The Experts below are selected from a list of 183 Experts worldwide ranked by ideXlab platform
Christopher A. Scholin - One of the best experts on this subject based on the ideXlab platform.
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On detection of pseudo‐nitzschia (bacillariophyceae) species using whole Cell Hybridization: sample fixation and stability
Journal of Phycology, 2000Co-Authors: Peter E. Miller, Christopher A. ScholinAbstract:Some species within the genus Pseudo-nitzschia H. Peragallo are associated with production of domoic acid, the agent responsible for amnesic shellfish poisoning (ASP). Identification and enumeration of particular Pseudo-nitzschia in natural populations is often difficult and time consuming because of the need for detailed morphological observations, which often require scanning or transmission electron microscopy. In earlier publications we described the development of large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for discriminating among a variety of Pseudo-nitzschia species collected from Monterey Bay, California. Probes are applied using whole Cell Hybridization and a custom filtration manifold, enabling rapid identification and quantification of target species in cultured as well as field samples. In this work we compared a variety of preservation techniques and assessed the stability of stored samples with respect to their reactivity towards the probes. Of the preservatives tested, a saline ethanol-based treatment gave the best results in terms of probes yielding a bright and uniform Cell label. Culture samples treated with this fixative continued to react well with the probes for at least 6 weeks post-fixation whether stored in the preservative or dried post-preservation, with samples being kept at either room temperature or −20° C. Likewise, field samples containing a variety of diatoms and dinoflagellate species stored in the saline ethanol solution at room temperature were also stable for at least 4–6 weeks, reacting brilliantly towards a positive control probe. After prolonged storage, however, Cell reactivity towards the probes diminished dramatically. Post-Hybridization, samples stored at 4° C were found to retain their fluorescence for at least 1 week. These results indicate a wider window of opportunity for Pseudo-nitzschia analysis using whole Cell Hybridization than previously reported. Sample collection, preservation, and probing protocols optimized for Pseudo-nitzschia are also applicable to a wide range of phytoplankton species. The time required to execute the whole Cell Hybridization protocol was reduced by premixing probe with Hybridization buffer. The premixed probe solutions as well as fixative and wash solutions are all stable at room temperature for at least 6 weeks. Application of two different species-specific probes, each labeled with a different fluorochrome, allowed detection of two species on a single filter. The latter could be adopted in the future to increase the rate of sample processing and decrease the cost of sample analysis.
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identification and enumeration of cultured and wild pseudo nitzschia bacillariophyceae using species specific lsu rrna targeted fluorescent probes and filter based whole Cell Hybridization
Journal of Phycology, 1998Co-Authors: Peter E. Miller, Christopher A. ScholinAbstract:Efforts to understand the ecologic and environmental parameters that govern harmful algal blooms (HABs) require rapid and specific identification of causative species. Traditional methods of species identification using light and electron microscopy are useful in this regard but are also time consuming, making routine analysis of a large number of samples difficult. Techniques that speed and ease the detection of HAB species as they occur in natural populations are therefore desirable. In this paper, we continue efforts to develop species-specific large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for a variety of Pseudo-nitzschia H. Peragallo species, a group of marine pennate diatoms that includes representatives linked to production of domoic acid and amnesic shellfish poisoning (ASP). A custom filter tube and filtration manifold that has utility for both whole Cell (in situ) Hybridization as well as preparing samples for scanning electron microscopy (SEM) is described. Filter-based whole Cell Hybridization was used to identify a variety of newly isolated Pseudo-nitzschia clones, and probe results were confirmed using SEM. Some isolates of P. pungens (Grunow) Hasle exhibited variable (intraclonal) reactivity toward the P. pungens-specific probe. Three isolates of P. subpacifica (Hasle) Hasle were found to cross-react with probes designed for P. fraudulenta (Cleve) Hasle and P. heimii Manguin. Four isolates did not react with any species-specific probes; this group comprised three distinct morphotypes whose fine-scale morphologic features did not agree with published descriptions of Pseudo-nitzschia species. Evaluation of the filter method using cultured Cells added to natural (whole water) samples indicated quantitative recovery of target species. Confirming results of probe assays using SEM was difficult when the target species was less than 10 4 Cells·L 21 in the presence of greater than 106 Cells·L 21 of other nontarget diatom species. A variety of Pseudo-nitzschia, including P. australis Frenguelli, P. fraudulenta, P. heimii, P. pseudodelicatissima (Hasle) Hasle, P. pungens, P. multiseries (Hasle) Hasle, and Nitzschia americana Hasle, were identified using whole Cell Hybridization in a variety of field samples containing mixed assemblages of plankton, and these results were confirmed using SEM. The filter tube method of applying probes was used onboard ship for near real-time identification and enumeration of a variety of Pseudonitzschia species.
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IDENTIFICATION AND ENUMERATION OF CULTURED AND WILD PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES‐SPECIFIC LSU rRNA‐TARGETED FLUORESCENT PROBES AND FILTER‐BASED WHOLE Cell Hybridization
Journal of Phycology, 1998Co-Authors: Peter E. Miller, Christopher A. ScholinAbstract:Efforts to understand the ecologic and environmental parameters that govern harmful algal blooms (HABs) require rapid and specific identification of causative species. Traditional methods of species identification using light and electron microscopy are useful in this regard but are also time consuming, making routine analysis of a large number of samples difficult. Techniques that speed and ease the detection of HAB species as they occur in natural populations are therefore desirable. In this paper, we continue efforts to develop species-specific large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for a variety of Pseudo-nitzschia H. Peragallo species, a group of marine pennate diatoms that includes representatives linked to production of domoic acid and amnesic shellfish poisoning (ASP). A custom filter tube and filtration manifold that has utility for both whole Cell (in situ) Hybridization as well as preparing samples for scanning electron microscopy (SEM) is described. Filter-based whole Cell Hybridization was used to identify a variety of newly isolated Pseudo-nitzschia clones, and probe results were confirmed using SEM. Some isolates of P. pungens (Grunow) Hasle exhibited variable (intraclonal) reactivity toward the P. pungens-specific probe. Three isolates of P. subpacifica (Hasle) Hasle were found to cross-react with probes designed for P. fraudulenta (Cleve) Hasle and P. heimii Manguin. Four isolates did not react with any species-specific probes; this group comprised three distinct morphotypes whose fine-scale morphologic features did not agree with published descriptions of Pseudo-nitzschia species. Evaluation of the filter method using cultured Cells added to natural (whole water) samples indicated quantitative recovery of target species. Confirming results of probe assays using SEM was difficult when the target species was less than 10 4 Cells·L 21 in the presence of greater than 106 Cells·L 21 of other nontarget diatom species. A variety of Pseudo-nitzschia, including P. australis Frenguelli, P. fraudulenta, P. heimii, P. pseudodelicatissima (Hasle) Hasle, P. pungens, P. multiseries (Hasle) Hasle, and Nitzschia americana Hasle, were identified using whole Cell Hybridization in a variety of field samples containing mixed assemblages of plankton, and these results were confirmed using SEM. The filter tube method of applying probes was used onboard ship for near real-time identification and enumeration of a variety of Pseudonitzschia species.
Josef Zeyer - One of the best experts on this subject based on the ideXlab platform.
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In situ Detection of Spores and Vegetative Cells of Bacillus megaterium in Soil by Whole Cell Hybridization
Systematic and Applied Microbiology, 2011Co-Authors: Kathrin Fischer, Ditmar Hahn, Wolfgang Hönerlage, Frank Sch Nholzer, Josef ZeyerAbstract:Summary Spores and vegetative Cells of Bacillus megaterium were detected in liquid culture and in soil by DAPI staining and whole Cell Hybridization with fluorescent oligonucleotide probes after special permeabilizanon. A SDS/DTT treatment (10 mg ml -1 SDS, 50 mM dithiothreitol at 65 °C for 30 minutes) significantly enhanced DAPI staining of spores. An additional lysozyme treatment (0.1% at 37°C for 20 minutes) made them even permeable for oligonucleotides as indicated by specific Hybridization of a bacterial probe. Vegetative Cells were permeable for DAPI, however, they required treatments with lysozyme (0.1% at 25 °C for 10 minutes) to allow permeation of oligonucleotides. The differential permeabilization of vegetative Cells by lysozyme treatment alone and of spores and vegetative Cells by the combination of SDS/DTT and subsequent lysozyme pretreatment facilitated quantification of spores and vegetative Cells of B. megaterium growing in soil. Counts for spores and vegetative Cells were generally higher (up to two orders of magnitude) than those obtained by colony forming units. The described approach offers a promising tool for quantitative autecological studies on bacilli is heterogeneous environments.
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Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-Cell Hybridization
FEMS Microbiology Ecology, 2001Co-Authors: Oliver Pelz, Antonis Chatzinotas, Annatina Zarda-hess, Wolf-rainer Abraham, Josef ZeyerAbstract:Polar lipid-derived fatty acids (PLFA) commonly found in sulfate-reducing bacteria were detected in high abundance in the sediment harvested from a monitoring well of a petroleum-hydrocarbon (PHC)-contaminated aquifer. Aquifer microcosms were incubated under sulfate-reducing conditions with [methyl-14C]toluene to determine the 14C-mass balances and with [methyl-13C]toluene to follow the flow of carbon from toluene into biomarker fatty acids. An aliquot was used to establish an aquifer-derived toluene-degrading sulfate-reducing consortium, which grew well in liquid medium. Whole-Cell Hybridization using 16S rRNA-targeted oligonucleotide probes specific for different phylogenetic levels within the sulfate-reducing bacteria was applied in order to characterize the sulfate-reducing populations in the original sediment, the aquifer microcosms, and the aquifer-derived consortium. In the aquifer microcosms, the 14C quantification revealed that 61.6% of the [methyl-14C]toluene was mineralized and 2.7% was assimilated. Following [methyl-13C]toluene depletion (
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Whole Cell Hybridization as a tool to study Frankia populations in root nodules
Physiologia Plantarum, 1997Co-Authors: Dittmar Hahn, Kornelia Zepp, Josef ZeyerAbstract:Molecular methods based on DNA or rRNA Hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising alternative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole Cell Hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial Cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin-labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes-specific insertion on the 23S rRNA are used. Digoxigenin-labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of Cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.
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Evaluation of a 23S rRNA Insertion as Target for the Analysis of Uncultured Frankia Populations in Root Nodules of Alders by Whole Cell Hybridization
Systematic and Applied Microbiology, 1997Co-Authors: Kornelia Zepp, Dittmar Hahn, Josef ZeyerAbstract:Summary An actinomycetes-specific insertion in domain III of the 23S rRNA was used as target for the analysis of uncultured Frankia populations in nodule homogenates of alders by whole Cell Hybridization. Fluorescent, Cy3-labeled oligonucleotide probes enabled detection of filaments and vesicles without permeabilization whereas detection of spores required a previous permeabilization with lysozyme. The signal intensity obtained on spores, however, remained quite low and their detection was not quantitative. Digoxigenin-labeled probes also allowed the detection of filaments, vesicles and spores of Frankia . However, for all Cell types a previous permeabilization with SDS/DTT and an additional incubation with lysozyme was necessary. Hybrid detection with antibody/alkaline phosphatase conjugates and NBT/BCIP as substrate resulted in clear images of filaments, vesicles and spores and in Cell numbers comparable to those obtained by fluorescent probes. The analysis of Frankia populations in nodule homogenates of different alders ( Alnus glutinosa, A. incana, A. viridis and A. nepalensis ) could be performed on subgroup-level with both oligonucleotide as well as in vitro transcript probes. The analysis revealed the presence of only one Frankia population in every nodule homogenate. Filaments and vesicles in nodules of the spore (-) type as well as filaments, vesicles and spores in nodules of the spore (+) type always belonged to the same group. Populations in nodules of the spore (-) type were usually identified as Frankia population belonging to group IV of the Alnus host infection group, with the exception of the Frankia population in nodules of A. nepalensis which belonged to group IIIa. Nodules of the spore (+) type contained Frankia populations either belonging to group IIIa or to group IV.
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In situ analysis of the bacterial community in the gut of the earthworm Lumbricus terrestris L. by whole-Cell Hybridization
Canadian Journal of Microbiology, 1995Co-Authors: Kathrin Fischer, Dittmar Hahn, Josef Zeyer, Otto Daniel, Rudolf AmannAbstract: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 signi...
Peter E. Miller - One of the best experts on this subject based on the ideXlab platform.
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On detection of pseudo‐nitzschia (bacillariophyceae) species using whole Cell Hybridization: sample fixation and stability
Journal of Phycology, 2000Co-Authors: Peter E. Miller, Christopher A. ScholinAbstract:Some species within the genus Pseudo-nitzschia H. Peragallo are associated with production of domoic acid, the agent responsible for amnesic shellfish poisoning (ASP). Identification and enumeration of particular Pseudo-nitzschia in natural populations is often difficult and time consuming because of the need for detailed morphological observations, which often require scanning or transmission electron microscopy. In earlier publications we described the development of large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for discriminating among a variety of Pseudo-nitzschia species collected from Monterey Bay, California. Probes are applied using whole Cell Hybridization and a custom filtration manifold, enabling rapid identification and quantification of target species in cultured as well as field samples. In this work we compared a variety of preservation techniques and assessed the stability of stored samples with respect to their reactivity towards the probes. Of the preservatives tested, a saline ethanol-based treatment gave the best results in terms of probes yielding a bright and uniform Cell label. Culture samples treated with this fixative continued to react well with the probes for at least 6 weeks post-fixation whether stored in the preservative or dried post-preservation, with samples being kept at either room temperature or −20° C. Likewise, field samples containing a variety of diatoms and dinoflagellate species stored in the saline ethanol solution at room temperature were also stable for at least 4–6 weeks, reacting brilliantly towards a positive control probe. After prolonged storage, however, Cell reactivity towards the probes diminished dramatically. Post-Hybridization, samples stored at 4° C were found to retain their fluorescence for at least 1 week. These results indicate a wider window of opportunity for Pseudo-nitzschia analysis using whole Cell Hybridization than previously reported. Sample collection, preservation, and probing protocols optimized for Pseudo-nitzschia are also applicable to a wide range of phytoplankton species. The time required to execute the whole Cell Hybridization protocol was reduced by premixing probe with Hybridization buffer. The premixed probe solutions as well as fixative and wash solutions are all stable at room temperature for at least 6 weeks. Application of two different species-specific probes, each labeled with a different fluorochrome, allowed detection of two species on a single filter. The latter could be adopted in the future to increase the rate of sample processing and decrease the cost of sample analysis.
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identification and enumeration of cultured and wild pseudo nitzschia bacillariophyceae using species specific lsu rrna targeted fluorescent probes and filter based whole Cell Hybridization
Journal of Phycology, 1998Co-Authors: Peter E. Miller, Christopher A. ScholinAbstract:Efforts to understand the ecologic and environmental parameters that govern harmful algal blooms (HABs) require rapid and specific identification of causative species. Traditional methods of species identification using light and electron microscopy are useful in this regard but are also time consuming, making routine analysis of a large number of samples difficult. Techniques that speed and ease the detection of HAB species as they occur in natural populations are therefore desirable. In this paper, we continue efforts to develop species-specific large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for a variety of Pseudo-nitzschia H. Peragallo species, a group of marine pennate diatoms that includes representatives linked to production of domoic acid and amnesic shellfish poisoning (ASP). A custom filter tube and filtration manifold that has utility for both whole Cell (in situ) Hybridization as well as preparing samples for scanning electron microscopy (SEM) is described. Filter-based whole Cell Hybridization was used to identify a variety of newly isolated Pseudo-nitzschia clones, and probe results were confirmed using SEM. Some isolates of P. pungens (Grunow) Hasle exhibited variable (intraclonal) reactivity toward the P. pungens-specific probe. Three isolates of P. subpacifica (Hasle) Hasle were found to cross-react with probes designed for P. fraudulenta (Cleve) Hasle and P. heimii Manguin. Four isolates did not react with any species-specific probes; this group comprised three distinct morphotypes whose fine-scale morphologic features did not agree with published descriptions of Pseudo-nitzschia species. Evaluation of the filter method using cultured Cells added to natural (whole water) samples indicated quantitative recovery of target species. Confirming results of probe assays using SEM was difficult when the target species was less than 10 4 Cells·L 21 in the presence of greater than 106 Cells·L 21 of other nontarget diatom species. A variety of Pseudo-nitzschia, including P. australis Frenguelli, P. fraudulenta, P. heimii, P. pseudodelicatissima (Hasle) Hasle, P. pungens, P. multiseries (Hasle) Hasle, and Nitzschia americana Hasle, were identified using whole Cell Hybridization in a variety of field samples containing mixed assemblages of plankton, and these results were confirmed using SEM. The filter tube method of applying probes was used onboard ship for near real-time identification and enumeration of a variety of Pseudonitzschia species.
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IDENTIFICATION AND ENUMERATION OF CULTURED AND WILD PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES‐SPECIFIC LSU rRNA‐TARGETED FLUORESCENT PROBES AND FILTER‐BASED WHOLE Cell Hybridization
Journal of Phycology, 1998Co-Authors: Peter E. Miller, Christopher A. ScholinAbstract:Efforts to understand the ecologic and environmental parameters that govern harmful algal blooms (HABs) require rapid and specific identification of causative species. Traditional methods of species identification using light and electron microscopy are useful in this regard but are also time consuming, making routine analysis of a large number of samples difficult. Techniques that speed and ease the detection of HAB species as they occur in natural populations are therefore desirable. In this paper, we continue efforts to develop species-specific large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for a variety of Pseudo-nitzschia H. Peragallo species, a group of marine pennate diatoms that includes representatives linked to production of domoic acid and amnesic shellfish poisoning (ASP). A custom filter tube and filtration manifold that has utility for both whole Cell (in situ) Hybridization as well as preparing samples for scanning electron microscopy (SEM) is described. Filter-based whole Cell Hybridization was used to identify a variety of newly isolated Pseudo-nitzschia clones, and probe results were confirmed using SEM. Some isolates of P. pungens (Grunow) Hasle exhibited variable (intraclonal) reactivity toward the P. pungens-specific probe. Three isolates of P. subpacifica (Hasle) Hasle were found to cross-react with probes designed for P. fraudulenta (Cleve) Hasle and P. heimii Manguin. Four isolates did not react with any species-specific probes; this group comprised three distinct morphotypes whose fine-scale morphologic features did not agree with published descriptions of Pseudo-nitzschia species. Evaluation of the filter method using cultured Cells added to natural (whole water) samples indicated quantitative recovery of target species. Confirming results of probe assays using SEM was difficult when the target species was less than 10 4 Cells·L 21 in the presence of greater than 106 Cells·L 21 of other nontarget diatom species. A variety of Pseudo-nitzschia, including P. australis Frenguelli, P. fraudulenta, P. heimii, P. pseudodelicatissima (Hasle) Hasle, P. pungens, P. multiseries (Hasle) Hasle, and Nitzschia americana Hasle, were identified using whole Cell Hybridization in a variety of field samples containing mixed assemblages of plankton, and these results were confirmed using SEM. The filter tube method of applying probes was used onboard ship for near real-time identification and enumeration of a variety of Pseudonitzschia species.
Dittmar Hahn - One of the best experts on this subject based on the ideXlab platform.
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Whole Cell Hybridization as a tool to study Frankia populations in root nodules
Physiologia Plantarum, 1997Co-Authors: Dittmar Hahn, Kornelia Zepp, Josef ZeyerAbstract:Molecular methods based on DNA or rRNA Hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising alternative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole Cell Hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial Cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin-labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes-specific insertion on the 23S rRNA are used. Digoxigenin-labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of Cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.
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Evaluation of a 23S rRNA Insertion as Target for the Analysis of Uncultured Frankia Populations in Root Nodules of Alders by Whole Cell Hybridization
Systematic and Applied Microbiology, 1997Co-Authors: Kornelia Zepp, Dittmar Hahn, Josef ZeyerAbstract:Summary An actinomycetes-specific insertion in domain III of the 23S rRNA was used as target for the analysis of uncultured Frankia populations in nodule homogenates of alders by whole Cell Hybridization. Fluorescent, Cy3-labeled oligonucleotide probes enabled detection of filaments and vesicles without permeabilization whereas detection of spores required a previous permeabilization with lysozyme. The signal intensity obtained on spores, however, remained quite low and their detection was not quantitative. Digoxigenin-labeled probes also allowed the detection of filaments, vesicles and spores of Frankia . However, for all Cell types a previous permeabilization with SDS/DTT and an additional incubation with lysozyme was necessary. Hybrid detection with antibody/alkaline phosphatase conjugates and NBT/BCIP as substrate resulted in clear images of filaments, vesicles and spores and in Cell numbers comparable to those obtained by fluorescent probes. The analysis of Frankia populations in nodule homogenates of different alders ( Alnus glutinosa, A. incana, A. viridis and A. nepalensis ) could be performed on subgroup-level with both oligonucleotide as well as in vitro transcript probes. The analysis revealed the presence of only one Frankia population in every nodule homogenate. Filaments and vesicles in nodules of the spore (-) type as well as filaments, vesicles and spores in nodules of the spore (+) type always belonged to the same group. Populations in nodules of the spore (-) type were usually identified as Frankia population belonging to group IV of the Alnus host infection group, with the exception of the Frankia population in nodules of A. nepalensis which belonged to group IIIa. Nodules of the spore (+) type contained Frankia populations either belonging to group IIIa or to group IV.
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In situ analysis of the bacterial community in the gut of the earthworm Lumbricus terrestris L. by whole-Cell Hybridization
Canadian Journal of Microbiology, 1995Co-Authors: Kathrin Fischer, Dittmar Hahn, Josef Zeyer, Otto Daniel, Rudolf AmannAbstract: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 signi...
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Detection of mRNA of nprM in Bacillus megaterium ATCC 14581 grown in soil by whole-Cell Hybridization
Archives of Microbiology, 1995Co-Authors: Wolfgang Hönerlage, Dittmar Hahn, Josef ZeyerAbstract:Transcripts of nprM , the gene encoding the major extraCellular protease of Bacillus megaterium ATCC 14581, were detected by both Northern blot analysis and whole-Cell Hybridization with digoxigenin-labeled in vitro ranscripts throughout the exponential growth phase and the early stationary phase. In Cells of the late stationary phase, only low amounts of transcripts were observed with the two techniques. No transcripts could be detected in spores. In soil the presence of mRNA of nprM could be demonstrated by whole-Cell Hybridization in growing Cells germinated from heat-activated spores until they reached the late transition state. No transcripts of nprM were detected in Cells containing forespores. Both Cells grown in pure culture and in soil had to be permeabilized with lysozyme to allow Hybridization with digoxigeninlabeled probes. These results demonstrate the applicability of nucleic-acid probing techniques to localize microbial processes in soil. The approach described of detecting mRNA in fixed bacterial Cells should facilitate in situ studies of gene transcription and specific activities in individual Cells in heterogeneous environmental systems.
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detection of mrna in streptomyces Cells by whole Cell Hybridization with digoxigenin labeled probes
Applied and Environmental Microbiology, 1993Co-Authors: Dittmar Hahn, Rudolf Amann, Josef ZeyerAbstract:Abstract Detection of mRNA of the thiostrepton resistance gene (tsr) harbored by plasmid pIJ673 in Streptomyces violacelatus was achieved by whole-Cell Hybridization with digoxigenin-labeled in vitro transcripts followed by an antibody-alkaline phosphatase detection of the digoxigenin reporter molecule. Prior to Hybridization, the Cells had to be permeabilized by lysozyme, the detergent Nonidet P-40, and toluene. The permeability of the S. violacelatus Cells for probes and the antibody-alkaline phosphatase conjugate was demonstrated by Hybridization with digoxigenin-labeled, 16S rRNA-targeted oligonucleotides.
Kornelia Zepp - One of the best experts on this subject based on the ideXlab platform.
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Whole Cell Hybridization as a tool to study Frankia populations in root nodules
Physiologia Plantarum, 1997Co-Authors: Dittmar Hahn, Kornelia Zepp, Josef ZeyerAbstract:Molecular methods based on DNA or rRNA Hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising alternative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole Cell Hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial Cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin-labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes-specific insertion on the 23S rRNA are used. Digoxigenin-labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of Cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.
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Evaluation of a 23S rRNA Insertion as Target for the Analysis of Uncultured Frankia Populations in Root Nodules of Alders by Whole Cell Hybridization
Systematic and Applied Microbiology, 1997Co-Authors: Kornelia Zepp, Dittmar Hahn, Josef ZeyerAbstract:Summary An actinomycetes-specific insertion in domain III of the 23S rRNA was used as target for the analysis of uncultured Frankia populations in nodule homogenates of alders by whole Cell Hybridization. Fluorescent, Cy3-labeled oligonucleotide probes enabled detection of filaments and vesicles without permeabilization whereas detection of spores required a previous permeabilization with lysozyme. The signal intensity obtained on spores, however, remained quite low and their detection was not quantitative. Digoxigenin-labeled probes also allowed the detection of filaments, vesicles and spores of Frankia . However, for all Cell types a previous permeabilization with SDS/DTT and an additional incubation with lysozyme was necessary. Hybrid detection with antibody/alkaline phosphatase conjugates and NBT/BCIP as substrate resulted in clear images of filaments, vesicles and spores and in Cell numbers comparable to those obtained by fluorescent probes. The analysis of Frankia populations in nodule homogenates of different alders ( Alnus glutinosa, A. incana, A. viridis and A. nepalensis ) could be performed on subgroup-level with both oligonucleotide as well as in vitro transcript probes. The analysis revealed the presence of only one Frankia population in every nodule homogenate. Filaments and vesicles in nodules of the spore (-) type as well as filaments, vesicles and spores in nodules of the spore (+) type always belonged to the same group. Populations in nodules of the spore (-) type were usually identified as Frankia population belonging to group IV of the Alnus host infection group, with the exception of the Frankia population in nodules of A. nepalensis which belonged to group IIIa. Nodules of the spore (+) type contained Frankia populations either belonging to group IIIa or to group IV.