TITLE: Phenotyping C. elegans behaviour on E. coli bacteria supplemented with  enterobactin, iron or paraquat
AUTHORS: Saul    Moore

[DESCRIPTION]
Testing whether Keio Collection E. coli mutants supplemented with exogenous enterobactin, iron(III) chloride, iron(III) sulfate or paraquat elicit a behavioural response when fed to C. elegans.

[STEPS]
SECTION: Preparing 6-well plates for imaging
1. Add 25mL fresh LB broth to each of 2 Falcon tubes, and inoculate with the BW25113 (Keio Collection parent strain) and the E. coli gene-deletion mutant of interest, respectively
SECTION: Preparing 6-well plates for imaging
2. Place inoculations to grow overnight in a shaking incubator (37°C, 200rpm)
SECTION: Preparing 6-well plates for imaging
4. Remove the imaging plates from 4°C and dry under a hood for 30 minutes to prevent condensation.
Remove the bacterial cultures from 4°C and leave on the bench for 30 minutes to acclimate to room temperature.
SECTION: Preparing 6-well plates for imaging
3. Prepare 1L NGM agar and pour 4mL into each well of 40 x 6-well plates (imaging plates). Leave to dry under a hood (~2 hours) until they lose between 3 - 5% of their original weight after pouring. Store at 4°C until seeding with bacterial lawns.
SECTION: Preparing 6-well plates for imaging
5. Pipette 30μL of bacterial culture into the centre of each well, taking care not to damage the agar with the pipette tip. Seed half of the 6-well plates with BW25113 control, and the other half with the desired test bacteria
SECTION: Preparing 6-well plates for imaging
6. Leave the seeded plates to dry for 20 minutes under the hood, then transfer to a 25°C incubator and leave to grow for a further 7 hours and 40 minutes (for a total of 8 hours lawn growth time), before storing at 4°C until tracking (max 2 days)
SECTION: Preparing 6-well plates for imaging
7. On the day of tracking, remove the seeded plates from 4°C and dry under a hood for 30 minutes to remove condensation
SECTION: Preparing 6-well plates for imaging
8. Approximately 1 - 2 hours prior to adding worms and imaging, exogenously add the following on top of the lawns in the desired wells, as per the experimental design, and leave to dry under a hood:
5uL of 1mg/mL enterobactin (in DMSO)
160ul of 1 and 4mM iron(III) chloride (in H2O)
160ul of 1 and 4mM iron(III) sulfate (in H2O)
160ul of 0.5, 1, 2 and 4mM paraquat dichloride (in H2O)

SECTION: Imaging with worm tracking rig (Hydra)
9. Prior to tracking, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also empty the dehumidifier waste water tray (see pre-imaging checklist)
SECTION: Imaging with worm tracking rig (Hydra)
10. Remove the plate of age-matched (N2 Bristol, Day1 adult) worms from 20°C incubator
SECTION: Imaging with worm tracking rig (Hydra)
11. Using a platinum pick, gently but swiftly transfer 10 worms onto the edge of the bacterial lawn of each well in a single imaging plate at a time
SECTION: Imaging with worm tracking rig (Hydra)
12. Quickly transport the 6-well plates to the imaging cave and place them under the rigs. Ensure that the plate is in the correct orientation for the recording so that the positions of each of the wells under the cameras is correct and matches the recorded treatment information in the metadata
SECTION: Imaging with worm tracking rig (Hydra)
13. Track worm behaviour on each well for a total of 36 minutes (at 25 fps), applying a 10-second blue-light stimulus at the 30th, 31st and 32nd minute timepoints