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

Abstract

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.

Differentiation of Two Very Similar Glaucomid Ciliate Morphospecies (Ciliophora, Tetrahymenida) by Fluorescence in Situ Hybridization with 18S rRNA Targeted Oligonucleotide Probes

“…11, 12). The possibility of a single mismatch discrimination by oligonucleotide probes is well known and has been demonstrated previously for bacteria (Amann, Glöckner, and Neef 1997) and for ciliates, too (Schmidt et al 2006). The weak signals, obtained after hybridization with probes glsc413 and glbr413 to their target organisms, might be explained by inaccessibility of the probes to the target molecule, a phenomenon that is also known from other organisms (Behrens et al 2003a, b).…”

Saprophytic growth of inoculated Frankia sp. in soil microcosms

“…In situ hybridization and concomitant DAPI staining have been shown to be adequate tools for the quantitative analysis of microorganisms in both aquatic and terrestrial environments in the past Amann et al, 1995Amann et al, , 1997. These tools have successfully been used to identify specific Frankia strains in root nodules, and to detect them after introduction into soil (Hahn et al, 1997.…”

Some multidisciplinary techniques used in MIC studies

“…Better fluorescent dyes can identify more cells even in oligotrophic aquatic samples than cultivation-dependent methods. There are several reasons which make rRNA targeted oligodeoxyribonucleotides particularly suitable for the in situ identification of microorganisms [17]. 16S rRNA is quite conservative but also having enough clues for evolution study.…”

“…There are three steps in the hybridization tests [17]: First, the morphology of the cell in the examined sample has to be stabilized and the cell walls and membranes have to be permeable for the penetration of the probes. This can be achieved with fixatives which are usually based on aldehydes and alcohols.…”