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. 1994 Sep;60(9):3228–3235. doi: 10.1128/aem.60.9.3228-3235.1994

Identification of Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-Targeted Polynucleotide Probes

Karlheinz Trebesius 1, Rudolf Amann 1,*, Wolfgang Ludwig 1, Klaus Mühlegger 2, Karl-Heinz Schleifer 1
PMCID: PMC201793  PMID: 16349377

Abstract

Polyribonucleotide probes (ca. 200 to 300 nucleotides in length) carrying multiple reporter molecules were produced by in vitro transcription with labeled UTP derivatives (fluorescein-12-UTP, 7-amino-4-methyl-coumarin-3-acetyl-6-UTP, tetramethylrhodamine-6-UTP, or digoxigenin-11-UTP). Despite their length, these molecules penetrated into whole fixed gram-negative cells and hybridized specifically to their target sites on the 23S rRNA. Fluorescence intensities were quantified for target and nontarget cells by the combination of a charge-coupled device videocamera and an image-processing system. Polyribonucleotide probes confer up to 26 times more fluorescence to target cells than oligonucleotide probes do. Probe sensitivity and specificity were strongly influenced by the stringency of hybridization. The use of differently labeled probes allowed the simultaneous detection of three populations. Identification of introduced test organisms in activated-sludge samples proved the applicability of this method for the in situ identification of microorganisms in complex microbial communities.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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