久9色情I久久免费在线I18禁裸男晨勃露j毛免费观看I熟女黑料51吃瓜I懂色AV一区在线观看I福利网址在线观看I国产美女精品视频免费观看I亚洲福利视频一区I久久深夜I国产黑丝avI看片地址I亚洲Free性ⅩⅹⅩⅹ护士I亚洲国产AV天堂I精品国产I国模冰莲大尺度I一区二区三区四区在线看I免费日p视频I91网在线看

1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min. The cells grown in a medium containing agar are concentrated by centrifugation, a solid pellet does not form. The soft pellet is re-suspended to desired cell concentration with agar-free supernatant.
2. Adjust the concentration of cells to 2 x 10⁶ - 10⁷/ml in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium.
   1. Add 1.0 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
   2. Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 9.0 ml of ice cold medium;
   3. Invert several times to dissolve the DMSO;
   4. Allow to warm to room temperature.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁶ - 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 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. Vials should not be stored above -55°C.
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 of ATCC medium 2154.
10. Incubate the culture on a 15° horizontal slant at 35°C.

Hibler. The morphology and incidence of the Trichomonads of swine, Tritrichomonas suis (Gruby & Delafond), Tritrichomonas rotunda, n. sp. and Trichomonas buttreyi, n. sp.. J. Protozool. 7: 159-171, 1960.

Felleisen RS, et al. Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences. J. Clin. Microbiol. 36: 513-519, 1998. PubMed: 9466768

Felleisen RS. Comparative genetic analysis of tritrichomonadid protozoa by the random amplified polymorphic DNA technique. Parasitol. Res. 84: 153-156, 1998. PubMed: 9493217

Felleisen RS. Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa. Parasitology 115: 111-119, 1997. PubMed: 10190167

Tachezy J, et al. Cattle pathogen Tritrichomonas foetus (Riedmuller, 1928) and pig commensal Tritrichomonas suis (Gruby & Delafond, 1843) belong to the same species. J. Eukaryot. Microbiol. 49: 154-163, 2002. PubMed: 12046599

Wei-Dong Xu, et al. Chromosome numbers of Tritrichomonas foetus and Tritrichomonas suis. Vet. Parasitol. 78: 247-251, 1998. PubMed: 9786624