Leishmania major (Yakimoff and Schokhor) Bray et al.
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
2. Incubate the culture vertically at 35°C with the cap screwed on tightly.
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.
1. Harvest cells from cultures that are at or near peak density. Aseptically transfer the broth overlay to a plastic centrifuge tube and adjust the concentration of cells to 2 x 107/ml in fresh medium (broth overlay). If necessary, cells may be concentrated by centrifugation at 800 x g for 5 min.
2. Prepare a 10% (v/v) solution of sterile DMSO in fresh medium (broth). Cool on ice.
3. 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 solution to the start of the freezing process should be no less than 15 min and no longer than 30 min.
4. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
5. 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
6. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -70°C.
7. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.
8. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate a 16 x 125 mm screw-capped test tube containing ATCC medium 807.
9. Incubate the culture vertically at 25°C. Observe the culture daily and transfer when numerous trophozoites are observed.
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Ray DK, et al. Efficacy of seven anthelmintics against Ancylostoma ceylanicum in the golden hamster, Mesocricetus auratus. Ann. Trop. Med. Parasitol. 72: 56-65, 1978. PubMed: 580701
Ann. Trop. Med. Parasitol. 75: 247-249, 1981.