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

Abstract

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. The procedures to prepare rabbit polyclonal antibodies to surface antigens of microbes, their conjugation to a fluorochrome, preparation of soil and root samples for examination, and use of purified antibody-fluorochrome conjugates to detect microorganisms by IFM are discussed. Selected examples are given to illustrate the use of IFM in autecological studies on Rhizobium and its association with higher plant roots, including its combined use with quantitative CMEIAS (Center for Microbial Ecology Image Analysis System) image analysis to measure this microbe's in situ spatial abundance and patterns of distribution on root surfaces. Finally, a brief discussion is given on the application of IFM to detect specific metabolic potentials of microbes in situ, using FA directed against the corresponding enzymes that catalyse those reactions. Finally, some recent developments of improved FISH techniques are discussed