An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R2 = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.

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

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.539-545.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 539-545 ◽

Cited By ~ 162

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.

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Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry

Applied and Environmental Microbiology ◽

10.1128/aem.02750-06 ◽

2007 ◽

Vol 73 (10) ◽

pp. 3283-3290 ◽

Cited By ~ 512

Author(s):

Michael Berney ◽

Frederik Hammes ◽

Franziska Bosshard ◽

Hans-Ulrich Weilenmann ◽

Thomas Egli

Keyword(s):

Flow Cytometry ◽

Outer Membrane ◽

Shigella Flexneri ◽

Epifluorescence Microscopy ◽

E Coli ◽

Flow Cytometric ◽

Freshwater Bacteria ◽

Serovar Typhimurium ◽

Nucleic Acid Stain ◽

Green Fluorescent

ABSTRACT The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

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

10.5194/acp-2018-1073 ◽

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.

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O-GlcNAcylation in early stages of chronic lymphocytic leukemia; protocol development for flow cytometry

Cancer Biomarkers ◽

10.3233/cbm-203049 ◽

2021 ◽

pp. 1-10

Author(s):

Viktória Temesfői ◽

Kinga Molnár ◽

Péter Kaltenecker ◽

Barbara Réger ◽

Árpád Szomor ◽

...

Keyword(s):

Flow Cytometry ◽

Lymphocyte Count ◽

Blood Parameters ◽

Cell Number ◽

Metabolic Changes ◽

Protein Glycosylation ◽

Lymphocytic Leukemia ◽

Tween 20 ◽

Protocol Development ◽

Flow Cytometric

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

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

Diagnostics ◽

10.3390/diagnostics11081320 ◽

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.

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Applying EFDC Explorer model in the Gallinas River, Mexico to estimate its assimilation capacity for water quality protection

Scientific Reports ◽

10.1038/s41598-021-92453-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Claudia Villota-López ◽

Clemente Rodríguez-Cuevas ◽

Franklin Torres-Bejarano ◽

Rodolfo Cisneros-Pérez ◽

Rodolfo Cisneros-Almazán ◽

...

Keyword(s):

Dissolved Oxygen ◽

Industrial Wastewater ◽

Socioeconomic Development ◽

Inorganic Compounds ◽

Organic Content ◽

Phosphorus Compounds ◽

Active Components ◽

Nitrogenous Substances ◽

Efficient Recovery ◽

Assimilation Capacity

AbstractSanitary and industrial wastewater discharged into rivers, is a general problem that occurs in most of the world and Mexico is not the exception, the main goal of this research is to determine based on simulations of pollutants concentrations, the assimilation capacity of the Gallinas River against discharges of agricultural and industrial wastewater from the cultivation and processing of sugar cane under two different hypothetical simulation scenarios, based on reproducing two well know scenarios. In sugarcane cultivation, large quantities of fertilizers are used whose main active components are based on nitrogen or phosphorus compounds, therefore, the wastewater resulting from sugarcane processing contains a high organic content from 20 to 40% of inorganic compounds, such as nitrogenous substances, organic acids, and phosphorous sulfates. For this reason, the physical–chemical variables of interest analyzed in this work are the PO$$_4$$ 4 (phosphate), NO$$_3$$ 3 (nitrate), and DO (dissolved oxygen). With the simulation results according to each scenery, it can be determined, that despite the continuous discharge of polluting elements, the Gallinas River has a good assimilation capacity thanks to reaeration processes that permit efficient recovery of the dissolved oxygen in the water column. Gallinas River is located in the region known as the Huasteca Potosina, this investigation is relevant for the region due to the River is of vital importance being the main tributary that allows socioeconomic development activities in this zone. To carry out the simulations, was used the Explorer Modeling System 8.4 (EFCD) model and was performed two samplings campaign along 15 km in the water body to calibrate the numerical model to represent the dry and wet seasons during May and September respectively named as calibration scenarios.

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Flow cytometric measurements as a proxy for sporulation intensity in the cultured macroalga Ulva (Chlorophyta)

Botanica Marina ◽

10.1515/bot-2020-0050 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Omri Nahor ◽

Cristina F. Morales-Reyes ◽

Gianmaria Califano ◽

Thomas Wichard ◽

Alexander Golberg ◽

...

Keyword(s):

Flow Cytometry ◽

Life Cycle ◽

Abiotic Factors ◽

Physiological Status ◽

Seaweed Cultivation ◽

Reproductive Growth ◽

Biotic And Abiotic Factors ◽

Flow Cytometric ◽

Abiotic Stimuli ◽

Fundamental Shift

Abstract Controlling the life cycle of the green macroalga Ulva (Chlorophyta) is essential to maintain its efficient aquaculture. A fundamental shift in cultivation occurs by transforming the thallus cells into gametangia and sporangia (sporulation), with the subsequent release of gametes and zoids. Sporulation occurrence depends on algal age and abiotic stimuli and is controlled by sporulation inhibitors. Thus, quantification of sporulation intensity is critical for identifying the biotic and abiotic factors that influence the transition to reproductive growth. Here, we propose to determine the sporulation index by measuring the number of released gametes using flow cytometry, in proportion to the total number of thallus cells present before the occurrence of the sporulation event. The flow cytometric measurements were validated by manually counting the number of released gametes. We observed a variation in the autofluorescence levels of the gametes which were released from the gametangia. High autofluorescence level correlated to phototactically active behaviour of the gametes. As autofluorescence levels varied between different groups of gametes related to their mobility, flow cytometry can also determine the physiological status of the gametes used as feedstock in seaweed cultivation.

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Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of Staphylococcus aureus andMicrococcus luteus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.4.827-834.2000 ◽

2000 ◽

Vol 44 (4) ◽

pp. 827-834 ◽

Cited By ~ 159

Author(s):

David J. Novo ◽

Nancy G. Perlmutter ◽

Richard H. Hunt ◽

Howard M. Shapiro

Keyword(s):

Staphylococcus Aureus ◽

Flow Cytometry ◽

Membrane Potential ◽

Membrane Permeability ◽

Micrococcus Luteus ◽

Flow Cytometric Analysis ◽

Bacterial Counts ◽

Flow Cytometric ◽

Permeability Parameter ◽

Particle Counts

ABSTRACT Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus andMicrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC2(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC2(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 μg/ml did not change permeability, while a tetracycline concentration of 4 μg/ml permeabilized 50% of the bacteria; 4 μg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

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Flow cytometric evaluation of bladder cancer: recommendations of the NCI flow cytometry network for bladder cancer

World Journal of Urology ◽

10.1007/bf00186094 ◽

1992 ◽

Vol 10 (1) ◽

pp. 63-67 ◽

Cited By ~ 11

Author(s):

R. L. Aamodt ◽

J. S. Coon ◽

A. Deitch ◽

R. W. deVere White ◽

L. G. Koss ◽

...

Keyword(s):

Flow Cytometry ◽

Bladder Cancer ◽

Flow Cytometric ◽

Flow Cytometric Evaluation

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A Teaching Database for Diagnosis of Hematologic Neoplasms Using Immunophenotyping by Flow Cytometry

Archives of Pathology & Laboratory Medicine ◽

10.5858/2008-132-829-atdfdo ◽

2008 ◽

Vol 132 (5) ◽

pp. 829-837

Author(s):

Andy N. D. Nguyen ◽

Jitakshi De ◽

Jacqueline Nguyen ◽

Anthony Padula ◽

Zhenhong Qu

Keyword(s):

Flow Cytometry ◽

World Health ◽

Hematologic Neoplasms ◽

Web Based ◽

Mathematical Algorithm ◽

Medical Centers ◽

Flow Cytometric ◽

Public Resource ◽

Immunologic Marker ◽

Health Organization

Abstract Context.—In the diagnosis of lymphomas and leukemias, flow cytometry has been considered an essential addition to morphology and immunohistochemistry. The interpretation of immunophenotyping results by flow cytometry involves pattern recognition of different hematologic neoplasms that may have similar immunologic marker profiles. An important factor that creates difficulty in the interpretation process is the lack of consistency in marker expression for a particular neoplasm. For this reason, a definitive diagnostic pattern is usually not available for each specific neoplasm. Consequently, there is a need for decision support tools to assist pathology trainees in learning flow cytometric diagnosis of leukemia and lymphoma. Objective.—Development of a Web-enabled relational database integrated with decision-making tools for teaching flow cytometric diagnosis of hematologic neoplasms. Design.—This database has a knowledge base containing patterns of 44 markers for 37 hematologic neoplasms. We have obtained immunophenotyping data published in the scientific literature and incorporated them into a mathematical algorithm that is integrated to the database for differential diagnostic purposes. The algorithm takes into account the incidence of positive and negative expression of each marker for each disorder. Results.—Validation of this algorithm was performed using 92 clinical cases accumulated from 2 different medical centers. The database also incorporates the latest World Health Organization classification for hematologic neoplasms. Conclusions.—The algorithm developed in this database shows significant improvement in diagnostic accuracy over our previous database prototype. This Web-based database is proposed to be a useful public resource for teaching pathology trainees flow cytometric diagnosis.

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