scholarly journals A critical evaluation of a flow cytometer used for detecting enterococci in recreational waters

2007 ◽  
Vol 5 (2) ◽  
pp. 295-305 ◽  
Author(s):  
Dawn N. King ◽  
Kristen P. Brenner ◽  
Mark R. Rodgers
Keyword(s):  
Flow Cytometry ◽  
Environmental Protection Agency ◽  
Detection System ◽  
Membrane Filter ◽  
Critical Evaluation ◽  
Flow Cytometer ◽  
Target Cells ◽  
Recreational Water ◽  
Recreational Waters ◽  
High Background

The current U. S. Environmental Protection Agency-approved method for enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens may occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect enterococci in bathing beach waters. The flow cytometer chosen for this study was a laser microbial detection system designed to detect labeled antibodies. A comparison of MF counts with flow cytometry counts of enterococci in phosphate buffer and sterile-filtered recreational water showed good agreement between the two methods. However, when flow cytometry was used, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations. The unspiked sample controls frequently had higher counts than the samples spiked with enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody and carryover to subsequent samples. For these reasons, this technology may not be suitable for enterococci detection in recreational waters. Improvements in research and instrument design that will eliminate high background and carryover may make this a viable technology in the future.

scholarly journals A novel genetic marker for the rapid detection of Bacteroides fragilis in recreational water as a human-specific faecal indicator

2011 ◽  
Vol 9 (2) ◽  
pp. 253-264 ◽  
Author(s):  
Chang Soo Lee ◽  
Jason W. Marion ◽  
Jiyoung Lee
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection System ◽  
Bacteroides Fragilis ◽  
Human Colon ◽  
Recreational Water ◽  
Recreational Waters ◽  
Pcr Products ◽  
Us Epa

Bacteroides spp. has gained substantial interest among the suggested potential candidates for alternative faecal indicators for untreated recreational waters by the US EPA. Interest in Bacteroides as a faecal indicator is based upon the relative abundance of selected members of the Bacteroides genus in the human colon and human faeces. In this study, we developed a real-time PCR detection system based on gyrase B subunit genes (gyrB) specific to Bacteroides fragilis. The gryB-based method was compared with previously described 16S rRNA-based real-time qPCR methods and evaluated for specificity, sensitivity and robustness in detecting B. fragilis from untreated recreational water impacted by human and non-human faecal sources. The new gyrB-based system only detected B. fragilis, whereas the 16S rRNA-based methods generated cross-amplifications with other Bacteroides and Prevotella species. We used a procedure of prefiltration, filtration, sonication and DNA concentration in order to improve the DNA extraction efficiency and the sensitivity of the real-time PCR while removing interference. The amplification and sequencing of PCR products generated by the gyrB-based method confirmed that gyrB-amplified sequences only contained B. fragilis. This rapid method is useful for quantifying faecal contamination and may assist beach and watershed managers in elucidating possible contamination sources.

A Rapid, Specific Membrane Filtration Procedure for Enumeration of Enterococci in Recreational Water

1998 ◽  
Vol 64 (2) ◽  
pp. 678-680 ◽  
Author(s):  
James W. Messer ◽  
Alfred P. Dufour
Keyword(s):  
Environmental Protection Agency ◽  
False Positive ◽  
Membrane Filtration ◽  
Membrane Filter ◽  
False Positive Rate ◽  
False Negative ◽  
False Negative Rate ◽  
Triphenyltetrazolium Chloride ◽  
Recreational Waters ◽  
Substrate Medium

ABSTRACT A two-step membrane filter (MF) method with mE medium, upon which the membrane must be incubated for 48 h and then transferred to a substrate medium to differentiate enterococci, is recommended by the U.S. Environmental Protection Agency to measure enterococci in fresh and marine recreational waters. The original mE medium was modified by reducing the triphenyltetrazolium chloride from 0.15 to 0.02 g/liter and adding 0.75 g of indoxyl β-d-glucoside per liter. The new MF medium, mEI medium, detected levels of enterococci in 24 h comparable to those detected by the original mE medium in 48 h, with the same level of statistical confidence. In addition, the use of mEI medium eliminated the need to transfer the membrane to a substrate medium to differentiate enterococci from other genera of the fecal streptococcal group. Colonies from mEI medium were examined to determine the rates of false-positive and false-negative occurrences. mEI medium had a false-positive rate of 6.0% and a false-negative rate of 6.5%. Interlaboratory testing of the MF method with mEI medium demonstrated that the relative reproducibility standard deviations among laboratories ranged from 2.2% for marine water to 18.9% for freshwater. The comparative recovery studies, specificity determinations, and multilaboratory evaluation indicated that mEI medium has analytical performance characteristics equivalent to those of mE medium. The simplicity of use and decreased incubation time with mEI medium will facilitate the detection and quantification of enterococci in fresh and marine recreational waters.

Acquisition of EELS spectra for high-resolution spectroscopy

1993 ◽  
Vol 51 ◽  
pp. 578-579
Author(s):  
Maoxu Qian ◽  
Mehmet Sarikaya ◽  
Edward A. Stern
Keyword(s):  
Energy Loss ◽  
Detection System ◽  
High Resolution Spectroscopy ◽  
High Signal ◽  
Short Report ◽  
Edge Structure ◽  
High Background ◽  
Gain Variation ◽  
Sufficient Energy ◽  
On Line

It is difficult, in general, to perform quantitative EELS to determine, for example, relative or absolute compositions of elements with relatively high atomic numbers (using, e.g., K edge energies from 500 eV to 2000 eV), to study ELNES (energy loss near edge structure) signal using the white lines to determine oxidation states, and to analyze EXELFS (extended energy loss fine structure) to study short range ordering. In all these cases, it is essential to have high signal-to-noise (S/N) ratio (low systematical error) with high overall counts, and sufficient energy resolution (∽ 1 eV), requirements which are, in general, difficult to attain. The reason is mainly due to three important inherent limitations in spectrum acquisition with EELS in the TEM. These are (i) large intrinsic background in EELS spectra, (ii) channel-to-channel gain variation (CCGV) in the parallel detection system, and (iii) difficulties in obtaining statistically high total counts (∽106) per channel (CH). Except the high background in the EELS spectrum, the last two limitations may be circumvented, and the S/N ratio may be attained by the improvement in the on-line acquisition procedures. This short report addresses such procedures.

Impact of the New Beckman Coulter Cytomics FC 500 5-Color Flow Cytometer on a Regional Flow Cytometry Clinical Laboratory Service

2004 ◽  
Vol 10 (2) ◽  
pp. 102-108 ◽  
Author(s):  
J. Luider ◽  
M. Cyfra ◽  
P. Johnson ◽  
I. Auer
Keyword(s):  
Flow Cytometry ◽  
Clinical Laboratory ◽  
Flow Cytometer ◽  
Color Flow ◽  
Laboratory Service ◽  
Regional Flow

scholarly journals Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1320
Author(s):  
Kristýna Pekárková ◽  
Jakub Soukup ◽  
Marie Kostelanská ◽  
Jan Širc ◽  
Zbyněk Straňák ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Extracellular Vesicles ◽  
Correlation Coefficients ◽  
Flow Cytometer ◽  
Liquid Biopsies ◽  
Physiological Conditions ◽  
Flow Cytometric ◽  
Term Births ◽  
And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

scholarly journals Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland

2019 ◽  
Vol 475 ◽  
pp. 112348 ◽  
Author(s):  
Hana Glier ◽  
Ingmar Heijnen ◽  
Mathieu Hauwel ◽  
Jan Dirks ◽  
Stéphane Quarroz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Feasibility Study ◽  
Flow Cytometer ◽  
Color Flow ◽  
Clinical Laboratories

scholarly journals Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014

2016 ◽  
Vol 21 (15) ◽  
Author(s):  
Aleksandra Jovanović Galović ◽  
Sanja Bijelović ◽  
Vesna Milošević ◽  
Ivana Hrnjaković Cvjetkovic ◽  
Milka Popović ◽  
...  
Keyword(s):  
Surface Waters ◽  
Water Samples ◽  
Genetic Material ◽  
Recreational Water ◽  
Recreational Waters ◽  
Quality Of Water ◽  
Viral Indicators ◽  
Urban Wastewater Treatment ◽  
Recreational Use

From August to September 2014 a water quality study was conducted on five popular public Danube beaches in Vojvodina, Serbia. To assess the safety of Danube water for bathing, physical, chemical, bacteriological tests were performed. While many parameters for monitoring the quality of water are regulated by law, there are neither national nor international legislations addressing the presence of viruses in recreational waters. In this study, we performed analyses that surpassed national requirements, and investigated if adenovirus, enterovirus or rotavirus genetic material was present in samples of recreational water collected for quality monitoring. Of 90 water samples obtained during the study, enterovirus material was not found in any sample, but adenovirus and rotavirus genetic materials were respectively detected in 60 and 31 samples. Statistical analyses showed a significant correlation between adenovirus DNA and total coliforms in the water. Even when water samples were adequate for recreational use, adenoviruses were detected in 75% (57/76) of such samples. Our results indicate that implementation of viral indicators in recreational water might be helpful to better assess public health safety. This might be particularly relevant in areas where urban wastewater treatment is insufficient and surface waters affected by wastewater are used for recreation.

Identification of the side population associated with ATP-binding cassette transporters activity using imaging flow cytometry

2021 ◽  
Vol 67 (2) ◽  
pp. 137-143
Author(s):  
A.M. Gisina ◽  
Y.S. Kim ◽  
K.N. Yarygin ◽  
A.Yu. Lupatov
Keyword(s):  
Flow Cytometry ◽  
Side Population ◽  
Flow Cytometer ◽  
Atp Binding ◽  
Tumor Resistance ◽  
Side Population Cells ◽  
Imaging Flow Cytometry ◽  
Atp Binding Cassette Transporters ◽  
Resistance To Chemotherapy ◽  
Atp Binding Cassette

DyeCycle Violet efflux, caused by ATP-binding cassette transporters activity, was analyzed in human colorectal adenocarcinoma cell lines SW480, HT-29, Caco-2 by neans of FACSAria III flow cytometer and ImageStreamX Mk II imaging flow cytometer. Along with similarity of cytometry data obtained on the two instruments, the use of imaging flow cytometry made it possible to characterize the morphology of side population cells, as well as morphology of other cell populations differing in the degree of dye accumulation. The population of cells, which are smaller than the side population cells and practically do not take the dye, is of the special interest. Probably, this population may contribute to the tumor resistance to chemotherapy.

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

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