Device for conditioning a filtration membrane for analysis by solid-phase cytometry on said filtration membrane, and associated methods

Abstract

The invention relates to a filtration device for use in solid-phase cytometry, said filtration device being configured to filter a biological sample likely to contain microorganisms and to allow its analysis, the filtration device comprising: a filtration membrane configured to filter the biological sample, the filtration membrane having an upper face and a lower face, characterized in that the filtration device further comprises at least: a coverslip arranged above the upper face of the filtration membrane, the coverslip being arranged so as to cover the entirety of the upper face of the membrane, an interstitial space formed between the coverslip and the upper face of the filtration membrane, the interstitial space being shaped to receive a fluid.

Description

[0001] Title: Device for conditioning a filtration membrane for analysis by solid phase cytometry on said filtration membrane and associated methods

[0002] Technical field of the invention

[0003] The invention relates to the technical field of solid phase cytometry and more particularly to the devices and methods linked to operations on the filtration membrane allowing such analysis.

[0004] Technological background of the invention

[0005] bioMérieux designed, developed, and markets the SCANRDI® method based on solid-phase cytometry analysis for the rapid microbiological control of filtered samples. This method is used in particular in the pharmaceutical field for the control of environments and production processes for sterile products (e.g., process water) as well as for release tests of finished products (e.g., sterility tests on injectable products).

[0006] This method is based on (i) reagents and consumables, including in particular a dedicated filtration membrane (detailed below) and fluorescent labeling reagents, (ii) an instrumental solid-phase cytometry platform, such as the SCANRDI® instrument marketed by the company bioMérieux, (iii) algorithms for detecting and counting microorganisms.

[0007] As explained above, one of the central elements of the analysis is a filtration membrane. The latter is porous with a pore density (~ 1.5 10 8 pores / cm 2 ) and a pore size of less than 0.4pm, allowing all microorganisms (excluding viruses) to be retained. Said filtration membrane is a so-called "surface" membrane where the objects are retained on the upper face and not in the volume of the membrane, and has a low thickness between 20pm and 30pm. Furthermore, the filtration membrane is preferably made of polymer: polyester (PET type) or polycarbonate, pierced by "track-etching" and colored black to reduce its intrinsic fluorescence (high for plastics) and thus increase the contrast of the detection of microorganisms by fluorescence.

[0008] The membrane is flexible and can be used as is as a consumable to be handled (placement / removal on a filtration support, on the pads, etc.) but can be advantageously pre-fixed and stretched in a suitable support, facilitating coupling with the other accessories necessary for the method, such as a filtration funnel, a support for a filtration ramp, an impregnation pad.

[0009] Furthermore, there are also surface filtration membranes based on inorganic materials, such as ceramics, which are natively rigid, with pores machined by lithography. Such membranes are generally not colored because they have very low intrinsic fluorescence.

[0010] The solid-phase cytometry method has excellent performance: it can detect the presence of a single microbial cell in a sample volume of up to several tens of ml in just 3 hours.

[0011] However, several factors limit its use, including: the fact that the protocol is essentially manual and quite tedious (see details below), the fact that the sample is transferred from one piece of equipment to another (filtration ramp, incubator, SCANRDI®, microscope), with possibilities of secondary contamination linked to the test itself, the fact that confirmation of a possible contaminant requires careful examination by fluorescence microscopy, which is not always easy because of the risk of photobleaching and associated phototoxicity, the fact that some detected microbes cannot subsequently grow back on a culture medium because of the overall stress induced by the method.

[0012] The solid phase cytometry method is carried out in several steps as illustrated in Figure 1: filtration 101, counterstaining 105, activation 106, labeling 102, detection 103 and confirmation 104. The steps of counterstaining 105, activation

[0013] 106 and confirmation 104 are optional. Optionally, a culture step

[0014] 107 can be implemented after confirmation 104.

[0015] The first step consists of filtering the entire volume of the sample to be analyzed through a filtration membrane, in a controlled environment: a biological safety cabinet (BSC) or under a laminar flow hood. To ensure that the entire sample passes through the membrane, a second sterile fluid is generally filtered behind it, to recover the residues attached to the filtration funnel. In a second step, and optionally, a counterstaining reagent is filtered, following the sample, to increase the contrast of the microorganisms. To be effective, this step must be carried out preferably from above the membrane.

[0016] Next comes the labeling step, possibly preceded by an activation phase to increase the effectiveness of the label. These two steps are preferably carried out from the lower face of the filtration membrane, with a medium placed in intimate contact under the filtration membrane. The reagent diffuses through the filtration membrane to the upper face of the filtration membrane. It can be assumed that a very thin layer of supernatant liquid forms on the surface of the membrane, a few microns or even tens of microns. Under the action of air, this layer can dry out, creating an additional stress factor for the microbes, whose viability and reproductive capacity must be maintained. On the other hand, too much liquid can disrupt the optical examination of the surface with a loss of focus.In general, the medium is soaked so that there is no visible liquid layer on the surface of the filtration membrane.

[0017] During the detection step, the filtration membrane is generally placed in intimate contact on a soaked pad to ensure its flatness and prevent drying. The detection of specific fluorescence signals from microorganisms can be done with different optical means, but in all cases, there is a more or less long exposure to an exciting light (Laser or LED) and the risks of drying and contamination are present.

[0018] Detection can be followed by a confirmation step, allowing high-resolution visualization, using a high-numerical-aperture microscope, of the detected objects and confirmation that they are indeed microbes. As with the detection step, and even more critically, it is important to control the flatness of the membrane and the possible thin layer of supernatant liquid on the surface in order to guarantee the quality of the acquired images (resolution and contrast).

[0019] If the presence of microorganisms is detected, and optionally, the membrane can finally be placed on agar or in a liquid culture medium in order to grow them to allow the most precise identification possible.

[0020] In the current state of the art, the filtration membrane is always exposed to the air, except during the transfer steps from one station to another where a protective cover may possibly be placed on the membrane, with no properties other than limiting the possible contamination of the sample by the environment or vice versa, the cover being removed for the detection step of the method. Subject of the invention

[0021] The invention aims to improve the existing system described above and in particular to: maintain the integrity of the sample throughout the analysis, from filtration to the detection result, or even the identification of contaminants, reduce the risks of pollution of the environment by the sample, reduce the risk of contamination of the sample during the process (secondary contamination), reduce the phototoxicity of the analysis, reduce photobleaching, promote post-analysis regrowth, allow easier handling of the membrane and in particular to place it upside down, top side down, in an inverted microscope, allow the use of immersion objectives and therefore improve the quality of the images during the confirmation step under the microscope (the use of immersion objectives for microscopic imaging with a very high numerical aperture allows better light collection and better image quality).

[0022] To this end, the subject of the invention is a device for conditioning a filtration membrane for analysis by solid phase cytometry, said conditioning device comprising: a filtration membrane configured to filter a biological sample, the filtration membrane having an upper face and a lower face, characterized in that the conditioning device further comprises at least: a lamella arranged above the upper face of the filtration membrane, the lamella being arranged so as to cover the entire upper face of the filtration membrane, an interstitial space formed between the lamella and the upper face of the filtration membrane, the interstitial space being shaped to receive a fluid.The invention aims to maintain the integrity of the sample by controlling the state of the upper face of the filtration membrane during the steps of the protocol thanks to the application of a coverslip and the interstitial space. This avoids pollution of the sample by the analysis environment (and mainly the equipment which is in direct contact), contamination of the environment (mainly equipment) by the sample, and thus limits cross-contamination from one sample to another. The pollution can be microbiological but also by particles and dust from the environment.

[0023] In addition, the lamella helps to limit any unexpected direct contact with the surface of the filtration membrane, friction for example, and to limit the effects of poor handling, such as for example, poor grip of the membrane, or the membrane falling, etc. and also helps to avoid contact with the surrounding air, thus limiting the drying out of the filtration membrane or oxidation.

[0024] Furthermore, the presence of a fluid in the interstitial space, with a determined thickness, is compatible with the optical examination of the surface of the filtration membrane and has a specific favorable dissipation effect limiting “photo-bleaching” during fluorescence detection.

[0025] The combined presence of the coverslip and the interstitial space of the interstitial fluid presents other advantages among which: the specific control of an environment favorable to the metabolism of microorganisms (aerobic or anaerobic), the prevention of the toxic effects of oxygen specific to fluorescence analysis, in particular "quenching" and phototoxicity, the possibility of using an immersion objective.

[0026] According to a characteristic of the invention, the coverslip has optical qualities allowing microscopic examination. More particularly, the coverslip is transparent and for example made of borosilicate glass or quartz, or even of polymer material (acrylate resins, crystal polystyrene). The choice of material depends in particular on the mechanical constraints to be supported, the size of the membrane to be covered and the refractive index of the interstitial fluid placed between the upper face of the membrane and the lower face of the coverslip (between 1.35 for water up to 1.52 for immersion oils). According to another characteristic of the invention, the coverslip has a thickness of between 100 μm and 300 μm.

[0027] According to a characteristic of the invention, the lamella is secured to the filtration membrane while maintaining the interstitial space.

[0028] According to a characteristic of the invention, the strip can be secured to the membrane by gluing and / or clipping and / or sealing.

[0029] According to a characteristic of the invention, the interstitial space has a predetermined thickness of between 100 pm and 500 pm and preferably at least 250 pm. Thanks to this space, it is possible to guarantee the filtration step (by limiting the capillary forces / fluidic resistance due to the lamella) and the optical examination after filtration.

[0030] The minimum limit of 100 pm thickness of the interstitial space is determined by the manufacturing constraints of the parts by injection / molding as well as by the intrinsic thickness of the filtration membrane itself, approximately 25 pm. In addition, in the context of the subsequent growth of the remaining microorganisms on the surface of the upper face of the filtration membrane, the thickness must be sufficient so as not to constrain the development of micro-colonies.

[0031] In the option where the lamella is positioned above the filtration membrane before the filtration step, the minimum thickness of the interstitial space is at least 250 μm, which ensures that filtration takes place uniformly over the entire filtration membrane, without risk of mechanical rupture, tearing of the filtration membrane or leakage. Indeed, with such a thickness, and in the context of a vacuum applied between the upper and lower face of the filtration membrane, the entire volume above the filtration membrane is first uniformly filled with interstitial fluid, thanks to the fluidic resistance of the filtration membrane which is greater than the capillary friction forces linked to the lamella, before the interstitial fluid then passes through the filtration membrane towards the area of ​​the filtration membrane under vacuum.

[0032] The maximum limit of 500 pm of interstitial space thickness is determined by optical constraints and in particular the maximum working distance achievable for standard high magnification objectives, around 1 mm, which must cover the thickness of the coverslip added to said thickness of the interstitial space. In addition, depending on the fluid used to fill this interstitial space, and in particular its absorption, scattering or fluorescence characteristics, this maximum limit will be further reduced to guarantee the best imaging quality.

[0033] Preferably, an interstitial space thickness of around 250 pm allows for a device that can be used throughout the analysis protocol, from filtration of the biological sample to the confirmation step under the microscope and beyond during possible culture allowing identification.

[0034] According to a feature of the invention, the filtration device comprises an interstitial fluid housed in the interstitial space, the fluid being a liquid buffer or a gel. Advantageously, the fluid is a liquid buffer optionally containing a surfactant, such as for example a polysorbate, also known under the trade name Tween®, in order to improve the wettability of said buffer.

[0035] According to a characteristic of the invention, the interstitial fluid is transparent and not very diffusive, which allows its compatibility with a high-resolution optical examination of the filtration membrane.

[0036] Advantageously, the interstitial fluid is a gel or liquid gelled by means of a gelling agent, for example Kappa Carrageenan. In this configuration, the liquid is introduced at a temperature between 45°C and 50°C and then gels rapidly at room temperature in the interstitial space.

[0037] According to a characteristic of the invention, the interstitial fluid may contain a reducing reagent in solution which aims to hydrolyze the fluorophore molecules present in the matrix, thereby increasing the contrast between the labeled microorganisms (fluorophore trapped in the cells) and the background.

[0038] According to a feature of the invention, the interstitial fluid can be supplemented with soluble nutrients to promote microbial growth.

[0039] According to a feature of the invention, the reagents added to the interstitial fluid can be encapsulated in microparticles allowing their controlled and programmed release throughout the protocol. Alternatively, the interstitial fluid can also be a mixture of oil, paraffin or water.

[0040] According to a characteristic of the invention, the filtration membrane can be made of polymer, polycarbonate or polyethylene terephthalate (PET) or of rigid composite material.

[0041] According to a feature of the invention, the filtration membrane can be made of polymer and pierced with the "track-etched" type process, which means that the pores are made in 2 steps: a first step where the plastic film is bombarded by high-energy particles in order to generate impacts on the surface ("track"), a second step where the plastic film is then subjected to a chemical attack ("etching") in a bath, where the pores are formed from the traces ("tracks") previously formed. In this process, the density of the pores is determined by the first step (density of the "tracks"), while the size of the pores is controlled by the conditions of the chemical attack.

[0042] According to a characteristic of the invention, the membrane can be made of ceramic and engraved / pierced by laser lithography.

[0043] According to a characteristic of the invention, the filtration membrane can be colored or metallized.

[0044] According to a characteristic of the invention, the filtration device comprises at least one support on which the filtration membrane is positioned, said support having at least one port communicating with the upper face of the filtration membrane.

[0045] According to a characteristic of the invention, the support comprises a plurality of ports.

[0046] Advantageously, the port(s) allow manipulations on the upper face of the filtration membrane, in particular filtration (by closing one of the two ports) or the introduction of an interstitial fluid after filtration.

[0047] According to a feature of the invention, at least one port of the support is configured to allow the filling of the interstitial space with the interstitial fluid. Advantageously, the support of the filtration membrane is a frame on which the filtration membrane is stretched and fixed. Advantageously, the frame is positioned on the perimeter of the filtration membrane.

[0048] According to a characteristic of the invention, the support of the filtration membrane has a lower housing provided under the filtration membrane.

[0049] Advantageously, the lower housing is open on the lower face 2b of the filtration membrane.

[0050] According to a characteristic of the invention, the support comprises a lower housing, formed under the filtration membrane, the lower housing being shaped to receive a functionalized cartridge intended to come into contact with the lower face of the membrane.

[0051] According to a characteristic of the invention, the filtration device comprises at least one functionalized cartridge, said functionalized cartridge being able to be a reagent or a culture medium or a medium for activating spores, or a cartridge for fluorescent labeling or a cartridge for deactivating the labeling and / or for keeping the filtration membrane soaked.

[0052] According to a characteristic of the invention, the cartridge contains the reagents in the form of a solution in a liquid matrix.

[0053] According to a characteristic of the invention, the cartridge is a pad soaked in reagent.

[0054] According to a characteristic of the invention, the cartridge contains the reagents in a gel matrix.

[0055] According to a feature of the invention, the cartridges can be brought into contact one after the other during the protocol; for example, the activation cartridge, then the labeling cartridge and finally the support cartridge ensuring flatness for detection. According to a feature of the invention, a single cartridge can be used for several steps, with a mixture of reagents.

[0056] According to a feature of the invention, the cartridge reagents are encapsulated in microparticles allowing controlled / programmed release. Thus, within a given time interval, or under the effect of a temperature gradient, the particles release the labeling substrate into the medium which then diffuses towards the filtration membrane and labels the microorganisms of interest. Such a cartridge makes it possible to combine the activation and labeling steps with a single cartridge.

[0057] Advantageously, the filtration device further comprises a cover which is clipped / glued onto the support 6, once the filtration step has been carried out. Such a filtration device according to the invention, once closed and before the detection step, makes it possible to keep the filtration membrane protected and leave it for several days in an incubator to allow the growth of microorganisms on the surface of the filtration membrane, which facilitates the identification of the microorganisms.

[0058] Advantageously, the reagent can be in liquid, gel or soaked pad form.

[0059] Advantageously, the device also includes a space containing gases, allowing the growth of microbes.

[0060] The present invention also relates to a method of microbiological analysis by solid phase cytometry using a filtration device according to the invention, the analysis method comprising at least the following steps: a step of filtration of the sample by the filtration membrane; a step of fluorescence labeling of the microorganisms likely to be present in the biological sample, a step of detection of the labeled microorganisms, the method being characterized by the fact that it comprises at least one step according to which the coverslip is positioned above the upper face of the filtration membrane of the filtration device.

[0061] According to one characteristic of the invention, the method comprises a step of confirming detection by fluorescence microscope, this step being carried out after the detection step or after the labeling step. According to one characteristic of the invention, the fluorescence labeling step consists of making the microorganisms fluorescent.

[0062] According to a characteristic of the invention, the fluorescence labeling step can be carried out with a fluorogenic substrate or a reagent or any other means making it possible to achieve fluorescence of the microorganism.

[0063] According to a characteristic of the invention, the sample is in liquid form so as to be filterable. Preferably, the sample is non-biological and is for example: wastewater, injectable chemicals, etc.

[0064] According to a characteristic of the invention, the lamella is positioned above the upper face of the filtration membrane of the filtration device before the filtration step. After this lamella positioning step, an additional step of adding an interstitial fluid into the interstitial space of the filtration device is carried out after the filtration step, directly after the filtration step or not.

[0065] Alternatively and according to a characteristic of the invention, the lamella is positioned above the upper face of the filtration membrane of the filtration device after the filtration step. After this lamella positioning step, the interstitial fluid is added into the interstitial space of the filtration device before the detection step, directly after the detection step or not.

[0066] According to a characteristic of the invention, the lamella can be secured to the membrane by clipping at any time during the analysis method, once a filtration funnel has been removed.

[0067] According to a characteristic of the invention, if the slide is positioned after the filtration step, the sample is injected directly onto the upper face of the filtration membrane via a filtration funnel.

[0068] According to a characteristic of the invention, if the slide is positioned before the filtration step, the sample is injected onto the upper face of the filtration membrane via the port(s) of the support of the filtration device.

[0069] According to a characteristic of the invention, during the filtration of the sample, whether the lamella is present or not, a depression can be applied to the lower face of the filtration membrane, which makes it possible to obtain homogeneous filtration over the entire surface of the filtration membrane, thereby improving the efficiency and speed of the filtration.

[0070] Advantageously, if the coverslip is present during filtration, the sample in liquid form fills the interstitial space before the suction effect causes it to pass through the pores of the filtration membrane.

[0071] According to a feature of the invention, the method may further include a step of adding a counter-staining solution, this addition being carried out by filtering said solution, after filtering the sample. As for the filtration of the sample, this step is carried out via the upper face of the membrane, either via a funnel or via the injection ports, if the coverslip is present.

[0072] This counterstaining step is an optional step that reduces the endogenous fluorescence of the sample and non-specific labeling. Indeed, the sample can correspond to different products, more or less complex (solution, suspension, emulsion) with possible prior preparation to make it filterable (centrifugation, mechanical lysis, chemical lysis). The sample, in addition to any microbes labeled in fluorescence, can therefore contain intrinsically fluorescent particles or debris.Even if some particles are not natively fluorescent, the exogenous labeling provided by the protocol may not be perfectly specific and may label objects other than microbes, either by direct conversion of the fluorogenic substrate into a fluorophore in an undesired site (not a microbial cell), or by efflux of the fluorophore generated inside a microbial cell through the membrane, by the action of efflux pumps; the fluorophore therefore labels the entire sample, and reduces the contrast. The counterstaining solution therefore masks / covers the sites before the labeling step to prevent / prevent access of the fluorophore.

[0073] According to a characteristic of the invention, the method may comprise a pre-marking step prior to the marking step, said pre-marking step constituting an activation to resuscitate the spores and guarantee optimal enzymatic activity. According to a characteristic of the invention, the activation and marking steps may be carried out with or without a coverslip present on the surface of the membrane.

[0074] According to a characteristic of the invention, the interstitial volume is filled with a fluid after the filtration and counter-staining step and before the activation and marking step.

[0075] According to a characteristic of the invention, the interstitial space is filled with an interstitial fluid after the activation and marking step and before the detection step.

[0076] According to a feature of the invention, the interstitial fluid can be injected via the ports of the device.

[0077] According to a characteristic of the invention, the interstitial space can be filled from the inner face of the membrane, via diffusion of the liquid through the pores of the membrane.

[0078] According to a characteristic of the invention, the optional confirmation step can be carried out using an immersion microscopy objective. Indeed, thanks to the presence of the coverslip above the filtration membrane, it is possible to use immersion microscopy objectives with a very high numerical aperture (from 40X and NA > 0.6) which therefore have a very high collection power and make it possible both to minimize the dose of light received by the sample and also to improve the resolution of the images. In the case of the use of an immersion objective, it is necessary to provide for the use of an immersion oil compatible with the material of the coverslip. Alternatively, the confirmation step can be carried out with an air objective, possibly corrected for the presence of the coverslip.

[0079] According to a characteristic of the invention, the air objective is a 60X, air, open at 0.7, with a long working distance (greater than or equal to 1 mm) allowing focusing over the majority of the range of the interstitial space and with correction of the coverslip (variable correction ring).

[0080] Brief description of the figures

[0081] The invention will be better understood from the following description, which relates to embodiments according to the present invention, given as non-limiting examples and explained with reference to the attached schematic figures. The attached schematic figures are listed below:

[0082] Figure 1 is a diagram of a method of microbiological analysis by solid phase cytometry according to the prior art,

[0083] Figure 2 is a sectional view of the filtration device according to the invention,

[0084] Figure 3 is an exploded sectional view of the filtration device according to the invention,

[0085] Figure 4 is a possible implementation diagram, with a gel interstitial layer and the thickness of interest for detection,

[0086] Figure 5 is a perspective view of a portion of the support and filtration membrane of the filtration device,

[0087] Figure 6 is an illustration of the method according to the invention.

[0088] Figure 7 is an alternative embodiment of the method according to the invention.

[0089] Detailed description

[0090] The invention will now be described in detail with reference to the figures listed above.

[0091] The filtration device 1 according to the invention is intended for use in solid-phase cytometry. The filtration device 1 comprises a filtration membrane 2 configured to filter a sample (not shown). As illustrated in FIG. 2 in particular, the filtration membrane 2 has an upper face 2a and a lower face 2b, the filtration of the biological sample taking place from the upper face 2a to the lower face 2b. The filtration membrane is fixed to a support 6. Ideally, the support 6 is arranged around the filtration membrane 2 as visible in FIG. 5. In this example illustrated in FIG. 5, the filtration membrane 2 is stretched and fixed to the support 6.

[0092] The support 6 of the filtration membrane 2 has a lower housing 8 arranged under the filtration membrane 2. The lower housing 8 is open on the lower face 2b of the filtration membrane 2. The lower housing 8 is configured to receive one or more functionalized cartridges 9, which will be described later.

[0093] As illustrated in Figures 2 and 3, the filtration device 1 further comprises a lamella 3 arranged above the upper face 2a of the filtration membrane 2. As can be seen, the lamella 3 is arranged so as to cover the entire upper face 2a of the filtration membrane 2, in particular to protect the latter. The lamella 3 is transparent to allow analysis. The lamella 3 is secured to the filtration membrane 2 while maintaining an interstitial space 4. The interstitial space 4 is formed between the lamella 3 and the upper face 2a of the filtration membrane 2, the interstitial space 4 being shaped to receive an interstitial fluid 5 as visible in Figure 2. The interstitial space 4 also makes it possible to accommodate the sample (not shown) before filtration. The interstitial space 4 is sealed when the lamella 3 is positioned above the filtration membrane 2.Indeed, the filtration device 1 comprises seals 10 to seal said interstitial space 4, as seen in particular in figures 2 and 4.

[0094] Further details of the filtration device 1 will become apparent in the light of the description of the method which will now be detailed.

[0095] The microbiological analysis method according to the invention is a solid phase cytometry analysis method.

[0096] To implement this analysis method, a filtration device 1 as described previously is used.

[0097] The first step 101 of the analysis method consists of filtering the sample to be analyzed. The second step 102 of the analysis method is a step of labeling the microorganisms likely to be present in the biological sample, the labeling step 102 consisting of labeling the microorganisms with a fluorogenic substrate. The analysis method comprises a third step, a step of detection 103 of the labeled microorganisms. The analysis method comprises a fourth, optional, step of confirmation 104 of detection by fluorescence microscope.

[0098] The method comprises a positioning step 110 in which the lamella 3 is positioned above the upper face 2a of the filtration membrane 2 of the filtration device 1 and a step of adding interstitial fluid 112 into the interstitial space 4. Depending on the steps of the method, these two steps can be carried out at different times.

[0099] For example, as illustrated in figure 6, the positioning step 110 of the coverslip 3 is carried out before the filtration 101, the addition step 112 is carried out after filtration 110 or after any step requiring the filling of the interstitial space 4, such as for example the step of introducing the sample 111 or the counter-coloring step 105 as illustrated in figure 6.

[0100] The sample introduction step 111, consisting of introducing the sample onto the upper face 2a of the filtration membrane 2, can be carried out via the port(s) 7 of the support 6 of the filtration device 1.

[0101] In the example illustrated in Figure 6, the coverslip 3 is positioned (step 110) before filtration 101, and the sample is introduced (step 111) after the positioning of the coverslip 3, which ensures optimal non-contamination of the biological sample. Following the filtration, an optional counter-staining step 105 can be implemented (the step is noted in dotted line in Figure 6 given its optional nature). When the counter-staining step 105 is implemented, the interstitial space 4 fills with counter-staining solution which is then filtered by the membrane. The interstitial fluid addition step 112 occurs after the counter-staining step 105. When the counter-staining step is not implemented, the interstitial fluid addition step 112 is implemented directly after filtration 110.

[0102] The step 112 of adding interstitial fluid can be carried out either: by first soaking the filtration membrane 2 on its lower face 2b with a cartridge 9 comprising the interstitial fluid 5 and therefore allowing the interstitial fluid 5 in liquid form to rise by capillarity via the pores of the filtration membrane 2 and fill the interstitial space 4, by wetting the lamella 3 with the interstitial fluid 5 in liquid form before placing it on the filtration membrane 2;by turning over the pair of filtration membrane 2 and lamella 3 to access the lower face 2b of the filtration membrane 2 and allowing the interstitial fluid 5 in liquid form to settle by gravity in the interstitial space 4, by injecting, once the lamella 3 has been deposited (step 110), the interstitial fluid 5 in liquid form via the port(s) 7 of the support 6 of the filtration membrane 2, by positioning the pair of filtration membrane 2 and lamella 3 vertically and injecting the interstitial fluid 5 in liquid form from bottom to top to expel the air bubbles. As illustrated in Figure 6, an optional pre-marking step 106 follows (the step is noted in dotted lines in Figure 6 given its optional nature), consisting of an activation to resuscitate the spores present in the sample and guarantee optimal enzymatic activity. In the present invention, the "spores";

[0103] The sample corresponds to a certain physiological state of microorganisms, with a very reduced metabolism. Then, the labeling step 102 of the microorganisms is implemented.

[0104] During the pre-marking 106 and marking 102 steps, functionalized cartridges 9 can be used as illustrated in FIG. 3. These functionalized cartridges 9, depending on the step, can be: a reagent 12 or a medium for activating spores 12, or a cartridge for fluorescent marking or a cartridge for deactivating the marking. The latter 9 are inserted into the support 6 of the filtration device 1 in order to be in contact with the lower face 2b of the filtration membrane 2 on which the microorganisms of the biological sample are retained.

[0105] The pre-marking 106 and marking 102 steps can also be implemented by a single functionalized cartridge having two types of particles (A) and (B) with different release times. Thus, the marking substrate can be diffused initially, then after a programmed marking time, a reagent which “bleaches” and extinguishes the marking, such as a strong reducing agent. With such a device, a single cartridge can be used, with a culture gel 12, which allows all 3 operations: activation, marking, stopping of the marking.

[0106] Finally, the microorganisms collected on the filtration membrane 2 are analyzed in two stages: the detection stage 103 and the confirmation stage 104.

[0107] It is also possible to add a microorganism culture step in order to increase the mass of microorganisms for more efficient detection purposes: a functionalized cartridge of culture media can be considered or the cultivation of the filtration membrane 2 is also considered. In this step (not shown), two types of cartridges can be considered depending on whether one wants to detect and grow aerobic or anaerobic germs. The gas 11 present in the functionalized cartridge 9, as illustrated in figures 2, 3 and 4, for aerobes contains oxygen and that for anaerobes is without oxygen.

[0108] The detection step 103 is carried out by fluorescence imaging, in Laser-Scanning and / or Full-Field and / or Image Mosaic mode. The detection confirmation step 104 can be carried out using an air objective or an immersion objective, the coverslip 3 allowing the use of these two objectives. Figure 4 shows a possible implementation diagram, with an interstitial gel layer and the thickness of interest for the detection, with 01 representing the focusing range of the objective and 02 the maximum working distance of the objective, the numerical reference 50 being the objective.

[0109] Figure 7 also represents the method of the invention but in which the lamella 3 is only added before analysis (steps 103 and 104).

[0110] Furthermore, prior to step 111 of introducing the biological sample, it is possible to prepare the biological sample so that it is filterable. Of course, the invention is not limited to the embodiments described and shown in the appended figures. Modifications remain possible, in particular from the point of view of the constitution of the various elements or by substitution of technical equivalents, without departing from the scope of protection of the invention.

Claims

CLAIMS 1. A filtration device intended for use in solid-phase cytometry, said filtration device being configured to filter a sample likely to contain microorganisms and allow its analysis, the filtration device comprising: a filtration membrane configured to filter the biological sample, the filtration membrane having an upper face and a lower face, characterized in that the filtration device further comprises at least: a lamella arranged above the upper face of the filtration membrane, the lamella being arranged so as to cover the entire upper face of the membrane, an interstitial space formed between the lamella and the upper face of the filtration membrane, the interstitial space being shaped to receive a fluid.

2. A filtration device according to claim 1, comprising an interstitial fluid housed in the interstitial space, the fluid being a liquid or a gel.

3. Filtration device according to any one of claims 1 or 2, in which the lamella is secured to the filtration membrane while maintaining the interstitial space.

4. Filtration device according to any one of claims 1 to 3, comprising at least one support on which the filtration membrane is positioned, said support having at least one port communicating with the upper face of the membrane.

5. Filtration device according to claim 4, in which the support comprises a lower housing, formed under the filtration membrane, the lower housing being shaped to receive a functionalized cartridge intended to come into contact with the lower face of the membrane.

6. Filtration device according to claim 5, comprising at least one functionalized cartridge, said functionalized cartridge being able to be a reagent or a culture medium or a medium for the activation of spores, or a cartridge for the fluorescent labeling or a label deactivation cartridge and / or to keep the filtration membrane soaked.

7. Method of microbiological analysis by solid phase cytometry using a filtration device according to any one of the preceding claims, the analysis method comprising at least the following steps: a step of filtration of the sample by the filtration membrane, a step of fluorescence labeling of the microorganisms likely to be present in the biological sample, a step of detection of the labeled microorganisms, the method being characterized in that it comprises at least one step according to which the coverslip is positioned above the upper face of the filtration membrane of the filtration device.

8. Method of microbiological analysis by solid phase cytometry according to claim 7, in which the coverslip is positioned above the upper face of the filtration membrane of the filtration device before the filtration step and an additional step of adding an interstitial fluid into the interstitial space of the filtration device is carried out after the filtration step.

9. Method of microbiological analysis by solid phase cytometry according to any one of claims 7 or 8, in which the sample is injected onto the upper face of the filtration membrane via the port(s) of the support of the filtration device.

10. Method of microbiological analysis by solid phase cytometry according to any one of claims 7 to 9, in which the coverslip is positioned above the upper face of the filtration membrane of the filtration device and the interstitial fluid is added into the interstitial space of the filtration device before the detection step.

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