scholarly journals Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments

2000 ◽  
Vol 66 (10) ◽  
pp. 4486-4496 ◽  
Author(s):  
Mark E. Fuller ◽  
Sheryl H. Streger ◽  
Randi K. Rothmel ◽  
Brian J. Mailloux ◽  
James A. Hall ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Detection Limit ◽  
Sediment Cores ◽  
Growth Conditions ◽  
Bacterial Transport ◽  
Epifluorescence Microscopy ◽  
Transport Studies ◽  
Subsurface Environments ◽  
High Throughput Method ◽  
Initial Work

ABSTRACT Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate,Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-105CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

scholarly journals Using flow cytometry and light-induced fluorescence technique to characterizethe variability and characteristics of bioaerosols in springtime at Metro Atlanta, Georgia

2018 ◽  
Author(s):  
Arnaldo Negron ◽  
Natasha DeLeon-Rodriguez ◽  
Samantha M. Waters ◽  
Luke D. Ziemba ◽  
Bruce Anderson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Acid Content ◽  
Fungal Spores ◽  
Epifluorescence Microscopy ◽  
Nucleic Acid Content ◽  
Rain Event ◽  
Bacterial Cells ◽  
Sampling System ◽  
Spores And Pollen

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA over a two-month period and showed clearly defined populations of DNA-containing particles: Low Nucleic Acid-content particles (bioLNA), High Nucleic Acid-content particles (HNA) being fungal spores and pollen. We find that daily-average springtime PBAP concentration (1 to 5 μm diameter) ranged between 1.4 × 104 and 1.1 × 105 m−3. The BioLNA population dominated PBAP during dry days (72 ± 18 %); HNA dominated the PBAP during humid days and following rain events, where HNA (e.g., wet-ejected fungal spores) comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that FBAP and total FCM counts are similar; HNA (from FCM) significantly correlated with ABC type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 μm diameter). However, the FCM bioLNA population, possibly containing bacterial cells, did not correlate to any FBAP type. The lack of correlation of any WIBS FBAP type with the bioLNA suggest bacterial cells may be more difficult to detect with autofluorescence than previously thought. Ιdentification of bacterial cells even in the FCM (bioLNA population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed highest concentration on a humid and warm day after a rain event (4/14), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days, and a bioLNA population dominating bioaerosol community during dry days.

Nanoplastic transport in aqueous environments: The role of chemo-electric properties and hydrodynamic forces for nanoplastic-mineral interaction.

2021 ◽  
Author(s):  
Sascha Müller ◽  
Jacek Fiutowski ◽  
Horst-Günter Rubahn ◽  
Nicole Rita Posth
Keyword(s):  
Specific Interaction ◽  
Chemical Processes ◽  
Flow Rates ◽  
Transport Studies ◽  
Subsurface Environments ◽  
Dissolved Species ◽  
Aqueous Environments ◽  
Interaction Scheme ◽  
Almost All ◽  
Mineral Interaction

The fate and transport characteristics of nanoplastic (NP) through different environmental systems is largely governed by physio-chemical processes and their specific interaction with environmental constituents (i.e., minerals, dissolved species, suspended particles). A hydrodynamic component present in almost all terrestrial and marine aqueous environments impact the physio-chemical processes micron-scale is largely overlooked in NP transport studies. Therefore, we tested the interaction behavior of nanosized plastic polystyrene particles of various coatings in the presence of minerals abundant in the Earth crust within a hydrodynamic continuum representing flow rates from groundwater to surface water systems. Our batch experiments show that particle-mineral adsorption is largely driven by the magnitude of opposite charge configurations, which is either produced by mineral type or specific nanoplastic surface coating. Zetapotential serves as a good predictor of adsorption between uncoated and carboxyl-coated polystyrene with minerals. It fails, however, to predict adsorption behavior between NH2 coated polystyrene and apatite or feldspars, due to the more complex and varying compositions of these minerals. Incorporating the hydrodynamic force component into the particle- mineral interaction scheme reproduces those adsorption trends at slow flowrates of 1e-04 m/d. However, increasing flow rates by a factor of 100 modifies charge-driven adsorption between minerals and plastics. This study highlights the unabating importance of hydrodynamic conditions when predicting nanoplastic transport in different subsurface environments, and has implications for nanoplastic behavior in both terrestrial and marine aqueous environments.

scholarly journals DNA Content and Nucleoid Distribution in Methanothermobacter thermautotrophicus

2005 ◽  
Vol 187 (5) ◽  
pp. 1856-1858 ◽  
Author(s):  
Alan I. Majerník ◽  
Magnus Lundgren ◽  
Paul McDermott ◽  
Rolf Bernander ◽  
James P. J. Chong
Keyword(s):  
Flow Cytometry ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Dna Content ◽  
Replication Initiation ◽  
Epifluorescence Microscopy ◽  
Single Chromosome ◽  
Dna Replication Initiation ◽  
Methanothermobacter Thermautotrophicus ◽  
Multiple Cells

ABSTRACT Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.

scholarly journals Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold

2001 ◽  
Vol 67 (2) ◽  
pp. 539-545 ◽  
Author(s):  
Feng Chen ◽  
Jing-rang Lu ◽  
Brian J. Binder ◽  
Ying-chun Liu ◽  
Robert E. Hodson
Keyword(s):  
Flow Cytometry ◽  
Image Analysis ◽  
Digital Image ◽  
Digital Image Analysis ◽  
Flow Cytometric Analysis ◽  
Epifluorescence Microscopy ◽  
Flow Cytometric ◽  
Viral Particles ◽  
Sybr Gold ◽  
Nucleic Acid Stain

ABSTRACT A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

Refining chronologies by dating pollen concentrates – new approach of separating pollen using flow cytometry

2020 ◽  
Author(s):  
Christoph Steinhoff ◽  
Nadine Pickarski ◽  
Thomas Litt
Keyword(s):  
Flow Cytometry ◽  
Radiocarbon Dating ◽  
Sediment Cores ◽  
Pollen Grains ◽  
Plant Macrofossils ◽  
Small Samples ◽  
Large Lakes ◽  
Promising Alternative ◽  
Ams Radiocarbon Dating ◽  
Short Period

<p>Radiocarbon dating of terrestrial plant-remains is a traditional method for precise age estimations of lake sediments. The absence of sufficient large plant macrofossils required for AMS dating in continental records, especially large lakes, demands for a satisfactory alternative, such as carbon-containing microfossils. Due to their ubiquitous presence in sedimentary archives pollen grains may be considered for dating. Nevertheless, the isolation and enrichment of pollen without a significant carbon contamination is still challenging. Even though commonly applied separation techniques can be used to remove the predominant portions of foreign particles, the undesirable transfer of these particles into the pollen concentrate cannot be excluded, yet. However, flow cytometry, as a highly promising alternative, offers the possibility to sort huge quantities of particles in a short period of time and to generate pure pollen concentrates from heterogeneous samples suitable for AMS radiocarbon dating.</p><p>In this study we present the approach to sort limnic sediment samples using flow cytometry. We are able to unequivocally identify pollen populations in the heterogeneous composition of the sediments and isolate them. The sediments analyzed were taken from the continental record of Lake Van (Eastern Anatolia). Annually laminated layers from the Holocene section of the sediment cores allow a precise temporal classification and validation of generated radiocarbon ages derived from fossil pollen. Although it is now possible to produce pollen concentrates without the contamination of foreign particles, the isolation of a sufficient quantity of pollen grains to generate reliable radiocarbon ages is still difficult. An increase pollen yield is required. Due to the limitation of the initial material, it is therefore especially necessary to improve the efficiency during the cytometric analysis.</p><p>Our results show the importance to steadily optimize the processing steps during chemical pretreatment, cytometric analysis as well as the radiocarbon dating itself. This facilitates the handling of the ultra-small samples and ensures precise age estimations of the pollen concentrates. Furthermore improving the laboratory routine for the enrichment of pollen will allow the analysis of vast amounts of samples in a short period of time. In consequence, dating pollen concentrates generated by flow cytometry can be used as a robust contribution and independent time control for existing chronologies in continental climate records.</p>

scholarly journals Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry

2010 ◽  
Vol 76 (15) ◽  
pp. 5088-5096 ◽  
Author(s):  
Mohiuddin M. Taimur Khan ◽  
Barry H. Pyle ◽  
Anne K. Camper
Keyword(s):  
Flow Cytometry ◽  
Pseudomonas Syringae ◽  
Phase 1 ◽  
Epifluorescence Microscopy ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Gram Negative ◽  
Analytical Technique

ABSTRACT An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different Gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

scholarly journals Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in Metro Atlanta, Georgia

2020 ◽  
Vol 20 (3) ◽  
pp. 1817-1838 ◽  
Author(s):  
Arnaldo Negron ◽  
Natasha DeLeon-Rodriguez ◽  
Samantha M. Waters ◽  
Luke D. Ziemba ◽  
Bruce Anderson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Acid Content ◽  
Aerosol Particles ◽  
Fungal Spores ◽  
Epifluorescence Microscopy ◽  
Nucleic Acid Content ◽  
Rain Event ◽  
Bacterial Cells ◽  
Biological Aerosol Particles

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation, and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA, over a 2-month period and showed clearly defined populations of nucleic-acid-containing particles: low nucleic acid-content particles above threshold (LNA-AT) and high nucleic acid-content particles (HNA) likely containing wet-ejected fungal spores and pollen. We find that the daily-average springtime PBAP concentration (1 to 5 µm diameter) ranged between 1.4×104 and 1.1×105 m−3. The LNA-AT population dominated PBAP during dry days (72±18 %); HNA dominated the PBAP during humid days and following rain events, where HNA comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that fluorescent biological aerosol particles (FBAP) and total FCM counts are similar; HNA (from FCM) moderately correlated with ABC-type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 µm diameter). However, the FCM LNA-AT population, possibly containing bacterial cells, did not correlate with any FBAP type. The lack of correlation of any WIBS FBAP type with the LNA-AT suggests that airborne bacterial cells may be more difficult to unambiguously detect with autofluorescence than currently thought. Identification of bacterial cells even in the FCM (LNA-AT population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed the highest concentration on a humid and warm day after a rain event (14 April 2015), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days and an LNA-AT population dominating the bioaerosol community during dry days.

Homogeneous immunofluorometric assays of alpha-fetoprotein with macroporous, monosized particles and flow cytometry

1993 ◽  
Vol 39 (10) ◽  
pp. 2174-2181 ◽  
Author(s):  
J Frengen ◽  
R Schmid ◽  
B Kierulf ◽  
K Nustad ◽  
E Paus ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Detection Limit ◽  
Correlation Coefficient ◽  
High Precision ◽  
Particle Concentration ◽  
Regression Line ◽  
Alpha Fetoprotein ◽  
Immunoradiometric Assay ◽  
Nonspecific Binding ◽  
Separate Particle

Abstract We evaluated two homogeneous immunofluorometric assays (IFMAs) of alpha-fetoprotein (AFP) based on new macroporous acrylate particles combined with flow cytometry. The standard IFMA, requiring 1 h of incubation, provided a working range from 1.8 to > 900 kIU/L (CV < 10%) and a detection limit of 0.6 kIU/L. Use of overnight incubation and a lower particle concentration extended the working range by 1 decade in the lower end. Analytical recoveries for the standard IFMA varied between 97% and 108%. The slope and y-intercept of the regression line correlating measurements by the standard IFMA and a routine immunoradiometric assay were not significantly different from 1 and 0, respectively (P > 0.5), and the correlation coefficient was 0.996. High precision and warning of spuriously high measurements were obtained by including in each sample separate particle types for detecting instrument instability and measuring nonspecific binding only.

Sign in / Sign up

Export Citation Format

Share Document