Method and device for detecting at least one microorganism based on staining kinetics and detection support

Abstract

The invention relates to a method for detecting at least one microorganism (2) present in a sample to be analyzed (3) on a detection support (1, 21), the microorganism being visualized by means of at least one cell marker (4). The method comprises first taking an image (I0, I'0) before a step of controlled release of the marker (4), bringing the marker into contact with the microorganism, and taking an image (I0, I'0) at least once after the contacting step. i ), thereby detecting the microorganism according to the change of its staining dynamics by comparative analysis between different images that have been taken. The invention also relates to an automated detection device (14) capable of implementing the method, and a detection support that can be used in the method and is part of the device (14).

Description

[0001] The present invention relates to the field of detection of microorganisms such as bacteria, yeast and mold types in solid phase.

[0002] The invention is primarily applicable in the field of industrial microbiology, such as, but not limited to, the pharmaceutical, biotechnological, cosmetic or food processing industries, or in clinical or environmental microbiology.

[0003] The present invention more particularly relates to a device and method for the very early detection of microorganisms in a solid phase on a suitable support without the need for culturing and propagating said microorganisms.

[0004] Many techniques are currently implemented to detect contaminants, such as bacteria, in test samples.

[0005] The oldest and most conventional method involves placing the sample (optionally after filtration) on the surface of a nutrient agar growth medium that is more or less selective for one or more microorganisms.

[0006] The medium is then incubated at a suitable temperature to allow the desired microorganisms to grow, usually for up to several days, after which the visible colonies are counted "manually" by an operator.

[0007] This method, although highly sensitive and allowing the detection of a single viable culturable contaminant in the original sample, has the disadvantage of requiring long incubation times before colonies formed on the agar can be detected by the naked eye.

[0008] Instead of solid agar, another possible method involves inoculation in a liquid medium in which, after incubation, microbial growth is detected, for example by turbidimetry, whereby the absorbance measured is proportional to the concentration of the microorganisms present in the medium.

[0009] A disadvantage of liquid phase detection techniques is that they cannot detect 6 or 10 7 Therefore, the sample must be incubated again for a variable period of time to reach this cell concentration.

[0010] It should also be noted that under stress (e.g., heat stress) or due to the presence of inhibitors in the sample, some microorganisms may take an unusually long time to multiply, further delaying the point at which they can be detected using conventional culture techniques.

[0011] Another method known in the art involves performing a polymerase chain reaction (also known as PCR amplification) to amplify DNA or RNA sequences and thereby determine the presence of specific microorganisms in a sample.

[0012] However, these methods have the disadvantage of requiring multiple nucleic acid strands, i.e., multiple contaminating microorganisms in the sample, usually at least dozens of microorganisms. Therefore, such methods are not as sensitive as conventional culture techniques based on microbial growth.

[0013] Therefore, in recent years, many methods have been developed and refined in order to increase the speed of microbial detection while attempting to maintain the sensitivity of conventional growth-based techniques.

[0014] Most of these methods are based on detecting the fluorescence emitted by specific fluorescent chemicals, fluorophores or fluorescent dyes, and using different staining techniques to associate them with the cellular functions or structures (nucleic acids, proteins, enzymes, etc.) of the microorganisms to be detected.

[0015] These methods require specific detection equipment to be implemented, such as a microscope or cytometer for "DEFT" (Direct Epi-fluorescence Filter Technique).

[0016] Therefore, flow cytometry techniques are now routinely used to detect and enumerate microorganisms present in a sample.

[0017] In this technique, cells or particles, which can be dyed with fluorophores and suspended in a liquid, are isolated one by one in a stream of single cells. They are then passed at high speed through a light source, such as a beam from a laser.

[0018] The light diffused and emitted by the cells, which is fluorescent in the case of staining with a fluorophore and excitation at a given wavelength, is directed by a system of mirrors and filters that constitute the optics of the cytometer to a set of light detectors. These detectors are in particular equipped with photomultiplier tubes, which convert the optical signal into an electrical signal that can then be analyzed by a computer to generate relevant data about, for example, the physical characteristics of the cells.

[0019] Current commercially available flow cytometers are capable of analyzing hundreds of cells or “events” per second in real time, and some of these devices can be configured to allow sorting of cells or particles based on their optical properties.

[0020] However, detection of rare events is often complicated, whether flow cytometry or microscopy techniques are implemented.

[0021] In reality, flow cytometry has limited sensitivity, estimated at 10 in the best case. 2 Up to 10 3 cells / mL suspension and is matrix-dependent.

[0022] In the "DEFT" technique, a sample to be analyzed is filtered through a membrane which retains the microorganisms that may be contained in said sample.

[0023] The microorganisms are then stained with a fluorescent dye that interacts with DNA or RNA (eg, acridine orange) and counted by visual analysis of multiple fields of view of the membrane using an epifluorescence microscope.

[0024] In order to obtain representative results in fluorescence microscopy, it is estimated that at least 10 4 It should be noted that the counting was performed on only a small portion of the membrane (less than 10% of the total size) and that a uniform distribution of cells was assumed rather than determined.

[0025] However, in certain applications of industrial or clinical microbiology, early detection of the presence of a single contaminant or several contaminants in a sample may be particularly important. Indeed, the products manufactured and / or transformed in this type of industry are particularly sensitive, in particular due to their final destination (food, consumer health, etc.). Therefore, it is necessary to ensure as quickly as possible that their microbiological quality is impeccable or, if this is not the case, it is necessary to be able to quickly identify the source of contamination.

[0026] For these reasons in particular, solid phase cytometry, also known as laser scanning cytometry, which is suitable for filterable samples has been developed in recent years. In particular, it is the subject of European patent document EP 0713087.

[0027] In this method, after a first step of filtering the sample on a suitable membrane, any microorganisms potentially present on said membrane are subjected to a staining step using a fluorophore.

[0028] The entire surface of the membrane is then scanned by a laser beam to excite the fluorophores and any living microorganisms potentially present in the original sample to be analyzed are counted by measuring the emitted fluorescence.

[0029] An apparatus named ChemScan RDI (registered trademark) is used to perform this solid phase cytometry method.

[0030] However, currently commercially available equipment for performing solid phase cytometry techniques, as well as the necessary reagents, are extremely expensive and require a significant investment. As a result, not all laboratories have access to this technique.

[0031] Furthermore, once detection and enumeration of microorganisms has been performed, toxic effects of the reagents used on cellular metabolism can hinder subsequent culturing for identification of the microorganisms.

[0032] It should also be noted that this solid phase cell counting method is only applicable to filterable matrices, and the time required from placing the sample on the solid support to obtaining the count result of the microorganisms present on the solid support is relatively long. In fact, depending on the application, the time required has been estimated to be at least 90 minutes and up to 4 hours.

[0033] Finally, whether using flow cytometry or solid phase cell counting technology, the false positive rate of detection is still relatively high. In fact, non-microbial inert particles (such as dust) may also emit natural fluorescence, or even combine with fluorescent dyes to emit interfering fluorescence.

[0034] This constitutes a real drawback of the methods available to date: depending on the field of application, samples that prove to be false positives require further checks, which are very time-consuming and can also have significant financial consequences, in particular if the production line (e.g., that of biopharmaceuticals) has to be shut down during the aforementioned checks.

[0035] Typically, methods for staining cells with fluorophores require sample handling by qualified personnel and require handling of fluorescent reagents to bring them into contact with the microorganisms to be detected, which inevitably creates the risk of contaminating the sample.

[0036] It should also be noted that all of these operations, as well as work performed prior to sample preparation (particularly including filtration, activation, staining and washing, etc.), inevitably require a significant amount of processing time by the operator, which similarly delays the detection of potential contamination.

[0037] The present invention proposes a method and a device for extremely early (if necessary almost instantaneous) detection of one or more microorganisms potentially present in an original sample to be analyzed, thereby providing the possibility of at least partially overcoming the above-mentioned disadvantages of the prior art.

[0038] The methods and devices of the present invention are based on the detection and analysis of staining kinetics of potential microorganisms using cellular markers delivered in a controlled manner without the need for time-consuming manual manipulations that are particularly prone to sample contamination.

[0039] For this purpose, the present invention relates to a method for detecting, on a solid detection support, at least one microorganism of the bacteria, yeast or mold type present in a sample to be analyzed, said microorganism being visualized by means of at least one cell marker, said method comprising at least the following steps, in order:

[0040] a) placing the sample to be analyzed on the solid detection support;

[0041] b) irradiating the detection support at least once with light radiation capable of visualizing the at least one cell marker;

[0042] c) using an optical imaging device, with a field of view of at least 10 mm long by at least 10 mm wide as a target, to collect at least one image I0 of the first stage P0 of the sample on at least a portion of the detection support;

[0043] d) performing a controlled release of the at least one cell marker through the detection support, thereby bringing the marker into contact with the microorganism;

[0044] e) after the controlled release step of the marker, in at least one subsequent stage Pi, using the optical device to acquire an image I of each portion of the detection support for which an image has been acquired in stage P0 i (i ≥ 1), thereby detecting the at least one microorganism based on a change in the staining kinetics of the microorganism;

[0045] f) for each portion of the detection support, the image I0 acquired at P0 and at least one subsequent stage P i Each image I collected i A comparative analysis is performed between the samples and a conclusion is drawn as to whether at least one microorganism is present in the sample.

[0046] Preferably, each time at P0, P i During the image acquisition phase, the detection support is locally illuminated with optical radiation.

[0047] In a particularly advantageous example embodiment, the method of the present invention comprises at least the following steps, in order:

[0048] a1) placing the sample to be analyzed on the solid detection support;

[0049] b1) locally irradiating the detection support with light radiation capable of visualizing the at least one cell marker;

[0050] c1) simultaneously with the irradiation step b1), using an optical imaging device, with a field of view of at least 10 mm long by at least 10 mm wide as a target, collecting at least one image I0, I'0 of the first stage P0 of the sample on at least a portion of the detection support;

[0051] d1) performing a controlled release of the at least one cell marker through the detection support, thereby bringing the marker into contact with the microorganism;

[0052] e1) locally irradiating said detection support with said light radiation capable of visualizing said at least one cell marker within a period of less than 5 seconds after said controlled release step d1) of said cell marker;

[0053] f1) simultaneously with the irradiation step e1), after the controlled release step of the marker, in at least one subsequent phase P1, acquiring, by means of the optical device, an image I1, I'1 of each portion of the detection support for which an image was acquired in phase P0;

[0054] g1) locally irradiating the detection support again with the light radiation at least once;

[0055] h1) simultaneously with the irradiation step g1), in at least one second subsequent stage P2, acquiring, by means of the optical device, images I2, I'2 of each portion of the detection support for which images were acquired in stages P0 and P1, in order to detect said at least one microorganism according to changes in the staining kinetics of said microorganism;

[0056] i1) for each portion of the detection support, a comparative analysis is performed between the image I0, I'0 acquired at P0 and the images I1, I'1, I2, I'2 acquired at at least two subsequent stages P1 and P2, and a conclusion is drawn as to whether at least one microorganism is present in the sample.

[0057] Particular non-limiting features of the method according to the invention:

[0058] - subjecting the detection support to n displacements along different paths in order to spatially divide said detection support into n+1 parts, in at least two phases P0 and P i wherein n is an integer less than or equal to 20;

[0059] - in the image acquisition steps c) and e) or c1), f1) and h1), using an optical imaging device with a resolution of at least 20 megapixels, preferably greater than 100 megapixels;

[0060] - if in step f) or i1) it is concluded that at least one microorganism is present in the sample, enumerating the microorganisms present in the sample;

[0061] - the cell marker is a fluorescent marker;

[0062] - the cell marker released in a controlled manner in step d) or d1) is encapsulated in a solid form (e.g. powder form) in an encapsulating element, and the detection support comprises a solvent for dissolving the marker;

[0063] - the cell marker is encapsulated in the encapsulation element in liquid form;

[0064] - the encapsulating element is thermosensitive and in step d) or d1), the controlled release of the cell marker encapsulated in the thermosensitive encapsulating element is carried out by subjecting the thermosensitive encapsulating element to a temperature increase capable of melting the thermosensitive encapsulating element, for example increasing the temperature from ambient temperature to a maximum temperature of 45°C;

[0065] - delivering the cell marker in a liquid solution to a solid detection support containing the sample to be analyzed in a controlled manner;

[0066] - placing a volume of 10 μL to 2,000 μL of the sample to be analyzed directly on the detection support;

[0067] - filtering the sample to be analyzed through a filter membrane with a cut-off threshold ranging from 0.2 μm to 0.45 μm, preferably with a cut-off threshold equal to 0.3 μm, the filter membrane being directly located in the detection support.

[0068] The invention also relates to a method for distinguishing dust and microorganisms on a detection support that has received a sample to be analyzed, characterized in that:

[0069] - after each image acquisition, measuring the brightness of the detected illuminated pixels or pixel clusters;

[0070] - comparing the brightness of each illuminated pixel or pixel cluster detected in the different images;

[0071] - identifying pixels or pixel clusters whose brightness is substantially constant in at least two consecutive images during the staining kinetics;

[0072] - Pixels or pixel clusters that change in brightness during the staining kinetics are analyzed and counted.

[0073] The present invention also relates to a device for detecting at least one microorganism of the bacteria, yeast or mold type present in a sample to be analyzed, the device being capable of implementing the detection method of the present invention and comprising at least:

[0074] - a solid detection support intended to receive said sample to be analyzed which may contain at least one microorganism;

[0075] - an element storing at least one cell marker of said microorganism;

[0076] - an element for releasing the cell marker in a controlled manner so that the marker comes into contact with the sample to be analyzed;

[0077] - irradiating an element of said detection support;

[0078] - an optical device for acquiring an image of at least a portion of the detection support, the optical device having a field of view of at least 10 mm long by at least 10 mm wide;

[0079] - Elements for storing in a memory, comparing and analyzing the images acquired by said optical device, in order to detect said at least one microorganism potentially present in said sample, based on changes in the staining kinetics of said at least one microorganism.

[0080] Preferably, the device of the present invention comprises an element for displacing the detection support below the optical imaging device.

[0081] Advantageously, in the detection device of the present invention:

[0082] - the solid detection support comprises at least one filter membrane resting on a glass fiber support disc;

[0083] -The element for storing cell markers is included in the detection support and consists of a microcapsule layer formed by the cell markers encapsulated in the encapsulation element, and the microcapsule layer is arranged between the glass fiber support disk and the filter membrane; in this case, the element for releasing the cell markers in a controlled manner includes a heating element and a temperature control and regulation element, which is capable of generating a temperature increase inside the device and capable of melting the encapsulation element encapsulating the cell markers.

[0084] - the cell marker storage element consists of a reservoir of a liquid cell marker solution separate from the detection support, and the detection device comprises an element for transferring the cell marker from the storage element to the detection support; in this case, the element for releasing the cell marker in a controlled manner may comprise at least one flow-controlled pump;

[0085] - the optical imaging device has a focal depth of about 0.5 mm or equal to 0.5 mm;

[0086] - the optical imaging device is based on a CMOS sensor camera,

[0087] - the optical imaging device has a field of view of 25 mm x 25 mm;

[0088] The optical imaging device has a resolution of at least 20 megapixels, preferably greater than 100 megapixels.

[0089] The present invention also relates to a detection support that can be implemented in the detection method of the present invention or can form an integral part of the detection device of the present invention, the detection support consisting of at least the following: a rigid substrate, wherein at least one liquid feed line and at least one liquid suction and discharge line pass through the rigid substrate, the feed line extending by means of an outlet extending from the substrate and, on the one hand, passing through a first orifice opened in a rigid permeable support element above the substrate and, on the other hand, through a second orifice opened in a flexible support disk made of glass fiber, the disk being located on the rigid permeable support element and supporting a filter membrane capable of retaining microorganisms with a cutoff threshold of 0.2 μm to 0.45 μm, the detection support also comprising a clamping ring, which is fixed to be integrated with the substrate and holds the substrate, the rigid permeable support element, the support disk and the filter membrane together.

[0090] The invention has many advantages. On the one hand, the method of the invention is based on the evaluation of the staining kinetics of microorganisms, making it possible to detect the presence of one or more living microorganisms in a sample almost immediately, within a few minutes or tens of minutes at most after the sample is prepared, without the need for culturing, while distinguishing these microorganisms from potential artifacts, such as dust, etc., in an extremely reliable manner.

[0091] On the other hand, the present invention requires only minimal handling of the sample and in any case does not require any handling of cell markers, which is complex, requires qualified personnel and entails a non-negligible risk of contaminating the sample.

[0092] Other features and advantages of the present invention will become apparent from the following detailed description of non-limiting embodiments of the present invention given with reference to the accompanying drawings, in which:

[0093] - Figure 1 Schematically showing different steps of two specific embodiments of placing a sample on a solid detection support, which steps can be performed in the method of detecting at least one microorganism of the present invention;

[0094] - Figure 2 The specific method for performing each step of the detection method of the present invention is schematically shown, and particularly comprises: placing a sample on a detection support, acquiring a P0 image before staining the sample, then sequentially acquiring a plurality of images from P1 to P4 after releasing the cell marker in a controlled manner, and performing comparative analysis on each image to generate an analysis result;

[0095] - Figure 3A and 3BSchematically showing two non-limiting example embodiments of a detection support that can be used in the method or device of the present invention, respectively, in a fully exploded view and in a perspective view through a middle section;

[0096] - Figure 4A and 4B A complete exploded view and a perspective view through a middle section of an embodiment of a detection support that can be used in the method or device of the present invention are schematically shown, respectively, Figure 3A and 3B The embodiments shown are different and are particularly preferred;

[0097] - Figure 5 A specific embodiment of the detection method of the present invention is shown, wherein a sample to be tested is brought into contact with an encapsulated cell marker in the form of a microcapsule, and the cell marker is released in a controlled manner under the action of an external stimulus, thereby allowing the detection of microorganisms potentially present in the sample;

[0098] - Figure 6 shows a series of image acquisitions of a Bacillus subtilis microcolony after a cultivation phase, the left image I0 having been acquired before the release of the cell marker, while the other images I1 to I5 have been acquired at regular intervals after the controlled release of said marker, thus allowing the staining kinetics of the Bacillus subtilis colony to be assessed through these consecutive image acquisitions;

[0099] - Figure 7 A series of five consecutive image acquisitions, indicated by the symbols I'0 to I'4, of a detection support on which, on the one hand, dust is present and, on the other hand, cells to be detected in a test sample, in this case yeast belonging to the species Candida albicans of the genus Candida, are present. According to the method of the invention, the support is photographed before releasing the cell marker (image I'0) and after releasing this marker in a controlled manner (images I'1 to I'4); the figure also shows three curves Pr1, Pr2, Pr3, Pr4, Pr5, Pr6, Pr7, Pr8, Pr9, Pr10, Pr11, Pr12, Pr13, Pr14, Pr15, Pr16, Pr17, Pr18, Pr19, Pr20, Pr21 2a and Pr 2b , which, in parallel with the acquisition of images I'0 to I'4, illustrate the staining of dust and yeast (Pr 2a and Pr 2b ) over time (shown as a grayscale representing the brightness of a pixel and coded from 0 (black) to 255 (white)), especially with the successive acquisition of images (20 seconds between two consecutive image acquisitions), showing that the light intensity emitted by the stained dust suddenly increases to a strong and stable intensity over time, while the light intensity emitted by the yeast gradually increases as the marker is assimilated by the cell until it reaches a peak of maximum intensity and then decreases in light intensity (also known as photobleaching).

[0100] As shown in the attached figure Figures 1 to 7 As shown, the present invention firstly relates to a method for detecting at least one microorganism 2 or a plurality of microorganisms 2 that are potentially but not necessarily present in a sample 3 to be tested on a solid detection support 1, 21.

[0101] In the detection method of the invention, the one or more microorganisms 2 (whether present in the form of single cells or in the form of microcolonies) are brought into contact with at least one cell marker 4 after a preliminary cultivation phase.

[0102] In the description below, the term "cell marker" should be understood as a substance that can visualize microorganisms potentially present in the original sample to be tested by luminescence, in particular by fluorescence, optionally by bioluminescence, or even by staining. It should be understood that the cell marker can preferably be a fluorescent marker that can only visualize living microorganisms or can distinguish between living microorganisms and non-living microorganisms, or can even only stain certain pathogenic microorganisms of interest.

[0103] It should be noted that the sample 3 to be tested may originate from, for example but not limited to, the pharmaceutical or food processing industry, or may consist of samples of human origin or samples of environmental origin in clinical or medical microbiology, and the microorganisms 2 to be detected in the sample 3 are mainly of the bacterial, yeast or mold type.

[0104] In the first step a) of the detection method of the present invention, the sample 3 is placed on the solid detection support 1 , 21 , and the solid detection support 1 , 21 preferably includes at least one filter membrane 5 , 25 .

[0105] exist Figure 1 In an exemplary embodiment of step a) of the method of the invention shown at the bottom, the sample 3 to be analyzed consists of a liquid matrix, the volume of which can be between 1 mL and 500 mL, which is filtered through a detection support 1 comprising at least one filter membrane 5, said support 1 being located on a device D suitable for filtration.

[0106] Said membrane 5 has a sufficient cut-off threshold to retain any microorganisms 2 potentially present in the sample 3 and to remove mainly residual liquid of said sample 3 on the one hand and optionally impurities contained therein having a size smaller than the cut-off threshold on the other hand.

[0107] Therefore, whatever the embodiment chosen for carrying out the first step a) of the method of the invention, the cut-off threshold of said membrane 5,25 is preferably between 0.20 and 0.45 μm. Even more preferably, the cut-off threshold of this membrane 5,25 is equal to or about 0.30 μm.

[0108] Such a cut-off threshold makes it possible to retain on the surface of the membrane 5 , 25 all microorganisms 2 of the bacteria, yeast and mold type which may be present in the sample 3 and whose sizes vary between about 0.5 and about 10 μm.

[0109] However, it should be noted that the use of such a cut-off threshold not only facilitates the separation of the microorganisms 2 from the rest of the sample 3, but also improves the retention of the majority of inert particles (such as dust) that were also initially present in said sample 3.

[0110] In a second exemplary embodiment of step a) of the method of the present invention (not shown in the figure), a liquid sample 3 with a volume between 1 and 500 mL is filtered through a filter membrane 5 on a suitable filtration device D, wherein the cut-off threshold of the membrane 5 is between 0.20 μm and 0.45 μm, preferably equal to 0.3 μm.

[0111] Once the sample 3 has been filtered, the filter membrane 5 is removed from the filtration device D to be placed on the detection device 1 .

[0112] The detection support 1 can also be supplemented with at least one support disk 13 made of a permeable material, such as glass fiber.

[0113] Attached Figure 3A and 3B , in particular, such a support disc 13 is shown, on which a filter membrane 5 which has been removed from the device D optionally rests, thereby forming a solid detection support 1 in this exemplary embodiment.

[0114] In a third exemplary embodiment applied to a smaller volume of sample 3 (typically 10 to 2,000 μL or less), the sample 3 is directly inoculated on a detection support 1, 21 comprising at least a membrane 5, 25 and a support disk 13, 23, such as Figure 1 as shown in the upper part of .

[0115] Then, preferably, the detection support 1 is inserted into a fully automatic detection device 14 capable of performing the method of the present invention. The device 14 will be described in more detail below.

[0116] It should be noted that in a particularly advantageous exemplary embodiment, the detection support 21 used in step a) of placing the sample 3 is configured to preferably allow a small volume of sample 3 of 10 to 2,000 μL to be placed on the filter membrane 25, or a larger volume of sample 3 greater than 2,000 μL (typically up to 100 mL) to be directly filtered without the need for a filtration device D dedicated to this operation and external to the detection device 14.

[0117] Even in the case of small volumes being placed on the filter membrane, the sample 3 can be filtered in an automated manner in the detection device 14 .

[0118] In the following description, reference will be made to Figure 4A and 4B The detection support 21 is described in detail.

[0119] It should be noted that in a conventional manner, these operations of processing and placing the sample 3 on the support 1, 21 can preferably be performed by an operator in a sterile atmosphere, for example by performing the operations in a biological safety cabinet BSC, such as Figure 1 Visible in.

[0120] In the attached Figure 2 In step b) of the detection method of the invention shown in , the detection support 1 , 21 comprising the sample 3 is irradiated at least once with light radiation 6 suitable for observing or visualizing the cell marker 4 .

[0121] In fact, in a specific exemplary embodiment of the detection method of the present invention, the detection support 1, 21 can be continuously irradiated throughout the execution of the method. However, as will be described in detail in the following description, it is more preferred to locally irradiate the detection support 1, 21 at the same time or almost at the same time as each image acquisition.

[0122] After the illumination step b) and in step c), at least one image I0, I'0 of the sample 3 is acquired in a first phase P0 on at least a portion of the detection support 1, 21. Two examples of images I0, I'0 can be seen in Figure 6 and Figure 7 middle.

[0123] The image acquisition performed during the method of the present invention is preferably performed with the aid of an optical imaging device 7 having an optical resolution greater than or equal to 20 megapixels, preferably greater than 100 megapixels, thereby enabling detection of stained microorganisms having a size of at least 0.5 μm on a predetermined surface.

[0124] It is particularly important that the optical device 7 actually has a relatively wide field of view, typically at least 10 mm long by at least 10 mm wide, and even more preferably 25×25 mm, so that for example the entire detection support 1 , 21 can be acquired in a single image.

[0125] It should also be noted that, preferably, the optical means 7 have a focal depth of + / - 0.5 mm in order to overcome any potential flatness defects of the membrane 5 , 25 of the detection support 1 , 21 .

[0126] Therefore, preferably, in step c), a single image I0 , I′0 of the entire surface of the detection support 1 , 21 is acquired.

[0127] In the method of the present invention, when the size of the detection support 1, 21 is too large to be viewed by a single image acquisition, and in order to be able to observe a wider field of view without investing in non-standard optical devices (which would result in a much more unfavorable economic situation), it is also possible to consider causing the detection support 1, 21 to be displaced n times along different paths, where n is an integer greater than 1 and less than or equal to 20, and even more preferably less than or equal to 10.

[0128] By means of these n displacements along different paths, the detection support 1, 21 is spatially divided into n+1 parts, at the image acquisition time, in particular at P0 and at least one subsequent phase P described in more detail below. i In the example, each such part is the object of at least one image acquisition, so the sum of the image acquisitions of these different parts makes it possible to obtain the image of each different stage P0, P i Observe the entire detection support1,21.

[0129] Indeed, the ability to view the entire support 1 , 21 , either once or multiple times, is of particular interest in order to ensure the detection of small numbers of bacteria or even single bacteria initially present in the sample 3 to be tested.

[0130] After acquisition of the image I0 , I′0 , step d) is performed to release at least one cell marker 4 in a controlled manner through the detection support 1 , 21 so as to bring said marker 4 into contact with microorganisms potentially present in the original sample 3 and placed on the support 1 , 21 .

[0131] A specific embodiment of the controlled release step d) is more specifically shown in Figure 5 middle.

[0132] Preferably, the cell marker 4 implemented in the method of the present invention is a fluorescent marker.

[0133] The person skilled in the art can advantageously select the marker 4 from all cell markers that are familiar to him and commonly used (eg commonly used in current cell counting techniques).

[0134] Thus, the cell marker can be selected, for example, from the following markers: DNA markers, such as acridine orange, DAPI (4',6-diamidino-2-phenylindole), PI (propidium iodide), DRAQ7* (deep red anthraquinone 7), 7-AAD (7-aminoactinomycin D), TO-PRO-1*, TO-PRO-3*, SYTOX*, LDS 751, ethidium bromide, eosin-5'-maleimide (EMA), amine-reactive markers, such as "Zombie Fixable*", "Fixable Viability Dye eFluor*", "LIVE / DEADFixable dead*", "Viobility Fixable*", "Horizon Fixable Viability*", "Ghost*", esterase activity markers, such as calcein AM (acetoxymethyl), cFDA (carboxyfluorescein diacetate), "CellTrackerGreen CMFDA*”, redox labels such as resazurin or tetrazolium salts, or cell markers in kits such as the “LIVE / DEADViability*” kit, the reagent names followed by an asterisk mentioned in this paragraph are registered trademarks.

[0135] Combinations of multiple cell markers from the above-mentioned cell markers may also be used.

[0136] It should be noted that in case fluorescent cell markers 4 are used in the method of the invention, the optical radiation 6 is selected from a laser beam and one or more light emitting diodes, the latter being particularly preferred.

[0137] In an alternative embodiment of the method, in step d), the one or more potential microorganisms 2 present may also be brought into contact with at least one non-fluorescent but visibly detectable cell marker 4 .

[0138] The cell marker 4 may further consist of a bioluminescent marker, and it should be understood that in this case, the method for detecting microorganisms of the present invention is implemented by removing one or more steps of irradiating the detection support 1, 21. In other words, except for the irradiation support, all other steps, alternative embodiments and exemplary embodiments of the method of the present invention described in detail in this specification are applicable to the method for detecting microorganisms using bioluminescence (if proven to be applicable and feasible), of course, which uses a suitable bioluminescent marker.

[0139] In any case, it is particularly advantageous that, in a first precise and non-limiting embodiment of the controlled release step d), the cell marker 4 to be released in a controlled manner in the method of the invention is encapsulated in an encapsulation element 8, thereby advantageously forming microcapsules, microbeads or microspheres 9 having a diameter of, for example, about 100 μm.

[0140] Preferably, the encapsulating element 8 is sensitive to the application of an external stimulus 10 , causing it to rupture and release the cell marker 4 .

[0141] Therefore, preferably, the encapsulating element 8 is formed of a thermosensitive lipid wax, and the release of the encapsulated marker 4 is achieved by gradually applying an elevated temperature to the encapsulating element 8 so that the encapsulating element 8 melts and releases the marker 4 to contact the potential microorganism 2 to be detected.

[0142] In this case, the temperature to which the heat-sensitive encapsulating element 8 is subjected may be gradually increased from the ambient temperature to a temperature not exceeding 45° C., exceeding this threshold value which may destroy certain microorganisms 2 .

[0143] In the present invention, the release of the cell marker 4 is preferably performed in an automated manner, thereby freeing it from any manual operation that constitutes a source of contamination, and is controlled so that the release occurs in sufficient amounts of marker 4 and with correct kinetics to stain the microorganisms 2 efficiently.

[0144] According to a first alternative embodiment, the cell marker 4 is encapsulated in liquid form, so that the microcapsule 9 contains, in addition to said marker 4, a solvent for dissolving it.

[0145] In a second alternative embodiment, the cell marker 4 is encapsulated in solid form, in particular in powder form. After the cell marker 4 is released, it is placed in the presence of a suitable dissolving solvent (eg water), so that staining of the microorganisms 2 can occur.

[0146] Preferably, in this alternative embodiment, the solvent for dissolving the solid cell marker 4 is contained in a glass fiber support disk 13 and the filter membrane 5 through which the sample 3 passes is arranged above said support disk 13 .

[0147] Thus, once the filter membrane 5 has been placed over the support disc 13 (after external filtration of the sample 3), or once the sample 3 has been placed and filtered directly on the filter membrane 5 in the detection support 1, whatever the volume of the sample 3, in the case where the layer of microcapsules 9 containing the cell markers 4 is located between the membrane 5 and the disc 13, the support disc 13 will be impregnated with sufficient solvent to allow the cell markers 4 to dissolve when they are released from their capsules 9 (for example by heating). This example is in Figure 3B Shown in.

[0148] exist Figure 3B In the configuration shown, the support disk 13 may also be impregnated with a suitable solvent and maintained at a temperature capable of melting the encapsulating layer 8, thereby releasing the encapsulated cell marker 4 in solid powder form and simultaneously dissolving it to reveal the potential presence of the microorganisms 2 on the membrane 5.

[0149] In any case, when a layer of microcapsules 9 containing cell markers 4 in solid or liquid form is located above the support disk 13, its main function, after rupture of said microcapsules 9, is to homogenize by diffusion the distribution of said markers 4 below the membrane 5, which is itself placed above the layer of microcapsules 9. Thus, the method of the invention will visualize each microorganism 2 that may be present at said membrane 5.

[0150] It should also be noted that in this embodiment of step d), and when step a) of placing a small volume (10 to 2,000 μL) of sample 3 is directly performed on the solid detection support 1 (which can also be formed by at least one supporting plate 13 of a layer surrounding the microcapsule 9 and the filter membrane 5), the supporting plate 13 also has the function of supporting the microcapsule 9 to prevent the microcapsule from being discharged together with the filtrate during the placement process.

[0151] exist Figure 3A In the method, a layer of microcapsules 9 is placed underneath a support disk 13, for example made of glass fiber, and when the cell markers 4 are released in a controlled manner, the disk 13 ensures capillary diffusion of the microcapsules 9 or cell markers 4 and a uniform distribution of these diffused microcapsules 9 underneath the filter membrane 5.

[0152] The embodiment of releasing the cell marker 4 in a controlled manner by destroying the encapsulating element 8 containing said marker 4, although proving to be particularly interesting, cannot be regarded as the only embodiment of step d) of the method according to the invention.

[0153] In fact, in a second embodiment of this step d), the cell marker 4 is directly delivered in the form of a liquid solution and in a controlled, automated manner, for example at a flow rate controlled by appropriate means (e.g. a flow control pump), so that it comes into contact with one or more microorganisms 2 present on the surface of the filter membrane 5, 25 of the detection support 1, 21.

[0154] In this case, whatever mode is chosen to bring the cell marker 4 into contact with one or more microorganisms 2 in a controlled manner, the marker 4 (whether it is preferably fluorescent or bioluminescent or other) preferably has minimal toxicity to the microorganisms 2 potentially present on the membrane 5.

[0155] Indeed, if contamination of the sample 3 is detected during the implementation of the method and in certain applications, in particular in the field of industrial pharmaceuticals, the ability to subsequently identify the detected microorganisms 2 may be of particular interest.

[0156] therefore, Figure 2 This identification step 12 shown can be carried out by culturing the sample 3 after detection, in particular by removing the filter 5 , 25 and placing it on a suitable nutrient agar to multiply the microorganisms, and then performing conventional identification methods well known to those skilled in the art, such as mass spectrometry.

[0157] In the method of the invention for detecting one or more microorganisms, at the end of the step of releasing at least one cell marker 4 in a controlled manner, in step e) and in at least one stage P0 after stage P1 i In the process, at least one image I is collected. i , where i is an integer greater than or equal to 1.

[0158] Figure 6 and Figure 7 Successive images I0 to I5 and I′0 to I′4 acquired during phase P0 and during a number of subsequent phases after P0 are shown, respectively.

[0159] Where appropriate, in at least one subsequent phase P i Each image I collected in i Divided into multiple parts of the detection support 1,21.

[0160] Such continuous image acquisition, i.e., acquiring one image (e.g., I0) before staining the microorganism 2 by controlled release of the cell marker 4, and acquiring at least one other image (I0) after the release. i ), making it possible to characterize the staining kinetics of these microorganisms 2, i.e. the changes in the behavior of said microorganisms, in particular, preferably changes in fluorescence, optionally changes in bioluminescence, which correspond to the first acquisition of the image I0 (without the marker 4) and to at least the second acquisition of the image I after the release of the marker. i Assimilation of marker 4 by microorganisms.

[0161] This makes it possible to distinguish the behavior of inert particles (eg dust which tends to fluoresce, for example naturally) from the behavior of microorganisms 2 which are effectively stained when the method is performed.

[0162] In fact, under the premise of using fluorescent cell markers 4 to visualize potential microorganisms 2, in the image I0 before the controlled release of the marker 4 and in at least one image I after the release, iInert particles or dust which naturally fluoresce and remain on the filter membrane 5 will be visible and detected, whereas the microorganisms 2 will only be visible in the image I i It is seen and detected in, in particular in, image I1 or I'1.

[0163] In the subsequent step f), the images I0 and I i , in particular at least I1 or I′1 is subjected to a comparative analysis, which will lead to a conclusion as to whether the microorganism 2 is present in the original sample 3 .

[0164] Considering the premise that the detection supports 1, 21 undergo n displacements along different paths during stage P0, i During this period, the support 1,21 undergoes n identical displacements again, so that at P0 and P i The images I0 and I collected respectively i The comparison can be performed in a subsequent comparative analysis step (step f)).

[0165] Furthermore, similar logic applies to subsequent images (if any).

[0166] In fact, Figure 6 and Figure 7 In a preferred embodiment shown, after the first acquisition of images I1, I'1 during the first subsequent stage P1, after a variable time, about a few seconds to tens of seconds, for example 10 to 30 seconds, images I2, I'2 are acquired again for each part of the support 1 whose images were acquired during stages P0 and P1.

[0167] Therefore, in the detection method of the present invention, after step d) of controlled release of the cell marker 4 and before step f) of comparative analysis of the images I0, I'0; I1, I'1; I2, I'2, etc., it is appropriate and advantageous to repeatedly collect the images I i Step e), image I i The continuous collection interval is 10 to 30 seconds (preferably equal to 20 seconds).

[0168] It goes without saying that each image is stored by the device 14 in a memory for subsequent processing operations, in particular for comparison.

[0169] Preferably, before the comparative analysis step f), step e) of the method is repeated three times in succession, so that during the subsequent image acquisition stages P1, P2 and P3, three images I1, I'1; I2, I'2; I3, I'3 are generated for each part of the detection support 1, 21 whose image has been acquired at P0.

[0170] Even more preferably, for each portion of the detection support 1, 21 that has been viewed at stage P0, five images from I1 to I5 or from I'1 to I'5 are generated (but image I'5 is not Figure 7 ).

[0171] Therefore, after placing the sample 3 on the detection support 1, 21 and also after the first acquisition of the image I0 in the stage P0, by releasing the cell marker 4 in a particularly original manner, it is assumed that the microorganisms 2 present in the sample 3 will be stained within a short time (about a few minutes), preferably as early as the single cell marker stage, which corresponds to the kinetics of the assimilation reaction of the microorganism 2 to the cell marker 4, thereby obtaining a rapid detection result by continuously acquiring images I0, I'0; I1, I'1, etc. of the stained supports 1, 2 during the various stages P0, P1, etc.

[0172] Depending on the nature of the cell marker 4 used in step d) of the method, the biochemical reaction of its assimilation by the microorganism 2 may in particular be an enzymatic reaction or a redox reaction, or even a reaction which inserts the marker between nucleic acid bases.

[0173] Characterization of the staining kinetics of microorganisms 2 potentially present in the sample 3 to be tested also makes it possible to quickly distinguish the behavior of inert particles or dust prone to naturally fluorescing from that of said microorganisms 2 , thus significantly reducing false positives compared to systems proposed in the prior art.

[0174] It is also clear from the above that implementation of the method of the invention allows the detection of single contaminants that may be contained in the original sample 3 , corresponding to CFUs (ie colony forming units, according to the terminology used by those skilled in the art).

[0175] The image I0; I'0 acquired during the first phase P0 and at least the image I i At the end of step f) of comparative analysis (eg images I1 ; I′ 1 ), a conclusion is drawn as to whether the sample 3 is contaminated, as already mentioned above in the description and which is advantageously achieved with the aid of an image detection and processing algorithm 11 .

[0176] It should also be noted that when the presence of at least one contaminating microorganism 2 is detected in analysis step f), a step g) of counting one or more microorganisms 2 present in the sample 3 and on the detection support 1 , 21 may be performed.

[0177] This optional step g) can also be performed by algorithm 11.

[0178] Therefore, the algorithm 11 must in particular allow the presence of microorganisms 2 in images I1, I'1, I2, I'2, etc. to be detected quickly and as accurately as possible by evaluating the changes in the behavior of said microorganisms 2 over time and by distinguishing them from any potential artifacts (in particular dust), and must then allow these microorganisms 2 to be counted.

[0179] Furthermore, the algorithm 11 is preferably capable of detecting elements (dust, microorganisms, etc.) having a size approximately equal to or smaller than the resolution of the sensor of the optical imaging device 7 .

[0180] This is one of the main advantages of the present invention: using a method based on the kinetics of assimilation of a cellular marker 4 by a microorganism 2, the optical resolution requirement is overcome to a certain extent.

[0181] In fact, the fluorescence intensity changes caused by the dyeing dynamics can be detected on a single pixel. Therefore, the size of the microorganism 2 does not have to be larger than the size of the pixel. In some cases, the light intensity emitted by a microorganism 2 that is smaller than a pixel is just enough to illuminate the pixel. Although this intensity may not be enough to detect the microorganism 2 with certainty, the intensity change during the dyeing may be sufficient for its detection.

[0182] Preferably, after the controlled release of the cell marker 4, an image acquisition I of the detection support 1, 21 is performed. i In step e), the time delay after releasing the marker 4 is usually 20 to 300 seconds, preferably 80 to 160 seconds in the case of staining with, for example, acridine orange.

[0183] This time delay after the controlled release of the marker 4, and therefore after the contact of said marker 4 with the microorganisms 2 potentially present on the detection support (after which the image I is acquired) i ) makes it possible to ensure that the marker 4 has been correctly assimilated by the microorganism 2.

[0184] In a very preferred exemplary embodiment, each image acquisition (including the initial acquisition of images I0, I'0 in step b in the first stage P0) is performed, and also in at least one subsequent stage P i The image I i Simultaneously or almost simultaneously with the collection of the cell markers 4, the detection support 1,21 is locally irradiated with light radiation 6 capable of visualizing the cell markers 4.

[0185] In other words, during the image acquisition phases P0 and P i In addition, the detection support 1,21 may not be irradiated with optical radiation 6.

[0186] It should be noted that, preferably, the method of the present invention comprises at least the following steps, in order:

[0187] a1) placing the sample 3 to be analyzed on the solid detection support 1, 21;

[0188] b1) locally irradiating the detection support 1, 21 with light radiation 6 capable of visualizing the at least one cell marker 4;

[0189] c1) simultaneously with the irradiation step b1), acquiring at least one image I0, I'0 of a first phase P0 of said sample 3 on at least a portion of said detection support 1, 21 using an optical imaging device 7 having a resolution at least equal to 20 megapixels and a field of view of at least 10 mm long and at least 10 mm wide;

[0190] d1) performing controlled release of the cell marker 4 or a series of cell markers 4 or reagents through the detection support 1, 21, so that the marker 4 is in contact with the microorganism 2;

[0191] e1) locally irradiating said detection support 1, 21 with said light radiation 6 capable of visualizing said at least one cell marker 4 within a period of less than 5 seconds after step d1) of controlled release of said cell marker 4;

[0192] f1) simultaneously with the irradiation step e1), after the controlled release step of the marker 4, in at least one subsequent phase P1, acquiring an image I1, I'1 of each portion of the detection support 1, 21 for which an image was acquired in phase P0;

[0193] g1) locally irradiating the detection support 1, 21 again at least once with the light radiation 6;

[0194] h1) simultaneously with the irradiation step g1), in at least one second subsequent phase P2, acquiring an image I2, I'2 for each portion of the detection support 1, 21 for which an image I0, I'0 was acquired in phase P0 and an image I1, I'1 was acquired in phase P1, so as to detect at least one microorganism 2 according to changes in the staining kinetics of the microorganism 2;

[0195] i1) For each portion of the detection support 1, 21, a comparative analysis is performed between the image I0, I'0 acquired at P0 and the images I1, I'1; I2, I'2 acquired at at least two subsequent stages P1 and P2, and a conclusion is drawn as to whether at least one microorganism 2 is present in the sample 3.

[0196] Steps g1) and h1) of acquiring the images I2, I'2 are preferably performed within 20 to 300 seconds, preferably 80 to 160 seconds, after step f1) of acquiring the images I1, I'1.

[0197] It should be noted that the alternative embodiments or preferred examples of steps a) to f) described above in the description also apply to the corresponding steps a1) to i1) of the method presented in the above paragraphs.

[0198] This approach is of particular interest because, on the one hand, it can effectively distinguish between:

[0199] - a first type of inert particles or dust, which naturally emit, for example, fluorescence or bioluminescence, and which will be observed in the images I0, I'0 acquired during the first phase P0 before the controlled release step d1) of the cell marker 4;

[0200] - a second type of inert particles or dust, which are not autofluorescent (and therefore not visible in images I0, I'0), but which quickly absorb the cell marker 4 after the controlled release of the cell marker 4, and which are therefore clearly visible in images I1, I'1;

[0201] - one or more microorganisms 2 potentially present in the original sample 3 to be analyzed, which are not visible in images I0, I'0, but can begin to appear in images I1, I'1 at low light intensity, and then become clearly visible in images I2, I'2 and potential subsequent images at higher light intensity. It will be understood that steps g1) and h1) can be repeated as many times as necessary to acquire images in subsequent stages P2, P3, etc.

[0202] Figure 7 A series of acquisitions of images I'0 to I'4 are shown, and some visible elements in these images (in particular images I'1 to I'4) are compared with the curves Pr1, Pr 2a and Pr 2b Matching, these curves show the variation with time of the light intensity of the element in question after the moment (abscissa 0) when the cell marker 4 is released in a controlled manner and comes into contact with the element in question (in this case dust and Candida albicans yeast present in the sample 3 and on the solid detection support 1, 21).

[0203] There are no visible elements in the image I'0 acquired before the controlled release step of the cell marker 4. This image I'0 therefore shows that there is no naturally luminescent dust in the original sample 3.

[0204] In the following images I'1 to I'4, some elements present in the sample 3 after staining can be seen with a strong and substantially constant light intensity over time. In this case, the comparison between the consecutive images I'1 to I'4 leads to the conclusion that this is dust that has absorbed the cell markers, and its curve Pr1 is mapped in association with the identified light points.

[0205] In the successive images I'1 to I'4 after staining, other elements can also be seen, in this case microorganisms, whose light intensity is shown in the curve Pr 2a and Pr 2b The light intensity gradually increases with time until it reaches a peak intensity, which corresponds to the gradual assimilation of the cell marker 4 by the microorganism 2 (in this case, the Candida albicans yeast), after which the light intensity also decreases in a gradual manner, which corresponds to the photobleaching phenomenon.

[0206] It should be noted that when naturally luminescent inert particles (eg dust) are placed on the detection support 1, 21 in the first step a1) of the method according to the invention, they will be present in the image I'0 and in all subsequent images I' i Therefore, by implementing the detection method of the present invention, this dust can also be distinguished from microorganisms 2.

[0207] The invention also relates to a device 14 for automatically detecting at least one microorganism 2 of the bacteria, yeast or mold type potentially present in a sample 3 to be tested and capable of executing the steps of the above-mentioned detection method.

[0208] Advantageously, the device 14 of the present invention comprises at least:

[0209] - a solid test support 1, 21 for receiving said sample 3 to be analyzed which may contain at least one microorganism 2. This support 1, 21 preferably consists of at least one filter membrane 5, 25 through which the sample 3 passes, and a support plate 13, 23 on which the membrane 5, 25 rests.

[0210] exist Figure 3A and 3B In the embodiment shown, the detection support 1 can be supplemented with a protective cover 16 and an element for holding all the elements of the support 1 on a rigid support plate 18, which holding element can be in the form of a hoop 17.

[0211] In another embodiment, Figure 4A and 4B More specifically, the detection support 21 comprising a filter membrane 25 resting on a glass fiber support disk 23 is supplemented with:

[0212] · A support element 24, which is rigid and in particular made of plastic material and is permeable by the presence of a plurality of circular perforations and / or oblong perforations, said rigid support element 24 being also referred to as drainer 24 in the description below; the latter having a shape and size similar or somewhat similar to that of the support plate 23 and the filter membrane 25.

[0213] a rigid substrate 26, made of, for example, a plastic material, through which, on the one hand, a feed line 26a for feeding a liquid (in this case, the cell marker 4) passes, said line 26a extending by means of an outlet 26b extending from said substrate 26 and passing through a first orifice 24a opened in the drainer 24 and a second orifice 23a opened in a support disk 23 made of, for example, fiberglass; and, on the other hand, a suction and discharge line 26c for the liquid passes through said rigid substrate 26, said line 26c being connected in an appropriate manner to a receiver for receiving the discharged residual liquid for subsequent treatment or destruction.

[0214] A clamping ring 27 which holds all the components of the detection holder 21 together so that they are integral with one another; for this purpose, the clamping ring 27 and the base 26 preferably have threads and are able to clamp all the components of the detection holder 21 .

[0215] • A gasket 28 which may be added to ensure a leak-proof seal to the detection support 21 .

[0216] - an element for storing at least one cell marker 4 of said microorganism 2 .

[0217] exist Figure 3A and 3B In one embodiment shown, a storage element 19 is included in the detection support 1. More specifically, such a storage element 19 is preferably located below the filter membrane 5 and preferably between the membrane 5 and the glass fiber support disk 13. Such a storage element 19 is also Figure 5 , and advantageously takes the form of a layer of microcapsules 9, each microcapsule 9 in the layer being formed by a cell marker 4 encapsulated in an encapsulation element 8. The storage element 19 can also be conveniently located below the glass fiber support disk 13, so that the filter membrane 5 rests directly on the support disk 13.

[0218] The detection device 14 includes Figure 4A and 4BIn another embodiment of the detection support 21 shown in , the element for storing the cell marker 4 consists of a reservoir of a liquid solution of the marker 4, which is separate from the detection support 21, so that the device 14 includes elements for conveying the cell marker 4 from the storage element to the detection support 21. These elements for conveying the cell marker 4 in liquid form are therefore connected by suitable connection means to the liquid feed line 26a of the substrate 26 of the detection support 21. Via the feed line 26a and then via the outlet 26b of the substrate 26 (which passes through the drain 24 via the orifice 24a and through the support disk 23 via the orifice 23a), the cell marker 4 in liquid form will in this case be conveyed directly to the top of the support disk 23 and below the filter membrane 25 which may contain the microorganisms 2 to be detected.

[0219] - Elements 15 for releasing said cell marker 4 in a controlled manner, so as to bring said marker 4 into contact with said sample 3 to be analyzed.

[0220] In an embodiment in which the element for storing at least one cell marker 4 takes the form of a layer of microcapsules 9, such an element may comprise, for example, an element for heating the detection support 1 and being connected to conventional elements for controlling and regulating the temperature, thereby applying an elevated temperature to melt the encapsulating element 8 and release the marker 4.

[0221] In another exemplary embodiment, the cell marker 4, preferably in liquid form, can be gradually transported from a separate reservoir from below (particularly from below the support plate 13, 23) to the detection support 1, 21 when appropriate, and diffuse by capillary action with the aid of the plate 13, 23, so that the marker 4 comes into contact with the microorganism 2 that can be retained on the membrane 5, 25. Figure 2 The controlled release element 15 shown in the figure in the form of a syringe may be composed of an element for aspirating the liquid marker 4 and an element for regulating the feeding flow rate (eg a flow control pump).

[0222] - an element 6 for illuminating the detection support 1 , 21 , which is capable of visualizing the cell marker 4 , in particular based on a laser beam or even more preferably based on a high-power light-emitting diode;

[0223] - Optical means 7 for capturing an image of at least a portion of the detection support 1, 21, located above the support 1, 21, the resolution of the optical means 7 preferably being at least equal to 20 megapixels, more preferably at least equal to 100 megapixels. The optical means 7 more preferably has a depth of focus of about 0.5 mm or equal to 0.5 mm, in order to overcome drawbacks related to the flatness of the detection support 1 to be observed. Advantageously, it consists of a camera with a CMOS sensor and has a field of view with dimensions of, for example, at least 10 mm long, at least 10 mm wide, even more preferably 25*25 mm.

[0224] In the case where the detection support 1, 21 must be divided into multiple parts to capture images before and after staining and view a wider field of view, the detection support 1, 21 can also cooperate with elements for displacing the detection support 1, 21 below the optical imaging device 7. These displacement elements are not shown in the figure.

[0225] - an element for storing in a memory for comparing and analyzing the images acquired by the optical device 7 in order to detect said at least one microorganism 2 potentially present in said sample 3 based on changes in the staining kinetics of said at least one microorganism 2. Such an analysis element preferably comprises an image detection and processing algorithm 11.

[0226] Figure 4A and 4B A particularly preferred embodiment of a solid detection support 21 is shown, which is used to receive the sample 3 to be analyzed that may contain at least one microorganism 2, and can be implemented in the detection method of the present invention, or can be part of a detection device 14 capable of implementing the steps of the method.

[0227] like Figure 4A and 4B As shown, the detection support 21 includes at least one filter membrane 25 for allowing the sample 3 to pass through, and a glass fiber support disk 23 on which the membrane 25 is placed.

[0228] The detection device is also provided with a rigid support element 24 or drain 24, which is permeable by a plurality of circular and / or rectangular perforations. A glass fiber support disc 23, located below the filter membrane 25, rests on the drain 24. Thus, the three elements, support disc 23, drain 24 and filter membrane 25, advantageously have a substantially circular shape and similar or slightly similar dimensions.

[0229] This rigid permeable support 24 notably maintains the flatness of the flexible element disc 23 and the membrane 25 supported thereby, said flexible element being able to deform when implementing the method of the invention, or by placing a sample 3 on the surface of said membrane 25, by aspirating residual liquid, etc. Furthermore, liquids can be drained through this support 24 thanks to the presence of a plurality of perforations.

[0230] The rigid support or ejector 24 itself is mounted on a rigid base 26 through which passes at least one liquid feed line 26a and through which passes at least one liquid suction and discharge line 26c.

[0231] The feed line 26a is used to transport the liquid cell marker 4 to the detection support 21. The line 26a also extends via an outlet 26b extending from the base 26, and passes through a first opening 24a opened in the drain 24 on the one hand, and through a second opening 23a opened in the glass fiber support disk 23 on the other hand, so that the liquid cell marker 4 is in direct contact with the filter membrane 25, and thus in contact with the microorganisms 2 that may be contained therein.

[0232] In addition to the feed line 26a, at least one suction and discharge line 26c for a liquid also passes through the substrate 26, whether the liquid is excess liquid cell marker 4 after contact with the filter membrane 25 or any other liquid, for example, residual liquid or washing liquid of the sample 3 after filtration through the membrane 25.

[0233] A clamping ring 27 holds all the components of the detection support 21 together so that they are integral with one another, and optionally a gasket 28 complements said support 21 .

[0234] It should be noted that, whatever the embodiment chosen for the detection support 1 , 21 , the filter 5 , 25 is advantageously dark, preferably black, in order to allow optimal visualization of the fluorescence or bioluminescence of the cell marker 4 by means of the contrast between the emitted light and the dark shadow of said filter 5 , 25 .

[0235] If the detection method or detection device 14 of the invention is implemented for the detection of cells or microorganisms 2 in the single-cell stage, membranes 5, 25 made of polyester or polycarbonate are preferred, while for the detection of cell clusters in the form of microcolonies, after the culture phase, membranes 5, 25 made of nitrocellulose or ester are preferred.

[0236] Generally speaking, if proven to be appropriate and feasible, the above-mentioned elements for the detection method can be applied to the detection device and the detection support 1, 21 of the present invention, and vice versa.

[0237] It goes without saying that the invention is not limited to the examples shown and described above, but that substitutions and modifications may be made thereto without departing from the scope of the invention.

Claims

1. A method for detecting at least one microorganism (2) of the bacteria, yeast or mold type present in a sample to be analyzed (3) on a solid detection support (1, 21), the method being a non-diagnostic method, the microorganism being visualized by means of at least one cell marker (4), the method being characterized in that it comprises at least the following steps, in order: a) placing the sample to be analyzed (3) on the solid detection support (1, 21); b) irradiating the solid detection support (1, 21) at least once with light radiation (6) capable of visualizing the at least one cell marker (4); c) acquiring, using an optical imaging device (7), at least one image (I0) of a first stage P0 of the sample (3) on at least a portion of the solid detection support (1, 21) with a field of view of at least 10 mm long by at least 10 mm wide as a target; d) performing a controlled release of the at least one cell marker (4) through the solid detection support (1, 21), thereby bringing the cell marker (4) into contact with the microorganism (2); e) after the controlled release step of the cell marker (4), in at least one subsequent stage Pi, using the optical imaging device (7) to capture an image (I) of each portion of the solid detection support (1, 21) for which an image has been captured in stage P0 i )(i≥1), thereby detecting the at least one microorganism (2) based on a change in the staining kinetics of the microorganism (2); f) for each portion of said solid detection support (1, 21), said image (I0) acquired at P0 and at least one subsequent stage P i Each image collected (I i ) and draw a conclusion on whether at least one microorganism (2) is present in the sample (3).

2. A method for detecting at least one microorganism (2) of the bacteria, yeast or mold type present in a sample to be analyzed (3) on a solid detection support (1, 21), the method being a non-diagnostic method, the microorganism being visualized by means of at least one cell marker (4), the method being characterized in that it comprises at least the following steps, in order: a1) placing the sample to be analyzed (3) on the solid detection support (1, 21); b1) locally irradiating the solid detection support (1, 21) with light radiation (6) capable of visualizing the at least one cell marker (4); c1) simultaneously with the irradiation step b1), using an optical imaging device (7), targeting a field of view of at least 10 mm long by at least 10 mm wide, collecting at least one image (I0, I'0) of the first stage P0 of the sample (3) on at least a portion of the solid detection support (1, 21); d1) performing a controlled release of the at least one cell marker (4) through the solid detection support (1, 21), thereby bringing the cell marker (4) into contact with the microorganism (2); e1) locally irradiating said solid detection support (1, 21) with said light radiation (6) capable of visualizing said at least one cell marker (4) within a period of less than 5 seconds after step d1) of controlled release of said cell marker (4); f1) simultaneously with the irradiation step e1), after the controlled release step of the cell marker (4), in at least one subsequent stage P1, using the optical imaging device (7) to acquire an image (I1, I'1) of each part of the solid detection support (1, 21) whose image was acquired in stage P0; g1) locally irradiating the solid detection support (1, 21) again at least once with the light radiation (6); h1) simultaneously with the irradiation step g1), in at least one second subsequent stage P2, using the optical imaging device (7) to acquire an image (I2, I'2) of each portion of the solid detection support (1, 21) for which an image has been acquired in stages P0 and P1, so as to detect at least one microorganism (2) based on changes in the staining kinetics of the microorganism (2); i1) For each part of the solid detection support (1, 21), a comparative analysis is performed between the image (I0, I'0) acquired at P0 and the images (I1, I'1; I2, I'2) acquired at at least two subsequent stages P1 and P2, and a conclusion is drawn as to whether at least one microorganism (2) is present in the sample (3).

3. The detection method according to claim 1 or 2, characterized in that: Each time at P0, P i When acquiring an image, the solid detection support (1, 21) is locally illuminated with optical radiation (6).

4. The detection method according to claim 1 or 2, characterized in that: The solid detection support (1, 21) is subjected to n displacements along different paths to spatially divide the solid detection support (1, 21) into n+1 parts, in at least two stages P0 and P i The image is collected from n, where n is an integer less than or equal to 20.

5. The detection method according to claim 1, characterized in that In the image acquisition steps c) and e), an optical imaging device (7) with a resolution at least equal to 20 megapixels is used.

6. The detection method according to claim 2, characterized in that: In the image acquisition steps c1), f1) and h1), an optical imaging device (7) with a resolution at least equal to 20 megapixels is used.

7. The detection method according to claim 1, characterized in that If the conclusion drawn in step f) is that at least one microorganism (2) is present in the sample (3), the microorganisms (2) present in the sample (3) are counted.

8. The detection method according to claim 2, characterized in that: If it is concluded in step i1) that at least one microorganism (2) is present in the sample (3), the microorganisms (2) present in the sample (3) are counted.

9. The detection method according to claim 1 or 2, characterized in that: The cell marker (4) is a fluorescent marker.

10. The detection method according to claim 1, characterized in that: The cell marker (4) released in a controlled manner in step d) is encapsulated in an encapsulating element (8).

11. The detection method according to claim 2, characterized in that: The cell marker (4) released in a controlled manner in step d1) is encapsulated in an encapsulating element (8).

12. The detection method according to claim 10 or 11, characterized in that: The cell marker (4) is encapsulated in a solid form in an encapsulating element (8), and the solid detection support (1, 21) contains a solvent for dissolving the cell marker (4).

13. The detection method according to claim 10 or 11, characterized in that: The cell marker (4) is encapsulated in the encapsulating element (8) in the form of solid powder, and the solid detection support (1, 21) contains a solvent for dissolving the cell marker (4).

14. The detection method according to claim 10 or 11, characterized in that: The cell marker (4) is encapsulated in liquid form.

15. The detection method according to claim 10 or 11, characterized in that: The encapsulating element (8) is thermosensitive and in step d) or d1), the controlled release of the cell marker (4) encapsulated in the thermosensitive encapsulating element (8) is carried out by subjecting the thermosensitive encapsulating element (8) to a temperature increase capable of melting the thermosensitive encapsulating element (8).

16. The detection method according to claim 15, characterized in that: The temperature was increased from ambient to a maximum temperature of 45°C.

17. The detection method according to claim 1 or 2, characterized in that: A cell marker (4) in a liquid solution is delivered in a controlled manner to the solid detection support (1, 21) containing the sample (3) to be analyzed.

18. The detection method according to claim 1 or 2, characterized in that: The sample to be analyzed (3) with a volume of 10 μL to 2,000 μL is placed directly on the solid detection support (1, 21).

19. The detection method according to claim 1 or 2, characterized in that: The sample to be analyzed (3) is filtered through a filter membrane (5, 25) with a cut-off threshold of 0.2 μm to 0.45 μm, and the filter membrane (5, 25) is directly located in the solid detection support (1, 21).

20. The detection method according to claim 1 or 2, characterized in that: In order to distinguish between microorganisms and dust on a solid detection support (1, 21) that has received a sample to be analyzed, the following steps are performed: - after each image acquisition, measuring the brightness of the detected illuminated pixels or pixel clusters; - comparing the brightness of each illuminated pixel or pixel cluster detected in the different images; - identifying pixels or clusters of pixels whose brightness remains substantially constant in at least two consecutive images during the staining kinetics; - Pixels or pixel clusters that change in brightness during the staining kinetics are analyzed and counted.

21. A device (14) for detecting at least one microorganism (2) of the bacteria, yeast or mold type present in a sample (3) to be analyzed, the device (14) being capable of performing the detection method according to any one of claims 1 to 20, characterized in that: It includes at least: - a solid detection support (1, 21) intended to receive said sample to be analyzed (3) which may contain at least one microorganism (2); - an element for storing at least one cell marker (4) of said microorganism (2); - an element (15) for releasing the cell marker (4) in a controlled manner so that the cell marker (4) comes into contact with the sample to be analyzed (3); - an element (6) for irradiating said solid detection support (1, 21); - an optical imaging device (7) for acquiring an image of at least a portion of the solid detection support (1, 21), the optical imaging device (7) having a field of view of at least 10 mm long by at least 10 mm wide; - an element for storing in a memory, comparing and analyzing the images acquired by the optical imaging device (7), so as to detect at least one microorganism (2) potentially present in the sample (3) based on changes in the staining kinetics of the at least one microorganism (2).

22. The detection device (14) according to claim 21, characterized in that It comprises elements for displacing the solid detection support (1, 21) below the optical imaging device (7).

23. The detection device (14) according to claim 21 or 22, characterized in that The solid detection support (1, 21) comprises at least one filter membrane (5, 25) resting on a glass fiber support disk (13, 23).

24. The detection device (14) according to claim 23, characterized in that The element (19) for storing the cell marker (4) is included in a solid detection support (1, 21) and consists of a layer of microcapsules (9) formed by the cell marker (4) encapsulated in an encapsulation element (8), and the layer of microcapsules (9) is arranged between the glass fiber support disk (13) and the filter membrane (5).

25. The detection device (14) according to claim 24, characterized in that The element for releasing the cell marker (4) in a controlled manner comprises a heating element and a temperature control and regulation element capable of generating a temperature increase inside the device (14) and capable of melting the encapsulating element (8) encapsulating the cell marker (4).

26. The detection device (14) according to claim 21 or 22, characterized in that The cell marker (4) storage element consists of a liquid cell marker (4) solution reservoir separated from a solid detection support (1, 21), and the detection device (14) includes an element for transporting the cell marker (4) from the storage element to the solid detection support (1, 21).

27. The detection device (14) according to claim 26, characterized in that The means for releasing the cell marker (4) in a controlled manner comprises at least one flow-controlled pump.

28. The detection device (14) according to claim 21 or 22, characterized in that The optical imaging device (7) has a focal depth of + / - 0.5 mm.

29. The detection device (14) according to claim 21 or 22, characterized in that The optical imaging device (7) is based on a CMOS sensor camera.

30. The detection device (14) according to claim 21 or 22, characterized in that The optical imaging device (7) has a field of view of 25 mm×25 mm.

31. The detection device (14) according to claim 21 or 22, characterized in that The resolution of the optical imaging device (7) is at least equal to 20 million pixels.

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