JP6692075B2 - Microorganism detection device, microorganism detection program, and microorganism detection method - Google Patents

Description

  The present invention relates to a microorganism detection device, a microorganism detection program, and a microorganism detection method for detecting whether or not a specimen contains microorganisms. More specifically, the present invention relates to a microorganism detecting device, a microorganism detecting program, and a microorganism detecting method that detect a microorganism colony in a short time and do not kill the microorganism at the time of detection.

BACKGROUND ART When manufacturing and shipping products such as processed foods, microbial tests are performed to confirm that they do not contain microorganisms such as bacteria. In this microbiological test, usually, foods to be detected are crushed, and then a sample of purified water that is agitated is adhered to or mixed with an agar plate, and thereafter, a microbe colony with a width of about 100 μm or more is visible so that microbes can be visually observed. As described above, the test is carried out in accordance with the agar plate culture test in which the test is carried out by culturing for a certain period of time and then visually inspecting for microbial colonies.
However, since this inspection method usually takes one day or more as a culture period, a method capable of performing an inspection in a shorter time is desired. On the other hand, some inspection methods have been proposed (for example, refer to Patent Document 1).

  In the microorganism inspection method described in Patent Document 1, a non-fluorescent substance that reacts with an enzyme of a microorganism to be inspected to generate a fluorescent substance is diffused in a plate medium, and the surface of the plate medium is irradiated with light to emit fluorescence. The number of microbial colonies to be inspected contained in the sample is measured from the number of locations. Further, the measurement of the number is performed by a scanner.

JP-A-07-147997

In the conventional microorganism inspection method as described in Patent Document 1, when contaminants such as scraps of foods and dust are attached, these contaminants emit autofluorescence that is confused with a fluorescent substance, There is a problem that it may be difficult to distinguish it from a microbial colony because it may emit fluorescence due to staining.
In addition, when the culture time is short, the microbial colonies are small, and the difference between the size of the microbial colonies and the size of contaminants is small, making it difficult to distinguish the two.
A method of detecting a microorganism in a short time by detecting an intracellular substance such as ATP is also known, but the cell is destroyed due to the detection of the intracellular substance. With such an inspection method, it becomes impossible to perform another test after inspecting one sample for microorganisms, and for example, the agar plate culture test cannot be performed subsequently. For this reason, it is necessary to prepare a plurality of specimens, and the inspection takes time.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microorganism detection device, a microorganism detection program, and a microorganism detection method that detect a microorganism colony in a short time and do not kill the microorganism at the time of detection. And

1. A microorganism detecting device for detecting a colony of a microorganism by culturing a microorganism by placing a substrate on which a sample is adhered in a medium, and then immersing the substrate in a fluorescent dye solution to perform staining, An imaging unit that irradiates the substrate on which the sample stained with a fluorescent stain is attached with excitation light, acquires an image of the substrate by capturing an image of the substrate, and, after dyeing the sample, at a predetermined cycle. The substrate image is acquired by the image capturing unit, the brightness of each section made up of one or a plurality of pixels on the board image in the predetermined cycle is obtained, and the brightness of each section is determined based on the background brightness of the board image. A brightness correction unit that calculates a corrected correction brightness, and the correction brightness exceeds a predetermined value and the change rate of the correction brightness exceeds a predetermined value with the passage of time in a predetermined inspection time after staining of the specimen. Was And a determining means and microbial colonies present in at least one of the compartments produced out of the imaging unit, the substrate acquires the substrate image while being accommodated in a container with a lid, The lid of the lidded container has a light-transmitting property, and a dew condensation preventing portion is formed on its inner surface or a dew condensation preventing tool is provided on the outside, and the substrate is placed on a medium after detection of the microbial colonies. A microorganism detection device characterized in that the culture can be continued by doing so.
2. The determination means determines that a microbial colony exists in the section in which the corrected luminance exceeds a predetermined value and the rate of change in the corrected luminance exceeds a predetermined value. The microorganism detection device described.
3. When the change rate of the corrected luminance has a positive value and then has a negative value, the determining means determines that a microbial colony exists in the section. Or 2. The microorganism detection device according to 1.
4 . It is a method for detecting a microorganism by detecting a colony of a microorganism by culturing a microorganism by placing a substrate on which a sample is adhered in a medium for a predetermined time and then immersing the substrate in a fluorescent dye solution to perform staining. An irradiation step of irradiating the substrate, to which the sample stained with the fluorescent stain is attached, with excitation light, and capturing an image of the substrate to obtain a substrate image; The board image is acquired in the imaging step in a cycle, the brightness of each section made up of one or a plurality of pixels on the board image in the predetermined cycle is obtained, and each section is based on the background brightness of the board image. A luminance correction step of calculating the corrected luminance by correcting the luminance of the sample, and that the corrected luminance exceeds a predetermined value and a change in the corrected luminance with a lapse of time in a predetermined inspection time after staining the sample. A determination step of determining that a microbial colony is present in the section in which at least one of the microbial colonies exceeds a predetermined value is generated, and the culture is performed by placing the substrate on a medium after the detection of the microbial colony. A method for detecting a microorganism, which enables continuation.
5 . The determining step includes the judges that the corrected luminance change rate and the corrected luminance exceeds a predetermined value is present microorganism colonies in said compartment exceeds a predetermined value 4. The method for detecting a microorganism as described.
6 . The determining step, the rate of change of the corrected luminance becomes a positive value, if followed by a negative value, the determining a microorganism colonies in said compartment is present 4. Or 5 . The method for detecting a microorganism according to 1.
7 . In the imaging step, the substrate image is acquired in a state where the substrate is housed in a lidded container, the lid of the lidded container has a light-transmitting property, and a dew condensation preventing portion is formed on an inner surface of the lid. the external dew condensation preventing device is provided 4. To 6 . The method for detecting a microorganism according to any one of 1.

According to the microorganism detection device of the present invention, the microorganisms are cultured by placing the substrate on which the sample is attached in the medium, and then the substrate is immersed in a fluorescent dye solution to stain the microorganisms to colonize the microorganisms. a microorganism detection apparatus for detecting the fluorescent staining agent of the excitation light is irradiated to the substrate adhered with stained the specimen by an imaging unit that acquires substrate image by imaging the substrate, wherein After the specimen is stained , the substrate image is acquired by the imaging unit in a predetermined cycle, the brightness of each section including one or a plurality of pixels on the board image in the predetermined cycle is obtained, and the background of the board image is displayed. A brightness correction unit that calculates a corrected brightness by correcting the brightness of each of the sections with the brightness as a reference, and a change rate of the corrected brightness exceeds a predetermined value with the lapse of time in a predetermined inspection time after staining of the specimen . The above The determination means for determining the presence of microbial colonies in the image is provided, so that the fluorescence brightness of the microbial colonies and the fluorescence brightness of the contaminants and the substrate background are about the same, or the area of the microbial colonies and the contaminants is large. It is possible to detect a microbial colony without erroneous detection even under a short-time culture condition where there is no difference in size.
In addition, the detection by this microorganism detection device is based on the imaging of fluorescence and does not destroy microorganisms when dyeing with a fluorescent staining agent.Therefore, culture for the agar plate culture test should be performed after detection by this device. You can continue. Accordingly, after the inspection of one sample by the present microorganism detection apparatus, the agar plate culture test and the like can be performed using the same sample, so that the verification of the detection result can be facilitated.

When the determination means determines that a microbial colony exists in the section in which the corrected luminance exceeds a predetermined value and the change rate of the corrected luminance exceeds a predetermined value, it determines from the magnitude of the corrected luminance and its temporal change. By discriminating between the microbial colony and the contaminant, the microbial colony can be detected with higher accuracy.
If the rate of change of the corrected luminance has a positive value and then has a negative value, the determining means determines that microbial colonies are present in the section, and after increasing the luminance, consumes the fluorescent stain. It is possible to reliably detect a microbial colony whose brightness decreases due to such factors.
The brightness correction means, when calculating the corrected brightness by subtracting the value of the brightness of the background from the value of the brightness of each of the sections on the substrate image, easily obtain the corrected brightness, microbial colonies and contamination. It is possible to distinguish the object.
The imaging unit acquires the substrate image in a state where the substrate is housed in a lidded container, the lid of the lidded container has a light-transmitting property, and a dew condensation preventing portion is formed on an inner surface of the lid. When the dew condensation prevention tool is provided outside, even if the lid of the lidded container becomes lower in temperature than the substrate and is liable to dew condensation, the dew condensation prevention unit can prevent dew condensation.

According to the microorganism detecting program for detecting the colonies of microorganisms, a function suitable for the microorganism detecting device can be realized on a computer.
According to the method for detecting a microorganism for detecting a colony of a microorganism, the fluorescence brightness of the microorganism colony and the fluorescence brightness of the contaminants and the substrate background are similar to each other, or the size of the area of the microorganism colony and the contaminants is large. Microorganism colonies can be detected without erroneous detection even under a short-term culture condition where there is no difference.

The present invention is further explained in the following detailed description with reference to the several drawings referred to, by way of non-limiting examples of typical embodiments according to the present invention, wherein like reference numerals are used in the drawings. Similar parts are shown through several figures.
It is a schematic diagram for demonstrating the structure of this microorganism detection apparatus. 3 is a graph showing changes in fluorescence brightness of microorganisms, contaminants, and a substrate during an imaging period, (a) shows a brightness value, (b) shows a corrected brightness value, and (c) shows a corrected change rate of the brightness. It is a perspective view which shows the external appearance of the container with a lid. It is a schematic cross section for demonstrating the structure of the container main body in a container with a lid. It is a schematic cross section for demonstrating the structure of the lid part in a container with a lid. It is a flow chart for explaining the procedure of microorganisms detection by a microorganisms pixel detection part. It is a flowchart for demonstrating the procedure of the microorganism detection by another microorganism pixel detection part. 6 is a graph showing changes in fluorescence brightness of microorganisms, contaminants, and a substrate during an imaging period. (A) shows a brightness value, (b) shows a corrected brightness value, and (c) shows a corrected brightness change rate. 6 is a graph showing changes in fluorescence brightness of microorganisms, contaminants, and a substrate during an imaging period. (A) shows a brightness value, (b) shows a corrected brightness value, and (c) shows a corrected brightness change rate. 6 is a graph showing changes in fluorescence brightness of microorganisms, contaminants, and a substrate during an imaging period. (A) shows a brightness value, (b) shows a corrected brightness value, and (c) shows a corrected brightness change rate. It is the image which imaged the board | substrate with the fluorescence microscope. It is a part of the board image taken by the imaging unit at the same position as in FIG. 11. It is an image showing the surface of the substrate which has been further cultured after completion of imaging by the present microorganism detection device.

  The matters shown here are for the purpose of exemplifying the exemplary embodiments and the embodiments of the present invention, and are the explanations that allow the principles and conceptual features of the present invention to be most effectively and effortlessly understood. It is mentioned for the purpose of providing what you think. In this respect, it is not intended to present the structural details of the invention more than to the extent necessary for a fundamental understanding of the invention, and the description taken in conjunction with the drawings may explain some aspects of the invention. It will be clear to those skilled in the art how it is actually implemented.

1. Microorganism detection device The microorganism detection device (1) according to the present embodiment detects a microorganism colony from an image of a substrate (6) to which a sample stained with a fluorescent stain and emitting fluorescence by excitation light is attached. The detection device includes an image pickup unit (2), a brightness correction unit (41), and a determination unit (46) (see FIG. 1). In addition, counting means (47) can be provided. The brightness correction means (41), the determination means (46) and the counting means (47) can be provided on the information processing section (4).

The sample is a target object for detecting the presence or number of colonies of microorganisms, for example, food, drinking water, cosmetics, raw water, drainage, industrial water, a liquid sample prepared by dissolving an environmental sample or the like in purified water. Can be mentioned. The microorganism to be detected by the microorganism detecting device (1) is not particularly limited, and forms colonies of E. coli, Vibrio parahaemolyticus, Salmonella, Listeria, Clostridium, Bacillus, etc. regardless of whether they are gram-positive, gram-negative, aerobic or anaerobic. Bacteria etc. can be mentioned.
The fluorescent stain may be any dye capable of selectively staining a microbial colony to be detected, and is appropriately selected depending on the microorganism to be detected. Examples of fluorescent dyes that utilize the activity of microorganisms include FDA dyes such as fluorescein diacetate that is dyed by the esterase activity of microorganisms, tetrazolium salt dyes that are dyed by respiratory activity such as CTC. it can. Examples of stains that nonspecifically bind to the nucleus or cell wall of microorganisms include propidium iodide, ethidium bromide, DAPI, methylene blue, safranine, fuchsin, crystal violet, and gentian violet. Further, the fluorescent substance may be metabolized from the enzyme derivative by an enzyme or the like produced by the microorganism to be detected without staining the microorganism to be detected. Examples of such enzymes include β-glucuronidase, β-galactosidase and β-glucosidase produced by Escherichia coli.

The substrate (6) only needs to be capable of adhering a sample, and examples thereof include a medium for culturing microorganisms in the sample and a filter material such as a membrane filter.
The time required for the microorganism detecting device (1) to be able to detect a microbial colony varies depending not only on the type of microorganism to be detected but also on the image capturing resolution of the image capturing unit (2) and the like. For example, when Escherichia coli is to be detected, usually, in 3 to 6 hours from the start of culture, the number of one E. coli becomes 100 to 10,000 and a microbial colony with a diameter of about 20 μm or more, and imaging using a general imaging device is performed. It is possible to discriminate using the section (2).

Hereinafter, the microorganism detection device 1 will be described in detail with reference to FIG. The microorganism detecting device 1 can be composed of an imaging unit 2 and an information processing unit 4. The information processing unit 4 includes a brightness correction unit 41, a determination unit 46, and the like.
(Imaging unit)
The image capturing unit 2 includes at least a light source 23 and an image sensor 28, and acquires a substrate image by capturing an image of the substrate 6 irradiated with the excitation light emitted from the light source 23 with the image sensor 28.
The specific configuration of the imaging unit 2 can be arbitrarily selected. For example, the mounting table 22 of the substrate 6, the light source 23 that emits the excitation light, the epi-illumination unit 24 that irradiates the mounting table 22 with the excitation light, and the substrate 6. It can be configured by the image pickup device 28 for picking up an image. The image capturing unit 2 can be provided with a temperature maintaining unit such as a heating unit for maintaining the reaction condition of the stain.
The mounting table 22 is a table for mounting or fixing the substrate 6, and the mounting method and the fixing method thereof are not particularly limited.
The light source 23 is a light source for emitting excitation light of fluorescence of a microbial colony to be detected, and its type is not particularly limited as long as it does not exhibit excessive photobactericidal properties due to wavelength or energy. For example, the light source 23 may be an LED lamp, a mercury lamp, a halogen lamp, a laser oscillator, or the like.
The epi-illumination unit 24 is for making the optical path of the light source for irradiating the substrate and the optical axis of the optical path of the image pickup device for imaging the substrate the same, and may be configured by combining a dichroic mirror, a prism and the like. it can.

The image pickup device 28 may be any means capable of picking up an image (substrate image) of the substrate 6 by capturing the wavelength of the fluorescence emitted from the microbial colony, and may be a CCD image sensor, a CMOS image sensor, a SIT image sensor, or the like. It can be illustrated. Further, the image pickup device may pick up an image in a plane by an area image sensor or may obtain an image by scanning with a linear image sensor.
Further, the wavelength of the fluorescence reaching the image pickup device 28 can be limited by arbitrarily combining a dichroic mirror, an optical filter and the like.

The image of the substrate 6 captured by the image capturing unit 2 may be captured as a plurality of still images or may be captured as a moving image.
Further, the substrate 6 to be imaged may be directly placed on the mounting table 22 to be imaged, or the substrate 6 may be placed in the container 7 and imaged in a protected state. When imaging is performed by the imaging unit 2 with the substrate 6 housed in the container 7, as illustrated in FIGS. 3 and 5, the lid 72 is normally covered with a translucent material, and the excitation light is emitted from above. It can be irradiated and imaged. In this case, it is preferable that the dew condensation preventing portion 75 is formed on the inner surface of the lid 72. The culturing temperature is often higher than room temperature, and the lid 72 of the container containing the substrate 6 to which the specimen adheres is cooled by the outside air, and the vapor in the container in contact with the inner surface of the lid 72 condenses to cover the lid 72 and cloud the image. This can be difficult.
The dew condensation prevention unit 75 may be provided with a means for preventing the dew condensation on the inner surface of the lid 72 by some means while maintaining the translucency of the lid 72. For example, a hydrophilic drug or a surfactant may be applied to the inner surface of the lid. In addition, hydrophilic plastic processing or the like may be performed to make the surface hydrophilic by giving a hydrophilic functional group to the inner surface of the lid 72 made of a translucent resin or changing the surface shape.
Further, instead of the dew condensation preventing portion 75, a dew condensation preventing tool may be provided outside the lid 72. The dew condensation prevention tool may include a glass heater having a light-transmitting property provided so as to cover the lid 72, a heater that can be moved to the outside of each optical path at the time of image capturing, and the like. Condensation on the inner surface of 72 can be prevented.
Further, as a method of preventing dew condensation on the lid 72, the substrate 6 is fixed to the container body 71, and the lid 72 is turned upside down so that the image is taken from the lid 72 located on the lower surface by using the image sensor 28. Can be mentioned.

(Brightness correction means)
The brightness correction unit 41 causes the imaging unit 2 to acquire a board image in a predetermined cycle after staining the sample, obtains the brightness of each section including one or a plurality of pixels on the board image in a predetermined cycle, and calculates the board image. It is a means for calculating the corrected brightness by correcting the brightness of each section with the background brightness as a reference.
The time required for the test depends on the type of microorganisms and the conditions such as the resolution of the imaging unit, but when Escherichia coli is the target, it is usually about 1 to 50 minutes after staining the sample. .. During the inspection time, the brightness correction unit 41 is configured to cause the imaging unit 2 to acquire a board image at a predetermined cycle and perform a predetermined process on the board image at each time point. The predetermined cycle may be an interval at which a change in the brightness of fluorescence from the microbial colony with the passage of time can be detected, and can be, for example, about 30 seconds to 10 minutes. Further, the cycle does not necessarily have to be constant, and may be changed depending on the elapsed time after staining the sample.

The section is a block that is a unit for detecting a microbial colony in a substrate image, and the size of the block may be appropriately selected. That is, one partition may be one pixel or may be a region including a plurality of pixels (for example, 2 × 2, 3 × 3, etc.). The size of one section is preferably smaller than the size of the microbial colony to be detected. This is because if the size of the section is larger than the size of the microbial colony to be detected, the background around the microbial colony is included in the section, and the detection cannot be performed accurately.
The brightness correction means 41 can divide the board image into the above-mentioned sections and acquire the brightness of each section. When one section is composed of a plurality of pixels, the average value of the brightness detected in each pixel can be used as the brightness of the section.

The brightness correction unit 41 is configured to calculate the corrected brightness in which the brightness of each section is corrected with reference to the background brightness of the board image (hereinafter referred to as background brightness). The background luminance is the luminance of a region in the substrate image where there are no microorganisms or foreign substances such as scraps of food and dust. The brightness correction means 41 can calculate the background brightness from the entire board image by various methods.
The specific method of calculating the background brightness is not particularly limited. Usually, the number of sections in which microorganisms and contaminants are present is smaller than the total number of sections in the board image. Therefore, for example, the brightness of the section having the largest number of sections having the same brightness value can be set as the background brightness. Specifically, it is possible to generate a histogram showing the number of pixels for each luminance of the board image, and set the portion having the largest mountain in the histogram as the background luminance. Further, the average value of the brightness of the entire board image can be used as the background brightness. In addition, the brightness of the appropriately selected section or the average value of the brightness of a plurality of sections may be used as the background brightness.

  The brightness correction unit 41 is configured to calculate the corrected brightness by correcting the brightness of each section of the entire board image with the background brightness calculated as described above as a reference. A specific correction method is not particularly limited, and for example, a value obtained by subtracting the background brightness from the brightness of each section can be set as the correction brightness (see FIGS. 2A and 2B). Further, the ratio of the brightness of each section to the background brightness may be the corrected brightness.

The brightness correction unit 41 can be configured to calculate the background brightness and the corrected brightness each time the board image is acquired at a predetermined cycle. The acquired board image data, the background brightness, and the corrected brightness for each section can be stored in the storage device provided in the information processing unit 4.
In addition, the brightness correction unit 41 stores the data of the board image acquired in a predetermined cycle in a storage device, and at each time based on the stored board image data at an appropriate timing during the inspection or after the inspection is completed. The background brightness and the corrected brightness for each section can be calculated.

(Determination means)
The determination unit 46 determines that a microbial colony is present in a section in which at least one of the corrected luminance exceeding the predetermined value and the corrected luminance change rate exceeding the predetermined value has occurred with the lapse of the imaging time. It is a means. To make this determination, it is necessary to distinguish between microbial colonies and contaminants.

  The sample on the substrate may contain impurities in addition to the microorganism to be detected. For example, FIG. 2A shows a change in fluorescence brightness on a substrate image with respect to the elapsed time after staining when microorganisms (1, 2, 3) and contaminants are present on the substrate. In this example, the fluorescence brightness of the compartments in which the microorganisms (1, 2, 3) are present once increase with time and then decrease. On the other hand, the fluorescence brightness of the section containing the contaminants monotonically increases with time. Also, the background brightness monotonically increases at a lower level with the passage of time.

FIG. 2B shows a change in the corrected luminance obtained by subtracting the background luminance from the fluorescent luminance of each section shown in FIG. 2A. The corrected luminances of the compartments in which the microorganisms (1, 2, 3) exist all increase once with the passage of time and then decrease. On the other hand, the corrected brightness of the section in which the contaminants are present is smaller than the corrected brightness of the section in which the microorganisms are present, and is a substantially constant value regardless of the passage of time.
FIG. 2C shows the rate of change in the corrected luminance of each section shown in FIG. 2B from the value one cycle (5 minutes) before. The rate of change in corrected luminance in units of one or a plurality of cycles is the rate of change in corrected luminance. Then, the rate of change in the corrected luminance of the section where the microorganisms (1, 2, 3) are present is a relatively large value, which becomes a positive value with the passage of time, and then becomes a negative value. On the other hand, the rate of change in the corrected luminance of the section containing the contaminants is much smaller than the rate of change in the corrected luminance of the section containing the microorganisms, and is almost zero.

From the changes in the fluorescence brightness of the microorganisms (1, 2, 3), the fluorescence brightness of the contaminants, and the background brightness as described above, the determination unit 46 determines that the amount of change in the corrected brightness is a predetermined value with the elapse of the imaging time (see FIG. When it exceeds S ₀ shown in 2 (b), it can be determined that microbial colonies are present in that section.
In addition, when the change rate of the corrected luminance exceeds a predetermined value (S ₁ shown in FIG. 2C) with the elapse of the imaging time, the determination unit 46 determines that a microbial colony exists in that section. You can
Further, when the corrected luminance exceeds the predetermined value and the rate of change in the corrected luminance exceeds the predetermined value as the imaging time elapses, the determination means 46 can determine that a microbial colony exists in that section. ..
Further, the judging means 46 can judge that the microbial colony exists in the section when the rate of change in the corrected luminance has a positive value and then has a negative value.

Depending on the type of microorganism and the staining conditions, the change in fluorescence brightness may be different from the change shown in FIG. 2 (described later), but microbial colonies can be detected by appropriately selecting the above determination method. Is possible.
As shown in FIG. 2, the fluorescent brightness of the microbial colony once increases and then decreases because the fluorescent stain is consumed by the microorganism. In this way, even when the fluorescence intensity of the microbial colony increases for a certain period of time and then decreases, it becomes possible to clearly distinguish the microbial colony from the impurities and the substrate even when the intensity is close to that of the impurities and the background. Is.

(Counting means)
The microorganism detecting device 1 may be provided with a counting means 47 for counting microbial colonies. As described above, the brightness correction unit 41 and the determination unit 46 can detect the section where the microbial colony exists from the substrate image. The counting means 47 is means for calculating the number and size of the microbial colonies.
A specific method for determining one microbial colony is not particularly limited, and when the sections determined to be microbial colonies are adjacent to each other, they can be collectively determined as one microbial colony. For example, when a plurality of sections that are determined to be microbial colonies are adjacent to each other and the magnitude of the corrected brightness and the value of the rate of change are within a certain range, these sections are combined into one microbial colony. Can be determined. Then, the counting means 47 can calculate the number of microbial colonies on the substrate image. Further, the counting means 47 can calculate the size (area) of the microbial colony from the number of sections or the number of pixels constituting one microbial colony.

  The above brightness correction means 41, determination means 46, and counting means 47 may be realized by either hardware or software. Usually, it can be realized by a program that operates on a computer having an interface with the imaging unit 2.

2. Microorganism detection program A microorganism detection program for detecting a colony of microorganisms has an image pickup function for obtaining a board image using the image pickup unit 2, a brightness correction function for realizing the brightness correction unit 41 on a computer, and the determination unit 46. And a determination function for realizing the above on a computer. Further, it is possible to provide a counting function for realizing the counting means 47 on a computer.
As shown in FIG. 6, the imaging function irradiates the substrate on which the specimen stained with the fluorescent dye is attached with the excitation light, and images the substrate to acquire a substrate image (S11).
The brightness correction function acquires the substrate image by the imaging function at a predetermined cycle after staining the specimen, obtains the brightness of each section made up of one or a plurality of pixels on the board image at a predetermined cycle, The brightness of the background is calculated (S12), and the corrected brightness is calculated by correcting the brightness of each section based on the brightness of the background (S13).
The determination function determines that a microbial colony exists in a section in which at least one of the corrected brightness exceeds a predetermined value and the change rate of the corrected brightness exceeds a predetermined value with the passage of time ( S14).
The counting function calculates the number and size of the detected microbial colonies (S15).

Further, FIG. 7 exemplifies the processing flow of the microorganism detection program that can be applied to a sample that causes a change in brightness as shown in FIG. 2 where the brightness once increases and then decreases.
The imaging step S21, the background luminance calculation step S22, the corrected luminance calculation step S23, and the counting step S27 are the same as steps S11, S12, S13, and S15 shown in FIG.

[a] First determination step As the first determination step S24, a difference between the corrected luminance of each section and the corrected luminance of the same section of the board image one cycle before is calculated, and when the difference is larger than a predetermined value, Detected as putative section A in which microbial colonies may exist.
[b] Second Judgment Step In the second judgment step S25, the corrected brightness of the same section of the board image one cycle before is subtracted from the corrected brightness of each section, and the difference is negative and the absolute value is a predetermined value. When it exceeds, it is detected as the presumed section B in which microbial colonies may exist.
[c] Third determination step In the third determination step S26, the section detected as the estimated section A and then the estimated section B is determined as the section C in which the microbial colony exists. That is, at the same position on the board image, the section that has been detected as the estimated section A in the past and then detected as the estimated section B is determined as the section C having the microbial colony.

3. Microorganism Detection Method Hereinafter, the microorganism detection method will be specifically described based on Examples.
The present microorganism detecting method detects a colony of a microorganism using the microorganism detecting apparatus 1 as shown in FIG.
The imaging unit 2 is provided in the housing 21, and includes a mounting table 22 for the substrate 6, an epi-illumination unit 24 arranged above the mounting table 22, and an imaging device arranged above the epi-illumination unit 24. 28, and a light source 23 arranged laterally of the epi-illumination unit 24. The inside of the housing 21 is kept at a constant temperature by heating means or the like.
The epi-illumination unit 24 uses the optical filter 26 and the dichroic mirror 25 to reflect the excitation light from the light source 23, which is incident from the side, to the mounting table 22 side below. Further, the fluorescence emitted from the mounting table 22 is transmitted through the optical filter 27 to the upper image pickup device 28. Further, the incident light from the side is limited to the wavelength required as the excitation light by the optical filter 26 and the dichroic mirror 25, and the incident light from below is limited to the wavelength near the fluorescence by the dichroic mirror 25 and the optical filter 27. It As the image pickup device 28, a camera using a CCD image sensor is used.
The information processing unit 4 is a computer device such as a personal computer and can execute the microorganism detection program.
The substrate 6 is mounted on the mounting table 22 while being accommodated in the lidded container 7. The container 7 with a lid is made of resin, and includes a container body 71 and a lid portion 72 that covers the container body 71, as shown in FIGS. The top surface of the lid 72 is transparent to excitation light and fluorescence, and the inner surface (the surface facing the container body 71) is formed by applying a surfactant or processing hydrophilic plastic. A dew condensation prevention unit 75 is provided.

First, a sample that is purified water in which a detection target is dissolved is filtered by a membrane filter that is the substrate 6. If the sample contains microorganisms by filtration, it adheres to the substrate 6. Then, the substrate 6 is placed on an agar plate medium on a plate and cultured in a constant temperature bath kept at 37 ° C. This culturing time is 3 to 6 hours for E. coli.
If Escherichia coli adheres to the substrate after the culturing time has passed, as shown in FIG. 11, the number of Escherichia coli becomes 100 to 10,000 and the colony has a width of 10 to 20 μm.
The substrate 6 after the culturing time is stored in a container body 71 containing a solution of a fluorescent dye and is dipped in the container body 71 for dyeing. After that, the lid 72 covers the lid and the lid 22 is placed.

After the specimen is stained as described above, the following imaging step, brightness correction step, determination step, and counting step are performed.
(Imaging process)
In the imaging step, the substrate 6 to which the specimen stained with the fluorescent dye is attached is irradiated with excitation light, and the substrate 6 is photographed by the imaging element 28 to obtain a substrate image. The wavelength of the excitation light is shorter than the wavelength of the fluorescence incident on the image sensor 28, and is generally 370 to 650 μm, and the light of the light source 23 is emitted from above the lidded container 7 via the epi-illumination unit 24.

(Brightness correction process)
In the brightness correction step, the board image is acquired by the imaging step (image acquisition step). The acquisition of the substrate image in the image acquisition step is repeatedly performed for 30 minutes at a 5-minute cycle after the sample is dyed to obtain a plurality of substrate images. An example of the captured board image is shown in FIG.
After a series of imaging is completed, the substrate 6 can be taken out from the container 7 with a lid, placed on the plate again and returned to the incubator to continue the culture.
In the brightness correction step, the brightness of each section made up of one or a plurality of pixels is calculated in the substrate image acquired every 5 minutes. For example, the board image is divided into a grid shape, a square block surrounded by each grid is defined as one section, and the average brightness of a plurality of pixels included in the section is defined as the brightness of the section. Then, the background brightness of each board image is calculated (background brightness calculation step). Next, the corrected luminance is calculated by correcting the luminance of each section on the basis of the calculated background luminance (corrected luminance calculation step).

In the background luminance calculation step, a histogram showing the number of pixels (sections) for each luminance of the imaged substrate image is generated, and the portion having the largest mountain in the histogram is calculated as the background luminance. Also, the background brightness is calculated for each board image.
Next, in the corrected luminance calculation step, the corrected luminance is obtained by correcting the luminance of each section of each board image with the background luminance. The corrected brightness of each section is a value obtained by subtracting the background brightness from the brightness of each section.

(Judgment process)
Then, as a determination step, it is determined whether or not a microbial colony exists in each section of each substrate image. As described above, when the correction luminance exceeds a predetermined value, the change rate of the correction luminance exceeds a predetermined value, the change rate of the correction luminance changes from a positive value to a negative value, etc. It can be determined that microbial colonies are present. It is possible to select or combine these determination conditions according to the type of microorganisms to be detected and determine whether or not microbial colonies are present. Further, when a plurality of sections determined to have microbial colonies are adjacent to each other, they can be treated as one microbial colony.
As described above, even when the size of the microbial colony is not different from the size of the contaminant, it is possible to clearly distinguish them. Further, even when the fluorescent brightness of the microbial colony section is not significantly different from the fluorescent brightness of the background of the contaminants or the substrate, the microbial colonies can be clearly distinguished.

(Counting process)
Then, a counting step of counting the number of microbial colonies on each substrate image can be performed. The number of microbial colonies can be the number of sections where microbial colonies are detected in each substrate image. When a plurality of sections determined to have microbial colonies are adjacent to each other, they can be treated as one microbial colony. Further, the size (area) of the microbial colony can be obtained from the number of sections forming one microbial colony.

  According to the present microorganism detection method, since the microorganism colonies are detected only by adding the fluorescent stain and irradiating the exciting light, the microorganisms on the substrate 6 are not easily killed, and the culture of the substrate 6 is continued after the implementation of the microorganism detection method. can do. By continuing the culture, the microbial colonies can be grown to a visually observable size as shown in FIG. 13, and the microbial colonies can be visually counted. Therefore, it is not necessary to prepare a plurality of culture media in order to detect microorganisms.

An example of measuring the change in brightness on the substrate image by the present microorganism detection method is shown in FIG. 2 and FIGS. However, the positions of the compartments for which the microorganisms, contaminants, and background brightness shown in each graph are obtained are the substrates at the time when all the imaging by the microorganism detection method is completed, observed by a fluorescence microscope as shown in FIG. The identified sections (see bright spots in Figure 12) were followed.
2 and 8 to 9, (a) shows changes in the fluorescence luminance of each section of the board image obtained by the image sensor 28. Further, (b) shows a change in corrected luminance obtained by correcting the luminance of each section with the background luminance. Further, (c) shows the rate of change in corrected luminance.

  FIG. 8 (and FIG. 2 described above) is a graph showing the change in the brightness of the substrate 6 that was stained with FDA (fluorescein diacetate) as a fluorescent stain after culturing for 4 hours. Here, as shown in FIGS. 11 and 12, each microorganism (4, 5) is a microbial colony whose position is specified by observing with a fluorescence microscope after the completion of measurement by the present microorganism detection method. Further, each of the contaminants 1 and 2 was made to adhere to the substrate 6 by including a cabbage piece having a size of about 100 μm in the sample and performing filtration through the substrate 6.

Referring to FIG. 8A, it is difficult to distinguish the microorganisms 4 and 5 in the sample, each contaminant, and the background of the substrate from the magnitude of the brightness before correction. However, looking at the change in the corrected brightness shown in FIG. 6B, the corrected brightness of the microorganisms 4 and 5 was significantly higher than that of the impurities 1 and 2 within about 5 minutes from the start of measurement, and the corrected brightness was increased. The change rate of is also large. On the other hand, the corrected brightness of the impurities 1 and 2 does not change significantly. Also, as can be seen from FIG. 7C, the corrected luminance change rates of the microorganisms 4 and 5 are outstanding in comparison with the impurities 1 and 2 within about 5 minutes from the start of measurement.
Therefore, with respect to the sample as shown in FIG. 8, it is possible to detect the microbial colony from the change in the corrected brightness calculated based on the substrate image in a short time after staining.

9 and 10 show a case where agar plate was used as the substrate 6 instead of a membrane filter, and after culturing for 5 hours, microbial colonies grown on the agar plate were directly stained using CTC which is a tetrazolium salt as a fluorescent stain. It is a graph which shows the change of the luminosity of a substrate image. On the substrate, cabbage pieces having a size of about 20 μm are attached as impurities.
As shown in FIG. 9A, it is difficult to distinguish the microorganisms 6 to 8 in the sample, the contaminants, and the background of the substrate from the magnitude of the brightness before correction. However, looking at the change in the corrected brightness shown in FIG. 6B, the corrected brightness of the microorganisms 6 to 8 is significantly higher than that of the impurities in about 10 minutes after the start of measurement. On the other hand, the corrected brightness of the impurities has not changed significantly. Also, as can be seen from FIG. 7C, the corrected luminance change rate of the microorganisms 6 to 8 is outstanding in comparison with impurities in about 10 minutes after the start of measurement.
Therefore, even with respect to the sample as shown in FIG. 9, it is possible to detect the microbial colony from the change in the corrected brightness calculated based on the substrate image in a short time after staining.

Further, also in the example shown in FIG. 10, the correction luminance of the microorganisms 9 to 11 greatly increases within about 5 minutes from the start of measurement ((b) and (c) in the figure). On the other hand, the corrected brightness of the impurities has not changed significantly.
Therefore, even with respect to the sample as shown in FIG. 10, it is possible to detect the microbial colony from the change in the corrected luminance calculated based on the substrate image in a short time after staining.

  The present invention is not limited to the embodiments detailed above, and various modifications and changes can be made within the scope of the claims of the present invention.

1; microorganism detection device,
2; Imaging unit, 21; Housing, 22; Mounting table, 23; Light source, 24; Epi-illumination unit, 25; Dichroic mirror, 26, 27; Optical filter, 28; Imaging device,
4; information processing unit, 41; brightness correction means, 46; determination means, 47; counting means,
6; substrate (membrane filter),
7; container with lid, 71; container body, 72; lid, 75; dew condensation prevention unit.

Claims (7)

A microorganism detecting device for culturing microorganisms by placing a substrate on which a sample is attached to a medium, and then detecting the colonies of microorganisms by immersing the substrate in a fluorescent stain solution and performing staining,
An imaging unit that irradiates excitation light to the substrate to which the sample stained with the fluorescent stain is attached, and captures a substrate image by imaging the substrate,
After dyeing the specimen, the imaging unit is caused to acquire the substrate image at a predetermined cycle, and the brightness of each section including one or a plurality of pixels on the substrate image at the predetermined cycle is obtained. Luminance correction means for calculating the corrected luminance by correcting the luminance of each section with the luminance of
The section in which at least one of the correction luminance exceeds a predetermined value and the change rate of the correction luminance exceeds a predetermined value over time in a predetermined inspection time after staining the specimen Determination means for determining that there is a microbial colony in
Equipped with
The imaging unit acquires the substrate image in a state where the substrate is housed in a container with a lid,
The lid of the lidded container has a light-transmitting property, and a dew condensation preventing portion is formed on the inner surface thereof, or a dew condensation preventing tool is provided on the outside,
A microorganism detecting apparatus, characterized in that the culture can be continued by placing the substrate on a medium after detecting the microorganism colony.   The microorganism detecting apparatus according to claim 1, wherein the determining unit determines that a microbial colony exists in the section in which the corrected luminance exceeds a predetermined value and the rate of change in the corrected luminance exceeds a predetermined value.   The microorganism detecting apparatus according to claim 1 or 2, wherein the determining unit determines that a microbial colony exists in the section when the rate of change in the corrected luminance has a positive value and then has a negative value. A method for detecting a microorganism by culturing microorganisms by placing a substrate on which a sample is adhered in a medium for a predetermined time, and then immersing the substrate in a fluorescent dye solution to perform staining to detect a colony of the microorganism. hand,
An imaging step of irradiating the substrate to which the sample stained with the fluorescent stain is attached with excitation light, and capturing the substrate to obtain a substrate image.
After staining the sample, the substrate image is acquired in the imaging step at a predetermined cycle, the brightness of each section including one or a plurality of pixels on the board image at the predetermined cycle is obtained, and the background of the board image is obtained. A luminance correction step of calculating a corrected luminance in which the luminance of each section is corrected with reference to the luminance of
The section in which at least one of the correction luminance exceeds a predetermined value and the change rate of the correction luminance exceeds a predetermined value over time in a predetermined inspection time after staining the specimen A determination step of determining that a microbial colony exists in
Equipped with
A method for detecting a microorganism, wherein the culture can be continued by placing the substrate on a medium after detecting the microorganism colony. The microorganism detection method according to claim 4 , wherein the determination step determines that a microbial colony exists in the section in which the corrected luminance exceeds a predetermined value and the rate of change in the corrected luminance exceeds a predetermined value. The microorganism detection method according to claim 4 or 5 , wherein in the determination step, when the rate of change in the corrected luminance has a positive value and then has a negative value, it is determined that a microbial colony exists in the section. The imaging step acquires the substrate image in a state where the substrate is accommodated in a container with a lid,
Lid of the lid container comprises a translucent, and microbial detection method according to any one of claims 4 to 6 condensation preventing device or the external dew condensation preventing portion is formed on the inner surface thereof is provided ..