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# Goal/Experiment:
The goal of this experiment is to screen 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.

# Antioxidant Rescue of *C. elegans* Behaviour on Keio E. coli Mutants (6-Well Plates)

DOI: [dx.doi.org/10.17504/protocols.io.j8nlkw5kdl5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkw5kdl5r/v1)

Author: Saul Moore  
Imperial College London, MRC London Institute of Medical Sciences (LMS)

## Abstract
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.

## Disclaimer
**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK**

## License
This is an open access protocol distributed under the terms of the Creative Commons Attribution License.

## Materials
- 6-well flat bottom plates ('imaging plates')
- 60mm Petri plates ('maintenance plates')
- 90mm Petri plates ('nursery plates')
- 50mL Erlenmeyer flasks

## Reagents
- 500mL LB broth
- 1L NGM agar (for ingredients, see protocol for making NGM agar)

## Preparing NGM Agar + Pouring Plates
1. Prior to screening, prepare the materials needed for screening *C. elegans* on selected Keio *E. coli* mutants:
    - 6-well plates (imaging plates)
    - 15 mL Falcon tubes
    - 50 mL Erlenmeyer flasks
    - 90 mm Petri plates (maintenance plates)
    - 150 mm Petri plates (nursery plates)
2. Make 1L normal Nematode Growth Media (NGM) agar, following the protocol: [Making normal NGM for imaging plates (Cabreiro Lab)](dx.doi.org/10.17504/protocols.io.6bhhaj6)
3. Pour 15 mL NGM agar into each 60 mm maintenance plate, and 35 mL NGM agar into each 90 mm nursery plate, following the protocol for Plate pouring [here](dx.doi.org/10.17504/protocols.io.6bhhaj6). Keep the remaining agar warm in a water bath set to 65°C, for pouring into 6-well imaging plates afterwards.
4. Using the Integra ViaFill, dispense 4 mL NGM agar into each well of the 6-well plates, following the protocol: [Dispensing agar into multiwell plates](dx.doi.org/10.17504/protocols.io.8g2ww7e)
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).

## Preparing Worms
9. Inoculate 10 mL 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: [Inoculating a Liquid Bacterial Culture](dx.doi.org/10.17504/protocols.io.dd8i2h6)
10. Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight.
11. Remove the BW culture from the shaking incubator and place in 4°C fridge until seeding.
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.
13. Using aseptic technique, seed the 60 mm maintenance plates each with approximately 250 µL of BW25113 culture.
14. Leave under hood until dry (with lids on, timing depends on humidity).
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.
16. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae.
17. Inoculate a further 10 mL LB broth with BW25113 bacteria for overnight culture (no Kanamycin), following the protocol [here](dx.doi.org/10.17504/protocols.io.5dbp2i6) and place in a shaking incubator at 37°C, 200 rpm.
18. After 24 hours, remove the culture from the incubator, and the 90 mm nursery plates from storage, and leave to acclimate on bench top for 30 minutes.
19. Seed the nursery plates each with approximately 1 mL of fresh BW25113 culture. Leave under hood until dry.
20. Wash the worms off the BW-seeded maintenance plates, into two 15 mL Falcon tubes.
21. Perform an egg prep on worms in the Falcon tubes, following the protocol: [Egg Prep for Bleach Synchronization (Cabreiro Lab)](dx.doi.org/10.17504/protocols.io.4h4gp3w)

## Preparing Bacteria
23. Fill 2 separate Erlenmeyer flasks with 25 mL LB. Add 50 µg/mL Kanamycin to one flask, and leave the other flask without Kanamycin for the BW25113 control.
24. Remove the required Keio frozen 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. 
    > **Safety Information**: To avoid damaging the bacterial stocks through repeated freeze-thawing, do not let the wells completely defrost. Just enough to be able to pick up some cells with the replicator.
25. Inoculate the Erlenmeyer flasks with the desired strains for antioxidant testing from Keio frozen stock plates, following the protocol: [Inoculating a Liquid Bacterial Culture](dx.doi.org/10.17504/protocols.io.dd8i2h6)
26. Incubate the cultures overnight at 37°C in a shaking incubator at 200 rpm.
27. Remove the overnight cultures from the incubator. Inoculate 2 more Erlenmeyer flasks for a second round of overnight cultures from the first, this time without Kanamycin (to avoid exposing the worms to the antibiotics), and incubate overnight at 37°C at 200 rpm.
28. After 24 hours, remove the cultures from the incubator and store at 4°C until used for experiments.

## Seeding Imaging Plates (6-Well)
29. Remove the imaging plates from 4°C storage.
30. Ensure that imaging plates have lost approximately 3-5% of their original weight (so that they are not too wet for imaging when seeded). Place under a hood or drying cabinet until they have.
31. Remove overnight cultures of Keio strains from 4°C storage. Using a pipette, seed 30 µL of bacterial culture into the wells of each 6-well imaging plate.
32. Place the seeded plates under a laminar flow 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).
33. After 8 hours, remove the plates from the incubator and store at 4°C.

## Adding Antioxidants (6-Well)
34. On the day of tracking, remove the seeded imaging plates from 4°C, and dry for 30 minutes under a laminar flow hood.
35. Remove the antioxidants from 4°C. Prepare 100 mM NAC or Vitamin C (in H2O).
36. Using a pipette, dispense 40 µL of antioxidant solution into each desired well of the 6-well imaging plates (for a final concentration of 1 mM in 4 mL agar).
37. Leave the plates to dry under a hood for a further 30 minutes. Record the weight of the plates after drying (as weight at imaging).

## Picking Worms + Hydra Tracking (6-Well)
38. 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).
39. Remove the nursery plates from the incubator.
40. Using a platinum worm pick, carefully pick 10 Day1 worms onto the edge of the lawns in each well of the 6-well imaging plates, then place in incubator at 20°C until tracking (at +4 hours on food).
41. 30 minutes prior to tracking with the Hydra rig (each run is performed every 20-30 minutes), remove 5 imaging plates from the 20°C incubator and leave to acclimate in the imaging cave.
42. Record worm behaviour on the bacterial food for 15 minutes at the 4-hour timepoint (25 fps, exposure: 25000 msec, blue-light stimulation).

## Post-Tracking
43. After tracking, discard the plates in a biological waste bin.
44. Check tracking checklist to ensure that all videos have been saved correctly: `'/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'`

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