of 40 adult C. elegans worms following serotonin or naloxone drug treatment. </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 ~4.30pm)]
Take drug plates prepared from the day before 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 ~4.30pm)]
Seed 20 μL of the bacteria culture onto the centre of each 35 mm imaging plate. Dry the plate in a laminar flow hood (setting II) for 30 minutes.
?. [Initialise the IVIS imaging system (Day 0 ~4.30pm)]
Initialise the IVIS system via the LivingImage software.
?. [Seed bacteria for imaging (Day 0 ~4.30pm)]
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 ~4.30pm)]
Measure bacteria culture optical density with a spectrophotometer for the record.
?. [Inoculate worms and drug exposure for experiment (Day 0 ~5.30pm)]
Retrieve imaging plates from the flow hood and label them with sample name.
?. [Initialise the IVIS imaging system (Day 0 ~4.30pm)]
Set up imaging parameters within the LivingImage 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: 6 minutes; 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 and drug exposure for experiment (Day 0 ~5.30pm)]
Harvest synchronised adult worms using 2 mL of M9 buffer and transfer them into a 15 mL tube. 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 and drug exposure for experiment (Day 0 ~5.30pm)]
Using a glass pipette with a medicine dropper bulb attached, transfer 40 starved worms to each imaging plate. Use a manual click counter to count 40 worms.
Try to keep the droplet small so it gets absorbed into the media in a reasonable amount of time (a couple of minutes), although this makes accurate counting more difficult so it's worth practicing worm transfer beforehand to find the optimal worm transfer techniques.
It's handy to have a P20 pipette in hand with a few microliters of M9 already inside. To remove excess worms, lower the tip near the worms and carefully suck worms back out. It's not usually precise as to how many worms are removed, so repeat counting with a manual counter again after removing worms.
?. [Inoculate worms and drug exposure for experiment (Day 0 ~5.30pm)]
Place the lid back onto the imaging plate. When all worms are transferred, put all the samples inside a cardboard box and then inside a styrofoam box, and take them to the IVIS imaging system.
Carry carefully, as some liquid in which worms are transferred may not have tried completely and may disturb the bacterial lawn if it spills over.
?. [Signal extraction and export (Day 1)]
After image sequence acquisition, read signal across the acquisition sequence using the LivingImage 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 samples inside the IVIS chamber inside the laser grid guidelines. Close the chamber door, and start acquisition via the software exactly 1 min after the vortex start.
?. [Image (Day 0)]
Vortex imaging samples for 10s on the lowest setting of a vortex mixer to disperse any existing aggregates in order to randomise the initial position of worms.
?. [Prepare drug plates (Day -1)]
Make up drug plates and store in the dark at  overnight.
4 °C
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?. [Inoculate worms and drug exposure for experiment (Day 0 ~5.30pm)]
Using a glass pipette with a medicine dropper bulb attached, transfer all worms onto an unseeded NGM plate in order to starve the worms for  .
?. [Inoculate worms and drug exposure for experiment (Day 0 ~5.30pm)]
After 1 hour, harvest the starved worms again using M9 into a 15 mL tube. Pellet worms by spinning at 1500 rpm/210 rcf (ascending 9, descending 7) and aspiring the supernatant leaving the loose pellet intact.
?. [Inoculate worms and drug exposure for experiment (Day 0 ~5.30pm)]
Let the worms be exposed to drugs for  at  .
20 °C
?. [Bleach synchronise worms (Day -7 to -4)]
Bleach synchronise animals from three 60mm 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 using a glass pipette. Incubate at 20 °C.
Monitor these plates until use to ensure worms do not run out of food.