Reagent, measurement method, and measurement device

Abstract

[Problem] To provide a reagent, a measurement method, and a measurement device 200 capable of quickly and accurately measuring a microorganism. [Solution] A reagent 1 according to the present invention causes a target microorganism 101 contained in a sample 100 to fluoresce, has E. coli 102 as the target microorganism 101, and selectively causes E. coli 102 in the sample 100 to fluoresce. The reagent 1 according to the present invention is obtained by binding [bacteriophage t4 and / or bacteriophage t4 DNA] 1A and a predetermined fluorescent reagent 1B. The [bacteriophage t4 and / or bacteriophage t4 DNA] 1A is caused to fluoresce by the predetermined fluorescent reagent 1B and the fluorescing [bacteriophage t4 and / or bacteriophage t4 DNA] 1A selectively infects the E. coli 102, which is the target microorganism 101 contained in the sample 100, thereby selectively taking in the E. coli 102 and causing the E. coli 102 to fluoresce.

Description

[Technical field]

[0001] The present invention relates to a reagent, a measurement method, and a measurement device, and more particularly to a reagent, a measurement method, and a measurement device that cause target microorganisms contained in a specimen to fluoresce. [Background technology]

[0002] Microbiological control of food has been a central factor in total quality control for some time. The Food Sanitation Act also stipulates standard values ​​for the number of general bacteria and the number of E. coli and other microorganisms for each food and food ingredient. Therefore, various food-related establishments take samples from food and food ingredients every day to test the number of general bacteria and the number of E. coli and other microorganisms. For technology to test the number of bacteria in food, the technology disclosed in Patent Document 1 can be referred to, for example. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2014-52976 A Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, testing has to rely on the culture method set by the government, which takes 48 hours and incurs huge costs. Companies that do not have their own testing system outsource all testing. However, these testing systems have a big problem. That is the time required for testing. Most foods are shipped immediately after production because freshness is important. In other words, even though the microbial test results for the products produced on the day are only available within 48 hours, the products are shipped and eaten before the test results are available. In the unlikely event that food poisoning occurs due to microbial contamination of foods shipped earlier, the cause will be identified later. This problem is a common theme worldwide. What is needed is to be able to ship products with peace of mind after the test results are available. In recent years, various simple test kits other than the culture method have been sold, but they take more than two hours and are simple, so there are problems with accuracy.

[0005] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a reagent, a measurement method, and a measurement device that are capable of measuring microorganisms quickly and accurately. [Means for solving the problem]

[0006] In order to achieve the above object, the reagent of the present invention is a reagent that causes a target microorganism contained in a specimen to fluoresce, and is characterized in that it selectively causes the target microorganism to fluoresce.

[0007] According to the present invention, by selectively making the target microorganisms fluorescent, it is possible to selectively measure the microorganisms, thereby enabling rapid and accurate measurement of the microorganisms.

[0008] By using Escherichia coli as the target microorganism and selectively making the Escherichia coli fluorescent in the specimen, it is possible to selectively measure Escherichia coli in the specimen, thereby enabling rapid and accurate measurement of Escherichia coli.

[0009] It is preferable that the bacteriophage t4 and / or bacteriophage t4 DNA is bound to a specific fluorescent reagent.

[0010] It is preferable to selectively infect Escherichia coli, which is a target microorganism contained in the sample, with bacteriophage t4 and / or bacteriophage t4 DNA bound to a specified fluorescent reagent, thereby causing the Escherichia coli to fluoresce.

[0011] The predetermined fluorescent reagent is preferably FDA (fluorescein diacetate) and / or PI (propidium iodide).

[0012] In order to achieve the above-mentioned object, the measurement method of the present invention is a method for measuring a target microorganism contained in a sample using a reagent that causes the target microorganism to fluoresce, and is characterized in that the target microorganism is selectively made fluorescent.

[0013] According to the present invention, by selectively causing the target microorganisms to fluoresce, it is possible to selectively measure the microorganisms in a specimen, thereby enabling rapid and accurate measurement of microorganisms.

[0014] The target microorganism is Escherichia coli, and the reagent selectively causes the Escherichia coli in the specimen to fluoresce, thereby enabling the Escherichia coli to be selected and measured in the specimen, thereby enabling rapid and accurate measurement of Escherichia coli.

[0015] The reagent is preferably a reagent in which a specific fluorescent reagent is bound to bacteriophage t4 and / or bacteriophage t4 DNA.

[0016] The reagent is a reagent combining bacteriophage t4 and / or bacteriophage t4 DNA with a specified fluorescent reagent, and it is preferable that the reagent is selectively infected with Escherichia coli, which is a target microorganism contained in the specimen, to cause the Escherichia coli to fluoresce.

[0017] The predetermined fluorescent reagent is preferably FDA (fluorescein diacetate) and / or PI (propidium iodide).

[0018] By impregnating a black filter with a reagent that selectively makes the microorganisms fluoresce, and then using the black filter impregnated with the reagent to perform a process that selectively makes the microorganisms fluoresce, the fluorescence of the microorganisms stands out against a black background, allowing the microorganisms to be measured quickly and accurately.

[0019] The black filter impregnated with the reagent and the specimen containing the microorganism is placed within a measurement circle marked on a slide glass, and then covered with a cover glass to prepare a preparation.

[0020] The measurement circles are marked on the glass slide in multiple numbers, and the black filter impregnated with a sample containing the microorganism is placed in each of the multiple marked measurement circles, so that multiple samples containing microorganisms can be measured in parallel, thereby improving the efficiency of the measurement.

[0021] The stage on which the slide is placed can be moved along the XY axes to capture an image of the slide.

[0022] A CMOS sensor can be used to capture images of the slide, correct the captured images, and analyze the captured images.

[0023] By correcting the captured image so as to correct differences in thickness and / or shape between the slides in the preparation, the differences can be corrected even if there is a difference in thickness and / or shape between the slides.

[0024] By correcting the captured image so as to correct the inclination of the stage, even if the stage is inclined, the inclination can be corrected so as to be corrected.

[0025] The target microorganism is recognized from the captured image based on predetermined recognition parameters, and nothing other than the target microorganism is recognized, thereby making it possible to efficiently recognize only the target microorganism.

[0026] The recognition parameters can include at least three of the shape of the target microorganism, the size of the target microorganism, the intensity of the fluorescence of the target microorganism, and the shade of the fluorescence of the target microorganism.

[0027] In order to achieve the above-mentioned object, the measurement device of the present invention is a measurement device that measures target microorganisms contained in a sample using a reagent that causes the target microorganisms to fluoresce, and is characterized in that it selectively causes the target microorganisms to fluoresce.

[0028] According to the present invention, by selectively making the target microorganisms fluorescent, it is possible to selectively measure the microorganisms, thereby enabling rapid and accurate measurement of the microorganisms.

[0029] The target microorganism is Escherichia coli, and the reagent selectively causes the Escherichia coli in the sample to fluoresce, thereby enabling the Escherichia coli to be selected and measured in the sample, thereby enabling rapid and accurate measurement of Escherichia coli.

[0030] The reagent is preferably a reagent in which a specific fluorescent reagent is bound to bacteriophage t4 and / or bacteriophage t4 DNA.

[0031] The reagent is a reagent combining bacteriophage t4 and / or bacteriophage t4 DNA with a specified fluorescent reagent, and it is preferable that the reagent is selectively infecting Escherichia coli, which is a target microorganism contained in the specimen, to cause the Escherichia coli to fluoresce.

[0032] The predetermined fluorescent reagent is preferably FDA (fluorescein diacetate) and / or PI (propidium iodide).

[0033] By impregnating a black filter with a reagent that selectively makes the microorganisms fluoresce, and then using the black filter impregnated with the reagent to perform a process that selectively makes the microorganisms fluoresce, the fluorescence of the microorganisms stands out against a black background, allowing the microorganisms to be measured quickly and accurately.

[0034] The black filter impregnated with the reagent and the specimen containing the microorganism is placed within a measurement circle marked on a slide glass, and then covered with a cover glass to prepare a preparation.

[0035] The measurement circles are marked on the glass slide in multiple numbers, and the black filter impregnated with a sample containing the microorganism is placed in each of the multiple marked measurement circles, so that multiple samples containing microorganisms can be measured in parallel, thereby improving the efficiency of the measurement.

[0036] The stage on which the slide is placed can be moved along the XY axes to capture an image of the slide.

[0037] A CMOS sensor can be used to capture images of the slide, correct the captured images, and analyze the captured images.

[0038] By correcting the captured image so as to correct differences in thickness and / or shape between the slides in the preparation, the differences can be corrected even if there is a difference in thickness and / or shape between the slides.

[0039] By correcting the captured image so as to correct the inclination of the stage, even if the stage is inclined, the inclination can be corrected so as to be corrected.

[0040] The target microorganism is recognized from the captured image based on predetermined recognition parameters, and nothing other than the target microorganism is recognized, thereby making it possible to efficiently recognize only the target microorganism.

[0041] The recognition parameters can include at least three of the shape of the target microorganism, the size of the target microorganism, the intensity of the fluorescence of the target microorganism, and the shade of the fluorescence of the target microorganism. Effect of the Invention

[0042] An object of the present invention is to provide a reagent, a measurement method, and a measurement device that enable rapid and accurate measurement of microorganisms. [Brief description of the drawings]

[0043] [Figure 1] FIG. 2 is a diagram showing a configuration of a reagent according to an embodiment of the present invention. [Diagram 2] FIG. 13 shows the state in which the reagent is taken up by E. coli. [Diagram 3] 1A to 1C are diagrams showing a method for preparing a slide used in a measuring method and a measuring device according to an embodiment of the present invention. [Figure 4] 5 is a view following FIG. 3, showing a method for preparing a slide used in the measuring method and measuring device according to an embodiment of the present invention. [Diagram 5]5 is a view following FIG. 4 showing a method for preparing a slide used in the measuring method and measuring device according to an embodiment of the present invention. [Figure 6] 5A to 5C are views showing a method for preparing a slide used in the measuring method and measuring device according to an embodiment of the present invention. [Figure 7] 7 is a view following FIG. 6 showing a method for preparing a slide used in the measuring method and measuring device according to an embodiment of the present invention. [Figure 8] 8 is a view following FIG. 7 showing a method for preparing a slide used in the measuring method and measuring device according to an embodiment of the present invention. [Figure 9] 2 is a diagram showing a configuration of a microscope device used in the measurement method and measurement device. FIG. [Figure 10] 13 is another diagram showing the configuration of the microscope apparatus used in the measurement method and measurement apparatus. FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

[0045] [Embodiment of Reagent] FIG. 1 is a diagram showing the configuration of a reagent according to an embodiment of the present invention, and FIG. 2 is a diagram showing the state in which the reagent has been taken up into E. coli.

[0046] As shown in Fig. 1, the reagent 1 according to the embodiment of the present invention is a reagent in which [bacteriophage t4 and / or bacteriophage t4 DNA] 1A and a predetermined fluorescent reagent 1B are mixed and bound in advance. Bacteriophage T4 is a species of bacteriophage that infects Escherichia coli 102 and is classified as a double-stranded DNA virus. Bacteriophage t4 DNA is the DNA of bacteriophage T4, and can be obtained, for example, by extracting DNA from bacteriophage T4.

[0047] That is, the reagent 1 is a reagent 1 that causes a target microorganism 101 contained in a specimen 100 shown in Fig. 2 to fluoresce, and can selectively cause the target microorganism 101 to fluoresce. In this embodiment, the target microorganism 101 is Escherichia coli 102, and the Escherichia coli 102 in the specimen 100 can be selectively made to fluoresce.

[0048] That is, as described above, the reagent 1 is a reagent in which [bacteriophage t4 and / or bacteriophage t4 DNA] 1A and a predetermined fluorescent reagent 1B are mixed and bound in advance, and the reagent 1 is selectively infected with E. coli 102, which is a target microorganism 101 contained in a specimen 100, and taken up by the E. coli 102, so that the E. coli 102 can emit fluorescence. In this embodiment, the predetermined fluorescent reagent 1B is a DNA fluorescent reagent, and can be FDA (fluorescein diacetate) and / or PI (propidium iodide). FDA (fluorescein diacetate) and / or PI (propidium iodide) can be used without culturing E. coli 102, and have a remarkable effect on, for example, AO (acrylic orange), which requires the E. coli 102 to be cultured in advance.

[0049] In this way, the reagent 1 selectively causes the target microorganisms 101 to fluoresce, thereby making it possible to selectively measure the microorganisms 101, and thus to rapidly and accurately measure the microorganisms 101.

[0050] In addition, by using Escherichia coli 102 as the target microorganism 101 and selectively causing the Escherichia coli 102 to fluoresce in the specimen 100, the Escherichia coli 102 can be selected and measured in the specimen 100, thereby enabling rapid and accurate measurement of the Escherichia coli 102.

[0051] [Embodiments of the measurement method and the measurement device] 3 to 8 are diagrams showing a method of preparing a slide for use in the measurement method and measurement apparatus according to an embodiment of the present invention, and FIGS. 9 and 10 are diagrams showing the configuration of a microscope apparatus for use in the same measurement method and measurement apparatus.

[0052] The above-mentioned reagent 1 is used in the measurement method and measurement device 200 according to the embodiment of the present invention. That is, the measurement method and measurement device 200 are capable of measuring a target microorganism 101 contained in a specimen 100 using a reagent 1 that causes the target microorganism 101 to fluoresce, and can selectively cause the target microorganism 101 to fluoresce. The measurement device 200 has a fluorescence processing device 60 and a microscope device 70, which will be described later.

[0053] In this measurement method and measurement device 200, the target microorganism 101 is Escherichia coli 102, and the reagent 1 can selectively cause the Escherichia coli 102 in the specimen to emit fluorescence.

[0054] As shown in FIG. 1, the reagent 1 is a reagent in which [bacteriophage t4 and / or bacteriophage t4 DNA] 1A is mixed in advance with a specific fluorescent reagent 1B and bound to it. Bacteriophage T4 is a species of bacteriophage that infects Escherichia coli 102 and is classified as a double-stranded DNA virus. Bacteriophage t4 DNA is the DNA of bacteriophage T4, and can be obtained, for example, by extracting DNA from bacteriophage T4.

[0055] That is, the reagent 1 is a reagent 1 that causes a target microorganism 101 contained in a specimen 100 shown in Fig. 2 to fluoresce, and can selectively cause the target microorganism 101 to fluoresce. In this embodiment, the target microorganism 101 is Escherichia coli 102, and the Escherichia coli 102 in the specimen 100 can be selectively made to fluoresce.

[0056] That is, as described above, the reagent 1 is a reagent in which [bacteriophage t4 and / or bacteriophage t4 DNA] 1A and a predetermined fluorescent reagent 1B are mixed and bound in advance, and the reagent 1 is selectively infected with E. coli 102, which is a target microorganism 101 contained in a specimen 100, and taken up by the E. coli 102, so that the E. coli 102 can emit fluorescence. In this embodiment, the predetermined fluorescent reagent 1B is a DNA fluorescent reagent, and can be FDA (fluorescein diacetate) and / or PI (propidium iodide). FDA (fluorescein diacetate) and / or PI (propidium iodide) can be used without culturing E. coli 102, and have a remarkable effect on, for example, AO (acrylic orange), which requires the E. coli 102 to be cultured in advance.

[0057] In this measurement method and measurement device 200, the E. coli 102 can be measured using a specific preparation 10. The method for producing the preparation 10 will be described as follows.

[0058] That is, first, as shown in FIG. 3, a black filter 20 is prepared. Next, as shown in FIG. 4, a black filter 20 is impregnated with a reagent 1 that selectively causes the E. coli 102 to fluoresce, and the black filter 20 impregnated with the reagent 1 can be used to perform a process that selectively causes the E. coli 102 to fluoresce.

[0059] More specifically, as shown in FIG. 4, a black filter 20 is impregnated with a reagent 1 that selectively causes E. coli 102 to fluoresce, and as shown in FIG. 5, the black filter 20 impregnated with the reagent 1 is impregnated with a specimen 100 that contains E. coli 102, thereby making it possible to perform a process that selectively causes E. coli 102 to fluoresce.

[0060] Next, a slide glass 30 is prepared, and a measurement circle 40 is marked on the slide glass 30 as shown in FIG.

[0061] Next, as shown in FIG. 7, a black filter 20 impregnated with a specimen 100 containing reagent 1 and E. coli 102 is placed within the marked measurement circle 40, and then, as shown in FIG. 8, it is covered with a cover glass 50 to prepare a specimen 10.

[0062] A plurality of measurement circles 40 are marked on the slide glass 30, and a black filter 20 impregnated with a specimen 100 containing E. coli 102 is placed in each of the plurality of marked measurement circles 40.

[0063] In the present measurement method and measurement device 200, the prepared slide 10 can be used to perform fluorescence processing by a fluorescence processing device 60, and images can be photographed, corrected, and analyzed by a microscope device 70, as shown in Figures 9 and 10. The microscope device 70 is a fluorescence microscope device.

[0064] That is, the fluorescence processing device 60 has an excitation light irradiating section 61 and a wavelength measuring section 62. The excitation light irradiating section 61 can irradiate the preparation 10 (more specifically, the plurality of black filters 20 impregnated with the reagent 1 and the E. coli 102) with excitation light of a predetermined wavelength. The wavelength measuring section 62 can measure the wavelength of fluorescence from the preparation 10 (more specifically, the plurality of black filters 20 impregnated with the reagent 1 and the E. coli 102) that has absorbed the excitation light.

[0065] The microscope device 70 has a stage 71 that moves along the XY axes. That is, in the microscope device 70, the stage 71 on which the slide 10 is placed can be moved along the XY axes to capture multiple images of the slide 10 (more specifically, multiple black filters 20 impregnated with the reagent 1 and E. coli 102) that has absorbed the excitation light.

[0066] More specifically, the microscope device 70 further includes a storage unit 72 , an imaging unit 73 , a correction unit 75 , and an analysis unit 74 .

[0067] The memory unit 72 can store data on the standard shape of the target microorganism 101, E. coli 102, the standard size of the target microorganism 101, E. coli 102, the standard fluorescence intensity of the target microorganism 101, E. coli 102, and the standard fluorescence shade of the target microorganism 101, E. coli 102.

[0068] In addition, the photographing unit 73 uses a CMOS sensor to photograph multiple images of the preparation 10 through each of the multiple black filters 20, and the correction unit 75 corrects the images photographed by the photographing unit 73, and the analysis unit 74 can qualitatively and / or quantitatively analyze the images photographed by the photographing unit 73.

[0069] Here, the correction unit 75 can correct the image captured by the imaging unit 73 so as to correct the difference in thickness and / or shape between the slide glasses 30 in the preparation 10.

[0070] In other words, the correction unit 75 can correct the image captured by the photographing unit 73 so as to correct the difference in thickness and / or shape of the slide glass 30 to be photographed relative to the thickness and / or shape of a standard slide glass 30.

[0071] The correction unit 75 can correct the image captured by the photographing unit 73 so that the inclination of the stage 71 is corrected. That is, the correction unit 75 can correct the image captured by the photographing unit 73 so that the inclination of the stage 71 with respect to a predetermined direction, more specifically, the horizontal direction, is corrected. The photographing unit 73 can also capture an image while enlarging or reducing the image by changing the distance from the preparation 10 multiple times. That is, the photographing unit 73 has a function of measuring the surface of the specimen 100 at a distance of three or more measurement points for each measurement of the thickness and individual difference in the shape of the slide glass 30 and the inclination of the stage 71 on which the slide glass 30 is placed, and capturing the image while correcting the individual difference (thickness and distortion) and inclination of the slide glass 30 with the zoom of the photographing unit 73 or the correction unit 75.

[0072] Furthermore, the analysis unit 74 can recognize the target microorganism 101, E. coli 102, from the image captured by the imaging unit 73 based on predetermined recognition parameters, and can perform analysis so as not to recognize anything other than the target microorganism 101, E. coli 102.

[0073] The recognition parameters may include, for example, at least three of the shape of the target microorganism 101, E. coli 102, the size of the target microorganism 101, E. coli 102, the fluorescence intensity of the target microorganism 101, E. coli 102, and the shade of the fluorescence of the target microorganism 101, E. coli 102.

[0074] That is, the analysis unit 74 reads out data on the standard shape of E. coli 102, which is the target microorganism 101, the standard size of E. coli 102, which is the target microorganism 101, the standard fluorescence intensity of E. coli 102, which is the target microorganism 101, and the standard fluorescence shade of E. coli 102, which is the target microorganism 101, as standard recognition parameters stored in the memory unit 72, and compares this read out data with the image captured by the imaging unit 73 (more specifically, the image corrected by the correction unit 75) to analyze whether the captured image (more specifically, the corrected image) matches at least three of the standard shape of E. coli 102, which is the target microorganism 101, the standard size of E. coli 102, which is the target microorganism 101, the standard fluorescence intensity of E. coli 102, which is the target microorganism 101, and the standard fluorescence shade of E. coli 102, which is the target microorganism 101, within a specified range.

[0075] If the captured image (more specifically, the corrected image) matches within a specified range at least three of the standard shape of E. coli 102, which is the target microorganism 101, the standard size of E. coli 102, which is the target microorganism 101, the standard fluorescence intensity of E. coli 102, which is the target microorganism 101, and the standard fluorescence shade of E. coli 102, which is the target microorganism 101, the analysis unit 74 can determine that the microorganism 101 in the captured image (more specifically, the corrected image) is E. coli 102, which is the target microorganism 101.

[0076] The inventor has revealed that the microorganism 101 in a captured image (more specifically, a corrected image) can be considered to be the target microorganism 101, E. coli 102, when the captured image (more specifically, the corrected image) matches within a specified range at least three of the standard shape of the target microorganism 101, E. coli 102, the standard size of the target microorganism 101, E. coli 102, the standard fluorescence intensity of the target microorganism 101, E. coli 102, and the standard fluorescence shade of the target microorganism 101, E. coli 102.

[0077] That is, in addition to microorganisms 101 such as E. coli 102, the specimen 100 may contain food residue, dirt, and other foreign matter, and even if fluorescent staining as described above is performed, it may be difficult to distinguish between microorganisms 101 and non-microorganisms. However, by recognizing the microorganisms using three or more recognition parameters as described above, it is possible to measure only microorganisms 101 such as E. coli 102 and not measure anything other than microorganisms 101.

[0078] In the image captured by the photographing unit 73, the shape of the target microorganism 101, E. coli 102, the size of the target microorganism 101, the fluorescence intensity of the target microorganism 101, and the fluorescence shade of the target microorganism 101, E. coli 102, are affected by differences in thickness and / or shape between the glass slides 30 in the preparation 10 and the inclination of the stage 71, causing the image to become blurred due to focus shifts. However, as described above, the correction unit 75 can correct the image captured by the photographing unit 73 so as to correct the differences in thickness and / or shape between the glass slides 30 in the preparation 10, and can correct the image captured by the photographing unit 73 so as to correct the inclination of the stage 71, so that such influences can be appropriately corrected to perform accurate image processing.

[0079] As described above, according to the measurement method and measurement device 200 of the present invention, it is possible to selectively cause the target microorganisms 101 to fluoresce, thereby enabling the microorganisms 101 to be selected and measured in the specimen 100, thereby enabling rapid and accurate measurement of the microorganisms 101.

[0080] In addition, the target microorganism 101 is Escherichia coli 102, and the reagent 1 selectively causes the Escherichia coli 102 in the specimen 100 to fluoresce, thereby making it possible to selectively measure the Escherichia coli 102 in the specimen 100, thereby enabling rapid and accurate measurement of the Escherichia coli 102.

[0081] Furthermore, by impregnating the black filter 20 with a reagent 1 that selectively makes the E. coli 102 fluoresce, and by carrying out a process to selectively make the E. coli 102 fluoresce using the black filter 20 impregnated with the reagent 1, the fluorescence of the E. coli 102 stands out against the black background, and the E. coli 102 can be measured quickly and accurately.

[0082] Furthermore, multiple measurement circles 40 are marked on the glass slide 30, and a black filter 20 impregnated with a specimen 100 containing E. coli 102 is placed in each of the multiple marked measurement circles 40, so that multiple specimens 100 containing E. coli 102 can be measured in parallel, thereby improving the efficiency of the measurement.

[0083] Furthermore, the correction unit 75 can correct the image captured by the photographing unit 73 so as to correct differences in thickness and / or shape between the glass slides 30 in the preparation 10, thereby correcting the differences even if there is a difference in thickness and / or shape between the glass slides 30.

[0084] Furthermore, the correction unit 75 can correct the image captured by the imaging unit 73 so that the inclination of the stage 71 is corrected, thereby making it possible to correct the inclination even if the stage 71 is tilted.

[0085] Furthermore, the correction unit 75 recognizes the target microorganism 101 from the image captured by the imaging unit 73 based on predetermined recognition parameters, and does not recognize anything other than the target microorganism 101, thereby enabling efficient recognition of only the target microorganism 101.

[0086] It is needless to say that the present invention is not limited to the above-described embodiment, and various modifications and applications are possible.

[0087] That is, in the above-described embodiment, the microorganism 101 is Escherichia coli 102, but the present invention can be applied to a microorganism 101 other than Escherichia coli 102 as appropriate.

[0088] In other words, in the above-described embodiment, the black filter 20 is impregnated with reagent 1 that selectively causes E. coli 102 to fluoresce, and the black filter 20 impregnated with reagent 1 is used to perform a process of selectively causing E. coli 102 to fluoresce. However, the required effect can also be achieved by impregnating the black filter 20 with reagent 1 that selectively causes microorganisms 101 other than E. coli 102 to fluoresce, and using the black filter 20 impregnated with reagent 1 to perform a process of selectively causing microorganisms 101 other than E. coli 102 to fluoresce.

[0089] In addition, in the above-described embodiment, a black filter 20 impregnated with a specimen 100 containing reagent 1 and E. coli 102 is placed in the measurement circle 40 marked on the slide glass 30 and covered with a cover glass 50 to prepare the preparation 10. However, the desired effect can also be achieved by placing a black filter 20 impregnated with a specimen 100 containing reagent 1 and a microorganism 101 other than E. coli 102 in the measurement circle 40 marked on the slide glass 30 and covering it with a cover glass 50 to prepare the preparation 10.

[0090] Furthermore, in the above-described embodiment, the target microorganism 101, E. coli 102, is recognized from the image captured by the imaging unit 73 based on predetermined recognition parameters, and nothing other than the target microorganism 101, E. coli 102, is recognized. The recognition parameters include at least three of the shape of the target microorganism 101, E. coli 102, the size of the target microorganism 101, the fluorescence intensity of the target microorganism 101, E. coli 102, and the fluorescence shade of the target microorganism 101, E. coli 102. However, the desired effect can also be achieved even if the target microorganism 101 is a microorganism 101 other than E. coli 102.

[0091] In other words, the desired effect can be achieved by simply recognizing the target microorganism 101 from the image captured by the imaging unit 73 based on predetermined recognition parameters, and not recognizing anything other than the target microorganism 101, and the recognition parameters can include at least three of the shape of the target microorganism 101, the size of the target microorganism 101, the fluorescence intensity of the target microorganism 101, and the shade of the fluorescence of the target microorganism 101.

[0092] Furthermore, in the above-described embodiment, a black filter 20 impregnated with a specimen 100 containing E. coli 102 is placed in each of the multiple marked measurement circles 40, but a black filter 20 impregnated with a specimen 100 containing E. coli 102 and a black filter 20 impregnated with a specimen 100 containing a microorganism 101 other than E. coli 102 may be placed in separate measurement circles 40, respectively, to simultaneously measure two or more types of microorganisms 101 (for example, measuring the general bacterial count and the E. coli count simultaneously). [Explanation of symbols]

[0093] 1: Reagents 1A: Bacteriophage t4 and / or bacteriophage t4 DNA 1B: Fluorescent Reagent 10: Preparation 20: Filter 30: Slide glass 40: Measuring circle 50: Cover glass 60: Fluorescence treatment device 61: Excitation light irradiation unit 62: Wavelength measurement section 70: Microscope equipment 71: Stage 72: Storage section 73: Photography Department 74:Analysis Department 75: Correction section 100: Sample 101: Microorganisms 102: Escherichia coli 200: Measuring equipment

Claims

[Claim 1] A method for measuring Escherichia coli, which is a target microorganism contained in a specimen, by using a reagent that selectively causes fluorescence of Escherichia coli, which is a target microorganism contained in a specimen, comprising: The reagent is a mixture of bacteriophage t4 and FDA (fluorescein diacetate) and / or PI (propidium iodide), A black filter is impregnated with the reagent, The black filter impregnated with the reagent is impregnated with a specimen containing Escherichia coli; The black filter impregnated with the reagent and the sample is placed on a measurement circle marked on a slide glass; performing a fluorescence treatment by irradiating the E. coli with excitation light of a predetermined wavelength by an excitation light irradiation unit of a fluorescence treatment device, and selectively infecting the E. coli with the reagent to cause the E. coli to fluoresce; taking a plurality of images of the black filters absorbing the excitation light by an imaging unit of the microscope device; The photographing unit photographs the image using a CMOS sensor, corrects the photographed image, and analyzes the photographed image; a correction unit of the microscope apparatus corrects the image so as to correct differences in thickness and / or shapes between the slide glasses, and corrects the image so as to correct the inclination of a stage on which the slide glasses are placed in the microscope apparatus with respect to a horizontal direction; an analysis unit of the microscope device recognizes E. coli, which is a target microorganism, from the image captured by the image capture unit based on a predetermined recognition parameter, while analyzing the image so as not to recognize anything other than the E. coli, thereby measuring the E. coli; The measurement method, wherein the recognition parameters of the analysis unit include at least three of the shape, size, fluorescence intensity, and fluorescence shade of the target microorganism, Escherichia coli.

Images