Only young cells (rings) can be frozen in glycerolyte medium** because their membranes are more robust.
1. Centrifuge ring stage culture for 5 min at 1800rpm in 50 ml centrifuge tube.
2. Aspirate supernatant using sterile Pasteur pipet.
3. Resuspend pellet gently in remaining supernatant.
4. Slowly add 5 volumes of glycerolyte medium (see below) to 3 volumes pellet dropwise via a syringe as follows:
A. Add the first volume of glycerolyte and allow the tube to stand for 5 mins. at room temperature.
B. Add the remaining 4 volumes of glycerolyte and gently agitate.
5. Aliquot mixture into Nunc screwtop freezing vials and store at 80oC overnight.
6. Plunge vials into liquid nitrogen (-196oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.
** To formulate glycerolyte medium, combine the following with distilled water to 100 ml: 57.00g glycerol, 1.60g sodium lactate (C3H5NaO3), 30.00mg potassium chloride (KCl), 1.38g sodium dihydrogen phosphate (NaH2PO4). Mix well and adjust pH to 6.8 using concentrated NaOH and/or HCl. Autoclave to sterilize, and store at 4oC.