Jan 22, 2025

Public workspaceSoil bacteria

DOI
dx.doi.org/10.17504/protocols.io.14egn9j7pl5d/v1
  • 1MRC Laboratory of Medical Sciences, Imperial College London
  • Behavioural Genomics
John Bergqvist
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DOI: dx.doi.org/10.17504/protocols.io.14egn9j7pl5d/v1
Protocol Citation: John Bergqvist 2025. Soil bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9j7pl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 21, 2025
Last Modified: January 22, 2025
Protocol Integer ID: 118775
Keywords: C. elegans, behaviour, soil bacteria, high-throughput
Abstract
The behaviour of C. elegans is dependent on its microbial environment. This protocol describes how to utilise the Tierpsy Tracking software to extract the unique behaviours of C. elegans upon being subjected to 96 different soil bacteria.

We expect varied behaviours driven by the soil bacteria identity.
Materials
Microbes
Soil bacteria
E. coli (OP50)

Materials
100 mm petri dishes
150 mm petri dishes
500 mL bottle
100 mL bottle
15 mL falcon tubes
Pasteur pipettes
Glass pipettes
Round-bottom 96-well plates
Square, flat-bottom 96-well plates
96-peg replicator
Breathe-Easy sealing membrane for 96-well plates
Aluminium plate sealing film for 96-well plates
Ice
Glass slides

Reagents
Pre- & post autoclave reagents for making Nematode Growth Medium (NGM).
M9 medium
5% Sodium hypochlorite
Sterile water
1M NaOH solution
LB broth
LB agar
99% glycerol

Equipment
Integra ViaFill dispenser
Integra ViaFill dispenser tubes (Large)
Integra ViaFlo 96
Integra ViaFlo 96 12.5 µL GRIPTIP pipette tips
15 mL falcon tube benchtop spinner
Scale
Bunsen burner (or Class II Microbiological Safety cabinet)
Autoclave
Tecan Spark Multimode Microplate Reader
LoopBio Motif - Video Recording System (6x12 megapixel camera array setup)

Before start
Ensure you grow enough worms to fill the volume required in the bottle to dispense the worms using the Integra ViaFill.
Day -10. Prepare worm population
Day -10. Prepare worm population
20m
20m
Pick 10 L4 worms onto 5x 100 mm normal NGM petri dishes seeded with 1500 mL of OP50 (OD = 1.0).
Put the petri dishes in the 20°C incubator to grow the worm population for bleaching (Day -6).
20m
Day -08. Prepare 96-well plates with normal NGM
Day -08. Prepare 96-well plates with normal NGM
4h
4h
Prepare cholesterol for use in making normal Nematode Growth Medium (NGM) following the protocol below:

Protocol
Preparing Cholesterol for use in NGM
NAME
Preparing Cholesterol for use in NGM
CREATED BY
Bonnie Evans

40m
Prepare 500 mL of normal NGM following the protocol below:
Protocol
Making normal NGM
NAME

Making normal NGM

CREATED BY
Bonnie Evans

3h
Dispense 200 µL of autoclaved normal NGM into 24x 96-well plates using the Integra ViaFill according to the protocol below:

Protocol
Dispensing agar into multiwell plates
NAME
Dispensing agar into multiwell plates
CREATED BY
Bonnie Evans


20m
Store the 96-well plates upside-down at 4°C until day of use (Day -01).
Day -06. Bleach worms
Day -06. Bleach worms
2h 30m
2h 30m
Bleach the adult worm population according to the following protocol:
Protocol
Bleach synchronisation of C. elegans
NAME
Bleach synchronisation of C. elegans
CREATED BY
Ida Barlow

2h 30m
Leave the worms spinning in M9 medium for two days, synchronising the worms to L1.
Day -05. Seed 150 mm plates with OP50
Day -05. Seed 150 mm plates with OP50
1h
1h
Add 2 mL of OP50 (OD=1.0) to 5x 150 mm normal NGM petri dishes. Leave plates without lid in safety cabinet until bacterial culture has dried.
1h
Day -04. Re-feed the L1-synchronised larvae
Day -04. Re-feed the L1-synchronised larvae
30m
30m
Using a glass pipette, add 4-5 drops of larvae to each of the 150 mm petri dishes. Leave plates in 20°C incubator until adding worms to 96-well plates (Day -1).
30m
Day -02. Dry 96-well plates
Day -02. Dry 96-well plates
2h 5m
2h 5m
Measure the weight of 3 plates (with lids on) and record average plate weight on day of pouring.
5m
Dry the plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on)
2h
Day -02. Make O/N cultures
Day -02. Make O/N cultures
2h 30m
2h 30m
Dispense 200 µL of LB broth into four round bottom 96-well plates.

  • Work near a bunsen burner or work in a Class II Microbiological safety cabinet.
15m
Take the 96-well plate of soil bacteria out of the -80°C freezer and place the plate in a bucket of ice.
Step case

O/N cultures for experiment
22 steps

Let the plate of soil bacteria thaw in the bucket of ice.
15m
Dip a 96-peg replicator in the thawed soil bacteria plate, swirl around in the wells, and put the pegs in LB-filled 96-well plate and swirl around. Carefully note the orientation of the soil bacteria plate and the LB-filled plate to ensure the well position is the same when adding the bacteria.

Repeat for three of the LB-filled round-bottom 96-well plates.

  • Work near a bunsen burner or work in a Class II Microbiological safety cabinet.
10m
Dispense 50 µL of OP50 (OD=1.0 in LB) into half of 96-well plate. Dip a 96-peg replicator in the OP50-filled 96-well plate followed by putting the pegs in the last of the four LB-filled round bottom 96-well plate.
10m
Add Breathe-Easy sealing membrane over each plate.
5m
Place the plates in a shaking incubator at 200 rpm at 28°C DurationOvernight .

Day -01. Add bacteria to square, normal NGM 96-well plates
Day -01. Add bacteria to square, normal NGM 96-well plates
3h 55m
3h 55m
Take the square, normal NGM 96-well plates out of the 4°C storage and spread them out on the bench, upside-down, to adapt them to room temperature.
1h 30m
Use the Tecan Spark Multimode Microplate Reader to read the optical density of the soil bacteria plates and the OP50 plate. Ensure all wells have bacterial growth, comparing the OD of the part of the plate with no bacterial innoculation.

  • 20s shaking (orbital, 180 rpm) prior to reading the optical density.
20m
Replace 100 µL of the bacterial cultures in all plates with 100 µL, diluting the cultures 1:1.
30m
Read the optical density of the plates after diluting the bacterial cultures.

  • 20s shaking (orbital, 180 rpm) prior to reading the optical density.
20m
Use the 96-tip Integra ViaFlo to pipette 10 µl of each inividual bacterial culture into their corresponding wells in the square, normal NGM 96-well plates.
30m
Place the bacteria-added square 96-well plates in a Class II Microbiological Safety Cabinet without lids for ~30-45 min to dry the wells.
45m
Day -01. Add worms to square, normal NGM 96-well plates with bacteria
Day -01. Add worms to square, normal NGM 96-well plates with bacteria
7h 10m
7h 10m
Check that worms are egg-laying (D1 adults). Wash the worms off the plates with M9 using pasteur pipettes into 15 mL Falcon tubes.
10m
Centrifuge for 2 min at 1500 rpm (RCF: 210, ascending 9; descending 7) - program 1.
2m
Replace the bacteria supernatant with new M9.
1m
Repeat step 27 and 28 two more times.
6m
After the final centrifugation, remove the supernatant and replace with new 8 mL of supernatant. Pour the worms into a 100 mL autoclaved bottle.
3m
Add another 8 mL of M9 to the tubes and transfer to the 100 mL bottle.
3m
Check the number of worms in 10 µL drop on a small glass slide. Adjust the volume of M9 through additional spinning and adding of new M9 to reach an average of between 10-20 worms per 10 µL drop.

  • The volume of M9 in the bottle where a drop of 10 µL has between 10-20 worms will determine the number of plates that can be done in the screen.

  • Volume required to prime the ViaFill tubes: 5 mL
  • Min. volume required to not intriduce bubbles in the tubes: 20 mL
  • Volume required per plate: 10 µL x 96 wells x 1 plate + 5 mL + 20 mL min. volume= 25.96 mL
  • Volume required for 20 plates: 10 µL x 96 wells x 20 plates + 5 mL + 20 mL min. volume= 44.2 mL

30m
While stirring Use the Integra ViaFill dispenser with LARGE tubing to dispense 10 µL drops of worms to each well of the square, bacteria-added 96-well plates once the bacterial culture has dried.

  • Check that worms have been added to the wells. Blockage can easily occur.
15m
Leave the worm-filled plates in a Class II Microbiological Safety Cabinet until wells are dry (~6h)

  • Wells closer to the edge dry quicker.
  • Check every 2 hours for dry wells and cover the wells along the edges with masking tape if needed to avoid agar cracking and reducing in size.
6h
Once dry, leave the paltes in the 20°C incubator DurationOvernight

Day 0. Record the plates at 24h after addition of worms
Day 0. Record the plates at 24h after addition of worms
1h
1h
At 24h after dispensing the worms into the wells of the square, bacteria-added 96-well plates, record the plates at 25 frames/second with a script that records:
  1. 5 min of pre-stimulation.
  2. 6 min of blue-light stimulation (three 10s bluelight stimulations spaced 100s apart).
  3. 5 min of post-stimulation.
1h