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1 Journal of Environmental Biotechnology Vol. 8, No. 2, 81 87, 2008 総説 ( 特集 ) 土壌環境中の細菌の検出法 Methods for the Detection of Bacteria in the Soil Environment * KAZUHIDE KIMBARA, SOU IIJIMA, YUMI SHIMOMURA, and AKIO TANI * TEL: FAX: * kimbara@rib.okayama-u.ac.jp Research Institute for Bioresources, Okayama University Chuo, Kurasiki, Okayama , Japan キーワード Key words: fluorescence, detection, bacteria, soil / はじめに 2. 固体上の細菌の観察法 2.1. フィルター培養法を用いた細菌細胞のコロニー形成方法 PCB Comamonas testosteroni TK102 TK102 1/3 LB 1/3LB PBS PBS PBS 10 3 cells/ml 0.2 μm 25 mm ADVANTEC 121 C 15 ADVANTEC 1 ml 1/3 LB 30 C 蛍光試薬を用いたコロニーの二重染色とスライドグラス試料の作成 Oregon Green 488 carboxylic acid diacetate succinimidyl ester Carboxy-DFFDA SE Molecular Probe Propidium Iodide PI PI DNA Carboxy-DFFDA SE PI 5 μm 4 μm PBS
2 82 30 C 30 4 C LSC LSC 2.3. 蛍光顕微鏡によるコロニーの観察 100 W BX 50 Olympus Carboxy- DFFDA SE nm nm PI nm 580 nm CCD DP70 Olympus DP manager Olympus 2.2. Carboxy- DFFDA SE PI ,6,14 1/3LB 3. 土壌中の細菌の観察法 2 3 LSC 3.1. LSC を用いた土壌試料中の細菌細胞を検出する方法の開発 LSC LSC LSC2 488 nm nm nm 650 nm 3 10 mm LSC
3 83 1 C. testosteroni TK102 2 TK102 3 LSC LSC LSC Carboxy-DFFDA SE SYTOX Orange Ethidium Bromide EtBr PI Ethidium homodimer-1 EthD-1 LDS LSC
4 検出法の利用 488 nm 488 nm 405 nm 543 nm 590 nm 633 nm 405 nm DAPI Hoechst33342 Hoechst33342 Total count 543 nm 590 nm ph FISH 11 FISH-MAR 15 CFU 4. 特定の細菌細胞を検出する試み stable isotope probing SIP 4 cross-feeding 8,13
5 蛍光性代謝産物を用いた細菌細胞の特異的検出 2,3-dihydroxybiphenyl HOPDA nm 6 PCB TK102 2,3-dihydroxybiphenyl 4 4-ethylbiphenyl 4-butylbiphenyl 2,3-dihydroxy-4-butylbiphenyl 46 2,3-dihydroxy-4- butylbiphenyl 2,3-dihydroxy-4-butylbiphenyl PCB 4-butylbiphenyl HPLC TK102 2,3-dihydroxy-4-butylbiphenyl 2,3-dihydroxy-4-butylbiphenyl 6 FCM 2,3-dihydroxy-4-butylbiphenyl 7 HOPDA ph nm ph nm ph nm HOPDA FCM 488 nm HOPDA
6 86 PCB TK102 HOPDA TK102 TK 密度勾配遠心法による土壌粒子の除去 2 2,3-dihydroxy-4-butylbiphenyl 2 Sorensen tween80 Histodenz Nycodenz ,10 Hisotdenz 4 7 8
7 tween80 Histodenz Histodenz 8 60 Hisodenz 5. 特定の細菌細胞を単離する試み 4 FCM PCB FCM Histodenz FCM HOPDA PCB 6. おわりに 文 1) ) Bakken, L. R Separation and purification of bacteria from soil. Appl. Environ. Microbiol. 49: ) Burmolle, M., L. H. Hansen and S. J. Sorensen Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry. Microbial Ecol. 50: ) Friedrich, M. W Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. Curr. Opin. Biotechnol. 17: ) Hiraoka, Y., and K. Kimbara Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry. Appl. Environ. Microbiol. 68: ) Hiraoka, Y., T. Yamada, K. Tone, Y. Futaesaku, and K. Kimbara Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: effect of metabolites on cell-cell separation. Appl. Environ. Microbiol. 68: ) Khan, A., and S. Walia Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83. Appl. Environ. Microbiol. 55: ) Manefield, M., R. Griffiths, N. P. McNamara, D. Sleep, N. Ostle, and A. Whiteley Insights into the fate of a 13C labeled phenol pulse for stable isotope probing (SIP) experiments. J. Microbiol. Methods. 69: ) Martin, M. M., and L. Lindqvist The ph dependence of fluorescein fluorescence. Luminescence 10: ) Norman, A., L. H. Hansen, and S. J. Sorensen A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments. Mutation Research. 603: ) Rogers, S. W., T. B. Moorman, and S. K. Ong Fluorescent in situ hybridization and microautoradiography applied to ecophysiology in soil. Soil Sci. Soc. Am. J. 71: ) Shimomura, Y., R. Ohno, F. Kawai, and K. Kimbara Method for assessment of viability and morphological changes of bacteria in the early stage of colony formation on a simulated natural environment. Appl. Environ. Microbiol. 72: ) Singleton, D. R., M. Hunt, S. N. Powell, R. Frontera-Suau, and M. D. Aitken Stable-isotope probing with multiple growth substrates to determine substrate specificity of uncultivated bacteria. J. Microbiol. Methods. 69: ) Yamada, T., Y. Shimomura, Y. Hiraoka, and K. Kimbara, K Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation. Appl. Microbiol. Biotechnol., 73: ) Wagner, M., P. H. Nielsen, A. Loy, J. L. Nielsen, and H. Daims Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr. Opin. Biotechnol. 17: 献
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