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.

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).

scholarly journals Cytological Evidence for Association of the Ends of the Linear Chromosome in Streptomyces coelicolor

2001 ◽  
Vol 183 (17) ◽  
pp. 5180-5186 ◽  
Author(s):  
Melody C. Yang ◽  
Richard Losick
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Genetic Map ◽  
Streptomyces Coelicolor ◽  
Evidence Based ◽  
Filamentous Bacterium ◽  
Cytological Evidence ◽  
Circular Chromosome ◽  
Linear Chromosome

ABSTRACT The chromosome of the filamentous bacterium Streptomyces coelicolor is linear, but the genetic map is circular. We present cytological evidence based on the use of fluorescence in situ hybridization showing that the ends of the chromosome frequently colocalize, in agreement with the idea that the ends are held together, effectively forming a circular chromosome. These observations provide a possible explanation for how a linear bacterial chromosome can exhibit a circular genetic map.

scholarly journals Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

2009 ◽  
Vol 75 (8) ◽  
pp. 2446-2452 ◽  
Author(s):  
Lachlan Speirs ◽  
Tadashi Nittami ◽  
Simon McIlroy ◽  
Sarah Schroeder ◽  
Robert J. Seviour
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Molecular Data ◽  
Phosphate Removal ◽  
Filamentous Bacterium ◽  
Eastern Australia ◽  
Intracellular Aggregates ◽  
Rrna Sequences ◽  
16S Rrna Sequences

ABSTRACT Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-β-hydroxyalkanoates or polyphosphate.

scholarly journals Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

2004 ◽  
Vol 70 (9) ◽  
pp. 5426-5433 ◽  
Author(s):  
Annelie Pernthaler ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
False Negative ◽  
Proteinase K ◽  
Hybridization Temperature ◽  
Fish Samples ◽  
Different Types ◽  
Pure Cultures

ABSTRACT We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.

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