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ABSTRACT
The present study was intended to provide new approaches based
on analytical flow cytometry to characterize microbial assemblages in marine
environments, namely their spatial and temporal ditributions, their viability
and their physiological state. Micro-organisms play an essential role in
the ocean with respect to organic matter production and mineralization.
In addition, they represent an important biomass. The study of microbial
assemblages implies several investigation scales.
Counting and characterizing micro-organisms together with biomass estimation
can be achieved at one site, basin or ocean scale. The short-term, seasonal
or annual variability of their distribution is investigated at hour-, month-
or year-scale respectively. Micro-organisms are very sensitive to environmental
conditions and every perturbation, either natural or of anthropic origin
may affect microbial assemblages in their composition, in species abundances
and/or cell physiological state. The monitoring of ultraphytoplankton (size
< 10 µm) was conducted over two years in the Bay of Marseilles.
This survey reinforces the need of extending present phytoplankton surveys
in coastal waters, run by optical microscopy and therefore restricted to
the size class > 20 µm, to the lower size class by using flow cytometry.
The objective to characterize micro-organisms as bioindicators remains
to be substantiated.
To better understand the functioning of the marine ecosystem, it is necessary
to investigate at the cell scale the different activities of the micro-organisms.
Usually, bulk activity measurements are referred to total counts which
implies the involvement of all cells. This is not correct for natural samples
because total counts may include ghost, dead or damaged cells. It is therefore
crucial to quantify the amounts of viable as well as dead cells. In this
thesis work, the double staining of nucleic acids developed by Barbesti
and coworkers (2000) for cultured bacteria in fresh water, was adapted to
natural samples from marine environments. This "live/dead" assay is essentially
a test of membrane integrity and its resolving power stems from the energy
transfer mechanism between the two nucleic acid fluorescent probes.
The heterogeneity of micro-organisms with respect to their viability
is also accompanied by heterogeneity in their metabolic activities. In this
domain too, the search for information at the cellular scale is crucial. This
is the case for instance with cell respiration, an index of the rate of organic
matter mineralization. For this activity, the need is all the more important
that there is no available direct method to determine the bulk respiration
rate in seawater samples. A promising breakthrough has been achieved in this
thesis work by establishing a linear relationship between the green fluorescence
of cultured Dunaliella tertiolecta (Chlorophyceae) stained with DIOC6(3),
a probe sensitive to the mitochondrial membrane potential, and the rate of
O2 uptake. The extension of this approach to different phytoplanktonic and
bacterial species representative of the marine environment remains to be
substantiated in order to establish a relatively direct method to measure
cell respiration in situ by using analytical flow cytometry.
Key words :
Ultraplankton ; flow cytometry ; monitoring ; bacterial viability ; cellular
respiration ; artificial neural networks (Kohonen Self-Organizing-Maps).
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