黄色小说视频-日本少妇高潮抽搐-国内自拍av-天堂中文资源在线-蜜桃视频在线入口www-91久久综合-亚洲视频在线一区-日日夜夜拍-国产精品资源在线观看-欧美激情国产精品免费-人妻少妇久久中文字幕-成人免费无码大片a毛片-香蕉污视频在线观看-日本电影成人-国内精品久久99人妻无码

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 1ºC 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 35ºC 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.

Al-Taqi M, Evans DA. Characterization of Leishmania spp. from Kuwait by isoenzyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 72: 56-65, 1978. PubMed: 635978

Miles MA, et al. Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi. Trans. R. Soc. Trop. Med. Hyg. 74: 243-252, 1980. PubMed: 7385303