TITLE: Antioxidant rescue of C. elegans behaviour on Keio E. coli mutants
AUTHORS: Saul    Moore

[DESCRIPTION]
Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour in the presence of antioxidants.


[STEPS]
SECTION: Preparing NGM agar + pouring plates
1. Prior to screening, prepare the materials needed for screening C. elegans on selected Keio E. coli mutants:
For a single experiment replicate (10 biological replicates of each mutant, screened in 2 runs with the laboratory's 'Hydra' imaging rig):

- 12 Whatman 96-square-well flat-bottom plates (aka. 'imaging plates')
- 25 Nunc™ 96-round-well round-bottom microwell plates (aka. 'culture' plates)

- 10 x 15mL Falcon tubes
- 2 x 50mL Erlenmeyer flasks

- 3 x 150mm Petri plates (aka. 'nursery plates')
- 3 x 90mm Petri plates (aka. 'maintenance plates')
SECTION: Seeding Petri plates + worm maintenance
9. Inoculate 10ml LB broth media with E. coli BW25113 (Keio background wild-type strain, used as negative control and for raising worms, no Kanamycin) in an Erlenmeyer flask for overnight culture following the protocol: 
 

SECTION: Inoculating from frozen stocks (96-well)
23. Fill all wells of the 10 culture plates with 200μL LB broth media. Add 50μg/ml Kanamycin to all wells except the wells for the wild-type BW25113 control strain. 
SECTION: Seeding imaging plates (96-well)
32. Remove the imaging plates from 4°C storage and record the average weight of 3 randomly selected plates
(Friday)
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
41. 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: Preparing NGM agar + pouring plates
2. Make 1L normal Nematode Growth Media (NGM) agar, following the protocol:
 

SECTION: Preparing NGM agar + pouring plates
4. Using the Integra ViaFill, dispense 200μL of NGM agar into each well of the 10 imaging plates, following the protocol: 
 

SECTION: Preparing NGM agar + pouring plates
5. Leave the plates on the lab bench (with lids on) until the agar has cooled and solidified (approximately 1 hour, timing depends on humidity)
SECTION: Preparing NGM agar + pouring plates
6. Measure the weight of 3 imaging plates (with lids on) and record average plate weight on day of pouring
SECTION: Preparing NGM agar + pouring plates
7. Dry the imaging plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on)
SECTION: Preparing NGM agar + pouring plates
8. Store the imaging plates upside-down at 4°C until used for experiments
SECTION: Preparing NGM agar + pouring plates
3. Pour 20ml NGM agar into each maintenance plate, and 50ml NGM agar into each nursery plate, following the protocol for Plate pouring (dx.doi.org/10.17504/protocols.io.6bhhaj6). 
Keep the remaining agar warm in a water bath set to 65°C, for dispensing into 96-well imaging plates afterwards
SECTION: Seeding Petri plates + worm maintenance
10. Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight
SECTION: Seeding Petri plates + worm maintenance
11. Remove the BW culture from the shaking incubator and place in 4°C fridge until seeding
SECTION: Seeding Petri plates + worm maintenance
12. Remove the plates from storage and the BW culture from the fridge, and leave on the bench for approximately 30 minutes to acclimate to room temperature
SECTION: Seeding Petri plates + worm maintenance
13. Using aseptic technique, seed the maintenance plates each with 400μL of BW25113 culture
SECTION: Adding antioxidants (96-well)
37. On the day of tracking, remove the seeded imaging plates from 4°C, and dry for 30 minutes under a hood
(Tuesday)
SECTION: Seeding Petri plates + worm maintenance
14. Leave under hood until dry (with lids on, timing depends on humidity)
SECTION: Seeding Petri plates + worm maintenance
15. Using a platinum pick, gently pick 30 adult N2 Bristol C. elegans onto each maintenance plate, and store in an incubator at 20°C (Monday)
SECTION: Seeding Petri plates + worm maintenance
16. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae (Tuesday)
SECTION: Seeding Petri plates + worm maintenance
17. Inoculate a further 10ml LB broth media with BW25113 bacteria for overnight culture, following the protocol in   and place in a shaking incubator at 37°C, 200 rpm (Wednesday)

SECTION: Seeding Petri plates + worm maintenance
18. After 24 hours, remove the culture from the incubator, and the nursery plates from storage, and leave to acclimate on bench top for 30 minutes (Thursday)
SECTION: Seeding Petri plates + worm maintenance
19. Seed the nursery plates each with 1mL of fresh BW25113 culture. Leave under hood until dry
SECTION: Seeding Petri plates + worm maintenance
20. Wash the worms off the BW-seeded maintenance plates, into two 15ml Falcon tubes (Friday)
SECTION: Seeding Petri plates + worm maintenance
21. Perform an egg prep on worms in the Falcon tubes, following the protocol: 
 

SECTION: Seeding Petri plates + worm maintenance
22. At around noon the next day, wash L1 larvae off the empty plate and re-feed onto the BW-seeded nursery plates using a glass Pasteur pipette. Aim to dispense around 1000 worms per plate. Incubate at 20°C (Saturday)    
SECTION: Inoculating from frozen stocks (96-well)
24. Remove the required stock plates from -80°C containing the strains for antioxidant testing. Gently remove the aluminium film and leave to partially thaw for a minute or so
 

SECTION: Inoculating from frozen stocks (96-well)
25. Inoculate individual vials, containing 4mL LB broth and 50μg/ml Kanamycin, with the desired strains for antioxidant testing from Keio frozen stock plates, following the protocol:
(Tuesday) 
 

SECTION: Inoculating from frozen stocks (96-well)
30. Optional: Make a template stock plate for -80°C storage with the strain layout desired: mix 200μL culture with 15% glycerol in each well

SECTION: Inoculating from frozen stocks (96-well)
31. Remove the overnight cultures from the incubator. Again, fill the wells of 10 culture plates with 200μL LB broth. But this time do not add Kanamycin. Inoculate the second round of overnight cultures from the first, using a 96-pin replicator, following the protocol: 
(Thursday)
 

SECTION: Seeding imaging plates (96-well)
33. Ensure that imaging plates have lost approximately 3-5% of their original weight. Place under a hood or drying cabinet until they have
SECTION: Seeding imaging plates (96-well)
34. Remove overnight cultures of Keio strains from 4°C storage. Using the Integra ViaFlo, seed 10μl of bacterial culture from the wells of each overnight culture plate into the corresponding wells of each imaging plate
 

SECTION: Seeding imaging plates (96-well)
35. Place seeded plates under a hood to dry for 20 minutes, then place in an incubator at 25°C (no shaking) for 7 hours 40 minutes (total lawn growth time: 8 hours)
SECTION: Seeding imaging plates (96-well)
36. After 8 hours, remove the plates from the incubator and store at 4°C
SECTION: Adding antioxidants (96-well)
40. Leave to dry under a hood for a further 30 minutes
SECTION: Adding antioxidants (96-well)
39. Using the Integra ViaFlo, dispense 7μL of antioxidant solution from the prepared treatment plate into each well of 10 imaging plates
 

SECTION: COPAS worm-sorting + Hydra tracking (96-well)
42. Remove the nursery plates from the incubator. Wash the worms off the plates into two 15ml Falcon tubes using approximately 10mL sterile PBS 'A' buffer
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
43. Fill up the tubes to 15ml with PBS 'A' and centrifuge at 1000rpm for 2 minutes
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
44. Remove the supernatant using a Pasteur pipette
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
45. Repeat steps    to    four more times to thoroughly rinse off any remaining control BW25113 bacteria
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
46. Re-suspend the worms and divide them equally into two 50ml Falcon tubes (for the COPAS), and fill them both up to approximately 40ml with PBS 'A'
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
47. Use the COPAS to dispense three Day1 adult worms into each well of the 10 imaging plates, following the protocol: 
 

SECTION: COPAS worm-sorting + Hydra tracking (96-well)
48. Leave the plates to dry under a hood for 30 minutes to 1 hour (until dry, timing depends on humidity), then place in incubator at 20°C until tracking (at +4 hours on food)
 

SECTION: COPAS worm-sorting + Hydra tracking (96-well)
49. 30 minutes prior to tracking with the Hydra rig (every 20 minutes, 2 runs in total), remove 5 imaging plates from the 20°C incubator and leave to acclimate in the imaging cave.
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
50. Record worm behaviour on the bacterial food for 15 minutes at the 4-hour timepoint 
(25 fps, exposure: 25000 msec, blue-light stimulation)
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
51. After tracking, discard the plates in a biological waste bin
SECTION: COPAS worm-sorting + Hydra tracking (96-well)
52. Check tracking checklist to ensure that all videos have been saved correctly: '/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'
SECTION: Inoculating from frozen stocks (96-well)
28. Using a multi-pipette, fill all wells of 10 x 96-well culture plates with 200μL LB broth. Fill all wells with 50μg/mL Kanamycin, except for wells designated for BW control strain.
(Wednesday)
SECTION: Inoculating from frozen stocks (96-well)
27. Also inoculate 10mL LB broth media in an Erlenmeyer flask with BW control, and place in a shaking incubator overnight at 37°C, 200 rpm
SECTION: Inoculating from frozen stocks (96-well)
26. Wet some tissue with MilliQ water, wrap the culture plates in the tissue, and incubate overnight at 37°C (no shaking)
 

SECTION: Inoculating from frozen stocks (96-well)
29. Remove the overnight cultures from the incubators. Using a sterile pipette tip (or inoculation loop), inoculate the wells of the culture plates with strains from the overnight culture vials, as per the desired plate layout. Inoculate the wells with no Kanamycin with the BW control strain. Incubate cultures overnight at 37°C.
SECTION: Adding antioxidants (96-well)
38. Remove the antioxidants from 4°C. Prepare an empty 96-well plate with 100μL of 10μg/mL antioxidant (in PBS 'A' buffer) in each well as per the desired experiment design layout for antioxidant treatment.