波多野结衣毛片-乳色吐息在线观看-国产伦子伦对白视频-性做久久久-狠狠干2019-黄色裸体片-美女无遮挡免费网站-国产91熟女高潮一区二区-懂色av蜜臀av粉嫩av分享-小h片在线观看-台湾佬在线-日韩激情在线播放-欧日韩不卡在线视频-波多野结衣中文字幕一区-天天操夜夜草

1. Harvest cells from a culture that is at or near peak density by centrifugation at 600 x g for 5 min. Pool the cell pellets into a single tube.
2. Adjust the concentration of cells to 2.0 x 10⁶/mL.  If the concentration is too low, centrifuge at 600 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. Prepare a 15% (v/v) sterile DMSO solution in ATCC medium 1034 as follows:  Add the required volume of DMSO to a glass screw-capped test tube and place on ice.  Allow the DMSO to solidify.  Add the required volume of refrigerated ATCC medium 1034.  Dissolve the DMSO by inverting several times.  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 10⁶ 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 60 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.
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just sufficiently to cover only 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 into a fresh tube or flask of ATCC medium 1034.
10. Incubate at 35°C with the cap screwed on tightly (incubate a test tube on a 15° horizontal slant).

Holbrook TW, et al. Activation of the alternative complement pathway by Naegleria fowleri. Infect. Immun. 30: 58-61, 1980. PubMed: 7439979

Daggett PM, Nerad TA. The biochemical identification of vahlkampfiid amoebae. J. Protozool. 30: 126-128, 1983. PubMed: 6864593

Darby CP, et al. Primary amebic meningoencephalitis. Am. J. Dis. Child. 133: 1025-1027, 1979. PubMed: 495593

Holbrook TW, Parker BW. Naegleria fowleri in chick embryos effects of embryo age and incubation temperature, and the infectivity of embryo-derived amebae for mice. Am. J. Trop. Med. Hyg. 28: 984-987, 1979. PubMed: 574367

Holbrook TW. Naegleria fowleri in the chick embryo. Trans. R. Soc. Trop. Med. Hyg. 73: 460-462, 1979. PubMed: 555075

Olomu N, et al. Demonstration of various acid hydrolases and preliminary characterization of acid phosphatase in Naegleria fowleri. J. Protozool. 33: 317-321, 1986. PubMed: 3018238