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CRYOPRESERVATION:?

Only young cells (rings) can be frozen in glycerolyte medium* because their membranes are more robust.

1.?? To harvest parasites, inject host with ketamine (0.1?0.2 ml).

2.?? Open chest cavity to expose heart and exsanguinate via cardiac puncture using Yaeger's anticoagulant** (see below), 1 volume anticoagulant to 4 volumes blood.???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Glass distilled HGla?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????

3.?? Centrifuge blood for 5 mins. at 1800 rpm in 50 ml centrifuge tube.

4.?? Aspirate supernatant using sterile Pasteur pipet.

5.?? Resuspend pellet gently in remaining supernatant.

6.?? 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.

7.?? Aliquot mixture into Nunc screw-capped freezing vials and place in a Nalgene 1°C 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.).

8.?? Plunge vials into liquid nitrogen (-196^oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.

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