Use of FISH in difficult environments: application to hydrolysed wheat straw
Résumé
FISH (Fluorescent In Situ Hybridisation) can be a very efficient tool for monitoring active microbial cells. In microbial ecology, this method has mainly been applied to water samples as the as the use is soils needs previous sample treatment. However, Bertaux et al. (J. Microbiol. Met. 69, 2007) successfully experimented the use of FISH in soils after cell extraction using a density gradient (Optiprep®), and the method of Lunau et al. (Env. Microbiol. 7, 2005) for extracting bacterial cells prior to SYBR® Green staining has yielded promising results although it has not yet been applied for FISH. In the present work, we focused on yet another complex matrix; hydrolysed wheat straw, and tested the efficiency of cell extraction by either Bertaux et al.'s method, Lunau et al.'s method or a simple sedimentation, on the recovery of bacterial cells (Thermatoga maritima) previously inoculated in a wheat straw suspension. T. toga was fluorescently marked with bacterial EUB and T. toga probes to assess the possibility of carrying out FISH after cell extraction. Our first results showed that Lunau's method was the most efficient for eliminating wheat straw debris and recovering the amount of cells inoculated within an error margin of 10% when stained with SYBR® Green. However, EUB probes only stained half of the cells extracted and stained with SYBR® Green in the Lunau extract and the T. toga probe only stained 20% of them when it should have stained between 85-95%. No cells were visualized after staining the Bertaux extract and less that 10% with the sedimentation extract. This methodological study reiterated the difficulty in using FISH in dense material and demonstrates the need to adapt new methods. We are currently testing different hybridization methods in the Lunau extract as this seems to be a promising method for hydrolysed wheat straw. This study has been conducted within the frame of the research project HYCOFOL_BV (Hydrogen production process from plant biomass by coupling dark and light fermentation) co-financed by the French National Research Agency (ANR).