Glycerolstocks How to Make Glycerol Stocks of Bacteria

Once you have cells that contain a plasmid or genomic insertion that you want to preserve for years, you want to make a glycerol stock of those cells. E. coli can survive at -80 degrees for years if you prepare the cells properly. What kills cells upon freezing is the formation of ice crystals rupturing the plasma membrane. Therefore, we add glycerol to prevent ice from forming.

Bacteria on plates stored at 4 degrees survive for about 1 month. Bacteria in liquid cultures survive about 2 days on the bench and about 1 week at 4 degrees. Plasmids degrade rapidly in cells left in liquid culture.

1. Grow a fresh overnight culture of 2 mL for each frozen stock you want to make. You can grow 3 mL and freeze down duplicate tubes.
2. Get a Nunc cryotube, cap, and colored cap insert. Write the part number on the color cap insert. Print a label and attach it to the Nunc cryotube. This should be done for each tube you intend to freeze.
   
3. Microwave the sterile glyceroal for 30 seconds. Do not stir it when completed. You want to pipet from the top, hot, less viscous glycerol.
4. While in the microwave, take a fresh razor blade and cut off the tip of a yellow pipet tip. You goal is to make a larger opening since glycerol is so viscous.
5. Pipet 150 µL of hot glycerol into each of the pre-labeled Nunc cryotubes.
6. Allow about 1 minute for the glycerol to cool. Then add 850 µL of the overnight bacterial culture. Put the cap on and shake VERY vigorously, or vortex vigorously. You need to mix the glycerol evenly and it is very viscous. Final glycerol concentration is now 15%.
7. As soon as you can, put the tube in one of the numbered freezer boxes and write down each tube's specific location (Box number, and position within the box).
8. Enter the information for each frozen tube into our online freezer stock database. It is VERY IMPORTANT to enter each tube into the database, even duplicates. We have too many tubes now and they are hard to locate.
   
9. When you want to retrieve some cells from the glycerol stocks, do not let them thaw. Use a sterile toothpick and scrape off some of the ice and put this onto your plate or into a liquid culture.