Colony counting device and colony counting method

Abstract

The aim is to provide a colony counting device and method that enable more accurate judgment regarding the validity of counting results and can efficiently count colonies even in samples where spread colonies have occurred. [Solution] A colony counter 10 for counting colonies, which are aggregates of microorganisms, comprises: a camera 13 for capturing a sample image from a petri dish 1 in which microorganisms are cultured; and a control unit 17 that detects spread colony ICs from the sample image based on the number of pixels obtained by binarizing the sample image captured by the camera 13, and determines the validity of the count result from the ratio of the total pixel value of the detected spread colony ICs to the area of ​​the petri dish 1.

Description

Technical Field

[0001] The present invention relates to a colony counting device such as a colony counter for counting the number of colonies, which are aggregates of microorganisms (bacteria), and a colony counting method.

Background Art

[0002] Conventionally, a culture method has been used for microbial limit tests of foods and water quality tests in drinking water or pools. The culture method is, for example, a method in which a Petri dish (also called a petri plate) with a diameter of 90 mm is filled with a culture medium, and microorganisms are uniformly inoculated over the entire petri dish. After smear or dilution of the specimen, the specimen is cultured at a constant temperature for a certain period of time to grow colonies (aggregates of microorganisms) so that they can be visually observed. Then, by counting the number of colonies, the degree of growth of microorganisms over time is examined.

[0003] As a method for counting the number of colonies, a method has been proposed in which a plurality of specimen images (digital images) in a time series taken at different times are compared, and colonies are extracted and counted from the changes in the specimen images (see Patent Documents 1-2 below).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in the culture method using a Petri dish, the growth rate of colonies varies depending on the type of microorganism to be cultured, and so-called spreading colonies (abnormally grown colonies) are likely to occur due to the influence of condensed water droplets.

[0006] In samples where such spread colonies had occurred, in some cases, slower-growing colonies were covered by faster-growing colonies. Therefore, in actual testing, determining the validity of the count results—specifically, how to count the number of colonies under the spread colonies—often relied on the experience of the tester, making it difficult to establish clear criteria.

[0007] The present invention has been made in view of the above, and its objective is to provide a colony counting device and colony counting method that enable more accurate judgment regarding the validity of the counting results and can efficiently count colonies even in the case of samples in which spread colonies have occurred. [Means for solving the problem]

[0008] To achieve the above objectives, an aspect of the present invention is a colony counting device for counting colonies, which are aggregates of microorganisms, comprising: an imaging unit for capturing a sample image from a sample in which microorganisms have been cultured; a detection unit for detecting colonies in the sample image that are larger than a predetermined count threshold based on the number of pixels obtained by binarizing the sample image captured by the imaging unit; and a determination unit for determining the validity of the number of colonies estimated from the sample image based on the ratio of the total number of pixels of the colonies detected by the detection unit that are larger than the count threshold to the total number of pixels of the culture vessel used to culture the microorganisms.

[0009] Another aspect of the present invention is a colony counting method for counting colonies, which are aggregates of microorganisms, comprising: a step of capturing a sample image from a sample in which microorganisms have been cultured using an imaging unit; a step of detecting colonies from the sample image that are larger than a predetermined count threshold based on the number of pixels obtained by binarizing the sample image captured by the imaging unit using a detection unit; and a step of determining whether the detection result of colonies larger than the count threshold detected by the detection unit is within a certain range, and determining the validity of the number of colonies estimated from the sample image using a determination unit. [Effects of the Invention]

[0010] According to the present invention, it is possible to make a more accurate judgment regarding the validity of the counting results, and a colony counting device and colony counting method can be provided that can efficiently count colonies even in the case of samples in which spread colonies have occurred. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram showing an example of the configuration of a colony counter according to an embodiment of the present invention. [Figure 2] The following diagrams schematically show examples of sample images to be tested by the colony counter according to this embodiment. Figure (a) shows a petri dish image illustrating the case where only normal colonies are present, Figure (b) shows a petri dish image illustrating the case where larger colonies have occurred, Figure (c) shows a petri dish image illustrating the case where colonies have spread to a part of the sample, Figure (d) shows a petri dish image illustrating the case affected by condensation droplets, and Figure (e) shows a petri dish image illustrating the case where colonies have spread to the entire surface. [Figure 3] This is a schematic diagram showing an example of a sample image to be examined by the colony counter according to this embodiment. [Figure 4] This flowchart illustrates the inspection method using the colony counter according to this embodiment. [Modes for carrying out the invention]

[0012] The following describes a colony counter to which the colony counting device and colony counting method according to an embodiment of the present invention are applied, with reference to the drawings. It should be noted that in this embodiment, the drawings are schematic representations of the invention and may differ from the actual device.

[0013] Embodiment Figure 1 shows a system for testing for microorganisms (bacteria) from a sample image in a petri dish 1 according to an embodiment of the present invention, and here a colony counter 10 that counts the number of colonies, which are aggregates of microorganisms, is shown as an example.

[0014] The colony counter 10 consists of a stage glass 12, a camera 13, LED ring light illumination 14, 15, a monitor 16, a control unit 17, a storage unit 18, and an operation unit 19, as shown in Figure 1, for example.

[0015] The stage glass 12 is used to automatically or manually set the petri dish 1 used for culturing microorganisms. On this stage glass 12, the glass or plastic petri dish 1 is set with the lid 3 attached to the dish body 2.

[0016] The imaging camera 13 captures the sample image (original image) of the petri dish 1 set on the stage glass 12 as a petri dish image (digital image). In the colony counter 10 according to this embodiment, for example, the imaging camera 13 reads the sample image of the microorganism after (or during) cultivation at a constant temperature over a predetermined period of time.

[0017] The LED ring light illumination 14 illuminates the petri dish 1, which is set on the stage glass 12, in a ring shape from above with predetermined LED light.

[0018] The LED ring light illumination 15 is configured to illuminate the petri dish 1 set on the stage glass 12 in a ring shape with predetermined LED light from below.

[0019] The monitor 16 displays the specimen image of the petri dish 1 taken by the imaging camera 13, the measurement result image described later, and the like.

[0020] The control unit 17 controls each part of the colony counter 10. The imaging camera 13, the monitor 16, the storage unit 18, and the operation unit 19 are connected to the control unit 17 as each part.

[0021] Further, the control unit 17 includes, for example, an arithmetic processing unit (not shown). Although details will be described later, it has a function of softly processing calculation processes such as the area value (number of pixels) of colonies in a specimen image (binary image), and count processes such as counting the number of normal colonies.

[0022] As a count process for counting the number of colonies, for example, an aggregate of microorganisms with a pixel number smaller than a threshold value (counting threshold value) of a predetermined number of pixels is counted as a normal colony, and an aggregate of microorganisms with a pixel number larger than the counting threshold value is counted as a so-called spreading colony other than that.

[0023] The storage unit 18 is a storage (storage file) that stores a program (software) for arithmetic processing, a processed image (measurement result image) subjected to calculation processing such as a specimen image and the area value of colonies, and the like.

[0024] The operation unit 19 is operated by an operator (such as a measurer) when counting the number of colonies, which are aggregates of microorganisms grown by culturing.

[0025] In the colony counter 10 according to this embodiment, for example, the monitor 16, control unit 17, storage unit 18, and operation unit 19 can be easily configured using a general-purpose PC (personal computer).

[0026] Here, we will specifically explain the so-called spread colony IC that occurs in microbial culture using Petri dish 1, due to differences in the type of microorganism (differences in growth rate) and the environmental conditions inside the Petri dish during culture.

[0027] Figures 2(a) to 2(e) show examples of sample images to be tested.

[0028] In other words, Figure (a) is a so-called normal petri dish image, illustrating a case where, for example, the measurement area 1A (100 pixels), which is equal to the area of ​​the inner diameter of petri dish 1, contains only countable normal colonies RC, and no spread colonies IC (0 pixels) have occurred. In this example, the area ratio of the calculated measurement area (100 - 0 pixels) to the area of ​​measurement area 1A is 100%.

[0029] Figure (b) is a petri dish image illustrating a case where, for example, a portion of measurement area 1A is covered by a so-called large, spreading colony IC (20 pixels) due to a rapidly growing microorganism. In this example, the calculated measurement area (100-20 pixels) accounts for approximately 80% of the area of ​​measurement area 1A.

[0030] Figure (c) is a petri dish image illustrating a case where, for example, a portion of measurement area 1A is covered by so-called spreading colony ICs (60 pixels in total) due to rapidly growing microorganisms. In this example, the calculated measurement area (100-60 pixels) accounts for approximately 40% of the area of ​​measurement area 1A.

[0031] Figure (d) is a petri dish image illustrating a case where, for example, the peripheral area of ​​measurement region 1A is covered by so-called spread colony ICs (80 pixels in total) due to the effects of condensation droplets. In this example, the area ratio of the calculated measurement region (100-80 pixels) to the area of ​​measurement region 1A is approximately 20%.

[0032] Figure (e) is a petri dish image illustrating a case where, for example, the entire measurement area 1A is covered by so-called spread colony IC (approximately 100 pixels) due to rapidly growing microorganisms. In this example, the area ratio of the calculated measurement area (100-100 pixels) to the area of ​​measurement area 1A is approximately 0%.

[0033] If the sample image is a normal petri dish image, such as the one shown in Figure 2(a), the number of countable normal colonies (RC) can be accurately counted.

[0034] Similarly, in the case of Figure 2(b), for example, about 80% of the calculation measurement area remains. By counting the number of colonies within that area and multiplying the counted number of colonies by the area ratio (100 / 80), the number of colonies present in measurement area 1A can be estimated with a certain degree of accuracy.

[0035] In contrast, if the sample image is a petri dish image, such as those shown in Figures 2(c) to 2(e), where at least a portion of the countable normal colonies RC are largely covered by spread colonies IC, the accuracy of estimating the number of colonies present within the measurement area 1A decreases.

[0036] Thus, the ratio of the number of pixels in the measurement area 1A to the number of pixels in the spread colony IC affects the validity of the count result (estimated number of colonies).

[0037] Figure 3 shows an example of a sample image (Petri dish image) to be tested in the colony counter 10 according to this embodiment.

[0038] The sample image shown in Figure 3 is an example where the number of pixels in measurement area 1A is "100", the total number of pixels for spread colony IC is "60", and the number of normal colony RC is "8".

[0039] In other words, in the case of this sample image, the area ratio of the calculation measurement area (100-60 pixels) to the area of ​​measurement area 1A is approximately 40%.

[0040] Regarding the validity of the counting results, if the recognition threshold is set at approximately 50% or more of the area ratio of the calculation measurement area to the area of ​​measurement area 1A, the calculation measurement area in Figure 3 is less than 50%, and therefore the counting results are judged to be invalid.

[0041] Furthermore, the estimated number of colonies, calculated by multiplying the count result of normal colonies (RC) (8) by the area ratio (100 / 40), is deemed to lack validity for the reasons mentioned above.

[0042] Next, referring to the flowchart shown in Figure 4, we will explain the counting process, which counts the number of normal colonies RC, as an inspection performed by the control unit 17.

[0043] For example, as shown in Figure 4, the operator first uses the control unit 19 to display the measurement list on the monitor 16 screen (step S11).

[0044] Next, the sample number and other information of Petri dish 1 to be tested are registered (Step S12). Next, one petri dish 1 corresponding to the measurement list is automatically or manually placed on the stage glass 12, and a petri dish image of the petri dish 1 is taken by the imaging camera 13 (step S13).

[0045] The petri dish image is then sent from the control unit 17 to the storage unit 18 and saved as the original image in a storage file, associated with the sample number (step S01).

[0046] Meanwhile, the petri dish image captured by the camera 13 is binarized by the control unit 17 (step S14) and displayed on the monitor 16 screen.

[0047] Then, on the Petri dish image displayed on the monitor 16 screen, the areas to be excluded as spread colony ICs, for example as shown in Figure 3, are drawn either automatically or manually as filled areas or contour enhancements (step S15).

[0048] Subsequently, the control unit 17 determines the number of pixels corresponding to the total area of ​​the expanded colony IC (step S16).

[0049] On the other hand, by excluding the area of ​​spread colony IC, measurement area 1A is established (step S17), and within this measurement area 1A, microbial aggregates smaller than or equal to a predetermined count threshold are counted as normal colony RC (step S18).

[0050] The petri dish image used for counting the normal colonies RC is then sent from the control unit 17 to the storage unit 18 and saved as a measurement result image (binary image) after colony counting, associated with the sample number, in a storage file (step S02).

[0051] Next, the area ratio between the calculation measurement area and the area of ​​the inner diameter of Petri dish 1 is determined from the number of pixels corresponding to the calculation measurement area and the total number of pixels corresponding to the area of ​​the inner diameter of Petri dish 1 (step S19).

[0052] If the area ratio is outside the predetermined recognition threshold, as shown in Figures 2(c) to 2(e) (NO in step S19), an identification flag is automatically added to the sample number obtained in step S12, indicating that the sample does not require recognition of the validity of the normal colony RC count result (step S22).

[0053] On the other hand, as shown in Figures 2(a) and 2(b), if the calculated area ratio is within a predetermined recognition threshold (YES in step S19), the product of the number of normal colonies (count result) and their area ratio is displayed in the measurement list (step S20).

[0054] Thus, the process from step S12 onwards is repeated (without step S21) until the measurement of Petri dish 1 corresponding to all sample numbers registered in the measurement list is completed (step S22).

[0055] As described above, this embodiment makes it possible to make more accurate judgments about the validity of the count results.

[0056] Specifically, the colony area value is calculated as the number of pixels, and the validity of the count result is judged based on the area ratio on the sample image being examined. This allows the validity of the count result to be judged based on clear criteria, without being influenced by the operator's experience. Therefore, even in sample images where spread colonies (IC) have occurred, anyone can efficiently count normal colonies (RC).

[0057] In particular, since it eliminates the need for complicated pretreatment such as applying agar to the surface of the sample during culture to suppress the occurrence of spread colony IC, it is possible to significantly improve the efficiency of the test.

[0058] Although embodiments of the present invention have been described above with reference to examples of the embodiments, these embodiments are merely examples, and the scope of the invention as described in the claims can be modified in various ways without departing from the spirit of the invention. [Explanation of Symbols]

[0059] 1…Petri dish (culture vessel) 1A…Measurement area 10…Colony counter (colony counting device) 12… Stage glass 13…Camera for filming (Filming Department) 14, 15… LED ring light lighting 16…Monitor 17...Control Unit (Detection Unit / Decision Unit) 18...Storage section RC... Normal Colony IC... Spread Colony

Claims

1. A colony counting device for counting colonies, which are aggregates of microorganisms, An imaging unit for capturing a sample image from a sample in which the aforementioned microorganisms have been cultured, A detection unit detects colonies in the sample image that are larger than a predetermined count threshold based on the number of pixels obtained by binarizing the sample image captured by the imaging unit, A determination unit that determines the validity of the number of colonies estimated from the sample image based on the ratio of the total number of pixels of colonies larger than the count threshold detected by the detection unit to the total number of pixels of the culture vessel used to cultivate the microorganism, A colony counting device characterized by being equipped with the following features.

2. The colony counting device according to claim 1, characterized in that the detection unit counts colonies smaller than the count threshold from the sample image as normal colonies.

3. The colony counting device according to claim 1, characterized in that the determination unit identifies the sample image as invalid when the ratio of the total number of pixels of colonies greater than the count threshold to the total number of pixels of the culture vessel is greater than a predetermined recognition threshold.

4. The colony counting device according to claim 1, characterized in that the determination unit subtracts the total number of pixels of colonies greater than the count threshold from the total number of pixels in the culture vessel, and identifies the sample image as invalid if the ratio of the subtraction result to the total number of pixels in the culture vessel is smaller than a predetermined recognition threshold.

5. The colony counting device according to claim 1, further comprising a storage unit that stores the sample image and a binary image obtained by binarizing the sample image in association with each other.

6. The colony counting device according to claim 5, characterized in that the storage unit associates the sample image with the binary image and further stores an identification flag for determining whether the sample image is valid or not.

7. A colony counting method for counting colonies, which are aggregates of microorganisms, The process involves the imaging unit capturing an image of the sample from which the microorganisms have been cultured, The detection unit detects colonies in the sample image that are larger than a predetermined count threshold based on the number of pixels obtained by binarizing the sample image captured by the imaging unit, The process involves a determination unit determining whether the detection result of colonies larger than the count threshold detected by the detection unit is within a certain range, and determining the validity of the number of colonies estimated from the sample image, A colony counting method characterized by comprising the following features.

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