How to detect microorganisms

Abstract

The present invention relates to a method and associated device for observing, detecting and optionally counting, if applicable, microbiological objects on a support, such as a membrane or solid growth medium. In particular, the method comprises the steps of: a) illuminating an area of ​​the support with at least two incident collimated light sources forming an angle (α) of at least 10° with respect to the normal of the support, b) acquiring an image of the area of ​​the support illuminated by the at least two incident collimated light sources by a receiving element having its optical acquisition axis along the normal of the support, and c) detecting the presence or absence of microbiological objects on the area of ​​the support by identifying on the acquired image the light reflected, scattered and / or diffused from the support and from the microbiological objects on the support.

Description

[Technical field]

[0001] The present invention relates to the field of microbiology, and more specifically to a method for observing, where applicable detecting and optionally enumerating microbiological objects on a support, such as a membrane or a solid growth medium. [Background technology]

[0002] Microorganisms need to be monitored in the environment (bath water or drinking water) or in raw materials and final products designed for consumption or use by humans and animals, e.g. foods, beverages, etc., medical devices, medicines or cosmetics. The presence of microorganisms can not only lead to deterioration or alteration of the product, but also to disease when consumed or administered to humans or animals. To ensure the safety of such products, microbiological tests are required to check the risk of contamination under normal conditions of use, thus preventing poisoning or epidemics. Especially for the medical device, pharmaceutical or cosmetic industry, the need for sterility is very important, and since product defects can occur at any stage of production, it is important to test the microbiological quality of products and raw materials throughout the supply chain.

[0003] Of course, microorganisms can also infect humans or animals, and detection and / or quantification of the microorganisms involved in an infectious disease is required in clinical diagnosis. For example, the diagnosis of a urinary tract infection (UTI) can be performed through quantitative urine culture. Traditionally, the presence of 1,000 or more bacteria per ml of urine is considered to represent significant bacteriuria, suggesting a UTI.

[0004] In all these industries, both for clinical use and for environmental monitoring, the contamination thresholds and the methods used may differ, however it is always important to reveal the presence of microorganisms at the earliest possible stage.

[0005] Louis Pasteur's method of culturing on petri dishes has been the most effective method for identifying and counting microorganisms since the 19th century. To this day, this technique still remains the reference method for detecting and counting microorganisms in many industries. The method is simple: extracting the microorganism from a sample, placing it on a dedicated petri dish containing a suitable medium, and counting the colonies when they become visible. Although reliable, the method is lengthy (up to several days depending on the target microorganism).

[0006] To shorten the length of the assay, it has been proposed to use indicators, such as chromogenic substrates, fluorescence-inducing substrates, or fluorescent or radiolabeled antibodies to detect the smaller sized microcolonies (e.g., International Patent Applications WO 96 / 14431, WO 2013 / 050598, WO 2008 / 118400). However, in most of these methods, the growth and indicator stages must be separated to obtain optimal results: the growth stage does not contain harmful indicators therein that slow growth in order to allow fast growing cells, and the indicator stage is specialized for color development and identification that efficiently allows the detection of the smaller sized microcolonies.

[0007] Over the years, many other attempts have been made to reduce the time required for the assay, in particular using culture-independent approaches based on immunological methods, nucleic acid amplification methods, or flow cytometry methods (using labeling with fluorescent antibodies or fluorescence-inducing substrates). However, these methods are still expensive, have a non-trivial workflow, require highly skilled experts and / or concentrated samples, and, among other things, are very difficult to distinguish between live and dead microorganisms. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO 96 / 14431 [Patent Document 2] International Publication No. 2013 / 050598 [Patent Document 3] International Publication No. 2008 / 118400 [Non-patent literature]

[0009] [Non-Patent Document 1] Ismail et al., Int J Environ Res Public Health. 2013 Nov 14;10(11):6169-83 Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, a need remains for rapid, sensitive, user-friendly, and cost-effective methods for detecting and / or enumerating microorganisms in a sample. [Means for solving the problem]

[0011] The present inventors provide herein a method that fulfills this need. More specifically, a novel method is provided that allows for the observation and, where applicable, detection of microbial colonies at the very early stages of growth, i.e., when the colonies are too small in diameter to be visible to the naked eye. This method does not require the use of any additional reagents, such as labeling agents, that facilitate detection.

[0012] In a first aspect, the present invention relates to a method for detecting a microbiological object on a support, comprising the steps of: a) illuminating an area of ​​the support with at least two incident collimated light sources that form an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being independently selected for each incident collimated light source; b) acquiring an image of the area of ​​the support illuminated by the at least two incident collimated light sources by a photodetector having its optical acquisition axis along a normal to the support; c) detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from the support and from microbiological objects on said support; The present invention relates to a method comprising the steps of:

[0013] Preferably, the microbiological object to be detected is selected from the group consisting of filamentous fungi, and colonies / microcolonies of bacteria, archaea, and yeasts, and combinations thereof, more preferably selected from the group consisting of filamentous fungi, and colonies / microcolonies of bacteria and yeasts, and combinations thereof, and even more preferably includes filamentous fungi, and colonies / microcolonies of bacteria and / or yeasts.

[0014] Preferably, the microbiological objects to be detected are individualizable or individualized microbiological objects.

[0015] Preferably, the microbiological objects to be detected are not labeled with compounds or moieties that produce a photonic signal.

[0016] Preferably, steps b) and c) are carried out multiple times in order to image several areas of said support, preferably in order to image the entire support.

[0017] The method may further comprise a step d) of combining the acquired images of said area to form a combined image.

[0018] The support may be a membrane filter, preferably made of mixed cellulose esters (MCE), polyvinylidene fluoride (PVDF), nitrocellulose, polytetrafluoroethylene, polycarbonate, or nylon, or a combination thereof, more preferably made of mixed cellulose esters (MCE), polyvinylidene fluoride (PVDF), polyestersulfone (PES), nitrocellulose, polytetrafluoroethylene, polycarbonate, or nylon, even more preferably made of mixed cellulose esters (MCE) or polyvinylidene fluoride (PVDF).

[0019] Alternatively, the support can be a solid growth medium.

[0020] In some embodiments, the support is in a container and is covered by a translucent lid.

[0021] In some embodiments, the method further comprises, after step b) and before step c), repeating steps a) and b) one or more times, each time using a different set of at least two incident collimated light sources forming an angle (α) of at least 10° with respect to the normal to the support, thereby obtaining multiple captured images of the area, and combining the captured images of the area to form a combined image of the area, wherein the numerical value of the angle (α) is selected independently for each incident collimated light source.

[0022] Preferably, in this embodiment, in step c) the presence or absence of a microbiological object on the area of ​​the support is detected by identifying light reflected, scattered and / or diffused from the support and from the microbiological object on the support on the combined image of the area.

[0023] In some embodiments, steps a) and b), or a), b) and b') are performed multiple times to image several areas of the support, preferably to image the entire support. The method may further comprise, after step b) or b'), step b''), combining the acquired images of the area to form a combined image of the area. In step c), the presence or absence of microbiological objects on the area of ​​the support may be detected by identifying light reflected, scattered and / or diffused from the support and from the microbiological objects on the support on the combined image of the area.

[0024] Preferably, steps a) and b) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times using 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different sets of at least two input collimated light sources, respectively. More preferably, steps a) and b) are repeated 1, 2, 3, 4, 5, 6, 7, or 8 times using 1, 2, 3, 4, 5, 6, 7, or 8 different sets of at least two input collimated light sources, respectively.

[0025] Preferably, the two sets of collimated input light sources differ in their number, their light type, their position, and / or the value of the angle α of each light source. More preferably, the two sets of collimated input light sources differ in their number, their position, and / or the value of the angle α of each light source.

[0026] Preferably, the light receiving element is a camera comprising an array of pixel sensors, preferably a CCD (charge coupled device) image sensor, or an active pixel sensor, such as a CMOS (complementary metal oxide semiconductor) image sensor.

[0027] Preferably, in step a) (the first time of step a) or any of its repetitions), the support is illuminated with a set of at least 3, preferably 3 to 24, more preferably 3 to 12, and even more preferably 3 to 6, incident collimated light sources. In particular, in step a) (the first time of step a) or any of its repetitions), the support may be illuminated with a set of 3 incident collimated light sources.

[0028] Preferably, the input collimated light sources are evenly distributed on a circle centered on the optical acquisition axis. In particular, the input collimated light sources of a set may be evenly distributed on one or several circles centered on the optical acquisition axis.

[0029] Each incident collimated light source forms an angle (α) with respect to the normal to the support that can be independently selected between 15° and 75° with respect to the normal to the support, preferably between 25° and 65° with respect to the normal to the support, and more preferably between 30° and 60° with respect to the normal to the support.

[0030] Preferably, the set comprises at least three input collimated light sources, and the value of the angle α is the same for all light sources of the set.

[0031] Preferably, the input collimated light source is selected from a collimated light emitting diode (LED), and a laser diode, and combinations thereof. More preferably, the input collimated light source is a collimated white LED.

[0032] The method may further comprise the step of counting and / or identifying the microbiological objects on the support from the acquired images or the combined image.

[0033] The method may further comprise, prior to step a), the steps of providing a sample to be tested, contacting said sample with a support, and incubating said support to allow growth of microorganisms, if present.

[0034] The method of the invention may be used to detect microorganisms in a sample or to test the sterility of a sample.

[0035] In particular, the sample may be obtained from a liquid, a solid or a gas. Preferably, the sample is selected from the group consisting of a biological sample, an environmental sample, a medical device or any part thereof, a food or beverage for human or animal consumption, a medicine or cosmetic, and ingredients of such a food, beverage, medicine or cosmetic.

[0036] In a second aspect, the present invention provides a device for detecting a microbiological object on a support, preferably a membrane filter or a solid growth medium, comprising: at least two collimated input light sources for illuminating the support, said at least two collimated input light sources forming an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being independently selected for each collimated input light source; a light receiving element for acquiring an image of an area of ​​the support illuminated by the at least two incident collimated light sources, the light receiving element having its optical acquisition axis along a normal to the support; means for detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from the support and from microbiological objects on said support; Optionally, means for holding and optionally moving the support; The present invention relates to a device comprising:

[0037] Preferably, the device comprises at least 3, preferably 3 to 50, more preferably 3 to 24, and even more preferably 3 to 12 input collimated light sources. Alternatively, the device may comprise at least 13 input collimated light sources, preferably 15 to 50, more preferably 15 to 30, and even more preferably 24 input collimated light sources.

[0038] Preferably, each incident collimated light source forms an angle (α) with respect to the normal to the support that is independently selected between 15° and 75°, preferably between 25° and 65°, more preferably between 30° and 60°.

[0039] Preferably, the value of the angle α is the same for incident collimated light sources that are symmetric about the optical acquisition axis.

[0040] In particular, the device (i) at least 3, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, incident collimated light sources having an angle (α) value between 45° and 55°; or (ii) at least 3, preferably 3, 4, 5, or 6, input collimated light sources with an angle (α) value between 30° and 40°, and at least 3, preferably 3, 4, 5, or 6, input collimated light sources with an angle (α) value between 45° and 55°, or (iii) at least 3, preferably 3, 4, 5, or 6 input collimated light sources having an angle (α) value between 25° and 35°, at least 3, preferably 3, 4, 5, or 6 input collimated light sources having an angle (α) value between 40° and 50°, and at least 6, preferably 7, 8, 9, 10, 11, or 12 input collimated light sources having an angle (α) value between 60° and 70°, or (iv) at least three, preferably three, input collimated light sources having an angle (α) value between 28° and 32°, at least three, preferably three, input collimated light sources having an angle (α) value between 33° and 37°, at least three, preferably three, input collimated light sources having an angle (α) value between 38° and 42°, at least three, preferably three, input collimated light sources having an angle (α) value between 48° and 52°, at least three, preferably three, input collimated light sources having an angle (α) value between 53° and 57°, and at least three, preferably three, four, five, six, seven, eight, nine, input collimated light sources having an angle (α) value between 60° and 64°. It may be equipped with.

[0041] Preferably, the device comprises at least 3 input collimated light sources, more preferably 4 to 12 input collimated light sources, said input light sources being evenly distributed on a circle centered on the optical acquisition axis and each input light source forming an angle (α) independently selected between 15° and 75° with respect to the normal to the support, preferably between 30° and 60° with respect to the normal to the support.

[0042] The present invention also relates to the use of the device of the invention for detecting and optionally counting and / or identifying microbiological objects on a support, preferably based on the method of the invention.

[0043] The present invention also relates to the use of the device of the present invention for detecting microorganisms in a sample or for testing the sterility of a sample, preferably using the method of the present invention. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 shows a schematic diagram of one embodiment of a device of the present invention. [Diagram 2]Figure 1 illustrates an illustration of the technical results of the present invention applied to the detection of bacteria, yeasts and filamentous fungi: Bacterial microcolonies with a size of about 200-500 μm in diameter (P. aeruginosa, E. coli, R. piketti), 500-700 μm (L. pneumophila, B. cepacia) or 1-2 mm (B. subtilis); Yeast cells with a size of about 500 μm in diameter (yeast fungus C. albicans) or filamentous fungi (A. brasiliensis) that fit into a window size of ≈5×5 mm. Each image shows the scale factor with a white line representing about 100 μm. Microorganisms were grown on PDVF membranes, black cellulose mixed ester (MCE) membranes, white MCE membranes, or black polyethersulfone (PES) membranes. [Diagram 3] Figure 1 illustrates an illustration of the technical results of the present invention as applied to the detection of bacteria, yeasts, and molds. The incidence angle α was set at 37°, 50°, or 60°. Each image shows a scale factor with a white line representing approximately 100 μm. Microorganisms were grown on PDVF membranes, black MCE membranes, or white MCE membranes. [Figure 4] Figure 1 illustrates an illustration of the technical results of the present invention, applied to the detection of bacteria, yeasts and moulds. The membrane was illuminated with white, UV, blue or red light. Among the possible reflected light, only those carrying the same colour as the illumination were collected. Microorganisms were grown on PDVF membranes or on black PES membranes. Each image shows the scale factor with a white line representing approximately 100 μm. [Diagram 5]Figure 1 illustrates an illustration of the technical results of the invention applied to the detection of bacteria, yeasts and moulds. In the reference condition "no lid", the membrane was not covered and was directly exposed to the incidence light. In the other conditions, a lid was added to the top of the membrane (between the membrane and the light illumination / reception system). The distance between the lid and the membrane was set to 1-3 mm. In such conditions, the incident light had to pass through the lid before reaching the sample and the reflected light had to pass through the lid before reaching the sensor. Three different materials for the lid were investigated: Lid 1: plastic from a Merck filtration unit (Merck Millipore, EZ-FIT filtration unit, Ref EFHVW10IS), Lid 2: UV grade fused silica (Chroma corp., thickness: 1 mm), Lid 3 = soda lime glass (Selba corp., thickness: 1.8-2 mm). Microorganisms were grown on PDVF membranes or on white MCE membranes. Each image shows scale with white lines representing approximately 100 μm. [Figure 6] Figure 1 shows an illustration of the technical results of the present invention as applied to the detection of bacteria, yeasts, and molds. Microorganisms were grown on PDVF, black MCE, white MCE, black PES, or nylon membranes. Each image shows the scale factor with the white line representing approximately 100 μm. [Figure 7-1] Figure 1 illustrates an illustration of the technical results of the present invention as applied to the detection of A. brasiliensis or E. coli. The microorganisms were detected either directly on the surface of the solid growth medium or on a PVDF or MCE membrane layered on the surface of the solid growth medium. [Figure 7-2] Continued from Figure 7. [Figure 8]Figure 1 shows the technical results of the invention applied to the detection of C. albicans. Microorganisms were grown on white MCE membranes (either gridded or non-gridded) and detected after 23, 25 or 28 hours of growth on solid growth medium. At each investigated time, the number of C. albicans detected was counted in standard condition 0 (3 LEDs, incidence angle α set to 50°), condition 1 (6 LEDs, incidence angle α set to 37°) or condition 2 (12 LEDs, 6 of which had incidence angle α set to 50°, for 6 of which α=37°) and the results are expressed as a percentage of detection by MICA relative to the number counted on the same membrane after re-incubation for 48 hours until colonies were visible to the naked eye. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The object of the present invention is to provide a method and a corresponding device for detecting and optionally counting microbiological objects, such as microorganisms or microbial colonies, present on a support at a very early stage of their growth. The inventors have demonstrated here that this method offers a very flexible solution for detecting a great variety of microorganisms on several types of supports and in various conditions, including closed containers to prevent contamination from the environment. This method further exhibits the advantage of allowing an early detection of such microbiological objects without requiring, but allowing, the use of any additional reagents, such as labeling agents or dyes, that facilitate the detection.

[0046] Thus, in a first aspect, the present invention relates to a method for detecting a microbiological object on a support, comprising: a) illuminating an area of ​​the support with at least two incident collimated light sources that form an angle (α) of at least 10° with respect to the normal of the support; b) acquiring an image of the area of ​​the support illuminated by the at least two incident collimated light sources by a photodetector having its optical acquisition axis along a normal to the support; c) detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from the support and from microbiological objects on said support; The present invention relates to a method comprising the steps of:

[0047] As used herein, the term "microbiological object" refers to a microorganism in isolated form (e.g., a filamentous fungus) or a cluster of microorganisms, i.e., a colony or microcolony of a microorganism. Preferably, the microbiological object has a dimension (e.g., diameter, length, or width) of at least 10 μm. More preferably, the microbiological object has a dimension (e.g., diameter, length, or width) of at least 10 μm and is invisible or nearly invisible to the naked eye. The microbiological object to be detected by the method of the present invention may comprise or consist of a microorganism in isolated form, in particular a filamentous fungus, a colony or microcolony, and a mixture thereof, i.e., a combination of (i) a microorganism in isolated form, (ii) a colony or microcolony.

[0048] The microbiological objects to be detected by the method of the invention are preferably individualizable or individualized microbiological objects. In particular, such objects are preferably not included in a microbial lawn. An individualizable microbiological object is an object whose outline can be visually distinguished from the outlines of other microbiological objects. An individualizable microbiological object may be isolated from other objects or may partially overlap with other objects, for example two overlapping colonies. An individualized microbiological object is an object isolated from other objects, for example an isolated colony or microcolony, or an isolated filamentous fungus. In a preferred embodiment, the microbiological objects to be detected by the method of the invention are individualizable or individualized (i) filamentous fungi and / or (ii) colonies / microcolonies of bacteria, archaea and / or yeasts. In particular, the microbiological objects to be detected are selected from the group consisting of filamentous fungi, and colonies / microcolonies of bacteria, archaea, and yeasts, and combinations thereof, and are preferably selected from the group consisting of filamentous fungi, and colonies / microcolonies of bacteria and yeasts, and combinations thereof. In particular, the microbiological objects to be detected may include filamentous fungi, and colonies / microcolonies of bacteria and / or yeasts.

[0049] In some preferred embodiments, the microbiological object comprises or consists of a microcolony. As used herein, the term "microcolony" refers to a colony that grows for hours or days, depending on the microorganism, and is invisible or nearly invisible to the naked eye. Generally, the diameter of a microcolony is less than 500 μm, preferably between 10 μm and 500 μm, more preferably between 30 μm and 500 μm, and even more preferably between 30 μm and 200 μm.

[0050] As used herein, the term "microorganism" refers to bacteria, archaea, or microscopic fungi. Preferably, the term refers to bacteria, yeasts (i.e., unicellular microscopic fungi), or filamentous fungi (i.e., multicellular and filamentous microscopic fungi). The microorganism to be detected may be selected from the group consisting of bacteria, archaea, and microscopic fungi, and combinations thereof. Preferably, the microorganism to be detected is selected from the group consisting of bacteria, and microscopic fungi, and combinations thereof. The microorganism to be detected may be a pathogenic or non-pathogenic microorganism. Preferably, the microorganism is a pathogenic microorganism, or a non-pathogenic microorganism that may, for example, deteriorate or degrade a product (e.g., food, beverage, medical device, pharmaceutical, or cosmetic product), or the environment (e.g., swimming pool, groundwater).

[0051] In some embodiments, the microorganisms to be detected include bacteria. Such bacteria are detectable in the form of colonies or microcolonies, preferably in the form of microcolonies. Such bacteria can be aerobic, anaerobic, or facultative anaerobic bacteria, and gram-negative or gram-positive bacteria. In particular, the microorganisms to be detected are those of the genus Aeromonas (e.g., Aeromonas hydrophila), Alcaligenes (e.g., Alcaligenes faecalis), Acinetobacter (e.g., Acinetobacter aceti, Acinetobacter baumanii), Alicyclobacillus (e.g., Alicyclobacillus acidiphilus, Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, Alicyclobacillus ketoconidioides ... acidoterrestris, Alicyclobacillus contaminans, Alicyclobacillus cycloheptanicus, Alicyclobacillus herbarius, Alicyclobacillus hesperidum), Asaia (e.g., Asaia siamensis), Bacillus (e.g., Bacillus subtilis), Brevundimonas (e.g., Brevundimonas diminuta), Burkholderia (e.g., Burkholderia cepacia),cepacia), Citrobacter (e.g., Citrobacter freundii), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., Cutibacterium acnes), Edwardsiella (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Enterococcus (e.g., Enterococcus faecalis), Escherichia (e.g., Escherichia coli), Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), Gluconobacter (e.g., Gluconobacter oxydans), Klebsiella (e.g., Klebsiella pneumoniae), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus nagelii, Lactobacillus plantarum), Legionella (e.g., Legionella pneumophila), pneumophila), Methylobacterium (e.g., Methylobacterium extorquens), Micrococcus(e.g., Micrococcus luteus), Moraxella (e.g., Moraxella osloensis), Ochrobactrum (e.g., Ochrobactrum anthropi), Pantoea (e.g., Pantoea agglomerans), Pediococcus (e.g., Pediococcus pentosaceus), Proteus (e.g., Proteus mirabilis), Pseudomonas (e.g., Pseudomonas aeruginosa), Ralstonia (e.g., Ralstonia pickettii), Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Shigella (e.g., Shigella sonnei), Sphingomonas (e.g., Sphingomonas paucimobilis), Staphylococcus (e.g., Staphylococcus aureus), aureus), Streptococcus (e.g., Streptococcus pneumoniae), Vibrio (e.g., Vibrio parahaemolyticus), Weissella(e.g., Weissella confusa), Yersinia (e.g., Yersinia enterocolitica), and combinations thereof.

[0052] In some embodiments, the microorganisms to be detected include yeasts or filamentous fungi. Yeasts can be detected in colony or microcolony form, preferably in microcolony form. Filamentous fungi can be detected in isolated form (detection of indigenous organisms). In particular, the microorganisms to be detected are those of the genera Aspergillus (e.g., Aspergillus brasiliensis), Candida (e.g., Candida albicans), Geotrichum (e.g., Geotrichum candidum), Penicillium (e.g., Penicillium chrysogenum, Penicillium variotii), Saccharomyces (e.g., Saccharomyces cerevisiae), Zygosaccharomyces (e.g., Saccharomyces cerevisiae), and the like. (e.g., Zygosaccharomyces bailii), or filamentous fungi, and combinations thereof.

[0053] The microbiological target to be detected may be contained in the sample to be analyzed. This sample may be obtained from a liquid (e.g. water, fruit juice, beer, wine, biological fluids such as urine, etc.), a solid (e.g. food, medicine or cosmetic products, medical devices, or any solid surface), or a gas (e.g. air). Depending on the form of the product / environment to be tested, the sample may be allowed to grow directly on the support or may be subjected to a preliminary step before it can grow on the support.

[0054] In particular, the sample may be a liquid specimen or a liquefied sample. As used herein, the term "liquefied sample" refers to a liquid sample obtained from a solid sample. In some cases, the solid sample may be dissolved or suspended in a liquid medium through physical and / or chemical treatment. Microorganisms may also be extracted from various surfaces or devices using any method known to those skilled in the art, such as swabbing, scraping (e.g., using a wipe), printing (e.g., by agar contact method), rinsing or immersion, or ultrasonication, particularly to remove biofilms (see, for example, Ismail et al., Int J Environ Res Public Health. 2013 Nov 14;10(11):6169-83, which is incorporated herein by reference).

[0055] Liquid samples may contain suspended matter. However, if necessary, residual suspended matter may be removed from the liquid medium using a suitable method, preferably one that minimizes loss of microorganisms, for example, by low speed centrifugation or filtration using a suitable pore size. The liquid medium used to suspend the solid sample may be any suitable solvent, such as sterile water, a buffer solution, or a liquid culture medium.

[0056] Optionally, the liquid or liquefied sample may be diluted (eg, serially diluted) or concentrated prior to step a), using any suitable method, such as centrifugation or filtration.

[0057] The sample analyzed using the methods of the present invention can be any sample in which it is desired to determine whether it is contaminated with a microorganism.

[0058] Examples of samples include, but are not limited to, biological samples (e.g., saliva, nasopharyngeal samples, urine, feces, blood, plasma, cerebrospinal fluid, or mucus samples), environmental samples (e.g., domestic, commercial, or industrial water, wastewater, cooling water, boiler water, ground water, recreational water, process water, water treatment unit effluent, soil, or other environmental material), medical devices or any parts thereof, foods or beverages for human or animal consumption (e.g., dairy products, raw materials, drinking water, fruit juice, beer, wine, water used in formulating products), pharmaceuticals or cosmetics, as well as ingredients of such foods, beverages, pharmaceuticals, or cosmetics.

[0059] Before being observed using the method of the invention, the microbiological object can be grown on a support. In particular, the sample can be contacted with the support to allow the growth of microorganisms, if present. The support can be a membrane filter or a solid growth medium.

[0060] As used herein, the term "growth medium" or "culture medium" refers in the context of the present invention to a nutrient medium used to grow microorganisms. A growth medium can be a defined medium (hence the exact molecular composition is known) synthesized from individual chemicals, or an undefined medium containing some complex components, such as yeast extract or casein hydrolysate, composed of a mixture of many chemical species in unknown proportions. The growth medium used in the method of the present invention can be a non-selective or selective growth medium. A non-selective growth medium is a general medium for bacterial growth, fungal growth, or both bacterial and fungal growth. A non-selective medium generally contains the nutrients required to support the growth of a wide variety of microorganisms. A selective growth medium is a medium used to grow only selected microorganisms. In fact, the composition of a selective medium ensures the growth of cells that have certain characteristics, such as antibiotic resistance, or the ability to synthesize certain metabolites, such as amino acids. This medium thus provides an environment that is more favorable for the growth of the target microorganism than non-target microorganisms that may be present in the sample.

[0061] The expression "solid growth medium" or "solid culture medium", as used herein, refers to a growth medium that allows microorganisms to form microcolonies on its surface, such as a medium that has a gel-like appearance or is in the form of a gel, where a gel is a colloidal system in which a porous network of interconnected particles extends throughout the volume of the liquid medium and allows nutrients to diffuse through the medium and become available to the microorganism. Preferably, a solid growth medium, as used herein, is prepared by adding to a liquid growth medium a sufficient amount of a gelling agent, such as agar, agarose, alginate, carrageenan, cellulose, gelatin, pectin, and combinations thereof. Generally, a solid growth medium contains a gelling agent, preferably agar, at a concentration of 0.5% to 3%, preferably 1% to 2.5%. Preferably, the solid growth medium used in the method of the present invention is an agar growth medium. The solid growth medium is poured into a container. This enclosure is any sterile container dedicated to microbial culture, in particular a conventional Petri plate.

[0062] In some embodiments in which the support is a solid growth medium, the method may further comprise, prior to step a), contacting the sample in the container with a solid growth medium containing nutrients that support the growth of the microorganism of interest.

[0063] In some embodiments where the support is a membrane filter, the method may further comprise, prior to step a), concentrating the microorganisms of the sample on a membrane filter and then contacting said membrane with a growth medium, e.g. a pad immersed in a solid growth medium or a liquid growth medium, preferably a solid growth medium. During an incubation period, nutrients are passed through the filter, allowing the growth of the microorganisms on the upper surface of the membrane.

[0064] Liquid, liquefied, or gas samples may be filtered / concentrated using a sterile membrane filter suitable for retaining the microorganisms contained in the sample, typically a microfiltration membrane filter with a pore size smaller than the target microorganism. The sample may be passed through the membrane using a filter funnel and a vacuum system. Preferably, the membrane filter has a nominal pore size of 1.2 μm or less, particularly a pore size of 0.22 μm to 1.2 μm, or a pore size of 0.22 μm to 0.8 μm. In some preferred embodiments, the membrane filter has a nominal pore size of 0.45 μm or less, particularly a pore size of 0.22 μm to 0.45 μm. The diameter of the filter may depend on the device used to filter the sample and the size of the container that contains the growth medium. For example, the container may be a Petri dish with a diameter of 100 mm, 90 mm, or 55 mm, and the membrane may have a diameter of 47 mm, 50 mm, or less.

[0065] The membrane filter may be made of any suitable material, such as mixed cellulose esters (MCE), polyvinylidene fluoride (PVDF), polyestersulfone (PES), nitrocellulose, polytetrafluoroethylene, polycarbonate, or nylon, or a combination thereof. Preferably, the membrane filter is made of mixed cellulose esters (MCE), polyvinylidene fluoride (PVDF), nitrocellulose, polytetrafluoroethylene, polycarbonate, or nylon. More preferably, the membrane filter is made of mixed cellulose esters (MCE) or polyvinylidene fluoride (PVDF). The membrane filter may be white or colored, such as black, and / or may be a grid membrane.

[0066] In a preferred embodiment, the sample dilution and / or filtration sample volume is 1 cm 2 of the membrane filter or solid growth medium surface. 2 The number of microbiological objects per sample is adjusted to a maximum of 1000, preferably a maximum of 300.

[0067] In some embodiments where the support is a membrane filter, the membrane can be kept on the growth medium during the illumination and image acquisition steps (steps a) and b), optionally repeated), or can be removed from the growth medium and placed on a sample holder before step a). Optionally, the membrane can be placed on a sample holder in a container, for example a Petri dish. The container can be an open container or can be closed with a translucent lid.

[0068] In some embodiments where the support is a solid growth medium, the support is a container, e.g. a Petri dish. Prior to step a) of the method, the container can be opened by removing the lid used during the culture period or can be kept closed, i.e. with a translucent lid.

[0069] In these embodiments, the semi-transparent lid is disposed between the microbiological object and the light receiving element, facing the microbiological object and not in contact with the object. Preferably, the semi-transparent lid is disposed at a distance of 1 mm to 10 cm, more preferably 1 mm to 5 cm, and even more preferably 1 mm to 1 cm from the microbiological object.

[0070] In step a) of the method of the invention, an area of ​​the support is illuminated with a set of at least two incident collimated light sources forming an angle (α) of at least 10° with respect to the normal to the support.

[0071] The incident light source is an electrically powered light source. The light source may illuminate the substrate in any pattern, such as a point, line, circle, or square pattern.

[0072] As used herein, the term "collimated light source" refers to a light source that produces parallel light beams. Collimated light therefore only minimally diffuses as it propagates. The terms "collimated light source", "incident collimated light source", "incident light source", and "light source" are used interchangeably in this document. The collimated incident light source is preferably selected from collimated light emitting diodes (LEDs) and laser diodes, and combinations thereof. More preferably, the collimated incident light source is a collimated LED.

[0073] The light source used in the present invention may emit monochromatic light, polychromatic light, or even white light. In particular, the light source may emit visible light (i.e., having a wavelength in the range of 400-700 nm), infrared light (i.e., having a wavelength longer than 700 nm), or ultraviolet light (i.e., having a wavelength shorter than 400 nm). Preferably, the light source emits light having a wavelength in the range of 320-750 nm, more preferably in the range of 400 nm-700 nm.

[0074] In one embodiment, each light source of the set emits light independently selected from the group consisting of white light, UV light (preferably having a wavelength in the range of 320-400 nm), green light (preferably having a wavelength in the range of 530-600 nm), blue light (preferably having a wavelength in the range of 430 nm-530 nm), and red light (preferably having a wavelength in the range of 600 nm-700 nm).

[0075] In a preferred embodiment, each light source of the set emits light independently selected from the group consisting of white light, UV light (preferably having a wavelength in the range of 320-400 nm), blue light (preferably having a wavelength in the range of 430 nm-530 nm), and red light (preferably having a wavelength in the range of 600 nm-700 nm).

[0076] The light sources of the set may emit the same type of light or different types of light. By way of example, each light source of the set may provide white light. Alternatively, at least one light source of the set may provide white light, while at least one other light source of the set may provide UV light. Preferably, each light source of the set emits the same type of light, preferably white light.

[0077] In a particular embodiment, the light sources of the set are selected from the group consisting of collimated white LEDs and collimated LEDs emitting UV light, blue light, or red light, and combinations thereof. Preferably, the light sources of the set are collimated white LEDs.

[0078] In a preferred embodiment, all light sources of the set are selected from the group consisting of collimated white LEDs and collimated LEDs emitting UV light, blue light, or red light, and combinations thereof. Preferably, all light sources of the set are collimated white LEDs.

[0079] In a preferred embodiment, the set of incident light is not filtered through an excitation filter, i.e. a filter commonly used in fluorescence microscopy and spectroscopy applications to select the excitation wavelength of light from the light source, before reaching the microbiological object or support.

[0080] As mentioned above, the method of the present invention does not require the use of any additional reagents, such as labeling agents or dyes, to facilitate detection. However, in some particular embodiments, the microbiological object may be labeled using such reagents, such as fluorescent dyes. Preferably, the microbiological object is unlabeled, i.e., not labeled with a labeling agent, i.e., a compound or moiety that generates a photon signal, such as a colored dye or a fluorescent dye. In a preferred embodiment, the method of the present invention does not involve the use of any reagents (e.g., substrates, probes, antibodies, dyes, etc.) to label the microbiological object.

[0081] In step a), the support is illuminated with at least 2 sets of light sources, preferably with at least 3 sets of light sources. In particular, the support may be illuminated with 3 to 50 sets of light sources, preferably with 3 to 24 sets of light sources, more preferably with 3 to 12 sets of light sources and even more preferably with 3 to 6 sets of light sources.

[0082] In a preferred embodiment, the support is illuminated with a set of three light sources.

[0083] Preferably, the light sources of a set are distributed on one or several circles around the optical acquisition axis, preferably evenly distributed (i.e. equidistant from each other). In some embodiments, the light sources are distributed on a circle around the optical acquisition axis, preferably evenly distributed. In some other embodiments, the light sources are distributed on several circles, preferably of different diameters, preferably 2, 3, 4, 5 or 6 circles around the optical acquisition axis, preferably evenly distributed. Preferably, the circles lie in a plane parallel to the plane of the support.

[0084] In one embodiment, the support is illuminated with a set of three light sources evenly distributed on a circle centered on the optical acquisition axis.

[0085] In some particular embodiments, the support is illuminated with an even number of light sources, and said light sources are symmetrical with respect to the optical acquisition axis.

[0086] The value of the angle α is selected independently for each light source of the set. Thus, the values ​​of the angles α of two light sources of a set may be the same or different. Preferably, each incident collimated light source of a set forms an angle (α) with respect to the normal to the support that is independently selected between 15° and 75°, preferably between 25° and 65° with respect to the normal to the support, more preferably between 30° and 60° with respect to the normal to the support.

[0087] In a special embodiment, the value of the angle α is identical for a set of light sources that are symmetric about the optical acquisition axis.

[0088] In a preferred embodiment, the value of the angle α is the same for all light sources in the set.

[0089] In a particularly preferred embodiment, the support is illuminated with a set of at least three light sources and the value of the angle α is the same for all light sources. In particular, in this embodiment, the value of the angle α can be between 25° and 65° with respect to the normal to the support.

[0090] The luminous flux emitted by each light source and the sum of the luminous flux emitted by all light sources can be easily adjusted by a person skilled in the art. Preferably, the sum of the luminous flux emitted by all light sources of the set is between 0.80 and 15 lumens / mm 2 and the luminous flux emitted by each light source is 0.10-5 lumens / mm 2 It is.

[0091] The area of ​​the support illuminated by the collimated light source can be a part of the support or the whole support. Preferably, the area of ​​the support illuminated by the collimated light source is a part of the support. In particular, said area is between 3 and 100 mm 2 , preferably 3 to 50 mm 2 , more preferably 25 to 50 mm 2 Typically, the total area of ​​the support can be 100 mm 2 ~10,000mm 2 , preferably 400 mm 2 ~8,000mm 2 , more preferably 450 to 2000 mm 2 It consists of:

[0092] The distance between the support and the light receiving element and the light source can be easily adjusted by a person skilled in the art, an operator, or automatically, for example, using computer image analysis software. In particular, this distance can be determined using triangulation. In general, the distance between the support and the light receiving element is 5 mm to 10 cm, preferably 5 mm to 5 cm, and more preferably 5 mm to 3 cm.

[0093] In step b) of the method of the present invention an image of said area of ​​said support illuminated by said at least two incident collimated light sources is acquired by a photodetector having its optical acquisition axis along a normal to the support.

[0094] The light receiving element can be a camera including an array of pixel sensors, preferably a CCD (charge-coupled device) image sensor, or an active pixel sensor, such as a CMOS (complementary metal oxide semiconductor) image sensor. Preferably, the light receiving element is a CMOS image sensor.

[0095] One of the advantages of the present invention is that it detects microbiological objects at a very early stage of growth, without the need for magnification. Therefore, preferably, the light receiving element is a non-magnifying system. However, the use of such a system is not excluded, and the images can be acquired, for example, at a magnification of x0.5 to x10, preferably x2 to x5.

[0096] In some embodiments, the method of the present invention further comprises, after step b) and before step c), b') repeating steps a) and b) one or more times, each time using a different set of light sources, thereby obtaining a plurality of captured images of said area, and combining said captured images of said area to form a combined image of said area.

[0097] Steps a) and b) may be repeated 1 to 20 times using 1 to 20 different sets of at least two input collimated light sources. In particular, steps a) and b) may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times using 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different sets of at least two input collimated light sources, respectively. Preferably, steps a) and b) are repeated 1, 2, 3, 4, 5, 6, 7, or 8 times using 1, 2, 3, 4, 5, 6, 7, or 8 different sets of at least two input collimated light sources, respectively. More specifically, steps a) and b) are repeated 1, 2, 3, 4 times using 1, 2, 3, or 4 different sets of at least two input collimated light sources, respectively.

[0098] Each set of light sources may be as defined above. All embodiments disclosed above for the set used in step a) are also considered for each set of light sources used in the repeating steps. In particular, each set may comprise at least two, preferably at least three light sources forming an angle (α) of at least 10° with respect to the normal to the support, the value of the angle (α) being selected independently for each light source. Preferably, the value of the angle α is the same for each light source of the same set.

[0099] Each set may contain 3 to 24 light sources, preferably 3 to 12 light sources, more preferably 3 to 6 light sources, and even more preferably 3 light sources. The number of light sources may be the same or different for each set. In particular, all sets may contain different numbers of light sources, although all sets may contain the same number of light sources or some sets may contain the same number of light sources.

[0100] Preferably, each set comprises at least three light sources and the value of the angle α is the same for all light sources of the set. Preferably, the value of the angle α is between 25° and 65° with respect to the normal to the support.

[0101] Each set of light sources includes a different combination of light sources and differs from the other sets in at least one light source, and preferably differs from all light sources.

[0102] The two sets of light sources may differ in their number, their type of light, their position, and / or the value of the angle α of each light source. Preferably, the two sets of light sources differ in their number, their position, and / or the value of the angle α of each light source. More preferably, the two sets of light sources differ at least in their position and / or the value of the angle α of each light source.

[0103] Preferably, all sets of light sources emit the same type of light, preferably white light, more preferably, all sets of light sources are collimated white LEDs.

[0104] An image of an area is acquired for each set of illuminants, and the images are then combined to obtain a combined image of the area.

[0105] It should be noted that the method of the invention does not exclude the possibility of repeating steps a) and b) once or several times with the same set of light sources.

[0106] In particular, the method of the present invention comprises: a) illuminating an area of ​​the support with a set of at least two, preferably at least three, incident collimated light sources that form an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being selected independently for each incident collimated light source; b) acquiring an image of said area of ​​said support illuminated by said set of at least two, preferably at least three, incident collimated light sources by a photodetector having its optical acquisition axis along a normal to the support; b') repeating steps a) and b) one or several times, each time using a different set of at least two, preferably at least three, incident collimated light sources forming an angle (α) of at least 10° with respect to the normal to the support, thereby obtaining a plurality of acquired images of said area, and combining said acquired images of said area to form a combined image of said area, the value of the angle (α) being selected independently for each incident collimated light source; may include.

[0107] In a particular embodiment, the method of the present invention comprises the steps of: a) illuminating an area of ​​the support with a first set of at least two, preferably at least three, incident collimated light sources that form an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being selected independently for each incident collimated light source; b) acquiring an image of said area of ​​said support illuminated by said first set of at least two, preferably at least three, incident collimated light sources by a photodetector having its optical acquisition axis along a normal to the support; b') repeating steps a) and b) using a second set of at least two, preferably at least three, incident collimated light sources that form an angle (α) of at least 10° with respect to the normal to the support, thereby obtaining a plurality of acquired images of said area, and combining the acquired images of said area to form a combined image of said area, the value of the angle (α) being selected independently for each incident collimated light source; may include.

[0108] Preferably, the angle (α) of each of the input collimated light sources in the first set is the same, more preferably, the angle (α) of each of the input collimated light sources in the first set is the same and is between 25° and 65°.

[0109] Preferably, the angle (α) of each of the second set of collimated input light sources is the same, more preferably, the angle (α) of each of the second set of collimated input light sources is the same and is between 25° and 65°.

[0110] The angle (α) value of each of the input collimated light sources of the first set and the angle (α) value of each of the input collimated light sources of the second set may be the same or different. In a special embodiment, the angle (α) value of each of the input collimated light sources of the first set and the angle (α) value of each of the input collimated light sources of the second set are the same, preferably between 25° and 55°, more preferably between 30° and 40°. In another special embodiment, the angle (α) value of each of the input collimated light sources of the first set and the angle (α) value of each of the input collimated light sources of the second set are different, preferably between 30° and 40° and between 45° and 55°, or vice versa.

[0111] In a particular embodiment, the method of the present invention comprises the steps of: a) illuminating an area of ​​the support with a first set of at least two, preferably at least three, incident collimated light sources that form an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being selected independently for each incident collimated light source; b) acquiring an image of said area of ​​said support illuminated by said first set of at least two, preferably at least three, incident collimated light sources by a photodetector having its optical acquisition axis along a normal to the support; b') repeating steps a) and b) one or more times with each of N sets of at least two, preferably at least three, incident collimated light sources, thereby obtaining a plurality of captured images of said area, and combining the captured images of said area to form a combined image of said area, wherein each light source of each set forms an angle (α) of at least 10° with respect to the normal to the support, the value of angle (α) being selected independently for each incident collimated light source; may include.

[0112] N is an integer and is 2 to 20, preferably 2 to 12, and more preferably 2 to 8, ie, selected from the group consisting of 2, 3, 4, 5, 6, 7, and 8.

[0113] Preferably, the angle (α) of each of the input collimated light sources in the first set is the same, more preferably, the angle (α) of each of the input collimated light sources in the first set is the same and is between 25° and 65°.

[0114] For each of the N sets, the angle (α) of each collimated input light source of the set may be the same or may be different. Preferably, the angle (α) of each collimated input light source of the set is the same. More preferably, the angle (α) of each collimated input light source of the set is the same and is between 25° and 65°.

[0115] In a particular embodiment, the value of the angle (α) of each incident collimated light source of a set is the same, preferably between 25° and 65°, and the value of the angle (α) is different for each of the N sets.

[0116] In another particular embodiment, the angle (α) of each input collimated light source of a set is the same, preferably between 25° and 65°, and two or more sets have the same angle (α) value.

[0117] In another particular embodiment, the angle (α) of each input collimated light source of the same set is the same, and four sets of light sources are used, two sets having an angle (α) value between 30° and 40°, and two sets having an angle (α) value between 45° and 55°. Preferably, each set comprises at least three light sources, preferably between 3 and 6 light sources, more preferably consisting of three light sources.

[0118] In another particular embodiment, the angle (α) value of each input collimated light source of a set is the same, and at least eight sets of light sources are used, two sets having an angle (α) value between 25° and 35°, two sets having an angle (α) value between 40° and 50°, and four sets having an angle (α) value between 60° and 70°. Preferably, each set comprises at least three light sources, preferably between 3 and 6 light sources, more preferably consisting of three light sources.

[0119] In another particular embodiment, the angle (α) value of each input collimated light source of the same set is the same, and at least eight sets of light sources are used, one set having an angle (α) value between 28° and 32°, one set having an angle (α) value between 33° and 37°, one set having an angle (α) value between 38° and 42°, one set having an angle (α) value between 48° and 52°, one set having an angle (α) value between 53° and 57°, and three sets having an angle (α) value between 60° and 64°. Preferably, each set comprises at least three light sources, preferably consisting of three light sources. Preferably, each set comprises at least three light sources, preferably between three and six light sources, more preferably consisting of three light sources.

[0120] In several embodiments including steps a), b) and b'), in step c) the presence or absence of a microbiological object on the area of ​​the support may be detected by identifying light reflected, scattered and / or diffused from the support and from the microbiological object on the support on the combined image of the area.

[0121] In some preferred embodiments, steps a) and b) (using one set of light sources) or steps a), b) and b') (repeated using several sets of light sources) are performed multiple times to image several areas of the support, preferably to image the entire support. Thus, the method may further comprise, after step b) or b'), step b'') of combining the acquired images of said areas to form a combined image of said area. In step c), the presence or absence of a microbiological object on said area of ​​the support may then be detected by identifying on said combined image of said area the light reflected, scattered and / or diffused from the support and from the microbiological object on said support.

[0122] Steps a) and b), or a) to c), may be performed multiple times to image several different areas of the support, preferably steps a) and b), or a) to c), are performed to image the entire support, i.e. are performed a sufficient number of times to image the entire support by combining the acquired images.

[0123] In particular, the steps of illuminating and image acquiring an area (steps a) and b)) and, optionally, when steps a) and b) are repeated with one or several different sets of light sources, the step of combining the acquired images of said areas may be performed multiple times in order to image several different areas of said support, preferably in order to image the entire support by combining the acquired images of the different areas.

[0124] The number of rounds depends on the size of the area to be imaged in each round and the size of the support. In general, steps a) and b), or steps a) to c), can be performed 10 to 500 times, preferably 50 to 400 times.

[0125] In particular, the steps of illuminating and image acquiring an area (steps a) and b)) and, optionally, when steps a) and b) are repeated with one or several different sets of light sources, the step of combining the acquired images of said area may be performed 10 to 500 times, preferably 50 to 400 times.

[0126] The method may further comprise the step of combining the acquired images of the different areas to form a combined image of the support. For each illuminated area an image may be acquired, said area image then being processed to form a combined image of the support. Alternatively, for each illuminated area several images may be acquired and combined, said combined image of the areas then being processed to form a combined image of the support. The combined image then constitutes a representation of the support in its entirety or in parts thereof. In a preferred embodiment step c) is performed on such combined image of the support, in its entirety or in parts thereof.

[0127] Image manipulation, such as combining acquired images of an area to form a combined image of the same area, or combining acquired images or combined images of different areas to form a combined image of the support, can be performed by any method routinely used by those skilled in the art, for example, image combining can be performed using ImageJ software.

[0128] The support is advantageously mounted on a mobile sample holder, which makes it possible to scan the surface of the support and to reconstruct a partial or complete image of said support. The width of the incident area (illuminated and imaged area) is preferably equal to or greater than the translation step between two successive positions of the support. Thanks to the mobile sample holder, the support can be scanned by linear and / or rotational movements of the sample relative to the receiving element.

[0129] The detection of the presence or absence of microbiological objects on the support is performed by identifying the light reflected, scattered and / or scattered from the support and from the microbiological objects on the support on the acquired image or the combined image. The microbiological objects add a certain degree of irregularity on the surface of the support, which is detectable due to the different reflection, scattering and / or scattering of light on the support and on the microbiological objects. The light reflected, scattered and / or scattered from the support and from the microbiological objects on the support is different from the photon signal emitted by the labeling agent, in particular the fluorescent signal emitted by the fluorescent dye, or the autofluorescence signal emitted by the microbiological objects. In particular, the light reflected, scattered and / or scattered from the support and from the microbiological objects on the support has the same wavelength range as the incident light. In a special embodiment, the incident collimated light source emits white light, and the light receiving element detects the white light reflected, scattered and / or scattered from the support and from the microbiological objects on the support. The same applies to other types of light (eg, blue light, red light, green light, UV light).

[0130] This detection can be performed by an operator or can be performed automatically by computer-implemented image processing software connected to the light receiving element, which can be any suitable software readily selected by one of skill in the art.

[0131] In particular, the detection of microbiological objects can be performed by detecting reflection spots resulting from surface irregularities caused by the microbiological objects, for example, this spot detection can be performed by (i) using the number of pixels per row or column of the array of pixels of the pixel sensor that receive a signal level above a threshold around the light intensity peak, (ii) using the maximum signal level of each row or column of the array of pixels of the pixel sensor, or (iii) using the signal level of the array of pixel sensors.

[0132] In some embodiments, the method of the invention further comprises, prior to step a), the steps of providing a sample to be tested, contacting said sample with a support, and incubating said support to allow growth of microorganisms, if present. The sample, support, and incubation conditions may be as defined above.

[0133] This detection may involve counting the microbiological objects and classifying / identifying them based on given criteria, such as their surface area or their morphology.

[0134] The method of the invention may therefore further comprise the step of counting and / or identifying the microbiological objects on the support from the acquired images or the combined image.

[0135] As used herein, the term "identification" does not necessarily require the determination of the genus and species of a given microbiological object. It may mean the classification of a microorganism in a taxonomic group (in any rank) or in a specific group (e.g. thermophilic acidophiles, acetic bacteria, lactic acid bacteria, yeasts or filamentous fungi). This identification may be possible through information (size, morphology, characteristics) obtained from the acquired image or the combined image, and / or simply through the detection of said object, especially if the cultivation step was carried out on a selective growth medium and / or under selective conditions (e.g. thermophilic conditions).

[0136] In some preferred embodiments, the method of the present invention is used to test the sterility of a sample or to detect microorganisms in a sample. In particular, before step a), the sample can be contacted with a support and incubated to allow the growth of microorganisms as detailed above. The presence of microbiological objects or the abundance of some microbiological objects is higher than a pre-determined threshold indicates that the sample is not sterile. The absence of microbiological objects or the abundance of some microbiological objects is lower than a pre-determined threshold indicates that the sample is sterile. This threshold can vary based on the product being tested and the applicable regulatory standards.

[0137] The method of the present invention allows the detection of microbiological objects with high reliability after incubation times generally of up to 4 days, preferably 3, 2, 1 or even less days, depending on the nature of the microbiological object to be detected.

[0138] In another aspect, the present invention relates to a device for carrying out the method of the present invention, in particular for detecting a microbiological object on a support, preferably a membrane filter or a solid growth medium, comprising: at least two collimated incident light sources for illuminating the support, said at least two collimated incident light sources forming an angle (α) of at least 10° with respect to a normal to the support; a light receiving element for acquiring an image of an area of ​​the support illuminated by the at least two incident collimated light sources, the light receiving element having its optical acquisition axis along a normal to the support; means for detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from the support and from microbiological objects on said support; Optionally, means for holding and optionally moving the support. The present invention relates to a device comprising:

[0139] The incident light source can be any collimated light source as defined above. In particular, the incident light source is an electrically powered light source. The incident light source can illuminate the support in any pattern, such as a point, a line, a circle, or a square.

[0140] The light source is preferably selected from collimated light emitting diodes (LEDs) and laser diodes, and combinations thereof. More preferably, the collimated incident light source is a collimated LED.

[0141] The light source used in the present invention may emit monochromatic light, polychromatic light, or even white light. In particular, the light source may emit visible light (i.e., having a wavelength in the range of 400-700 nm), infrared light (i.e., having a wavelength longer than 700 nm), or ultraviolet light (i.e., having a wavelength shorter than 400 nm). Preferably, the light source emits light having a wavelength in the range of 320-750 nm, more preferably in the range of 400 nm-700 nm.

[0142] In one embodiment, each light source emits light independently selected from the group consisting of white light, UV light (preferably having a wavelength in the range of 320-400 nm), green light (preferably having a wavelength in the range of 530-600 nm), blue light (preferably having a wavelength in the range of 430 nm-530 nm), and red light (preferably having a wavelength in the range of 600 nm-700 nm).

[0143] In a preferred embodiment, each light source emits light independently selected from the group consisting of white light, UV light (preferably having a wavelength in the range of 320-400 nm), blue light (preferably having a wavelength in the range of 430 nm-530 nm), and red light (preferably having a wavelength in the range of 600 nm-700 nm).

[0144] The light sources may emit the same type of light or different types of light. By way of example, each light source may provide white light. Alternatively, at least one light source may provide white light while at least one other light source may provide UV light. Preferably, each light source emits the same type of light, preferably white light.

[0145] In a particular embodiment, the at least two incident collimated light sources are selected from the group consisting of collimated white LEDs and collimated LEDs emitting UV light, blue light, or red light, and combinations thereof. Preferably, the at least two incident collimated light sources are collimated white LEDs.

[0146] In a preferred embodiment, all of the light sources are selected from the group consisting of collimated white LEDs and collimated LEDs emitting UV light, blue light, or red light, and combinations thereof. Preferably, all of the light sources are collimated white LEDs.

[0147] The device may comprise at least 3 input collimated light sources, preferably 3 to 50, more preferably 3 to 24, and even more preferably 3 to 12. In a preferred embodiment, the device may comprise at least 4 input collimated light sources, preferably 4 to 50, more preferably 4 to 24, and even more preferably 4 to 12. In another preferred embodiment, the device comprises at least 13 input collimated light sources, preferably 15 to 50, more preferably 15 to 30, and even more preferably 24 input collimated light sources.

[0148] Preferably, the light sources are distributed on one or several circles around the optical acquisition axis, preferably evenly distributed (i.e. equidistant from each other). In some embodiments, the light sources are distributed on a circle around the optical acquisition axis, preferably evenly distributed. In some other embodiments, the light sources are distributed on several circles, preferably of different diameters, preferably 2, 3, 4, 5 or 6 circles around the optical acquisition axis, preferably evenly distributed. Each circle is in a plane parallel to the plane of the support. Preferably, each circle includes at least 3 light source positions, preferably 3 to 12 light source positions, more preferably 3 to 9 light source positions. In a preferred embodiment, each light source arranged on the same circle has the same value of the angle α.

[0149] In some particular embodiments, the device comprises an even number of light sources, and said light sources are symmetrical about the optical acquisition axis.

[0150] The value of the angle α is selected independently for each light source. Thus, the values ​​of the angles α of two light sources of a device may be the same or different. Preferably, each incident collimated light source forms an angle (α) with respect to the normal to the support that is independently selected between 15° and 75°, preferably between 25° and 65°, more preferably between 30° and 60° with respect to the normal to the support.

[0151] In a special embodiment, the value of the angle α is the same for light sources that are symmetric about the optical acquisition axis.

[0152] In a special embodiment, the value of the angle α is the same for all light sources.

[0153] In another particular embodiment, the device comprises at least 3, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 light sources with an angle (α) value between 45° and 55°. Preferably, the device comprises 12 incident collimated light sources with an angle (α) value between 45° and 55°. The light sources may be distributed, preferably evenly distributed, on one or several circles around the optical acquisition axis. Preferably, the light sources are distributed, preferably evenly distributed, on one circle around the optical acquisition axis.

[0154] In another particular embodiment, the device comprises at least 3, preferably 3, 4, 5 or 6 light sources with an angle (α) value between 30° and 40° and at least 3, preferably 3, 4, 5 or 6 light sources with an angle (α) value between 45° and 55°. Preferably, the device comprises 6 light sources with an angle (α) value between 30° and 40° and 6 light sources with an angle (α) value between 45° and 55°. The light sources may be distributed, preferably evenly distributed, on one or several circles around the optical acquisition axis. Preferably, the light sources are distributed, preferably evenly distributed, on two circles around the optical acquisition axis. In particular, a first circle may include the positions of light sources with an angle (α) value between 30° and 40° and a second circle may include the positions of light sources with an angle (α) value between 45° and 55°.

[0155] In another particular embodiment, the device comprises at least 3, preferably 3, 4, 5 or 6 light sources with an angle (α) value between 25° and 35°, at least 3, preferably 3, 4, 5 or 6 light sources with an angle (α) value between 40° and 50°, and at least 6, preferably 7, 8, 9, 10, 11 or 12 light sources with an angle (α) value between 60° and 70°. Preferably, the device comprises 6 light sources with an angle (α) value between 25° and 35°, 6 light sources with an angle (α) value between 40° and 50°, and 12 light sources with an angle (α) value between 60° and 70°. The light sources may be distributed, preferably evenly distributed, on one or several circles around the optical acquisition axis. Preferably, the light sources are distributed, preferably evenly distributed, on three circles around the optical acquisition axis. In particular, a first circle may include light source positions having angle (α) values ​​between 25° and 35°, a second circle may include light source positions having angle (α) values ​​between 40° and 50°, and a third circle may include light source positions having angle (α) values ​​between 60° and 70°.

[0156] In another particular embodiment, the device comprises at least 3, preferably 3 light sources with an angle (α) value between 28° and 32°, at least 3, preferably 3 light sources with an angle (α) value between 33° and 37°, at least 3, preferably 3 light sources with an angle (α) value between 38° and 42°, at least 3, preferably 3 light sources with an angle (α) value between 48° and 52°, at least 3, preferably 3 light sources with an angle (α) value between 53° and 57° and at least 3, preferably 3, 4, 5, 6, 7, 8, 9 light sources with an angle (α) value between 60° and 64°. Preferably, the device comprises 3 light sources with an angle (α) value between 28° and 32°, 3 light sources with an angle (α) value between 33° and 37°, 3 light sources with an angle (α) value between 38° and 42°, 3 light sources with an angle (α) value between 48° and 52°, 3 light sources with an angle (α) value between 53° and 57° and 9 light sources with an angle (α) value between 60° and 64°. The light sources may be distributed, preferably evenly distributed, on one or several circles around the optical acquisition axis. Preferably, the light sources are distributed, preferably evenly distributed, on six circles around the optical acquisition axis. In particular, the first circle may include light source positions having an angle (α) value between 28° and 32°, the second circle may include light source positions having an angle (α) value between 33° and 37°, the third circle may include light source positions having an angle (α) value between 38° and 42°, the fourth circle may include light source positions having an angle (α) value between 48° and 52°, the fifth circle may include light source positions having an angle (α) value between 53° and 57°, and the sixth circle may include light source positions having an angle (α) value between 60° and 64°.

[0157] The luminous flux emitted by each light source and the total luminous flux emitted by all light sources can be easily adjusted by a person skilled in the art. Preferably, the total luminous flux emitted by all light sources is 0.80 to 15 lumens / mm 2 The luminous flux emitted by each light source is 0.10 to 5 lumens / mm 2 It is.

[0158] In a preferred embodiment, the device does not include any excitation filters, ie, filters for selecting the excitation wavelength of light from the incident light source.

[0159] The light receiving element is as defined above. In particular, the light receiving element can be a camera including an array of pixel sensors, preferably a CCD (charge-coupled device) image sensor, or an active pixel sensor, such as a CMOS (complementary metal oxide semiconductor) image sensor. Preferably, the light receiving element is a CMOS image sensor.

[0160] The light receiving element can be a magnifying or non-magnifying system. The magnification can be comprised between ×0.5 and ×10, preferably between ×2 and ×5. Preferably, the light receiving element is a non-magnifying system.

[0161] The light source and the light receiving element are mounted together on a support structure so that they have a defined and fixed positional relationship relative to one another.

[0162] The device comprises means for detecting the presence or absence of a microbiological object on the support.

[0163] The device preferably comprises means for combining images, in particular for combining acquired images of the same area, and for combining acquired images of several areas or combined images. Detection of the presence or absence of a microbiological object on the support can thus be performed on an acquired image of an area of ​​the support, on a combined image of an area of ​​the support (combination of several acquired images of the same area) or on a combined image of several areas of the support.

[0164] The light receiving element may be connected to a detection unit, i.e. a computer implemented image processing software known per se, which receives the image information acquired from the light receiving element and processes this information to detect any microbiological objects.

[0165] Thus, by detecting the light reflected, scattered and / or diffused from the support and from the microbiological objects on said support, it is possible to distinguish the light reflected, scattered and / or diffused from the support and from the microbiological objects on said support, and thus to detect the presence or absence of the microbiological objects on said support. The microbiological objects add a certain degree of irregularity on the surface of the support, which is detectable due to the different reflection, scattering and / or diffusion of light on the support and on the microbiological objects. In fact, the irregularities caused by the microbiological objects have a specific structure and result in a special reflection pattern.

[0166] The light reflected, scattered and / or scattered from the support and from the microbiological objects on said support is different from the photon signal emitted by the labeling agent, in particular the fluorescent signal emitted by the fluorescent dye, or the autofluorescence signal emitted by the microbiological objects. In particular, the light reflected, scattered and / or scattered from the support and from the microbiological objects on said support has the same wavelength range as the incident light. In a special embodiment, the incident collimated light source emits white light, and the light receiving element detects the white light reflected, scattered and / or scattered from the support and from the microbiological objects on said support. The same applies to other types of light (e.g. blue light, red light, green light, UV light).

[0167] In particular, the detection of microbiological objects can be performed by detecting reflection spots resulting from surface irregularities caused by the microbiological objects, for example, this spot detection can be performed by (i) using the number of pixels per row or column of the array of pixels of the pixel sensor that receive a signal level above a threshold around the light intensity peak, (ii) using the maximum signal level of each row or column of the array of pixels of the pixel sensor, or (iii) using the signal level of the array of pixel sensors.

[0168] Optionally, the device may further comprise means for holding and optionally moving the support. Preferably, the device comprises a mobile sample holder, as described above, which allows scanning of the support surface. Thanks to the mobile sample holder, the support can be scanned by linear and / or rotational movement of the sample relative to the light receiving element.

[0169] FIG. 1 illustrates one embodiment of a device of the present invention. This exemplary device comprises: two incident collimated light sources (3) for illuminating the support (2) forming an angle (α) of at least 10° with respect to the normal of the support; a photodetector (4) for acquiring an image of an area of ​​said support illuminated by said at least two incident collimated light sources, said photodetector having its optical acquisition axis along a normal to said support; - means (not shown) for detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from the support and from microbiological objects on said support; A sample holder (1) for holding a support; Equipped with The light source and the light receiving element are mounted together on a support structure (5) so that they have a defined and fixed positional relationship relative to one another.

[0170] In a further aspect, the present invention also relates to the use of the device of the present invention for detecting and optionally counting and / or identifying microbiological objects on a support, preferably based on the method of the present invention.

[0171] In particular, the present invention relates to the use of the device of the present invention for testing the sterility of a sample or for detecting microorganisms in a sample, preferably according to the method of the present invention.

[0172] All embodiments described above for the method and device of the present invention are also contemplated in this aspect.

[0173] As used herein, a range defined by the term "X to Y" is inclusive of the limits of the range, i.e., includes the value X and the value Y.

[0174] All references cited in this description are incorporated by reference in this application. Other features and advantages of the present invention will become more apparent in the following examples, which are presented for illustrative purposes and are not limiting in any way. EXAMPLES

[0175] Example 1 Materials and Methods: Microbial preparation For A. brasiliensis, BioBall® from Biomerieux (BioBall® Multishot 550 Aspergillus brasiliensis SKU number: 56001) containing the correct number of microorganisms was diluted in 0.9% NaCl to obtain a defined number of microorganisms per experimental condition loaded onto the filtration membrane (targets ≈ 100 / membrane density). The membranes were layered on SDA agar plates (5 g / L casein pancreatic digest, 5 g / L animal tissue pepsin digest, 40 g / L dextrose, 15 g / L agar, pH 5.6 ± 0.2) and the microorganisms were grown for 17 or 18 hours at 32.5°C. Alternatively, the membrane was layered onto R2A agar plates (0.5 g / L casein acid hydrolysate, 0.5 g / L dextrose, 0.3 g / L dipotassium phosphate, 0.024 g / L magnesium sulfate, 0.5 g / L proteose peptone, 0.3 g / L sodium pyruvate, 0.5 g / L soluble starch, 0.5 g / L yeast extract, 15 g / L agar, pH 7.2±0.2) and the organisms were grown at 22.5° C. for 38 or 44 hours.

[0176] Candida albicans (ATCC® 10231™), Pseudomonas aeruginosa (ATCC® 10145™), Escherichia coli (ATCC® 8739™), Bacillus subtilis (ATCC® 6633™), Burkholderia cepacia (ATCC® 25608™), Ralstonia picketii (ATCC® 27511™), and Legionella pneumophila (CIP 105349) from previously quantified frozen stocks were diluted in 0.9% NaCl to obtain defined numbers of organisms per experimental condition. The resulting suspension was filtered onto a membrane and the microorganisms were grown on solid growth media, i.e., SDA, R2A, TSA (15 g / L tryptone, 5 g / L Soja papain peptone, 5 g / L sodium chloride, 15 g / L agar, pH 7.3 ± 0.2), or GVPC (glycine-vancomycin-polymyxin-cyclohexamide purchased from Oxoid, catalogue reference: PO5074A) agar plates, at temperature T for time t.

[0177] The membranes used in the examples were: PVDF membrane (Durapore HVWG04700, Merck Millipore), MCE black membrane (HABG047, Merck Millipore), MCE white gridded membrane (HAWG047, Merck Millipore), MCE white non-gridded membrane (GE 10401670), PES membrane (metricel 66585, Pall laboratory), and nylon membrane (nylaflo 66608, Pall laboratory).

[0178] [Table 1]

[0179] For each sample, growth was stopped by removing the membrane from the solid growth medium and samples were stored at 4°C until imaging.

[0180] Sample preparation The membrane was layered onto a sample holder (1) made of black anodized aluminum along with three drops of sterile water (approximately 50-250 μl) (to rehydrate the membrane and keep it moist) and the cassette was then placed into the imaging position as illustrated in FIG.

[0181] Imaging System Configuration The membrane was imaged through a device as illustrated in FIG. 1, where the membrane (2) was placed on a sample holder (1) and illuminated with an incident collimated LED (3) forming an angle (α) with respect to the normal of the support. The incident area of ​​the membrane was imaged by a CMOS image sensor (4) with its optical acquisition axis along the normal of the membrane. Light reflected / scattered and / or diffused from the membrane and / or microbiological objects on the membrane was detected.

[0182] Acquisition parameters (light source intensity of 0.7-11 lumens / LED and camera exposure time of 10-300 ms) were adjusted to maximize the signal-to-noise ratio. A multiband emission filter (435±20 nm, 546±10 nm, and 690±50 nm bandpass) was placed between the CMOS image sensor and the sample. The CMOS sensor used in the examples was Sony IMX178LLJ-C (IDS Imaging Camera UI-3880CP-M-GL_R2 or UI-3880CP-C-GL_R2).

[0183] In the standard configuration, the membrane was illuminated by three collimated LEDs emitting white light, equidistant from each other, and centered on the optical acquisition axis. The angle of incidence α was set to 50°.

[0184] In the examples described below, several parameters of this setup were varied: number of LEDs, value of angle α, and wavelength of incident light. Further parameters were investigated, such as membrane type and the presence / absence of a translucent lid on the membrane.

[0185] result Change in number of LEDs The membrane was illuminated by collimated LEDs emitting white light, equidistant from each other, and centered on the optical acquisition axis. The angle of incidence α was set to 50°. The number of LEDs varied between 3, 6, or 12, and several types of membranes were tested (PVDF membranes, white and black MCE membranes, PES membranes). The individual LED intensities were reduced as the number of LEDs increased in order to keep the overall intensity constant (total light reaching the membrane was within the same order of magnitude as in the condition with 3 LEDs). For each of these conditions, the acquisition parameters (camera exposure time and light intensity) were manually adjusted to generate similar membrane backgrounds (same order of magnitude of gray levels regardless of condition).

[0186] Examples of images acquired under each condition are shown in Figure 2. These results reveal that a reflective spot can be found on the microorganisms regardless of the number of LEDs. This spot is well defined as a microorganism with a spherical type shape (e.g., C. albicans or Pseudomonas aeruginosa, etc.). An imperfect spherical type shape may cause a missing spot. However, the use of multiple light sources increases the amount of information, thus enabling efficient detection of each type of microorganism.

[0187] Change in the value of angle α The membrane was illuminated by six collimated LEDs emitting white light, equidistant from each other, and centered on the optical acquisition axis. The incidence angle α was set at 37°, 50°, or 60°, and several types of membranes were tested (PVDF membrane, white and black MCE membranes).

[0188] Examples of images acquired in each condition are shown in Figure 3. These results reveal that, regardless of the angle of incidence, a reflection spot is found on the microorganism. A small angle of incidence (37°) leads to a reflection spot located near the center of the microorganism. Indeed, on a flat surface (probably found at the center of the microcolony), light with a small angle of incidence is only slightly deflected towards the sensor: collection of the reflected light is possible. In contrast, on a hard surface (probably found at the edge of the microorganism), the light is deflected far away from the sensor: collection of the reflected light is not possible.

[0189] On the other hand, a large angle of incidence (e.g. 60°) results in a reflection spot located near the edge of the microorganism. Indeed, on a flat surface (likely to be found at the center of the microcolony), the light is deflected far away from the sensor: collection of the reflected light is not possible. In contrast, on a hard surface (likely to be found at the edge of the microorganism), light with a small angle of incidence is only deflected slightly towards the sensor: collection of the reflected light is possible.

[0190] These results show that three angle values ​​(37°, 50°, and 60°) allow efficient detection of each type of microorganism, and in some cases detection can be improved by adjusting the incident angle based on the structure of the microorganism to be detected, or by using a combination of different incident angles.

[0191] Wavelength change The membrane was illuminated by three collimated LEDs equidistant from each other and centered on the optical acquisition axis. The angle of incidence α was set at 50° and two types of membranes were tested (PVDF and PES membranes). The LEDs emitted white light, UV light (365±10 nm), blue light (495±20 nm), or red light (645±30 nm). In the "white light" condition, the sensor collected light within 435±20 nm, 546±10 nm, and 690±50 nm bandpasses. In the "UV light" condition, the sensor collected light within a 365±10 nm bandpass. In the "blue light" condition, the sensor collected light within a 482±35 nm bandpass. In the "red light" condition, the sensor collected light within a 640±75 nm bandpass.

[0192] Examples of images acquired under each condition are shown in Figure 4. These results indicate that each type of microorganism can be effectively detected regardless of the light / wavelength.

[0193] The presence of a translucent lid The membrane, covered by a translucent lid, was illuminated by 12 collimated LEDs emitting white light, equidistant from each other and centered on the optical acquisition axis. The angle of incidence α was set at 50° and two types of membranes were tested (PVDF and white MCE membrane). Three materials for the lid were investigated: plastic from a Merck filtration unit, UV grade fused silica (Chroma corp., thickness: 1 mm), soda lime glass (Selba corp., thickness: 1.8-2 mm).

[0194] Examples of images acquired under each condition are shown in Figure 5. These results indicate that even if the membrane is covered with a semi-transparent lid, and regardless of the material of the lid, each type of microorganism can be effectively detected.

[0195] Membrane Type The method of the invention was investigated for different types of membranes: PDVF, black MCE, white MCE, black PES, or nylon membranes. For each condition, the membrane was illuminated by six collimated LEDs emitting white light, equidistant from each other, and centered on the optical acquisition axis. The incidence angle α was set to 37°.

[0196] Examples of images acquired under each condition are shown in Figure 6. These results show that each type of microorganism can be effectively detected on all membranes tested.

[0197] Detection on solid growth media E. coli (ATCC® 8739™) was diluted from a glycerol stock. The resulting suspension was either spread onto TSA agar plates (15 g / L tryptone, 5 g / L Soja papain peptone, 5 g / L sodium chloride, 15 g / L agar, pH 7.3±0.2) or filtered onto a membrane (PVDF, or white MCE). The membrane was overlaid onto the TSA plate. In both cases, the bacteria were grown for 8 hours at 37° C.

[0198] A. brasiliensis (BioBall® Multishot 550 Aspergillus brasiliensis SKU number: 56001 from Biomerieux containing the correct number of organisms) was diluted in 0.9% NaCl and spread onto R2A plates (5 g / L casein pancreatic digest, 5 g / L animal tissue pepsin digest, 40 g / L dextrose, 15 g / L agar, pH 5.6 ± 0.2) or loaded onto PVDF filtration membranes at a density of ≈100 objects / membrane and layered onto R2A plates. In both cases, organisms were grown for 44 h at 22.5°C.

[0199] A. brasiliensis or E. coli microorganisms were then detected either directly on the surface of the solid growth medium or on a membrane layered on the surface of the solid growth medium (Petri dish 90 mm in diameter). In both cases, the samples were layered on a sample holder (1) made of black plastic.

[0200] Examples of images acquired in each condition are shown in Figure 7. These results show that detection of microorganisms can be performed directly on solid growth media or on a membrane, even if the membrane is held on the growth media.

[0201] Use of different composition conditions to minimize the incubation time required for detection of microorganisms The method of the invention was investigated for the detection of C. albicans over different time periods. Microorganisms were grown on white MCE membranes (either gridded or non-gridded) in contact with SDA solid growth medium, and growth was stopped at different times (23, 25, or 28 hours). At each investigated time, samples were imaged under different conditions: standard condition 0 (3 LEDs, incidence angle α set to 37°), or condition 1 (6 LEDs, incidence angle α set to 37°), or condition 2 (12 LEDs: 6 of them set to α=37° and the other set of 6 set to α=50°). For each condition, the membrane was illuminated by collimated LEDs emitting white light, equidistant from each other, and centered on the optical acquisition axis.

[0202] For each condition, the number of microorganisms detected on the membrane after imaging was quantified (operator count, N), and the membranes were allowed to grow again for 48 h until colonies were visible to the naked eye (CFU count). The detection rate (%) was calculated as the ratio of N / CFU count.

[0203] The percentages obtained for each condition are shown in Figure 8. The results show that some compositional conditions allow for early detection of microorganisms.

[0204] Example 2 Materials and Methods Detection of microorganisms associated with contamination of raw materials or final products originating from the pharmaceutical and cosmetic industries Aspergillus brasiliensis ATCC 16404™, Saccharomyces cerevisiae ATCC 9763™, Zygosaccharomyces bailii DSM 70492, Penicillium chrysogenum ATCC 18502™, Candida albicans ATCC 10231™, and Penicillium variotii ATCC 18502™ were loaded into PVDF, black, or white MCE filtration membranes depending on the strain. The membranes were layered on SDA agar plates (5 g / L casein pancreatic digest, 5 g / L animal tissue pepsin digest, 40 g / L dextrose, 15 g / L agar, pH 5.6 ± 0.2). The plates were then incubated at 22.5° C. for 24-50 hours (depending on the strain and membrane type).

[0205] Acinetobacter baumannii ATCC 19606™, Aspergillus brasiliensis ATCC 16404™, Bacillus subtilis ATCC 6633™, Brevundimonas diminuta ATCC 19146™, Burkholderia cepacia ATCC 25608™, Candida albicans ATCC 10231™, Clostridium sporogenes ATCC 19404™, Cutibacterium acnes ATCC 6919™, Enterobacter aerogenes ATCC 35028™, Enterococcus faecalis ATCC 29212™, Escherichia coli ATCC 8739™, Klebsiella pneumoniae ATCC® 13883™, Kocuria rhizophila ATCC® 9341™, Methylobacterium extorquens ATCC® 43645™, Proteus mirabilis ATCC® 29906™, Pseudomonas aeruginosa ATCC® 9027™, Ralstonia picketii ATCC® 27511™, Serratia marcescens ATCC® 14756™, Shigella zoneii ATCC® 25931™, Staphylococcus aureus ATCC® 6538™, Streptococcus pneumoniae ATCC® 49619™, Vibrio parahaemolyticus ATCC® 17802™, Yersinia enterocolitica ATCC® 9610™ were loaded onto PVDF, black or white MCE filtration membranes depending on the strain. The membranes were layered onto TSA agar plates (15 g / L tryptone, 5 g / L Soja papain peptone, 5 g / L sodium chloride, 15 g / L agar, pH 7.3 ± 0.2). The plates were then incubated at 32.5°C for 6 to 72 hours (depending on the strain and membrane type).

[0206] Detection of bacteria associated with uncomplicated and complicated UTI Escherichia coli ATCC® 8739™, Klebsiella pneumoniae ATCC® 13883™, P. mirabilis ATCC® 29906™, E. faecalis ATCC® 19433™, and Staphylococcus aureus ATCC® 6538™, which account for 89% of uncomplicated and complicated UTIs, were loaded onto PVDF and black MCE filtration membranes. The membranes were layered on Columbia blood agar plates (23 g / L peptone, 1 g / L starch, 5 g / L sodium chloride, 50 ml / L sheep blood, 14 g / L agar, pH 7.3 ± 0.2) for all strains and on Mac Conkey agar plates (20 g / L peptone, 10 g / L lactose, 1.5 g / L bile salts, 0.001 g / L crystal violet, 0.05 g / L neutral red, 5.0 g / L sodium chloride, 15.0 g / L agar, pH 7.1 ± 0.2) for coliforms (E. coli and K. pneumoniae). Columbia agar plates were incubated at 37°C for 7 h 30 min to 10 h 45 min (depending on the strain). Mac Conkey agar plates were incubated at 32.5°C for 10-11 hours (depending on strain and membrane type).

[0207] Detection of microorganisms involved in contamination of primary water and pharmaceutical water (purified water and WFI water) The gram-negative bacteria used in this example were Acinetobacter baumannii ATCC 19606™, Aeromonas hydrophila ATCC 35654™, Brevundimonas diminuta ATCC 19146™, Burkholderia cepacia ATCC 25608™, Citrobacter freundii ATCC 8090™, Edwardsiella tarda ATCC 15947™, Enterobacter aerogenes ATCC 35028™, Ochrobactrum anthropi CIP 82.115, Klebsiella pneumoniae ATCC 13883™, Methylobacterium extorquens CIP 106787, Moraxella osloensis ATCC 25608™, and 100% ethanolamine. 19976®, Pantoea agglomerans ATCC® 27155™, Proteus mirabilis ATCC® 29906™, Pseudomonas aeruginosa ATCC® 10145™, Ralstonia picketii ATCC® 27511™, Salmonella typhimurium ATCC® 13311™, Serratia marcescens ATCC® 13880™, Shigella zonei ATCC® 25931™, Sphingomonas paucimobilis ATCC® 29837™, Stenotrophomonas maltophilia ATCC® 13637™, Vibrio parahaemolyticus ATCC® The gram-positive bacteria were Bacillus subtilis ATCC 6633™, Enterococcus faecalis ATCC 19433™, Staphylococcus aureus ATCC 6538™. The yeasts and filamentous fungi tested were Aspergillus brasiliensis ATCC 16404™ and Candida albicans ATCC 10231™. This selection covers at least 84% of the microbial contaminants of major water systems and 77% of the most widely recognized contaminants of purified water systems and pharmaceutical WFI systems.Strains were loaded onto PVDF and black or white MCE filtration membranes (depending on the strain). The membranes were layered onto Reasoner's 2A agar (R2A) plates (0.5 g / L yeast extract, 0.5 g / L proteose peptone, 0.5 g / L casein hydrolysate, 0.5 g / L glucose, 0.5 g / L starch, 0.3 g / L dipotassium phosphate, 0.024 g / L magnesium sulfate anhydrous, 0.3 g / L sodium pyruvate, 15.0 g / L agar, final pH 7.2 ± 0.2). R2A plates were incubated at 32.5°C for 24–48 h (depending on the strain).

[0208] Detection of microorganisms involved in deterioration or contamination in the food industry (raw materials and finished products) general Acinetobacter baumannii ATCC 19606™, Bacillus subtilis ATCC 6633™, Brevundimonas diminuta ATCC 19146™, Enterobacter aerogenes ATCC 35028™, Enterococcus faecalis ATCC 29212™, Kocuria rhizophila ATCC 9341™, Shigella zoneii ATCC 25931™, Streptococcus pneumoniae ATCC 49619™, Vibrio parahaemolyticus ATCC 17802™, and Yersinia enterocolitica ATCC 9610™ were loaded onto the PVDF filtration membrane. The membrane was layered on PCA agar plates (5 g / L tryptone, 2.5 g / L yeast extract, 1 g / L dextrose, 15 g / L agar, pH 7.0 ± 0.2) and the plates were incubated at 35°C for 18 to 27 hours (depending on the strain).

[0209] Soft drinks The filamentous fungi used in this example are Aspergillus brasiliensis ATCC 16404™ and Penicillium variotii ATCC 18502™. The acetic acid bacteria used are Acetobacter aceti ATCC 15973™, Gluconoacetobacter liquefaciens ATCC 14835™, Asaea siamensis DSM 15972, and Gluconobacter oxydans ATCC 19357™, and the lactic acid bacteria strains used are Lactobacillus plantarum ATCC 8014™, Weissella confusa ATCC 10881™, and Lactobacillus casei ATCC 393™. Strains were loaded onto PVDF, black or white MC filtration membranes (depending on the strain). The membranes were then plated on OSA (10 g / L casein peptone, 3 g / L dipotassium hydrogen phosphate, 4 g / L D(+)-glucose, 5 g / L orange extract, 3 g / L yeast extract, 17 g / L agar, pH 5.5 ± 0.2), PDA (4 g / L potato infusion, 20 g / L dextrose, 17 g / L agar, pH 5.6 ± 0.2), or YM agar plates (10 g / L glucose, 3 g / L malt extract, 5 g / L peptone, pH 5.5 ± 0.2) for Aspergillus brasiliensis and Penicillium variotii. For lactic acid bacteria, the strains were layered on YM agar plates (2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 20 g / L D(+)-glucose, 0.1 g / L magnesium sulfate, 0.05 g / L manganous sulfate, 5 g / L meat extract, 5 g / L sodium acetate, 10 g / L universal peptone, 5 g / L yeast extract, 12 g / L agar, pH 5.7). The OSA plates were incubated at 30°C for 40 hours. The PDA plates were incubated at 25°C for 40 hours. The YMA plates were incubated at 25°C or 30°C for 15 to 72 hours (depending on the strain and membrane type).The MRS agar plates were incubated at 30°C for 24 to 26 hours.

[0210] Fruit juice Alicyclobacillus strains including Alicyclobacillus acidoterrestris ATCC® 49025™, Alicyclobacillus acidocaldarius ATCC® 27009™, Alicyclobacillus acidiphilus DSM 14558, Alicyclobacillus cycloheptanicus ATCC® 49028™, Alicyclobacillus hesperidum DSM 12766, Alicyclobacillus hervarius DSM 13609, and Alicyclobacillus contaminans DSM 17975 were loaded onto PVDF or black MCE filtration membranes (depending on the strain). The membrane was layered on a BAT agar plate (2 g / L yeast extract, 5 g / L D(+) glucose, 0.25066 g / L calcium chloride, 0.5 g / L magnesium sulfate, 0.2 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 0.00018 g / L zinc sulfate, 0.00016 g / L copper sulfate, 0.00015 g / L manganese sulfate, 0.00030 g / L sodium molybdate dihydrate, 18 g / L agar-agar, pH 3.8-4.2). The BAT agar plate was incubated at 45°C for 24 hours.

[0211] wine Saccharomyces cerevisiae (environmental strain), Zygosaccharomyces bailii (environmental strain), Lactobacillus naugeli (environmental strain), and Pediococcus pentosaceus ATCC® 33316™ were loaded onto PVDF filtration membranes. The membranes were layered onto tomato juice agar plates (20 g / L tomato juice, 10 g / L peptone, 10 g / L peptonized milk, 12 g / L agar, pH 6.1 ± 0.2). The tomato juice agar plates were incubated at 25°C and 30°C for 18 to 72 hours (depending on the strains and culture conditions).

[0212] Sample preparation The membrane was layered onto a sample holder (1) made of black anodized aluminum along with three drops of sterile water (approximately 50-250 μl) (to rehydrate the membrane and keep it moist) and the cassette was then placed into the imaging position as illustrated in FIG.

[0213] Imaging System Configuration The membrane was imaged through a device as illustrated in FIG. 1, where the membrane (2) was placed on a sample holder (1) and illuminated by three collimated LEDs (3) emitting white light, equidistant from each other and centered on the optical acquisition axis and forming an angle (α) of 50° with respect to the normal to the support. The incident area of ​​the membrane was imaged by a CMOS image sensor (4) with its optical acquisition axis along the normal to the membrane. Light reflected / scattered and / or diffused from the membrane and / or from microbiological objects on the membrane was detected.

[0214] Acquisition parameters (light source intensity 3-7 lumens / LED and camera exposure time 100 ms for microorganisms loaded on PVDF and black MCE membranes, and light source intensity 1-5 lumens / LED and camera exposure time 40-50 ms for microorganisms loaded on MCE white membrane) were adjusted to maximize the signal-to-noise ratio. A multiband emission filter (435 ± 20 nm, 546 ± 10 nm, and 690 ± 50 nm bandpass) was placed between the CMOS image sensor and the sample.

[0215] result

[0216] [Table 2A]

[0217] [Table 2B]

[0218] [Table 2C]

[0219] [Table 2D]

[0220] [Table 2E]

[0221] [Table 2F]

[0222] [Table 2G]

[0223] Example 3 Materials and Methods: Preparation of microorganisms For A. brasiliensis, BioBall® from Biomerieux (BioBall® Multishot 550 Aspergillus brasiliensis SKU number: 56001) containing the correct number of microorganisms was diluted in 0.9% NaCl to obtain a defined number of microorganisms per experimental condition loaded onto the filtration membrane (targets ≈ 100 / membrane density). The membranes were layered on SDA agar plates (5 g / L casein pancreatic digest, 5 g / L animal tissue pepsin digest, 40 g / L dextrose, 15 g / L agar, pH 5.6 ± 0.2) and the microorganisms were grown for 38 h at 22.5 °C.

[0224] Aspergillus brasiliensis (ATCC® 16404™), Candida albicans (ATCC® 10231™), Escherichia coli (ATCC® 8739™), Pseudomonas aeruginosa (ATCC® 9027™), Methylobacterium extorquens (NBRC 15911, ATCC® BAA-2500™), Ralstonia pickettii (ATCC® 27511™), Brevundimonas diminuta (ATCC® 19146™), Burkholderia cepacia (ATCC® 25416™), Staphylococcus aureus (ATCC® 25416™), Staphylococcus aureus (ATCC® 25416™), Staphylococcus aureus (ATCC® 25416™), Staphylococcus aureus (ATCC® 25416™), Staphylococcus pneumoniae (ATCC® 25416™), Staphylococcus aureus (ATCC® 25416™), Staphylococcus pneumoniae (ATCC® 25416™), Staphylococcus aureus (ATCC® 25416™), Staphylococcus pyogenes ... 6538™), Staphylococcus epidermidis (ATCC® 12228™), Pseudomonas fluorescens (ATCC® 17386™), and Bacillus subtilis (ATCC® 6633™) were diluted in 0.9% NaCl to obtain a defined number of microorganisms per experimental condition (targets ≈ 100 / membrane density). The resulting suspension was filtered onto an MCE gridded white membrane (HAWG047, Merck Millipore) and the microorganisms were grown on solid growth medium, i.e., SDA, R2A, or TSA agar plates, at temperature T for t time. The conditions used to grow the microorganisms on the filtration membranes are shown in Table 3. For each sample, growth was stopped by removing the membrane from the solid growth medium, and the samples were stored at 4 °C until imaging.

[0225] Sample preparation The membrane was layered onto a sample holder (1) made of black anodized aluminum along with three drops of sterile water (approximately 50-250 μl) (to rehydrate the membrane and keep it moist) and the cassette was then placed into the imaging position as illustrated in FIG.

[0226] Imaging System Configuration The membrane was imaged through a device as illustrated in FIG. 1, where the membrane (2) was placed on a sample holder (1) and illuminated with several incident collimated LEDs (3) emitting white light, each LED forming an angle (α) with respect to the normal to the support. The incident area of ​​the membrane was imaged by a CMOS image sensor (4) with its optical acquisition axis along the normal to the membrane. Light reflected / scattered and / or diffused from the membrane and / or from microbiological objects on the membrane was detected.

[0227] The device used for the detection of Aspergillus brasiliensis was equipped with six light sources with a numerical angle (α) of 37°, evenly distributed on a circle centered on the optical acquisition axis.

[0228] The device used for the detection of Candida albicans and bacteria had six light sources with an angle (α) of 37° evenly distributed on a circle centered on the optical acquisition axis, and six light sources with an angle (α) of 50° evenly distributed on another circle centered on the optical acquisition axis.

[0229] Acquisition parameters (light source intensity between 0.7 and 11 lumens / LED and camera exposure time between 100 and 300 ms) were adjusted to maximize the signal-to-noise ratio. A multiband emission filter (435 ± 20 nm, 546 ± 10 nm, and 690 ± 50 nm bandpass) was placed between the CMOS image sensor and the sample. The CMOS sensor used in this example was a Sony IMX178LLJ-C (IDS Imaging Camera UI-3880CP-M-GL_R2 or UI-3880CP-C-GL_R2).

[0230] The detection rate (%) was assessed by comparison with the number of colonies obtained under control conditions, i.e. after sufficient time had passed for colonies to become visible on the agar plate (with / without membrane).

[0231] result Detection of Aspergillus brasiliensis Two experimental configurations were tested.

[0232] In the first configuration, the membrane was illuminated simultaneously by six collimated LEDs equidistant from each other and centered on the optical acquisition axis. The incidence angle α was set to 37°. Images acquired in this condition were then used for the detection of microorganisms. This configuration revealed that 69% of Aspergillus brasiliensis microorganisms were detectable.

[0233] In the second configuration, the membrane was illuminated successively by a first set of three collimated LEDs equidistant from each other and centered on the optical acquisition axis, and a second set of three collimated LEDs, different from the first set, equidistant from each other and centered on the optical acquisition axis. The incidence angle α was set to 37°. The images acquired with the first and second sets of three LEDs were then combined and used for the detection of the microorganisms. It was found that with this configuration 100% of the Aspergillus brasiliensis microorganisms could be detected.

[0234] Detection of Candida albicans Two experimental configurations were tested.

[0235] In a first configuration, the membrane was illuminated simultaneously by 6 collimated LEDs equidistant from each other, centered on the optical acquisition axis and with an angle α of 37°, and 6 collimated LEDs equidistant from each other, centered on the optical acquisition axis and with an angle α of 50°. Images acquired in this condition were then used for the detection of microorganisms. With this configuration, it was found that 64% of Candida albicans microorganisms could be detected.

[0236] In a second configuration, the membrane was illuminated sequentially by four sets of LEDs: a first set of three collimated LEDs with an angle α of 37°, a second set of three other collimated LEDs with an angle α of 37°, a third set of three collimated LEDs with an angle α of 50°, and a fourth set of three other collimated LEDs with an angle α of 50°. Images acquired with each of these four sets were then combined and used in the detection of microorganisms. With this configuration, it was found that 100% of the Candida albicans microorganisms could be detected.

[0237] Bacteria detection The second configuration detailed above for the detection of Candida albicans was used to detect different bacteria, the results of which are shown in Table 3 below.

[0238] [Table 3] [Explanation of symbols]

[0239] 1 Sample holder 2 Support / Membrane 3 Collimated Incident Light Source / Collimated Incident LED 4 Photodetector / CMOS image sensor 5 Support structure

Claims

1. 1. A method for detecting a microbiological object on a support, comprising: a) illuminating an area of ​​the support with a set of at least two incident collimated light sources that form an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being independently selected for each incident collimated light source; b) acquiring an image of the area of ​​the support illuminated by the set of at least two incident collimated light sources by a light receiving element having its optical acquisition axis along a normal to the support; c) detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from said support and from microbiological objects on said support; A method comprising:

2. 2. The method of claim 1, wherein the microbiological object to be detected is selected from the group consisting of filamentous fungi, colonies / microcolonies of bacteria, archaea, and yeasts, and combinations thereof.

3. 2. The method of claim 1, wherein the microbiological object to be detected is selected from the group consisting of filamentous fungi, and bacterial and yeast colonies / microcolonies, and combinations thereof.

4. 2. The method of claim 1, wherein the microbiological objects to be detected include filamentous fungi and bacterial and / or yeast colonies / microcolonies.

5. 10. The method of claim 1, wherein the microbiological object to be detected is an individualizable or individualized microbiological object.

6. 10. The method of claim 1, wherein the microbiological object to be detected is not labeled with a compound or moiety that produces a photon signal.

7. The method of claim 1, wherein the support is a membrane filter.

8. 8. The method of claim 7, wherein the membrane filter is made of mixed cellulose esters (MCE), polyvinylidene fluoride (PVDF), polyester sulfone (PES), nitrocellulose, polytetrafluoroethylene, polycarbonate, or nylon, or a combination thereof.

9. 8. The method of claim 7, wherein the membrane filter is made of mixed cellulose esters (MCE), polyvinylidene fluoride (PVDF), nitrocellulose, polytetrafluoroethylene, polycarbonate, or nylon.

10. 8. The method of claim 7, wherein the membrane filter is made of mixed cellulose esters (MCE) or polyvinylidene fluoride (PVDF).

11. The method of claim 1, wherein the support is a solid growth medium.

12. The method of claim 1 , wherein the support is in a container and can be covered by a translucent lid.

13. 10. The method of claim 1, wherein steps b) and c) are performed multiple times to image several areas of the support.

14. The method of claim 13 , further comprising step d) combining the acquired images of the area to form a combined image.

15. 2. The method of claim 1, further comprising the step b') after step b) and before step c), repeating steps a) and b) one or more times, each time using a different set of at least two incident collimated light sources that form an angle (α) of at least 10° with respect to a normal to the support, thereby obtaining a plurality of acquired images of the area, and combining the acquired images of the area to form a combined image of the area, wherein the numerical value of the angle (α) is selected independently for each incident collimated light source.

16. 16. The method of claim 15, wherein in step c) the presence or absence of a microbiological object on the area of ​​the support is detected by identifying light reflected, scattered and / or diffused from the support and from the microbiological object on the support on the combined image of the area.

17. 10. The method of claim 1, wherein steps a) and b) are performed multiple times to image several areas of the support.

18. 18. The method of claim 17, further comprising, after step b), step b'') of combining the acquired images of the area to form a combined image of the area.

19. 19. The method of claim 18, wherein in step c) the presence or absence of a microbiological object on the area of ​​the support is detected by identifying light reflected, scattered and / or diffused from the support and from the microbiological object on the support on the combined image of the area.

20. 16. The method of claim 15, wherein steps a) and b) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times using 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different sets of at least two input collimated light sources, respectively.

21. 16. The method of claim 15, wherein steps a) and b) are repeated 1, 2, 3, 4, 5, 6, 7, or 8 times using 1, 2, 3, 4, 5, 6, 7, or 8 different sets of at least two input collimated light sources, respectively.

22. 16. The method of claim 15, wherein the two sets of incident collimated light sources differ in their number, their light type, their position, and / or the value of the angle α of each light source.

23. 16. The method of claim 15, wherein the two sets of incident collimated light sources differ in their number, their position, and / or the value of the angle α of each light source.

24. 16. The method of claim 1 or 15, wherein the set comprises at least three incident collimated light sources.

25. 16. The method of claim 1 or 15, wherein the set comprises 3 to 24 incident collimated light sources.

26. 16. The method of claim 1 or 15, wherein the set comprises between 3 and 12 incident collimated light sources.

27. 16. The method of claim 1 or 15, wherein the set comprises 3 to 6 incident collimated light sources.

28. 16. The method of claim 1 or 15, wherein the set comprises three incident collimated light sources.

29. 16. The method according to claim 1 or 15, wherein the set of incident collimated light sources are evenly distributed on one or several circles centered on the optical acquisition axis.

30. 16. The method of claim 1 or 15, wherein each incident collimated light source forms an angle (α) with the normal to the support that is independently selected between 15° and 75°.

31. 16. The method of claim 1 or 15, wherein the set comprises at least three incident collimated light sources and the value of the angle α is the same for all light sources of the set.

32. 16. The method of claim 1 or 15, wherein the incident collimated light source is selected from a collimated light emitting diode (LED), and a laser diode, and combinations thereof.

33. 16. The method of claim 1 or 15, wherein the incident collimated light source is a collimated white LED.

34. The method of claim 1 or 15, wherein the light receiving element is a camera including a pixel sensor or an array of active pixel sensors.

35. 16. The method of claim 1 or 15, further comprising the step of counting and / or identifying microbiological objects on the support from the acquired images or the combined images.

36. Before step a), providing a sample to be tested; contacting the sample with the support; incubating the support to allow growth of microorganisms, if present; 16. The method of claim 1 or 15, further comprising:

37. 16. The method of claim 1 or 15, used to detect microorganisms in a sample or to test the sterility of a sample.

38. 37. The method of claim 36, wherein the sample is obtained from a liquid, solid, or gas.

39. 37. The method of claim 36, wherein the sample is selected from the group consisting of a biological sample, an environmental sample, a medical device or any part thereof, a food or beverage for human or animal consumption, a pharmaceutical or cosmetic product, and ingredients of such a food, beverage, pharmaceutical or cosmetic product.

40. 1. A device for detecting a microbiological object on a support, comprising: at least two collimated incident light sources for illuminating the support, the at least two collimated incident light sources forming an angle (α) of at least 10° with respect to a normal to the support, the value of the angle (α) being independently selected for each collimated incident light source; a light receiving element for acquiring an image of an area of ​​the support illuminated by the at least two incident collimated light sources, the light receiving element having its optical acquisition axis along a normal to the support; means for detecting the presence or absence of microbiological objects on said area of ​​said support by identifying on said acquired image light reflected, scattered and / or diffused from said support and from microbiological objects on said support; Optionally, means for holding and optionally moving said support; 1. A device comprising:

41. 41. The device of claim 40, comprising at least three incident collimated light sources.

42. 41. The device of claim 40, comprising at least 13 incident collimated light sources.

43. 41. The device of claim 40, wherein each incident collimated light source forms an angle (α) with the normal to the support that is independently selected between 15° and 75°.

44. 41. The device of claim 40, wherein the value of the angle α is the same for incident collimated light sources that are symmetric about the optical acquisition axis.

45. 41. The device of claim 40, comprising at least three incident collimated light sources with angle (α) values ​​between 45° and 55°.

46. 41. The device of claim 40, comprising at least three incident collimated light sources with an angle (α) value between 30° and 40° and at least three incident collimated light sources with an angle (α) value between 45° and 55°.

47. 41. The device of claim 40, comprising at least three collimated incident light sources with an angle (α) between 25° and 35°, at least three collimated incident light sources with an angle (α) between 40° and 50°, and at least six collimated incident light sources with an angle (α) between 60° and 70°.

48. 41. The device of claim 40, comprising at least three collimated incident light sources having an angle (α) value between 28° and 32°, at least three collimated incident light sources having an angle (α) value between 33° and 37°, at least three collimated incident light sources having an angle (α) value between 38° and 42°, at least three collimated incident light sources having an angle (α) value between 48° and 52°, at least three collimated incident light sources having an angle (α) value between 53° and 57°, and at least three collimated incident light sources having an angle (α) value between 60° and 64°.

49. 41. The device of claim 40, comprising at least three collimated incident light sources, the incident light sources being evenly distributed on a circle centered on the optical acquisition axis and each incident light source forming an angle (α) with a normal to the support that is independently selected between 15° and 75°.

50. 50. Use of a device according to any one of claims 40 to 49 for detecting and optionally enumerating and / or identifying microbiological objects on a support.

51. Use of a device according to any one of claims 40 to 49 for detecting microorganisms in a sample or for testing the sterility of a sample.

Images