2010 Chapters (Layout Features)

Analysis of Phosphatase Activity from Aquatic Heterotrophic Bacteria at the Single Cell Level by Flow Cytometry: Example of a Development Achieved in the Regional Flow Cytometry Platform for Microbiology (Precym) Hosted by the Oceanology Center of Marseille

Gregori, Gerald; Denis, Michel; Duhamel, Solange; Van Wambeke, France

Marine microbes play a very important role in biogeochemical cycles. Because of their ­biodiversity, the diversity of their metabolisms, and their ­physiological heterogeneity (live/dead, active/inactive cells), it is necessary to study them at the single cell level.

Flow cytometry has become a technique of choice to reach this goal, providing a fast, reliable, and multiparametric analysis of individual cells. However, the cost of such a technique makes flow cytometry out of reach for small institutes or laboratory units. It also requires a qualified and well-trained operator.

To make this technique available to all the microbiologists of the “Provence Alpes Côte d’Azur” Region, a Regional Flow Cytometry Platform (PRECYM), hosted by the Oceanology Centre of Marseille, was created by a consortium of six laboratories. Its goal is to provide researchers with the possibility to consider new approaches and to guarantee optimal technical, economic, and scientific exploitations of these instruments.

An example of such a technical development is described in this chapter. The ELF97 phosphatase substrate has been shown to be a performing tool to assess the phosphorus status of phytoplankton populations.

Recently, this technique has been successfully applied to marine heterotrophic bacteria in culture samples in which ELF-labeling was observed by microscopy.

In this study, we describe a new protocol for ­ELF-labeling of heterotrophic bacteria that allow detection by flow cytometry. ELF-labeled samples were stored in liquid nitrogen for up to 4 months before analysis without any significant loss of total or ELF-labeled cell abundance and of relative ELF ­fluorescence intensity.

This method enables studying ectoenzymatic alkaline phosphatase activity of heterotrophic bacteria at the single cell level in environments even with low cell abundance. It has been successfully applied in both fresh and marine samples.

This approach is set to improve our understanding of the physiological response of heterotrophic bacteria to phosphorus limitation.