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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 (0.1-0.2 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.     
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:
   
   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.

Boyer JM, et al. Lipid composition and activity of a lytic factor isolated from Plasmodium berghei. Infect. Immun. 25: 805-809, 1979. PubMed: 387594

D'Antonio LE, et al. Malaria vaccine antigen(s): detergent solubilization, partial isolation, and recovery of immunoprotective activity. Infect. Immun. 43: 442-444, 1984. PubMed: 6360908

Alger NE, et al. Plasmodium berghei NK65 in the inbred A-J mouse: variations in virulence of P. berghei demes. J. Protozool. 18: 598-601, 1971. PubMed: 5133123

Alger NE, Keshavarz-Valian H. Plasmodium berghei: the effects of suppressor factor on vaccination. Int. J. Parasitol. 14: 301-307, 1984. PubMed: 6381347

D'Antonio LE, et al. Model system for study of artificially induced resistance to malaria. Nature 223: 507-509, 1969. PubMed: 5796953

D'Antonio LE, et al. Malaria resistance: artificial induction with a partially purified plasmodial fraction. Science 168: 1117-1118, 1970. PubMed: 5441687

D'Antonio LE. Plasmodium: a resume of the isolation of the vaccine fraction by the French pressure cell technique. Exp. Parasitol. 31: 75-81, 1972. PubMed: 5010607