How to make Bacterial Glycerol Stocks (protocol)

  Bacterial glycerol stocks are incredibly important, if you’re planning on storing plasmids for an extended period of time. This way, when you want to make more plasmid DNA, the plasmid will already be in your desired bacterial strain and you will not need to obtain more competent cells and retransform.

  Bacteria on an LB agar plate can normally be stored for a few weeks in the fridge. Storing the bacteria in a tube and adding glycerol before freezing will stabilize the bacteria, preventing damage to the cell membrane and keeping cells alive. This allows you to store a glycerol stock at -80°C for many years.

To get started, you’ll need to grow your cultures overnight.

Materials Required:

  •  2 ml screw cap or cryovial
  • 50% glycerol Liquid Culture with Bacterial Growth
  • 1000μL pipette and tips
  • 70% isopropanol
  • Sterile loop or autoclaved toothpicks
  • Dry Ice And
  • LB Agar Plate

Procedure :

First, you will need to prepare your 50% glycerol solution solution. You can do this by diluting 50 ml glycerol into 100 ml Distilled water.

  • Label the tubes by using a marker and writing directly onto the tube.
  • (Note - It is important that you do this before freezing, because frozen tubes are difficult to write on, and labels can easily unstick from tubes at low temperatures.) Snap top tubes are not recommended, because they can open unexpectedly at very cold temperatures.
  • Once you have bacterial culture and your diluted glycerol solution, add 500 μl 50% glycerol and culture to 500 μl of the overnight to a 2ml  screw top tube, or cryovial. This gives you a final glycerol concentration of 25% for your glycerol stock.
  • Shake the tube five to six times to thoroughly mix the glycerol with the bacterial culture. Once there are no layers present and you see one uniform solution, the tube is ready to be frozen.
  • Place the glycerol stock tube in a freezer at -80°C.
  • Your stock is now stable for years, as long as it is kept at -80°C.
  • Keep in mind that multiple freeze and thaw cycles will greatly reduce the shelf life of your stock.

Recovering bacteria from your glycerol stock :

To recover your bacteria, remove the stock tube from the freezer, and open it.

  • Then, use a toothpick, sterile loop or pipettetip to scrape some of the frozen bacteria off the top.
  • Do not let the glycerol stock thaw! Place it on dry ice whilestreaking to prevent the stock from completely thawing, preserving its shelf life.
  • Now, streak the bacteria onto an LB agar plate.
  • Grow your bacteria overnight at the appropriate temperature. You can check growth conditions, includingcopy number and growth temperature, on your plasmid’s information page. The next day you will be able to start anovernight culture for plasmid DNA preparation.