In recent years, methods of molecular microbiology have been used for the investigation of soil microbial diversity. Fluorescence in situ hybridization (FISH) represents a method which allows a specific staining and enumeration of soil microorganisms by using fluorescent-labelled oligonucleotide probes. However, the detection of FISH-stained cells is often affected by strong autofluorescence of the background, especially in samples of the top soils. In this study a more efficient FISH-approach coupled with catalyzed reporter deposition (CARD) was adapted to soils. Due to tyramide signal amplification (TSA) the fluorescence intensity has been considerably increased at the target binding site of a probe. Six different soils were investigated to evaluate the effect of sample preparation and pre-treatments, TSA, and the procedure of detection. The results show that both cell permeabilization and TSA are two important factors which improve in situ hybridization of soil microorganisms. Soils with higher clay contents have shown better results when prepared on polycarbonate filters rather than on glass slides. Using specific fluorescence filter systems and dye combinations the detection of hybridized cells was extensively increased compared with the application of monolabelled oligonucleotide probes in regular FISH-analysis. As a result, CARD-FISH-stained cells were suitable for automated counting using digital image analysis. Nevertheless, the counterstain with DAPI had to be analyzed manually as it was strongly affected by autofluorescence. (C) 2008 Elsevier Ltd. All rights reserved.
