Method for the detection and quantification by flow cytometry of fungi and bacteria contained in a divided solid containing cellulosic organic matter

Abstract

The invention relates to a method for the simultaneous detection and quantification of fungi and bacteria present in a divided solid containing cellulosic organic matter, according to which a solid-liquid extraction is carried out of said divided solid, a sample of said liquid obtained is introduced into a flow cytometer and a biparametric histogram is established giving for each point the intensity of the diffracted light and the intensity of the reflected and refracted light so as to discriminate a first group of points attributable to fungi and a second group of points attributable to bacteria.According to the invention, before introduction into said cytometer, at least one first fluorochrome capable of binding to DNA is added to said sample, said sample is further excited so as to obtain fluorescence of said first fluorochrome and said biparametric histogram is also established for the points corresponding to particles emitting fluorescence in the emission wavelength of said fluorochrome for the first group of points and for the second group of points, for each of said groups a cloud of points is determined which corresponds to a fluorescence intensity due to the first fluorochrome greater than that of the other points of said group and optionally the points of said cloud are counted, which correspond respectively to the cells of fungi or bacteria.

Description

Title of the invention: Method for detecting and quantifying by flow cytometry fungi and bacteria contained in a divided solid containing cellulosic organic matter Technical field

[0001] The present invention relates to a flow cytometry method which makes it possible to simultaneously detect and quantify fungi and bacteria present in a solid in a divided state containing organic matter. Prior art

[0002] A growing medium, whether natural like topsoil or artificial like potting soil, is a complex environment in which many species of bacteria and fungi live. The same is true for composts, manures and other amendment substrates. These bacteria and fungi can be useful for plant growth or, on the contrary, be pathogenic. It is also known that the activity of soil microorganisms (especially microbes and fungi) also has an effect, in the presence of oxygen, on the emission of CO2 by respiration and on that of methane. In all cases, we see that it is important to be able to quickly quantify the bacteria and fungi in the soil.

[0003] The publication entitled "A rapid flow cytometry method to assess bacterial abundance in agricultural soil" by M. Bressan and published in the journal Applied Soil Ecology in 2015 (88 (2015) pages 60-68) indicates that the number of bacterial cells contained in a soil can be determined by flow cytometry using the fluorochrome SYBR Green. This publication suggests that the fluorochrome SYTOX-green could also be used. The difficulty of the analysis lies in the treatment of the soil sample because the bacteria are often very attached to the soil particles. This publication indicates that the soil sample is diluted in an 85% sodium chloride solution and filtered at 0.22pm. The mixture is homogenized by means of a vortex for 5 minutes at full speed. After centrifugation to remove the larger particles, the supernatant is analyzed by flow cytometry. This method is not applied to fungi.

[0004] The publication entitled "Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry" by B. Khalili published on July 16, 2021 in the journal American Society for Microbiology indicates that it is possible to quantify the population of bacteria contained in a soil sample by flow cytometry. The bacteria are extracted with a surfactant added to the Nicodenz® medium (which comes from a benzoic acid derivative on which three aliphatic chains have been grafted hydrophilic). This publication applies to a soil sample to which E. coli cells have been added. It is therefore not a real soil sample. In addition, the publication is silent regarding fungi.

[0005] The publication entitled "Schrôdinger's microbes: Tools for distinguishing the living from the dead in microbial ecosystems" by JB Emerson and published in 2017 in the journal Microbiome (DOI 10.1186 / s40168-017-0285-3) reviews all the techniques that can be used to determine whether a microorganism is alive or not. It is noted that certain fluorochromes do not allow the distinction between living and dead organisms. This is, for example, the case of propidium iodide in the case of particular proteobacteria and mycobacteria. The publication indicates that reagents can also be used for yeasts. In the case of yeasts, a fluorescent marker is used that binds to chitin (which forms the cell wall of the fungi) coupled with a marker that only fluoresces when the cell membrane and metabolic function are intact.This publication does not specifically aim at the study of soils, which are complex environments.

[0006] The publication entitled "Specific and Rapid Enumeration of Viable but Non-culturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry" by MM Taimu Khan published in August 2010 in the journal APPLIED AND ENVI-RONMENTAL MICROBIOLOGY, (pages 5088-5096 Vol. 76, No. 15) indicates that it is possible to enumerate latent bacterial cells by flow cytometry. The bacteria studied are: Escherichia coli O157:H7, Pseudomonas ae-ruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium; the fluorescent markers used are: SYTO 9®, SYTO 13®, SYTO 17®, SYTO 40® and propidium iodide. This publication does not address soils and does not report on fungi.

[0007] The publication entitled "Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry" published in the journal FEMS microbiology Ecology in 2004 (volume 51, Page 123-132) indicates that it is possible to determine the quantity of Pseudomonas - a bacterium useful for plant growth - by marking the bacterium with a fluorescent gene.

[0008] The publication entitled "Flow cytometry as a tool to assess the effects of gamma radiation on the viability, growth and metabolic activity of fungal spores" by N. Mesquita and published in the journal International Biodeterioration & Biodegradation in 2013 (Volume 84, page 250-257) indicates that it is possible to determine the viability of spores of Penicillium chrysogenum, Aspergillus nidulans and Aspergillus niger by flow cytometry. The markers used are propidium iodide and dihydroethidium. Dihydroethidium intercalates into the DNA of these cells, staining the nucleus in red. This publication only focuses on the spores and not the fungi themselves.

[0009] The publication entitled “The filamentous fungus Pénicillium chrysogenum analysed via flow cytometry—a fast and statistically sound insight into morphology and viability” by L. Veiter & al, published in the journal applied microbial and cell physiology in 2019 (103: 6725-6735) indicates that it is possible to extract P. chrysogenum from its culture medium with a saline solution containing 50 g / l of a CaCl2 solution with a concentration of 2.65 g / l, and having a concentration of KC1 of 0.2 g / l, KH2PO4 of 0.2 g / l, MgCI*6 H2O of 0.1 g / l, NaCl of 8 g / l and 0.764 g / l of Na2HPO4 + 2H2O. The cells are stained with propidium iodide, which can distinguish cells with damaged cell walls. Fluorescein diacetate is also used to determine the metabolic activity of the cells. This method is used for culture media of less complex fungi.It is not applicable to soils, which are too complex and for which the fungi, particularly the hyphae, are closely linked to the particles of the environment.

[0010] Technical problems to be solved

[0011] A first aim of the present invention is to propose a flow cytometry method which makes it possible to simultaneously quantify the fungi and bacteria present in a solid in a divided state, in particular a soil sample, containing organic matter.

[0012] Another aim of the present invention is to propose a method which also makes it possible to simultaneously determine for bacteria and for fungi the state of their cells, namely, latent, living or dead.

[0013] Another aim of the present invention is to propose a method which makes it possible to take into account the hyphae contained in the substrate.

[0014] Brief description of the invention

[0015] The present invention relates to a method for the simultaneous detection and quantification of fungi and bacteria present in a divided solid containing cellulosic organic matter, according to which a solid-liquid extraction of said divided solid is carried out, a sample of said liquid obtained, optionally diluted, is introduced into a flow cytometer and a bi-parametric histogram is established giving for each point the intensity of the diffracted light and the intensity of the reflected and refracted light so as to discriminate a first group of points attributable to fungi and a second group of points attributable to bacteria.Typically, according to the invention, at least one first fluorochrome capable of binding to DNA is added to said sample before introduction into said cytometer, said sample is further excited so as to obtain fluorescence from said first fluorochrome, said bi-parametric histogram is also established for the points corresponding to particles emitting fluorescence in the wavelength. emission of said fluorochrome for the first group of points and for the second group of points and for each of said groups a cloud of points is determined which corresponds to a fluorescence intensity due to the first fluorochrome greater than that of the other points of said group and possibly the points of said cloud are counted, which correspond respectively to the cells of fungi or bacteria.

[0016] The first fluorochrome being capable of binding to DNA, it makes it possible to distinguish microorganisms from the background noise formed by suspended particles.

[0017] Advantageously, said first fluorochrome is capable of binding to the DNA of living cells and to the DNA of cells whose wall is permeable, which correspond to dead cells.

[0018] Although both fungi and bacteria possess DNA, it was not clear that a single fluorochrome would separate them from background noise.

[0019] Indeed, it should be noted that the cells of fungi are distinguished from those of bacteria. Fungi are eukaryotes while bacteria are prokaryotes. In addition, the wall of fungi is composed of chitin while it is composed of peptidoglycans in bacteria. The permeability of the membrane to various fluorochromic markers is therefore not the same in bacteria and in fungi.

[0020] The solid may contain cellulose and cellulosic organic materials.

[0021] The solid may also contain mineral materials such as clays or sand, for example.

[0022] According to a particular embodiment, the solid is made up of cellulosic organic materials.

[0023] According to an advantageous embodiment which can be combined with each of the other embodiments, at least one second and at least one third fluorochrome are also added to said sample before introduction into said cytometer, said second fluorochrome is capable of binding only to the DNA of cells whose wall is permeable and said third fluorochrome becomes fluorescent by reaction with the esterases contained in the living cells and, for at least one of said groups, a bi-parametric histogram is established giving the intensity of the fluorescence due to the second fluorochrome and the intensity of the fluorescence due to the third fluorochrome, three clouds of points are determined on said histogram: a first cloud of points for which the fluorescence of the third fluorochrome is the strongest,a second cloud of points for which the fluorescence due to the second fluorochrome is the weakest and a third cloud of points and we deduce that the first cloud of points corresponds to the living bacteria / fungi, the second cloud of points corresponds to the fungi / bacteria in the latent state and the third cloud of points corresponds to the dead fungi / bacteria, possibly by counting the points in each cloud, we determine the number of bacteria or fungi and their state, respective.

[0024] As explained above, it is surprising that a mixture of three fluorochromes manages to distinguish the life states (latent, dead, alive) of fungi and bacteria. Indeed, fungi and bacteria have different metabolisms which often do not allow them to be analyzed together. In addition, it should be noted that the method also takes into account hyphae, which are counted as one event (one point).

[0025] Advantageously, said first fluorochrome is chosen from fluorochromes capable of binding to the DNA of cells and having a maximum fluorescence absorption wavelength equal to or greater than 599nm and equal to or less than 657nm and a maximum fluorescence emission wavelength equal to or greater than 619nm and equal to or less than 678nm and a quantum yield equal to or greater than 0.16 and equal to or less than 0.39.

[0026] In particular, said first fluorochrome may be chosen from fluorochromes capable of binding to the DNA of cells and having a maximum fluorescence absorption wavelength of 652 nm and a maximum fluorescence emission wavelength of 676 nm and a fluorescence quantum yield on DNA of 0.27 and fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657 nm and a maximum fluorescence emission wavelength of 673 nm and a fluorescence quantum yield on DNA of 0.17.

[0027] Such fluorochromes are marketed under the names SYTO62® and SYTO 63® by the company Thermofischer.

[0028] Advantageously, said second fluorochrome is chosen from fluorochromes having a maximum fluorescence absorption wavelength of 547nm and a maximum fluorescence emission wavelength of 570nm and a fluorescence quantum yield on DNA of 0.9 and in that said third fluorochrome is chosen from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate, mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate and 5,6-carboxylate fluorescein diacetate of suc-cinimidyl of the following general formula:

[0029] [Formula 1]

[0030] HvO" ' O ÇX

[0031] Advantageously, the second fluorochrome is the fluorochrome marketed by the company Thermofischer under the name SYTOX-orange.

[0032] Advantageously, the third fluorochrome is the cFDA mixture.

[0033] Advantageously, the first fluorochrome is SYTO62®, the second fluorochrome is SYTOX-orange and the third fluorochrome is the cFDA mixture

[0034] The divided solid containing cellulosic materials can be chosen from topsoil, potting soil, compost, manure, mulch, humus and mixtures thereof, in particular two-by-two mixtures thereof.

[0035] The fungi or fungi and the bacteria or bacteria contained in the divided solid are not limited according to the invention. They may be fungi and bacteria capable of degrading cellulose but also other bacteria or fungi originating from animals. Indeed, when the solid is, for example, manure or contains it, it is likely to also contain fecal bacteria.

[0036] Thus, the method of the invention makes it possible to detect and quantify at least one fungi and preferably a mixture of fungi chosen from microscopic fungi capable of forming mycorrhizae, imperfect fungi, Lichtheimia corymbifera co-rymbilfer mucor, mucor mucedo, yeasts, and hyphae of microscopic fungi and at least one bacterium or a mixture of bacteria chosen from cytophaga spp, streptomyces spp, Bacillus radicola, Candida albicans, Phycomycetes Rhizopus, Penicillium spp, Aspergillus spp, verticillium, Helminthosporium, Fusarium, Cladosporium, actynomycetes, Nitrosomonas, Nitrosocoocus, Azotobacter, Clostridium spp, Rhizobium, acidobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Alphaproteobacteria and Betaproteobacteria,, Saccharomyces spp, Pseudomonas spp, Staphylococcus aureus. E. coli, Micrococcus luteus, Bacillus megaterium, Bacillus polymyxa and Enterococcus faecium.

[0037] It should be noted that the method also makes it possible to detect and quantify hyphae, which are groups of cells not separated by walls. Since the detected fluorescence can be correlated with the quantity of DNA, the method also makes it possible to quantify hyphae, which are counted as several cells in depending on the number of nuclei they contain.

[0038] Advantageously, the solid-liquid extraction is carried out with an aqueous sodium chloride solution containing from 7 to 12g / L of sodium chloride and in particular 8.5g / L of sodium chloride and filtered with a filter having a cut-off threshold of 0.22pm.

[0039] It is also surprising that despite the absence of surfactant, it is possible according to the invention to extract fungi and bacteria which are both intimately bound to the particles of the divided solid. Moreover, this method also makes it possible, surprisingly, to extract hyphae, which are intimately bound physicochemically to the particles of the solid.

[0040] Advantageously, said divided solid is sieved before extraction so as to retain only fragments of a size less than or equal to 2 mm. It is likely, although the Applicant is not bound by this explanation, that the sieving already makes it possible to prepare the extraction of the fungi and bacteria and already separates them slightly from the particles of the divided solid.

[0041] Advantageously, said sample is diluted a first time to 1 / 10 or 1 / 100 then diluted to 5 / 100. Such dilution facilitates detection and quantification.

[0042] The present invention also relates to a fluorochromic mixture containing a first fluorochrome chosen from fluorochromes capable of binding to the DNA of cells and having a maximum fluorescence absorption wavelength equal to or greater than 599nm and equal to or less than 657nm and a maximum fluorescence emission wavelength equal to or greater than 619nm and equal to or less than 678nm and a quantum yield equal to or greater than 0.16 and equal to or less than 0.39 and mixtures thereof and in particular from fluorochromes capable of binding to the DNA of cells and having a maximum fluorescence absorption wavelength of 652 nm and a maximum fluorescence emission wavelength of 676 nm and a fluorescence quantum yield on DNA of 0.27 and fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657nm and a maximum fluorescence emission wavelength of 673nm and a fluorescence quantum yield on DNA of 0.17 and mixtures thereof, a second fluorochrome chosen from fluorochromes having a maximum fluorescence absorption wavelength of 547nm and a maximum fluorescence emission wavelength of 570nm and a fluorescence quantum yield on DNA of 0.9 and a third fluorochrome chosen from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate, mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate and suc-cinimidyl fluorescein 5,6-carboxylate diacetate of the following general formula: ,

[0043] [Formula 2]

[0044]

[0045] Advantageously, the mixture contains a concentration of third fluorochrome greater than the concentration of second and first fluorochrome and in that it contains a concentration of first fluorochrome greater than that of said second fluorochrome.

[0046] The inventors have in fact demonstrated that such a mixture could, with a single analysis with a flow cytometer, allow the detection, quantification and determination of the state of life (latent, dead, alive) of fungi and bacteria.

[0047] Definitions

[0048] The terms “divided state” designate, within the meaning of the invention, a powder, granules, fibers, particles and their mixtures.

[0049] The terms "cellulosic organic materials" designate, within the meaning of the invention, cellulose, hemicellulose, organic materials resulting from the biological degradation of cellulose and / or hemicellulose and mixtures of at least two of these compounds. In particular, these terms designate a mixture of cellulose, hemicellulose and materials resulting from the biological degradation of cellulose and hemicellulose.

[0050] The term "topsoil" means any soil from humus-bearing surface horizons or deep horizons that can be mixed with organic matter of plant origin, organic amendments and / or mineral matter. The term encompasses, for the purposes of the invention, sandy topsoil, silty topsoil, calcareous topsoil, humus-bearing topsoil and loam topsoil, which is a mixture of these four soils.

[0051] The term “compost” designates the substrate obtained by the decomposition of organic plant and / or animal matter by organisms living in or on the soil.

[0052] The term “potting soil” designates a mixture of topsoil and / or compost and / or manure.

[0053] The term “humus” refers to the upper layer of soil present under trees, particularly in forests.

[0054] The term "mulch" means a divided material comprising or consisting of wood chips and / or bark and / or needles and / or dried grass clippings and / or dead leaves and / or hay and / or chaff and / or sawdust and / or hemp chaff and / or flax chaff and / or fragmented branch wood.

[0055] The term "simultaneous" means that the results can be obtained by a single pass of the same sample in a flow cytometer.

[0056] The term "fungi" encompasses all microscopic fungi and in particular microscopic fungi capable of degrading cellulose and / or hemicellulose, the hyphae of microscopic fungi and imperfect fungi.

[0057] The term "bacteria" designates according to the invention all the bacteria that degrade cellulose and / or hemicellulose and the bacteria originating from the animal tract (vegetarian or carnivorous).

[0058] The acronym FSC refers to the signal corresponding to the diffracted light (FSC: Forward Scatter); this signal depends on the size and surface of the particle analyzed;

[0059] The acronym SSC (Side Scatter) refers to the signal corresponding to reflected and refracted light; this signal depends on the granularity and cellular complexity of the particle analyzed.

[0060] The acronym SSC-H refers to the signal intensity corresponding to reflected and refracted light;

[0061] The acronym SSC-A refers to the intensity per unit area of ​​the signal corresponding to reflected and refracted light;

[0062] The acronyms FSC-H and FSC-A refer, respectively, to the intensity and the intensity per unit area of ​​the signal corresponding to the diffracted light;

[0063] The acronym cFDA or c-FDA denotes a mixture of 5-carboxy fluorescein diacetate and 6-carboxy fluorescein diacetate.

[0064] Throughout the application, the maximum fluorescence values ​​in absorption and emission defining the fluorochromes are determined in the presence of DNA with a ratio of approximately 100 base pairs and in particular 100 base pairs of nucleic acid for a fluorochrome molecule in a Tris medium of pH 7.5 and EDTA concentration equal to ImM.

[0065] Throughout the application, the fluorescence quantum yield that defines the fluorochromes is measured in the presence of DNA and expressed relative to the yield determined for cresyl violet in methanol.

[0066] Other characteristics and advantages of the invention will emerge on reading the description which follows, with reference to the appended figures, which illustrate:

[0067] [Fig. 1]: [Fig. 1] is a bi-parametric histogram indicating for each point the intensity of the fluorescence emitted by the fluorochrome SYTO63® and the intensity of the reflected and refracted light;

[0068] [Fig.2]: [Fig.2] is a bi-parametric histogram indicating for each point in the TOTAL window visible in [Fig.l] the intensity of the diffracted light and the intensity of reflected and refracted light;

[0069] [Fig.3]: [Fig.3] is a bi-parametric histogram indicating for each point of the “bacteria” window visible in [Fig.2], the fluorescence intensity due to the SYTOX-orange fluorochrome and the fluorescence intensity due to the cFDA mixture; and

[0070] [Fig.4]: [Fig.4] is a bi-parametric histogram indicating for each point of the “mushroom” window visible in [Fig.2], the fluorescence intensity due to the SYTOX-orange fluorochrome and the fluorescence intensity due to the cFDA mixture; and

[0071] [Fig.5]: Represents photographs of fungal hyphae detected by the cytometer in flow and indicated as an event (a point) on the results histogram; their state (latent, alive, dead) can be determined by the method according to the invention.

[0072] EXAMPLES

[0073] In the example below, the term “mushroom” is synonymous with fungi.

[0074] Mixture of fluorochromes

[0075] Physiological water (osmosed / ultra pure water + NaCl at 8.5g / L) is prepared then autoclaved and filtered before use (0.22pm filter). SYTOX®-Orange (Thermofischer, 5pM) and 5,6 carboxyfluorescein diacetate (c-FDA) are diluted in DMSO (respectively 12.5pM and 2g / L in final concentrations) then stored in the freezer; SYTO 63® (Thermofischer, 5mM) is directly used in the labeling solution. The final concentrations of fluorochromes in the labeling mixture are 7.5pM of SYTO 63®, 0.05pM of SYTOX®-Orange (SYTOX-gold) and 10 mg / L of c-FDA (21.7 pM) in the sodium chloride solution.

[0076] Sample preparation

[0077] A sample of 2.5 + / - 0.1 g of soil (topsoil) or fresh compost is sieved to 2 mm; 22.5 mL of filtered physiological saline is then added. The solid-liquid extraction is then carried out using a rotary shaker for 15 min. The sample is then left to settle overnight at room temperature. The supernatant is diluted 1 / 10 (soil) or 1 / 100 (compost), the dilutions are vortexed, a second dilution of 5 / 100 is finally carried out in the labeling mix, the whole is vortexed. The labeling incubates for approximately 30 min in the dark before analysis.

[0078] Material used

[0079] The cytometer used is an ATTUNE® NXT acoustic focusing cytometer (thermofischer scientific). This flow cytometer is equipped with 3 lasers: blue (488 nm), green (532 nm) and red (637 nm). The cytometer can be equipped with an automatic sampler for reading 96-well microplates. This equipment is equipped with an acoustic flow focusing system allowing a flow rate of up to 1000 pL / min to be used.

[0080] The flow rate is set at 25 pL / min. The latter is slowed down when the microbial load is high. The data are collected on the channels FSC, SSC, BL1 (525 / 50) for c-FDA, GL1 (575 / 36) for SYTOX-orange and RL1 (670 / 14) for SYTO 62 or 63. The voltage values ​​for each of these channels are respectively 320V, 340V, 460V, 480V and 570V. Different values ​​can be applied.

[0081] There is no compensation problem to correct in this configuration.

[0082] Processing of results

[0083] In order to separate microorganisms from background noise, SYTO63® is used, so positive SYTO63® events are considered microorganisms. SYTO62® can also be used.

[0084] Triple labeling of the sample and a "gatting" strategy (determination of windows) allowed the separation of bacteria and fungi in soils and compost. Initially, the majority of microorganisms were separated from the background noise by the SSC-H / RL1-H plot (670 / 14 nm) (see [Fig. 1]). The points for which fluorescence was obtained corresponded to microorganisms since the SYTO63® fluorochrome binds to DNA and not to particles suspended in the sample.

[0085] Then, thanks to the SSC-H / FSC-H plot applied to the TOTAL window visible in [Fig.l] and including bacteria and fungi (microorganism containing DNA), the bacteria and fungi are separated due to their morphological and structural differences. We therefore define two windows visible in [Fig.2]: a CHAMPI window which contains the points attributable to the fungi and a BACT window which contains the points attributable to the bacteria. These windows could be validated by comparison between the results obtained in cytometry, microscopy and on Petri dishes. The analyses carried out in microscopy and on Petri dishes allow a determination of the windows also serving as calibration for the same type of substrate, for example.

[0086] The different states of the microorganisms studied (total population, Bacteria and Fungi) are obtained by crossing the data obtained by the different fluorochromes on each of the determined windows. Thus for each window corresponding either to the fungi (fungus) or to the bacteria, we produce the bi-parametric histograms giving the fluorescence intensities emitted for each of the two fluorochromes which are SYTOX-orange and the cFDA mixture. The “dead” state corresponds to a fluorescence emission due to SYTOX-Orange and an absence of fluorescence due to the cFDA mixture (SYTOX-or + / cFDA -). The “latent living state (non-culturable living VNC)” corresponds to an absence or weak fluorescence emission due to SYTOX-orange and an absence or weak fluorescence emission due to the cFDA mixture (SYTOX-Orange - / c-FDA -). The “active / vital living” state corresponds to the points showing an absence of fluorescence emission due to SYTOX orange and a fluorescence emission due to the cFDA mixture (SYTOX-Orange - / c-FDA +).

[0087] The method of the invention thus makes it possible to carry out at the same time, with the same sample and with a single passage in the cytometer: the separation of the microorganisms of interest (fungi and bacteria) from the background noise, the separation of the living microorganisms from the dead microorganisms, within the population of living microorganisms, separating the physiologically active microorganisms from the dormant microorganisms (latent state);

[0088] It also provides all the information about vitality and viability at the same time.

[0089] The method is rapid, with active labeling in approximately 15 minutes, inexpensive and industrializable for high-throughput analysis.

[0090] Furthermore, as can be seen in the photographs of [Fig.5], the method makes it possible to extract the hyphae, to detect them by flow cytometry by labeling and to determine their state of life (latent, dead, active) via triple labeling.

[0091] Naturally, the invention is described in the above by way of example. It is understood that those skilled in the art are able to carry out different variant embodiments of the invention without departing from the scope of the invention.

Claims

1. Claims Method for the simultaneous detection and quantification of fungi and bacteria present in a divided solid containing cellulosic organic matter, according to which a solid-liquid extraction is carried out on said divided solid, a sample of said liquid obtained, optionally diluted, is introduced into a flow cytometer and a bi-parametric histogram is established giving for each point the intensity of the diffracted light and the intensity of the reflected and refracted light so as to discriminate a first group of points attributable to fungi and a second group of points attributable to bacteria, characterized in that at least one first fluorochrome capable of binding to DNA is added to said sample before introduction into said cytometer, in that said sample is further excited so as to obtain the fluorescence of said first fluorochrome,in that said bi-parametric histogram is also established for the points corresponding to particles emitting fluorescence in the emission wavelength of said fluorochrome for the first group of points and for the second group of points and in that for each of said groups a cloud of points is determined which corresponds to a fluorescence intensity due to the first fluorochrome greater than that of the other points of said group and in that possibly the points of said cloud are counted,which correspond respectively to fungal or bacterial cells and in that said first fluorochrome is chosen from fluorochromes capable of binding to the DNA of the cells and having a maximum fluorescence absorption wavelength equal to or greater than 599nm and equal to or less than 657nm and a maximum fluorescence emission wavelength equal to or greater than 619nm and equal to or less than 678nm and a quantum yield equal to or greater than 0.16 and equal to or less than 0.39 and in particular from fluorochromes capable of binding to the DNA of the cells and having a maximum fluorescence absorption wavelength of 652 nm and a maximum fluorescence emission wavelength of 676 nm and a fluorescence quantum yield on DNA of 0,27 and fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657nm and a maximum fluorescence emission wavelength of 673nm and a fluorescence quantum yield on DNA of 0.17.,

2.

3. Method according to claim 1, characterized in that at least one second and at least one third fluorochrome are also added to said sample before introduction into said cytometer, in that said second fluorochrome is capable of binding only to the DNA of cells whose wall is permeable and a third fluorochrome which becomes fluorescent by reaction with the esterases contained in the living cells and in that for at least one of said groups a bi-parametric histogram is established giving the intensity of the fluorescence due to the second fluorochrome and the intensity of the fluorescence due to the third fluorochrome, two or three clouds of points are determined on said histogram: a first cloud of points for which the fluorescence of the third fluorochrome is the strongest,a second cloud of points for which the fluorescence due to the second fluorochrome is the weakest and a third cloud of points and it is deduced that the first cloud of points corresponds to the living bacteria / fungi, the second cloud of points corresponds to the fungi / bacteria in the latent state and the third cloud of points corresponds to the dead fungi / bacteria, by counting the points in each cloud, the number of bacteria or fungi and their respective state are determined. Method according to claim 1 or 2, characterized in that said second fluorochrome is chosen from fluorochromes having a maximum absorption wavelength in fluorescence of 547nm and a maximum emission wavelength in fluorescence of 570nm and a fluorescence quantum yield on DNA of 0.9 and in that said third fluorochrome is chosen from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate,mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate and succinimidyl fluorescein 5,6-carboxylate diacetate of the following general formula: [Formula 3], 0 i H3C^G

4. Method according to any one of the preceding claims, characterized in that said divided solid containing cellulosic materials is chosen from topsoil, potting soil, compost, manure, mulch, humus and mixtures thereof, in particular mixtures thereof in pairs.

5. Method according to any one of the preceding claims, characterized in that it makes it possible to detect and quantify at least one fungi and preferably a mixture of fungi chosen from microscopic fungi capable of forming mycorrhizae, imperfect fungi, Lichtheimia corymbifera corymbilfer mucor, mucor mucedo, yeasts, and hyphae of microscopic fungi and at least one bacterium or a mixture of bacteria chosen from cytophaga spp, streptomyces spp, Bacillus radicola, Candida albicans, Phycomycetes Rhizopus, Penicillium spp, Aspergillus spp, verticillium, Helminthosporium, Fusarium, Cladosporium, actynomycetes, Nitrosomonas, Nitrosocoocus, Azotobacter, Clostridium spp, Rhizobium, acidobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Alphaproteo-bacteria and Betaproteobacteria,, Saccharomyces spp, Pseudomonas spp, Staphylococcus aureus. E.coli, Micrococcus luteus, Bacillus me-gaterium, Bacillus polymyxa and Enterococcus faecium.

6. Method according to any one of the preceding claims, characterized in that the solid-liquid extraction is carried out with an aqueous sodium chloride solution containing from 7 to 12g / L of sodium chloride and in particular 8.5g / L of sodium chloride and filtered with a filter having a cut-off threshold of 0.22pm.

7. Method according to any one of the preceding claims, characterized in that said divided solid is sieved before extraction so as to retain only fragments of a size less than or equal to 2 mm.

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