Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes.

A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine differentBrucella clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate betweenBrucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp. On the basis of differences in fluorescence intensities, no discrimination was established betweenBrucella spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions withBrucella spp. and with a total of 17 clinical isolates not belonging to the genus Brucella. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification ofBrucella spp.

Download Full-text

Identification of “Candidatus Thioturbo danicus,” a Microaerophilic Bacterium That Builds Conspicuous Veils on Sulfidic Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8929-8933.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8929-8933 ◽

Cited By ~ 20

Author(s):

Gerard Muyzer ◽

Esengül Yildirim ◽

Udo van Dongen ◽

Michael Kühl ◽

Roland Thar

Keyword(s):

Molecular Analysis ◽

New Genus ◽

Oligonucleotide Probes ◽

Cell Hybridization ◽

Whole Cell ◽

Appl Environ

ABSTRACT Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the ε-subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “Candidatus Thioturbo danicus.”

Download Full-text

Rapid Identification of Bacteria in Blood Cultures by Using Fluorescently Labeled Oligonucleotide Probes

Journal of Clinical Microbiology ◽

10.1128/jcm.38.2.814-817.2000 ◽

2000 ◽

Vol 38 (2) ◽

pp. 814-817 ◽

Cited By ~ 73

Author(s):

G. J. Jansen ◽

M. Mooibroek ◽

J. Idema ◽

H. J. M. Harmsen ◽

G. W. Welling ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Species ◽

Fluorescein Isothiocyanate ◽

Negative Control ◽

Oligonucleotide Probes ◽

Coagulase Negative Staphylococci ◽

Cell Hybridization ◽

Variable Regions ◽

Gram Negative ◽

Whole Cell

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used: Streptococcus spp., Enterococcus faecalis, Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and the Enterobacteriaceae family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for S. aureusand the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.

Download Full-text

Discrimination of Alexandrium andersoni and A. minutum (Dinophyceae) using LSU rRNA-targeted oligonucleotide probes and fluorescent whole-cell hybridization

Phycologia ◽

10.2216/06-11.1 ◽

2007 ◽

Vol 46 (2) ◽

pp. 168-177 ◽

Cited By ~ 13

Author(s):

Nicolas Touzet ◽

Robin Raine

Keyword(s):

Oligonucleotide Probes ◽

Cell Hybridization ◽

Whole Cell ◽

Lsu Rrna

Download Full-text

In situ analysis of the bacterial community in the gut of the earthwormLumbricus terrestrisL. by whole-cell hybridization

Canadian Journal of Microbiology ◽

10.1139/m95-092 ◽

1995 ◽

Vol 41 (8) ◽

pp. 666-673 ◽

Cited By ~ 54

Author(s):

Kathrin Fischer ◽

Dittmar Hahn ◽

Otto Daniel ◽

Josef Zeyer ◽

Rudolf I. Amann

Keyword(s):

Bacterial Community ◽

Lumbricus Terrestris ◽

In Situ Analysis ◽

Oligonucleotide Probes ◽

Dapi Staining ◽

Cell Hybridization ◽

Whole Cell ◽

Hind Gut ◽

Number Of Bacteria

The bacterial community in the gut of the earthworm Lumbricus terrestris was analyzed by whole-cell hybridization with 16S rRNA targeted oligonucleotide probes. Whole-cell hybridization protocols using fluorescence-, peroxidase-, or digoxigenin-labeled oligonucleotide probes facilitated detection of significant fractions of bacterial cells stained with 4′,6-diamidino-2-phenylindole (DAPI) in the fore-, mid-, and hind-gut and cast of the earthworm. The application of peroxidase- and digoxigenin-labeled probes, however, was hampered by several methodological drawbacks: the requirement of enzymatic permeabilization, the diffuse images of stained cells, and the incompatibility with DAPI staining used as control. Quantitative analysis of the bacterial community was also influenced by its considerable variability in different individual earthworms. Though the number of bacteria detected by DAPI staining as well as by whole-cell hybridization with the fluorescent eubacterial probe Eub338 generally showed a significant increase in the number of bacteria towards the end of the gut, a decrease in bacterial numbers could be found in some earthworms. In situ analysis of the bacterial community in the fore-, mid-, and hind-gut of one individual earthworm by whole-cell hybridization with the fluorescent eubacterial probe Eub338 recorded 15, 30, and 25% of DAPI-stained bacteria, respectively. In the cast 37% of the bacteria were detected. Similar to counts obtained by DAPI and by whole-cell hybridization with probe Eub338, the number of bacteria belonging to the α-, β-, and γ-subgroups of proteobacteria increased significantly towards the end of the gut and remained high in the cast. While the most significant difference in the counts of bacteria belonging to the α-subgroup was obtained between the hind-gut and cast, bacterial populations of the β- and γ- subgroups of proteobacteria increased most prominently between the fore- and hind-gut.Key words: digoxigenin, fluorescent probes, in situ detection, Lumbricus terrestris, rRNA, whole-cell hybridization.

Download Full-text