Protocol 12

Protocol 12 : Glycerol storage

Aim: Safely store cells at -80°C

Following steps will be performed under a class II PSM

Glycerol Freezing Solution (50%)

Prepare a 50% glycerol stock solution by mixing 5ml glycerol with 5ml distilled, sterile water.
Mix well to make sure that you see one uniform solution, and there are no layers present.

Bacteria storage

Mix 50 µL of the overnight culture of bacteria of interest with 50 µL 50% glycerol solution (25% glycerol final concentration) into a 2 mL cryotube
NB : Snap top tubes are not recommended as they can open unexpectedly at -80°C
Label both the lid and the tube of the cryotube. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube. Store at -80°C

Bacteria recovery

Open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw.
Either streak the bacteria onto an LB agar plate (see culture medium preparation protocol) and grow your bacteria overnight at the appropriate culture condition. The next day you will be able to start an overnight culture for plasmid DNA prep the following day.
Or scrub a bit of bacteria cell and put it into a 500mL sterile Erlenmeyer containing 100mL of sterile liquid LB medium. Cover with aluminum paper and place the Erlenmeyer in a rotative incubator at 37 Celsius degrees, 120rpm.