青青草原av-午夜视频免费看-免费久久久-中国av片-欧美一级欧美三级在线观看-火影忍者羞羞漫画-成av人片在线观看www-国产日本亚洲-欧美视频在线免费-日本韩国在线-在线日韩中文字幕-国产成人三级在线播放-久久福利在线-老司机免费精品视频-男人操女人逼逼视频-av大片免费-欧美精品第二页-操校花视频-欧美插插视频-优优色综合

1. Harvest cells from a culture that is at or near peak density. To detach cells from the wall of the culture tubes place on ice for 10 minutes. Invert tubes several times until the majority of the cells are in suspension. Centrifuge tubes at 800 x g for 5 minutes.
2. Adjust the concentration of cells to 2 x 10⁷/mL in fresh medium.
3. Before centrifuging prepare a 24% (v/v) solution of sterile DMSO in fresh medium containing 8% (w/v) sucrose. The solution is prepared as follows:
   1. Add 1.05 g sucrose to 10 mL of fresh medium and filter sterilize through a 0.2 µm filter;
   2. Add 2.4 mL of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
   3. Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 7.6 mL of ice cold medium prepared in step 3a. The final concentration will be 24% (v/v) DMSO and 8% (w/v) sucrose;
   4. Invert several times to dissolve the DMSO;
   5. Allow to warm to room temperature.
4. Mix the cell preparation and the cryoprotective agent, prepared in step 3, in equal portions. Thus, the final concentration will equal 12% (v/v) DMSO + 4% sucrose (w/v) and 10⁷ cells/mL. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no longer than 30 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 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 -1°C/min.)
7. 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.
8. 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.
9. 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 13 mL ATCC Medium 2695.
10. Incubate the culture on a 15º horizontal slant at 35°C.

Nash TE, et al. Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J. Infect. Dis. 152: 64-73, 1985. PubMed: 2409186

Nash TE, Keister DB. Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia. J. Infect. Dis. 152: 1166-1171, 1985. PubMed: 4067331

Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin. Microbiol. Rev. 15: 329-341, 2002. PubMed: 12097242

Miller RL, et al. Identification of Giardia lamblia Isolates Susceptible and Resistant to Infection by the Double-Stranded RNA Virus. Exp. Parasitol. 66: 118-123, 1988. PubMed: 3366209