国产精品成人久久I97成人精品视频在线播放I综合色综合色I福利视频在线看I美女精品网站I久久综合久久伊人

1. Prepare a 10% (v/v) sterile DMSO solution in fresh ATCC Medium 355. 
2. Transfer a culture at peak density to centrifuge tubes and centrifuge at 525 x g for 5 minutes.
3. Remove the supernatant and resuspend the cells in ATCC medium 355 to a concentration of 2 x 10⁶ to 2 x 10⁷ cells/ml.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 10⁶ and 10⁷ cells/ml and 5% (v/v) DMSO.
5. Distribute the cell suspension in 0.5 ml aliquots into 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).  The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
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 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 sufficient to cover the frozen material. Do not agitate the vial.
9. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5 ml of fresh ATCC medium 355 in a 16 x 125 mm screw-capped test tube. Incubate upright at 25°C with caps screwed on tightly.

Hanson and McGhee. The biology and morphology of Crithidia acanthocephali n. sp., Leptomonas leptoglossi n. sp., and Blastocrithidia euschisti n. sp.. J. Protozool. 8: 200-204, 1961.

Figueiredo EN, et al. Enzymes of the ornithine-arginine metabolism of trypanosomatids of the genus Crithidia. J. Protozool. 25: 546-549, 1978.

Conchon I, et al. Trypanosomatids, other than Phytomonas spp., isolated and cultured from fruit. J. Protozool. 36: 412-414, 1989.

Clark CG. Riboprinting: A tool for the study of genetic diversity in microorganisms. J. Eukaryot. Microbiol. 44: 277-283, 1997. PubMed: 9225441

Camargo EP, et al. Proteolytic activities in cell extracts of trypanosomatids. J. Parasitol. 64: 1120-1121, 1978. PubMed: 739304

Goncanlves De Lima VM, et al. Comparison of six isoenzymes from 10 species of Crithidia. J. Protozool. 29: 397-401, 1982.

Teixeira MM, Camargo EP. Monoclonal antibodies for the identification of trypanosomatids of the genus Phytomonas. J. Protozool. 36: 262-264, 1989.

Roitman I, et al. Nutrition of trypanosomatids Crithidia acanthocephali and Crithidia harmosa ribose and adenosine: Substrates for Crithidia acanthocephali. J. Protozool. 32: 490-492, 1985.

Galinari S, Camargo EP. Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase. Exp. Parasitol. 46: 277-282, 1978. PubMed: 569594

Fish WR, et al. The cyclopropane fatty acid of trypanosomatids. Mol. Biochem. Parasitol. 3: 103-115, 1981. PubMed: 7254247

Freymuller E, Camargo EP. Ultrastructural differences between species of trypanosomatids with and without endosymbionts. J. Protozool. 28: 175-182, 1981. PubMed: 7024533

Alfieri SC, Camargo EP. Trypanosomatidae: isoleucine requirement and threonine deaminase in species with and without endosymbionts. Exp. Parasitol. 53: 371-380, 1982. PubMed: 6806116

Teng SC, et al. A new non-LTR retrotransposon provides evidence for multiple distinct site-specific elements in Crithidia fasciculata miniexon arrays. Nucleic Acids Res. 23: 2929-2936, 1995. PubMed: 7659515

Camargo EP, et al. Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids. J. Protozool. 29: 251-258, 1982. PubMed: 6284925

Cho J, Eichinger D. Crithidia fasciculata induces encystation of Entamoeba invadens in a galactose-dependent manner. J. Parasitol. 84: 705-710, 1998. PubMed: 9714198