Apparatus for conditioning a filtration membrane on the membrane for analysis by solid phase cytometry, and associated methods

Abstract

The present invention relates to a filtration device for use in solid phase cytometry, configured to filter a biological sample that may contain microorganisms to enable its analysis, the filtration device comprising a filtration membrane configured to filter the biological sample, the filtration membrane having an upper surface and a lower surface, the filtration device further comprising at least a cover glass arranged above the upper surface of the filtration membrane, the cover glass being arranged to cover the entire upper surface of the membrane, and a gap formed between the cover glass and the upper surface of the filtration membrane, the gap being shaped to receive a fluid.

Description

[Technical Field]

[0001] The present invention relates to the technical field of solid phase cytometry, and more particularly to devices and methods relating to operations on filtration membranes that enable such analyses. [Background technology]

[0002] bioMerieux designs, develops, and markets the SCANRDI™ method, based on solid-phase cytometric analysis, for the rapid microbiological control of filterable samples. This method is used in particular in the pharmaceutical sector for environmental and process control for the production of sterile products (e.g., process water) and for release testing of final products (e.g., sterility testing of injectable products).

[0003] The method is based on (i) reagents and consumables, particularly specialized filtration membranes (described in detail below) and fluorescently labeled reagents, (ii) a solid-phase cytometry instrument platform such as the SCANRDI™ instrument marketed by bioMerieux, and (iii) algorithms for detecting and enumerating microorganisms.

[0004] As explained above, one of the central elements of the analysis is the filtration membrane. This membrane is porous, with a pore density (approximately 1.5 × 10 8 hole / cm 2 ) and a pore size of less than 0.4 μm, allowing all microorganisms (except viruses) to be retained. The filtration membrane is called a "surface" membrane, in which objects are retained on the upper surface rather than within the membrane's volume, and has a thin thickness of between 20 μm and 30 μm. Furthermore, the filtration membrane is preferably made of polymers: polyester (PET) or polycarbonate, perforated by track etching, and colored black to reduce its inherent fluorescence (high for plastic materials) and therefore increase the contrast of the detection of microorganisms by fluorescence.

[0005] The membranes are flexible and can be used as consumables that can be handled (placed / removed onto a filtration support or pad, etc.) or, advantageously, can be pre-mounted and stretched onto a suitable support, facilitating connection to other accessories required for the method, such as filtration funnels, filtration manifold supports, impregnated pads, etc.

[0006] Alternatively, there are surface filtration membranes based on inorganic materials such as ceramics that are rigid in nature, with lithographically machined pores. Such membranes have very low intrinsic fluorescence and are therefore generally uncolored.

[0007] Solid-phase cytometry is highly efficient and can detect the presence of single microbial cells in sample volumes of up to tens of milliliters in as little as three hours. However, its use is influenced by several factors, in particular: - The fact that the protocol is manual in nature and somewhat tedious (see details below) - The fact that samples are transferred from one instrument to another (filtration manifold, incubator, SCANRDI™, microscope) with the possibility of cross-contamination associated with the test itself - the fact that identification of any contaminants requires careful examination by fluorescence microscopy, which is not always easy due to the risk of photobleaching and associated phototoxicity; - the fact that some of the detected microorganisms are unable to grow again on the medium afterwards due to the overall stress caused by the method; is limited by

[0008] 1, solid-phase cytometry is performed in several steps: filtration 101, counterstaining 105, activation 106, labeling 102, detection 103, and confirmation 104. The counterstaining step 105, activation step 106, and confirmation step 104 are optional. Optionally, an incubation step 107 may be performed after confirmation 104.

[0009] The first step consists of filtering the entire volume of the sample to be analyzed through a membrane filter in a controlled environment, i.e., in 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 usually subsequently filtered to collect any residue adhering to the filter funnel.

[0010] In a second step, a counterstaining reagent can optionally be subsequently filtered through the sample to enhance the contrast of the microorganisms. To be effective, this step must be performed primarily from above the membrane.

[0011] The next step is labeling, possibly preceded by an activation step to enhance the effectiveness of the labeling. These two steps are preferably performed from the underside of the filtration membrane using a medium placed closely underneath the membrane. The reagent diffuses through the membrane to the upper surface. It can be assumed that a very thin layer of supernatant is formed on the membrane surface, several microns or even tens of microns thick. When exposed to air, this layer may dry out, creating additional stress factors for the microorganisms, but their viability and reproductive ability must be maintained. On the other hand, if the liquid layer is too large, it may cause a loss of focus and interfere with optical inspection of the surface. Generally, the medium is immersed so that there is no visible liquid layer on the membrane surface.

[0012] During the detection step, the filtration membrane is generally placed in close contact with a soaking pad to ensure it is flat and prevent it from drying out. The specific fluorescent signal from the microorganisms can be detected using various optical means, but in all cases there is a relatively long exposure to the excitation light (laser or LED), which carries the risk of drying out and contamination.

[0013] Detection is followed by a confirmation step, which makes it possible to observe the detected objects at high resolution using a high numerical aperture microscope to confirm that they are microorganisms. As well as the detection step, and even more precisely, it is important to control the flatness of the film on the surface and, if present, the thin layer of supernatant liquid, in order to guarantee the quality (resolution and contrast) of the acquired images.

[0014] If the presence of microorganisms is detected, the membrane can then optionally be placed on agar or in liquid medium to grow them so that they can be identified as accurately as possible.

[0015] In the prior art, currently, the filtration membrane is always exposed to air, except possibly during the transfer step from one station to another, when a protective cover may be placed on the membrane, its only property being to limit the possibility of contamination of the sample by the environment, or vice versa, when the cover is removed for the detection step of the method. Summary of the Invention

[0016] The present invention aims to improve the above-mentioned existing systems, and in particular to: - Maintaining sample integrity throughout the analysis, from filtration to detection results or identification of contaminants; - reducing the risk of contamination of the environment with the sample; - reducing the risk of contamination of samples during processing (cross-contamination); - reducing the phototoxicity of the assay; - reducing photobleaching, - promoting post-analysis regrowth; allowing easier handling of the membrane in an inverted microscope, in particular allowing the membrane to be placed upside down with its upper surface facing downwards; - Allows the use of immersion objectives during the microscopic verification step, thus improving the quality of the images (the use of immersion objectives for microscopic imaging with very high numerical apertures allows for better light collection and better image quality) The purpose is to:

[0017] To this end, the present invention provides an apparatus for conditioning a filtration membrane for analysis by solid phase cytometry, the conditioning apparatus comprising: a filtration membrane configured to filter a biological sample, the filtration membrane having an upper surface and a lower surface; The conditioning device further comprises at least a cover glass arranged above the upper surface of the filtration membrane, the cover glass being arranged so as to cover the entire upper surface of the filtration membrane; a gap formed between the cover glass and the upper surface of the filtration membrane, the gap being shaped to receive a fluid; and characterized in that it comprises Regarding the device.

[0018] The present invention aims to maintain the integrity of the sample by controlling the state of the upper surface of the filtration membrane during the steps of the protocol through the application of a cover glass and a gap. This avoids contamination of the sample by the analytical environment (mainly the equipment with which it is in direct contact) and contamination of the environment (mainly the equipment) by the sample, thus limiting cross-contamination from one sample to another. Contamination can be microbiological, but can also result from particles and dust in the environment.

[0019] Furthermore, the cover glass makes it possible to limit accidental direct contact with the surface of the filtration membrane, e.g., friction, and to limit the effects of poor handling, e.g., if the membrane is not properly gripped or falls, while also making it possible to avoid contact with the surrounding air, thus limiting drying or oxidation of the filtration membrane.

[0020] Furthermore, the presence of a fluid within a gap having a predetermined thickness is compatible with optical inspection of the surface of the filtration membrane and has a specific beneficial dissipative effect that limits "photobleaching" during fluorescence detection.

[0021] The combined presence of the cover slip and the gap and interstitial fluid is: -Specific control of the medium (aerobic or anaerobic) favored by the microbial metabolism - Prevention of the toxic effects of oxygen specific to fluorescence analysis, in particular quenching and phototoxicity - possibility of using immersion objectives Other advantages include:

[0022] According to one feature of the present invention, the cover glass has optical properties that allow microscopic examination. More specifically, the cover glass is transparent and is made of, for example, borosilicate glass or quartz, or a polymer (acrylate resin, crystalline polystyrene). The material selected depends, in particular, on the mechanical constraints to be taken into account, the size of the membrane to be covered, and the refractive index of the interstitial fluid placed between the upper surface of the membrane and the lower surface of the cover glass (1.35 or more for water, and up to 1.52 for immersion oil).

[0023] According to another feature of the present invention, the cover glass has a thickness of 100 μm to 300 μm.

[0024] According to one feature of the invention, the cover glass is firmly secured to the filtration membrane while maintaining a gap.

[0025] According to one feature of the invention, the cover glass may be firmly secured to the membrane by adhesive bonding and / or clipping and / or sealing.

[0026] According to one feature of the invention, the gap has a predetermined thickness of 100 μm to 500 μm, preferably at least 250 μm, which makes it possible to ensure the filtering step (by limiting the capillary force / fluid resistance due to the cover glass) and the optical inspection after filtering.

[0027] The minimum gap thickness limit of 100 μm is determined by the constraints of manufacturing parts by injection / molding and the inherent thickness of the filtration membrane itself, i.e., approximately 25 μm. Furthermore, the thickness must be sufficient so as not to limit the development of microcolonies in the event of subsequent growth of residual microorganisms on the upper surface of the filtration membrane.

[0028] In the case where a cover glass is placed above the filtration membrane before the filtering step, the minimum gap thickness is at least 250 μm, which ensures that filtering occurs uniformly across the entire filtration membrane without the risk of mechanical damage, tearing of the filtration membrane, or leakage. Specifically, in the case of a recess having such a thickness and applied between the upper and lower surfaces of the filtration membrane, the fluid resistance of the filtration membrane, which is greater than the capillary friction force connected to the cover glass, ensures that the entire volume above the filtration membrane is first uniformly filled with interstitial fluid before the interstitial fluid passes through the filtration membrane and is directed toward the recessed region of the filtration membrane.

[0029] The maximum gap thickness limit of 500 μm is determined by optical constraints, in particular the maximum achievable working distance of a standard high-magnification objective of approximately 1 mm, which must cover the thickness of the cover glass plus the thickness of the gap. Furthermore, depending on the fluid used to fill the gap, in particular its absorption, scattering, or fluorescence properties, this maximum limit can be further reduced to ensure the best imaging quality.

[0030] Advantageously, a gap thickness of about 250 μm makes it possible to have a device that can be used throughout the entire analytical protocol, from the filtration of the biological sample to the confirmation step under the microscope and during any subsequent incubation aimed at identification.

[0031] According to one aspect of the invention, the filtration device includes interstitial fluid contained within the interstitial spaces, the fluid being a liquid buffer or gel.

[0032] Advantageously, this liquid is a liquid buffer optionally containing a surfactant, such as polysorbate, also known under the trade name Tween®, to improve the wetting properties of the buffer.

[0033] According to one feature of the present invention, the interstitial fluid is transparent with low diffusion capacity, making it compatible with high-resolution optical inspection of the filtration membrane.

[0034] Advantageously, the interstitial fluid is a gel or liquid that has been gelled using a gelling agent, such as Kappa Carrageenan, in which the liquid is introduced at a temperature of 45°C to 50°C and then rapidly gels within the interstitial space at room temperature.

[0035] According to one feature of the invention, the interstitial fluid may contain a reducing reagent in solution whose purpose is to hydrolyze fluorophore molecules present in the matrix, thereby increasing the contrast between the labeled microorganisms (intracellularly trapped fluorophores) and the background.

[0036] According to one feature of the invention, the interstitial fluid may be supplemented with soluble nutrients to promote microbial growth.

[0037] According to one feature of the present invention, reagents added to the interstitial fluid may be encapsulated in microparticles that allow for their controlled and programmed release throughout the protocol.

[0038] Alternatively, the interstitial fluid may be a mixture of oil, paraffin, or water.

[0039] According to one feature of the invention, the filtration membrane may be made from a polymer, polycarbonate or polyethylene terephthalate (PET), or a rigid composite material.

[0040] According to one feature of the present invention, filtration membranes may be made from polymers and perforated by a "track-etched" process, meaning that the pores are created in two steps: first, by bombarding a plastic film with high-energy particles to create impacts on the surface ("tracks"), and then, in a second step, by chemical attack ("etching") in a bath, where the pores are formed from the previously created tracks. In this process, the pore density is determined by the first step (track density), and the pore size is controlled by the etching conditions.

[0041] According to one feature of the invention, the membrane is made from ceramic and can be etched / drilled by laser lithography.

[0042] According to one feature of the invention, the filtration membrane may be colored or metallized.

[0043] According to one aspect of the invention, a filtration device comprises at least one support on which a filtration membrane is positioned, the support having at least one port in communication with an upper surface of the filtration membrane.

[0044] According to one aspect of the invention, the support comprises a plurality of ports.

[0045] Advantageously, the ports allow procedures to be carried out on the upper surface of the filtration membrane, in particular filtration (by closing one of the two ports) or the introduction of interstitial fluid after filtration.

[0046] According to one feature of the invention, at least one port in the support is configured to allow the gap to fill with interstitial fluid.

[0047] Advantageously, the support for the filtration membrane is a frame onto which the filtration membrane is stretched and attached. Advantageously, the frame is positioned at the periphery of the filtration membrane.

[0048] According to one aspect of the invention, the support for the filtration membrane has a lower housing disposed below the filtration membrane.

[0049] Advantageously, the lower housing opens onto the lower face 2b of the filtering membrane.

[0050] According to one feature of the invention, the support comprises a lower housing formed below the filtration membrane, the lower housing being shaped to receive a functionalized cartridge intended to contact the underside of the membrane.

[0051] According to one feature of the invention, the filtration device comprises at least one functionalized cartridge, possibly a cartridge for a reagent or a culture medium or a medium for activating spores, or a cartridge for a fluorescent label, or a cartridge for inactivating a label and / or for keeping the filtration membrane submerged.

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

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

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

[0055] According to one aspect of the invention, the cartridges may be contacted one after the other during the protocol, for example, an activation cartridge, then a labeling cartridge, and finally a support cartridge to ensure flatness for detection.

[0056] According to one aspect of the invention, a single cartridge may be used for several steps with a mixture of reagents.

[0057] According to one feature of the present invention, the reagents in the cartridge are encapsulated in microparticles that allow for controlled / programmed release. Thus, within a given time interval or under the influence of a temperature gradient, the particles release the labeled substrate into the medium, which then diffuses toward the filtration membrane and labels the target microorganisms. Such a cartridge allows for the combination of activation and labeling steps using a single cartridge.

[0058] Advantageously, the filtration device further comprises a cover which is clipped / adhesively bonded onto the support 6 once the filtering step has been performed. Once closed and before the detection step, such a filtration device according to the invention leaves the filtration membrane protected and can be left in an incubator for several days to allow microorganisms to grow on the surface of the filtration membrane, facilitating their identification.

[0059] Advantageously, the reagent may be in the form of a liquid, gel, or soaked pad.

[0060] Advantageously, the device also includes a space containing gas, allowing for the growth of microorganisms.

[0061] The present invention also relates to a method for microbiological analysis by solid phase cytometry using a filtration device according to the invention, which method of analysis comprises at least the following steps: - filtering the sample through a filtration membrane; - fluorescently labeling microorganisms that may be present in a biological sample; - detecting the labeled microorganisms; Including, The method is characterized in that it comprises at least one step according to which a cover glass is positioned above the upper surface of the filtration membrane of the filtration device.

[0062] According to one feature of the invention, the method includes a step of confirming the detection by fluorescence microscopy, which step is performed after the detecting step or after the labeling step.

[0063] According to one feature of the invention, the fluorescent labeling step comprises making the microorganisms fluorescent.

[0064] According to one feature of the invention, the fluorescent labeling step may be carried out using a fluorogenic substrate or reagent, or any other means making it possible to achieve fluorescence of the microorganisms.

[0065] According to one aspect of the invention, the sample is in liquid form so as to be filterable. Preferably, the sample is non-biological, such as wastewater, injectable chemicals, etc.

[0066] According to one aspect of the invention, a cover slip is positioned over the upper surface of the filtration membrane of the filtration device prior to the filtering step. After this step of positioning the cover slip, an additional step of adding interstitial fluid to the interstitial fluid gap of the filtration device is performed after the filtering step, immediately after the filtering step, or otherwise.

[0067] Alternatively, according to one aspect of the invention, a cover slip is positioned over the upper surface of the filtration membrane of the filtration device after the filtering step. After this cover slip positioning step, interstitial fluid is added to the interstitial space of the filtration device before the detecting step, immediately after the detecting step, or in other ways.

[0068] According to one feature of the invention, the cover slip can be securely fastened to the membrane by clipping at any time during the analytical method once the filtration funnel has been removed.

[0069] According to one feature of the invention, when a cover slip is positioned after the filtering step, the sample is injected directly onto the top surface of the filtering membrane through the filtering funnel.

[0070] According to one feature of the invention, when a cover slip is positioned prior to the filtering step, the sample is injected onto the upper surface of the filtering membrane through a port in the support for the filtering funnel.

[0071] According to one feature of the present invention, during sample filtration, depressions can be applied to the underside of the filtration membrane, whether or not a cover glass is present, making it possible to achieve uniform filtration across the entire surface of the filtration membrane, thereby improving the efficiency and speed of filtration.

[0072] Advantageously, if a cover slip is present during filtration, the suction effect causes the liquid form of the sample to fill the gaps before it passes through the pores of the filtration membrane.

[0073] According to one aspect of the invention, the method can further include the step of adding a counterstain solution, which is performed by filtering the solution after filtering the sample. As with filtering the sample, this step is performed through the top surface of the membrane either through a funnel, if a coverslip is present, or through an injection port.

[0074] This counterstaining step is an optional step that allows for the reduction of endogenous sample fluorescence and nonspecific labeling. Specifically, samples can correspond to various more or less complex products (solutions, suspensions, emulsions), possibly with prior preparation (centrifugation, mechanical lysis, chemical lysis) to make them filterable. Therefore, in addition to any fluorescently labeled microorganisms, the sample may contain intrinsically fluorescent particles or debris. Even if some particles are not naturally fluorescent, the exogenous labeling provided by the protocol may not be completely specific and may label non-microorganisms either by directly converting the fluorogenic substrate to a fluorophore at undesired sites (not microbial cells) or by the action of efflux pumps, which allow fluorophores generated inside microbial cells to escape through the membrane. Thus, the fluorophore labels the entire sample, reducing contrast. Therefore, the counterstaining solution masks / covers sites before the labeling step, preventing / obstructing fluorophore access.

[0075] According to one feature of the invention, the method may comprise a pre-labeling step prior to the labeling step, which pre-labeling step constitutes activation to reactivate the spores and ensure optimal enzyme activity.

[0076] According to one aspect of the invention, the activation and labeling steps can be performed with or without a coverslip present on the surface of the membrane.

[0077] According to one aspect of the invention, the interstitial volume is filled with fluid after the filtration and counterstaining steps and before the activation and labeling steps.

[0078] According to one feature of the invention, the gap is filled with interstitial fluid after the activation and labeling steps and before the detecting step.

[0079] According to one feature of the invention, interstitial fluid may be infused through a port in the device.

[0080] According to one feature of the invention, the gaps may be filled from the inner surface of the membrane via diffusion of liquid through the pores of the membrane.

[0081] According to one feature of the present invention, the optional verification step may be performed using an immersion microscope objective. Indeed, thanks to the presence of a cover glass above the filtration membrane, it is possible to use an immersion microscope objective with a very high numerical aperture (40x or more and NA≧0.6), and therefore with a very high collection power, which allows both to minimize the light dose received by the sample and to improve the image resolution. When using an immersion objective, it is necessary to use an immersion oil that is compatible with the cover glass material.

[0082] Alternatively, the verification step may be performed using an air objective, possibly corrected for the presence of a cover slip.

[0083] According to one feature of the invention, the air objective is a 60x objective with a 0.7 numerical aperture used with air, with a long working distance (1 mm or more) that allows focusing over most of the gap range, as well as compensation for the cover glass (variable compensation collar).

[0084] The invention will be better understood from the following description of embodiments according to the invention, given by way of non-limiting example and illustrated with reference to the accompanying schematic drawings, of which: [Brief explanation of the drawings]

[0085] [Figure 1] FIG. 1 is a diagram of a method for microbiological analysis by solid phase cytometry according to the prior art. [Figure 2] 1 is a cross-sectional view of a filtering device according to the present invention; [Figure 3] 1 is an exploded cross-sectional view of a filtering device according to the present invention; [Figure 4] 10 is a diagram of a possible embodiment with a gel interstitial layer and a desired thickness for detection. FIG. [Figure 5] FIG. 2 is a perspective view of a support and a portion of a filtration membrane of a filtration device. [Figure 6] 1 is a diagram of a method according to the invention; [Figure 7] 3 is an alternative embodiment of the method according to the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0086] The present invention will now be described in detail with reference to the above-listed figures.

[0087] A filtration device 1 according to the present 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 shown in particular in FIG. 2 , the filtration membrane 2 has an upper surface 2 a and a lower surface 2 b, and the biological sample is filtered from the upper surface 2 a to the lower surface 2 b. The filtration membrane is attached to a support 6. Ideally, the support 6 is arranged around the filtration membrane 2, as can be seen in FIG. 5 . In this example shown in FIG. 5 , the filtration membrane 2 is stretched and attached to the support 6.

[0088] The support 6 for the filtration membrane 2 has a lower housing 8 provided below the filtration membrane 2. The lower housing 8 is open to the lower surface 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.

[0089] As shown in FIGS. 2 and 3 , the filtration device 1 further includes a cover glass 3 arranged above the upper surface 2 a of the filtration membrane 2. As can be seen from the figures, the cover glass 3 is arranged to cover the entire upper surface 2 a of the filtration membrane 2, particularly to protect the filtration membrane 2. The cover glass 3 is transparent to enable analysis. The cover glass 3 is firmly fixed to the filtration membrane 2 while maintaining a gap 4. The gap 4 is formed between the cover glass 3 and the upper surface 2 a of the filtration membrane 2, and the gap 4 is shaped to receive interstitial fluid 5, as shown in FIG. 2 . The gap 4 also allows for the accommodation of a pre-filtration sample (not shown). The gap 4 is sealed when the cover glass 3 is positioned above the filtration membrane 2. Specifically, the filtration device 1 includes a seal 10 for blocking and sealing the gap 4, as can be seen particularly in FIGS. 2 and 4 .

[0090] Further details of the filtration device 1 will become apparent in light of the method description detailed herein.

[0091] The microbiological analysis method according to the invention is a method for analysis by solid phase cytometry.

[0092] To carry out this analysis method, a filtration device 1 as described above is used.

[0093] The first step 101 of the analytical method consists of filtering the sample to be analyzed. The second step 102 of the analytical method is to label microorganisms likely to be present in the biological sample, where labeling step 102 consists of labeling the microorganisms with a fluorogenic substrate. The analytical method includes a third step 103, detecting the labeled microorganisms. The analytical method includes a fourth, optional step of confirming detection 104 by fluorescence microscopy.

[0094] The method includes a positioning step 110 in which a cover glass 3 is positioned above the upper surface 2a of the filtration membrane 2 of the filtration device 1, and a step 112 in which interstitial fluid is added into the gap 4. Depending on the process steps, these two steps may be performed at different times.

[0095] For example, as shown in FIG. 6, step 110 of positioning the cover slip 3 occurs before filtration 101, and step 112 of adding occurs after filtration 101 or any step requiring filling of the gap 4, such as step 111 of introducing the sample or counterstaining step 105, as shown in FIG.

[0096] The sample introduction step 111, which consists of introducing the sample to the upper surface 2a of the filtration membrane 2, may be performed via a port 7 in the support 6 of the filtration device 1.

[0097] In the example shown in Figure 6, the cover slip 3 is positioned before filtration 101 (step 110), and the sample is introduced after positioning the cover slip 3 (step 111), which ensures optimal non-contamination of the biological sample. Following filtration, an optional counterstaining step 105 may be performed (this step is optional and is therefore indicated by a dotted line in Figure 6). When counterstaining step 105 is performed, the gap 4 is filled with a counterstaining solution, which is then filtered by the membrane. A step 112 of adding interstitial fluid is performed after counterstaining step 105. If a counterstaining step is not performed, the step 112 of adding interstitial fluid is performed immediately after filtration 101.

[0098] The step 112 of adding interstitial fluid comprises: firstly by immersing the filtration membrane 2 on its underside 2b with a cartridge 9 containing interstitial fluid 5, thus causing the interstitial fluid 5 in liquid form to rise by capillary action through the pores of the filtration membrane 2 and fill the gaps 4, by wetting the cover glass 3 with interstitial fluid 5 in liquid form before placing it on the filtration membrane 2, by inverting the pair formed by the filtration membrane 2 and the cover glass 3 to access the underside 2b of the filtration membrane 2 and allowing the interstitial fluid 5 in liquid form to settle by gravity into the gaps 4, Once the cover glass 3 is deposited (step 110), by injecting interstitial fluid 5 in liquid form through a port 7 in the support 6 of the filtration membrane 2, - by positioning the pair formed by the filter membrane 2 and the cover glass 3 vertically and injecting the interstitial fluid 5 in liquid form from bottom to top, releasing air bubbles; It may be performed in either of the following ways.

[0099] As shown in Figure 6, the next step is an optional pre-labeling step 106, which consists of activating any spores present in the sample to reactivate them and ensure optimal enzyme activity (this step is optional and therefore is shown in dotted lines in Figure 6).

[0100] The sample corresponds to a particular physiological state of the microorganisms in which the metabolism is severely reduced. Next comes the step 102 of labeling the microorganisms.

[0101] During the pre-labeling step 106 and the labeling step 102, functionalized cartridges 9 can be used as shown in Figure 3. Depending on the step, these functionalized cartridges 9 can contain reagents 12 or media 12 for spore activation, or cartridges for fluorescent labels, or cartridges for label inactivation. These cartridges 9 are inserted into the support 6 of the filtration device 1 so as to contact the lower surface 2b of the filtration membrane 2 on which the microorganisms of the biological sample are retained.

[0102] The pre-labeling step 106 and the labeling step 102 may also be performed using a single functionalized cartridge with two types of particles (A) and (B) with different release times. Thus, a labeling substrate may be initially diffused, followed by a reagent that "bleaches" and eliminates the labeling, such as a strong reducing agent, after a programmed labeling time. Such a device may use a single cartridge with culture gel 12, which allows all three operations: activation, labeling, and labeling termination.

[0103] Finally, the microorganisms trapped on the filtration membrane 2 are analyzed in two steps: a detection step 103 and a confirmation step 104 .

[0104] A microbial culture step can be added to increase the mass of microorganisms for more efficient detection; a functionalized cartridge of culture medium may be envisaged, or a filtration membrane 2 may also be envisaged. In this step (not shown), two types of cartridges may be envisaged, depending on whether the goal is to detect and grow aerobic or anaerobic bacteria. As shown in Figures 2, 3 and 4, the gas 11 present in the functionalized cartridge 9 contains oxygen in the aerobic case and does not contain oxygen in the anaerobic case.

[0105] The detecting step 103 is performed by laser scanning and / or fluorescence imaging in full field and / or image mosaic mode.

[0106] The detection verification step 104 may be performed using an air objective or an immersion objective, the cover slip 3 allowing the use of both of these objectives.

[0107] FIG. 4 shows a diagram of a possible embodiment with a gel gap layer and a desired thickness for detection, where O1 represents the focusing range of the objective lens, O2 represents the maximum working distance of the objective lens, and reference number 50 indicates the objective lens.

[0108] FIG. 7 also shows the method of the present invention, but with only the cover slip 3 added before analysis (steps 103 and 104).

[0109] Furthermore, prior to step 111 of introducing the biological sample, the biological sample can be prepared so that it is filterable.

[0110] Naturally, the invention is not limited to the embodiments described and shown in the attached drawings: modifications are still possible, in particular with regard to the configuration of the various elements or the substitution of technical equivalents, without departing from the scope of protection of the invention.

Claims

1. 1. A filtration device for use in solid phase cytometry configured to filter a sample potentially containing microorganisms to enable its analysis, said filtration device comprising: a filtration membrane configured to filter the biological sample, the filtration membrane having an upper surface and a lower surface; The filtering device further comprises at least a cover glass arranged above the upper surface of the filtration membrane, the cover glass being arranged to cover the entire upper surface of the membrane; a gap formed between the cover glass and the upper surface of the filtration membrane, the gap being shaped to receive a fluid; characterized in that it comprises Filtration device.

2. 10. The filtration device of claim 1, comprising interstitial fluid contained within said gaps, said fluid being a liquid or a gel.

3. 3. The filtration device of claim 1, wherein the cover glass is firmly secured to the filtration membrane while maintaining the gap.

4. 4. The filtration device of claim 1, further comprising at least one support on which the filtration membrane is positioned, the support having at least one port in communication with the upper surface of the membrane.

5. 5. The filtration device of claim 4, wherein the support comprises a lower housing formed below the filtration membrane, the lower housing being shaped to receive a functionalized cartridge intended to contact the lower surface of the membrane.

6. 6. The filtration device of claim 5, comprising at least one functionalized cartridge, possibly a cartridge for a reagent or culture medium or a medium for activating spores, or a cartridge for a fluorescent label, or a cartridge for inactivating a label and / or for keeping the filtration membrane submerged.

7. A method for microbiological analysis by solid phase cytometry using a filtration device according to any one of claims 1 to 6, said analysis method comprising at least the following steps: filtering the sample through the filtration membrane; fluorescently labeling the microorganisms possibly present in the biological sample; detecting the labeled microorganism; Including, the method comprising at least one step according to which the cover glass is positioned above the upper surface of the filtering membrane of the filtering device. method.

8. 8. The method for microbiological analysis by solid phase cytometry of claim 7, wherein, prior to the filtering step, the cover glass is positioned above the upper surface of the filtration membrane of the filtration device, and, after the filtering step, an adding step is performed of adding interstitial fluid into the gap of the filtration device.

9. 9. A method for microbiological analysis by solid phase cytometry according to claim 7 or 8, wherein the sample is injected onto the upper surface of the filtration membrane through one or more ports in the support of the filtration device.

10. 10. A method for microbiological analysis by solid phase cytometry according to any one of claims 7 to 9, wherein prior to the detecting step, the cover glass is positioned above the upper surface of the filtration membrane of the filtration device and the interstitial fluid is added into the gap of the filtration device.

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