Dataset Information

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

ABSTRACT: Marine fungus-like eukaryotic unicellular protists (thraustochytrids) are considered to play an important role in the marine microbial food web. However, their abundance, distribution, and relative biomass in coastal waters have not yet been examined in detail. By using a flow cytometry method (FCM) for the rapid enumeration of thraustochytrids in nearshore and offshore stations along the Gulf of Bohai, China, we herein expanded current knowledge on their ecological significance. The FCM method allows for the rapid detection and quantification of prokaryotic and eukaryotic cells, but is rarely applied to the enumeration of small eukaryotic protists. Epifluorescence microscopy (EpiM) has been commonly used for the direct detection and enumeration of thraustochytrids; however, this method is time-consuming and inapplicable to a large-scale analysis of complex seawater samples. There is no available FCM method to track the abundance and biomass of thraustochytrids in marine habitats. The FCM enumeration of thraustochytrids in seawater samples ranged between 400 and 4,080 cells mL-1 with a biomass range of 8.15-83.96 ?g C L-1. The thraustochytrid biomass contributed 10.9% to 98.1% of the total biomass of the heterotrophic microbial community comprising bacterioplankton and thraustochytrids. Their overall abundance in nearshore stations was significantly different from that in offshore stations (P<0.5). The present results provide an optimized method for the rapid detection and enumeration of thraustochytrids in seawater and facilitate large-scale studies of the ecological role of thraustochytrids in the microbial food web of coastal waters.

SUBMITTER: Duan Y

PROVIDER: S-EPMC6031391 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

Duan Yingbo Y Sen Biswarup B Xie Ningdong N Paterson James S JS Chen Zixi Z Wang Guangyi G

Microbes and environments 20180616 2

Marine fungus-like eukaryotic unicellular protists (thraustochytrids) are considered to play an important role in the marine microbial food web. However, their abundance, distribution, and relative biomass in coastal waters have not yet been examined in detail. By using a flow cytometry method (FCM) for the rapid enumeration of thraustochytrids in nearshore and offshore stations along the Gulf of Bohai, China, we herein expanded current knowledge on their ecological significance. The FCM method ...[more]

PMID: 29910220

Similar Datasets

Project description:Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs. Click to hear the ISTH Academy's webinar on microvesicles SUMMARY: Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC-optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side-scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MPs' clinical relevance at a multicenter level.

Project description:BackgroundNucleic acids can fold into non-canonical secondary structures named G-quadruplexes (G4s), which consist of guanine-rich sequences stacked into guanine tetrads stabilized by Hoogsteen hydrogen bonding, π-π interactions, and monovalent cations. G4 structure formation and properties are well established in vitro, but potential in vivo functions remain controversial. G4s are evolutionarily enriched at distinct, functional genomic loci, and both genetic and molecular findings indicate that G4s are involved in multiple aspects of cellular homeostasis. In order to gain a deeper understanding of the function of G4 structures and the trigger signals for their formation, robust biochemical methods are needed to detect and quantify G4 structures in living cells. Currently available methods mostly rely on fluorescence microscopy or deep sequencing of immunoprecipitated DNA or RNA using G4-specific antibodies. These methods provide a clear picture of the cellular or genomic localization of G4 structures but are very time-consuming. Here, we assembled a novel protocol that uses the G4-specific antibody BG4 to quantify G4 structures by flow cytometry (BG-flow).ResultsWe describe and validate a flow cytometry-based protocol for quantifying G4 levels by using the G4-specific antibody BG4 to label standard cultured cells (Hela and THP-1) as well as primary cells obtained from human blood (peripheral blood mononuclear cells (PBMCs)). We additionally determined changes in G4 levels during the cell cycle in immortalized MCF-7 cells, and validated changes previously observed in G4 levels by treating mouse macrophages with the G4-stabilizing agent pyridostatin (PDS).ConclusionWe provide mechanistic proof that BG-flow is working in different kinds of cells ranging from mouse to humans. We propose that BG-flow can be combined with additional antibodies for cell surface markers to determine G4 structures in subpopulations of cells, which will be beneficial to address the relevance and consequences of G4 structures in mixed cell populations. This will support ongoing research that discusses G4 structures as a novel diagnostic tool.

Project description:Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.

Project description:Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria-host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

Dataset Information

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

Publications

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets