Combined Microautoradiography and Fluorescence in situ Hybridization (MAR-FISH) for the Identification of Metabolically
Microautoradioactivity (MAR) in combination with fluorescence in situ hybridization (FISH) is a powerful method to obtain information about the ecophysiology of probe-defined single cells in mixed microbial communities. Here we give an overview of how to
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4094
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Experimental Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4095 Sampling and Incubation Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4095 Fixation and Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4096 FISH Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4097 Autoradiographic Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4098 Development and Microscopic Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4099 Solutions and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4099 Time Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4100 Trouble Shooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4100
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Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4101
K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_319, # Springer-Verlag Berlin Heidelberg, 2010
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Combined Microautoradiography and Fluorescence in situ Hybridization
Abstract: Microautoradioactivity (MAR) in combination with fluorescence in situ hybridization (FISH) is a powerful method to obtain information about the ecophysiology of probedefined single cells in mixed microbial communities. Here we give an overview of how to use MAR-FISH in various ecosystems and provide a detailed protocol for MAR-FISH, which includes sampling, incubation with radiotracers, the MAR procedure in combination with FISH and other staining techniques, microscopy, and trouble shooting.
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Introduction
Microautoradiography (MAR) is a powerful tool first used in microbial ecology in the 1960s (Brock and Brock, 1966, 1968). The method is based on assimilation of radiolabeled substrates by individual cells, which can then be visualized by exposure to a radiation-sensitive silver halide emulsion placed over the radiolabeled bacteria and subsequently, processed by standard photographic procedures. The radiotracers used are typically soft beta-emitters, 3H, 14C and 33 P that allows formation of silver grains on top of the labeled bacteria and can be clearly visualized by bright field or phase contrast microscopy. Low e
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