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

1. To harvest parasites, inject host with ketamine (0.5-1.0 ml 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 pipette.
5. Resuspend pellet gently in remaining supernatant.
6. Slowly add 5 volumes of glycerolyte medium to 3 volumes pellet drop wise via a syringe as follows:
   1. Add the first volume of glycerolyte and allow the tube to stand for 5 mins. at room temperature.
   2. 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^°C) the next day and store in liquid nitrogen or liquid nitrogen vapor.

Coatney GR. The primate malarias. Washington, DC: Government Printing Office; 1971.

Collins WE, et al. Studies of comparative infectivity of fifteen strains of Plasmodium vivax to laboratory-reared anopheline mosquitoes, with special reference to Anopheles culicifacies. J. Parasitol. 72: 521-524, 1986. PubMed: 3537255