Mar 04, 2024
Creating Bacterial Glycerol Stocks for Long-term Storage
- Bonnie Evans1,2
- 1MRC Laboratory of Medical Sciences;
- 2Imperial College London
- Behavioural Genomics
Protocol Citation: Bonnie Evans 2024. Creating Bacterial Glycerol Stocks for Long-term Storage. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdwr7lmk/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: March 04, 2024
Last Modified: March 04, 2024
Protocol Integer ID: 96114
Keywords: term storage bacterial glycerol stock, creating bacterial glycerol stock, glycerol stock of bacteria, bacterial glycerol stock, frozen bacteria, glycerol stock, bacteria on lb agar plate, glycerol, addition of glycerol, bacteria, term storage
Abstract
Bacterial glycerol stocks are important for long-term storage. Bacteria on LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years.
Troubleshooting
Safety warnings
- Snap top tubes are not recommended as they can open unexpectedly at -80°C
- Be sure to label both the lid and the tube of a glycerol stock before you place the sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube!
- Try not to freeze/thaw your glycerol stock too many times as this will reduce the shelf life. Placing the glycerol stock on dry ice while streaking onto LB agar will prevent it from thawing completely.
Before start
Make the 50% glycerol solution by diluting 100% glycerol in dH20 and autoclaving.
Steps
Grow an overnight culture of the selected strain
Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryotube and gently mix
Shake the glycerol 5-6 times before freezing.
Expected result
One uniform solution, no layers present
Freeze the glycerol stock tube at -80°C.
To recover bacteria from your glycerol stock, open the tube and use a sterile loop or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock thaw!