1. Prepare a 30% (v/v) sterile glycerol solution in Alsevers solution.
2. Draw approximately 0.5 mL of anticoagulant solution (Yaegers or heparin, etc.) into a syringe and move it back and forth over the length of the syringe, several times.xa0 Remove all air bubbles.xa0 Draw blood by cardiac puncture into the syringe from a host animal that has reached or is near peak parasitemia.xa0 If clotting occurs during extraction of blood, insufficient heparin was used.xa0xa0
3. Mix the heparinized blood with the 30% glycerol solution in a 2:1 ratio.xa0 If any clotting has occurred do not use.xa0 After mixing, the final concentration of cryoprotectant solution will be 10% (v/v).xa0 The mixture should be placed in a 4°C ice bath.xa0 The time from the mixing of the cell preparation and glycerol stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
4. Dispense in 0.5 mL aliquots into 1.0 mL toxa02.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).xa0 Filled ampules should be placed in a 4°C ice bath.xa0 Do not immerse ampules to the level of the vial cap.
5. Plunge ampules from 4°C into liquid nitrogen.xa0 The frozen preparations may be stored in a mechanical freezer until needed, however storage in either the vapor or liquid phase of a nitrogen refrigerator is recommended for the longest viability.
6. To thaw a frozen ampule, place in a 35°C water bath, until thawed (2 to 3 min).xa0 Immerse the ampule just sufficient to cover the frozen material.xa0 Do not agitate the ampule.
7. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected hamster.xa0 Follow the protocol for maintenance in vivo.xa0 The course of infection may be longer or shorter than usual depending on percent recovery of the parasite from the frozen state.

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M Eberhard, personal communication

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