Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes

Like many other parts of microbial ecology, subsurface microbiology has entered the molecular age. As one example of various powerful molecular techniques, fluorescently labeled rRNA-targeted nucleic acid probes today allow an in situ identification of individual microbial cells in their natural habitats. The technique relies on the specific hybridization of the nucleic acid probes to the naturally amplified intracellular rRNA. Fluorescently labeled, rRNA-targeted oligonucleotide probes are perfect tools for many areas of microbial ecology since they can monitor specific populations in environmental samples based on constant genotypic features and not on variable phenotypic features like morphology. In case of immobilized communities like biofilms, exact spatial distributions of microorganisms can be analyzed on a micrometer scale. Recent technical improvements have increased the number of potential applications considerably. Today, better fluorescent dyes enable identification of routinely more than 50% of the cells even in oligotrophic aquatic samples in which the visualization of small cells with low numbers of ribosomes had been problematic. This compares favorably with the usually less than 1% of microorganisms which can be characterized based on cultivation-dependent methods.

[1]  Yves Van de Peer,et al.  Database on the structure of large ribosomal subunit RNA , 1994, Nucleic Acids Res..

[2]  R. Amann In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes , 1995 .

[3]  R. Amann,et al.  Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization , 1996, Applied and environmental microbiology.

[4]  K. Schleifer,et al.  Identification in situ and phylogeny of uncultured bacterial endosymbionts , 1991, Nature.

[5]  K. Schleifer,et al.  Phylogenetic Diversity and Identification of Nonculturable Magnetotactic Bacteria , 1992 .

[6]  K. Schleifer,et al.  Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge , 1994, Applied and environmental microbiology.

[7]  T. Dobzhansky Genetics and the Origin of Species , 1937 .

[8]  K. Schleifer,et al.  In situ visualization of high genetic diversity in a natural microbial community , 1996, Journal of bacteriology.

[9]  D. Stahl,et al.  Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences , 1993, Applied and environmental microbiology.

[10]  S. R. Parker,et al.  Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters. , 1990, Cytometry.

[11]  B Flesher,et al.  Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology , 1988, Applied and environmental microbiology.

[12]  Ross A. Overbeek,et al.  The ribosomal database project , 1992, Nucleic Acids Res..

[13]  Frank Oliver Glöckner,et al.  An in situ hybridization protocol for detection and identification of planktonic bacteria , 1996 .

[14]  N. Pace,et al.  Microbial ecology and evolution: a ribosomal RNA approach. , 1986, Annual review of microbiology.

[15]  Ross A. Overbeek,et al.  The Ribosomal Database Project (RDP) , 1996, Nucleic Acids Res..

[16]  K. Schleifer,et al.  Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure , 1993, Applied and environmental microbiology.

[17]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[18]  K. Schleifer,et al.  Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms , 1996, Applied and environmental microbiology.

[19]  R. Amann,et al.  Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms , 1992, Applied and environmental microbiology.

[20]  D. Stahl Evolution, Ecology and Diagnosis: Unity in Variety , 1986, Bio/Technology.

[21]  K. Schleifer,et al.  Diversity of Magnetotactic Bacteria , 1995 .