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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 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 ?80^oC overnight.

6.???? Plunge vials into liquid nitrogen (-196^oC) 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 4^oC.