Abstract
A major goal of microbial ecology is to study the abundance, localization, and activities of microorganisms in situ in order to understand ecophysiological roles that the microorganisms play in complex natural ecosystems. In fact, in typical microbial habitats such as biofilms, sediments, and microbial aggregates, resources and physicochemical conditions are dynamically changing with time and across even a very tiny distance because of metabolic activities and substrate transport limitation. To directly correlate microbial identity (16S rRNA-based phylogeny) to the specific metabolic function of individual cells within such complex and heterogeneous microbial habitats, several new molecular-based techniques have been developed in the last decade. These techniques exploit in situ simultaneous phylogenetic identification and metabolic capabilities of even uncultured microorganisms without the need to isolate them in culture. Microautoradiography is a powerful but "rather old" tool, with which the in situ uptake of specific radiolabeled substrates by individual cells can be determined. Fluorescence in situ hybridization (FISH) is a new molecular-based technique that allows the in situ phylogenetic identitification of individual cells. However, FISH cannot provide sufficient information on metabolic capabilities, because phylogeny and phenotype are rarely congruent. Recently, microautoradiography and FISH have been successfully combined to further improve the complementary strengths of the two methods. Microautoradiography combined with FISH (MAR-FISH) can be used to simultaneously examine the phylogenetic identity and the relative or actual specific activity of microorganisms within a complex microbial community at a single-cell level. This article overviews the principle, experimental protocol and application of the MAR-FISH technique, as well as current developments of other analytical techniques for in situ microbial functions (metabolic activities) from a single-cell level to community levels.
