最新中文字幕-精品国产99-国产精品福利在线观看-粉嫩av一区二区三区-日韩精品久久久久-美丽的姑娘国语视频-亚洲欧洲色-国产不卡毛片-三级黄毛片-免费福利在线-日韩av三区-欧美成年人网站-欧洲美女与动交ccoo-成人涩涩软件-秋霞影院午夜老牛影院-婷婷久久伊人-呦呦视频在线观看-www.天天综合-日韩精品高清视频-国产欧美综合在线-欧美日韩一区二区三区在线视频-a猛片免费播放-91九色蝌蚪国产-国产欧美日韩专区-嫩模被强到高潮呻吟不断

1. Harvest the cells from a culture that is at or near peak density by centrifuging at 900 x g for 5 minutes.
2. If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 10⁶ and 2 x 10⁷ cells/ml with fresh medium.  If the concentration is too low, centrifuge at 900 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
3. While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.  Allow the DMSO to solidify.  Add the required volume of refrigerated medium.  Dissolve the DMSO by inverting the tube several times.
   NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 10⁶ and 10⁷ cells/ml and 7.5% (v/v) DMSO. 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 vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules 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 are stored in either the vapor or liquid phase of a nitrogen freezer.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min).  Immerse the vial just sufficiently to cover the frozen material. Do not agitate the vial.
9. Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 6-8 ml of ATCC Medium 1171 diluted to 5% strength in artificial seawater (ASW).
10. Screw the cap on tightly and incubate on a 15° horizontal slant at 25°C.
11. Follow the protocol for maintenance of culture.

Panek T, et al. Diversity, evolution and molecular systematics of the Psalteriomonadidae, the main lineage of anaerobic/microaerophilic heteroloboseans (Excavata: Discoba). Protist, in press (doi:10.1016/j.protis.2011.11.002), 2011.