ANALYSIS OF BACTERIAL COMMUNITY STRUCTURE IN BULK SOIL BY IN-SITU HYBRIDIZATION

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) ol igonucleotide probes was used to analyze bacterial community structure in ethanol-or paraformaldehyde fixed bulk soil after homogenization o f soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 +/- 7%, and in paraformaldehyde 41 +/- 8% of the 4', 6-diamidino-2-phenylindole(DAPI)-stained cells were detected w ith the bacterial probe Eub338. The yield could not be increased by en zymatic and/or chemical pretreatments known to enhance the permeabilit y of bacterial cells for probes. However, during storage in ethanol fo r 7 months, the detectability of bacteria increased in both ethanol-an d paraformaldehyde-fixed samples to up to 47 +/- 8% due to an increase in the detection yield of members of the alpha-subdivision of Proteob acteria from 2 +/- 1% to 10 +/- 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic gr oups such as the alpha-, beta-, gamma-, and delta-subdivisions of Prot eobacteria, gram-positive bacteria with a high G+C DNA content, bacter ia of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the alpha- and delta-subdivision of Proteobacteria and the planctomycetes were predom inant. Here, members of the alpha-subdivision of Proteobacteria accoun ted for approximately 10 +/- 3% of DAPI-stained cells, which correspon ded to 44 +/- 16 x 10(8) cells (g soil, dry wt.)(-1), while members of the delta-subdivision of Proteobacteria made up 4 +/- 2% of DAPI-stai ned cells [17 +/- 9 x 10(8) cells (g soil, dry wt.)(-1)]. A large popu lation of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 +/- 3% of DAPI-stained cells [32 rt 12 x 10(8) cells (g soil, dry wt.)(-1)]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, whi ch to date has never been isolated from terrestrial environments.