Image processing process applied to colony counters in microbiology

Abstract

To provide a device that is capable of simplifying the use of colony counting machines.SOLUTION: In a colony counting device (30) for colonies present in a sample, the device comprises a space configured to receive a support arranged to receive a solid nutrient medium and the sample, and includes first and second detection mechanisms formed by a single detection mechanism (35), wherein a processing unit (36) is configured to selectively control, in a bright field configuration, substitution of a dark diffusing surface (24) with a light diffusing surface (14), turning off of an electroluminescent diode (23) in the dark field configuration, turning on of an electroluminescent diode (13) in the bright field configuration, and in a dark field configuration, substitution of the light diffusing surface (14) with the dark diffusing surface (24), turning off of the electroluminescent diode (13) in the bright field configuration, and turning on of the electroluminescent diode (23) in the dark field configuration.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to the field of detection of microorganisms in growth media. The present invention is particularly applicable to microbiological analysis and quality control. The present invention can also be used in the fields of environment, agro-processing, pharmacology, cosmetology, and research and development.

[0002] The invention particularly relates to image processing methods applied to colony counters in microbiology. [Background technology]

[0003] Colony counts are still often performed today by manual counting of colonies visually observed in inoculated cultures.

[0004] Nevertheless, in the state of the art, there exist colony counting devices that are based on acquiring images of a sample containing a pre-seeded medium and then processing the acquired images to determine the number of colonies.

[0005] Thus, European Patent Specification No. 2807484 is known which describes a microbial detection device including a scanner-type detection system arranged to scan an area including the upper surface of the entire inoculated sample positioned side-by-side on the surface of a tray.

[0006] The images are then analyzed by image processing software to identify and count the colonies. A drawback of this type of instrument is that each sample to be analyzed is exposed to illumination for the entire time required for acquisition. Therefore, this type of device cannot be used when counting light-sensitive microcolonies or when using light-sensitive markers.

[0007] Another drawback of this type of device is that the medium support must be the same for all samples unless the device has optics that allow for focal length adjustment. If the device has optics that allow for focal length adjustment, the time required to acquire the entire sample will increase significantly.

[0008] French Patent No. 3 062 133 seeks to overcome the aforementioned drawbacks by proposing an instrument and a method capable of reducing the time required to count the final number of colonies present in a medium inoculated with a sample and of reducing the rate of counting errors.

[0009] However, sometimes complex preconditioning must be performed on the instrument depending on the microcolony.

[0010] Currently, several known light condition adjustments are used on colony counters, in particular those known as dark field microscopy.

[0011] In darkfield observation, the illumination of the sample can be on the opposite side of the objective (transmission type), on the same side (reflective type) or even to the side. For example, darkfield observation can be obtained with a light placed closely below on a black background. This type of observation has good performance because it can illuminate all small particles or diffuse elements, but it makes many artifacts appear, for example textile fibers or dust.

[0012] White background or brightfield transillumination type adjustments are also used. This adjustment allows the detection of any obstacles that absorb light while highlighting shadows. Brightfield transillumination is very robust since it does not create excessive flashes or reflections, but it lacks sensitivity in some cases, for example when bacteria are too small or lack contrast.

[0013] On the colony counters cited above, the user must adjust several parameters and create adjustments that define the type of algorithm and lighting for counting the colonies. These steps complicate the use of these machines. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] European Patent No. 2807484 [Patent Document 2] French Patent No. 3062133 Summary of the Invention [Problem to be solved by the invention]

[0015] An object of the present invention is to simplify the use of colony counting devices, also called colony counters.

[0016] Another object of the present invention is to improve counting performance, especially for environmental counting, where the entire bacterial flora is sought and highly diverse colonies need to be counted on the same sample. [Means for solving the problem]

[0017] According to a first aspect of the invention, there is provided a method for counting colonies present in a sample placed in a support arranged to accommodate said sample and a solid nutrient medium, comprising the steps of: - generating a brightfield image by a first detection mechanism from light transmitted through the support and collected by the first collection mechanism in a brightfield configuration; - generating a dark field image by a second detection mechanism from light scattered from the support and collected by the first collection mechanism in a dark field configuration; - generation by a processing unit of a so-called fused image obtained by the combination of two image operands, one of which is an inverted image of a bright-field or dark-field image, respectively, and the other image operand is a dark-field or bright-field image, respectively; - counting the number of colonies by a processing unit from the processing of the fused image; A method has been proposed which includes:

[0018] The first and second detection mechanisms may be the same or distinctly different. The detection mechanisms may be, for example, matrix photoelectric sensors.

[0019] The processing unit configured to generate the fused image and the processing unit configured to count the number of colonies may be identical or distinctly different, the processing unit may for example be a computing unit equipped with a storage means.

[0020] Advantageously, the generation of the fused image can be obtained by a linear combination, preferably an addition, of two image operands. Of course, multiple image operands may be used, each of which may be of bright field or dark field type. It is possible to use other illuminations, for example illumination from above, which performs well in the context of opaque media. The processing of the fused image can include thresholding the fused image based on a predefined threshold.

[0021] According to another possibility, the processing of the images can be realized by means of a neural network driven in order to count the number of colonies from the fused image.

[0022] According to a second aspect of the invention, there is provided a device for counting the colonies present in a sample, comprising a space adapted to accommodate a solid nutrient medium and a support arranged to accommodate said sample, the device comprising: - a first detection mechanism arranged to generate a bright field image from said space in a bright field configuration, i.e. from light transmitted through said sample, if present in the support, and collected by said first collection mechanism; - a second detection mechanism arranged to generate a dark field image from said space in a dark field configuration, i.e. from light scattered through the sample, if present in the support, and collected by said first collection mechanism; a processing unit arranged to generate a so-called fused image, which is obtained by the combination of two image operands, one of which is an inverted image of a bright field or a dark field image, respectively, and the other image operand is a dark field or a bright field image, respectively; - A unit for counting the number of colonies from the processing of fusion images and An apparatus including the following has been proposed:

[0023] Advantageously, the first and second detection mechanisms are formed by one detection mechanism, and the processing unit is arranged to selectively control, in the bright field configuration, replacement of the diffuse dark surface by a diffuse bright surface, turning off the electroluminescent diode for the dark field configuration, turning on the electroluminescent diode for the bright field configuration, and, in the dark field configuration, replacement of the diffuse bright surface by the diffuse dark surface, turning off the electroluminescent diode for the bright field configuration, turning on the electroluminescent diode for the dark field configuration.

[0024] According to one possibility, the first and second detection arrangements are formed by one detection arrangement which further comprises a diffusion device forming a bright surface and at the same time a dark surface for the bright field configuration.

[0025] Other characteristics and advantages of the invention will become apparent during the course of the following detailed description, in which reference should be made to the accompanying drawings, in which: [Brief description of the drawings]

[0026] [Figure 1]FIG. 1 is a side view of a brightfield observation adjustment according to the present invention. [Diagram 2] FIG. 2 is a graphical representation of the luminous intensity levels of pixels in the adjustment according to FIG. [Diagram 3] FIG. 3 is a side view of a dark field adjustment according to the present invention. [Figure 4] FIG. 4 is a graphical representation of the luminous intensity levels of the pixels in the adjustment according to FIG. [Diagram 5] FIG. 5 is a graphical representation of the inversion of FIG. [Figure 6] FIG. 6 is a graph of pixel intensity levels of the overlay of the diagrams of FIGS. [Figure 7] FIG. 7 is a schematic diagram of one embodiment of an apparatus according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Since the embodiments described below are in no way limiting, it will be possible to consider variations of the invention that include only a selection of features described and then separated from other described features, in particular if this selection of features is sufficient to provide a technical advantage or to distinguish the invention from the previous state of the art, including at least one feature, preferably functional, that does not include structural details or that is accompanied by only a portion of structural details if this selection alone is sufficient to provide a technical advantage or to distinguish the invention from the previous state of the art.

[0028] In the following description, elements having the same structure or similar function will be referred to by the same reference numerals.

[0029] Here, a method for counting the number of colonies present in a sample will be described.

[0030] Referring to FIG. 1, a brightfield observation instrument 10 is depicted diagrammatically.

[0031] The imaging apparatus 10 comprises a support 11 arranged to receive a Petri dish 12 containing the sample to be analysed.

[0032] The observation device 10 is further equipped with a number of electroluminescent diodes 13 , or LEDs, an abbreviation for Light-Emitting Diode in English, a white surface diffuser 14 and a matrix photoelectric sensor 15 .

[0033] A plurality of electroluminescent diodes 13 and a diffuser 14 are both positioned on the opposite side of the matrix photoelectric sensor relative to the Petri dish 12 .

[0034] The different devices are arranged in such a way that the matrix photoelectric sensor 15 and the Petri dish 12 are in the cone 16 of light diffused by the diffuser 14. The cone is represented in the figure by a dotted line.

[0035] Similarly, when the electroluminescent diode 13 is illuminated in the absence of any colonies in the Petri dish, the image formed by the matrix photoelectric sensor 15 is that of a white background.

[0036] The light intensity of a pixel located at x and y on the detection surface of the matrix photoelectric sensor 15 can be written as Ifc(x,y).

[0037] Thus, a first collection mechanism, a matrix photoelectric sensor 15, is arranged to collect light transmitted by the sample in a bright-field configuration and generate a bright-field image.

[0038] FIG. 2 illustrates the intensity levels of pixels along a column (or row) of the detection surface of a matrix photoelectric sensor 15 with an optical axis.

[0039] The light intensity I is represented on the ordinate, while the different pixels of a column (or row) are represented on the abscissa.

[0040] As can be considered here, in the absence of colonies, the light intensity has a maximum value approximately equal to 255, denoted as Icmax.

[0041] Temporarily, the light intensity has other values denoted as Ifc1, Ifc2, Ifc3, and Ifc4, which are lower than Ifcmax although they may vary depending on the case.

[0042] On the graph, the following order relationship is pointed out: Ifc1 < Ifc2 < Ifc3 < Ifc4

[0043] Here, with Ifc1 < Ifcmax, the value Ifc1 indicates the presence of shielding of the light directly received by the matrix photoelectric sensor 15.

[0044] It can be inferred that a colony exists at the position x1 corresponding to the value Ifc1.

[0045] Since the values Ifc3 and Ifc4 are close to Ifcmax, it can be inferred that no colonies exist at the positions x3 and x4.

[0046] Since the value Ifc2 is intermediate between the values of Ifc1 and Ifc3 or Ifc4 respectively, the presence of a colony at the position x2 corresponding to Ifc2 may be doubtful. In fact, the position x2 may correspond, for example, to the lack of opacity of the Petri dish rather than the presence of a colony.

[0047] It is considered that a determination can be made by threshold processing by determining a threshold Ifcs < Ifcmax, where an intensity Ifc above that is not considered to suggest the presence of a colony, and an intensity Ifc below that is considered to suggest the presence of a colony.

[0048] On the graph, the following order relationship is pointed out: Ifc1 < Ifcs < Ifc2 < Ifc3 < Ifc4

[0049] In this case, the following would be determined: presence of a colony at x1, absence of colonies at x2, x3 and x4.

[0050] In accordance with the present invention, it is not useful to define such a threshold.

[0051] Referring to FIG. 3, a dark field observation instrument 20 is shown diagrammatically.

[0052] The observational apparatus 20 comprises a support 21 arranged to receive a Petri dish 22 containing the nutrient medium and the seeded sample to be analysed.

[0053] More specifically, with respect to the Petri dish 22, a number of elements corresponding to positions x1, x2, x3, x4 are referenced to generally represent the substance of the contents of the Petri dish.

[0054] As can be better understood below, positions x1 and x4, represented by black circles, indicate the presence of large colonies, position x2 indicates the presence of more subdued colonies, while position x3 represents an artefact.

[0055] The observation device 20 is further equipped with a number of electroluminescent diodes 23, or LEDs, an abbreviation for Light-Emitting Diode in English, a diffuser 24 with a black surface and a matrix photoelectric sensor 25 with an optical axis.

[0056] A plurality of electroluminescent diodes 23 and a diffuser 24 are both positioned on the opposite side of the matrix photoelectric sensor relative to the Petri dish 22 .

[0057] Each of the electroluminescent diodes 23 has an emission axis that is at an angle of more than 60° to the optical axis of the collector 25 .

[0058] The different devices are arranged in such a way that the matrix photoelectric sensor 25 is not in the cone of light, and the Petri dish 12 is in the cone of light 16 diffused by the diffuser 14. The cone is represented in the figure by a dotted line.

[0059] Similarly, when the electroluminescent diode 23 is illuminated in the absence of any colonies in the Petri dish, the image formed by the matrix photoelectric sensor 25 is an image of a black background since no light rays reach the sensor 25 directly.

[0060] The light intensity of a pixel located at x and y on the detection surface of the matrix photoelectric sensor 25 can be written as Idf(x,y).

[0061] Thus, a second collection mechanism, a matrix photoelectric sensor 25, is arranged to collect light scattered by the sample in a dark field configuration and generate a dark field image.

[0062] FIG. 4 illustrates the intensity levels of pixels along a column (or row) of the sensing surface of the matrix photoelectric sensor 25. As shown in FIG.

[0063] The light intensity Idf is represented on the ordinate, while the different pixels of a column (or row) are represented on the abscissa.

[0064] On the graph, the following order relationship is noted: Idf1=Idf4>Idf2>Idf3

[0065] The values ​​Idf1 and Idf4 indicate the presence of direct light scattering and its reception by the matrix photoelectric sensor 25.

[0066] Since the value Idf3 is close to 0, it can be inferred that there is no colony at position x3.

[0067] The presence of a colony at position x2 corresponding to Idf2 may be doubtful since the value Idf2 is intermediate between Idf1 and 0. In fact, position x2 may for example correspond to a lack of opacity of the Petri dish rather than the presence of a colony.

[0068] The determination could be made by thresholding, by determining a threshold value Idfs above which the intensity Idf is considered to indicate the presence of a colony and below which the intensity Idf is considered to indicate the absence of a colony.

[0069] On the graph, the following order relationship is noted: Idf1=Idf4>Idfs>Idf2>Idf3

[0070] In this case, the following would be determined: presence of colonies at x1 and x4, absence of colonies at x2, x3.

[0071] In accordance with the present invention, it is not useful to define such a threshold.

[0072] Moreover, it is pointed out that depending on the technique used, the results are not the same. - Brightfield observation adjustment: presence of colonies at x1, absence of colonies at x2, x3 and x4; - Darkfield observation adjustment: colonies present at x1 and x4, no colonies present at x2 and x3

[0073] According to the invention, an inverted dark field graph Idfinv is generated by a processing unit (not shown), as represented in FIG.

[0074] The inverted graph Idf inv can be obtained by a transformation such as Idf inv (x, y) = Idf max - Idf (x, y) where Idf max = 255.

[0075] Then, according to the invention, a so-called fused image If is generated by the processing unit by addition of the images Idf inv and Ifc, which can be written as If(x,y) = Idf inv(x,y) + Ifc(x,y). Such a fused image may present the graph represented in FIG.

[0076] In the image If obtained in this manner, four positions x1, x2, x3, and x4 can be observed which correspond to the four positions x1 to x4 in the image Ifc and the four positions x1 to x4 in the image Idf.

[0077] On the graph, the following order relationship is noted: If1=If4 <If2<If3

[0078] Similarly, it is possible to take x1 and x4 as markers for the location of the colony, whereas this was not the case when only the images obtained in bright field were considered: by correlating the dark field and bright field results, it is possible to improve the interpretation.

[0079] It is believed that the determination can be made by thresholding by determining a threshold value Ifs below which the intensity If is deemed to indicate the presence of a colony and above which the intensity If is deemed to indicate the absence of a colony.

[0080] On the graph, the following order relationship is noted: If1=If4 <If2<Ifs<If3

[0081] In this case, the following would be determined: presence of colonies at x1 and x4 and x2, absence of a colony at x3.

[0082] The processing unit may be configured to count the number of colonies from thresholding the fused image.

[0083] In the above examples, an inverse image of the image obtained in the dark field configuration was used, it is of course also possible to determine an inverse image of the image obtained in the bright field configuration and add this to the image obtained in the dark field.

[0084] In the above examples, for reasons of simplicity, the variation of intensity along only one dimension has been represented.

[0085] Naturally, the variation of the intensity levels can be illustrated by a surface rather than by a curve. In fact, the sensors 15 and 25 are matrix sensors which form an image in two dimensions.

[0086] In the above examples, the pixels represent variations in intensity, but the invention can be extended to color variations as well.

[0087] More generally, a fused image can be obtained by the combination of two image operands, one of which is an inverted bright-field or dark-field image, respectively, and the other of which is a dark-field or bright-field image, respectively.

[0088] Therefore, for example, if C1 and C2 of the same sign are, for example, 0 to 1, and the sum of the two absolute values ​​of C1 and C2 is equal to 1, then If(x,y) can be written as C1 × Ifc(x,y) + C2 × Idfinv(x,y).

[0089] As illustrated by FIG. 7, one embodiment of the present invention is depicted which envisages an apparatus 30 for counting colonies present in a sample, including a detection mechanism, here designated 35, and a processing unit 36 ​​which are identical according to the two configurations.

[0090] The processing unit 36 ​​is arranged to selectively control: - In bright field configuration, Replacement of the diffuse dark surface 24 by the diffuse light surface 14, ◯ Turning off the electroluminescent diode 23, Lighting of the electroluminescent diode 13, Creation of a bright field image from light transmitted by the sample and collected by the detection arrangement 35. - In dark field configuration, Replacement of the diffuse light surface 14 by a diffuse dark surface 24, 〇 Turning off the electroluminescent diode 13, Lighting of the electroluminescent diode 23, Creation of a dark field image from light scattered by the sample and collected by the detection mechanism 35.

[0091] According to one possibility, the diffuse bright and the diffuse dark surfaces can be realized by the same surface, with a material allowing an alternating transition from one mode to the other, and then it is no longer necessary to carry out the replacement step described above.

[0092] The processing unit 36 ​​is further configured to generate a so-called fused image and to count the number of colonies.

[0093] Naturally, different features, aspects, variants and embodiments of the invention can be combined with each other according to various combinations, unless they are mutually inconsistent or exclusive. In particular, all variants and embodiments described above can be combined with each other. [Explanation of symbols]

[0094] 10 Brightfield observation equipment 11 Support 12 Petri dish 15 First detection mechanism 20 Dark-field observation equipment 21 Support 22 Petri dish 25 Second detection mechanism 30 Colony counting device 35 Detection mechanism 36 Processing Unit

Claims

1. In the counting device (30) for counting colonies present in a sample, the first and second detection mechanisms are formed by one detection mechanism (35) further including a bright surface (14) and an electroluminescent diode (13) for a bright field configuration, and a dark surface (24) and an electroluminescent diode (23) for a bright field configuration, and the processing unit (36) is arranged to selectively control, in the bright field configuration, the replacement of the diffuse dark surface (24) with the diffuse bright surface (14), the turning off of the electroluminescent diode (23) for the dark field configuration, and the turning on of the electroluminescent diode (13) for the bright field configuration, and, in the dark field configuration, the replacement of the diffuse bright surface (14) with the diffuse dark surface (24), the turning off of the electroluminescent diode (13) for the bright field configuration, and the turning on of the electroluminescent diode (23) for the dark field configuration, of the counting device (30).

2. 2. The counting device (30) of claim 1, wherein the first and second detection mechanisms are formed by a single detection mechanism (35) that further includes a diffusion device that forms a bright surface (14) for the bright field configuration and simultaneously a dark surface (24).

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