Plasmodium relictum Grassi and Feletti

1. Place the frozen vial in a 37°C water bath until mixture is completely thawed.
2. Aseptically transfer the contents to a sterile syringe and inject into wing vein of host organism.
3. Make a smear if required (see below).

1. Aseptically transfer 0.05–0.1 mL of freshly-drawn blood into an eppendorf tube.

2. Spin down the eppendorf tube at high speed and aspirate the supernatant.

3. Mix the pellet and place a drop of the suspension on a glass slide. Spread the drop into a thin film with the edge of another glass slide. Air dry for 3 mins. at room temperature.

4. Fix air-dried blood film by rinsing with methyl alcohol. Air dry for 3 mins. at room temperature.

5. Stain blood films in 5% Giemsa solution for 15 mins. Rinse with distilled water, air dry.

6. Observe using light microscopy at 1000X magnification to determine parasitemia of culture.

1. To harvest parasites, inject host with ketamine (0.1–0.2 mL).
2. Exsanguinate via the wing vein using a sterile syringe and Yaeger's anticoagulant, 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.

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