Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization

2019 ◽  
Vol 53 (12) ◽  
pp. 6895-6905
Author(s):  
Yijing Zhu ◽  
Yayi Wang ◽  
Yuan Yan ◽  
Hao Xue
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Flow Cytometric Analysis ◽  
Anammox Bacteria ◽  
Flow Cytometric ◽  
Catalyzed Reporter Deposition

scholarly journals Endoscopic ultrasound and endobronchial ultrasound-guided fine-needle aspiration of deep-seated lymphadenopathy: Analysis of 1338 cases

CytoJournal ◽  
2012 ◽  
Vol 9 ◽  
pp. 14 ◽  
Author(s):  
Amberly L Nunez ◽  
Nirag C Jhala ◽  
Andrew J Carroll ◽  
Fady M Mikhail ◽  
Vishnu V.B. Reddy ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Fine Needle Aspiration ◽  
Flow Cytometric Analysis ◽  
Needle Aspiration ◽  
Endobronchial Ultrasound ◽  
Ultrasound Guided ◽  
Fine Needle ◽  
Flow Cytometric

Background: We retrospectively studied 1338 samples of lymph nodes obtained by endoscopic and endobronchial ultrasound-guided fine needle aspiration biopsy (EUS and EBUS-FNAB) with an objective of characterizing the utility of this diagnostic modality in the assessment of deep-seated lymphadenopathy. The secondary aims were to establish the utility in the diagnosis of lymphoma and to determine the number of passes required to obtain adequate cellularity for flow cytometric analysis. Materials and Methods: On-site assessment was performed by a cytopathologist using Diff-Quik (American Scientific Products, McGraw Park, IL) stain. In addition, Papanicolaou and immunohistochemical stains were performed and additional samples were sent for flow cytometric analyses (n = 145). The final cytologic diagnosis was correlated with surgical pathology diagnosis and/or clinical follow-up. In select cases, fluorescence in situ hybridization analysis with specific probes was performed on Diff-Quik smears. Results: Both morphology as well as ancillary studies (flow cytometry or immunohistochemical stain and/or fluorescence in situ hybridization) show that EUS and EBUS-FNA are effective techniques to detect and stage intrathoracic and intra-abdominal tumors. Operating characteristics show that these are highly sensitive (89%) and specific (100%) techniques for the diagnosis of lymphoma. At least two passes provided an average of 5.66 million cells (range, 0.12-62.32 million) for lymphoma cases. Conclusions: EUS and EBUS-FNA are powerful modalities to stage malignancies and at least two passes can provide adequate cells for flow cytometric analysis. We also demonstrate that fluorescence in situ hybridization analysis can be performed on Diff-Quik-stained and mounted smears.

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).

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