青青草原av-午夜视频免费看-免费久久久-中国av片-欧美一级欧美三级在线观看-火影忍者羞羞漫画-成av人片在线观看www-国产日本亚洲-欧美视频在线免费-日本韩国在线-在线日韩中文字幕-国产成人三级在线播放-久久福利在线-老司机免费精品视频-男人操女人逼逼视频-av大片免费-欧美精品第二页-操校花视频-欧美插插视频-优优色综合

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 10⁷ cysts/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 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 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 sufficiently 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.

Moffat JF, Tompkins LS. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect. Immun. 60: 296-301, 1992. PubMed: 1729191

King CH, et al. Survival of coliforms and bacterial pathogens within protozoa during chlorination. Appl. Environ. Microbiol. 54: 3023-3033, 1988. PubMed: 3223766

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

Cirillo JD, et al. Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect. Immun. 62: 3254-3261, 1994. PubMed: 8039895

Steinert M, et al. Resuscitation of viable nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl. Environ. Microbiol. 63: 2047-2053, 1997. PubMed: 9143134

Cirillo JD, et al. Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect. Immun. 65: 3759-3767, 1997. PubMed: 9284149

Neumeister B, et al. Multiplication of different Legionella species in mono mac 6 cells and in Acanthamoeba castellanii. Appl. Environ. Microbiol. 63: 1219-1224, 1997. PubMed: 9097418

Helbig JH, et al. Detection of intracellular growth of Legionella pneumophila in protozoa by antigen quantification using ELISA. Zentralbl. Hyg. Umweltmed. 194: 392-397, 1993. PubMed: 8397687

Buck SL, Rosenthal RA. A quantitative method to evaluate neutralizer toxicity against Acanthamoeba castellanii. Appl. Environ. Microbiol. 62: 3521-3526, 1996. PubMed: 8795247

Grimm D, et al. Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe. Appl. Environ. Microbiol. 64: 2686-2690, 1998. PubMed: 9647849

Essig A, et al. Infection of Acanthamoeba castellanii by Chlamydia pneumoniae. Appl. Environ. Microbiol. 63: 1396-1399, 1997. PubMed: 9097437

Cirillo JD, et al. Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect. Immun. 67: 4427-4434, 1999. PubMed: 10456883

Segal G, Shuman HA. Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. Infect. Immun. 67: 2117-2124, 1999. PubMed: 10225863

Hales LM, Shuman HA. The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii. J. Bacteriol. 181: 4879-4889, 1999. PubMed: 10438758

Neumeister B, et al. Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes. Appl. Environ. Microbiol. 66: 914-919, 2000. PubMed: 10698751

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

Miltner EC, Bermudez LE. Mycobacterium avium grown in Acanthamoeba castellanii is protected from the effects of antimicrobials. Antimicrob. Agents Chemother. 44: 1990-1994, 2000. PubMed: 10858369

Khan NA, et al. Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba. J. Clin. Microbiol. 38: 2858-2861, 2000. PubMed: 10921939

Polesky AH, et al. Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis. Infect. Immun. 69: 977-987, 2001. PubMed: 11159993

Dietrich C, et al. Flagellum of Legionella pneumophila positively affects the early phase of infection of eukaryotic host cells. Infect. Immun. 69: 2116-2122, 2001. PubMed: 11254565

Steenbergen JN, et al. Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc. Natl. Acad. Sci. USA 98: 15245-15250, 2001. PubMed: 11742090

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

Zusman T, et al. Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. J. Bacteriol. 184: 67-75, 2002. PubMed: 11741845

Cirillo SLG, et al. Role of the Legionella pneumophila rtxA gene in amoebae. Microbiology 148: 1667-1677, 2002. PubMed: 12055287

Abd H, et al. Survival and growth of Francisella tularensis in Acanthamoeba castellanii. Appl. Environ. Microbiol. 69: 600-606, 2003. PubMed: 12514047

Boulanger CA, Edelstein PH. Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation. Appl. Environ. Microbiol. 61: 1805-1809, 1995. PubMed: 7646019

Khan NA, et al. Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays. Curr. Microbiol. 43: 204-208, 2001. PubMed: 11400071

Khan NA. Pathogenicity, morphology, and differentiation of Acanthamoeba. Curr. Microbiol. 43: 391-395, 2001. PubMed: 11685503

Khan NA, Paget TA. Molecular tools for speciation and epidemiological studies of Acanthamoeba. Curr. Microbiol. 44: 444-449, 2002. PubMed: 12000996

Marolda CL, et al. Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae. Microbiology 145: 1509-1517, 1999. PubMed: 10439391

Hilbi H, et al. Icm/dot-dependent upregulation of phagocytosis by Legionella pneumophila. Mol. Microbiol. 42: 603-617, 2001. PubMed: 11722729

Hales LM, Shuman HA. Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect. Immun. 67: 3662-3666, 1999. PubMed: 10377156

Steinert M, et al. Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells. FEMS Microbiol. Ecol. 15: 299-308, 1994.

Wintermeyer E, et al. Sequence determination and mutational analysis of the lly locus of Legionella pneumophila. Infect. Immun. 62: 1109-1117, 1994. PubMed: 8112844

Kohler R, et al. Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila. Mol. Gen. Genet. 262: 1060-1069, 2000. PubMed: 10660067

Helbig JH, et al. Immunolocalization of the Mip protein of intracellularly and extracellularly grown Legionella pneumophila. Lett. Appl. Microbiol. 32: 83-88, 2001. PubMed: 11169048

Heuner K, et al. Influence of the alternative sigma(28) factor on virulence and flagellum expression of Legionella pneumophila. Infect. Immun. 70: 1604-1608, 2002. PubMed: 11854250

Wintermeyer E, et al. Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells. Infect. Immun. 63: 4576-4583, 1995. PubMed: 7591108

Segal Gil, et al. Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila. Mol. Microbiol. 34: 799-809, 1999. PubMed: 10564519

Gal-Mor O, Segal G. The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii. Microb. Pathog. 34: 187-194, 2003. PubMed: 12668142

Hagele S, et al. Legionella pneumophila kills human phagocytes but not protozoan host cells by inducing apoptotic cell death. FEMS Microbiol. Lett. 169: 51-58, 1998. PubMed: 9851034

Steinert M, et al. Regrowth of Legionella pneumophila in a heat-disinfected plumbing system. Zentralbl. Bakteriol. 288: 331-342, 1998. PubMed: 9861677