Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol-or paraformaldehyde fixed bulk soil after homogenization of 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 with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol-and paraformaldehyde-fixed samples to up to 47 +/- 8% due to an increase in the detection yield of members of the alpha-subdivision of Proteobacteria from 2 +/- 1% to 10 +/- 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the alpha-, beta-, gamma-, and delta-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria 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 predominant. Here, members of the alpha-subdivision of Proteobacteria accounted for approximately 10 +/- 3% of DAPI-stained cells, which corresponded 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-stained cells [17 +/- 9 x 10(8) cells (g soil, dry wt.)(-1)]. A large population 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, which to date has never been isolated from terrestrial environments.