PFA treatment of OP50
Bonnie Evans
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.
Steps
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
Follow steps 1 to 12
In the fume hood, add 16% PFA stock to the culture to give a 0.5% concentration
Incubate culture for 2 hours at 37C 200rpm
Divide culture into an even number of 50mL Falcons with equal volume
Centrifuge for 20 minutes at 4000 rcf 4C
Pour off supernatant into a waste flask
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
