Can someone please advise the best protocol to heat kill OP50 while maintaining the bacteria's integrity as a food source? Also, for how long can heat killed Ecoli be stored at 4 degrees celsius? Thanks!
Hi!
We inactivate E.coli pellets (made in 50 mL falcon tube by spinning down E.coli suspension at 4000xg for 30 minutes and discarding the supernatant) with 2 freeze thaw cycles, using liquid nitrogen and a water bath set to 70C. This works most of the time. We use these inactivated pellets for feeding worms in 96-well plates (E. coli suspension made up in S-media with Pen/Strep, Nystatin and cholesterol) or when testing drugs on NGM plates (made up in s-media complete, without cholesterol).
I hope that helps, good luck with your experiment! Let me know if something is unclear.
Best wishes,
Franzi
A few minutes on the UV box does it (determine the former based on the strength of the latter).
How do you control if the inactivation was successful? Do you inoculate LB-Medium at 37°C over night and check for growth or will it never work 100% and there will always be growth?
Freezing-thawing and heating are procedures that are sub-optimal for E. coli inactivation.
With heat treatment the protein denaturation causes stress phenotypes within the nematode after having eaten the OP50
For freezing-thawing, you actually puncture the membrane and removing nutrients from the OP50 batch. Inactivation is there however, you will not have the same consistency in nutrition quality.
SOLUTION: When we produced large batches of OP50 for our research, we happen to find the solution to inactivate OP50 while keeping the structure intact. Using a freeze-drying method reduces the viability drastically but it takes a long time to get the process under control.
As we got many requests if we could also make this for our fellow researchers, we are now supplying this to anyone in need of high quality freeze-dried OP50 for their experiments.
you can send a message to [email protected] for more information.
Good luck with your experiments!
Bas
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