Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems

Lebaron, Philippe; Parthuisot, Nathalie; Catala, Philippe

doi:10.1128/aem.64.5.1725-1730.1998

Cited by 246 publications

(134 citation statements)

References 18 publications

(29 reference statements)

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“…Depending on the design, alignment and cleanliness, some flow cytometers can resolve cell numbers solely from light scatter characteristics and by triggering on light scatter. However, depending on the level of interference by signals derived from the instrument or the sample, the cells need to be stained, for example using nucleic acid dyes such as ethidium bromide (EB), propidium iodide (PI), Syto-9, Syto-13 or 4 0 ,6-diamidino-2-phenylindole (DAPI, Völsch et al, 1990;Monfort & Baleux, 1992;Lebaron et al, 1998b;Comas-Riu & Vives-Rego, 1999;Servais et al, 1999;Troussellier et al, 1999;Vives-Rego et al, 1999;Vogt et al, 2005). Even minimized chip-based microfluidic devices (on-chip FCM) are available that are described to count bacterial numbers within relatively short time ranges (six parallels in 30 min for 2000 cells each, Sakamoto et al, 2005).…”

Section: Cell Count Measurementsmentioning

confidence: 99%

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities

2010

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The still poorly explored world of microbial functioning is about to be uncovered by a combined application of old and new technologies. Bacteria, especially, are still in the dark with respect to their phylogenetic affiliations as well as their metabolic capabilities and functions. However, with the advent of sophisticated flow cytometric and cell sorting technologies in microbiological labs, there is now the possibility to gain this knowledge at the single-cell level without cumbersome cultivation approaches. Cytometry also facilitates the understanding of physiological diversity in seemingly likewise acting populations. Both individuality and diversity lead to the complex and concerted actions of microbial consortia. This review provides an overview of the state of the art in the field. It deals with the handling of microorganisms from the very beginning (i.e. sampling, and detachment and fixation procedures) and goes on to discuss the pitfalls and problems in analysing cells without any further treatment. If information cannot be gained by specific staining procedures, phylogenetic technologies, transcriptomic and proteomic approaches may be options for achieving advanced insights. All in all, flow cytometry will be a mediator technology to gain a deeper insight into the heterogeneity of populations and the functioning of microbial communities.

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Section: Cell Count Measurementsmentioning

confidence: 99%

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities

2010

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“…All samples were thawed at~4°C and immediately analysed with a BD Biosciences LSR-II flow cytometer. The biological component of the particulate loads was stained with SGII, which markedly increases the green fluorescence (530 nm) of particles containing nucleic acids excited by a 488 nm (blue) argon ion laser; SG II is considered the most appropriate stain for differentiating and enumerating microbes in freshwater samples (Lebaron et al, 1998;Weinbauer et al, 1998). Following Lebaron and colleagues (1998), 2 ml of 1:10 000 SYBR Green II stock solution was diluted in 1 ml Phosphate Buffered Saline (pH 7.4), from which 1 ml was added to 2 ml of sample in a sterile vial and incubated for 30 min in the dark at~23°C prior to FCM analysis.…”

Section: Flow Cytometrymentioning

confidence: 99%

Microbial cell budgets of anArctic glacier surface quantified using flow cytometry

Irvine‐Fynn

Edwards

Newton

et al. 2012

Environmental Microbiology

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Uncertainty surrounds estimates of microbial cell and organic detritus fluxes from glacier surfaces. Here, we present the first enumeration of biological particles draining from a supraglacial catchment, on Midtre Lovénbreen (Svalbard) over 36 days. A stream cell flux of 1.08 × 10(7) cells m(-2) h(-1) was found, with strong inverse, non-linear associations between water discharge and biological particle concentrations. Over the study period, a significant decrease in cell-like particles exhibiting 530 nm autofluorescence was noted. The observed total fluvial export of ~7.5 × 10(14) cells equates to 15.1-72.7 g C, and a large proportion of these cells were small (< 0.5 μm in diameter). Differences between the observed fluvial export and inputs from ice-melt and aeolian deposition were marked: results indicate an apparent storage rate of 8.83 × 10(7) cells m(-2) h(-1). Analysis of surface ice cores revealed cell concentrations comparable to previous studies (6 × 10(4) cells ml(-1)) but, critically, showed no variation with depth in the uppermost 1 m. The physical retention and growth of particulates at glacier surfaces has two implications: to contribute to ice mass thinning through feedbacks altering surface albedo, and to potentially seed recently deglaciated terrain with cells, genes and labile organic matter. This highlights the merit of further study into glacier surface hydraulics and biological processes.

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“…The more recently developed £uorochromes, which can be excited with the 488 nm line of an air-cooled argon ion laser and result in a much higher quantum yield, show less interference with phototrophic pigments and have improved the analysis. Examples of these £uorochromes are: SYTO 13 [38^40]; YOYO-1, YO-PRO-1 and Pico Green [38,41]; TOTO and TO-PRO [42]; and the SYTO and SYBR series [43]. Recent intercalibrations between epi£uorescence and £ow cytometric counts have been published, showing good correlations in coastal waters [27,44].…”

Section: Bacterioplanktonmentioning

confidence: 99%

Current and future applications of flow cytometry in aquatic microbiology

Vives-Rego

2000

FEMS Microbiology Reviews

127

182

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Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

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Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems

Cited by 246 publications

References 18 publications

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities

Microbial cell budgets of anArctic glacier surface quantified using flow cytometry

Current and future applications of flow cytometry in aquatic microbiology

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