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CRYOPRESERVATION:?

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 (-196^oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.

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