This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Bacterial Glycerol Stock.html
Bacterial Glycerol Stock


  1. Agar plate with isolated colonies
  2. LB media
  3. Glycerol
  4. Sterile flasks with ventilating caps or 50 ml tubes
  5. Screw-cap freezer vials
  6. Shaker incubator
  7. 5mL serological pipettes
  8. Micropipette and sterile tips
  9. Ethanol-dry ice bath
  10. -80oC freezer


  1. Fill 5mL media each into the flask/tube, aseptically.
  2. Add antibiotics if needed.
  3. Pick a single colony from agar plate and disperse into the media.
  4. Grow overnight culture of bacteria at 37oC and 300rpm.
  5. Add sterile glycerol (autoclaved) to 15% v/v final.
  6. Vortex vigoriously and dispense 1ml aloiquots in screw-cap freezer vials.
  7. Quick freeze on Ethanol-dry ice bath.
  8. Transfer to -80oC freezer for long term storage.