Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides

2008 ◽  
Vol 73 (2) ◽  
pp. 142-147 ◽  
Author(s):  
J.A. Dijk ◽  
P. Breugelmans ◽  
J. Philips ◽  
P.J. Haest ◽  
E. Smolders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fish Detection ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

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).

Assessment of Microbial Adhesion in Mixed Cultures to Sulfide Minerals Using CARD-FISH Techniques

2009 ◽  
Vol 71-73 ◽  
pp. 83-86 ◽  
Author(s):  
A. Echeverría ◽  
Cecilia Demergasso
Keyword(s):  
Fluorescent In Situ Hybridization ◽  
Culture Medium ◽  
Sulfide Minerals ◽  
Acidithiobacillus Thiooxidans ◽  
Mineral Surfaces ◽  
Dapi Staining ◽  
Factors Affecting ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

The adhesion of microorganisms to minerals is one of the least understood aspects of bioleaching processes. Mineral and microorganism type are determinant factors affecting the capacity of a microbial community to form biofilms in these processes. In this paper the adhesion capacity of different microorganisms to different copper sulfide minerals generally present in bioleaching processes was studied. The minerals, chalcosite, chalcopyrite and pyrite were used and the composition of the microbial population which adhered to the mineral surfaces was determined using the Catalyzed Reporter Deposition Fluorescent In Situ Hybridization (CARD-FISH) technique. This technique involves the fluorescent marking of the cells using specific DNA probes and their observation with a confocal microscope. Three probes were used against the microorganisms: Acidithiobacillus ferooxidans, Acidithiobacillus thiooxidans and Leptospirillum. Polished fragments of the three minerals were placed in a tube with a culture medium which was inoculated with cells from a mixed culture capable of growing at 25°C. The adhered microorganisms were counted with CARD-FISH and compared with the total count which was carried out with DAPI staining. The results show that microorganisms adhered indistinctly to pyrite and chalcopyrite but not chalcosite. It was also observed that in pyrite 60% of the adhered microorganisms were Acidithiobacillus thiooxidans, 35% Acidithiobacillus ferrooxidans, and 1 % Leptospirillum. The remaining 4% were unidentified microorganisms.

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.

scholarly journals An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

2003 ◽  
Vol 69 (5) ◽  
pp. 2928-2935 ◽  
Author(s):  
Raju Sekar ◽  
Annelie Pernthaler ◽  
Jakob Pernthaler ◽  
Falk Warnecke ◽  
Thomas Posch ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Cell Walls ◽  
Cell Loss ◽  
Permeabilized Cell ◽  
Optimized Protocol ◽  
High Concentrations ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml−1) followed by achromopeptidase (60 U ml−1) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.

The Use of CARD-FISH to Evaluate the Quantitative Microbial Ecology Involved in the Continuous Bioleaching of a Cobaltiferrous Pyrite

2007 ◽  
Vol 20-21 ◽  
pp. 565-568 ◽  
Author(s):  
E. Díaz ◽  
E. González-Toril ◽  
Catherine Joulian ◽  
R. Amils
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Microbial Populations ◽  
Powerful Method ◽  
High Fluorescence ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
Slow Growing ◽  
Signal Intensities

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is a powerful method with a growing number of applications in the quantitative evaluation of microbial populations of complex ecosystems. CARD-FISH is an improvement over traditional fluorescence in situ hybridization (FISH) especially suitable for aquatic habitats with small, slow growing, or starving bacteria, in which the signal intensities of hybridized cells is frequently below detection limits or lost in high fluorescence background of dense mineral matrixes. In this work we report the development of protocols and probes for the identification and quantification of the microorganisms involved in the continuous bioleaching of a cobaltiferrous concentrate using a four tank-leaching reactor operated by BRGM. After steady state was reached, samples were taken to identify and quantify the microorganisms present in the each of the tanks used in the process.

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