Monitoring of microbial diversity by fluorescence in situ hybridization and fluorescence spectrometry

2003 ◽  
Vol 47 (5) ◽  
pp. 133-138 ◽  
Author(s):  
V. Ivanov ◽  
S.T.-L. Tay ◽  
J.-H. Tay
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Environmental Samples ◽  
Specific Binding ◽  
Oligonucleotide Probes ◽  
Simple Method ◽  
Total Binding ◽  
Unlabeled Probe ◽  
Microbial Groups

The goal of the research was the development of a simple method to quantify microbial groups in environmental samples. Fluorescence intensity was measured in the sample before and after whole cell fluorescence in situ hybridization with rRNA-targeted, fluorochrome-labeled oligonucleotide probes. To determine specific and non-specific binding of different oligonucleotide probes the following approaches have been used: (1) incubation of the sample with probes at two different temperatures; (2) hybridization of labeled probe in the presence of unlabeled probe; (3) incubation of the sample with labeled specific probe or labeled nonsense probe. Specific binding (hybridization) of the probe was calculated as the difference between total binding and non-specific binding of the probe. Specific binding was 40-50% of total binding in the environmental samples tested. The ratio of the specific binding of different probes may be used to quantify the ratio of different microbial groups in the environmental samples. This quantification is suitable for the microbiological monitoring of microbial aggregates because it is a simple technique and the results can be measured by a portable fluorometer.

scholarly journals The Polymorphisms of Oligonucleotide Probes in Wheat Cultivars Determined by ND-FISH

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1126 ◽  
Author(s):  
Tianheng Ren ◽  
Maojie He ◽  
Zixin Sun ◽  
Feiquan Tan ◽  
Peigao Luo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Wheat Genome ◽  
Oligonucleotide Probes ◽  
Wheat Cultivars ◽  
Fish Signal

Non-denaturing fluorescence in situ hybridization (ND-FISH) has been used to distinguish wheat chromosomes and to detect alien chromosomes in the wheat genome. In this study, five different oligonucleotide probes were used with ND-FISH to examine 21 wheat cultivars and lines. These oligonucleotide probes distinguished 42 wheat chromosomes and also detected rye chromatin in the wheat genome. Moreover, the signal patterns of the oligonucleotide probes Oligo-pTa535-1 and Oligo-pSc119.2-1 showed high polymorphism in the wheat chromosomes. A total of 17.6% of the A group chromosomes, 25.9% of the B group chromosomes and 8.9% of the D group chromosomes showed obvious mutations when they were compared to the standard ND-FISH signal patterns, and most of them were Oligo-pSc119.2-1 mutants. The results suggested that these polymorphisms could be induced by the crossing of wheat cultivars. The results provided more information for the further application of oligonucleotide probes and ND-FISH.

scholarly journals Automated Image Analysis for Quantitative Fluorescence In Situ Hybridization with Environmental Samples

2007 ◽  
Vol 73 (9) ◽  
pp. 2956-2962 ◽  
Author(s):  
Zhi Zhou ◽  
Marie Noëlle Pons ◽  
Lutgarde Raskin ◽  
Julie L. Zilles
Keyword(s):  
Image Analysis ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Soil Sample ◽  
Environmental Samples ◽  
Swine Manure ◽  
Automated Image Analysis ◽  
Cell Aggregates ◽  
Manual Counting

ABSTRACT When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4′,6′-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.

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).

Composition of the bacterial sludge in occluded biliary stents characterized by fluorescence in situ hybridization with Rna-based oligonucleotide probes

2003 ◽  
Vol 124 (4) ◽  
pp. A246
Author(s):  
Alexander Swidsinski ◽  
Petra Schlien ◽  
Sonja Swidsinski ◽  
Guido Schachschal ◽  
Vera Loening Baucke ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Oligonucleotide Probes ◽  
Biliary Stents

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 Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis

2011 ◽  
Vol 19 (7) ◽  
pp. 901-909 ◽  
Author(s):  
Shelagh Boyle ◽  
Matthew J. Rodesch ◽  
Heather A. Halvensleben ◽  
Jeffrey A. Jeddeloh ◽  
Wendy A. Bickmore
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Parallel Synthesis ◽  
Massively Parallel ◽  
Oligonucleotide Probes ◽  
High Complexity

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.

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