EP3861125A1

EP3861125A1 - Method and device for detecting at least one microorganism according to the labelling kinetics thereof, and detection support

Info

Publication number: EP3861125A1
Application number: EP19779504.0A
Authority: EP
Inventors: Joseph Pierquin
Original assignee: Redberry
Current assignee: Redberry
Priority date: 2018-10-05
Filing date: 2019-10-04
Publication date: 2021-08-11
Also published as: FR3086951A1; CN112823211A; JP2022502058A; WO2020070265A1; US20210277440A1; FR3086951B1

Abstract

The invention relates to a method for detecting, on a detection support (1, 21), at least one microorganism (2) present in a sample to be analysed (3) and revealed by means of at least one cell marker (4). This method comprises a first taking of an image (I₀, I'₀) before a step of controlled release of said marker (4) and bringing this marker into contact with said microorganism, and at least one taking of an image (Ι_i) following this contacting step, so as to detect said microorganism according to the change in the labelling kinetics thereof, by means of a comparative analysis between the different images that have been taken. The invention also relates to an automated detection device (14) capable of implementing the method and to a detection support capable of being used in the method and which is part of the device (14).

Description

METHOD AND DEVICE FOR DETECTING AT LEAST ONE MICROORGANISM ACCORDING TO ITS MARKING KINETICS, AND DETECTION MEDIUM

The present invention relates to the field of detection of microorganisms of the bacteria, yeast and mold type in the solid phase.

The present invention will find its application mainly in the field of industrial microbiology, for example, but not limited to, in the pharmaceutical, biotechnological, cosmetic or food industries, or even in clinical or environmental microbiology.

The invention relates more particularly to a device and a method allowing extremely early detection of microorganisms in solid phase, on suitable supports, and this without necessarily requiring culturing of said microorganisms with a view to their multiplication.

Many techniques are implemented at present to allow the detection of contaminants, for example bacteria, in a sample which must be tested.

The most conventional and oldest method consists in depositing a sample, possibly after filtration, on the surface of an agar and nutritive growth medium, the latter being more or less selective of one or more types ( s) microorganisms.

Said medium is then incubated at the temperature suitable for the growth of the microorganism sought, for a period which can often go up to several days, before an operator proceeds to a “manual” counting of the colonies visible to the naked eye.

Such a method, although very sensitive and allowing the detection of a single viable and cultivable contaminant in the initial sample, has the disadvantage of requiring a relatively long incubation period to allow detection with the naked eye of colonies that have formed on the agar.

In place of a solid agar, it is also possible to inoculate a liquid medium in which microbial growth will be detected, after incubation, by

SUBSTITUTE SHEET (RULE 26) turbidimetry for example, the absorbance measured being proportional to the concentration of microorganisms present in the medium.

The disadvantage of liquid phase detection techniques is that they do not allow microbial concentrations below 10 ⁶ or 10 ⁷ cells / mL to be detected. Thus, the sample must again be incubated for a variable time in order to reach such a cell concentration.

It should also be noted that certain microorganisms, under the effect of stress, for example thermal stress, or else due to the presence of inhibiting agents in a sample, can take a particularly long time to multiply, further prolonging the moment. where they will be detectable by traditional cultivation techniques.

It is also known, in the prior art, to carry out a polymerase chain reaction, also called PCR amplification, to determine the presence of specific microorganisms within a sample, by amplification of a DNA sequence or RNA.

However, these methods have the drawback of requiring several strands of nucleic acid, that is to say several contaminating microorganisms in a sample, and generally at least several tens of microorganisms. Such methods are therefore less sensitive than traditional culture techniques based on microbial growth.

Thus, in recent years, in order to increase the speed of detection of microorganisms while trying to maintain the sensitivity of conventional techniques based on growth, many methods have been developed and developed.

Most of these methods are based on the detection of fluorescence emitted by specific and fluorescent chemicals, fluorophores or fluorochromes, which are coupled, by different labeling techniques, to cellular structures or functions (nucleic acids, proteins , enzymes, etc.) of the microorganisms to be detected. These methods require the use of specific detection devices, such as microscopes for the so-called "DEFT" technique (Direct Epifluorescence Filter Technique) or even cytometers.

Thus, the flow cytometry technique is commonly used today to detect and count microorganisms present in a sample.

In this technique, cells or particles, which can be labeled with a fluorophore and suspended in a liquid, are isolated one behind the other in the form of a single cell flow. They are then routed one by one, and at high speed, through a light source, for example a light beam from a laser.

The light scattered and emitted by the cells, which is fluorescent in the case of labeling with a fluorophore after excitation of the latter at a given wavelength, is directed, by a system of mirrors and filters constituting the optical device from the cytometer, to a set of light detectors. These detectors incorporate, in particular, photomultipliers which will make it possible to convert the light signals into electrical signals which can then be analyzed by a computer in order to generate data relating, for example, to the physical characteristics of the cells.

The flow cytometers currently available on the market are capable of analyzing several hundred cells, or "events" per second, in real time, and some of these devices can be configured so as to allow sorting of the cells or particles into based on their optical properties.

However, the detection of rare events is often complicated, either by the implementation of flow cytometry, or by the microscopic technique.

Indeed, flow cytometry has a limited sensitivity, evaluated, in the best of cases, between 10 ² and 10 ³ cells / ml of suspension, and dependent on the matrix. In the "DEFT" technique, a sample to be analyzed is filtered through a membrane allowing the retention of microorganisms likely to be contained in said sample.

The microorganisms are then marked with a fluorochrome, for example acridine orange which interacts with DNA or RNA, then counted by visual analysis of several fields of the membrane using an epifluorescence microscope.

In the case of fluorescence microscopy, it is estimated that at least 10 ⁴ cells must be present, on a membrane having a diameter of 25 mm, to obtain representative results, knowing that the counting is carried out only on a small part of the membrane (less than 10% of the total size), and that a homogeneous distribution of the cells is assumed and not certain.

However, in certain applications of industrial or clinical microbiology, it may prove to be particularly important to detect, at an early stage, the presence of a single contaminant or of a few contaminants in a sample. Indeed, products manufactured and / or processed in this type of industry are particularly sensitive, in particular because of their destination (food, consumer health, etc.). Therefore, it is essential to ensure, as quickly as possible, that their microbiological quality is beyond reproach, or, if this is not the case, to be able to quickly identify the source of the contamination.

It is in particular for these reasons that the solid phase cytometry method, also called scanning cytometry, which has been applied to filterable samples, has been developed in recent years and which is in particular the subject of European patent EP 0. 713 087.

In this method, after a first step of filtering a sample on a suitable membrane, the microorganisms possibly present on said membrane are subjected to a labeling step using a fluorophore. The entire surface of the membrane is then scanned with a laser beam to excite the fluorophore and count any microorganisms present and viable in the initial sample to be analyzed, by measuring the fluorescence emitted.

The device known under the name ChemScan RDI (registered trademark), implements this method of solid phase cytometry.

However, the devices currently available on the market for implementing the solid phase cytometry technique, like the necessary reagents, are particularly expensive and require significant investment. Consequently, this technique is not accessible to all laboratories.

In addition, once the microorganisms have been detected and counted, the toxic effects of the reagents used, on cell metabolism, are capable of preventing a subsequent culture, for the purpose of identification, of said microorganisms.

It should also be noted that this solid phase cytometry method is only applicable to filterable matrices, and that the time required, from depositing the sample on a solid support until obtaining the results with the enumeration of microorganisms present on said solid support, is relatively large; the time is indeed estimated between, at least, 90 min, and can go, depending on the application, up to 4 hours.

Finally, whether it is the flow cytometry technique or else solid phase cytometry, the rate of false positives detected remains relatively high. In fact, inert particles, for example dust, other than microorganisms are also capable of emitting natural fluorescence, or even of combining with fluorochrome to emit parasitic florescence.

This is a real disadvantage of existing methods, since, depending on the fields of application, a sample proving to be false positive must necessarily be the subject of further investigations, costly in particular in terms of time and which may also have significant financial repercussions, in particular if a production chain (of biopharmaceutical products, for example), were to be stopped during the said investigations.

In general, the methods implementing labeling of the cells with a fluorophore require manipulation of the sample by qualified personnel, as well as manipulation and bringing the fluorescent reagent into contact with the microorganisms to be detected, inevitably causing a risk of contamination of said sample.

Ά also note that all of these operations, as well as the upstream work of sample preparation, including in particular the steps of filtration, activation, marking, washing, etc. inevitably require significant handling time by an operator, which consequently delays the time of detection of any contamination.

The invention offers the possibility of overcoming, at least in part, the various drawbacks mentioned above of the state of the art by proposing a method and a device for the extremely early detection, almost instantaneous if necessary, of microorganism ( s) possibly present in a starting sample to be analyzed.

The method and the device of the invention are based on a detection and an analysis of the labeling kinetics of possible microorganisms by means of a cellular marker delivered in a controlled manner and without time-consuming manual operation and likely in particular to cause contamination. of the sample.

Ά for this purpose, the present invention relates to a method of detection, on a solid detection support, of at least one microorganism, of the bacteria, yeast or mold type, present in a sample to be analyzed and revealed by at least one cell marker, said method comprising, at least, the following steps, taken in order: a) depositing said sample to be analyzed on said solid detection support;

b) illuminating said detection support at least once via light radiation capable of revealing said at least one cellular marker;

c) At least one image I _{0 is taken} in a first phase P _{0 of} said sample on at least a portion of said detection medium by means of an optical device for taking pictures targeting an observation field of at least 10 mm long and at least 10 mm wide;

d) a controlled release of said at least one cell marker is carried out, through the detection support, for bringing said marker into contact with said microorganism;

e) An image I _± (i³l) of each portion of the detection medium which has been the subject of an image at P ₀ is taken using said optical device in at least one subsequent phase Pi following the release step. monitoring said marker so as to detect said at least one microorganism according to the evolution of the labeling kinetics of said microorganism;

f) A comparative analysis is carried out, by portion of the detection medium, between said image I ₀ taken at P ₀ and each image I _± taken in at least one subsequent phase P ± and we conclude as to the presence or not of at least one microorganism in said sample.

Preferably, said detection medium is punctually illuminated via light radiation each time an image is taken at P ₀ , P ±.

In a particularly advantageous embodiment, the method of the invention comprises, at least, the following steps, taken in order:

al) depositing said sample to be analyzed on said solid detection support; bl) said detection medium is punctually illuminated via light radiation capable of revealing said at least one cell marker;

cl) Simultaneously with the illumination step b1), at least one image I ₀ , I'o is taken in a first phase P _{0 of} said sample on at least a portion of said detection medium by means of an optical device for shots aimed at a field of view at least 10 mm long by at least 10 mm wide;

dl) a controlled release of said at least one cellular marker is carried out, through the detection support, for bringing said marker into contact with said microorganism;

el) In a period of less than 5 seconds after step dl) of controlled release of said cell marker, said detection medium is punctually illuminated via said light radiation capable of revealing said at least one cell marker;

fl) Simultaneously with the illumination step el), an image Ii is taken, by means of said optical device, I'i of each portion of the detection medium which has been the subject of an image at P ₀ in at at least one subsequent phase Pi following the step of controlled release of said marker;

gl) said detection medium is again punctually, at least once, via said light radiation;

hl) Simultaneously with the illumination step gl), an image I ₂ , I ′ ₂ of each portion of the detection medium which has been the subject of an image at P ₀ is taken, by means of said optical device. at Pi in at least a second subsequent phase P ₂ so as to detect said at least one microorganism according to the evolution of the labeling kinetics of said microorganism;

ii) A comparative analysis is carried out, by portion of the detection medium, between said image I ₀ , I'o taken at P ₀ and said images Ii, I'i; I ₂ , I ' ₂ taken in at least two subsequent phases Pi and P ₂ and it is concluded as to the presence or not of at least one microorganism in said sample.

According to particular and non-limiting characteristics of the method of the invention:

the detection support is subjected to n displacements according to distinct paths with a view to a spatial division of said detection support into n + 1 portions subject to shots in at least two phases P ₀ and Pi, n being an integer less than or equal to 20;

- an optical device is used during the image taking steps c) and e) or cl), fl) and hl) having a resolution at least equal to 20 megapixels, and preferably greater than 100 megapixels;

if it is concluded that at least one microorganism is present in said sample during step f) or ii), a count is made of the microorganisms present in said sample;

- said cell marker is a fluorescent marker; said cell marker released in a controlled manner during step d) or dl) is enveloped in an encapsulation means in solid form, for example in the form of a powder, and said detection support comprises a delivery solvent in solution of said marker;

said cell marker is enveloped in an encapsulation means in liquid form;

- Said encapsulation means is heat-sensitive and, in step d) or dl), a controlled release of the cell marker wrapped in said heat-sensitive encapsulation means is carried out by subjecting the latter to an increase in temperature capable of making melting said heat-sensitive encapsulation means, for example by increasing the temperature from room temperature to a maximum temperature of 45 ° C;

the cell marker in liquid solution is conveyed in a controlled manner to said solid detection support containing the sample to be analyzed; a volume between 10 and 2000 μL of said sample to be analyzed is directly deposited on said detection support, said sample to be analyzed is filtered through a filtering membrane having a cut-off threshold of between 0.2 and 0.45 μm, preferably a cutoff threshold equal to 0.3 μm, said filter membrane being positioned directly in said detection support;

The present invention also relates to a method for distinguishing microorganisms from dust on the detection supports having received the samples to be analyzed, characterized by:

measurement after each shot of the brightness of the pixels or groups of luminescent pixels detected;

comparison of the brightness of each pixel or group of luminescent pixels detected on the different shots; identification of the pixels or groups of pixels whose brightness does not change appreciably in at least two consecutive shots during the marking kinetics;

analysis and counting of pixels or groups of pixels whose brightness changes during the marking kinetics.

The 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, capable of implementing the detection method of the invention, and comprising at least:

a solid detection support intended to receive said sample to be analyzed capable of containing at least one microorganism;

means for storing at least one cell marker of said microorganism;

means for controlled release of said cell marker for bringing said marker into contact with said sample to be analyzed;

means for illuminating said detection medium;

an optical device for taking pictures of at least a portion of said detection medium, said optical device with a field of view of at least 10 mm long and at least 10 mm wide;

means for memorizing, comparing and analyzing the shots taken by the optical device so as to detect said at least one microorganism possibly present in said sample according to the evolution of the labeling kinetics of said at least one microorganism.

Preferably, the device of the invention comprises means for moving said detection support under said optical device for taking pictures.

In the detection device of the invention, advantageously:

- Said solid detection support comprises at least one filtering membrane resting on a fiberglass support disc;

said cell marker storage means is included in the detection support and consists of a layer of microcapsules made up of said cell marker wrapped in an encapsulation means, said layer of microcapsules being disposed between said glass fiber support disc and said filter membrane; in this case, said means for controlled release of the cell marker comprises heating means and means for controlling and regulating the temperature capable of operating a temperature increase within the device and melting said encapsulation means enveloping the cell marker;

- Said cell marker storage means consists of a reserve of liquid cell marker solution separate from the detection medium and said detection device comprises means for conveying said cell marker from said storage means to said detection medium; in this case, said means of controlled release of the cellular marker can comprise at least one pump with controlled flow; - Said optical device for taking pictures has a depth of field of the order of or equal to 0.5 mm;

- said optical device for taking pictures is based on a camera with CMOS sensors;

- Said optical device for taking pictures has a field of vision of 25 mm * 25 mm;

- Said optical device for taking pictures has a resolution at least equal to 20 megapixels and preferably greater than 100 megapixels.

The invention also relates to a detection support capable of being implemented in the detection method according to the invention or entering into the composition of the detection device according to the invention and composed, at least, of a rigid base through which at least one liquid supply duct and at least one liquid suction and discharge duct, said supply duct being extended by a chimney projecting from said base and passing through, on the one hand , a first orifice formed in a rigid and permeable support means surmounting said base and, on the other hand, a second orifice formed in a flexible support disc made of glass fibers, said disc being positioned on said rigid and permeable support means and supporting a filter membrane having a cut-off threshold of 0.2 and 0.45 µm, suitable for the retention of microorganisms, said detection support further comprising a clamping ring fixedly attached to said insert ase and holding together said base, said rigid and permeable medium support, said support disc and said filter membrane.

The present invention has many advantages. On the one hand, the present method, based on the evaluation of the labeling kinetics of microorganisms, makes it possible to detect almost immediately, within minutes or tens of minutes at most following the preparation of a sample, and without necessarily requiring cultivation, the presence or absence of living microorganism (s) in said sample, while discriminating, in an extremely reliable manner, these microorganisms from possible artefacts, such as dust or the like. On the other hand, the invention requires only a few manipulations of the sample, and in any case no manipulation of the cell marker, a complex operation involving the use of qualified personnel and presenting a non-negligible risk of contamination of said sample.

Other characteristics and advantages of the invention will emerge from the detailed description which follows of nonlimiting embodiments of the invention, with reference to the appended figures in which:

- Figure 1 illustrates, schematically, the different steps of two particular embodiments of depositing a sample on a solid detection support, these steps can be implemented in the method of detecting at least one microorganism according to the invention;

- Figure 2 shows, schematically, a particular embodiment of implementation of the different steps of the detection method of the invention, including in particular the deposition of a sample on a detection medium, taking a image P ₀ before marking said sample and then taking several successive images Pi to P ₄ after controlled release of a cellular marker, and a comparative analysis of the different images to arrive at an analysis result; Figures 3Ά and 3B each schematically illustrate an exploded and perspective view, respectively in full and in a median section, of two nonlimiting exemplary embodiments of a detection support that can be implemented in the method or in the device the invention;

Figures 4Ά and 4B each schematically illustrate an exploded and perspective view, respectively in full and in a median section, of an embodiment different from those shown in Figures 3A and 3B and particularly preferred of a support detection that can be implemented in the method or in the device of the invention;

FIG. 5 represents a particular embodiment of the detection method of the invention in which the sample to be tested is brought into contact with an encapsulated cell marker, in the form of microcapsules, and released in a controlled manner under the action of an external stimulus to allow the detection of microorganisms possibly present in the sample;

- Figure 6 shows a series of shots of Bacillus subtilis microcolonies, following a culture phase, the shot on the left I ₀ having been carried out before the release of a cell marker while the other shots Ii at I ₅ were carried out at regular time intervals following the controlled release of said marker, allowing the evaluation of a labeling kinetics of B. subtilis colonies by these successive shots;

FIG. 7 illustrates a series of five successive shots, referenced I ' ₀ to I' ₄ , of a detection support on which are present, on the one hand, a dust and, on the other hand, cells to detect in a test sample, in this case yeasts belonging to the genus Candida and to the species albicans. Said support is photographed before the release of a cell marker (image I ' ₀ ) and following the controlled release of this marker (image I'i to I' ₄ ) according to the method of the invention; said figure also shows three profiles Pr, Pr _2a and Pr _2b illustrating graphically, in parallel with the shots I ' ₀ to I' ₄ , a variation in light intensity (expressed in gray levels, representing the brightness of a pixel and coded from 0 (black) to 255 (white)), depending on the marking, emitted by dust absorbing the cellular marker (Pr ^ and by a yeast (Pr _2a and Pr _2b ) as a function of time, more particularly as a function shots successive, two consecutive shots being spaced 20 seconds apart, demonstrating a sudden increase in the light intensity emitted by the dust after marking to a strong and stationary 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 before a decrease in light intensity, also called photobleaching.

As shown in Figures 1 to 7 of the accompanying drawings, the present invention relates, firstly, to a method of detection, on a solid detection support 1, 21, of at least one microorganism 2, or d 'A plurality of microorganisms 2, present (s), optionally but not necessarily, in a sample 3 which should be tested.

In the detection method of the invention, the said microorganism (s) 2, whether they are present in a single-cell form or in the form of microcolonies after a prior culture phase, are brought into contact with at least one cell marker 4.

In the following description, the term “cell marker” is intended to mean a substance capable of demonstrating by light emission, in particular fluorescence, possibly by bioluminescence, or even by coloring, the microorganisms optionally present in a starting sample to be tested. , it being understood that this cellular marker can preferably be a fluorescent marker capable of demonstrating only viable microorganisms or of differentiating viable microorganisms from non-viable microorganisms, or even capable of only marking certain pathogenic microorganisms of interest.

Note that the sample 3 to be tested can come, for example but not limited to, from a pharmaceutical or food industry, or consist of a sample either of human origin in clinical or medical microbiology, or of environmental origin, and microorganisms 2 to detect in said sample 3 are of the bacteria, yeast or mold type, mainly.

In a first step a) of the detection method according to the invention, the sample 3 to be tested is deposited on said solid detection support 1, 21 preferably comprising at least one filtering membrane 5, 25.

In an exemplary embodiment of step a) of the method of the invention, shown in the lower part of FIG. 1, the sample 3 to be analyzed consists of a liquid matrix, with a volume which can range from 1 ml at 500 mL, filtered through a detection support 1 comprising, at least, a filter membrane 5, said support 1 being positioned on a device D suitable for filtration.

Said membrane 5 has an adequate cut-off threshold for retaining the microorganisms 2 possibly present in the sample 3 and mainly eliminating on the one hand, the residual liquid of said sample 3 and, possibly, on the other hand, impurities contained in the latter and smaller than the cutoff threshold.

Thus, and whatever the embodiment chosen for the implementation of the first step a) of the method of the invention, said membrane 5, 25 preferably has a cutoff threshold of between 0.20 and 0.45 pm. Even more preferably, the cutoff threshold of this membrane 5, 25 is equal to, or of the order of, 0.30 μm.

Such a cut-off threshold makes it possible to retain, on the surface of said membrane 5, all the microorganisms 2 of the bacteria, yeast and mold type, the size of which varies between approximately 0.5 and approximately 10 μm, and which are liable to be present in sample 3.

Note, however, that the use of such a cut-off threshold promotes, in addition to the separation of microorganisms 2 from the rest of the sample 3, retention of the majority of inert particles (such as dust) which are also present at 'origin, in said sample 3. In a second embodiment of step a) of the method of the invention, not shown in the figures, the liquid sample 3, of volume between 1 and 500 ml, is filtered through a filter membrane 5 on an appropriate filtration device D, said membrane 5 having a cutoff threshold of between 0.20 and 0.45 μm and preferably equal to 0.3 μm.

Once the filtration of the sample 3 has been carried out, the filtering membrane 5 is extracted from the filtration device D to be deposited on the detection device 1.

Said detection support 1 can also be supplemented at least by a support disc 13 made of a permeable material, for example fiberglass.

Such a support disc 13 on which is brought to rest, possibly, the filter membrane 5 after removal of the latter from the device D to form, in this embodiment, the solid detection support 1, is illustrated in particular in the figures 3Ά and 3B attached.

In a third exemplary embodiment, which is applied to a sample 3 of a lower volume, typically between 10 and 2000 μl, or even less, one proceeds to a direct inoculation of said sample 3 on the detection support 1, 21 comprising at least minus the membrane 5, 25 and the support disc 13, 23 as illustrated in the upper part of FIG. 1.

Then, preferably, said detection medium 1 is inserted into a fully automated detection device 14 capable of implementing the method of the invention. Said device 14 will be described in more detail later.

Ά note that, in a particularly advantageous embodiment, step a) of depositing the sample 3 implements a detection support 21 configured to allow, quite preferably, either the deposition of a small volume of sample 3 between 10 and 2000 μL on a filter membrane 25, ie direct filtration of a larger volume of sample 3 and greater than 2000 μL, typically up to a volume of 100 ml, without going through a filtration device D dedicated to this operation and outside the detection device 14.

Filtration of the sample 3, even in the case where a small volume is deposited on the filtering membrane, can be envisaged, in an automated manner, in the detection device 14.

The detection support 21 will be described in detail in the following description, with reference to Figures 4Ά and 4B.

Ά note that, in a conventional manner, these operations of handling and depositing the sample 3 on the support 1, 21 can preferably be carried out by an operator in a sterile atmosphere, for example by carrying out said operations under a microbiological safety station PSM , visible in Figure 1.

In a step b) of the detection method according to the invention, illustrated in the attached FIG. 2, the detection support 1, 21 containing said sample 3 is illuminated at least once by means of light radiation 6 capable to the visualization or the revelation of said cell marker 4.

Indeed, in a particular embodiment of the detection method of the invention, continuous illumination of the detection support 1, 21 throughout the implementation of said method is possible. However, it is even more preferable to carry out an illumination of the detection support 1, 21 punctual and simultaneous or almost simultaneous with each of the image captures, which will be detailed later in the description.

According to this illumination step b), at least one image I ₀ , I'o, of the sample 3 is taken during a step c) on at least a portion of said detection support 1, 21, in a first phase P ₀ , two examples of images I ₀ , I'o being visible in FIGS. 6 and 7.

The taking of images during the process of the invention is preferably carried out by means of an optical device 7 for taking pictures having a higher optical resolution or equal to 20 megapixels, preferably greater than 100 megapixels, allowing detection of labeled microorganisms having a size at least equal to 0.5 μm on a predetermined surface.

In particular, said optical device 7 in fact has a relatively wide field of observation, typically at least 10 mm long by at least 10 mm wide, and, more preferably still, this field of observation is 25 * 25mm, so that you can, for example, take the entire detection medium 1, 21 in a single image.

It should also be noted that, preferably, said optical device 7 has a depth of field of +/- 0.5 mm in order to overcome any flatness defects of the membrane 5, 25 of said detection support 1, 21.

Thus, most preferably, during step c), a single image I ₀ , I'o of the entire surface of the detection support 1, 21 is taken.

In the present method, it is also envisaged, when the size of the detection medium 1, 21 is too large for it to be viewed by means of a single image capture, and to be able to observe a wider field without invest in a non-standard optical device under economic conditions which are therefore much less favorable, than subjecting said detection medium 1, 21 to n displacements according to distinct paths, n being an integer greater than 1 and less than or equal to 20, and more preferably still less than or equal to 10.

By means of these n displacements of separate paths, a spatial division of said detection medium 1, 21 is allowed into n + 1 portions, each of said portions being the subject of at least one shot at the instants of taking pictures. , in particular at P ₀ and during at least one subsequent phase P _± described in more detail below, the sum of the shots of the different portions then allowing a visualization of the entire detection support 1, 21 to each of the different phases P ₀ , P _± .

Indeed, it is particularly advantageous to be able to view the entirety of said support 1, 21, whether in one alone or several times, in order to ensure the detection of a small number of germs, or even of a single germ present at the start in the sample 3 to be tested.

Following the capture of image I ₀ , I'o proceed to a step d) of controlled release of at least one cell marker 4, through said detection support 1, 21, so that said marker 4 comes into contact microorganisms possibly present in the starting sample 3 and deposited on the support 1, 21.

A particular embodiment of this step d) of controlled release is more particularly illustrated in FIG. 5.

Preferably, the cell marker 4 used in the method of the invention is a fluorescent marker.

This marker 4 can be advantageously selected by a person skilled in the art from all the cellular markers well known to him and commonly used, for example, in current cytometry techniques.

Thus, said 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, bromide ethidium, eosin 5'maleimide (EMA), amine reactive markers such as "Zombie Fixable *", "Fixable Viability Dye eFluor *", "LIVE / DEAD Fixable dead *", "Viobility Fixable * "," Horizon Fixable Viability * "," Ghost * ", esterase activity markers such as calcein AM (acetoxy methyl), cFDA (Carboxy-fluorescein diacetate)," CellTracker Green CMFDA * ", redox markers like resazurin or tetrazolium salts, or cell markers in kits like "LIVE / DEAD Viability *" kits, the names of the reagents mentioned in the pr this paragraph and followed by an asterisk being filed as a trademark. It is also conceivable to use a plurality of cellular markers in combination among those mentioned above.

Ά note that, in the case of the implementation, in the method of the invention, of a fluorescent cellular marker 4, the light radiation 6 is selected from a laser beam and one, or more, light-emitting diode (s) (s), the latter solution being moreover particularly preferred.

In an alternative embodiment of the present method, it is also envisaged, during step d), to bring the possible microorganism (s) 2 into contact with at least one non-fluorescent but visible and detectable cell marker 4.

Said cell marker 4 may also consist of a bioluminescent marker, it being understood that in this case, the method for detecting microorganisms of the invention is implemented by eliminating the illumination step (s) of the detection support 1, 21. In other words, with the exception of the illumination of the support, all the other steps, variants and examples of embodiment of the method of the invention detailed in the present description are applicable to a method for detecting microorganisms by bioluminescence, if this proves to be suitable and conceivable, by implementing, of course, a suitable bioluminescent marker.

In any case, in a particularly advantageous manner, in a first precise and non-limiting embodiment of step d) of controlled release, the cell marker 4 which it is desired to release in a controlled manner during the method of the invention is wrapped in an encapsulation means 8 thus advantageously forming a microcapsule, a microbead or a microsphere 9 having a diameter for example of the order of 100 μm.

Preferably, the encapsulation means 8 is sensitive to the application of an external stimulus 10 leading to its rupture and the release of the cell marker 4.

Thus, most preferably, the encapsulation means 8 consists of thermosensitive lipid waxes and the release of the encapsulated marker 4 is carried out by gradually applying a rise in temperature to said encapsulation means 8 so that the latter melts and releases the marker 4 on contact with potential microorganisms 2 to be detected.

In this case, the temperature to which said heat-sensitive encapsulation means 8 is subjected can gradually be increased from an ambient temperature to a temperature not exceeding 45 ° C., threshold beyond which destruction of certain microorganisms 2 could occur. .

In the invention, preferably, the release of the cellular marker 4 is carried out in an automated manner, consequently making it possible to dispense with any manual operation which is a source of contamination, and is controlled so as to be done with the adequate quantity of marker 4 and good kinetics for effective labeling of microorganisms 2.

According to a first variant, the cell marker 4 is encapsulated in liquid form, the microcapsule 9 then comprising, in addition to said marker 4, a solvent for dissolving the latter.

In a second variant embodiment, the cell marker 4 is encapsulated in solid form, in particular in the form of a powder. Following the release of the cell marker 4, the latter is then placed in the presence of an appropriate dissolving solvent, for example water, so that the labeling of the microorganisms 2 can be carried out.

Most preferably, in this alternative embodiment, the solvent for dissolving the solid cell marker 4 is contained in the support disc 13 made of glass fiber and the filtering membrane 5 through which the sample 3 has previously been passed is disposed above said support disc 13.

Thus, once the filter membrane 5 has been deposited above the support disc 13 (after external filtration of the sample 3), or once the sample 3 has been deposited and filtered directly on the filter membrane 5 in the support detection 1, regardless of the sample volume 3, a layer of microcapsules 9 containing the cellular marker 4 being positioned between said membrane 5 and said disc 13, the support disc 13 is soaked with adequate solvent so as to allow, once the cellular marker 4 released from its capsule 9 (for example by increasing temperature), dissolving it. This example is illustrated in Figure 3B.

In the configuration of FIG. 3B, it is also conceivable that the support disc 13 can be imbibed with an appropriate solvent and maintained at a temperature capable of melting the encapsulation layer 8 thus releasing the cell marker 4 encapsulated in the form solid powder by simultaneously putting it in solution so that it reveals the possible presence of microorganisms 2 on the membrane 5.

Anyway, when a layer of microcapsules 9 containing the cell marker 4 in solid or liquid form is positioned above the support disc 13, the latter then has the main function, after rupture of said microcapsules 9, to homogenize , by diffusion, the distribution of said marker 4 under the membrane 5, itself placed above the layer of microcapsules 9. Thus, each microorganism 2 likely to be present at said membrane 5 will be revealed by the method of the invention.

Note also that, in this embodiment of step d) and when the step of depositing a) of the sample 3 of small volume (10 to 2000 μL) is carried out directly on the solid detection support 1, may again be composed of at least one support disc 13 and a filter membrane 5 framing a layer of microcapsules 9, said support disc 13 also has a support function for said microcapsules 9 to prevent them from s' evacuate with the filtered liquid at the time of removal.

In FIG. 3A, the layer of microcapsules 9 is placed under the support disc 13, for example made of glass fibers, and said disc 13 ensures the diffusion by capillarity of said microcapsules 9 or of the cell marker 4 once it released in a controlled manner, as well as a homogeneous distribution of these microcapsules 9 diffused under the filtering membrane 5.

Controlled release of a cellular marker 4 by means of destruction of the encapsulation means 8 in which said marker 4 is contained, although proving to be particularly advantageous, should not be considered as the only embodiment of step d) of the method of the invention.

In fact, in a second embodiment of this step d), the cell marker 4 is conveyed directly in the form of a liquid solution, and in a controlled and automated manner, for example at a controlled rate through appropriate means, such as a controlled flow pump, for contacting with the microorganism (s) 2 present on the surface of the filter membrane 5, 25 of the detection support 1, 21.

However, whatever the mode of release chosen for a controlled contact of the cell marker 4 with the microorganism (s) 2, said marker 4, whether it is preferably fluorescent, or else bioluminescent or not , most preferably has minimal toxicity with respect to microorganisms 2 potentially present on the membrane 5.

Indeed, in the event that contamination of the sample 3 is detected during the implementation of the present method, and in certain applications, in particular in the field of industrial pharmacy, it may prove to be particularly advantageous to be able to subsequently identify the microorganism 2 detected.

Such an identification step 12, illustrated in FIG. 2, can thus be carried out by culturing the sample 3 subsequent to detection, in particular by removing the filtering membrane 5, 25 and depositing that -this on an appropriate nutrient agar allowing the multiplication of microorganisms, then by the implementation of a conventional identification method and well known to those skilled in the art, for example by mass spectrometry. In the method of detecting microorganism (s) according to the invention, following the step of controlled release of at least one cell marker 4, one takes, during a step e), and during at least one phase P _± subsequent to phase P ₀ , at least one image I _± , i being an integer greater than or equal to 1.

In FIGS. 6 and 7, a succession of images respectively I ₀ to I ₅ and I ' ₀ to I' ₄ , taken during phase P ₀ and during several phases subsequent to P ₀ , are illustrated.

Each image I _± taken during at least one subsequent phase P _± is, if necessary, cut into portions of said detection medium 1, 21.

Such a succession of image shots, one (for example I ₀ ) being carried out before the labeling of microorganisms 2 by controlled release of the cellular marker 4 and at least one other (I _± ) after this release, makes it possible to characterize a kinetics of labeling of these microorganisms 2, in other words an evolution of the behavior, and in particular preferentially of the fluorescence, possibly of bioluminescence, of said microorganisms, corresponding to an assimilation of the marker 4 by the latter, between the first image taking I ₀ without marker 4 and at least the second image taken I _± after release of the latter.

This makes it possible to differentiate the behavior of inert particles, for example dust, capable of emitting, for example, natural fluorescence, of microorganisms 2 actually marked during the implementation of the process.

Indeed, in this hypothesis where possible microorganisms 2 are revealed by means of a fluorescent cell marker 4, inert particles or dust which would be naturally fluorescent and which have been retained on the filtering membrane 5 will be visible and detectable on the image I ₀ before controlled release of said marker 4 and on at least one image Ii after this release, while said microorganisms 2 will only be visible and detectable on the image! , especially on image Ii or I'i. The subsequent step f), during which a comparative analysis of said images I ₀ and I _± , in particular at least Ii or I 'i, is carried out, will make it possible to conclude as to the presence or absence of microorganisms 2 in the starting sample 3.

Now taking up the hypothesis that, during phase P ₀ , said detection support 1, 21 has been subjected to n displacements along separate paths, this support 1, 21 is again subjected, during phase P _± , at n identical displacements so that the images I ₀ and Ii taken respectively at P ₀ and Pi can be compared in a later step of comparative analysis (step f)) ·

In addition, similar logic applies to subsequent images, if any.

Indeed, in a preferred embodiment, illustrated in FIGS. 6 and 7, according to a first image capture Ii, I'i during a first subsequent phase Pi, after a variable period of time, of the order of a few seconds to a few tens of seconds, for example between 10 and 30 seconds, an image I ₂ , I ′ _{2 is again taken} of each portion of said support 1 having been taken from image during phases P ₀ and Pi.

Thus, in the detection method of the invention, it is conceivable and advantageous to repeat several times step e) of image taking I _± , after step d) of controlled release of the cell marker 4 and before l step f) of comparative analysis of the images I ₀ , I'0; Ii, I'i; I ₂ , I ' ₂ , etc., the successive image captures Ii being spaced apart by a period of time between 10 and 30 seconds, preferably 20 seconds.

Each image is clearly heard memorized by the device 14, so that subsequent processing, in particular comparison, can be implemented.

Most preferably, step e) of the present method is repeated three times successively before the comparative analysis step f), so as to generate three images Ii, I'i; I ₂ , I '₂; I ₃ , I ' ₃ , of each portion of the detection support 1, 21 having is the subject of an image at P ₀ during subsequent phases of taking images respectively Pi, P ₂ and P ₃ .

Even more preferably, five images are generated from Ii to I ₅ or from I to I ' ₅ (the image I' ₅ not being shown in FIG. 7) of each of the portions of the detection support 1, 21 having been viewed during phase P ₀ .

Thus, by releasing, in a particularly original way, the cellular marker 4 after the deposition of the sample 3 on the detection support 1, 21 and also following a first shot I ₀ in phase P ₀ , the microorganisms 2 hypothetically present in said sample 3 will be marked, preferably from the unicellular stage, in a short period of time, of the order of a few minutes, corresponding to the kinetics of the reactions of assimilation of the cellular marker 4 by the microorganism 2, thus allowing a result of rapid detection by taking images I ₀ , I'o; Ii, I'i, etc., successive of the support 1, 21 after the marking during different phases P ₀ , Pi, etc.

Depending on the nature of the cellular marker 4 used in step d) of the method, the biochemical reaction of assimilation thereof by a microorganism 2 can be, in particular, either an enzymatic reaction, or an oxido reaction. -reduction, or even an intercalation reaction of said marker between the bases of a nucleic acid.

The characterization of a labeling kinetics of the microorganisms 2 potentially present in the sample 3 to be tested also makes it possible to quickly differentiate the behavior of inert or dust particles, capable of emitting fluorescence naturally, from that of said microorganisms 2, resulting in a significant reduction in false positives in comparison with the systems proposed in the prior art.

It also appears from the above that the implementation of the method of the invention allows the detection of a single contaminant corresponding to a CFU, that is to say a Colony-forming Unit, according to the terminology of the man of the trade, which can be contained in the starting sample 3. The conclusion as to the contamination or not of said sample 3 is given following step f) of comparative analysis between the image I ₀ ; I ' ₀ taken during the first phase P ₀ , and at least the image I _± (for example the image Ii; I'i), already mentioned previously in the description and which is implemented, advantageously, by means an image processing and detection algorithm 11.

Note also that, when it is detected, during this analysis step f), the presence of at least one contaminating microorganism 2, a step g) of counting the microorganism (s) 2 present (s) in the sample 3 and on the detection support 1, 21 can be implemented.

This optional step g) can also be carried out by algorithm 11.

This algorithm 11 must therefore make it possible in particular to detect as accurately and quickly as possible, the presence or absence of microorganisms 2 on the images Ii _, I'i; I _2, I ' ₂ etc., by evaluating the evolution of the behavior of said microorganisms 2 over time, and by discriminating them from possible artefacts, in particular dust, and then counting these microorganisms 2.

In addition, most preferably, the algorithm 11 is able to detect elements (dust, microorganisms, etc.), the dimensions of which are of the order of, or even less than, the resolution of the sensors of the optical device 7 for taking pictures. .

This is one of the preponderant advantages of the invention: the use of a method based on the kinetics of assimilation of the cellular marker 4 by the microorganisms 2 makes it possible to overcome, to a certain extent, the optical resolution requirement.

Indeed, the variation in fluorescence intensity due to the labeling kinetics can be detected on a single pixel. The size of the microorganism 2 is therefore not necessarily greater than the size of a pixel. In certain cases, the intensity of light emitted by a microorganism 2 of size less than one pixel is just sufficient to illuminate said pixel. If this intensity may be insufficient to reliably detect the microorganism 2, the variation in intensity during labeling may be sufficient to detect it.

Most preferably, step e) of taking an image I _± of the detection support 1, 21 according to the controlled release of the cellular marker 4, is carried out according to a time offset according to the release of said marker 4 typically comprised between 20 and 300 seconds , preferably between 80 and 160 seconds in the case of labeling with acridine orange for example.

Such a time difference, according to the controlled release of the marker 4, and therefore according to the contacting of said marker 4 with a microorganism 2 possibly present at the level of the detection support, observed before taking the image I _± , makes it possible to s' ensure that said microorganism 2 has correctly assimilated said marker 4.

In a very preferred embodiment, the detection support 1, 21 is punctually illuminated via light radiation 6 capable of revealing the cellular marker 4, and this simultaneously, or almost simultaneously, with each of the images taken. , both the initial image capture I ₀ , I'o of step b) in a first phase P ₀ , and simultaneously with the image capture I _± which takes place in at least one subsequent phase R _± .

In other words, apart from the image taking phases P ₀ and Pi, the detection support 1, 21 may not be illuminated by the light radiation 6.

Note that, most preferably, the method of the invention has at least the following steps, taken in 1 order:

al) The sample 3 to be analyzed is deposited on said solid detection support 1, 21;

bl) said detection support 1, 21 is punctually illuminated via a light radiation 6 capable of revealing said at least one cell marker 4;

cl) Simultaneously with the illumination step b1), at least one image I ₀ , I'o is taken in a first phase P _{0 of} said sample 3 on at least a portion of said detection support 1, 21 by means of an optical device 7 for taking pictures having a resolution at least equal to 20 megapixels and a field of observation at least 10 mm long by at least 10 mm wide;

dl) A controlled release of the cell marker 4, or even of a succession of cell markers 4 or reactive agents, is carried out, through the detection support 1, 21, for bringing said marker 4 into contact with said microorganism 2;

el) In a period of time less than 5 seconds after step dl) of controlled release of said cell marker 4, said detection medium 1, 21 is punctually illuminated via said light radiation 6 capable of revealing said at least one cell marker 4 ;

fl) Simultaneously with the illumination step el), an image Ii, I'i is taken of each portion of the detection support 1, 21 having been the subject of an image I ₀ , I'o to P ₀ in at least one subsequent phase Pi following the step of controlled release of said marker 4;

gl) once again, at least once, said detection support 1, 21 is illuminated via said light radiation 6;

hl) Simultaneously with the illumination step gl), an image I ₂ , I ' ₂ of each portion of the detection support 1, 21 having been the subject of an image I ₀ , I' ₀ to P is taken ₀ and I ₁ I'i to Pi in at least a second subsequent phase P ₂ so as to detect said at least one microorganism 2 according to the evolution of the labeling kinetics of said microorganism 2;

ii) A comparative analysis is carried out, by portion of the detection support 1, 21, between said image I ₀ , I'o taken at P ₀ and said images Ii, I'i; I ₂ , I ' ₂ taken in at least two subsequent phases Pi and P ₂ and we conclude as to the presence or not of at least one microorganism 2 in said sample 3.

The steps gl) and hl) for taking the image I ₂ , I ′ ₂ are preferably implemented within a period of 20 and 300 seconds, preferably between 80 and 160 seconds, depending on step f1) during which the image Ii, I'i is taken.

Ά note that the variants or preferred embodiments or examples of steps a) to f) described previously in the description are also applicable to the corresponding steps a) to ii) of the method presented in the paragraph above.

Such a method is particularly interesting, because it allows, on the one hand, to discriminate effectively:

- A first category of inert particles or dust which emit, for example, a fluorescence or a bioluminescence in a natural way, and which will be displayed on the image I ₀ , I 'o taken in a first phase P ₀ , before the step dl) controlled release of cell marker 4;

- A second category of inert particles or dust, which are not autofluorescent (and therefore not visible on the image I ₀ , I 'o) but which rapidly absorb the cell marker 4 after controlled release thereof, these dusts will then clearly visible on the image Ii, I'i;

- The microorganism (s) 2 possibly present in the initial sample 3 which should be analyzed, and which will not be visible on the image I ₀ , I 'o, which can start to appear on the image Ii, I' i, with a weak light intensity, and which are then clearly visible, with a higher light intensity, on the images I ₂ , I ' ₂ and following optionally, it being understood that steps gl) and hl) can be repeated as many times as desired to take images during subsequent phases P ₂ , P3, etc.

FIG. 7 illustrates a series of images taken I ' ₀ to I' ₄ , and relates some of the elements visible on these images, in particular on the images I'i to I ' ₄ , with the profiles Pr _j. , Pr _2a and Pr _2b illustrating the variation in light intensity of the elements in question as a function of time following the moment (0 on the axis of abscissa) where the cell marker 4 is released in a controlled manner and brought into contact with said elements, in this case dust and yeast Candida albicans present in the sample 3 and on the solid detection support 1, 21.

On image I ' ₀ , taken before the step of controlled release of the cellular marker 4, no element is visible. It therefore follows from this image I ' _{0 that} no dust emitting light naturally is present in said starting sample 3.

On the following images I'i to I ' ₄ , after marking, it is visible that certain elements present in said sample 3 have a high light intensity and substantially constant over time, depending on the marking step. In this case, the comparison of successive images I'i to I ' ₄ makes it possible to conclude that this is a dust having absorbed the cell marker and whose profile Pr _! is represented graphically in connection with the identified light point.

On the succession of images I'i to I ' ₄ after marking, other elements are also displayed, in this case microorganisms, and whose light intensity, represented on the profiles Pr _2a and Pr _2b , increases by gradually over time until a peak in intensity is reached, corresponding to a gradual assimilation of the cellular marker 4 by a microorganism 2 (Candida albicans yeasts in this case), before this light intensity also gradually decreases, which corresponds to the phenomenon of photobleaching.

Note that, on the assumption that, on the detection support 1, 21, an inert particle, for example a dust, emitting light naturally, would have been deposited during the first step a1) of the method of the invention, it would have been visible on image I ' _{0 as} well as on all the following images I' _± . Such dust could therefore also have been differentiated from a microorganism 2, by the implementation of the detection method according to the present invention.

The present invention also relates to an automated detection device 14 for at least one microorganism 2, of the type bacteria, yeast or mold, possibly present in a sample 3 to be tested, and capable of implementing the steps of the detection method described above.

Advantageously, the device 14 according to the invention comprises at least:

a solid detection support 1, 21 intended to receive said sample to be analyzed 3 capable of containing at least one microorganism 2. This support 1, 21 is preferably constituted by at least one filtering membrane 5, 25, through which the l 'sample 3, and a support disc 13, 23 on which the membrane 5, 25 rests.

In one embodiment, illustrated in FIGS. 3A and 3B, the detection support 1 can be completed by a protective cover 16 and by a means for holding all of the elements of the support 1 on a rigid support plate 18, said holding means which may be in the form of a hoop 17.

In another embodiment, illustrated more precisely in FIGS. 4A and 4B, the detection support 21, comprising a filtering membrane 25 resting on a support disc 23 made of fiberglass, is completed by:

• a rigid support means 24, in particular of plastic, and permeable, by the presence of a plurality of perforations of circular shape and / or of perforations of oblong shape, said rigid support means 24 also being referred to, in the following description drain 24; it has a shape and dimensions similar or almost similar to those of the support disc 23 and the filter membrane 25;

• a rigid base 26, made for example of plastic material, crossed, on the one hand, by a conduit 26a for supplying a liquid, in this case the cellular marker 4, said conduit 26a being extended by a chimney 26b making projection from said base 26 and passing through a first orifice 24a formed in the drain 24 and a second orifice 23a formed in the support disc 23, for example made of glass fibers and, on the other hand, by a suction and discharge conduit 26c of a liquid; this conduit 26c is connected, through appropriate means, to a container for receiving the residual liquid discharged for subsequent treatment or destruction;

• a clamping ring 27 holding, in an integral manner, all of the components of the detection support 21; for this purpose, preferably, said clamping ring 27 as well as the base 26 have a thread and said clamping ring 27 is capable of enclosing all of the components of the detection support 21;

• a seal 28 which can be added to guarantee the tightness of the detection support 21.

means for storing at least one cell marker 4 of said microorganism 2.

In one embodiment, illustrated in FIGS. 3Ά and 3B, the storage means 19 is included in the detection support 1. More precisely, such a storage means 19 is preferably positioned under the filter membrane 5, and preferably between said membrane 5 and the support disc 13 made of glass fibers. Such a storage means 19 is also shown in FIG. 5 and is advantageously in the form of a layer of microcapsules 9, each microcapsule 9 of the layer consisting of the cell marker 4 wrapped in an encapsulation means 8. It It is also conceivable that this storage means 19 is positioned under the support disc 13 made of glass fibers on which the filter membrane 5 then rests directly.

In another embodiment, in which the detection device 14 includes the detection support 21 illustrated in FIGS. 4Ά and 4B, the cell marker storage means 4 consists of a reserve of liquid marker solution 4, this reserve being distinct from said detection medium 21 and said device 14 then comprising means for conveying the cellular marker 4 from said storage means to said detection medium 21. These means for conveying the marker cell 4 in liquid form are then in connection, through appropriate connecting means, with the liquid supply conduit 26a from the base 26 of the detection support 21. Through the supply conduit 26a then the chimney 26b of the base 26 which crosses the drain 24 through the orifice 24a and the support disc 23 through the orifice 23a, the cell marker 4 in liquid form will, in this case, be routed directly on top of said support disc 23, under the filter membrane 25 capable of containing microorganisms 2 to be detected.

means 15 for controlled release of said cell marker 4 for bringing said marker 4 into contact with said sample 3 to be analyzed.

Such means may include, for example, in the embodiment where the means for storing at least one cell marker 4 is in the form of a layer of microcapsules 9, means for heating the detection support 1, associated to conventional temperature control and regulation means, in order to apply an increase in temperature to melt the encapsulation means 8 and release the marker 4.

In another exemplary embodiment, the cell marker 4, preferably in liquid form, can be progressively conveyed at the appropriate time from a separate reserve to the detection support 1, 21 from below it, in particular under the support disc. 13, 23 and be diffused by capillarity by means of this disc 13, 23 for bringing the marker 4 into contact with the microorganisms 2 which can be retained on the membrane 5, 25. In this case, the means 15 of controlled release, illustrated very schematically in the form of an injection syringe in Figure 2, may consist of means for aspiration of the liquid marker 4 and means for regulating the delivery rate, for example a pump with controlled flow;

a means of illumination 6 of said detection support 1, 21 capable of revealing the cell marker 4, in particular based on a laser beam or, more preferably still, high power light-emitting diodes;

an optical device 7 for taking pictures of at least a portion of said detection support 1, 21 and positioned above this support 1, 21 said optical device 7 preferably having a resolution at least equal to 20 megapixels, more preferably at least equal to 100 megapixels. This optical device 7 also preferably has a depth of field of the order of 0.5 mm, or equal to 0.5 mm in order to overcome the flatness of the detection support 1 which must be viewed. It advantageously consists of a camera with CMOS sensors and has a field of vision of dimensions for example at least 10 mm long by at least 10 mm wide, and, more preferably still, 25 * 25 mm .

The detection support 1, 21 can also cooperate with means for moving said support 1, 21 under this optical device 7 for taking pictures, in the event that the detection support 1, 21 must be divided into several portions for the shooting before and after marking and for the observation of a wider field. These displacement means are not shown in the figures.

means for memorizing, comparing and analyzing the shots taken by the optical device 7 so as to detect said at least one microorganism 2 possibly present in said sample 3 according to the evolution of the marking kinetics of said at least one microorganism 2. Such means of analysis preferably consist of an image processing and detection algorithm 11.

FIGS. 4Ά and 4B illustrate a particularly preferred embodiment of a solid detection support 21, intended to receive said sample to be analyzed 3 capable of containing at least one microorganism 2, and which can be used in the detection method according to the invention or be part of the detection device 14 capable of implementing the steps of this method. As visible in these FIGS. 4A and 4B, this detection support 21 is composed of at least one filtering membrane 25 through which the sample 3 is passed and of a support disc 23 made of fiberglass on which the membrane 25 rests .

Such a detection device is also provided with a support means 24, or drain 24, which is rigid and permeable by means of a plurality of perforations of circular shape and / or perforations of oblong shape. On this drain 24 rests the support disc 23 made of glass fibers itself surmounted by said filter membrane 25. Thus, these three elements, support disc 23, drain 24 and filter membrane 25 have, advantageously, a shape, essentially circular, as well as similar or almost similar dimensions.

Such a rigid and permeable support 24 makes it possible, in particular, to maintain the flatness of the flexible elements that it supports, disc 23 and membrane 25, liable to be subjected to deformations during the implementation of the method of invention, by depositing the sample 3 on the surface of said membrane 25, by suctioning the residual liquid, etc. In addition, due to the presence of numerous perforations, a discharge of liquid is possible through this support 24.

Said rigid support or drain 24 is itself mounted on a rigid base 26 traversed by at least one conduit 26a for supplying a liquid and by at least one suction and discharge conduit 26c for a liquid.

Said inlet conduit 26a has the function of conveying the cellular marker 4 at the level of the detection support 21, said conduit 26a being moreover extended by a chimney 26b projecting from said base 26 and passing through, on the one hand, a first orifice 24a formed in the drain 24 and, on the other hand, a second orifice 23a formed in the support disc 23 made of glass fibers so as to bring the liquid cellular marker 4 into immediate contact with the filtering membrane 25 and therefore microorganisms 2 which it potentially contains.

In addition to the supply duct 26a, the base 26 is also traversed by at least one suction and discharge duct 26c of a liquid, either a surplus of cell marker 4 liquid after contacting the latter with the filter membrane 25, or any other liquid, for example the residual liquid of the sample 3 after filtration by the membrane 25 or even a washing liquid.

A clamping ring 27 integrally securing all the components of the detection support 21, as well as, optionally, a seal 28, complete said support 21.

Note that, whatever the embodiment adopted with regard to the detection support 1, 21, the filter membrane 5, 25 advantageously has a dark color, preferably a black color, in order to allow optimal revelation fluorescence or bioluminescence of the cell marker 4, by contrast between the light emitted and the dark shade of said filter membrane 5, 25.

If the detection method or the detection device 14 of the invention is implemented with the objective of detecting cells or microorganisms 2 at the unicellular stage, a membrane 5, 25 made of polyester or polycarbonate will be preferred, while , for the detection of clusters of cells in the form of microcolonies, after a culture phase, a membrane 5, 25 of cellulose ester or nitrate will be preferred.

In general, the elements which have been described above for the detection method can be applied to the detection device and to the detection supports 1, 21 according to the invention, and vice versa, if this proves to be suitable and conceivable.

Of course, the invention is not limited to the examples illustrated and described above which may have variants and modifications without departing from the scope of the invention.

Claims

1. Method of detection, on a solid detection support (1, 21), of at least one microorganism (2), of the bacteria, yeast or mold type, present in a sample to be analyzed (3) and revealed by at least a cell marker (4), said method being characterized in that it comprises, at least, the following steps, taken in order:

a) depositing said sample (3) to be analyzed on said solid detection support (1, 21);

b) Illuminating said detection support at least once

(1) via light radiation (6) capable of revealing said at least one cell marker (4);

c) At least one image (I ₀ ) is taken in a first phase P _{0 of} said sample (3) on at least a portion of said detection support (1, 21) by means of an optical device (7) for taking views aimed at a field of view at least 10 mm long by at least 10 mm wide;

d) a controlled release of said at least one cellular marker (4) is carried out, through the detection support (1, 21), for bringing said marker (4) into contact with said microorganism (2);

e) An image (Ii) (i³l) of each portion of the detection support (1, 21) having been the subject of an image at P ₀ in at least one is taken by means of said optical device (7) subsequent phase P _± following the step of controlled release of said marker (4) so as to detect said at least one microorganism (2) according to the evolution of the labeling kinetics of said microorganism (2); f) A comparative analysis is carried out, by portion of the detection support (1, 21), between said image (I ₀ ) taken at P ₀ and each image (Ii) taken in at least one subsequent phase Pi and we conclude as to the presence or absence of at least one microorganism (2) in said sample (3).

2. Detection method according to the preceding claim, characterized in that the said detection medium (1, 21) is punctually illuminated via a light radiation (6) at each image taking at P ₀ , P _± .

3. Detection method according to any one of the preceding claims, characterized in that it comprises, at least, the following steps, taken in 1 order:

al) depositing said sample (3) to be analyzed on said solid detection support (1, 21); bl) The detection support (1, 21) is punctually illuminated via light radiation (6) capable of revealing the at least one cellular marker (4) cl) Simultaneously with the illumination step bl), at least an image (I ₀ , I'o) in a first phase P _{0 of} said sample (3) on at least a portion of said detection support (1, 21) by means of an optical device (7) for taking pictures aimed a field of view at least 10 mm long by at least 10 mm wide;

dl) a controlled release of said at least one cell marker (4) is carried out, through the detection support (1, 21), for bringing said marker (4) into contact with said microorganism (2); el) In a period of time less than 5 seconds after step dl) of controlled release of said cell marker (4), said detection medium (1, 21) is punctually illuminated via said light radiation (6) capable of revealing said at least one cell marker (4);

fl) Simultaneously with the illumination step el), an image (Ii, I'i) of each portion of the detection support (1, 21) having made the image is taken, by means of said optical device (7). object of an image at P ₀ in at least one subsequent phase Pi following the step of controlled release of said marker (4);

gl) once again, at least once, said detection support (1, 21) is illuminated via said light radiation (6);

hl) Simultaneously with the illumination step gl), an image (I ₂ , 1 · '2) of each portion of the detection support (1, 21) having been made is taken, by means of said optical device (7). the object of an image at P ₀ and at Pi in at least a second subsequent phase P ₂ so as to detect said at least one microorganism (2) according to the evolution of the labeling kinetics of said microorganism (2);

ii) A comparative analysis is carried out, by portion of the detection support (1, 21), between said image

(I ₀ , I '0) taken at P ₀ and said images (Ii, I'i; I ₂ , I' 2) taken in at least two subsequent phases Pi and P ₂ and we conclude as to the presence or not of d 'at least one microorganism (2) in said sample (3).

4. Detection method according to any one of the preceding claims, characterized in that the detection support (1, 21) is subjected to n displacements according to separate paths with a view to a spatial division of said detection support (1, 21) in n + 1 portions subject to shots in at least two phases P ₀ and Pi, n being an integer less than or equal to 20.

5. Detection method according to any one of the preceding claims, characterized in that, during the image taking steps c) and e) or cl), fl) and hl), an optical device is used ( 7) of shots having a resolution at least equal to 20 megapixels, and preferably greater than 100 megapixels.

6. Detection method according to any one of the preceding claims, characterized in that, if it is concluded that at least one microorganism (2) is present in said sample (3) during step f) or it), a count of the microorganisms (2) present in said sample (3) is carried out.

7. Detection method according to any one of the preceding claims, characterized in that said cell marker (4) is a fluorescent marker.

8. Detection method according to any one of the preceding claims, characterized in that said cell marker (4) released in a controlled manner during step d) or dl) is enveloped in an encapsulation means (8) .

9. Detection method according to the preceding claim, characterized in that said cell marker (4) is enveloped in an encapsulation means (8) in solid form, for example in the form of a powder, and in that said detection support (1) comprises a solvent for dissolving said marker (4).

10. Detection method according to claim 8 characterized in that said cell marker (4) is encapsulated in liquid form.

11. Detection method according to one of claims 8 to 10, characterized in that said encapsulation means (8) is thermosensitive and, in step d) or dl), a controlled release of the cell marker is carried out. (4) wrapped in said heat-sensitive encapsulation means (8) by subjecting the latter to an increase in temperature capable of melting said heat-sensitive encapsulation means (8).

12. Detection method according to the preceding claim, characterized in that the temperature is increased from ambient temperature to a maximum temperature of 45 ° C.

13. Detection method according to any one of claims 1 to 7, characterized in that the cell marker (4) is conveyed in liquid solution in a controlled manner up to said solid detection support (21) containing the sample. (3) to analyze.

14. Detection method according to any one of the preceding claims, characterized in that a volume between 10 and 2000 μL of said sample (3) to be analyzed is directly deposited on said detection support (1, 21).

15. Detection method according to any one of claims 1 to 13, characterized in that said sample (3) to be analyzed is filtered through a filter membrane (5, 25) having a cut-off threshold of 0 , 2 and 0.45 µm, preferably a cutoff threshold equal to 0.3 µm, said filter membrane (5, 25) being positioned directly in said detection support (1, 21).

16. Detection method according to any one of the preceding claims, characterized in that to distinguish the microorganisms from the dust on the detection supports (1, 21) having received the samples to be analyzed, the following steps are carried out:

- measurement after each shot of the brightness of the pixels or groups of luminescent pixels detected;

- comparison of the brightness of each pixel or group of luminescent pixels detected on the different shots;

- identification of pixels or groups of pixels whose brightness does not change appreciably in at least two consecutive shots during the marking kinetics;

- analysis and counting of pixels or groups of pixels whose brightness changes during the marking kinetics.

17. Detection device (14) of at least one microorganism (2), of the bacteria, yeast or mold type, present in a sample to be analyzed (3), capable of implementing the method of the preceding claims, characterized in that that it includes, at least:

- a solid detection support (1, 21) intended to receive said sample to be analyzed (3) capable of containing at least one microorganism (2);

- means for storing at least one cell marker (4) of said microorganism (2);

- means (15) for controlled release of said cell marker (4) for bringing said marker (4) into contact with said sample (3) to be analyzed;

- an illumination means (6) of said detection support (1, 21);

- an optical device (7) for taking pictures of at least a portion of said detection support (1, 21), said optical device (7) having a field of observation of at least 10 mm in length over at least 10 mm wide; means for memorizing, comparing and analyzing the shots taken by the optical device (7) so as to detect said at least one microorganism (2) possibly present in said sample (3) according to the evolution of the kinetics of labeling said at least one microorganism (2).

18. Detection device (14) according to the preceding claim, characterized in that it comprises means for moving said detection support (1, 21) under said optical device (7) for taking pictures.

19. Detection device (14) according to any one of claims 17 or 18, characterized in that said solid detection support (1, 21) comprises at least one filtering membrane (5, 25) resting on a support disc ( 13, 23) made of fiberglass.

20. Detection device (14) according to any one of claims 17 to 19, characterized in that said storage means (19) of the cell marker (4) is included in the detection support (1) and consists of a layer of microcapsules (9) consisting of said cell marker (4) wrapped in an encapsulation means (8), said layer of microcapsules (9) being disposed between said support disc (13) made of glass fibers and said filter membrane ( 5).

21. Detection device (14) according to the preceding claim, characterized in that said means for controlled release of the cell marker (4) comprises heating means and means for controlling and regulating the temperature capable of effecting an increase in the temperature within the device (14) and melt said encapsulation means (8) enveloping cell marker (4).

22. Detection device (14) according to any one of claims 17 to 19, characterized in that said cell marker storage means (4) consists of a reserve of liquid cell marker solution (4) distinct from the support of detection (21) and that said detection device (14) comprises means for conveying said cell marker (4) from said storage means to said detection medium (21).

23. Detection device (14) according to the preceding claim, characterized in that said release means controlled cell marker (4) comprises at least one pump with controlled flow.

24. Detection device (14) according to any one of claims 17 to 23, characterized in that said optical device (7) for taking pictures has a depth of field of the order of or equal to 0.5 mm .

25. Detection device (14) according to any one of claims 17 to 24, characterized in that said optical device (7) for taking pictures is based on a camera with CMOS sensors.

26. Detection device (14) according to any one of claims 17 to 25, characterized in that said optical device (7) for taking pictures has a field of vision of 25 mm * 25 mm.

27. Detection device (14) according to any one of claims 17 to 26, characterized in that said optical device (7) for taking pictures has a resolution at least equal to 20 megapixels and preferably greater than 100 megapixels.

28. Detection support (21) able to be implemented in the detection method according to any one of claims 1 to 15 or entering into the composition of the detection device (14) according to any one of claims 17 to 27 characterized in that it is composed, at least, of a rigid base (26) crossed by at least one conduit for supplying (26a) a liquid and at least one suction and evacuation conduit ( 26c) of a liquid, said supply conduit (26a) being extended by a chimney (26b) projecting from said base (26) and passing through, on the one hand, a first orifice (24a) formed in a support means (24) rigid and permeable surmounting said base (26) and, on the other hand, a second orifice (23a) formed in a flexible glass fiber support disc (23), said disc

(23) being positioned on said rigid and permeable support means (24) and supporting a filter membrane (25) having a cut-off threshold of 0.2 and 0.45 pm, suitable for the retention of microorganisms (2), said support detection

(21) further comprising a clamping ring (27) fixed integrally to said base (26) and holding together said base (26), said rigid and permeable medium support

(24), said support disc (23) and said filter membrane (25).