Immunofluorescence microscopy and fluorescence in situ hybridization combined with CMEIAS and other image analysis tools for soil- and plant-associated microbial autecology

This chapter describes the application of fluorescent molecular probes used with immunofluorescence microscopy (IFM) and fluorescence in situ hybridization (FISH) techniques for studies of microbial autecology, with an emphasis on soil and root-associated microbes. Proper filter sets must be used to match the specific optical requirements for excitation and emission of fluorescent light by different fluorochromes. Fluorescein isothiocyanate (FITC), and to a lesser extent tetramethyl rhodamine isothiocyanate (TRITC), are common fluorochromes for single labeling experiments of IFM. There are two general approaches in immunofluorescence staining: direct and indirect. Both approaches involve the production, in an immunologically competent animal (e.g., rabbit), of a primary specific antibody against the antigen of interest. The exciting innovations in image analysis technology featured in Center for Microbial Ecology Image Analysis System (CMEIAS) v. 3.0 software will undoubtedly enhance the ecological analysis of in situ bacterial colonization using immunofluorescence and other discriminating microscopy techniques operating at single cell resolution. A large online probeBase database provides an overview of more than 700 published oligonucleotide probes and their characteristics for prokaryotic rRNAs suitable for FISH. The potential ability of FISH-MAR techniques to target the ecological niche for physiological groups of microorganisms in environmental samples may help to close the gap to the general enzymatic measurements, which are also very much increased in sensitivity. CMEIAS can extract an abundance of quantitative information on microbial community structure from the multiprobe FISH image.

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