Image processing method applied to colony counting in microbiology

Abstract

Counting colonies present in a sample which is placed in a support, the method comprising: generating a bright field image by a first detection means 35 (Fig. 1; 15) from the light transmitted from sa

Description

Technical Field The present invention relates to the field of the detection of micro-organisms in a growth medium. The invention especially applies to microbiological analysis and to quality control. The invention also finds use in the fields of environment, agri-food, pharmacology, cosmetics and research and development. The present invention particularly concerns an image processing method applied to colony counting in microbiology. Prior art Colony counting is still carried out to a not inconsiderable extent today by manually counting visually observed colonies in a seeded culture medium. Nevertheless, in the prior art, there are devices for counting colonies by capturing images of a sample comprising a pre-seeded culture medium, then determining the number of colonies by processing the acquired images. Thus, the document EP 2807484 describes a device for detecting micro-organisms, said device comprising a detection system, such as a scanner that is designed to scan a zone that comprises the upper surfaces of a group of seeded samples, said samples being positioned side by side on the surface of a plate. The images are then analysed by an image processing software in order to identify and count the colonies. This type of apparatus has the drawback that each sample being analysed is illuminated during the time required for the data acquisition. Consequently, it is not possible to use this type of device to count photosensitive microcolonies or when photosensitive markers are employed. Another drawback of this type of device is that it does not possess an optical system that allows the focal distance to be adjusted; the supports for the culture medium have to be identical for all samples. If the device possessed an optical system to allow the focal distance to be adjusted then the acquisition time required for the group of samples would be significantly increased. The document FR 3062133 proposes to overcome the cited drawbacks by means of an apparatus and a method which decrease the time required to count the definitive number of colonies in the seeded culture medium of a sample as well as reducing the error count rates. However, depending on the micro-colonies, somewhat complicated prior adjustments have to be carried out on the apparatus. Currently, several known adjustments to the lighting conditions are employed on colony counters, in particular the adjustment known as “dark field microscopy”. In darkfield microscopy the sample can be illuminated from behind the objective (by transmission), from the same side (by reflection) or from the side. For example, the dark field microscopy may be obtained by a grazing light mounted below with a black background. This type of imagery is efficient as it enables very small particles or diffuse elements to be illuminated, but it reveals many artefacts, for example textile fibres or dust. An adjustment of the white background type or microscopy by bright field transmission is also employed. This adjustment enables the detection of all obstacles that absorb light, enabling a shadow to be seen. Microscopy by bright field transmission is very robust because it does not create too much radiance or reflections, but lacks sensitivity in certain cases, for example when the bacteria are too small or lack contrast. On the abovementioned colony counters the operators have to adjust certain parameters and create an adjustment where they fix the illumination and the algorithm type to count the colonies. These steps complicate the use of these machines. An object of the invention is to simplify the use of machines for colony counting -also called colony counters. Another object of the invention is to improve the counting performance, especially for environmental counting, where all bacterial flora are searched for, and where a large variety of colonies have to be counted on the same sample. Disclosure of the invention According to a first embodiment of the invention, a method is provided to count colonies present in a sample that is placed in a support designed to receive said sample and a solid nutrient medium, comprising: - generating by detection means a bright field image from the light transmitted from said support in a bright field mode and collected by said detection means, - generating by the detection means a dark field image from the light diffused from said support in a dark field mode and collected by said detection means and, 09 06 25 - generating a “fusion” image with a processing unit obtained by combining two operand images, wherein one combination is an inverse image of the bright field image combined with the dark field image, and the other combination is an inverse image of the dark field image combined with the bright field image, 5 wherein the generation of the fusion image is obtained by linearly combining the two operand images by addition, - enumerating the colonies by the processing unit from the processed fusion image. One detection means may, for example, be a dot matrix photoelectric sensor. 10 The processing unit configured to generate the fusion image and the processing unit configured to enumerate the colonies may be identical or different. The processing unit may, for example, be a calculation unit equipped with storage means. The generation of the fusion image is advantageously obtained by linearly combining the two operand images, preferably by addition. A plurality of operand 15 images may, of course, be utilised, wherein each operand image can be a bright field or dark field type. Other types of lighting may be used, for example by illuminating from above, which is efficient in the case of an opaque culture medium. The fusion image may be processed by thresholding the fusion image as of a predefined threshold. According to another possibility, the image may be processed by means of a neural 20 network configured to enumerate the number of colonies from a fusion image. According to a second embodiment of the invention a device is provided to count colonies present in a sample and equipped with a site configured to receive a support designed to receive a solid nutrient medium and said sample, comprising: o detection means (35) having a first detection mode configured to generate a 25 bright field image from the light transmitted from said site in a bright field mode and collected by said detection means, and o a second detection mode configured to generate a dark field image from the light diffused from said site in a dark field mode and collected by said detection means, 30 o a processing unit (36) configured to generate a “fusion” image obtained by combining two operand images, wherein one of the operand images is an inverse image of the bright field image, respectively the dark field image, and the other operand image is the dark field image, respectively the bright field image, and wherein the generation of the fusion image is obtained by linearly combining the two 35 operand images by addition, - the processing unit being also configured to enumerate a number of colonies from the fusion image. Advantageously, the processing unit is set up to control selectively in a bright field mode, the replacement of the dark diffusing surface by a bright diffusing surface, switching off the light emitting diodes for a dark field mode, switching on the light emitting diodes for a bright field mode; in a dark field mode the replacement of the bright diffusing surface by the dark diffusing surface, switching off the light emitting diodes for a bright field mode, switching on the light emitting diodes for a dark field mode. According to one possibility, the first and the second detection means also possesses a diffusing device that forms both a bright surface for a bright field surface mode and a dark surface. Brief description of the figures Other characteristics and advantages of the invention will become evident on reading the following detailed description that can be understood by referring to the attached drawings: - [Fig. 1 ] Figure 1 is a side view of an adjustment of bright field imaging according to the invention, - [Fig. 2] Figure 2 is a graphical representation of the brightness levels of the pixels in an adjustment according to Figure 1, - [Fig. 3] Figure 3 is a side view of an adjustment of dark field imaging according to the invention, - [Fig. 4] Figure 4 is a graphical representation of the brightness levels of the pixels in an adjustment according to Figure 3, - [Fig. 5] Figure 5 is a graphical representation of a negative of Figure 4, - [Fig. 6] Figure 6 is a graphical representation of the brightness levels of the pixels from the overlay of the representations of Figure 2 and Figure 5, - [Fig. 7] Figure 7 is a schematic representation of an embodiment of a device according to the invention. Description of the embodiments As the embodiments described below are not exhaustive, in particular it is possible to consider variants of the invention which comprise only a selection of the described characteristics, subsequently isolated from the other described characteristics, if this selection of characteristics is sufficient to provide a technical advantage or to differentiate the invention from the prior art. This selection comprises at least one characteristic, preferably functional without structural details, or with only a part of the structural details if this part alone is sufficient to provide a technical advantage or to differentiate the invention from the prior art. In the following description those elements having an identical structure or analogous functions will be designated by the same references. We now describe a method to count colonies present in a sample. With reference to Figure 1, a bright field imaging apparatus 10 is represented schematically. The imaging apparatus 10 possesses a support 11, arranged to receive a Petri dish 12 comprising a sample to be analysed. The imaging apparatus 10 is also equipped with a plurality of light emitting diodes (LEDs) 13, a white surface diffuser 14 and a matrix photoelectric sensor 15. Both the plurality of light emitting diodes 13 and the diffuser 14 are located on the side opposite to the matrix photoelectric sensor with respect to the Petri dish 12. The different devices are arranged such 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 dotted lines. Also, when the light emitting diodes 13 are lit up, then in the absence of a colony in the Petri dish the image formed by the matrix photoelectric sensor 15 is an image with a white background. The luminous intensity of a pixel disposed at x and y on the detection surface of the matrix photoelectric sensor 15 can be defined as Ifc (x, y). The first collection element, which is the matrix photoelectric sensor 15, is therefore arranged to collect the light transmitted by the sample in a bright field and generates a bright field image. Figure 2 illustrates an intensity level of the pixels in a column (or a row) of the detection surface of the matrix photoelectric sensor 15 having an optical axis. The luminous intensity I is represented on the ordinate-axis whereas the different pixels of the column (or row) are represented on the abscissa-axis. As can be observed, in the absence of a colony, the luminous intensity has a maximum value, designated as Icmax, substantially equal to 255. Occasionally, the luminous intensity shows other values, assigned Ifc1, Ifc2, Ifc3 and Ifc4, possibly different, but less than Ifcmax. The sequential order is depicted on the graph as follows: Ifc1 <Ifc2 <Ifc3 <Ifc4. The value Ifc1 indicates the presence of an occultation of the light received directly by the matrix photosensitive sensor 15, with here Ifc1 <Ifcmax. One may presume that a colony is present at position x1 corresponding to the value Ifc1. As the values Ifc3 and Ifc4 are close to Ifcmax one may presume that colonies do not exist at the positions x3 and x4. As the value Ifc2 lies between Ifc1 and that of Ifc3, respectively Ifc4, there may be some doubt regarding the presence of a colony at the position x2 that corresponds to Ifc2. Indeed, the position x2 may, for example, correspond to an opacity defect of the Petri dish, and not to the presence of a colony. The decision could be made by means of thresholding, in that a threshold lfcs<lfcmax is determined, above which an intensity Ifc is not considered to reveal the presence of a colony and below which an intensity Ifc is considered to reveal the presence of a colony. The sequential order is depicted on the graph as follows: Ifc1 <Ifcs <Ifc2 <Ifc3 <Ifc4. In this case the following decision would be taken: presence of a colony at x1, absence of colonies at x2, x3 and x4. According to the invention there is no need to fix such a threshold. With reference to Figure 3, a dark field imaging apparatus 20 is represented schematically. The imaging apparatus 20 is equipped with a support 21, arranged to receive a Petri dish 22 comprising a nutrient medium and a seeded sample to be analysed. Referring more particularly to the Petri dish 22, a plurality of elements corresponding to the positions x1, x2, x3, x4 are referenced to schematically represent the reality of the contents of the Petri dish. As will be better understood later on, the positions x1 and x4, represented by black disks indicate the presence of significant colonies; the position x2 indicates the presence of a less significant colony, whereas the position x3 represents an artefact. 10 02 25 The imaging apparatus 20 is also equipped with a plurality of light emitting diodes (LEDs) 23, a black surface diffuser 24 and a matrix photoelectric sensor 25 possessing an optical axis. Both the plurality of light emitting diodes 23 and the diffuser 24 are located on the side opposite to the matrix photoelectric sensor with respect to the Petri dish 22. Each light emitting diode 23 has a light emitting axis forming an angle greater than 60° with respect to the optical axis of the detector 25. The different devices are arranged such that the matrix photoelectric sensor 25 is not in the cone of light 26 from the light emitting diode 23 and the Petri dish 22 is in the cone 26 of light diffused by the diffuser 24. The cone 26 is represented in the Figure by dotted lines. Also, when the light emitting diodes 23 are lit up, then in the absence of a colony in the Petri dish the image formed by the matrix photoelectric sensor 25 is an image with a black background, because no light wave directly reaches the detector 25. The luminous intensity of a pixel disposed at x and y on the detection surface of the matrix photoelectric sensor 25 can be defined as Ifc (x, y). The collection element, which is the matrix photoelectric sensor 25, is therefore arranged to collect the light diffused by the sample in a darkfield mode and generates a dark field image. Figure 4 illustrates an intensity level of the pixels in a column (or a row) of the detection surface of the matrix photoelectric sensor 25. The luminous intensity Idf is represented on the ordinate whereas the different pixels of the column (or row) are represented on the abscissa. The sequential order is depicted on the graph as follows: Idf 1 = Idf4 >Idf2 >Idf3 The values Idf1 and Idf2 directly indicate the presence of diffused light and its capture by the matrix photoelectric sensor 25. As the value Idf3 is close to 0, one may presume that a colony does not exist at the position x3. As the value Idf2 lies between Idf1 and 0, there may be some doubt regarding the presence of a colony at the position x2 that corresponds to Idf2. Indeed, the position x2 may, for example, correspond to an opacity defect of the Petri dish, and not to the presence of a colony. The decision could be made by means of thresholding, in that a threshold Idfs is determined, above which an intensity Idf is considered to reveal the presence of a colony and below which an intensity Idf is considered to reveal the absence of a colony. The sequential order is depicted on the graph as follows: Idf 1 = Idf4 >Idfs >Idf2 >Idf3. In this case the following decision would be taken: presence of a colony at x1 and x4, absence of colonies at x2, x3. According to the invention there is no need to fix such a threshold. In addition, it should be noted that depending on the technique used the conclusions are not the same: - in the bright field imaging mode a colony is present at x1, colonies are absent at x2, x3 and x4. - in the dark field imaging mode a colony is present at x1 and x4, colonies are absent at x2, x3. According to the invention, and as illustrated in Figure 5, an inverted graph Idfinv of the dark field is generated by a processing unit (not shown). Idfinvl, Idfinv2, Idfinv3 and Idfinv4 correspond to the inverted values of idf1,2, 3, and 4 respectively. Idfinvmax is the maximum value of Idfinv. The inverted graph Idfinv can be obtained for example by a transformation such as Idfinv (x, y) = Idfmax - Idf (x, y) with, for example ldfmax= 255. Then, according to the invention, a ‘fusion’ image If is generated by the processing unit by adding the image Idfinv and the image Ifc; wherein If (x, y) = Idfinv (x, y) + Ifc (x, y). The graph shown in Figure 6 illustrates such a fusion image. Ifmax is the maximum value of If. The thus obtained image shows four positions x1, x2, x3, x4 corresponding to the four positions x1 to x4 of the image Ifc and to the four positions x1 to x4 of the image Idf. The sequential order is depicted on the graph as follows: If1 = If4 <If2 <If3 In addition, it is possible to accept that x1 and x4 mark the position of a colony, whereas this was not the case when only the bright field image was considered: combining the dark field results with the bright field results permits an improvement in decision making. The decision could be made by means of thresholding, in that a threshold Ifs is determined, below which an intensity If is considered to reveal the presence of a colony and above which an intensity If is considered to reveal the absence of a colony. The sequential order is depicted on the graph as follows: If 1 = If4 <If2 <Ifs <If3. In this case the following decision would be taken: presence of a colony at x1 and x4, as well as at x2, absence of colonies at x3. The processing unit can be set up to enumerate the colonies by thresholding the fusion image. In the just described example, the inverted image of that obtained in the dark field mode was used. Of course, the inverted image of that obtained in the bright field mode could have been used for the determination by adding it to that obtained in the dark field mode. In the just described example, for reasons of simplicity, the variations of intensity have been represented in one dimension only. Of course, the variations of intensity could have been illustrated with a surface rather than with a curve. In fact, the sensors 15 and 25 are matrix sensors that form two-dimensional images. In the example just described, the variations of intensity have been represented by pixels. The invention can be extended to variations in colour. More generally, a fusion image can be obtained by combining two operand images, wherein one combination of the operand images is an inverse image of the bright field image with the dark field image, and the other combination of operand image is the inverse of the dark field image, with the bright field image. Accordingly, one may write, for example: If (x, y) = C1 * Ifc (x, y) + C2 * ldfinv(x, y) with C1 and C2 of the same sign, for example 0 and 1, and where the sum of the two absolute values of C1 and C2 is equal to 1. As illustrated by Figure 7, an embodiment according to the invention is depicted which provides for a device 30 to count colonies present in a sample comprising a detection means 35 which is the same for the two modes, processing unit 36. The processing unit 36 is set up to selectively control: - in a bright field mode: o the replacement of the diffusing dark surface 24 by the diffusing bright surface 14, o switching off the light emitting diodes 23, o switching on the light emitting diodes 13, o generating a bright field image from light transmitted by the sample and collected by the detection means 35. - in a darkfield mode: o the replacement of the diffusing bright surface 14 by the diffusing dark surface 24, o switching off the light emitting diodes 13, o switching on the light emitting diodes 23, o generating a dark field image from light diffused by the sample and collected by the detection means 35. According to one possibility, the diffusing bright surface and the diffusing dark surface may be produced by the same surface, in a material that permits the passage alternatively from one mode to the other. The above cited replacement steps would then no longer need to be implemented. The processing unit 36 is also configured to generate the “fusion” image and to enumerate the colonies. Of course, the different characteristics, forms, variants and embodiments of the invention can be combined with one another according to diverse combinations insofar as they are not incompatible or exclusive to one another. In particular, all the above described variants and embodiments can be combined together. 09 06 25

Claims

1. Method for counting colonies present in a sample that is placed in a support designed to receive said sample and a solid nutrient medium, comprising:- generating by sole detection means (35) a bright field image from the light transmitted from said support in a bright field mode and collected by first detection means,- generating by the detection means (35) a dark field image from the light diffused from said support in a dark field mode and collected by said detection means and,- generating a “fusion” image with a processing unit (36) obtained by combining two operand images, wherein one combination is an inverse image of the bright field image combined with the dark field image, and the other combination is an inverse image of the dark field image combined with the bright field image, wherein the generation of the fusion image is obtained by linearly combining the two operand images by addition,- enumerating the colonies by the processing unit (36) from a processed fusion image.

2. Method according to any of the preceding claims wherein the processing of the fusion image comprises a thresholding of the fusion image as of a predefined threshold.

3. Device (30) to count colonies present in a sample and equipped with a site configured to receive a support designed to receive a solid nutrient medium and said sample, comprising:- Sole detection means (35) configured to generate a bright field image from the light transmitted from said site in a bright field mode and collected by said detection means, and- the detection means (35) configured to generate a dark field image from the light diffused from said site in a dark field mode and collected by said detection means,- a processing unit (36) configured to generate a “fusion” image obtained by combining two operand images, wherein one combination is an inverse image of the bright field image combined with the dark field image, and the other combination is an inverse image of the dark field image combined with the bright field image, and wherein the generation of the fusion image is obtained by linearly combining the two operand images by addition,- the processing unit being also configured to enumerate a number of colonies from the fusion image.09 06 254. Device (30) as claimed in claim 3, for counting colonies present in a sample wherein the detection means (35) also comprise a bright surface (14) and light emitting diodes (13) for the bright field mode, a dark surface (24) and light emitting diodes (23) for the dark field mode, and wherein the processing unit (36) is set up to control selectively in a bright field mode, the replacement of the dark diffusing surface (24) by the bright diffusing surface (14), switching off the light emitting diodes (23) for a dark field mode, switching on the light emitting diodes (13) for a bright field mode; in a dark field mode the replacement of the bright diffusing surface (14) by the dark diffusing surface (24), switching off the light emitting diodes (13) for a bright field mode, switching on the light emitting diodes (23) for a dark field mode.

5. Device (30) for counting according to claim 3 or claim 4 wherein the detection means also comprise a diffusing device that forms both a bright surface (14) for a bright field mode and a dark surface (24).

Images