Freezing Bacterial Cells

Categorized: Microbiological Techniques

  1. Grow the strain of bacteria to be frozen, in 5 to 10 mls of appropriate media, until an OD600 = 0.6-0.8 is reached OR aseptically (using a sterile swab) scrape bacteria from a freshly grown (24 – 48 hr) plate and resuspend in 2 to 5 mls of a rich broth such as Mueller Hinton or LBNS.
  2. Using a sterile pipette, add dimethyl sulfoxide (DMSO) so the final concentration is 7% (i.e. 70ul DMSO per 1 ml of cell suspension). Note: DMSO is considered a sterile solution but can be filter sterilized using a 0.22 um nylon filter.
  3. Vortex.
  4. Aseptically pipet 1.0 to 1.5 mls of bacterial suspension (with DMSO) into each cryovial. Make sure the cryovial is labelled with a waterproof, cryogenic pen (black color lasts better in the freezer). Freeze at -70oC.