Flow cytometric analysis of phytoplankton viability following viral infection (original) (raw)
Related papers
Journal of Phycology, 1999
Lagerheim, were followed during viral infection using flow cytometry. Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P. pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures. DNA analyses were performed using the nucleic acid-specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells.
Viral Control of Phytoplankton Populations-a Review1
The Journal of Eukaryotic Microbiology, 2004
Phytoplankton population dynamics are the result of imbalances between reproduction and losses. Losses include grazing, sinking, and natural mortality. As the importance of microbes in aquatic ecology has been recognized, so has the potential significance of viruses as mortality agents for phytoplankton. The field of algal virus ecology is steadily changing and advancing as new viruses are isolated and new methods are developed for quantifying the impact of viruses on phytoplankton dynamics and diversity. With this development, evidence is accumulating that viruses can control phytoplankton dynamics through reduction of host populations, or by preventing algal host populations from reaching high levels. The identification of highly specific host ranges of viruses is changing our understanding of population dynamics. Viral-mediated mortality may not only affect algal species succession, but may also affect intraspecies succession. Through cellular lysis, viruses indirectly affect the fluxes of energy, nutrients, and organic matler, especially during algal bloom events when biomass is high. Although the importance of viruses is presently recognized, it is apparent that many aspects of viral-mediated mortality of phytoplankton are still poorly understood. It is imperative that future research addresses the mechanisms that regulate virus infectivity, host resistance, genotype richness, abundance, and the fate of viruses over time and space.
An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2014
Preservation of cells, choice of fixative, storage, and thawing conditions are recurrent issues for the analysis of phytoplankton by flow cytometry. We examined the effects of addition of the surfactant Pluronic F68 to glutaraldehyde-fixed photosynthetic organisms in cultures and natural samples. In particular, we examined cell losses and modifications of side scatter (a proxy of cell size) and fluorescence of natural pigments. We found that different marine phytoplankton species react differently to the action of Pluronic F68. In particular, photosynthetic prokaryotes are less sensitive than eukaryotes. Observed cell losses may result from cell lysis or from cell adhesion to the walls of plastic tubes that are commonly used for flow cytometry analysis. The addition of the surfactant, Pluronic F68, has a positive effect on cells for long-term storage. We recommend to modify current protocols for preservation of natural marine planktonic samples, by fixing them with glutaraldehyde 0....
Immuno flow cytometry in marine phytoplankton research
Scientia Marina, 2000
The developments in the combination of flow cytometry and immunology as a tool to identify, count and examine marine phytoplankton cells are reviewed. The concepts of immunology and flow cytometry are described. A distinction is made between quantitative and qualitative immunofluorescence. Quantitative immunofluorescence, the identification and enumeration of phytoplankton cells, is the research area that has advanced rapidly in the past decade, and is reviewed extensively. Key steps of quantitative immunofluorescence, fixation and immunolabel intensity, are discussed in more detail. Qualitative immunofluorescence is a new, hardly explored but highly interesting development in which qualitative-physiological-variables related to e.g. nutrient limitation or primary production are measured in individual cells instead of phytoplankton populations as a whole. Several combinations of immunological probes, both for species identification and for physiological measurements, are proposed. A special case of qualitative immunofluorescence is the measurement of phytoplankton toxins in single cells from natural populations. It is anticipated that the future use of semiconductor nanocrystals or "quantum dots" as fluorophores will greatly enhance signal detection in flow cytometry, and hence in both quantitative and qualitative immunofluorescence applications.
Quantification of aquatic viruses by flow cytometry
2010
For many laboratories, flow cytometry is becoming the routine method for quantifying viruses in aquatic systems because of its high reproducibility, high sample throughput, and ability to distinguish several subpopulations of viruses. Comparison of viral counts between flow cytometry and epifluorescence microscopy typically shows slopes that are statistically not distinguishable from 1, thus confirming the usefulness of flow cytometry. Here we describe in detail all steps in the procedure, discuss potential problems, and offer solutions.
Flow cytometry as a tool for the study of phytoplankton
Scientia Marina, 2000
An overview is presented on flow cytometry as a tool for counting, analysis and identification of phytoplankton species and groups. The paper covers basics on the analysis technique and instrumentation such as the measuring principle, the type of instrument, limitations and pitfalls with phytoplankton samples and sample handling and preprocessing. Possibilities of the measured entities are discussed, roughly divided in light scatter and related parameters, the endogenous fluorescence and exogenous fluorescence, followed by a discussion on the actual applications such as phytoplankton abundance analysis, ecology and physiology research and monitoring of particle size and biomass. In addition to a limited literature review, we tried to assess how flow cytometry is used in routine laboratory practice and monitoring operations. Therefore, a questionnaire was sent out via email to 47 scientists at 43 institutes known to us as involved in flow cytometric analysis of phytoplankton. In total, 19 scientists responded. Specific survey results are included in italic print whereas some more general answers were integrated in the overview.
Simultaneous measurement of grazing and viral lysis of phytoplankton v2
protocols.io, 2015
Sampling and physicochemical variables Sampling and physicochemical variables In July-August of 2009, 32 stations were sampled in the Northeast Atlantic Ocean during the shipboard expedition of STRATIPHYT (changes in vertical stratification and its impacts on phytoplankton communities) (Figure 1). Water samples were collected from at least 10 separate depths in the top 250-m water column using GO-flow (General Oceanics, Miami, FL, USA), 10-liter samplers mounted on an ultra-clean (trace-metal free) system equipped with CTD (Sea-Bird Electronics,
Flow cytometric applicability to evaluate UV inactivation of phytoplankton in marine water samples
Marine Pollution Bulletin, 2015
Disinfection of microbes is of importance to prevent the spread of pathogens and non-indigenous species in the environment. Here we test the applicability of using flow cytometry (FCM) to evaluate inactivation of the phytoplankter Tetraselmis suecica after UV irradiation and labeling with the esterase substrate 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). Non-irradiated and UV irradiated samples were analyzed with the plate count technique and FCM for 24 days. The numbers of colony forming units were used as a standard to develop a FCM protocol. Our protocol readily distinguishes live and dead cells, but challenges were encountered when determining whether UV damaged cells are dying or repairable. As damaged cells can represent a risk to aquatic organisms and/or humans, this was taken into account when developing the FCM protocol. In spite of the above mentioned challenges we argue that FCM represents an accurate and rapid method to analyze T. suecica samples.
Flow Cytometric Applicability of Fluorescent Vitality Probes on PHYTOPLANKTON1
Journal of Phycology, 2011
The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2¢,7¢-dichlorofluorescein diacetate [H 2 DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC 4 ] and SYTOX-Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold-water, 26 temperate, and four warm-water species. The combined live ⁄ dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live ⁄ dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein-AM and H 2 DCFDA (P < 0.001). Of the two membrane probes, DIBAC 4 (3) stained rhodophytes and euglenophytes much better than SYTOX-Green. The 13 algal strains best stainable (high live ⁄ dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC 4 (3), and SYTOX-Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.