scholarly journals Effect of Signal Compounds and Incubation Conditions on the Culturability of Freshwater Bacterioplankton

2003 ◽  
Vol 69 (4) ◽  
pp. 1980-1989 ◽  
Author(s):  
Alke Bruns ◽  
Ulrich Nübel ◽  
Heribert Cypionka ◽  
Jörg Overmann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Eutrophic Lake ◽  
Homoserine Lactone ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number ◽  
Culture Independent ◽  
Effective Signal

ABSTRACT The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months. Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 μM) of the signal compounds N-(oxohexanoyl)-dl-homoserine lactone, N-(butyryl)-dl-homoserine lactone, cyclic AMP (cAMP), or ATP. cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts. Microautoradiography with [2,8-3H]cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells. The bacterial cAMP uptake systems had a very low Km value of ≤1 nM. Analysis of the cultured bacteria by 16S rRNA gene fingerprinting showed that different bacterial phylotypes were recovered in the presence and in the absence of cAMP. Consequently, the addition of cAMP caused a stimulation of otherwise nonculturable bacteria. Phylogenetically different bacteria were also recovered at different temperatures and oxygen partial pressures. Throughout the study period, mainly members of the β-subclass of the Proteobacteria were cultivated. In addition, some members of the Actinomycetales were enriched. Quantification by culture-independent fluorescence in situ hybridization demonstrated that β-Proteobacteria and Actinomycetales also dominated the natural bacterioplankton assemblage. Sequence comparison revealed that two members of the Actinomycetales which reached high numbers in the natural bacterioplankton assemblage could actually be enriched by our cultivation approach.

scholarly journals Abundance and Phylogenetic Affiliation of Iron Reducers in Activated Sludge as Assessed by Fluorescence In Situ Hybridization and Microautoradiography

2002 ◽  
Vol 68 (9) ◽  
pp. 4629-4636 ◽  
Author(s):  
Jeppe Lund Nielsen ◽  
Stefan Juretschko ◽  
Michael Wagner ◽  
Per Halkjær Nielsen
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Most Probable Number ◽  
Probable Number ◽  
Iron Reducing Bacteria ◽  
Reducing Conditions ◽  
Phylogenetic Affiliation ◽  
Reducing Bacteria

ABSTRACT Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 × 108 cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4′,6′-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.

scholarly journals Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection

2009 ◽  
Vol 75 (5) ◽  
pp. 1450-1455 ◽  
Author(s):  
Bledar Bisha ◽  
Byron F. Brehm-Stecher
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Adhesive Tape ◽  
Selective Agar ◽  
Culture Independent

ABSTRACT A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.

scholarly journals Karyotype and Fluorescence In Situ Hybridization Analysis of 15 Lilium Species from China

2017 ◽  
Vol 142 (4) ◽  
pp. 298-305 ◽  
Author(s):  
Guangxin Liu ◽  
Xiaoling Zhang ◽  
Yue Lan ◽  
Haoyang Xin ◽  
Fengrong Hu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Diploid Species ◽  
Hybridization Analysis ◽  
Rrna Gene ◽  
Fish Analysis ◽  
Lilium Species ◽  
Three Samples ◽  
Lilium Lancifolium

Karyotype comparison and fluorescence in situ hybridization (FISH) were conducted to analyze the wild Lilium species distributed in China. The karyotype results revealed that all species except Lilium lancifolium (2n = 3X = 36) were diploid and had two pairs of metacentric or submetacentric chromosomes. The karyotypes of all species are similar. FISH analysis revealed that there are 5–12 45S rRNA gene loci dispersed on the chromosomes of the 14 diploid species, and 15 45S rRNA gene loci were detected in the triploid species L. lancifolium. Most of the FISH signals were detected on the long arms and the centromeric regions. Three samples of L. brownii [Hubei, China (lat. 31°28′N, long. 110°23′E); Liaoning, China (lat. 40°07′N, long. 124°19′E); and Guangxi, China (lat. 25°06′N, long. 107°27′E)] showed very similar chromosome patterns in both the karyotype and the FISH analyses, further demonstrating that these samples belonged to the same species. L. brownii is widely distributed in China from latitude 25°06′N to 40°07′N, indicating that it is highly adaptable to the environment.

Detection of WWE2-relatedLentisphaeraeby 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

2010 ◽  
Vol 56 (10) ◽  
pp. 846-852 ◽  
Author(s):  
Rim Driss Limam ◽  
Théodore Bouchez ◽  
Rakia Chouari ◽  
Tianlun Li ◽  
Insaf Barkallah ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Fluorescence In Situ Hybridization ◽  
Landfill Leachate ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Victivallis Vadensis

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries

2002 ◽  
Vol 4 (11) ◽  
pp. 713-720 ◽  
Author(s):  
Andreas Schramm ◽  
Bernhard M. Fuchs ◽  
Jeppe L. Nielsen ◽  
Mauro Tonolla ◽  
David A. Stahl
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Fluorescence In Situ Hybridization ◽  
Rrna Gene ◽  
Clone Libraries

Molecular analyses of the diversity in marine bacterioplankton assemblages along the coastline of the northeastern Gulf of Mexico

2010 ◽  
Vol 56 (10) ◽  
pp. 853-863 ◽  
Author(s):  
Ola A. Olapade
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gulf Of Mexico ◽  
Fluorescence In Situ Hybridization ◽  
Community Diversity ◽  
Rrna Gene ◽  
Marine Bacterioplankton ◽  
Northeastern Gulf Of Mexico

Bacterial community diversity in marine bacterioplankton assemblages were examined in 3 coastal locations along the northeastern Gulf of Mexico (GOM) using 16S rRNA gene libraries and fluorescence in situ hybridization approaches. The majority of the sequences (30%–60%) were similar to the 16S rRNA gene sequences of unknown bacteria; however, the operational taxonomic units from members of the Cyanobacteria, Proteobacteria, and Bacteroidetes were also present at the 3 GOM sites. Overall, sequence diversity was more similar between the Gulf sites of Carrabelle and Ochlockonee than between either of the Gulf sites and Apalachicola Bay. Fluorescence in situ hybridization analyses revealed the quantitative predominance of members of the Alphaproteobacteria subclass and the Cytophaga – Flavobacterium cluster within the bacterioplankton assemblages. In general, the study further reveals the presence of many bacterial taxa that have been previously found to be dominant in coastal marine environments. Differences observed in the representation of the various bacterial phylogenetic groups among the GOM coastal sites could be partly attributed to dynamic variations in several site-specific conditions, including intermittent tidal events, nutrient availability, and anthropogenic influences.

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