Numération bactérienne en épifluorescence par la méthode couplée DAPI-INT : application à un cas concret

Abstract

Un protocole simple de coloration par la méthode couplée DAPI-INT en microscopie à épifluorescence, associé à une lecture des préparations par caméra et vidéo-Imprimante, permet la mesure de paramètres biologiques impliqués directement dans les processus de dégradation de la matière organique en rivière : numérations de microflore totale, nombre de cellules physiologiquement actives (réduisant l'INT), nombre de cellules avec réserves de PHB, dimensions cellulaires et répartition en classes de taille aboutissant à l'évaluation de la biomasse carbonée.Tous les comptages et mesures sont réalisés à partir de clichés d'imprimante thermique, ce qui assure une reproductibilité et une comparaison entre échantillons plus objective qu'à partir de seules observations directes. Cette méthode permet de différer soit les comptages (échantillons fixés au formol après incubation à l'INT), soit surtout les mesures de dimensions cellulaires (archivage des clichés pour une exploitation ultérieure).Son application au cas de la rivière Charente montre une évolution particulière de la biomasse bactérienne en aval des rejets de l'agglomération d'Angoulême. La mise en évidence de cellules de grande taille (biovolume moyen de 0,3 µm 3 ) et d'une population plus active pourrait traduire une modification physiologique de micro-organismes autochtones réagissant à des conditions de milieu particulières (rejets d'effluents carencés en azote). The increasing appreciation of heterotrophic bacterioplankton as an important and dynamic compartment in the carbon cycle of aquatic ecosystems has led to the development of methods allowing reproducible and reliable direct bacterial counts. Thus, since the 1970's, enumeration of bacteria by epifluorescence microscopy has become a widely used method for the estimation of cell number and biovolume. Cell counts are made using either the acridine orange (Hobbie et al. 1977) or the DAPI (Porter 1980) procedure on fixed samples. A tracer of cellular respiratory activity (INT) can be linked to either of these two methods before the samples are killed by formalin. Two observations of the same field, one in transmitted light, the other by epifluorescence, allow a determination of the percentage of active bacterioplankton (Tabor and Neihof 1982, Dufour 1990).However, the various authors working with these procedures have modified the protocol and up to now there has not been a standard method. Thus, before using it for the first time, numerous verifications about filters, concentration of dye solution and methods for estimating cell volume are necessary.In this study a simultaneous comparison, between three brands of filters, two dye solutions and three methods for numeration and cell volume estimation (by direct count, by photographs and by video graphic printer) has been performed.All the tests have been clone with samples collected from rivera or lakes. In comparison to laboratory cultures, natural bacteria are observed with difficulty on account of their small size and especially because natural organic matter absorbs dye solution and so interferes with counts. We prefer to use DAPI rather than acridine orange, because DAPI is colorless in direct light, so it doesn't interfere with enumeration of respiring bacteria (red crystal inside cell). To obtain the best contrast for observation in epifluorescence microscopy, a very fast drying in the open air and a structure less troublesome for the observation in direct light, we have chosen Millipore black filters. A camera associated with a video printer gives better results compared to the photographs : no fluorescent "halo" around the tell image and no waste of time waiting for development of the film. All counts and measures were made from video graphic printer photographs, which assures reproductibility and more objective comparisons between samples, compared to direct counts (with eyes) that are tiring and subjective. Finally, we can defer counting (samples fixed in formol after INT incubation) or cell size measurements (analysis of recorded photos) to a later time.On the basis of this tests, the DAPI-INT method in fluorescence microscopy, associated with the reading of slides with a camera and a video-printer, allows one to measure biological parameters directly implicated in the degradation of river organic matter : abundance of total microflora, number of physiologically active cells (reducing INT) and cells with PHB reserves, cell sizes and size range distribution used to evaluate carbon biomass. The application of this method to the river Charente (April to November 1991) shows a particular fate of bacterial biomass downstream from the city of Angouleme. A multifactor analysis of variance with a multiple range analyses (Tukey) shows that bacterial cells collected downstream from the effluent discharge of the Angouleme wastewater treatment plant are the largest (average cell volume up to 0.3 µm 3 ) and the more active cells. Similar trends have also been observed by Servais and Garnier (1989) downstream from the Paris wastewater treatment plant (Achères) on the Seine river. These authors suggested that allochtonous populations associated with the urban sewage were different from the autochtonous populations (higher biovolume). To verify this hypothesis, a sampling campaign (June 92) was performed on five stations along an eight kilometers river reach. There weren't large size bacteria downstream from the effluent discharge of Frégeneuil, Fléac and St Michel wastewater treatment plants. We Pound the largest bacteria at Nersac. Thus it seems that these large bacteria don't came from wastewater treatment plant. The increase of autochtonous bacterial size could be interpretated as a nutritional stress, the increase of the C/N ratio preventing cellular division (nitrogen deficient sewages).