Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides.
rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals (clay minerals) and organic material (up to 8%) did not significantly interfere with signal expression after hybridization. Background signals were mainly caused by autofluorescence of organic material. Non-specific binding of labelled oligonucleotides to soil particles was not observed. In situ detection of introduced cells of Pseudomonas cepacia in a sandy loam spiked with a mixture of selected soil micro-organisms was possible after hybridization with a specific probe. Analysis of natural bacterial populations in soil, however, was not possible by in situ hybridization without activation of these micro-organisms by adding nutrients. Growing cells, e.g. Streptomyces scabies hyphae growing in amended soil, were easily detected.
Hahn
D
D
Agricultural University Wageningen, Department of Microbiology, The Netherlands.
Amann
R I
RI
Ludwig
W
W
Akkermans
A D
AD
Schleifer
K H
KH
D001419:Bacteria / Q000302:isolation & purification*; D001483:Base Sequence; D002455:Cell Division; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011549:Pseudomonas / Q000737:chemistry / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000032:analysis; D012338:RNA, Ribosomal, 23S / Q000032:analysis*; D012235:Rhodamines; D012988:Soil Microbiology; D013302:Streptomyces / Q000737:chemistry / Q000302:isolation & purification
D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012235:Rhodamines; C009434:tetramethylrhodamine isothiocyanate