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 Yaegers 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.xa0 (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.

Branton MBStudies of clonal populations of NK65 Plasmodium berghei Ph.D. thesis, Univ. Illinois, 1978