JP2008005754A - Microorganism measuring system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism measuring system capable of comprehending whether the microorganism is live or dead and using a microorganism detecting method of a simple detecting mechanism, because there are a problem that whether microorganisms in a sample is live or dead is not comprehended in the conventional microorganism detecting method and a problem that as the conventional method requires to obtain fluorescence spectrums so a complex mechanism such as a complex coherent optical system using plural mirrors for obtaining fluorescence spectrum is required. <P>SOLUTION: The microorganism measuring system comprises a first excitation light source 1, a second excitation light source 2, a first spectral filter 3, a second spectral filter 4, a photographic means 5, a luminescent point detecting means 12 detecting luminescent points from obtained fluorescent images, a luminous point classifying means 13 classifying the detected luminescent points to live organisms, dead organisms and impurities, a luminous point measuring method 14 measuring number of luminous points and a personal-computer 15 having a function for displaying a number of luminous points. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a measurement system that acquires a fluorescence image of a microorganism that has undergone fluorescence staining, detects the microorganism using the fluorescence image, and measures the number of microorganisms.

Conventionally, as a method for detecting this type of microorganism, the one disclosed in Patent Document 1 is known.
Hereinafter, this type of microorganism detection method will be described.

In this method, using one kind of DNA-binding fluorescent dye, a fluorescence spectrum is obtained for each pixel from a fluorescence spectrum image including microorganisms in a stained sample, and a waveform component of the fluorescence spectrum of the obtained sample is compared with a control. A microorganism detection method is described in which the presence of microorganisms is detected by comparing waveform components of a microorganism fluorescence spectrum, a contaminant fluorescence spectrum, and a background fluorescence spectrum.
JP 2002-291499 A

  However, in the conventional microorganism detection method such as Patent Document 1 described above, even if the presence of the microorganism can be grasped, it is not known whether the microorganism in the specimen is alive or dead, and a fluorescence spectrum is acquired. Therefore, there is a problem that a complex mechanism such as an interference optical system using multiple mirrors is necessary to acquire a fluorescence spectrum, and are microorganisms present in the specimen alive? It is required to clarify whether it is dead, and to simplify the mechanism for detection, that is, to simplify the structure.

  The present invention solves such a conventional problem, and is provided with a function of performing fluorescence separation using a spectral filter by staining with two types of fluorescent reagents in order to discriminate between the life and death of microorganisms. An object of the present invention is to provide a microorganism measurement system using a microorganism detection method with a simple structure.

  In order to achieve the above object, the microorganism measurement system of the present invention fluorescently stains microorganisms in a specimen using two types of DNA-binding fluorescent staining reagents, and detects microorganisms using images of the microorganisms that emit fluorescence. In the measuring device, two types of excitation light sources for fluorescence of a specimen stained with a total bacterial fluorescent reagent for staining all microorganisms and a dead bacterial fluorescent reagent for staining dead microorganisms, and fluorescence emission Only a wavelength longer than the excitation light wavelength can be obtained by combining the two types of spectral filters for separating the fluorescence of the point, the photographing means for photographing the fluorescent image, and the two types of excitation light sources and the two types of spectral filters. Fluorescence point detection means that detects fluorescence emission points from fluorescence images taken through a spectral filter that passes through, and separates the detected emission points into live microorganisms, dead microorganisms, and contaminants A light emitting point classification unit, and a light emitting point measuring means for measuring a fractionated emission points is obtained by the arrangement for measuring the number of microorganisms.

  With this configuration, it is possible to distinguish between live microorganisms, dead microorganisms, and contaminants, and it is possible to provide a microorganism measurement system with a simple apparatus configuration.

  The other means is configured to perform the light emitting point detection means based on the luminance value of the photographed image.

  With this configuration, it is possible to provide a microorganism measuring system in which the light emission points are digitized.

  In addition, the other means is configured to include a position correction means for correcting the deviation of the coordinates of the light emitting points in the three types of fluorescent images taken.

  With this configuration, it is possible to provide a microorganism measurement system that can accurately grasp the emission point and can accurately measure living microorganisms, dead microorganisms, and contaminants.

  Another means uses a filtration filter as a means for collecting microorganisms.

  With this configuration, a microorganism measurement system that can measure microorganisms in a stationary state can be provided.

  In addition, another means is configured such that a structure holding a filter for filtering microorganisms can be attached.

  With this configuration, it is possible to provide a microorganism measuring system in which a filter obtained by filtering microorganisms can be easily attached to a measuring device for measuring microorganisms.

  In addition, another means uses two types of excitation light as LEDs.

  With this configuration, a microorganism measuring system with a simple power circuit configuration can be provided.

  Another means is configured to use two types of excitation light as blue light and green light.

  With this configuration, it is possible to provide a microorganism measuring system capable of generating green fluorescence and red fluorescence.

  The other means is configured such that the two types of spectral filters are a spectral filter that transmits green light and a spectral filter that transmits red light.

  With this configuration, it is possible to provide a microorganism measurement system that can separate green fluorescence and red fluorescence.

  Another means is that the photographing means is a CCD camera.

  With this configuration, it is possible to provide a microorganism measurement system that can capture a fluorescence emission point as an image.

  The other means is configured to capture a noise image of the imaging element before capturing the fluorescent image.

  With this configuration, it is possible to provide a microorganism measurement system that can perform measurement that is hardly affected by noise.

  Further, the other means is configured to photograph a plurality of places on the collection surface where microorganisms are collected.

  With this configuration, it is possible to provide a microorganism measurement system with improved accuracy of the estimated value of the microorganism measurement result.

  Another means is a structure in which metal deposition is performed on the surface of the filter for collecting microorganisms.

  With this configuration, it is possible to provide a microorganism measurement system that is not easily affected by reflection by excitation light.

  In addition, the other means is configured to include a microorganism number estimation means A for estimating the number of microorganisms per filter based on the ratio of the image capturing area and the area of the entire filter collecting the microorganisms.

  With this configuration, it is possible to provide a microorganism measuring system that can estimate the number of microorganisms per filter without measuring the area of the entire filter.

  Moreover, it is set as the structure provided with the microorganisms number estimation means B which estimates the number of microorganisms per ml from the quantity of the collected sample.

  With this configuration, it is possible to provide a microorganism measuring system that can unify the level for comparing the number of measured microorganisms from the entire sample.

  In addition, the other means is configured to include a microorganism number estimation means C for estimating the number of microorganisms in the sample before dilution from the dilution ratio of the sample diluted before collection.

  With this configuration, it is possible to provide a microorganism measurement system that can estimate the number of microorganisms in the entire measured sample.

  Another means is configured to perform fluorescent staining of the collected microorganisms on the collected filter.

  With this configuration, since it is not necessary to put a staining reagent into the solution, it is possible to provide a microorganism measuring system that can stain a reagent with a small amount and a high reagent concentration.

  In addition, the other means is configured to have a function of generating a warning sound if the measurement result of microorganisms is equal to or greater than a specified value.

  With this configuration, it is possible to provide a microorganism measurement system that can know by voice that the measurement result is equal to or greater than the specified value.

  The other means is such that the order of staining with the staining reagent is such that after staining with the total bacteria reagent, staining with the dead bacteria reagent is performed.

  With this configuration, it is possible to provide a microorganism measuring system that can enhance the staining with a reagent.

  The other means is such that the order of staining with the staining reagent is such that the staining with the dead bacteria reagent is performed and then the staining with the total bacteria reagent is performed.

  With this configuration, it is possible to provide a microorganism measuring system capable of enhancing the dyeability depending on the type of reagent.

  Another means is configured such that staining with a staining reagent is performed by staining with a reagent in which a total bacteria reagent and a dead bacteria reagent are mixed.

  With this configuration, a microorganism measuring system with a simple staining procedure can be provided.

  According to the microorganism counting apparatus of the present invention, an effect that it is possible to determine whether a microorganism detected in a sample to be measured is alive or dead, and an optical interference system using a mirror for taking a fluorescence spectrum, It is possible to provide a microorganism measurement system having an effect that a simple microorganism detection method having a structure that does not require a complicated mechanism can be realized.

  The invention described in claim 1 of the present invention is a total bacterial fluorescent reagent for staining all microorganisms in an apparatus for fluorescently staining microorganisms using a fluorescent staining reagent and detecting and measuring microorganisms using fluorescently emitted images of the microorganisms. And two types of excitation light sources for fluorescence of the specimen stained with a dead germ fluorescent reagent that stains dead microorganisms, two types of spectral filters for separating the fluorescence of the emission points that have emitted fluorescence, Luminous inspection that captures three types of fluorescent images using a combination of imaging means for capturing fluorescent images, the two types of excitation light, and the two types of spectral filters, and detects fluorescent emission points from the captured fluorescent images. A light emitting point separating means for separating the detected light emitting points into living microorganisms, dead microorganisms, and foreign substances, and a light emitting point measuring means for measuring the separated light emitting points. Profit Simple microorganism detection method with a structure that does not require a complicated mechanism such as an interference optical system, and the fluorescence emission images of all microorganisms by the staining reagent, the fluorescence emission images of dead microorganisms, and the autofluorescent foreign matter in the specimen. This has the effect that the luminescent image can be distinguished and photographed.

  Further, the invention described in claim 2 has an effect that the light emission point can be quantified by adopting a configuration in which the means for detecting the same light emission point of the three types of fluorescent light emission images is performed based on the luminance value of the photographed image.

  In addition, the invention according to claim 3 is provided with a function of correcting the coordinate deviation of the light emitting point due to the refraction of light in the three types of fluorescent images, so that the positional deviation of the light emitting point can be eliminated and the same light emission It has the effect that a point can be detected with high accuracy.

  The invention according to claim 4 has the effect that the microorganism can be fixed on the filtration filter by using the filtration filter as a means for collecting the microorganism.

  Further, the invention according to claim 5 has an effect that a specimen containing a microorganism can be easily attached to a microorganism measuring device by making it possible to attach a structure holding a filter for filtering microorganisms.

  Further, the invention described in claim 6 has the effect that the two types of excitation light provided in the microorganism measuring device are LEDs, so that fluorescence can be emitted with a low current.

  The invention according to claim 7 has an effect that two types of fluorescent reagents can be made to emit light by using blue and green LEDs as the two types of excitation light provided in the microorganism measuring apparatus.

  Further, the invention according to claim 8 can separate only the fluorescent light emitting component by using the two types of spectral filters provided in the microorganism measuring apparatus as a spectral filter that transmits green light and a spectral filter that transmits red light. It has the action.

  The invention described in claim 9 has the effect that the photographed means can be processed as electronic data by using a CCD camera as the photographing means.

  Further, the invention described in claim 10 has an effect that noise due to the influence of the image sensor on the captured fluorescent image can be removed by capturing the noise image of the image sensor before capturing the fluorescent image. .

  Further, the invention described in claim 11 has an effect that the surface on which the microorganisms are collected can be measured at a plurality of locations by photographing the collection surfaces on which the microorganisms are collected at a plurality of locations.

  In addition, the invention described in claim 12 has an effect that fluorescence emission on the filter surface can be suppressed by performing gold deposition on the filter surface for collecting microorganisms.

  Further, the invention described in claim 13 is provided with the microorganism number estimation means A for estimating the number of microorganisms per filter based on the ratio of the image capturing area to the total area of the filter collecting the microorganisms. This has the effect that the number of microorganisms in the filtered specimen can be estimated without performing photographing measurement over the entire filter surface, which is a collecting surface.

  In addition, the invention according to claim 14 can easily compare the measurement results with other samples by providing the microorganism number estimation means B for estimating the number of microorganisms per ml from the amount of collected samples. It has the action.

  Further, the invention according to claim 15 is provided with the microorganism number estimation means C for estimating the number of microorganisms before dilution from the dilution ratio of the collected sample, so that the total number of microorganisms contained in other samples can be calculated. Has the effect of understanding.

  The invention according to claim 16 has the effect that the fluorescent staining of the collected microorganism is performed on the collected filter, so that the staining can be performed without changing the concentration of the fluorescent staining reagent.

  The invention according to claim 17 has the function of improving the recognition of the abnormality in the measurement result by having a function of generating a warning sound if there is an abnormality in the measurement result of the microorganism.

  The invention according to claim 18 is characterized in that the staining of the total bacterial staining reagent is achieved by performing the staining with the dead bacteria staining reagent after the staining with the total bacterial staining reagent in the order of staining with the staining reagent. It has the effect that it can be improved.

  Further, the invention according to claim 19 is characterized in that the dyeing property of the dead bacteria staining reagent is obtained by performing the staining with the dead bacteria staining reagent after the staining with the death bacteria staining reagent is performed. It has the effect | action that can be improved.

  The invention according to claim 20 has the effect that the staining time can be shortened by staining the staining with the staining reagent as a staining in which the total bacteria staining reagent and the dead bacteria staining reagent are mixed.

(Embodiment 1)
FIG. 1 is an example of a configuration diagram of a microorganism measurement system according to Embodiment 1 of the present invention.

  As shown in FIG. 1, the microorganism measurement system includes a first excitation light source 1 for causing fluorescence emission, a second excitation light source 2, and a first spectral filter 3 for separating excitation light and fluorescence emission. Second spectroscopic filter 4, photographing means 5 for photographing a fluorescent image, objective lens 6 for collecting fluorescence emitted from the specimen, fluorescently stained specimen, stage 7 for installing the specimen, and moving stage A control board for controlling the drive unit 8 for switching and the spectral filter switching means 9 for switching between the first spectral filter 3 and the second spectral filter 4 for separating excitation light and fluorescence emission 10 for detecting a light emitting point from a control signal such as a light source point command, a fluorescent image photographing command, a filter switching command, and a photographed image. Luminescent point detecting means 12, luminescent point sorting means 13 for classifying the detected luminescent points into live microorganisms, dead microorganisms, and foreign substances, luminescent point measuring means 14 for measuring the number of luminescent spots, and luminescent points The personal computer 15 having a function of displaying the number of the computers is provided.

  In addition, the function of the microorganism measuring system in this configuration is to irradiate the specimen that has been fluorescently stained with the total bacteria fluorescent reagent and the dead bacteria fluorescent reagent with the excitation light from the first excitation light source 1, and to apply the first spectral filter 3. The fluorescent image A is taken through the second spectral filter 4 and then the fluorescent image B is taken. Then, the excitation light is switched to the second excitation light source 2 and the second excitation light source 2 is taken. The fluorescence image C is photographed through the second spectral filter 4 and the emission point of these fluorescence image A, fluorescence image B, and fluorescence image C is detected by the emission point detection means 12. The detected luminescent spots are sorted by the luminescent spot sorting means 13 for sorting the living microorganisms, dead microorganisms, and foreign substances, and the luminescent spots measuring means 14 for measuring the sorted luminescent spots, Dead microorganisms, total number of microorganisms, It is obtained to calculate the coarse product number.

  In this configuration, the photographing means 5 uses a camera using a CCD image sensor or a camera using a MOS image sensor.

  As the first excitation light source 1, an ultraviolet light excitation light source that combines an ultraviolet LED and a filter that transmits only ultraviolet light, a blue light excitation light source that combines a blue LED and a filter that allows only blue light to pass, and a second excitation light source 2 Is configured using a blue light excitation light source combining a blue LED and a filter that allows only blue light to pass through, a green light excitation light source that allows only green LED and green light to pass through, and the like.

  In the present embodiment, both the first excitation light source 1 and the second excitation light source 2 are LEDs, but a laser may be used instead of the LEDs.

  The first spectral filter 3 passes only blue light, the blue spectral filter passes green light only, the green spectral filter passes only green light, and the second spectral filter 4 passes green light only green spectral filter passes only red light. A red spectral filter or the like is used. As an example of a combination of the first excitation light source 1, the second excitation light source 2, the first also the spectral filter 3, and the second spectral filter 4, the first excitation light source 1 is excited with blue light. The light source, the first spectral filter 3 with a green spectral filter, the second excitation light source 2 with a green light excitation light source, the second spectral filter 4 with a red spectral filter, and other examples include The excitation light source 1 is an ultraviolet light excitation light source, the first spectral filter 3 is a blue light spectral filter, the second excitation light source 2 is a blue light excitation light source, and the second spectral filter 4 is a green spectral filter. Method, and the like.

  The excitation light is designed not to emit light having a wavelength component that passes through the combined spectral filter, and the spectral filter is designed not to pass the excitation light component of the combined excitation light source. For example, specifically, the excitation light and the spectral filter have the following wavelength characteristics. The blue light excitation light source is an excitation light source that emits light having a wavelength of 450 nm to 490 nm, the ultraviolet light excitation light source is an excitation light source that emits light between 350 nm and 400 nm, and the green light excitation light source is light from 490 nm to 540 nm. It is an excitation light source that irradiates. In addition, the spectral filter has a characteristic of allowing light of 410 nm to 490 nm to pass through, the green light spectral filter has a characteristic of passing a component having a wavelength of 500 nm to 560 nm, and the red light spectral filter of 570 nm. There is also a configuration in which a spectral filter having a characteristic of passing a wavelength component of ˜700 nm is passed.

  The switching between the first spectral filter 3 and the second spectral filter 4 that appears in this example is performed by the spectral filter switching means 9, which uses an electromagnetic magnet such as a solenoid coil, When not energized, the first spectral filter 3 is used, and the electromagnetic wave is switched to the second spectral filter 4.

  The control board 10 serves to control the power supply of the first excitation light source 1, the second excitation light source 2, the imaging means 5, the spectral filter switching means 9, and the on / off of each part. is there.

  As an example of the light emission point detection means 12, the luminance value of the photographed fluorescent image is used. This can be realized by detecting the luminance value of the light emitting point as an aggregate of pixels having a predetermined luminance value or more. For example, the predetermined value of the luminance value is 10, and an aggregate of 10 or more pixels is detected as one light emission point.

  As an example of the light emission point discriminating means 13, the sample is stained using a fluorescent staining reagent in which all microorganisms emit green light with blue excitation light and a fluorescent staining reagent in which only microorganisms dead by green excitation light emit red light. Then, the specimen is fluorescent using the first excitation light source 1 as a blue light excitation light source, a fluorescent image A is taken using the blue light spectral filter as the first spectral filter 3, and then the first excitation light source 1 is used. The spectral filter is switched to the second spectral filter 4, and the fluorescent image B is taken using the red spectral filter as the second spectral filter 4, and then the excitation light is switched to the second excitation light source 2. Then, the second excitation light source 2 is used as a green light excitation light source, and the fluorescence image C is obtained by photographing through the second spectral filter 4.

  At this time, as the fluorescent staining reagent, SYTO24, SYTO9 can be used as the total bacterial fluorescent reagent, and SYTOX / ORENGE, propidium iodide, etc. can be used as the dead bacterial fluorescent reagent.

  In this case, the luminescent spots shown in the fluorescent image A are measured as all microorganisms, the luminescent spots shown in the fluorescent image B are measured as impurities, and the luminescent spots shown in the fluorescent image C are dead. Measure as By subtracting the number of emission points of the fluorescence image C from the number of emission points of the fluorescence image A, living microorganisms can be separated.

  As the luminescent point measuring means 14 for measuring the luminescent point from the image, the luminescent point detected by the luminescent point detecting means and the number of microorganisms and contaminants sorted by the luminescent point sorting means 13 are obtained for each fluorescent image. It is done by doing.

  The display function of the measurement result is performed on a liquid crystal screen attached to the personal computer 15 or having a function.

  The role of the drive unit 8 that drives the stage 7 is to move the specimen by a certain distance in order to photograph the whole specimen because the field of view of the fluorescent image to be photographed is a part of the whole specimen. With this configuration, a plurality of fluorescent images can be taken.

  In this way, the detection of microorganisms is not a complicated mechanism such as an interference optical system using a mirror, but it can be done with a simple configuration of two types of light sources, a camera, and two types of spectral filters. It is possible to provide a microorganism measuring system that can be separated into microorganisms and contaminants and that can measure the number of living microorganisms, dead microorganisms, and contaminants in a specimen.

(Embodiment 2)
The same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 2 is an example of a configuration diagram of the microorganism measurement system according to the second embodiment of the present invention. As shown in FIG. 2, the light emitting point position correcting means 16 is added to FIG.

  The light emitting point position correcting means 16 is realized by the following method. The captured fluorescent image A, fluorescent image B, and fluorescent image C are fluorescent with the first excitation light source 1 and the second excitation light source 2, respectively, and passed through the first spectral filter 3 and the second spectral filter 4, respectively. However, using fluorescent particles that can capture fluorescence, fluorescent image shooting, fluorescent image A, fluorescent image B, and fluorescent image C are captured, and the displacement of each fluorescent image is superimposed to detect coordinate shifts. Then, position correction is performed to move the coordinates by the amount of deviation, and correction is performed so that the coordinates are aligned with any one of the fluorescent images A, B, and C. By correcting the positions of the fluorescent images A, B, and C in this way, if a fluorescent image of a fluorescently stained specimen is taken, if there are points that are not taken at the coordinates of the respective images, the fluorescent points are taken. Based on the fluorescent image, it is possible to determine whether the luminescent spot is a bacterium, a living bacterium, a dead bacterium, or a contaminant.

  The position correction means at this time can be performed only with the fluorescent image A and the fluorescent image C.

  The fluorescent particles used here are preferably R0100 manufactured by Moritex Corporation.

  Also, when preparing a specimen, microorganisms are filtered, and a filter is used when filtering. As an example, the filter 17 has a hole diameter of 0.4 μm. By using this hole diameter, it is possible to collect and measure even smaller microorganisms.

  Further, by setting the hole diameter of the filtration filter 17 to a small hole diameter of about 0.4 μm, a staining reagent can be placed on the filtration filter 17 and fluorescent staining of microorganisms can be performed on the filtration filter. It becomes like this.

  In addition, by depositing a metal thin film on the filtration filter 17, it is possible to suppress autofluorescence on the surface of the filtration filter 17, and there is a difference in luminance between the emission point of the fluorescence image and the surface of the filtration filter 17, thereby recognizing more fluorescence. Can be easily configured.

  At this time, the metal to be used is preferably a metal that is less likely to be self-fluorescent, such as gold, titanium, or copper. The thickness of the deposited thin film is preferably 30 nm or more in order to suppress the autofluorescence of the filtration filter 17 itself. However, increasing the thickness of the thin film leads to an increase in the cost of the filtration filter 17, so the thinnest film thickness that can suppress autofluorescence is the optimum film thickness.

  In addition, the following three methods can be used for fluorescent staining of microorganisms. The order of staining with the total bacterial fluorescent reagent that stains all microorganisms and the dead bacterial fluorescent reagent that stains dead microorganisms is the first method, and the appropriate amount of total bacterial fluorescent reagent is placed on the filtration filter 17 that has filtered the microorganisms. After staining, the total bacterial fluorescent reagent is filtered once, and then stained with an appropriate amount of dead fluorescent fluorescent reagent in the same procedure. If you want to capture the total bacteria as a clearer image at the time of measurement, the first method is preferred because all the microorganisms can be easily stained and emit more intense fluorescence by staining with the total bacteria fluorescent reagent first. .

  In this case, the appropriate amount is about 100 μl.

  As a second method, after putting a suitable amount of the dead germ fluorescent reagent on the filtration filter 17 that has filtered the microorganisms and staining, the dead germ fluorescent reagent is once filtered, and then using the same amount of the total germ fluorescent reagent in the same procedure. Perform staining. If you want to capture dead bacteria as a clearer image at the time of measurement, the second method can be used because the dead microorganisms can be easily dyed and emit more intense fluorescence by staining with the dead bacteria fluorescent reagent first. Good.

  An appropriate amount in this case is about 100 μl.

  As a third method, total bacterial fluorescence staining and dead bacterial fluorescence staining are performed simultaneously. This is a method in which the total bacterial fluorescence staining and the dead bacterial fluorescence staining are performed simultaneously on the filtration filter 17 in which microorganisms are filtered, or each reagent is applied in an appropriate amount without filtering. This method is characterized in that the reagent staining time can be halved compared to the first method and the second method. Next, staining is performed with an appropriate amount of the total bacterial fluorescent reagent in the same procedure. The third method is better when you want to measure as soon as possible.

  An appropriate amount in this case is about 50 μl. At this time, the concentration of each reagent is preferably double that of the first and second methods.

  By adopting such a configuration, small microorganisms can be collected, and fluorescence emission can be captured neatly, that is, fluorescence can be easily recognized. Therefore, microorganisms with improved measurement accuracy than the microorganism measurement system described in Embodiment 1. A measurement system can be provided.

(Embodiment 3)
The same parts as those in the first or second embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 3 is an example of a configuration diagram of the stage 7 in which the filter filter holding unit 18 can be attached to the structure holding the filter 17 according to the third embodiment of the present invention.

  The filtration filter holding unit 18 includes a structure for sandwiching the filtration filter 17, a structure for holding the filtration filter 17, and a structure for determining the position of the filtration filter holding unit 18, and the stage 7 has a structure for receiving the filtration filter 17. The stage is provided with a structure for receiving the filtration filter holding portion 18 and a stage 7 having a groove for fitting a projection for determining the position of the filtration filter holding portion 18. When the filtration filter holding unit 18 is mounted, the filtration filter holding unit 18 is connected to the filtration filter holding unit 18 when the filtration filter 17 is received by the filtration filter receiving unit. In order to increase the flatness, it has a structure with a slight gap and no direct contact. The gap is 10 μm to 200 μm.

  By doing so, the distance between the sample and the camera can be kept constant, an image with a stable focus accuracy can be acquired, and the measurement accuracy can be stabilized.

  The same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

(Embodiment 4)
The same parts as those in any of Embodiments 1 to 3 are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 4 is an example of a configuration diagram of the microorganism measurement system according to the fourth embodiment of the present invention. As shown in FIG. 4, a noise image photographing means 19 and a noise image removing means 20 are added to FIG.

  The noise image photographing means 19 is performed by acquiring a noise image by the photographing means 5 without irradiating excitation light according to a signal from the personal computer 15.

  At this time, the exposure time of the image photographed by the photographing means 5 is longer than that at the time of fluorescent image photographing. For example, if the fluorescent image capturing time is 6 seconds, the noise image capturing means 19 sets the exposure time to 10 seconds. By doing in this way, the fixed noise on the image generated from the photographing means 5 can be photographed, and the noise on the fluorescent image can be specified by comparing with the fluorescent images A, B, and C. it can.

  The noise removing unit 20 compares the captured fluorescent image with the noise image captured by the noise image capturing unit 19 and deletes the noise emission point existing in the noise image from the fluorescent image. . As an example of the deletion method, a fluorescence image and a noise image are overlapped and the images are subtracted.

  Thus, by taking a noise image and removing the light emission point of the noise from the fluorescence image, a microorganism measurement system with a high measurement system can be provided.

(Embodiment 5)
The same parts as those in any of Embodiments 1 to 4 are denoted by the same reference numerals, and detailed description thereof is omitted. FIG. 5 is an example of a configuration diagram of the microorganism measurement system according to the fifth embodiment of the present invention. As shown in FIG. 4, a configuration in which a microorganism number estimation means A <b> 21 for estimating the number of microorganisms from the area ratio of the entire filtration filter 17 and the measured fluorescence image area is added to FIG. 4.

  The microorganism estimation means A21 according to this configuration is realized as follows.

  The entire area of the filtration filter 17 is input from the personal computer 15 in advance, the ratio with the area of the photographed fluorescence image is calculated, the number of microorganisms in the entire area of the photographed fluorescence image is measured, and the measurement result is filtered. This is done by multiplying the ratio of the total area of the filter 17 and the total area of the image to calculate the total number of microorganisms in the total area of the filtration filter 17.

  The calculation formula is as follows.

Total number of microorganisms (in total area of filtration filter 17)
= (Total area of filtration filter 17 / total area of fluorescent image capturing) * Number of microorganisms in the entire area of fluorescent image capturing By using this method, the number of microorganisms in the entire area of filtration filter 17 can be estimated. Since the number of microorganisms in the specimen to be examined can be determined without measuring the entire area of the filtration filter 17, a microorganism measuring system that can quickly grasp the number of microorganisms in the specimen can be provided.

(Embodiment 6)
The same parts as those in any of Embodiments 1 to 5 are denoted by the same reference numerals, and detailed description thereof is omitted. FIG. 6 is an example of a configuration diagram of the microorganism measurement system according to the sixth embodiment of the present invention. As shown in FIG. 6, a configuration in which a microorganism number estimation means B22 for estimating the number of microorganisms in 1 ml is added to FIG.

  The microorganism estimation means B22 according to this configuration is realized as follows.

  By inputting the amount of the sample to be filtered from the personal computer 15 in advance and calculating the number of microorganisms in the total area of the filtration filter 17 inferred from the measurement result to be converted to the amount per 1 ml from the filtered amount. Is called.

  The calculation formula is as follows.

Number of microorganisms per ml = (estimated number of microorganisms in the total area of the filtration filter 17) * (1 ml / ml of filtration)
By estimating the number of microorganisms of 1 ml by such calculation, it is possible to evaluate the contamination amount of various specimens based on a certain standard, and to provide a microorganism measurement system that can easily compare the contamination amount of microorganisms.

(Embodiment 7)
The same parts as those in any of Embodiments 1 to 6 are denoted by the same reference numerals, and detailed description thereof is omitted. FIG. 7 is an example of a configuration diagram of the microorganism measurement system according to the seventh embodiment of the present invention. As shown in FIG. 7, a configuration in which a microorganism number estimation means C23 for estimating the number of microorganisms in a collected sample is added to FIG.

  The microorganism estimation means C23 according to this configuration is realized as follows.

  When the collected sample is diluted and used, the diluted ratio (dilution ratio) is input to the personal computer 15 before measurement, and the filtration amount obtained by filtering the number of microorganisms in the entire area of the filtration filter 17 inferred from the measurement result From this, it is calculated by converting to the amount per ml and multiplying the result by the dilution ratio.

  The calculation formula is as follows.

The number of microorganisms in the collected sample = (estimated value of the number of microorganisms in the entire area of the filtration filter 17) * (1 ml / ml of filtration) * dilution rate The number of microorganisms in the sample collected by this calculation can be estimated, By knowing the number of microorganisms in the sample, it is possible to provide a microorganism measurement system that can directly check the microorganism contamination status of the sample.

(Embodiment 8)
The same parts as those in any of Embodiments 1 to 7 are denoted by the same reference numerals, and detailed description thereof is omitted. FIG. 8 is an example of a configuration diagram of the microorganism measurement system according to the eighth embodiment of the present invention. As shown in FIG. 8, a warning generating means 24 is added to FIG.

  The warning generation means 24 according to this configuration is realized as follows.

  As the number of microorganisms measured by the luminescent point measuring means 14, the microorganism number estimating means A21, the microorganism number estimating means B22, and the microorganism number estimating means C23, a numerical value that can be determined to be abnormal is input from the personal computer 15 in advance. When the value exceeds the specified light emission point area upper limit value, the warning generation means 24 generates a warning sound from the personal computer 15 or displays a message notifying the abnormality on the screen of the personal computer 15 Done.

  For example, if the upper limit of the number of microorganisms in this case is 300,000, the number of microorganisms estimated A21. According to the microorganism estimation means of the microorganism estimation means B22 and the microorganism estimation means C23, it is determined whether there is an excess of 300,000 or not, and a warning display according to each means is performed by voice or screen. To.

  By providing the warning generating means 24 in this way, it is possible to provide a microorganism measurement system that can quickly confirm an abnormality when there is an abnormality in the measurement result and can quickly deal with the sample.

  The measurement system of the present invention can acquire fluorescent images of microorganisms that have been subjected to fluorescent staining, and can detect microorganisms using the fluorescent images, such as bacteria and fungi in food factories, pharmaceutical factories, hospitals, etc. It can also be applied to uses such as microbial detection.

Configuration diagram of the microorganism measurement system of Embodiment 1 of the present invention The block diagram of the microorganisms measurement system of Embodiment 2 of this invention The block diagram of the filter mounting structure of Embodiment 3 of this invention Configuration diagram of the microorganism measurement system of Embodiment 4 of the present invention Configuration diagram of microorganism measurement system according to Embodiment 5 of the present invention The block diagram of the microorganisms measurement system of Embodiment 6 of this invention Configuration diagram of microorganism measurement system according to Embodiment 7 of the present invention Configuration diagram of the microorganism measurement system according to the eighth embodiment of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st excitation light source 2 2nd excitation light source 3 1st spectral filter 4 2nd spectral filter 5 Imaging | photography means 6 Objective lens 7 Stage 8 Drive part 9 Spectral filter switching means 10 Control board 11 Microorganism measuring device main body 12 Light emission Point detection means 13 Light emission point classification means 14 Light emission point measurement means 15 Personal computer 16 Light emission point position correction means 17 Filtration filter 19 Noise image photographing means 20 Noise image removal means 21 Microorganism number estimation means A
22 Microorganism number estimation means B
23 Means C for estimating the number of microorganisms
24 Warning generation means

Claims (20)

In a device that detects and measures microorganisms using fluorescent staining reagents, and detects and measures microorganisms using fluorescent images of microorganisms, the total bacteria fluorescent reagent that stains all microorganisms and the dead bacteria fluorescence that stains dead microorganisms Two types of excitation light sources for causing a specimen stained with a reagent to fluoresce, two types of spectral filters for separating the fluorescence of the fluorescent emission point, and imaging means for taking a fluorescent image, A light emission point detecting means for detecting a fluorescent light emission point from three types of fluorescent images photographed through a spectral filter that passes only a wavelength longer than the excitation light wavelength by a combination of the two types of excitation light sources and the two types of spectral filters The luminescent point is classified into living microorganisms, dead microorganisms, and foreign substances, and the luminescent point measuring means for measuring the luminescent points is provided. Microbial measurement system for measuring a. 2. The microorganism measuring system according to claim 1, wherein means for detecting a light emitting point in the three types of fluorescent images is performed based on the luminance value of the photographed image. The microorganism measuring system according to claim 1, further comprising: a light emitting point position correcting unit that corrects a deviation of the coordinates of the light emitting point in the three types of fluorescent images. 4. The microorganism measuring system according to claim 1, wherein a filtration filter is used as means for collecting microorganisms. The microorganism measuring system according to any one of claims 1 to 4, wherein a structure holding a filter for filtering microorganisms can be attached. The microorganism measuring system according to claim 1, wherein two types of excitation light are LEDs. The microorganism measurement system according to claim 1, wherein the two types of excitation light are blue light and green light. The microorganism measuring system according to any one of claims 1 to 7, wherein the two types of spectral filters are a spectral filter that allows green light to pass through and a filter that allows red light to pass through. 9. The microorganism measuring system according to claim 1, wherein the photographing means is a CCD camera. The microorganism measuring system according to claim 1, wherein a noise image of the imaging element is captured before capturing a fluorescent image. The microbe measurement system according to any one of claims 1 to 10, wherein a plurality of capture surfaces on which microbes are collected are photographed. The microorganism measuring system according to claim 1, wherein metal deposition is performed on the surface of the filter for collecting microorganisms. The microorganism measuring system according to any one of claims 1 to 12, further comprising microorganism number estimating means A for estimating the number of microorganisms per filter based on a ratio of an image photographing area and an area of the whole filter collecting microorganisms. The microorganism measuring system according to any one of claims 1 to 13, further comprising microorganism number estimating means B for estimating the number of microorganisms per ml from the amount of collected sample. The microorganism measuring system according to any one of claims 1 to 14, further comprising microorganism number estimating means C for estimating the number of microorganisms in the sample before dilution from the dilution ratio of the sample diluted before collection. The microorganism measuring system according to any one of claims 1 to 15, wherein fluorescent staining of collected microorganisms is performed on a collected filtration filter. The microorganism measurement system according to any one of claims 1 to 16, further comprising warning generation means for generating a warning sound if there is an abnormality in the microorganism measurement result. The microorganism measuring system according to any one of claims 1 to 17, wherein the staining sequence is stained with a total bacterial fluorescent reagent and then stained with a dead bacterial fluorescent reagent. The microorganism measuring system according to any one of claims 1 to 18, wherein the staining sequence is stained with a dead bacteria fluorescent reagent and then stained with a total bacteria fluorescent reagent. The microorganism measuring system according to any one of claims 1 to 19, wherein the staining with the staining reagent is staining with a reagent in which a total bacterial fluorescent reagent and a dead bacterial fluorescent reagent are mixed.

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