Flow cytometric applicability to evaluate UV inactivation of phytoplankton in marine water samples (original) (raw)
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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.
Flow cytometric analysis of phytoplankton viability following viral infection
Aquatic Microbial Ecology, 2001
Two flow cytometric assays using physiological probes were used on the phytoplankton species Phaeocystis pouchetii and Micromonas pusilla to examine the assays' utility in detecting viral infections. Dead cells were detected using the membrane impermeant nucleic-acid dye SYTOX-Green, which stains algal cells that have lost their membrane integrity. Live cells were detected using the membrane permeant dye Calcein-AM, which is hydrolyzed by intracellular esterases into a green fluorescent charged form. We found that both assays are easy to use, are reproducible and can indeed be used as markers of the viability of individual phytoplankton cells following infection by viruses. Cell death rates up to 0.8 d -1 for P. pouchetii and 0.5 d -1 for M. pusilla were calculated. The first day postinfection, death rates determined by the Calcein-AM assay were typically twice as high as those determined by the SYTOX-Green assay. Both viability tests were found to assess the physiological status of noninfected P. pouchetii cells, independent of viral infection. The optimal choice of viability assay depended on the phytoplankton species studied. Compared with existing assays, the protocols described permit examination of infected phytoplankton in more detail, yielding insight into the heterogeneity of the algal population.
Detection of toxic phytoplankton species by immunochemical particle analysis based on flow cytometry
Netherlands Journal of Aquatic Ecology, 1994
Particulate suspended matter in oceanic, coastal, and estuarine regions can be specifically marked immunochemically with a fluorescent probe using antisera recognizing antigens present on their surface. Of the particulate matter, phytoplankton is a major component. Toxic species that may form harmful blooms can be a direct threat to aquaculturing, tourism, sea-life and man. In order to detect such species in natural fixed phytoplankton populations, immunochemical tagging has been combined with flow cytometric evaluation. Microa~gal cells can be labeled with a fluorescent probe (fluorescein isothiocyanate, FITC, is recommended). Labeled cells are counted using a flow cytometer. This method has proved to be applicable in a monitoring programme in the North Sea.
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.
The regrowth of phytoplankton cultures after UV disinfection
Marine Pollution Bulletin, 2013
This study addresses how cultures of three phytoplankton species-Chaetoceros calcitrans, Chlorella autotrophica and Phaeocystis globosa-can recover from the effects of UV-C exposure if the cells are placed in a rich medium. Flow cytometry and pulse amplitude modulation (PAM) were used to determine cell recovery after UV treatment. The recovery of C. calcitrans was complete 9 days after treatment. For C. autotrophica, the recovery was noticeable 5 days after treatment. P. globosa only recovered if the UV dose did not exceed 7.3 Â 10 5 lWs/cm 2. The recovery of the UV-treated cultures introduced to a regrowth medium, compared with the recovery of the irradiated cultures kept in their original environment, had two main characteristics: cell recovery was slower but was more efficient. This pattern of recovery has very important implications for real ballast water management systems because such systems discharge treated water into the environment.
Flow cytometry has been used in oyster feeding studies at Delaware State University for counting and sorting of marine micro-algal cells to determine the filtration activities of American oyster (Crasostrea virginica). This study was conducted to develop a simple protocol for accurate quantification of several cultured algae, using a known quantity of fluorescent microspheres as an internal calibration standard. While using flow cytometry to analyze algal samples in filtration studies on the oysters (C. virginica), we observed shifts in algae populations in both preserved and unpreserved samples over time. Because of these shifts, we felt it was important to determine the most efficient way to quantify algae accurately used in the filtration study. Two algal species, Isochrysis spp. (TISO) and Tetraselmis chui (PLY-429) cultured in F/2 media under the laboratory conditions at 12/12 hour light/dark cycles at 24°C room temperature at a set light intensity (130 umol), were used in this...
The advantages of flow cytometry in comparison to fluorometric measurement in algal toxicity test
2012
Fluorometric measurements in algal toxicity tests are very commonly used as surrogate parameters for algal biomass. Although, fluorometry is a powerful technique, we have demonstrated that it is not suitable for determination of toxic effects of chemicals, which alter the fluorescence spectra. We recommend the flow cytometry as the best technique for detecting algal and/or cyanobacterial cell count and fluorescence per cell. Flow cytometry has many advantages: little volume of algal/cyanobacterial sample required, suitable also for little algal/cyanobacterial cells, distinction between live and dead cells. Furthermore, flow cytometry reveals the early changes in fluorescence spectra as a consequence of the specific chemicals presence or stress, even though the cell count is not yet affected (an early marker for ecotoxicology testing).
Marine Ecology Progress Series, 1993
Ten methods to preserve phytoplankton populations for flow cytometric analyses were tested. These methods were differentiated by the rate of freezing and thawing, and the use or non-use of cryoprotectants (DMSO and/or glycerol) and chemical fixation. After freezing, the samples were stored in liquid nitrogen. These methods were tested on 3 freshwater and marine algal species. Different intensity parameters and 2 properties were considered. Firstly the number of cells lost, which was more significant with rapid freezing and chemical fixation, and less significant with the addition of cryoprotectants. Secondly, the preservation of both light scattering and fluorescence, which was better with slow freezing than with cryoprotectants. Slow freezing followed by chemical fixation appeared to be the best protocol studied and even i f glycerol addition without chemical fixation seemed to be overall the best method, implying the use of cryoprotectant, all these techniques had to be tested on a case by case basis, particularly when phycocyanin and chlorophyll fluorescence were studied.