scholarly journals Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization

2004 ◽  
Vol 70 (10) ◽  
pp. 6210-6219 ◽  
Author(s):  
Raju Sekar ◽  
Bernhard M. Fuchs ◽  
Rudolf Amann ◽  
Jakob Pernthaler
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Rrna Gene ◽  
Fluorescent Reporter ◽  
Bacterial Populations ◽  
Flow Sorting ◽  
Powerful Approach ◽  
Card Fish ◽  
Sequence Types

ABSTRACT We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the β-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of β-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the β-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

scholarly journals A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy)

2020 ◽  
Vol 8 (6) ◽  
pp. 936 ◽  
Author(s):  
Claudia Leoni ◽  
Mariateresa Volpicella ◽  
Bruno Fosso ◽  
Caterina Manzari ◽  
Elisabetta Piancone ◽  
...  
Keyword(s):  
Ecological Model ◽  
Salinity Range ◽  
Rrna Gene ◽  
Hypervariable Regions ◽  
Cell Counts ◽  
Precious Resource ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
The 16S Rrna Gene

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial “Candidatus Aquiluna” (~19% at 14.5% salinity). Interestingly, “Candidatus Aquiluna” had not been identified before in thalassohaline waters.

scholarly journals On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 966
Author(s):  
Zuzana Chumová ◽  
Terezie Mandáková ◽  
Pavel Trávníček
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Crucial Role ◽  
Evolutionary History ◽  
Essential Role ◽  
Grass Species ◽  
Polyploid Species ◽  
Anthoxanthum Odoratum ◽  
European Populations

Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed the origin of sweet vernal grass (Anthoxanthum odoratum) using flow cytometry, DNA sequencing, and in situ hybridization-based cytogenetic techniques. An allotetraploid and polytopic origin of the species has been verified. The chromosome study reveals an extensive variation between the European populations. In contrast, an autopolyploid origin of the rarer tetraploid vernal grass species, A. alpinum, has been corroborated. Diploid A. alpinum played an essential role in the polyploidization of both European tetraploids studied.

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides

2008 ◽  
Vol 73 (2) ◽  
pp. 142-147 ◽  
Author(s):  
J.A. Dijk ◽  
P. Breugelmans ◽  
J. Philips ◽  
P.J. Haest ◽  
E. Smolders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fish Detection ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

scholarly journals Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters

2021 ◽  
Vol 12 ◽  
Author(s):  
Sarah Zecchin ◽  
Simona Crognale ◽  
Patrizia Zaccheo ◽  
Stefano Fazi ◽  
Stefano Amalfitano ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Inorganic Arsenic ◽  
16S Rrna Gene Sequencing ◽  
Northern Italy ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Main Driver ◽  
Rrna Gene Sequencing ◽  
Arsenite Oxidizing Bacteria ◽  
Card Fish

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

Detection of t(14;18)(q32;q21) in B-Cell Chronic Lymphocytic Leukemia

2005 ◽  
Vol 129 (3) ◽  
pp. 410-411
Author(s):  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Susanne Schnittger ◽  
Claudia Schoch
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Chronic Lymphocytic Leukemia ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Lymphoma Therapy ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Cytomorphologic testing and multiparameter flow cytometry are the mainstays in diagnosing B-cell chronic lymphocytic leukemia, whereas fluorescence in situ hybridization that targets the translocation t(14;18)(q32;q21) often is used to identify follicular lymphoma. Therapy is highly diverse between both diseases. We describe a case with cytomorphologically and immunologically proven B-cell chronic lymphocytic leukemia in which t(14;18)(q32;q21) was found.

scholarly journals Ultrasensitive RNA In Situ Hybridization for Kappa and Lambda Light Chain mRNA in Marginal Zone Lymphoma Demonstrates Comparable Performance to Flow Cytometry

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S109-S109
Author(s):  
Michael Franklin ◽  
Chelsey Deel ◽  
Mohammad Vasef
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
B Cell ◽  
Light Chain ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Ffpe Tissue ◽  
Light Chains ◽  
Light Chain Restriction

Abstract Objectives Evaluation of light chain restriction is critical to establish clonality in B-cell lymphoproliferative disorders (LPDs). Immunohistochemistry (IHC) and in situ hybridization (ISH) are commonly used to assess light chain restriction in formalin-fixed, paraffin-embedded (FFPE) tissues. However, except for cases with plasma cell differentiation, these techniques often fail to identify immunoglobulin light chains. An ultrasensitive technique, RNAscope, has been recently introduced that can identify light chains in cases of B-cell LPDs. We analyzed the utility of this ultrasensitive method in detection of clonality and correlated with flow cytometry results when available. Methods A tissue microarray was constructed using 1.6-mm diameter tissue punches of 31 FFPE tissue blocks from 27 cases that were previously characterized as marginal zone lymphoma (MZL) by a combination of morphology, IHC, and/or flow cytometry. Cases included 8 nodal and 19 extranodal MZLs. In two cases, additional blocks were included to assess reproducibility. For ultrasensitive ISH RNAscope assay, 4-µm thickness tissue sections were hybridized using kappa and lambda probes, incubated overnight, counterstained with hematoxylin, cover-slipped, and reviewed blindly without knowledge of prior flow cytometry results. Results Of 18 cases with evaluable staining, 15 were clonal and 3 were polytypic. Flow cytometry was available in 14 of these 18 cases with concordance in 13 of 14 (93%). The discordant case was polytypic by flow cytometry but kappa restricted by RNAscope. The false-negative flow results could be due to sampling issues. In six cases, staining failed and could not be evaluated. Conclusion Ultrasensitive RNAscope is a reliable assay in the detection of clonality in FFPE tissue, particularly where fresh tissue is not available for flow cytometry. In addition, our results confirm and further expand prior observations that RNAscope is a highly sensitive and specific assay with high concordance with flow cytometry.

Sign in / Sign up

Export Citation Format

Share Document