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 10⁷/mL in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium (broth or liquid overlay from blood agar).xa0 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 1ºC freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.)xa0xa0
7. Store in either the vapor or liquid phase of a nitrogen refrigerator.
8. To thaw a frozen ampule, place it in a 35ºC water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed.
9. Immediately after thawing, aseptically transfer contents to a screw-capped borosilicate glass test tube containing an appropriate blood-agar medium (i.e., ATCC Medium 807).xa0 Alternatively, inoculate 10 mL complete ATCC medium 2736 in a T-25 flask or screw-capped test tube.xa0
10. Incubate culture tubes vertically at 20-25°C with the caps screwed on tightly; flask cultures should be incubated flat.
11. Maintain as described above.

Lanotte G, et al. Application de la methode numerique a la taxonomie du genre Leishmania Ross, 1903. A propos de 146 souches originnaires de lAncien Monde. Utilisation des allozymes. Corollaires epidemiologiques et phyletiques. Ann. Parasitol. 56: 575-592, 1981.