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 in situ physiology of Nostocoida limicola II, a filamentous bacterial morphotype in bulking activated sludge, using fluorescence in situ hybridization and microautoradiography

2006 ◽  
Vol 54 (1) ◽  
pp. 47-53 ◽  
Author(s):  
E.M. Seviour ◽  
K. Eales ◽  
L. Izzard ◽  
M. Beer ◽  
E.L. Carr ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Filamentous Bacterium ◽  
Anaerobic Conditions ◽  
Pure Cultures

The in situ physiology of the actinobacterial bulking and foaming filamentous bacterium “Nostocoida limicola” II was studied by fluorescence in situ hybridization/microautoradiography. Substrate assimilation patterns of pure cultures of this bacterium were different to those seen in activated sludge biomass samples. There was no evidence to suggest that “N. limicola” II preferred hydrophobic substrates, but evidence was produced to support the view that it is metabolically active under anaerobic conditions in activated sludge.

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

1996 ◽  
Vol 42 (10) ◽  
pp. 1061-1071 ◽  
Author(s):  
Marc E. Frischer ◽  
Peter J. Floriani ◽  
Sandra A. Nierzwicki-Bauer
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Differential Sensitivity ◽  
Probe Design ◽  
Oligonucleotide Probes ◽  
Whole Cells ◽  
Gene Probes ◽  
Wide Range

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [γ-32P] ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.Key words: 16S rRNA, oligonucleotide probes, in situ hybridization.

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.

scholarly journals Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

2002 ◽  
Vol 68 (6) ◽  
pp. 3094-3101 ◽  
Author(s):  
Annelie Pernthaler ◽  
Jakob Pernthaler ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Heterotrophic Bacteria ◽  
Cell Loss ◽  
Bacterial Cells ◽  
Detection Rates ◽  
Cell Counts ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml−1 at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.

scholarly journals Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria

2006 ◽  
Vol 72 (1) ◽  
pp. 918-922 ◽  
Author(s):  
Belinda C. Ferrari ◽  
Niina Tujula ◽  
Kate Stoner ◽  
Staffan Kjelleberg
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Soil Bacteria ◽  
Membrane System ◽  
Catalyzed Reporter Deposition ◽  
Real Time Identification ◽  
Card Fish ◽  
Growing Strategy ◽  
Slow Growing

ABSTRACT Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition.

scholarly journals Two Major Archaeal Pseudomurein Endoisopeptidases: PeiW and PeiP

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-4 ◽  
Author(s):  
Ganesh Ram R. Visweswaran ◽  
Bauke W. Dijkstra ◽  
Jan Kok
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Isolation ◽  
Permeability Ratio ◽  
Archaeal Species ◽  
Protoplast Preparation ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

PeiW (UniProtKB Q7LYX0) and PeiP (UniProtKB Q77WJ4) are the two major pseudomurein endoisopeptidases (Pei) that are known to cleave pseudomurein cell-wall sacculi of the members of the methanogenic ordersMethanobacterialesandMethanopyrales. Both enzymes, originating from prophages specific for some methanogenic archaeal species, hydrolyze the (Ala)-Lys bond of the peptide linker between adjacent pseudomurein layers. Because lysozyme is not able to cleave the pseudomurein cell wall, the enzymes are used in protoplast preparation and in DNA isolation from pseudomurein cell-wall-containing methanogens. Moreover, PeiW increases the probe permeability ratio and enables fluorescence in situ hybridization (FISH) and catalyzed reporter deposition (CARD-) FISH experiments to be performed on these methanogens.

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