Only young cells (rings) can be frozen in glycerolyte medium** because their membranes are more robust.
1. Centrifuge ring-stage culture for 5 mins. at 1000 x g 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 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 screw-capped freezing vials and place in a Nalgene 1C cooling apparatus. Place the apparatus at -80°C overnight and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.).
6. Plunge vials into liquid nitrogen (-196oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.