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 (0.5?1.0ml depending on host size / weight).
2.???? Exsanguinate via the femoral vein using a sterile syringe and Yaeger's anticoagulant**, 1 volume anticoagulant to 4 volumes blood.? Note: Unless there is an exchange transfusion, no more than 10% of estimated total blood volume should be collected over a 2 wk. period.
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 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.).
8.???? Plunge vials into liquid nitrogen (-196oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.
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