Sep 25, 2025
Bacterial glycerol stocks
- Jordan Ward1
- 1UC, Santa Cruz
Protocol Citation: Jordan Ward 2025. Bacterial glycerol stocks. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zrkkgqe/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: Working
We use this protocol and it's working
Created: September 24, 2025
Last Modified: September 25, 2025
Protocol Integer ID: 228096
Keywords: bacterial glycerol stocks ward lab protocol
Funders Acknowledgements:
NIH
Grant ID: R35GM158317
Abstract
Ward lab protocol for making bacterial glycerol stocks
Troubleshooting
Start with bacteria struck for isolation on an LB+agar plate with the appropriate antibiotic (if strain carries an antibiotic resistance marker).
Pick a single, isolated colony into 5 ml of LB with the appropriate antibiotic. Shake 16 hours at 37ºC.
Ensure that sterile 60% glycerol is prepared. Mix 300 ml of 100% glycerol with 200 ml of milliQ water. We typically do this in a 1 L graduated cylinder with a small stir bar added. Mix on a magnetic stir plate. Sterilize by filtering through a Millipore Stericup vacuum filter, or similar vacuum filtration system. Alternatively, transferring to a 500 ml bottle and autoclaving on a liquid cycle also works.
In a sterile cryovial, add 500 µl of sterile 60% glycerol and 500 µl of the overnight culture. Label the tube and number it. We maintain a database of glycerol stocks where each tube has a unique numeric identifier. Store tube at -80ºC.