Leishmania infantum Nicolle

1. To thaw a frozen ampule, place in a 35°C water bath until thawed (2-3 min). Immerse the ampule just sufficiently to cover the frozen material.  Do not agitate the ampule.
2. 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).  Alternatively, inoculate 10 mL complete ATCC medium 2736 in a T-25 flask or screw-capped test tube.
3. Incubate culture tubes vertically at 20-25°C with the caps screwed on tightly; flask cultures should be incubated flat.

1. When the culture has reached or is near peak density, gently mix the culture by aspirating with a sterile pipette and aseptically transfer 0.1-0.5 mL to a new culture vessel containing fresh medium (broth cultures generally require larger inocula than cultures grown in tubes of blood agar).
2. Incubate culture tubes vertically at 20-25°C with the caps screwed on tightly; flask cultures should be incubated flat.
3. Transfer the culture every 3-4 days as described in step 1.  The transfer interval will depend on the quantity of the inoculum and the quality of the medium.  This should be empirically determined by examining the culture on a daily basis until the growth cycle has stabilized.
   Note:  Some fastidious strains may not proliferate well in older growth medium made with blood. If growth is poor, wash cells in an osmotically-balanced saline solution, centrifuge to concentrate as indicated below, and use growth media made with fresh blood.

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).  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, aseptically transfer contents to a screw-capped borosilicate glass test tube containing an appropriate blood-agar medium (i.e., ATCC Medium 807).  Alternatively, inoculate 10 mL complete ATCC medium 2736 in a T-25 flask or screw-capped test tube. 
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.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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