Combination of fluorescent in situ hybridization and microautoradiography - a new tool for structure-function analyses in microbial ecology

A new microscopic method for simultaneously determining in situ the identit ies, activities, and specific substrate uptake profiles of individual bacte rial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligo nucleotide probes and microautoradiography. This method was evaluated by us ing defined artificial mixtures of Escherichia coli and Herpetosiphon auran tiacus under aerobic incubation conditions with added [H-3]glucose. Subsequ ently, we were able to demonstrate the potential of this method by visualiz ing the uptake of organic and inorganic radiolabeled substrates ([C-14]acet ate, [C-14]butyrate, [C-14]bicarbonate, and P-33(i)) in probe-defined popul ations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and withou t nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the up take of labeled substrates under the different experimental conditions used . Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within act ivated sludge flocs, cryosectioned sample material was examined with a conf ocal laser scanning microscope. The combination of in situ rRNA hybridizati on techniques, cryosectioning, microautoradiography, and confocal laser sca nning microscopy provides a unique opportunity for obtaining cultivation-in dependent insights into the structure and function of bacterial communities .