TITLE: Keio Acute Response Antioxidant Rescue 
AUTHORS: Saul    Moore

[DESCRIPTION]
Phenotyping the acute behavioural response of Caenorhabditis elegans (N2 Bristol) to E. coli single-gene deletion mutants (BW25113), in both the presence and absence of antioxidants (Trolox, NAC, vitamin C and resveratrol). 

Videos are recorded at 25 fps on the laboratory’s (Hydra) imaging rig, immediately after worms are picked onto imaging plates, for a total of 45 minutes at 25 fps, with blue-light stimulus delivered for 10 seconds in 5 minute intervals throughout the recording. 

[BEFORE_START]
Prepare NGM agar media and fill maintenance plates for raising N2 Bristol C. elegans on BW25113 background strain 

[GUIDELINES]
Read through the whole protocol before starting, as the sections are not in chronological order

[STEPS]
SECTION: Preparing maintenance plates
1. Make 500mL normal Nematode Growth Media (NGM) agar, following the protocol 
 

SECTION: Preparing maintenance plates
2. Under a hood, pour 20ml NGM agar into each of 10 x 60mm Petri plates (maintenance plates), and leave to dry for apprximately 1 hour. Once dry, store at 4°C until seeding bacterial lawns
SECTION: Preparing maintenance plates
3. Inoculate an overnight culture of E. coli BW25113 bacteria in an Erlenmeyer flask containing 50ml LB broth, and place in at 37°C in a shaking incubator (200rpm)
 

SECTION: Preparing maintenance plates
4. Remove the BW culture from the shaking incubator and store at 4°C until seeding
SECTION: Preparing maintenance plates
5. Remove the maintenance plates and the BW25113 culture from 4°C storage, and leave on the bench for approximately 30 minutes to acclimate to room temperature
SECTION: Preparing maintenance plates
6. Seed the maintenance plates each with 200μL of BW25113 culture, using aseptic technique working under a microbiological hood
SECTION: Preparing maintenance plates
7. Leave under hood until dry (approximately 30 min to 1 hour timing depends on humidity)
SECTION: Preparing worms
8. Using a platinum pick, gently pick 30 L4-stage N2 Bristol C. elegans onto each maintenance plate, and store in an incubator at 20°C (Monday)
SECTION: Preparing worms
9. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae (Tuesday)

SECTION: Preparing worms
10. Bleach-synchronise the worms by performing an egg prep, following the protocol:
(Friday)
 

SECTION: Preparing worms
11. At around noon the next day, wash L1 larvae off the empty plate with a few mL of M9 using a glass Pasteur pipette, and re-feed onto BW-seeded maintenance plates. Incubate at 20°C.
(Saturday)
SECTION: Preparing bacteria
12. Prepare two Erlenmeyer flasks with 50mL LB for inoculating a liquid bacterial culture of BW control and BW25113ΔfepD bacteria. Add 50mg/mL Kanamycin to BW25113ΔfepD flask
(Friday)
SECTION: Hydra Tracking
26. 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)
(Tuesday)
 

SECTION: Hydra Tracking
27. Remove the seeded imaging plates from 4°C and leave on bench top (with lids on) for 30 minutes to acclimate to room temperature
SECTION: Hydra Tracking
28. Remove the plate of age-matched (Day1 adult) worms from 20°C incubator
SECTION: Hydra Tracking
29. Using an eyebrow hairpick, gently but swiftly transfer 10 worms onto the edge of the bacterial lawn of each of 6 imaging plates
SECTION: Hydra Tracking
30. Quickly transport the 6-well plates to the imaging cave and place them under the rigs (recording the respective camera positions of each of the wells in the metadata along with the relevant treatment information)
SECTION: Hydra Tracking
31. Track worm behaviour on the different food + antioxidant treatment conditions for a total of 45 minutes (25 fps), applying a 10-second bluelight stimulus every 5 minutes
SECTION: Preparing imaging plates
18. Make 250mL normal Nematode Growth Media (NGM) agar, following the protocol in   
(Thursday)
 



SECTION: Preparing imaging plates
25. Leave the plates for at least 2 hours after adding antioxidants before picking worms onto the plate and tracking
SECTION: Preparing bacteria
14. Leave overnight in a shaking incubator (37°C, 200rpm)
SECTION: Preparing bacteria
13. Inoculate from a single colony picked from streaked LB plates stored at 4°C, following the protocol:
 

SECTION: Preparing bacteria
15. The next day, remove the bacterial cultures from the shaking incubator, and prepare another two Erlenmeyer flasks each with 50mL LB, for inoculating a second round of overnight cultures. Only this time, do not add Kanamycin.
(Saturday)
SECTION: Preparing bacteria
16. Inoculate the second round of cultures by pipetting approximately 50μL bacterial culture from the first overnight culture. Leave in a shaking incubator overnight (37°C, 200rpm)

SECTION: Preparing bacteria
17. The next day, remove the overnight cultures from the shaking incubator, and store at 4°C until seeding imaging plates the following day
(Sunday)
SECTION: Preparing imaging plates
19. Pour 3.5mL NGM agar into each well of 10 x 6-well plates, and leave to cool under a hood. Then store at 4°C until seeding lawns


SECTION: Preparing imaging plates
20. Remove imaging plates from 4°C, and dry under a hood for 30 minutes to remove condensation. Remove the bacterial cultures from 4°C and leave on the bench for 30 minutes to acclimate to room temperature. 
(Monday)
SECTION: Preparing imaging plates
21. Pipette 30μL of bacterial culture into the centre of each well in the 6-well imaging plates, taking care not to damage the agar with the pipette tip. Seed half of the 6-well plates with BW control, and the other half with BW25113ΔfepD lawns

SECTION: Preparing imaging plates
22. Leave the seeded plate 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 for tracking the next day

SECTION: Preparing imaging plates
23. On the day of tracking, remove the seeded plates from 4°C and dry under a hood for 30 minutes to remove condensation
(Tuesday)
SECTION: Preparing imaging plates
24. Prepare the antioxidants (see Materials section for details of antioxidant preparation). Pipette 200μL antioxidant solution onto the top of the lawns of each well, to yield a final concentration of 10μg/mL antioxidant (in 4mL NGM agar). Leave under a hood to dry for approximately 30 minutes