Structure and function of nitrifying biofilms as determined by molecular techniques and the use of microelectrodes

2002 ◽  
Vol 46 (1-2) ◽  
pp. 233-241 ◽  
Author(s):  
S. Okabe ◽  
H. Naitoh ◽  
H. Satoh ◽  
Y. Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Bacterial Community ◽  
Spatial Organization ◽  
Domestic Wastewater ◽  
Rotating Disk ◽  
Molecular Techniques ◽  
Bacterial Populations ◽  
Steady State Condition ◽  
Synthetic Media

The phylogenetic diversity of a nitrifying bacterial community of two types of nitrifying biofilms, a domestic wastewater biofilm and an autotrophic nitrifying biofilm grown on rotating disk reactors (RDR), was characterized by 16S ribosomal DNA (rDNA)-cloning analysis. Thereafter, successional development of nitrifying the bacterial community within both biofilms was visualized in situ by fluorescent in situ hybridization (FISH) with a set of fluorescently labeled 16S rRNA-targeted DNA probes. In situ hybridization revealed that Nitrosomonas ureae was the numerically dominant species of the ammonia-oxidizing population in the domestic wastewater biofilm and that a population shift from N. urea to N. europaea and N. eutropha occurred when the culture medium was switched to the synthetic media from the domestic wastewater. After reaching the steady-state condition, microprofiles of NH4+, NO2−, NO3−, and O2 in the biofilms were measured by use of microsensors, and the spatial distributions of in situ nitrifying activities were determined. The relationship between the spatial organization of nitrifying bacterial populations and the in situ activity of these populations within the biofilms was discussed. Microelectrode measurements revealed that the active ammonia-oxidizing zone was vertically separated from the active nitrite-oxidizing zone. This vertical separation became more evident with increase of the substrate C/N ratio, leading to deterioration of nitrification efficiency. The combined use of these techniques made it possible to relate in situ nitrifying activity directly to the occurrence of nitrifying bacterial populations.

Structure and function of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes

2000 ◽  
Vol 42 (12) ◽  
pp. 21-32 ◽  
Author(s):  
S. Okabe ◽  
Y. Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Spatial Organization ◽  
Settling Tank ◽  
Outer Part ◽  
Domestic Wastewater ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Bacterial Populations ◽  
Nitrite Oxidizing Bacteria

Time dependent development of the spatial organization of NH4+- and NO2−-oxidizing bacterial populations in a domestic wastewater biofilm and in an autotrophic nitrifying biofilm were investigated by fluorescent in situ hybridization (FISH) with a set of 16S rRNA-targeted oligonucleotide probes. Population dynamics of nitrifying bacteria in the biofilms were correlated with the biofilm performance. In situ hybridization indicated that Nitrosomonas spp. (excluding probe NEU stained NH4+-oxidizing bacteria: i.e., N. marina-lineage, N. europaea-lineage, N. eutropha, and N. halophila) and Nitrospira-like bacteria were the numerically dominant nitrifying species in the domestic wastewater biofilm. However, probe NEU stained NH4+-inoxidizing bacteria became dominant populations in the autotrophic nitrifying biofilm (which were initially cultured with the primary settling tank effluent) after switching to the synthetic media. This population shift might be attributed to the effect of NO2−-–N accumulation and higher growth rates of N. europaea-lineage and N. eutropha, outcompeting other Nitrosomonas spp. in the synthetic medium. This evidence indirectly supports that N. europhaea has been most commonly isolated and studied in most of the previous researches. For the spatial organization of NH4+- and NO2−-oxidizing bacterial populations, bacteria of the genus Nitrobacter could not be detected, instead Nitrospira-like bacteria were found as the main nitrite-oxidizing bacteria in both biofilms. Whereas most of the ammonia-oxidizing bacteria were found throughout the biofilms, the location of nitrite-oxidizing bacteria was restricted to the active nitrite-oxidizing zone, which was detected in the inner part of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of NO2−-oxidizing bacteria, as determined with FISH. These observations have considerable significance to our understanding of microbial nitrification occurring in wastewater treatment processes and in the natural environment.

scholarly journals In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

1999 ◽  
Vol 65 (7) ◽  
pp. 3182-3191 ◽  
Author(s):  
Satoshi Okabe ◽  
Hisashi Satoh ◽  
Yoshimasa Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Spatial Organization ◽  
Outer Part ◽  
Domestic Wastewater ◽  
Ammonia Oxidizing Bacteria ◽  
Bacterial Populations ◽  
Nitrite Oxidizing Bacteria ◽  
Microelectrode Measurements ◽  
Inner Parts

ABSTRACT We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.

scholarly journals New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1819
Author(s):  
Tatyana Karamysheva ◽  
Svetlana Romanenko ◽  
Alexey Makunin ◽  
Marija Rajičić ◽  
Alexey Bogdanov ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sex Chromosomes ◽  
Interphase Nucleus ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Dna Probes ◽  
B Chromosomes ◽  
Apodemus Flavicollis ◽  
Yellow Necked Mouse

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

scholarly journals Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

1991 ◽  
Vol 39 (11) ◽  
pp. 1495-1506 ◽  
Author(s):  
P M Motte ◽  
R Loppes ◽  
M Menager ◽  
R Deltour
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Spatial Organization ◽  
Higher Plants ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Thin Sections ◽  
Cytoplasmic Dna ◽  
Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

scholarly journals Human Ring Chromosomes – New Insights for their Clinical Significance

2013 ◽  
Vol 16 (1) ◽  
pp. 13-19 ◽  
Author(s):  
R.S. Guilherme ◽  
E Klein ◽  
A.B. Hamid ◽  
S Bhatt ◽  
M Volleth ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Clinical Signs ◽  
Ring Chromosome ◽  
Signs And Symptoms ◽  
Molecular Techniques ◽  
Ring Chromosomes ◽  
Multicolor Banding ◽  
Clinical Signs And Symptoms ◽  
Clinical Problems

Abstract Twenty-nine as yet unreported ring chromosomes were characterized in detail by cytogenetic and molecular techniques. For FISH (fluorescence in situ hybridization) previously published high resolution approaches such as multicolor banding (MCB), subcentromere-specific multi-color-FISH (cenM-FISH) and two to three-color-FISH applying locus-specific probes were used. Overall, ring chromosome derived from chromosomes 4 (one case), 10 (one case), 13 (five cases), 14, (three cases), 18 (two cases), 21 (eight cases), 22 (three cases), X (five cases) and Y (one case) were studied. Eight cases were detected prenatally, eight due developmental delay and dysmorphic signs, and nine in connection with infertility and/or Turner syndrome. In general, this report together with data from the literature, supports the idea that ring chromosome patients fall into two groups: group one with (severe) clinical signs and symptoms due to the ring chromosome and group two with no obvious clinical problems apart from infertility.

Quality Assessment of Genetic Markers Used for Therapy Stratification

2003 ◽  
Vol 21 (11) ◽  
pp. 2077-2084 ◽  
Author(s):  
I.M. Ambros ◽  
J. Benard ◽  
M. Boavida ◽  
N. Bown ◽  
H. Caron ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Tumor Cell ◽  
Quality Assessment ◽  
Genetic Markers ◽  
Molecular Genetic ◽  
Molecular Techniques ◽  
Cell Content ◽  
Therapeutic Decisions ◽  
Therapy Stratification

Purpose: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. Materials and Methods: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. Results: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. Conclusion: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

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