FLOW CYTOMETRIC ANALYSES OF VIRAL INFECTION IN TWO MARINE PHYTOPLANKTON SPECIES, MICROMONAS PUSILLA (PRASINOPHYCEAE) AND PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)

Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) 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. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis. The present study provides insight into basic virus–algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.     

Candidatus Prosiliicoccus vernus,a spring phytoplankton bloom associated member of the Flavobacteriaceae

Microbial degradation of algal biomass following spring phytoplankton blooms has been characterised as a concerted effort among multiple clades of heterotrophic bacteria. Despite their significance to overall carbon turnover, many of these clades have resisted cultivation. One clade known from 16S rRNA gene sequencing surveys at Helgoland in the North Sea, was formerly identified as belonging to the genus Ulvibacter. This clade rapidly responds to algal blooms, transiently making up as much as 20% of the free-living bacterioplankton. Sequence similarity below 95% between the 16S rRNA genes of described Ulvibacter species and those from Helgoland suggest this is a novel genus. Analysis of 40 metagenome assembled genomes (MAGs) derived from samples collected during spring blooms at Helgoland support this conclusion. These MAGs represent three species, only one of which appears to bloom in response to phytoplankton. MAGs with estimated completeness greater than 90% could only be recovered for this abundant species. Additional, less complete, MAGs belonging to all three species were recovered from a mini-metagenome of cells sorted via flow cytometry using the genus specific ULV995 fluorescent rRNA probe. Metabolic reconstruction indicates this highly abundant species most likely degrades proteins and the polysaccharide laminarin. Fluorescence in situ hybridisation showed coccoid cells, with a mean diameter of 0.78 mm, with standard deviation of 0.12 μm. Based on the phylogenetic and genomic characteristics of this clade, we propose the novel candidate genus Candidatus Prosiliicoccus, and for the most abundant and well characterised of the three species the name Candidatus Prosiliicoccus vernus.     

Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Optical plankton analyser: a flow cytometer for plankton analysis, I: Design considerations

The design criteria for a flow cytometer (FCM) for the analysis of field samples of phytoplankton are described. The criteria are based on the occurrence of a wide variety of particle sizes in field samples, normally at low concentrations. The instrument should be able to analyse cells and colonies from 0.5 to 500 microns diameter and of over 2,000 microns length. A minimum flow rate of 4 microliters.s-1 was calculated from natural plankton concentrations. Commercially available FCMs are not suited to measure this range of sizes at this rate. Further limitations of standard FCMs are uneven illumination or incomplete processing of long signals. In addition, long filamentous colonies can break into small fragments caused by too high acceleration in the standard flow cuvette. Recognition of these limitations is of importance for the flow cytometry of phytoplankton. The new design was developed to avoid these limitations. A dynamic range 5 to 6 decades could be accomplished by a combination of logarithmic amplifiers, a slit-shaped focal spot, and a pulse integration system that can process long pulses. Multilaser capability to identify different phytoplankton species, a low fluid shear cuvette, and a trigger gate-extension for inhomogeneously fluorescent algal filaments were included in the design.     

Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye.

The in vitro response of murine lymphocytes to allogeneic and mitogenic stimulation has been studied by using the nontoxic fluorescent DNA probe, Hoechst 33342, and a fluorescence flow cytometer-cell sorter. Under appropriate conditions, two peaks of fluorescent intensity, not related to cellular DNA content, can be seen. As early as 12 hr after culture set up, lymphocyte activation can be identified. It appears that Hoechst-labeled lymphocytes of higher fluorescence intensity represent those cells activated by allogeneic stimulation whereas cells obtained from the lower intensity peak are nonresponding lymphocytes.     

Flow cytometry, microscopy, and DNA analysis as complementary phytoplankton screening methods in ballast water treatment studies

Ballast water is the main vector for marine invasions. To minimize the spread of invasive species, the International Maritime Organization (IMO) has adopted the Ballast Water Management Convention which requires the installation of shipboard ballast water treatment systems (BWTS). During BWTS tests, the phytoplankton abundance and species composition were followed after treatment with both filtration and ultraviolet radiation. Although the installation fulfilled the IMO criteria after a 5-day holding time in a model ballast tank, the ultimate effectiveness of the treatment was further tested in long-term (20 days) incubation experiments under optimal phytoplankton growth conditions. Application of flow cytometry, microscopy, and DNA sequencing to these incubation samples gave an indication of the phytoplankton species that might be introduced by ballast water discharge—despite treatment. Phytoplankton was reliably quantified using flow cytometry, while fast identification was best done using microscopy. Some groups that contained potentially toxic species could not be identified at species level using microscopy; for these species, identification using genetic techniques was necessary. It is concluded that if long-term incubation experiments are used as an additional tool in testing BWTS effectiveness, a combination of phytoplankton screening methods can be applied depending on the detail of information that is required.     

Cell-specific beta-N-acetylglucosaminidase activity in cultures and field populations of eukaryotic marine phytoplankton.

It is widely appreciated that eukaryotic marine phytoplankton can hydrolyze a variety of compounds within the dissolved organic matter (DOM) pool in marine environments. Herein, cultures and field populations of marine phytoplankton were assayed for beta-N-acetylglucosaminidase activity, a terminal enzyme of chitin degradation. A traditional bulk assay, which can assess hydrolytic rate, but is not cell-specific, was complemented with a cell-specific assay that images the activity associated with single cells using an enzyme labeled fluorescence (ELF) substrate. beta-N-acetylglucosaminidase activity was widespread across various taxa of marine phytoplankton, and activity was observed both under controlled culture conditions and in field populations. The number of cells with enzyme activity varied with the nutritional physiology of the test species in three of the 17 cultures tested. In these three cases the number of cells with activity in the low nutrient medium was higher than in nutrient replete medium. Taken together, these data suggest that a broad group of marine phytoplankton may be a relevant part of chitin-like DOM degradation and should be incorporated into conceptual models of chitin cycling in marine systems.     

Flow cytometric evaluation of sperm parameters in relation to fertility potential

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.     

Refining cryptophyte identification: matching cell fixation methods to FISH hybridisation of cryptomonads

The Division Cryptophyta, Class Cryptophyceae, contains ecologically important microalgae that are found in all aquatic habitats. The identification of the Cryptophyta is challenged by a need to examine species in the scanning electron microscope or transmission electron microscope to visualise features needed to identify its species. Molecular verification is becoming increasingly important for this group because of its polymorphic haploid and diploid cells of the same species with different morphologies. Thus, for routine monitoring programmes, this group is not usually identified beyond the level of class and that is done only if the samples are routinely examined with a fluorescent microscope or with flow cytometry, and the cryptophytes are counted based on the natural orange fluorescent of their phycobilin pigments. In order to use rRNA probes, the cells must be fixed for permeabilisation of the cell membrane for probe penetration. Here, we present a test of routine fixation methods to determine the fixation that is most compatible for use in fluorescent in situ hybridisation methods with fluorescent microscopy and flow cytometer to facilitate cryptomonad identification.     

Study and application of flow injection spectrofluorimetry with a fluorescent probe of 2-(2-pyridil)-benzothiazoline for superoxide anion radicals

This paper presents an automatic spectrofluorimetric method (flow injection spectrofluorimetry) using a novel fluorescent probe named H. Py. Bzt (2-(2-pyridil)-benzothiazoline) for determining superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by infrared and (1)H nuclear magnetic resonance spectra. It could specially identify and trap O(2)(*-) and was oxidized by O(2)(*-) to form a strong fluorescence product. Based on this reaction, the flow injection spectrofluorimetric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species because the probe can be oxidized only by O(2)(*-) excluding H(2)O(2). As a kind of simple, rapid, precise, sensitive and automatic technique, it was applied to measurement of SOD activity in scallion, garlic, and onion with satisfactory results.     

Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa

In this study we used a new method to detect reactive oxygen species (ROS) induced damage at the level of the sperm plasma membrane in fresh and frozen-thawed stallion sperm. Lipid peroxidation (LPO) in sperm cells was assessed by a fluorescent assay involving the labeling of stallion sperm with the LPO reporter probe C11-BODIPY(581/591). The peroxidation dependent spectral emission shift of this membrane probe could be localized using inverted spectral confocal microscopy and quantified on living and deteriorated sperm cells using flow cytometry. Mass spectrometric analysis of the main endogenous lipid class, phosphatidylcholine (PC), was carried out to determine the formation of hydroxy- and hydroperoxyphosphatidylcholine in fresh sperm cells. Peroxidation as reported by the fluorescent probe corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of LPO. This allowed us to correlate endogenous LPO with localization of this process in the living sperm cells. In absence of peroxidation inducers, only relatively little peroxidation was noted in fresh sperm cells whereas some mid-piece specific probe oxidation was noted for frozen-thawed sperm cells. After induction of peroxidation in fresh and frozen-thawed sperm cells with the 0.1 mM of lipid soluble ROS tert-butylhydrogen peroxide (t-BUT) intense probe oxidation was produced in the mid-piece, whereas the probe remained intact in the sperm head, demonstrating antioxidant activity in the head of fresh sperm cells. At higher levels of t-BUT, probe peroxidation was also noted for the sperm head followed by a loss of membranes there. Frozen-thawed sperm were more vulnerable to t-BUT than fresh sperm. The potential importance of the new assays for sperm assessments is discussed.     

OPTIMIZATION OF AN IMMUNOFLUORESCENT ASSAY OF THE INTERNAL ENZYME RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RUBISCO) IN SINGLE PHYTOPLANKTON CELLS1

Immunochemical probes are widely used to identih different species and to quantify and understand the role that different antigens play within cells. We optimized a single-cell immunofluorescent assay for the carbon fixation enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) in order to quantify the enzyme by flow cytometry in phytoplankton cells. The criteria for optimization of the immunofluorescent assay for Rubisco in single cells included maximization of Rubisco immunogenicity, minimization of Rubisco diffusion out of the cells, minimization of cell breakage, and maximization of the cell labeling. Several fixatives (cross-linkers and denaturing) and permeabilizing agents were tested on 26 species of phytoplankton. The only fixative / permeabilizing agent that fulfilled the criteria established for the assay was 96% ethanol. Phytoplankton cells collected from the field needed further treatment with a strong oxidant to permeabilize ethanolfixed cells and thus allow the antibody probe to access the Rubisco antigen. This study should have a general applicability to the study of other soluble photosynthetic antigens in single phytoplankton cells.     

Development of a PNA probe for the detection of the toxic dinoflagellate Takayama pulchella

A peptide nucleic acid (PNA) probe was developed to detect the toxic dinoflagellate, Takayama pulchella TPXM, using fluorescent in situ hybridization (FISH) combined with epifluorescent microscopy and flow cytometry. The PNA probe was then used to analyze HAB samples from Xiamen Bay. The results indicated that the fluorescein phosphoramidite (FAM)-labeled probe (PNATP28S01) [Flu]-OO ATG CCA TCT CAA GA, entered the algal cells easily and bound to the target species specifically. High hybridization efficiency (nearly 100%) was observed. Detection by epifluorescence microscopy and flow cytometry gave comparable results. The fluorescence intensity of the PNA probe hybridized to T. pulchella cells was remarkably higher than that of two DNA probes used in this study and than the autofluorescence of the blank and negative control cells. In addition, the hybridization condition of the PNA probe was easier to control than DNA probes, and when applied to field-collected samples, the PNA probe showed higher binding efficiency to the target species than DNA probes. With the observed high specificity, binding efficiency, and detection signal intensity, the PNA probe will be useful for monitoring harmful algal blooms of T. pulchella.     

Flow cytometry: instrumentation and application in phytoplankton research

In flow cytometry, light scattering and fluorescence of individual particles in suspension is measured at high speed. When applied to planktonic particles, the light scattering and (auto-)fluorescence properties of algal cells can be used for cell identification and counting. Analysis of the wide size spectrum of phytoplankton species, generally present in eutrophic inland and coastal waters, requires flow cytometers specially designed for this purpose. This paper compares the performance in phytoplankton research of a commercial flow cytometer to a purpose built instrument. It reports on the identification of phytoplankton and indicates an area where flow cytometry may supersede more conventional techniques: the analysis of morphological and physiological characteristics of subpopulations in phytoplankton samples.     

Monitoring cell‐specific neutral lipid accumulation in Phaeodactylum tricornutum (Bacillariophyceae) with Nile Red staining – a new method for FlowCAM

With the fluorescent stain Nile Red (NR), phytoplankton lipid accumulation can be monitored quickly and in situ. In the light of recent results in phytoplankton diversity research, there is also a need for cell‐ and species‐specific lipid measurement techniques. The objective of this work was to investigate whether cell‐specific phytoplankton lipid accumulation could be monitored with the image‐based particle analyzer FlowCAM? and NR staining. Applying Phaeodactylum tricornutum as a model species, we compared the FlowCAM method to two established lipid quantification methods: spectrofluorometric NR fluorescence measurement and total lipid analysis by gas chromatography. The experiment was carried out in batch cultures under nitrogen limitation to induce lipid accumulation. We showed significant correlation between the three different lipid quantification methods confirming the applicability of the novel FlowCAM method in cell‐specific and near real‐time lipid quantification. Furthermore, with the method described here, the lipid content of taxonomically distinguished cells can eventually be measured from multispecies cultures, opening several new possibilities to study species‐specific responses to stress conditions and the complementarity effect.     

Sensitive detection of RNAs in single cells by flow cytometry.

A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.     

Flow cytometry to assess biochemical pathways in heat-stressed Cronobacter spp. (formerly Enterobacter sakazakii)

Aims: Using a flow cytometry (FC)‐based approach in combination with four selected fluorescent probes, the biochemical pathway activated following the adaptation of Cronobacter spp. to lethal heat stress was investigated. This approach assessed the physiological changes induced in four strains of Cronobacter spp. Methods and Results: Using the commercially available live/dead viability assessment fluorescence probes, live, injured or dead bacterial cells were studied. Cellular respiration and membrane potential were evaluated using the dye‐labelled probe 3,3′‐dihexylocarbocyanine iodide, metabolic activity was evaluated using a fluorescein diacetate (FDA) probe, intracellular pH changes were measured using a carboxy‐fluorescein diacetate succinimidyl ester probe, and reactive oxygen species were measured using a hydroethidine fluorescent probe. Adaptation to lethal heat stress induced physiological changes that potentially improve the survival of Cronobacter spp. Conclusions: These data showed that in situ assessment of physiological behaviour of lethally stressed cells using multiparameter FC is a useful, rapid and sensitive tool to study and assess the viability and physiological state of Cronobacter cells. Significance and Impact of the Study:  This study shows that FC is a valuable tool in the study of physiological aspects of increased survival because of sublethal adaptation to heat.     

Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: a possible role of alanine residue in tryptophan-rich undecapeptide

8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pH(i)) indicator. In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH-independent wavelength of 415 nm. A simplified correction of the fluorescent signals was applied, enabling to show for this species a good near-neutral pH(i) maintenance capacity in a pH 3.9 medium. Octanoic acid, which is known to have toxic effects on yeast, decreased the pH(i) and increased the 260-nm-absorbing compounds leakage. However, this acid inhibited the fluorescent probe efflux linearly with its concentration suggesting a pH(i)-dependent efflux of pyranine from cells.     

Microbiological monitoring in the biodegradation of sewage sludge and food waste

AIM: To study the microbiology of intensive, in-vessel biodegradation of a mixture of sewage sludge and vegetable food waste. METHODS AND RESULTS: The biodegradation was performed in a closed reactor with the addition of a starter culture of Bacillus thermoamylovorans SW25 under conditions of controlled aeration, stirring, pH and temperature (60 degrees C). The content of viable bacterial cells, determined by flow cytometry, increased from 5 x 108 g-1 of dry matter to 61 x 108 g-1 for 6 days of the process and then dropped to the initial value at the end of the process. The reductions of organic matter, 16S rRNA of methanogens and coenzyme F420 fluorescence during 10 days of the treatment were 67, 54 and 87% of the initial values, respectively. The biodegradability of the organic matter decreased during the 10 days of the treatment from 3.8 to 1.3 mg CO2 g-1 of organic matter per day. The treatment of sewage sludge and food waste at 60 degrees C did not remove enterobacteria, which are the agents of intestinal infections, from the material. The percentage of viable enterobacterial cells, determined by fluorescent in situ hybridization (FISH) with Enterobacteriaceae-specific oligonucleotide probe and flow cytometry, varied from 1 to 14% of the viable bacterial cells. CONCLUSIONS: The mixture of sewage sludge and food waste can be degraded by the aerobic thermophilic bacteria; the starter culture of Bacillus thermoamylovorans SW25 can be used to perform this process; and enterobacteria can survive under treatment of sewage sludge and food waste at 60 degrees C for 13 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that FISH with an oligonucleotide probe can be used to study not only the growth but also the degradation of biomass. Obtained results could be used to design the bioconversion of sewage sludge and food waste into organic fertilizer.