Only young cells (rings) can be frozen in glycerolyte medium* because their membranes are more robust.
1. To harvest parasites, inject host with ketamine (0.51.0ml 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.