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1. Harvest cells from a culture which is at or near peak density by centrifugation at 1,300 g for 5 min.
2. Adjust concentration of cells to 2 x 10^7/mL in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium (broth). The DMSO solution when first prepared will warm up due to chemical heat. The solution should be allowed to return to room temperature prior to use.
4. Mix the cell preparation and the DMSO solution in equal portions. The final concentration will be 10⁷ cells/mL and 5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no more than 15 min.
5. Dispense in 0.5 mL aliquots into 1.0-2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the ampules 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, do not leave in the water bath, aseptically transfer the contents of the ampule into a fresh tube of ATCC medium 431, or alternatively, inoculate 10 mL complete ATCC medium 2736 in a screw-capped test tube.
10. Incubate vertically at 25°C with the cap screwed on tightly.
11. Maintain as described above.

Kuwahara SS, et al. Occurrence of a 5,7-diene sterol in Leishmania donovani. J. Gen. Microbiol. 66: 375-378, 1971. PubMed: 5093419