Jan 17, 2024
PFA treatment of OP50
- 1MRC LMS, Imperial College London
Protocol Citation: Bonnie Evans 2024. PFA treatment of OP50. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyq71vpk/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: February 08, 2023
Last Modified: January 17, 2024
Protocol Integer ID: 76681
Abstract
Paraformaldehyde (PFA), a polymer of formaldehyde, is an organic solution that permeabilizes bacterial cells making them no longer viable/metabolically active. However, PFA does not cause the lysis of inner cell structure, meaning the bacteria are still edible for the worms. Adapted from Beydoun et al. 2021.
Materials
Paraformaldehyde Solution, 16% (15799150) - stored in safety cabinet
PFA solution
PFA solution
In the fume hood, break off the head of the ampoule of 16% paraformaldehyde (PFA)
Using a plastic Pasteur pipette, decant the solution into a brown glass bottle. Dispose of Pasteur pipette in Biobin in fume hood.
Leave the ampoule on the plastic tray in the fume hood to allow any remaining PFA to evaporate. Dispose of in sharps bin.
Store the PFA stock solution double contained at 4°C
PFA treatment
PFA treatment
Follow steps 1 to 12
Protocol
NAME
Growing overnight culture of OP50 as worm food
CREATED BY
Bonnie Evans
In the fume hood, add 16% PFA stock to the culture to give a 0.5% concentration
Safety information
If a spill occurs within the fume hood, the spill can be considered minor and can be cleaned up using paper towels. Clean any contaminated surface with cold water at least two times. Place the contaminated paper towels into a sealable bag for disposal as hazardous waste.
Incubate culture for 2 hours at 37C 200rpm
Divide culture into an even number of 50mL Falcons with equal volume
Note
It is important to use sterile technique to prevent the introduction of live bacteria into the culture
Centrifuge for 20 minutes at 4000 rcf 4C
Pour off supernatant into a waste flask
Safety information
Treat with Virkon when finished
Using a stripette, replace with an equal volume of M9 and re-suspend pellet
Repeat steps 6 to 8 twice more
Vortex Falcons to fully re-suspend pellet and combine solutions in a larger container (should be the same total volume as the original culture).
Measure OD600 using M9 as a blank
Using an inoculation loop, streak the PFA-treated culture on LB agar and incubate overnight at 37C to check for contamination/live bacteria
Store PFA culture at 4C
Once PFA killing has been confirmed as successful, aliquot into 50mL Falcons and store at 4C
Safety information
Dispose via hazardous waste