人妻少妇偷人精品无码丨色婷婷av久久久久久久丨欧美xxxx做受性欧美88丨欧洲女人牲交视频免费丨亚洲精品久久久av无码专区

1. To achieve the best results set up cultures with several different inocula (e.g. 0.25 mL, 0.5 mL, 1.0 mL).  Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.
2. If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 10⁶ and 2 x 10⁷cysts/mL with fresh medium.  If the concentration is too low, centrifuge at 600 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 before the freezing process is begun should be no less than 15 min and no longer than 30 min.
5. Dispense in 0.5 mL aliquots into 1.0 mL to 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 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 to 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 712 in a T-25 tissue culture flask or plastic 16 x 125 mm screw-capped test tube.  Incubate at 25°C.

Ahn KS, Henry R Jr.. S-adnenosylmethionine synthetase gene and the control of cellular growth and development. Curr. Top. Bot. Res. 1: 115-123, 1993.

Alfieri SC, et al. Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites. J. Parasitol. 86: 220-227, 2000. PubMed: 10780536

Alves JMP, et al. Random amplified polymorphic DNA probes as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba. Braz. J. Med. Biol. Res. 33: 19-26, 2000.

Anger C, et al. A quantitative bacterial plaque assay for the enumeration of viable Acanthamoeba cells. Rev. Infect. Dis. 13 suppl.5: S396, 1991. PubMed: 2047673

Berk SG, et al. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp.. Appl. Environ. Microbiol. 64: 279-286, 1998. PubMed: 9435080

Borazjani RN, et al. Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp. Appl. Environ. Microbiol. 66: 1057-1061, 2000. PubMed: 10698771

Bottone EJ, et al. Differential binding capacity and internalisation of bacterial substrates as factors in growth rate of Acanthamoeba spp.. J. Med. Microbiol. 40: 148-154, 1994. PubMed: 8107064

Daggett PM, et al. Distribution and possible interrelationships of pathogenic and nonpathogenic Acanthamoeba from aquatic environments. Microb. Ecol. 8: 371-386, 1982.

Daggett PM, et al. A molecular approach to the phylogeny of Acanthamoeba. Biosystems 18: 399-405, 1985. PubMed: 4084681

Fischer-Stenger K, et al. Separation of soluble amoebicidal and tumoricidal activity of activated macrophages. J. Protozool. 39: 235-241, 1992. PubMed: 1560419

Flores BM, et al. Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry. J. Clin. Microbiol. 28: 1999-2005, 1990. PubMed: 2229384

Fritsche TR, et al. Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses. J. Clin. Microbiol. 31: 1122-1126, 1993. PubMed: 8501212

Gatti S, et al. In vitro effectiveness of povidone-iodine on Acanthamoeba isolates from human cornea. Antimicrob. Agents Chemother. 42: 2232-2234, 1998. PubMed: 9736540

Gautom RK, et al. Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources. J. Clin. Microbiol. 32: 1070-1073, 1994. PubMed: 7913095

Hall J, Voelz H. Bacterial endosymbionts of Acanthamoeba sp.. J. Parasitol. 71: 89-95, 1985. PubMed: 3981353

John DTOpportunistically pathogenic free-living amebaeIn: John DTParasitic protozoa2nd ed.3San DiegoAcademic Presspp. 143-246, 1993

Kong HH, et al. Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga. Korean J. Parasitol. 33: 331-340, 1995. PubMed: 8591011

Kong HH, et al. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea. J. Clin. Microbiol. 40: 1199-1206, 2002. PubMed: 11923331

Lai S, et al. Non-radioactive DNA probe and polymerase chain reaction procedures for the specific detection of Acanthamoeba. Mol. Cell. Probes 8: 81-89, 1994. PubMed: 8028612

Marciano-Cabral F, Toney DM. The interacion of Acanthamoeba spp. with activated macrophages and with macrophage cell lines. J. Eukaryot. Microbiol. 45: 452-458, 1998. PubMed: 9703682

Mitro K, et al. Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga. Exp. Parasitol. 78: 377-385, 1994. PubMed: 8206136

Newsome AL, et al. Isolation of an amoeba naturally harboring a distinctive Legionella species. Appl. Environ. Microbiol. 64: 1688-1693, 1998. PubMed: 9572937

Noble JA, et al. Phagocytosis affects biguanide sensitivity of Acanthamoeba spp. Antimicrob. Agents Chemother. 46: 2069-2076, 2002. PubMed: 12069957

Pettit DA, et al. In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study. J. Parasitol. 82: 769-777, 1996. PubMed: 8885887

Powell EL, et al. Identification of antigens of pathogenic free-living amoebae by protein immunoblotting with rabbit immune and human sera. Clin. Diagn. Lab. Immunol. 1: 493-499, 1994. PubMed: 8556491

Qureshi MN, et al. Inhibition of Acanthamoeba species by Pseudomonas aeruginosa rationale for their selective exclusion in corneal ulcers and contact lens care systems. J. Clin. Microbiol. 31: 1908-1910, 1993. PubMed: 8349772

Reveiller FL, et al. Isolation of a unique membrane protein from Naegleria fowleri. J. Eukaryot. Microbiol. 48: 676-682, 2001. PubMed: 11831777

Schuster FL. Comparative effects of selected azole compounds on trophic and cystic stages of Acanthamoeba polyphaga. J. Eukaryot. Microbiol. 40: 563-569, 1993. PubMed: 8401469

Simione FP Jr., et al. The use of plastic ampoules for freeze preservation of microorganisms. Cryobiology 14: 500-502, 1977. PubMed: 891238

Visvesvara GS, Balamuth W. Comparative studies on related free-living and pathogenic amebae with special reference to Acanthamoeba. J. Protozool. 22: 245-256, 1975. PubMed: 807717

Visvesvara GS, et al. Isolation, identification, and biological characterization of Acanthamoeba polyphaga from a human eye. Am. J. Trop. Med. Hyg. 24: 784-790, 1975. PubMed: 811126

Tachibana H, et al. Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-kDa surface antigen. Parasitol. Res. 83: 435-439, 1997. PubMed: 9197389

Toney DM, Marciano-Cabral F. Resistance of Acanthamoeba species to complement lysis. J. Parasitol. 84: 338-344, 1998. PubMed: 9576508