Dénombrements directs des bactéries des milieux aquatiques par microscopie en épifluorescence : comparaison entre un système d'analyse d'images automatisé (Mudicam®) et l'observation visuelle

La technique de comptage par microscopie en épitluorescence est la méthode la plus performante pour dénombrer la totalité des bactéries présentes dans les milieux aquatiques. Cependant cette technique est longue, fastidieuse et subjective. Afin d'automatiser et de rendre objectif le dénombrement, le microscope à épifluorescence est couplé à un analyseur d'images. Si les systèmes d'analyse d'images sont utilisés pour les mesures de taille des bactéries aquatiques, très peu d'études font état de comparaison entre les dénombrements par analyse d'image et ceux réalisés de façon traditionnelle. Cet article présente les résultats des dénombrements de souches bactériennes de référence et de bactéries des milieux aquatiques, par la technique de microscopie en épifluorescence des cellules bactériennes marquées au DAPI, réalisés simultanément par observation microscopique visuelle (visuel) et par analyse d'images automatisée (automatique).Le système d'analyse d'images est composé d'une caméra vidéo (Lhesa LH40036) de sensibilité de 510-4 lux, d'une carte de numérisation (512 x 512 pixels, 8 bits, cyclope v 2.32, Digital vision) d'un micro-ordinateur 80-386 et d'un logiciel de dénombrement (Mudicam®. EAU). Le système est couplé à un microscope en épilluorescence Olympus BH2.Les dénombrements ont été réalisés d'une part sur des suspensions de souches bactériennes de référence (n = 30) à différents états physiologiques et sur des échantillons d'eaux (n = 50) d'origines diverses (fleuve, eaux saumâtre, marine et résiduaire). La comparaison des deux méthodes est réalisée par un modèle de régression linéaire et une analyse de variance. Les tests statistiques associés permettent de conclure à une bonne concordance entre les deux méthodes. A partir de l'ensemble des dénombrements réalisés, 18 d'entre eux pris au hasard ont été dénombrés de façon manuelle par deux opérateurs et par le système d'analyse d'image. Il apparaît que les différences de comptage les plus élevées correspondent aux dénombrements effectués par chacun des deux opérateurs. Ceci met en évidence que non seulement le système d'analyse d'image permet une quantification rapide des abondances bactériennes, mais en outre il supprime la subjectivité de l'opérateur tout en réalisant des dénombrements aussi précis.Direct counting by epifluorescence microscopy is the best method available to determine total counts of aquatic bacteria. However, microscopic observation is tedious and time-consuming. A more rapid and certainly less subjective way of counting bacteria is to combine epifluorescence microscopy with an image analysis system. Surprisingly, although image analysis is now a relatively common method to measure the size of aquatic bacteria, very few studies have been devoted to the validation of total counts by image-analysis systems. In this paper, we present data on simultaneous determination of total counts of 4'6-diamidino-2-phenylindole (DAPI) stained bacteria by visual means and by image-analysed (Mudicam® system) epifluorescence microscopy methods.The Olympus microscope BH2 is equipped for epifluorescence with a 100 W Hg lamp and a 100x oil immersion objective (Apo UVFL 160/1.3). The image analysis system consists et a high performance (5 x 10-4 lux) video camera (Lhesa LH40036) and an image processor which digitalizes the video image in a grey scale extending from 0 (black) to 255 (white) into a binary image with 512 x 512 pixels (8 bit, cyclope v 2.32, Digital Vision), and image analysis software (MUDICAM®. EAU). The samples were stained with DAPI (final concentration 2.5 µg/ml) and filtered through polycarbonate inters (0.22µm, Nuclepore Corporation). The surface area of the video image is 76 x 111 µm2.The analysed samples come from culture collections of different bacterial strains (n = 30) submitted to different conditions and incubation times to obtain various physiological states (Table 1). The nature water samples were collected from several aquatic ecosystems : Rhône river, Mediterranean sea, Thau lagoon and Montpellier sewage waters (n = 50). The bacterial abundances ranged from 105 to 108 cells/ml and the size range of the cells varied from 0.63 to 17 µm2. Comparisons between the image analysis and visual counts were made on the basic of thirty fields per filter. The image analysis counts are based on a two step procedure. The video image of each microscopie field is first numerised and stored on a hard disk (153 Mo). When all the fields have been stored, the digitized images are submitted to an automatic thresholding which allows background substraction. Automatic counting of bacterial cells is then performed on the basis of object specifications defined by the operator. These specifications concern the minima and maxima values of the area (expressed in pixel numbers) and the fluorescence (expressed in gray levels) of the objects. The MUDICAM®EAU software also provides the mean number of cells per millilitre and the associated variance.Average concentrations and confidence limits are shown in Table 2 for bacterial collection strain cultures and in Table 3 for water samples. When we compared visual and image analysis counts by- linear regression, the ability of the image analysis system to enumerate bacterial cells was clearly demonstrated. With bacterial culture (Fig. 2) and with water samples (Fig. 3), the coefficients of correlation were respectively r = 0.997 and r = 0.996 (p = 0.0001). The slopes of the models are not significantly different from unity and the Y-intercepts are not different from zero. Moreover we have compared the total visual counts of two experimenters and the image-analysed counts on eighteen random samples (Table 4). The variance analysis shows that there is no difference between the three methods, with mean value of 6.09, 6.08 and 6.11 for the image-analysed method, experimenter n° 1 and experimenter n° 2, respectively. While non significant, the greatest difference in counts was obtained between the two experimenters.If may be concluded that the image analyser tested for total counts by epifluorescence microscopy is a precise and rapid procedure for the determination of total bacterial counts. This method may be standardized and its automation allows the analysis of many samples, an important advantage in ecological studies. Storage of the samples also allows one to treat a posteriori some complementary aspects of the total count, such as the double staining of bacteria. The image analyser tested is appropriate for bacterial ecology studies which require epifluorescence microscopy

Utilisation du bouillon sélénite F modifié pour dénombrer Salmonella dans les milieux aquatiques

Ce travail a pour objet de présenter une nouvelle méthode de dénombrement des Salmonella dans différents types d'eaux (de rivière, saumâtre, eaux usées brutes et épurées) basée sur l'utilisation d'un milieu d'enrichissement au sélénite additionné de Novobiocine et de Pril et sur une adaptation de la technique du N.P.P. à l'ensemencement d'échantillons de grands volumes après filtration permettant de quantifier les très faibles concentrations de Salmonella. Les vérifications des performances de cette méthode d'isolement et de quantification sont basées sur l'étude des croissances de différentes souches de Salmonella et d'autres espèces bactériennes dans le milieu d'enrichissement modifié, ainsi que sur les résultats quantitatifs et qualitatifs fournis par cette méthode lorsqu'elle est appliquée à des échantillons d'eau en provenance de l'environnement aquatique. Ces résultats montrent notamment que la méthode proposée est suffisamment sensible pour détecter 1 à 2 Salmonella dans 10 litres d'eau analysée et qu'elle ne parait pas exclure de sérotypes, du moins parmi ceux les plus fréquemment isolés en France. L'efficacité de la méthode standardisée API Z pour l'identification enzymatique du genre Salmonella a été également testée en référence aux résultats de la sérotypie.This paper presents a new method for enumerating Salmonellae in environmental waters (freshwater, brackishwater, sewage and treated waters) using the F Selenite enrichment broth modified by the addition of Novobiocin and Pril, and an adaptation of the M.P.N. method for the inoculation of large amounts of water after filtration to improve the enumeration of low concentrations of Salmonellae. The verification of the performance of this detection and enumeration method are based on the study of the growth of different Salmonellae species and of others bacterial species in the modified enrichment broth, and on the quantitative and qualitative results obtained by the application of this methodology to aquatic environmental samples. These results show on one hand, that the sensitivity of the proposed method allows to enumerate 1 to 2 Salmonellae in 10 liter samples, and on the other hand that this method to net exclude any serovar from those which are the most frequently isolated in France. The efficiency of the API Z standardized method for the Salmonellae enzymatic identification versus serological identification was also verified

Isotopic and velocity distributions of Bi produced in charge-pickup reactions of 208Pb at 1 A GeV

Isotopically resolved cross sections and velocity distributions have been measured in charge-pickup reactions of 1 A GeV 208Pb with proton, deuterium and titanium target. The total and partial charge-pickup cross sections in the reactions 208Pb + 1H and 208Pb + 2H are measured to be the same in the limits of the error bars. A weak increase in the total charge-pickup cross section is seen in the reaction of 208Pb with the titanium target. The measured velocity distributions show different contributions - quasi-elastic scattering and Delta-resonance excitation - to the charge-pickup production. Data on total and partial charge-pickup cross sections from these three reactions are compared with other existing data and also with model calculations based on the coupling of different intra-nuclear cascade codes and an evaporation code.Comment: 20 pages, 12 figures, background information on http://www-w2k.gsi.de/kschmidt

Determination of the freeze-out temperature by the isospin thermometer

The high-resolution spectrometer FRS at GSI Darmstadt provides the full isotopic and kinematical identification of fragmentation residues in relativistic heavy-ion collisions. Recent measurements of the isotopic distribution of heavy projectile fragments led to a very surprising new physical finding: the residue production does not lose the memory of the N/Z of the projectile ending up in a universal de-excitation corridor; an ordering of the residues in relation to the neutron excess of the projectile has been observed. These unexpected features can be interpreted as a new manifestation of multifragmentation. We have found that at the last stage of the reaction the temperature of the big clusters subjected to evaporation is limited to a universal value. The thermometer to measure this limiting temperature is the neutron excess of the residues.Comment: 8 pages, 6 figures, corrected some misprints in the abstract, to be published in "Yadernaya Fizika" as a proceeding of the "VII International School Seminar on Heavy-Ion Phyics", Dubna (Russia), May 27 - June 1, 200

Entrance-channel Mass-asymmetry Dependence of Compound-nucleus Formation Time in Light Heavy-ion Reactions

The entrance-channel mass-asymmetry dependence of the compound nucleus formation time in light heavy-ion reactions has been investigated within the framework of semiclassical dissipative collision models. the model calculations have been succesfully applied to the formation of the $^{38}$Ar compound nucleus as populated via the $^{9}$Be+$^{29}$Si, $^{11}$B+$^{27}$Al, $^{12}$C+$^{26}$Mg and $^{19}$F+$^{19}$F entrance channels. The shape evolution of several other light composite systems appears to be consistent with the so-called "Fusion Inhibition Factor" which has been experimentally observed. As found previously in more massive systems for the fusion-evaporation process, the entrance-channel mass-asymmetry degree of freedom appears to determine the competition between the different mechanisms as well as the time scales involved.Comment: 12 pages, 3 Figures available upon request, Submitted at Phys. Rev.

Measurement of nuclide cross-sections of spallation residues in 1 A GeV 238U + proton collisions

The production of heavy nuclides from the spallation-evaporation reaction of 238U induced by 1 GeV protons was studied in inverse kinematics. The evaporation residues from tungsten to uranium were identified in-flight in mass and atomic number. Their production cross-sections and their momentum distributions were determined. The data are compared with empirical systematics. A comparison with previous results from the spallation of 208Pb and 197Au reveals the strong influence of fission in the spallation of 238U.Comment: 20 pages, 10 figures, background information at http://www-wnt.gsi.de/kschmidt

Evaporation residues produced in spallation of 208Pb by protons at 500A MeV

The production cross sections of fragmentation-evaporation residues in the reaction Pb+p at 500A MeV have been measured using the inverse-kinematics method and the FRS spectrometer (GSI). Fragments were identified in nuclear charge using ionisation chambers. The mass identification was performed event-by-event using the B-rho - TOF - Delta-E technique. Although partially-unresolved ionic charge states induced an ambiguity on the mass of some heavy fragments, production rates could be obtained with a high accuracy by systematically accounting for the polluting ionic charge states. The contribution of multiple reactions in the target was subtracted using a new, partly self-consistent code. The isobaric distributions are found to have a shape very close to the one observed in experiments at higher energy. Kinematic properties of the fragments were also measured. The total and the isotopic cross sections, including charge-pickup cross sections, are in good agreement with previous measurements. The data are discussed in the light of previous spallation measurements, especially on lead at 1 GeV

Cross-sections of spallation residues produced in 1.A GeV 208Pb on proton reactions

Spallation residues produced in 1 GeV per nucleon $^{208}$Pb on proton reactions have been studied using the FRagment Separator facility at GSI. Isotopic produc- tion cross-sections of elements from $_{61}$Pm to $_{82}$Pb have been measured down to 0.1 mb with a high accuracy. The recoil kinetic energies of the produced fragments were also determined. The obtained cross-sections agree with most of the few existing gamma-spectroscopy data. Data are compared with different intra nuclear-cascade and evaporation-fission models. Drastic deviations were found for a standard code used in technical applications.Comment: 4 pages, 3 figures, accepted for publication in Phys. Rev. Lett. Revised version May 12, 200

Measurement of residual nucleus cross sections and recoil energies in p + Fe collisions at 300, 500, 750, 1000 and 1500 MeV

The production of residual nuclei in p + Fe collisions has been measured at GSI on the FRS facility by means of the reverse kinematic techniques at 300, 500, 750, 1000 and 1500 MeV/A. The cross-sections larger than 0.01 mb of all isotopes with Z larger than 8 have been obtained. Velocity distributions were also measured. Comparisons to models describing spallation reactions and some empirical formulae often used in astrophysics are presented. These data are directly used to calculate impurety production and DPAs in a thin window as foreseen in spallation sources or accelerator-driven systems