Precym will host the “BD Biosciences Marine Biology Workshop” which will take place on Thursday April 2 at the “Université de la Méditeranée” in Marseille, France.
BD Biosciences scientists together with guest speakers will share their knowledge and expertise of Flow Cytometry and will demonstrate how Analytical Cytometry can be applied to marine sciences.
Additionally there will be scientific presentations by leading Oceanographers. In the afternoon the new BD Influx™ Mariner will be presented and its capabilities will be tested by analyzing samples that have been brought by the guest speakers.
During the Workshop, the following talks will be presented :
CYTOMETRIC CHARACTERIZATION OF AQUATIC BACTERIA :
FROM IDENTIFICATION TO PHYSIOLOGICAL STATUS ASSESSMENT
Gérald Grégori (Ph.D)
Université de la Méditerranée, Centre d’Océanologie de Marseille (OSU)
Laboratoire de Microbiologie, Géochimie et Ecologie Marines CNRS UMR 6117
163 Avenue de Luminy – Case 901- Bât TPR1 – Entrée F, 13288 Marseille Cedex 9, France
The goal of this presentation is to illustrate the qualitative and quantitative measurements that can be assessed by Flow Cytometry with particular reference to aquatic prokaryotes (bacteria and archaea). Because of their wide biodiversity, their high abundances, and their large variety of metabolic activities which makes them ubiquitous, these micro organisms represent a key component in both fresh and marine environments. To improve our understanding of the heterogeneity and complexity of the bacterial populations, and their functional role, their analysis must be done at the single cell level and requires a non-invasive interrogation of the cells. The development of analytical flow cytometry and cell sorting, together with advances in the field of fluorescent probes have enabled this rapid and semi-automatic technique to provide a powerful means of measurement of a wide range of cell characteristics (cellular content, membrane integrity, viability, cellular and enzymatic activities, etc.).
This presentation will concentrate on how to study bacteria by flow cytometry at the single cell level using scatter and various fluorochromes in order to gain accurate data representation of populations studied in the laboratory or within heterogeneous natural samples.
CYTOMETRIC DIVERSITY OF PHYTOPLANKTON :
FROM IDENTIFICATION TO PHYSIOLOGICAL STATUS ASSESSMENT
Dr. Raffaella Casotti (Ph.D)
Laboratory of Functional Ecology and Evolution
Stazione Zoologica “A. Dohrn”, Villa Comunale, I80121 Napoli, Italy
Flow cytometry has greatly contributed to our actual knowledge of aquatic trophic food webs through the exploration of plankton communities, leading to the assessment of the importance and relevance of picophotoautotrophs in the oceans. Apart from counting of phytoplankton and bacteria, the analysis of variations in autofluorescence, scatter and the use of fluorescent stains has demonstrated its power in elucidating the physiology and adaptations of organisms to the natural environment both in natural samples and in culture. We routinely use flow cytometry to assess the toxic effects of diatom-produced polyunsaturated aldehydes on viability and metabolic activity of phytoplankton. By using a specific stain we were also able to show the involvement of NO in stress-response mechanisms of Phaeodactylum tricornutum. Perspective work will greatly profit from increase in sensitivity and high sorting capability so to apply additional techniques, such as the molecular ones, on isolated cells and also to apply our findings to natural populations during blooms.
DIVERSITY OF SMALL PHOTOSYNTHETIC EUKARYOTES IN MARINE WATERS :
COUPLING FLOW CYTOMETRY SORTING WITH PHYLOGENETIC ANALYSES.
Daniel Vaulot (Ph.D)
Shi, Dominique Marie, Mikihide Demura, Masanobu Kawachi
Station Biologique de Roscoff, UMR 7144 CNRS et UPMC, BP 74, 29680 Roscoff, France
Small photosynthetic eukaryotes play a key role in the production of marine waters. However molecular approaches have failed to fully explore their diversity because 18S rRNA gene clone libraries constructed from natural samples are often dominated by heterotrophic groups. To circumvent this, we developed an approach consisting in physically sorting by flow cytometry photosynthetic cell populations based on their size and pigment fluorescence. Genes from the sorted cells are then amplified and cloned. We have applied this approach to coastal waters from the English Channel and to open ocean oligotrophic waters from the South East Pacific. Nuclear 18S rRNA gene clone libraries constructed from such sorted populations exhibit very little contamination from sequences of heterotrophic organisms. Analyses have revealed the importance of novel groups such as Prasinophyceae clade IX or marine Chrysophyceae. By coupling this approach with whole genome amplification, the number of cells that need to be sorted can be lowered considerably (down to 10), resulting in samples where a single genotype dominates for which it should be possible to perform metagenomics analyses targeting uncultured eukaryotic groups.
THE BDTM INFLUX CELL SORTER FOR MARINE MICROBIOLOGY
Ger van den Engh (PhD)
VP Cell Analysis
Marine microbes can be identified and counted with flow cytometers. Marine particles often have unusual optical properties compared to the cells seen in traditional samples. Detecting unknown fluorescence spectra, polarization properties and scattering by very small particles put special requirements on the detection system. In addition, particle identification requires genome analysis at the single cell level. To meet these requirements the BD Influx Mariner sorter has been equipped with special detectors and sort modules. I will describe these special modules and present data that demonstrate how these technical features are best applied to marine studies. I will also reveal some aspects of our plans to develop a submersible flow cytometer for long-term deployment in the ocean.
STRUCTURAL STUDIES ON LIVE PICOPLANKTON – TOWARDS A MOLECULAR-LEVEL DESCRIPTION OF A LIVING CELL
Laboratory of Molecular Biophysics, Institute of Cell and Molecular Biology, Uppsala University,
Box 596, Husargatan 3, SE 75124 Uppsala, Sweden
Life fits into a sphere of about 300 nanometer diameter as demonstrated by photosynthetic picoplankton from marine sources. Understanding life necessitates understanding the structure of an organism at the relevant length-scales and over a broad time domain. More than sixteen orders of magnitude in time and four orders of magnitude in space have to be considered to describe the tiniest cells in molecular terms. New experimental methods, including flash-diffraction imaging with extremely intense coherent X-ray pulses from X-ray lasers, bring these living cells within the scope of detailed structural analysis for the first time. Initial results indicate molecular-scale imaging may be achieved on unmodified living cells with a time resolution of a few femtoseconds. The talk will survey progress in ultra-fast coherent imaging. A facet of the project is that specific cells need to be selected and injected in a well controlled manner into the intense X-ray beam. Instruments based on cell sorters and spraying techniques need to be developed to achieve this.