国产精品国产精品一区精品国产自在现偷99精品国产在热2019国产拍偷精品网国产精品视频全国免费观看,国产精品v欧美精品v日韩精品青青精品视频国产久久国产精品久久精品国产亚洲精品国产精品国产欧美精品一区二区三区,国产精品第一页国产亚洲精品国产福利国产精品自拍国产精品视频在线观看亚洲国产精品一区二区久久国产精品国产三级国产专不,国产精品视频大陆精大陆国产国语精品2019精品国产品对白在线285年香蕉精品国产高清自在自线隔壁老王国产在线精品在线观看精品国产福利片,国产三级精品三级在专区精品国产自在现偷国产精品一区二区三区国产日韩精品欧美一区喷水亚洲精品国产精品国自产国产在线精品一区二区不卡

Cryoprotective Solution
DMSO, 1.5 ml
Fresh growth medium w/o bacteria, 8.5 ml

Note: Care must be taken to maintain this organism at 4°C at all times.  Media and cell suspension should be kept on ice during entire process.

1. Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.  Chill to 4°C.
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 800 x g for 5 min.
3. Adjust the concentration of cells at least 2 x 10⁶/ml in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
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. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place the vial in a 35°C water bath.  Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.  Do not leave in water bath after thawing. Aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml chilled ATCC medium 2417 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^®  700831).
9. Incubate at 4°C with the cap screwed on tightly.
10. Once the culture is established, vigorously agitate the flask and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 2417.
11. Follow the protocol for maintenance of culture.

Wainright PO, et al. Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260: 340-342, 1993. PubMed: 8469985

Leadbeater BSC, et al. A new genus, Helgoeca gen. nov., for a nudiform choanoflagellate. Eur. J. Protistol. 44:227-237, 2008. PubMed: 18403187

Carr M, et al. Molecular phylogeny of choanoflagellates, the sister group to Metazoa. Proc. Natl. Acad. Sci. USA. 105: 16641-16646, 2008. PubMed: 18922774