Add ingredients in the sequence listed. Filter-sterilize.

1. Harvest the parasites from spleen tissue homogenized in a balanced salt solution (i.e., Tyrodes Salt soln. or similar), approximately 5.0 mL solution per spleen.
2. Transfer the cell homogenate to a 15 mL plastic centrifuge tube and spin at approximately 1300 x g for 10 min.
3. Pool the cell pellets and adjust the concentration to 2.0 - 4.0 x 10⁷ cells/mL with a fresh solution of Tyrodes Salt Solution. *If the concentration is too low centrifuge at 1300 x g for 10 min and resuspend in the volume of Tyrodes Salt Solution required to yield the desired concentration.
4. Mix the cell preparation and 10% (v/v) DMSO in equal portions. The final concentration will be 1.0 - 2.0 x 10⁷ cells/mL and 5% DMSO. The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.
5. Dispense in 0.5 mL aliquots to 1.0-2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)
7. Store in either the vapor or liquid phase of a nitrogen refrigerator.
8. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. Do not agitate the ampule. Do not leave ampule in water bath after thawed.
9. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected Golden hamster at least 8 weeks old. Follow the protocol for maintenance in vivo.

Stauber LA. Characterization of strains of Leishmania donovani. Exp. Parasitol. 18: 1-11, 1966. PubMed: 5909670