Hello
I made a glycerol stock for my E. coli, but after 3 months of storage at -80°C, I took some to regrow and got zero bacterial growth. I'm not sure why this happened.
We used pure 100% glycerol and diluted it with water to make a 10% solution. I took 500 μL of this solution and mixed it with 500 μL of liquid bacterial culture to make a total volume of 1000 μL. I'm not sure if my steps were correct.
How to make glycerol stock for E. coli?
To make glycerol stock for E. coli, you will need the following materials:
- A culture of E. coli in liquid medium (such as LB broth) that has been grown overnight at 37°C with shaking
- 50% (v/v) glycerol solution (sterile)
- Cryogenic vials or screw-cap tubes (sterile)
- A freezer (-80°C)
The steps are as follows:
1. Label the vials or tubes with the name and date of the culture.
2. Transfer 1 ml of the culture into each vial or tube using a sterile pipette.
3. Add 1 ml of 50% glycerol solution to each vial or tube and mix well by vortexing or shaking.
4. Seal the vials or tubes tightly and store them in the freezer (-80°C).
5. To revive the culture, thaw a vial or tube at room temperature and inoculate a fresh liquid medium with a small amount of the glycerol stock. Grow the culture at 37°C with shaking until it reaches the desired optical density.
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