scholarly journals Quantification of Different Eubacteriumspp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

2000 ◽  
Vol 66 (1) ◽  
pp. 375-382 ◽  
Author(s):  
Andreas Schwiertz ◽  
Gwenaelle Le Blay ◽  
Michael Blaut
Keyword(s):  
16S Rrna ◽  
Intestinal Bacteria ◽  
Dry Weight ◽  
Oligonucleotide Probes ◽  
Cross Hybridization ◽  
Fecal Samples ◽  
Cell Counts ◽  
Species Specific ◽  
Type Strains

ABSTRACTSpecies-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify differentEubacteriumspecies in human fecal samples. Probes were directed atEubacterium barkeri,E. biforme,E. contortum,E. cylindroides(two probes),E. dolichum,E. hadrum,E. lentum,E. limosum,E. moniliforme, andE. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers.E. biforme,E. cylindroides,E. hadrum,E. lentum, andE. ventriosumcould be determined. All otherEubacteriumspecies for which probes had been designed were under the detection limit of 107cells g (dry weight) of feces−1. The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration ofEubacteriumspecies in feces.

scholarly journals Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

2001 ◽  
Vol 67 (10) ◽  
pp. 4850-4857 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Manigee Derakshani ◽  
Werner Liesack
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Fluorescence Signal ◽  
Type I ◽  
Oligonucleotide Probes ◽  
Type Ii ◽  
Cell Counts ◽  
Wet Weight

ABSTRACT Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or theMethylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in aSphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 106 cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 104 type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 106 type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus andMethylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion ofMethylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 106 cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population ofMethylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

1998 ◽  
Vol 64 (9) ◽  
pp. 3336-3345 ◽  
Author(s):  
Alison H. Franks ◽  
Hermie J. M. Harmsen ◽  
Gerwin C. Raangs ◽  
Gijsbert J. Jansen ◽  
Frits Schut ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Blot Hybridization ◽  
Dry Weight ◽  
Oligonucleotide Probes ◽  
Fecal Flora ◽  
Dot Blot ◽  
Clostridium Histolyticum

ABSTRACT Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the speciesBacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and theClostridium lituseburense groups. Another probe was designed for the genera Streptococcus andLactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectalegroup. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectalegroup-specific probe detected a mean of 7.2 × 1010cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, andStreptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

scholarly journals A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

2020 ◽  
Author(s):  
Michael Lukumbuzya ◽  
Jannie Munk Kristensen ◽  
Katharina Kitzinger ◽  
Andreas Pommerening-Röser ◽  
Per Halkjær Nielsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Oligonucleotide Probes ◽  
Cross Hybridization ◽  
Approaches To Study ◽  
In Situ Detection ◽  
Probe Set ◽  
Weak Target

AbstractAmmonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.

scholarly journals In Situ Estimation of Carbon Balance of In Vitro Sweetpotato and Tomato Plantlets Cultured with Varying Initial Sucrose Concentrations in the Medium

2002 ◽  
Vol 127 (6) ◽  
pp. 963-970 ◽  
Author(s):  
Chieri Kubota ◽  
Makiko Ezawa ◽  
Toyoki Kozai ◽  
Sandra B. Wilson
Keyword(s):  
Carbon Balance ◽  
Ipomoea Batatas ◽  
Dry Weight ◽  
Photon Flux ◽  
Sugar Uptake ◽  
Relative Contribution ◽  
Carbon Balances ◽  
Species Specific

The effects of initial sucrose (suc) concentrations in the medium (S0) on the carbon balance and growth of sweetpotato [Ipomoea batatas (L.) Lam. `Beniazuma'] and tomato (Lycopersicon esculentum Mill. `HanaQueen') plantlets were studied under controlled environmental conditions. Plantlets were cultured with 0, 7.5, 15, or 30 g·L-1 of S0 under high photosynthetic photon flux (160 to 200 μmol·m-2·s-1) and CO2 enriched (1400 to 2050 μmol·mol-1) conditions. Net photosynthetic rate per leaf area (Pl) decreased and dry weight per plantlet (Wd) increased with increasing S0, but did not differ significantly between S0 of 7.5 to 30 g·L-1 for sweetpotato or 15 to 30 g·L-1 for tomato. Carbon influxes and effluxes of the plantlets by metabolism of medium suc and/or photosynthesis, and respiration were estimated based on measurements of in situ and steady state CO2 exchange rates and sugar uptake during culture. At S0 from 7.5 to 30 g·L-1, photosynthesis was responsible for 82% to 92% and 60% to 67% of carbohydrate assimilation for sweetpotato and tomato, respectively. Estimated carbon balances of plantlets based on the estimated and actual increases of moles of carbon in plant tissue demonstrated that in situ estimation of carbon balance was reasonably accurate for sweetpotato at S0 of 0 to 15 g·L-1 and for tomato at S0 of 0 g·L-1 and that the actual contribution of photosynthesis for tomato at high S0 might be lower than the values estimated in the present experiment. Results showed that initial suc concentration affected the relative contribution of photosynthesis on their carbon balances and that the responses were species specific. The failure of validation at S0 in a range specific to each species suggested the need for further study on carbon metabolism of in vitro plantlets cultured with sugar in the medium.

scholarly journals Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

2006 ◽  
Vol 72 (3) ◽  
pp. 2110-2117 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Timofei A. Pankratov ◽  
Svetlana E. Belova ◽  
Irina S. Kulichevskaya ◽  
Werner Liesack
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Significant Proportion ◽  
Peat Bog ◽  
Rrna Gene ◽  
Bacterial Phyla ◽  
Cell Counts ◽  
Novel Strategy

ABSTRACT The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 107 and 1.1 × 107 cells g−1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

Oligonucleotide probes based on 16S rRNA sequences for the identification of four Azospirillum species

1995 ◽  
Vol 41 (12) ◽  
pp. 1081-1087 ◽  
Author(s):  
Md Mahbubul Kabir ◽  
Denis Faure ◽  
Jacqueline Haurat ◽  
Philippe Normand ◽  
René Bally ◽  
...  
Keyword(s):  
16S Rrna ◽  
Oligonucleotide Probe ◽  
Oligonucleotide Probes ◽  
Probe Sequence ◽  
Bacterial Isolation ◽  
Nontarget Organisms ◽  
Isolation Techniques ◽  
Species Specific ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Partial sequences of the 16S rRNA molecules of nine strains belonging to four Azospirillum species were used to design species-specific oligonucleotide probes. Azospirillum strains sequences were analyzed and three homologous fragments containing 16 nucleotides were determined. These three probes were found to be characteristic of A. lipoferum (Al), A. irakense (Ai), and A. brasilense/amazonense species (Aba) and of few nontarget organisms. The specificity of these three probes was tested both against sequences in the GenBank data base and in numerous colony hybridization experiments. As a few non-target organisms hyridized with the different Azospirillum probes, the use of these probes in bulk soil hybridization is not permitted. However, their use together with specific isolation techniques is validated.Key words: Azospirillum, bacterial isolation, hybridization, oligonucleotide probe, sequence analysis, 16S rRNA.

scholarly journals Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

2008 ◽  
Vol 74 (16) ◽  
pp. 5068-5077 ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
L. Safak Yilmaz ◽  
Daniel R. Noguera ◽  
Holger Daims ◽  
Michael Wagner
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Oligonucleotide Probes ◽  
Pure Cultures ◽  
Technical Modifications ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

The species-specific effects of sublethal concentrations of cadmium on freshwater phytoplankton communities in a Canadian Shield lake

1985 ◽  
Vol 63 (11) ◽  
pp. 1997-2003 ◽  
Author(s):  
Danny C. Reinke ◽  
Frank DeNoyelles Jr.
Keyword(s):  
Cadmium Concentration ◽  
Continuous Cultures ◽  
Experimental Lakes Area ◽  
Batch Cultures ◽  
Freshwater Phytoplankton ◽  
Specific Effects ◽  
Cell Counts ◽  
Phytoplankton Communities ◽  
Species Specific

The species-specific responses of natural phytoplankton communities to low cadmium concentrations were measured in Lake 239 (Experimental Lakes Area, northwestern Ontario). Both in situ and laboratory 5-L continuous-flow cultures, and 5-L and 100-mL cultures were used. Asterionella formosa, Dinobryon sertularia, and Dinobryon bavaricum showed dramatic negative sensitivity to low cadmium concentrations (5–100 μg/L), while Rhabdoderma gorskii and Elakatothrix sp. consistently increased in numbers at the same cadmium concentrations. In all experiments, some species exhibited no apparent effect to cadmium addition as measured by cell counts. The "bottle effect" of each technique was evaluated by comparing the community similarity valves of the control cultures to the lake samples and showed the in situ continuous cultures to be most similar to the lake followed by the laboratory continuous cultures, the in situ 5-L batch cultures, the 5-L laboratory cultures, and the 100-mL batch cultures. Replicate cadmium cultures, all techniques, were more similar to each other than the lake samples. The similarity of the cadmium cultures to the lake sample or control cultures decreased with increased cadmium concentration and incubation time.

Sign in / Sign up

Export Citation Format

Share Document