JP2006055161A - Method for detecting microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a microorganism, capable of rapidly and accurately detecting the microorganism. <P>SOLUTION: This method for measuring microorganisms capable of rapidly and accurately detecting microorganisms in a specimen is obtained by using a microscope, fluorescent, atomic force microorganism detecting means 15 by processing observation data in detail of an outline and surface profile of the microorganism obtained by using an atomic force microscope observation means 5 having a mechanism for observing a part identical to a part observed by a microscope observation means 3 not requiring fluorescent dyeing, or by processing measurement data obtained by using the atomic force microscope observation means 5 having a mechanism for observing a position identical to a part observed by a fluorescent observation means 11 with fluorescent dyeing. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microorganism detection method for detecting microorganisms.

  Conventionally, as this kind of microorganism detection method and microorganism detection apparatus, the one disclosed in Patent Document 1 is known.

  Hereinafter, this type of microorganism detection method and microorganism detection apparatus will be described with reference to FIG.

As a microorganism detection method, a method for detecting specific microorganisms collected from various environments by fluorescence emission obtained from a fluorescently stained sample 101 is described. The microorganism detection apparatus includes a fluorescence detection means 102 and an analysis apparatus 103. Things are listed.
JP 2003-144193 A

  In such a conventional method for detecting microorganisms, fluorescent staining has to be performed in order to detect microorganisms, and there is a problem that it takes time to perform staining. When foreign substances that are specifically stained in the fluorescent reagent are mixed, there is a problem that microorganisms cannot be detected and measured accurately, and it is required to improve the detection speed, accuracy, and measurement accuracy of microorganisms. ing.

  The present invention solves such a conventional problem, a method capable of detecting earlier than microorganisms without fluorescent staining, and a method of detecting only microorganisms more accurately by excluding foreign matters during fluorescent staining. The purpose is to provide.

  In order to achieve the above object, the microorganism detection method of the present invention comprises a microscope observation means, an atomic force microscope observation means, a position observed with the microscope observation means, and a position observed with the atomic force microscope observation means. Microscopic atomic force isotope observation means, imaging means for imaging the state observed with a microscope, information obtained from a microscope observation image and information obtained from an atomic force observation microscope observation means And a microscopic atomic force microbe detection means for detecting microbes.

  By this means, the same position as that of the microscope observation means is observed by the atomic force microscope observation means, so that it is possible to accurately determine whether the microorganism is a foreign substance or a foreign substance from the detailed information of the specimen. It is possible to provide a microorganism detection method that can rapidly detect microorganisms without intervention.

  Further, the other means includes a fluorescence observation means, a fluorescence atomic force isotope observation means capable of observing the same position as the position observed with the fluorescence observation means and the position observed with the atomic force microscope observation means, and the fluorescence observation means Fluorescence image photographing means for imaging the state observed in, and fluorescence atomic force microorganism detection means for analyzing the information obtained from the fluorescence observation image and the information obtained from the atomic force microscope observation means to detect microorganisms It is a configuration.

  Thereby, it is possible to accurately determine whether the microorganism is a foreign substance or not based on the information on the observation point obtained from the fluorescence observation means and the information obtained from the atomic force microscope observation means, thereby improving the detection accuracy of the microorganism. A method for detecting microorganisms can be provided.

  Further, the other means includes the microscope observation means, the fluorescence observation means, the atomic force microscope observation means, the position observed with the microscope observation means, the position observed with the fluorescence microscope observation means, and the interatomic position. It is obtained from the microscope fluorescence atomic force isotope observation means that makes the position observed with the force microscope observation means the same, the imaging means for imaging the state observed with the microscope, the information obtained from the microscope observation image, and the fluorescence observation image And information obtained from the atomic force observation microscope observation means are analyzed, and a microscope fluorescence atomic force microorganism detection means for detecting microorganisms is provided.

  Thereby, it is possible to more accurately determine whether the microorganism is a foreign substance or not by the information obtained from the microscope observation means, the fluorescence observation means, and the atomic force microscope observation means, and the detection accuracy of the microorganism can be improved. A method for detecting microorganisms can be provided.

  Another means is that the above-mentioned microscope atomic force microorganism detecting means detects microorganisms in both the outer shape or surface shape of the observation object or the outer surface shape.

  As a result, it is possible to more accurately determine whether the microorganism is a foreign substance or not based on the outer shape information of the observation point obtained by the microscope observation means and the outer shape and surface shape information obtained by the atomic force microscope observation means. A microorganism detection method that can be further improved can be provided.

  In another means, the fluorescent atomic force microorganism detecting means is configured to detect microorganisms by both fluorescence intensity, outer shape, surface shape, or outer shape.

  As a result, it is possible to more accurately determine whether the microorganism is a foreign substance or not based on the luminance and outline information of the observation point obtained by the fluorescence microscope and the outline and surface shape information obtained by the atomic force microscope observation means, and the detection accuracy of the microorganism It is possible to provide a method for detecting a microorganism that can further improve

  In addition, another means is configured to detect the microscope fluorescence atomic force microorganism detecting means by the fluorescence intensity, the external shape, the surface shape, or the external surface shape.

  As a result, it is possible to determine whether the microorganism is a foreign substance or not by the outline information of the observation point obtained by the microscope observation means, the brightness and outline information of the observation point obtained by the fluorescence microscope, and the outline and surface shape information obtained by the atomic force microscope observation means. It is possible to provide a microorganism detection method capable of accurately determining and improving the detection accuracy of microorganisms.

  Further, the other means includes a light wavelength spectrum analyzing means for analyzing a spectrum of a light wavelength emitted from an observation point observed by the microscope observation means and the fluorescence observation means, and information obtained from the microscope observation means, the fluorescence Microorganisms are detected based on fluorescence emission information obtained from the observation means, information obtained from the atomic force microscope observation means, and light wavelength analysis information obtained from the light wavelength spectrum analysis means.

  Thus, the information obtained from the microscope observation means, the information obtained from the fluorescence observation means, the information obtained from the atomic force microscope observation means, and the light wavelength analysis information obtained from the light wavelength spectrum analysis means Therefore, it is possible to provide a microorganism detection method that can more accurately determine whether the microorganism is a foreign substance or not, and can further improve the detection accuracy of the microorganism.

  In addition, the other means is configured to include a plurality of light wavelength spectrum analyzing means capable of analyzing a plurality of points of the light wavelength spectrum analyzing means for performing spectrum analysis of the light wavelength.

  Thereby, since the said light wavelength multiple location analysis means can obtain the light wavelength analysis information of the multiple location in the microscope 1 visual field, determination of a detection target can be performed at once, and microorganisms can be detected rapidly, A method for detecting microorganisms can be provided.

  The other means is configured such that the photographing means is a monochrome CCD camera.

  Thereby, it is possible to provide a microorganism detection method capable of numerically expressing photographed image information and enabling image analysis by a computer.

  The other means is configured such that the photographing means is a color CCD camera.

  Thereby, color information can be added to photographed image information, and a more accurate microorganism detection method can be provided.

  In addition, the other means is configured to include an image recognition means for collating an image observed by the microscope observation means, the fluorescence observation means, the light wavelength analysis means, and the atomic force microscope observation means with a specific image. Is.

  This provides a microorganism detection method that can more accurately determine whether a microorganism is a foreign substance or not by collating it with image processing recognition processing such as the outer shape and surface shape of the specimen, and can further improve the detection accuracy of the microorganism. it can.

  In addition, the other means is configured to include a hardness detection means for detecting the outer shape, the surface shape, and the hardness.

  Thereby, it is possible to provide a microorganism detection method that can more accurately determine whether a microorganism is a foreign substance from the difference in the outer shape, surface shape, and hardness of the specimen.

  In addition, the other means is configured to include charge detection means for detecting the presence or absence of charge or the strength.

  Thereby, it is possible to provide a microorganism detection method that can more accurately determine whether a microorganism is a foreign substance from the difference in charge amount of the specimen.

  Another means is that the fluorescent staining reagent is a staining reagent that emits light from living microorganisms.

  Thereby, the microorganism detection method which can detect a living microorganism more correctly can be provided by making a living microorganism fluorescent.

  Another means is that the fluorescent staining reagent is a staining reagent that emits light from dead microorganisms.

  Thereby, the microorganism detection method which can detect a dead microorganism more correctly can be provided by making a dead microorganism fluorescence.

  In another means, the fluorescent staining reagent is a reagent that emits light from all microorganisms.

  Thereby, the microorganisms detection method which can detect all the microorganisms in a sample more correctly by making all the microorganisms fluorescence can be provided.

  In addition, the other means is configured to include the differential interference observation means in the microorganism detection method.

  Thereby, since the specimen is observed by differential interference, a microorganism detection method that can detect microorganisms that are transparent and difficult to detect more accurately can be provided.

  ADVANTAGE OF THE INVENTION According to this invention, the microorganisms detection method which has the effect that microorganisms can be detected quickly and with high precision can be provided.

  In addition, according to the present invention, it is possible to provide a microorganism detection method having an effect that the number of microorganisms can be quickly grasped with high accuracy by quickly detecting and measuring microorganisms.

  The invention according to claim 1 of the present invention comprises a microscope observation means capable of magnifying and observing a specimen, and a microscope atomic force isotope capable of observing the same position as that of the magnification observation with the microscope with an atomic force microscope observation means. By having a structure equipped with a microscope atomic force microbe detection means for analyzing microorganisms by analyzing information obtained from a value observation means and an imaging means for imaging the state observed with a microscope and an atomic force observation microscope observation means, Microbes can be produced without staining, and the observation magnification can be changed from low magnification to high magnification without replacing the specimen, so that observation can be easily performed with a wide field of view at low magnification, and the identified observation points are located at the same position. Since it can be observed at a high magnification, it has an effect that a more magnified and fine observation can be performed.

  The invention according to claim 2 of the present invention includes a fluorescence observation means capable of observing a specimen labeled with a fluorescent staining reagent, a position observed with the fluorescence observation means, and a position observable with the atomic force microscope observation means. Fluorescent atomic force isotope observation means that can observe the same position, photographing means for imaging the state observed with the fluorescence observation means, information obtained from the fluorescence observation image and information obtained from the atomic force microscope observation means are analyzed However, it is difficult to observe normally because the microorganisms are transparent because many of them are transparent, but it is difficult to observe by fluorescent emission and fluorescent emission. It has the effect that it can be dulled and that the light emission point can be further enlarged and observed.

  The invention according to claim 3 of the present invention includes the microscope observation means, the fluorescence observation means, the atomic force microscope observation means, the position observed with the microscope observation means, and the position observed with the fluorescence microscope observation means. Microscope fluorescence atomic force isotope observation means for making the position observed by the atomic force microscope observation means the same, photographing means for imaging the state observed with the microscope, information obtained from the microscope observation image and fluorescence observation Analyzing the information obtained from the image and the information obtained from the atomic force observation microscope observation means, and comprising a microscope fluorescence atomic force microorganism detection means for detecting microorganisms, it is possible to obtain a non-stained specimen and a fluorescent staining specimen. That can be observed with the same device, the color information of the observation object by natural light, the state of brightness, the light emission color obtained by fluorescent emission of microorganisms, the light emission luminance, etc. And that can be observed with more information more observation points has the effect that it is observed by larger scale light emitting points.

  According to a fourth aspect of the present invention, the microscope atomic force microorganism detecting means according to the first aspect of the present invention is configured to detect the outer shape, the surface shape, or both the outer shape and the surface shape. Microorganisms can be detected by utilizing the consistency of shape data.

  According to the fifth aspect of the present invention, the fluorescence atomic force microorganism detecting means according to the second aspect of the present invention is configured to detect the fluorescence intensity and the external shape or the surface shape or both the external shape and the surface shape. Microorganisms can be detected by utilizing the consistency of strength, outer shape data, and surface shape data.

  The invention of claim 6 of the present invention is configured to detect the fluorescence fluorescence atomic force microbial microorganism detection means of the invention of claim 3 by the fluorescence intensity and the outer shape or the surface shape or both the outer shape and the surface shape. Microorganisms can be detected by utilizing the consistency of fluorescence intensity, outer shape data, and surface shape data.

  According to a seventh aspect of the present invention, there is provided a light wavelength spectrum for analyzing a spectrum of a light wavelength emitted from an observation point by the microscope observation means and the fluorescence observation means with respect to the invention according to any one of the first to sixth aspects. The configuration including the analyzing means has an effect that the reflected light or transmitted light of the microorganism to be detected or the spectrum analysis of the fluorescence wavelength can be performed.

  The invention according to claim 8 of the present invention is configured to have a plurality of light wavelength spectrum analyzing means for spectrally analyzing the light wavelength with respect to the invention according to claim 7, so that the light wavelength and fluorescence wavelength of the microorganism to be detected are detected. The spectrum analysis can be performed simultaneously at a plurality of locations.

  According to the ninth aspect of the present invention, since the photographing means according to any one of the first to eighth aspects is configured as a monochrome CCD camera, image information such as luminance value and largeness can be expressed numerically. It has the action.

  According to a tenth aspect of the present invention, the photographing means according to any one of the first to eighth aspects is configured as a muller CCD camera. Information can also be expressed numerically.

  The invention according to claim 11 of the present invention comprises an image recognition means for collating an image observed by the microscope observation means, the fluorescence observation means light wavelength analysis means, and the atomic force microscope observation means with a specific image. As a result, the microorganism can be compared with the specific image.

  Invention of Claim 12 of this invention has the effect | action that the hardness of microorganisms can be measured by setting it as the structure provided with the measurement means of hardness in the invention in any one of Claims 1 thru | or 11.

  Invention of Claim 13 of this invention has the effect | action which can measure the charge of microorganisms by having set it as the structure provided with an electric charge detection means in the invention in any one of Claim 1 thru | or 12.

  According to the fourteenth aspect of the present invention, the living microorganism is fluorescent by using the fluorescent staining reagent according to any one of the second, third, fifth to fourteenth aspects as a reagent for staining the living microorganism. It has the effect of being observable.

  According to the fifteenth aspect of the present invention, since the fluorescent staining reagent according to the second, third, fifth to thirteenth aspects is used as a reagent for staining a dead microorganism, the dead microorganism can be observed with fluorescence. Have.

  The invention described in claim 16 of the present invention has the effect that all the microorganisms can be observed with fluorescence by using the fluorescent staining reagent described in claims 2, 3, 5 to 14 as a reagent for staining all the microorganisms. .

  The invention according to claim 17 of the present invention is the differential interference observation in which, in the inventions of claims 1, 3, 4, 6 to 14, two lights are generated by utilizing the deflection of light and observed using the interference. The configuration including the means differential interference observation means has an effect of facilitating observation of a transparent specimen.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1)
1 and 2 are examples of a configuration diagram according to the first embodiment. FIG. 1 shows an example of the overall flow of the microorganism detection method, and FIG. 2 shows an example of the equipment configuration of the microorganism detection method. The flow of the configuration will be described next. Microscope observation means 3 for magnifying and observing the specimen 1 with the objective lens 2, and an observation point for observing the specimen surface with the atomic force microscope observation means 5 and the microscope observation means 3 for tracing the specimen surface with a very thin probe 4. The atomic force microscope observation means 5 and the observation means obtained by the atomic force microscope observation means 5 are imaged using a CCD camera or the like. And an image processing function 8 for extracting data from the microscopic image, and a data processing function 9 for processing atomic force microscope observation data obtained from the atomic force microscope observation means 5, the image processing function 8 and the data processing function 9 includes a microscope atomic force microorganism detecting means 10 for detecting microorganisms from the photographed image using the processing result 9.

  In the above configuration, the observation point observed by the microscope observation means 3 that does not require fluorescent staining is made the same as the observation point of the atomic force microscope observation means 5 by the microscope atomic force isotope observation means 6. The magnification can be changed without replacing the specimen, for example, from 10 to 1000 times with the microscope observation means, and 500 to 100,000 times with the atomic force microscope observation means 5 from the low magnification to the high magnification. Allows observation with a wide field of view, making it easy to identify the observation point, and the same position can be observed at a high magnification as it is, so it can be easily enlarged further, and detailed observation of subtle differences in shape and differences in surface shape It will be possible. Further, a microscope observation image can be imaged by the photographing means 7 using a CCD or a photomultiplier tube, and the image data and the shape data from the atomic force microscope observation means 5 are respectively detected by the microscope atomic force microbe. By being able to process by means 10 and not performing fluorescent staining, it is possible to quickly determine whether microorganisms or foreign substances are accurate.

  Here, the microbe detection method by the microscopic atomic force microbe detection means 10 means that, as shown in FIG. 3, the microscopic image obtained by the radiography means 7 is obtained by observing the size, actual size, brightness, and observation of the observation point. Information on points such as shading, color, and brightness is converted into data using the image processing function 8, and atomic force microscope observation data such as outer shape, surface shape, and thickness obtained from the atomic force microscope observation means 5 is further processed. By using the data obtained by the image processing and the data obtained by the data processing, the microbial data is registered in the microscope atomic force microorganism detecting means 10 in advance, and the data is collated with the registered data. It is a method that tries to detect microorganisms. Examples of registered data include microbe outer dimension data, thickness data, and surface shape data.

  Further, as an example of the microscope atomic force isotope observation means 6, the probe 4 is attached to one of attachment parts having a revolver structure to which the objective lens 2 used in the microscope observation means 3 is attached, and the attachment part is rotated. Thus, the probe 4 can observe the same position as that observed by the microscope observation means 3. The probe 4 sends observation data to the atomic force microscope observation means 5 through a cable. Furthermore, when observing with the atomic force microscope observing means 5, the center of the field of view of the objective lens 2 of the microscope observing means 3 so that the center of the observation point of the microscope observing means 3 and the probe 4 of the atomic force microscope observing means 5 coincide. Are adjusted so that the observation point of the microscope observation means 3 and the observation point of the atomic force microscope observation means 5 are at the same position.

  The microscope observation means 3 here is an optical microscope, an example of which is an upright microscope, and an example of the atomic force microscope observation means 5 is an atomic force microscope (AFM), Scanning probe microscopes (SPM) and intermolecular force microscopes are available.

  Examples of specimens containing microorganisms to be detected include relatively large plankton and river water and alcoholic beverages containing relatively large microorganisms such as yeast.

  In addition, the specimen 1 can also be detected by moving the observation table, that is, scanning, to observe the whole specimen and to observe and detect all the microorganisms on the specimen.

  Moreover, as another example of the microscope atomic force isotope observation means 6, it can also be performed by moving the observation table.

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

  4 and 5 are examples of a configuration diagram of the second embodiment.

  FIG. 4 shows an example of the entire flow of the microorganism detection method, and FIG. 5 shows an example of the equipment configuration of the microorganism detection method. The flow of the configuration will be described next. 4 and 5 show the fluorescence atomic force isotope instead of the microscope observation means 3 and the fluorescence atomic force isotope observation means 6 in the microorganism detection method shown in FIGS. In this configuration, the observation means 12 and the fluorescent atomic force microorganism detection means 13 instead of the microscope atomic force microorganism detection means 10 are provided. This fluorescence observation means 11 is fluorescently stained for the specimen 1 and has the same configuration as a fluorescence microscope equipped with an excitation light source, a spectral filter, an objective lens, and the like, and is equipped with a mechanism for irradiating excitation light and taking out fluorescence, thereby allowing fluorescence observation. Is. Further, the fluorescent atomic force isotope observing means 12 includes a mechanism for setting the observation point observed by the fluorescent observation means 11 and the observation point observed by the atomic force microscope observation means 5 to the same position. The position can be set by the same method as in the first embodiment. Moreover, as shown in FIG. 6, the fluorescence atomic force microorganism detection means 13 uses the microscope image in FIG. 3 as a fluorescence image. In other words, the fluorescence photographed image taken by the photographing means 7 is converted into data by the image processing function 8 such as the size, size, brightness, density of the observation point, fluorescence color, luminance information of the observation point, and the atomic force microscope observation means 5. Data obtained from atomic force microscope observation data such as outer shape, surface shape, thickness, etc. obtained from the data is processed. Using the data obtained by image processing and the data obtained by data processing, the microbe data is preliminarily stored in the microscope. Microorganisms that have been registered in advance by registering in the interstitial microorganism detection means 10 and comprehensively processing not only the external shape information but also luminance information and fluorescent color information by collating data with the registered data. Since it can be compared with the data, microorganisms can be detected with higher accuracy.

  Examples of the registration data include microorganism external dimension data, thickness data, surface shape data, and color information in fluorescence observation.

  An advantage of using the fluorescence observation means 11 is that microorganisms can be more selectively detected by fluorescent staining. That is, since the fluorescent staining reagent emits fluorescence when it binds to the DNA of the microorganism, it can be determined whether or not it is a microorganism based on whether the fluorescence is emitted or not. It is difficult to observe small and transparent microorganisms with the microscope observation means 3, but these microorganisms can be clearly observed by fluorescent staining. Microorganisms can be detected sooner and more accurately.

  As an example of fluorescent staining, a reagent that stains only living microorganisms, a reagent that stains only dead microorganisms, and a reagent that stains all microorganisms can be used.

  When a reagent that stains only living microorganisms is used, living microorganisms can be detected more accurately.

  When a reagent that stains only dead microorganisms is used, dead microorganisms can be detected more accurately.

  When a reagent that stains all microorganisms is used, all microorganisms can be detected more accurately.

  All the microorganisms, dead microorganisms, and all microorganisms in one specimen are prepared by mixing the reagent that stains all microorganisms and the reagent that stains dead microorganisms to produce the fluorescently stained specimen 1. It is also possible to detect living microorganisms by subtracting dead microorganisms from them.

  Here, the microorganism refers to a smaller one such as Escherichia coli or Staphylococcus aureus in addition to those described in the first embodiment.

  The reagent that stains living microorganisms refers to 6-carboxyfluorescein diacetate, and the reagent that stains dead microorganisms refers to propidium iodide, etc., and the reagent that stains all microorganisms. 4 ', 6-diamidino-2-phenylindole dihydrochloride, etc.

  A living microorganism means a microorganism having a proliferation ability, a dead microorganism means a microorganism having a hole in the outer wall of the microorganism, and a whole microorganism means a living microorganism and a dead microorganism. This is a combination of

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

  7 and 8 are examples of configuration diagrams of the third embodiment.

  FIG. 7 shows an example of the overall flow of the microorganism detection method, and FIG. 8 shows an example of the equipment configuration of the microorganism detection method. The flow of the configuration will be described next.

  In the microorganism detection method shown in FIG. 1, a fluorescence observation means 11 is added, and a microscope fluorescence atomic force isotope observation means 14 instead of the microscope atomic force isotope observation means 6 and a microscope atomic force microorganism detection means 10 are substituted. The microscopic fluorescent atomic force microorganism detecting means 15 is provided. The microscope fluorescence atomic force isotope observation means 14 is a mechanism that positions the observation point observed by the microscope observation means 3, the observation point observed by the fluorescence observation means 11, and the observation point observed by the atomic force microscope observation means 5 at the same position. The observation point can be set at the same position by the same method as in the first embodiment.

  In addition, as shown in FIG. 9, the microscopic image and the fluorescent image captured by the photographing means 7 are observed point size, size, brightness, density of observation point, microscopic microscope observation color, fluorescent color, luminance information, etc. Is processed into data by the image processing function 8, and further, the atomic force microscope observation data such as the outer shape, surface shape and thickness obtained from the atomic force microscope observation means 5 is processed, and the data obtained by the image processing and the data Using the data obtained by the processing, microorganism data is registered in advance in the microscopic fluorescence atomic force microorganism detecting means 15 and collated with the registered data, so that not only the outer shape information but also luminance information and Since the fluorescent color information is comprehensively processed and can be compared with previously registered microorganism data, microorganisms can be detected with higher accuracy.

  Examples of the registered data include microbe external dimension data, thickness data, surface shape data, and color information in microscopic observation and fluorescent observation.

  In this way, by combining the microscope observation means 3, the fluorescence observation means 11, and the atomic force microscope observation means 5 as one instrument, it is possible to observe both the unstained specimen and the fluorescence stained specimen with the same instrument, and observation with natural light. The observation point can be observed with more information based on information such as the color information of the object, lightness shade, and the information such as the emission color and emission luminance obtained by fluorescent emission of the microorganism, and the emission point is further expanded. This observation has the advantage that microorganisms can be detected more quickly and more accurately.

(Embodiment 4)
The same parts as those in the first to third embodiments are denoted by the same reference numerals, and detailed description thereof is omitted.

  10 and 11 are examples of configuration diagrams of the fourth embodiment. FIG. 4 shows a configuration in which the light wavelength analysis means 16 is provided in the microorganism detection method shown in FIG.

  An example of the optical wavelength analysis means 16 is a spectroscopic measurement device. The light wavelength analyzing means 16 takes in light directly by the microscope observing means 3 or the fluorescence observing means 11 and spectrally separates the taken light. An optical fiber 17 is used for introducing the measurement light into the optical wavelength analyzing means 16.

  In the above configuration, after the fluorescence observation with the microscope observation means 3 and the fluorescence observation means 11, the fluorescence generated from the fluorescently stained specimen and the reflected or transmitted light emitted during the observation of the microscope observation means 3 are transmitted via the optical fiber 17. Then, the light wavelength analyzing means 16 decomposes it into a wavelength spectrum. The spectrum of each wavelength can be observed by the light wavelength analyzing means 16, and it can be determined whether the light emitted from the microorganism by fluorescence staining or the light emitted from the foreign substance by autofluorescence is determined by the position (wavelength) and intensity of the spectrum. Since it is possible to determine whether a microorganism is a foreign substance or not without using the atomic force microscope observation means 5, it is possible to detect a microorganism earlier. In addition, foreign substances other than microorganisms can be excluded using the light wavelength, and light wavelength information increases as observation point information, thereby enabling more accurate microorganism detection.

  In addition, by using the light wavelength multi-location analyzing means, it is possible to analyze the light wavelengths at a plurality of locations and to quickly identify the observation target location within the observation image field.

  As an example of the optical wavelength multi-point analyzing means, a plurality of optical fibers 17 are configured, the light captured by each optical fiber 17 is decomposed by a plurality of spectrometers, and a plurality of positions corresponding to the number of optical fibers 17 are provided. Can be observed.

(Embodiment 5)
The same parts as those in the first to fourth embodiments are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 12 is an example of a configuration diagram of the fifth embodiment. FIG. 12 shows a configuration in which an image recognition means 18 is added to the microorganism detection method shown in FIG.

  In the above configuration, the image recognition means 18 is provided as one of the image processing functions 8 in the microscope fluorescence atomic force microorganism detection means 15 as shown in FIG. Image recognition by the image recognizing means 18 is performed by using an image taken by the photographing means 7 and an image created from data measured by the atomic force microscope observing means 5 in advance in the image recognizing means 18. The data is registered, the registered microorganism image data, the image photographed by the photographing means 7 and the image created from the data measured by the atomic force microscope observation means 5 are compared, and the shapes match. Choose what to do as microorganisms. The image recognition means 18 includes a microcomputer programmed as described above. As described above, the use of the image recognition means 18 enables more accurate microorganism detection. The data registered in advance includes a microscope magnified observation image, a fluorescence observation image, an atomic force microscope observation image, and the like of a microorganism to be detected.

(Embodiment 6)
The same parts as those in the first to fifth embodiments are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 14 is an example of a configuration diagram of the sixth embodiment. FIG. 14 shows a configuration in which the microbe detection method shown in FIG. 5 includes the hardness measuring means 19 for measuring the hardness of the specimen and a charge measuring means 20 for measuring the presence or absence of the charge of the specimen or the strength of the charge. It is a thing.

  The hardness measuring means 19 presses and separates the probe 4 from the observation object, and the probe 4 applies the repulsive force applied to the probe 4 from the observation object when pressed, and the attractive force applied to the probe 4 when separated. It transmits to the observation means 5, and based on the data at that time, it is determined that the repulsive force is strong, it is determined that it is hard, and if the repulsive force is weak, it is determined that it is soft. Moreover, the viscosity of the surface of the observation object can be measured by the attractive force at the time of separation.

  Therefore, by measuring the hardness of the observed object, a hard object can be a foreign substance, and a soft object can be a microorganism, and the foreign object and the microorganism can be easily distinguished. In addition, by knowing the viscosity of the observation object, it can be determined whether there is any viscous material on the surface, and a specimen containing microorganisms that produce secretions can easily understand the presence of microorganisms.

  FIG. 15 is a graph of microbe hardness measurement. The X-axis is the amount of push-in, and the Y-axis is the repulsive force received from the microorganisms during pushing or pulling. FIG. 16 is a graph obtained by measuring the hardness of polystyrene as a foreign material. The X axis and Y axis are set in the same manner as in FIG. In FIG. 15, since the surface is soft, the repulsive force received by the probe is different between when it is pushed in and when it is pulled away. In FIG. 16, the surface is hard and the surface receives a certain force without being dented. Sometimes it becomes a graph of the same shape. In this way, the difference between microorganisms and foreign substances can be distinguished by hardness.

  The charge measuring means 20 measures the charge of the observation object by measuring the charge having the opposite polarity to the observation object generated by the electrostatic induction when the probe 4 approaches the observation object. It is possible to determine whether it is a microorganism or a foreign substance based on the value of. In this case, when the value of the charge is large, there is a high possibility that it is a foreign substance.

  Furthermore, the accuracy of determining microorganisms is further improved by comprehensively evaluating data on hardness, charge, and viscosity.

  In the above configuration, not only the outer shape and the surface shape, but also the hardness and charge of the specimen are measured, so that microorganisms can be detected with higher accuracy.

  In the sixth embodiment, both the hardness and electric charge of the specimen are measured, but it is also possible to detect microorganisms by measuring only one of the hardness and electric charge of the specimen.

  Moreover, it can be judged whether it is a microorganism or a foreign substance by measuring not only electric charge but also magnetism.

(Embodiment 7)
The same parts as those in the first to sixth embodiments are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 17 is an example of a configuration diagram of the seventh embodiment. FIG. 17 shows a configuration in which differential interference observation means 21 is provided in the microorganism detection method shown in FIG.

  In the above-described configuration, the differential interference observation means 21 is used after the microscope magnified observation to create two lights using the deflection of the light, and the differential interference observation is performed using the interference. By performing differential interference observation after microscope magnification observation, the microscope observation means 3 can easily confirm a transparent specimen that is difficult to confirm, and can detect microorganisms with higher accuracy. The differential interference observation means 21 is for observing by replacing the difference in the height of the sample with a difference in color, making two lights using the deflection of the light, and observing using the interference, and is transparent. Such as differential interference microscopes.

  The microorganism detection method of the present invention has an effect that it is possible to detect whether a microorganism is a foreign substance more quickly or accurately when the microorganism is measured using a microscope, fluorescence observation, or an atomic force microscope. Or it is useful as a highly accurate detection method.

Configuration diagram of an example of the microorganism detection method of Embodiment 1 of the present invention Configuration diagram of an example of the microorganism detection method Configuration diagram of an example of the microscopic atomic force microorganism detecting means Configuration diagram of an example of the microorganism detection method of Embodiment 2 of the present invention Configuration diagram of an example of the microorganism detection method Configuration diagram of an example of the fluorescent atomic force microorganism detecting means The block diagram of an example of the microorganisms detection method of Embodiment 3 of this invention Configuration diagram of an example of the microorganism detection method Configuration diagram of an example of means for detecting fluorescence atomic force microorganisms of the microscope Configuration diagram of an example of the microorganism detection method of Embodiment 4 of the present invention Configuration diagram of an example of the microorganism detection method Configuration diagram of an example of the microorganism detection method of Embodiment 5 of the present invention Configuration diagram of an example of the image recognition means Configuration diagram of an example of the microorganism detection method of Embodiment 6 of the present invention Hardness measurement graph of the microorganism Hardness measurement graph of polystyrene Configuration diagram of an example of the microorganism detection method of Embodiment 7 of the present invention Configuration diagram of the conventional microorganism detection method

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Specimen 2 Objective lens 3 Microscope observation means 4 Probe 5 Atomic force microscope observation means 6 Microscope atomic force isotope observation means 7 Imaging means 8 Image processing function 9 Data processing function 10 Microscope atomic force microorganism detection means 11 Fluorescence observation Means 12 Fluorescence atomic force isotope observation means 13 Fluorescence atomic force microorganism detection means 14 Microscope fluorescence atomic force isotope observation means 15 Microscope fluorescence atomic force microorganism detection means 16 Optical wavelength analysis means 17 Optical fiber 18 Image recognition means 19 Hard Measuring means 20 charge measuring means 21 differential interference observation means

Claims (17)

A microscope observing means, an atomic force microscope observing means, a microscope atomic force isotope observing means that makes the position observed by the microscope observing means and the position observed by the atomic force microscope observing means the same, and a microscope Microorganism detection comprising imaging means for imaging the observed state and microscopic atomic force microbe detection means for analyzing microorganisms by analyzing information obtained from microscopic observation images and information obtained from atomic force observation microscopic observation means Method. Fluorescence observation means, fluorescence atomic force isotope observation means for making the position observed by the fluorescence observation means and the position observed by the atomic force microscope observation means the same, and fluorescence for imaging the state observed by the fluorescence observation means A microorganism detection method comprising: an image photographing unit; and a fluorescent atomic force microorganism detection unit that analyzes information obtained from a fluorescence observation image and information obtained from an atomic force microscope observation unit to detect a microorganism. Observation with the microscope observation means, the fluorescence observation means, the atomic force microscope observation means, the position observed with the microscope observation means, the position observed with the fluorescence microscope observation means, and the atomic force microscope observation means Microscope atomic force isotope observation means to make the same position, imaging means for imaging the state observed with a microscope, information obtained from a microscope observation image, information obtained from a fluorescence observation image, and atomic force observation A microorganism detection method comprising a microscope fluorescence atomic force microorganism detection means for analyzing information obtained from a microscope observation means and detecting microorganisms. 2. The microorganism detection method according to claim 1, wherein the microscopic atomic force microorganism detecting means detects microorganisms in both the outer shape or surface shape of the observation object or the outer surface shape. The microorganism detecting method according to claim 2, wherein the fluorescent atomic force microorganism detecting means detects the fluorescence intensity, the outer shape, the surface shape, or both of the outer surface shape. 4. The microorganism detecting method according to claim 3, wherein the microscopic fluorescent atomic force microorganism detecting means detects the fluorescence intensity, the outer shape, the surface shape, or both of the outer surface shape. A light wavelength analyzing means for analyzing a spectrum of a light wavelength emitted from an observation point observed by the microscope observing means and the fluorescence observing means; information obtained from the microscope observing means; and fluorescence emission obtained from the fluorescence observing means The microorganism detection method according to any one of claims 1 to 6, wherein a microorganism is detected based on information, information obtained from the atomic force microscope observation means, and light wavelength analysis information obtained from the light wavelength analysis means. The microorganism detection method according to claim 7, further comprising: a light wavelength multi-site analyzing unit having a plurality of light wavelength analyzing units as analysis sites. The microorganism detection method according to claim 1, wherein the photographing means is a monochrome CCD camera. The microorganism detecting method according to claim 1, wherein the photographing means is a color CCD camera. The image recognition means for collating the image observed by the microscope observation means, the fluorescence observation means, the light wavelength analysis means, and the atomic force microscope observation means with a specific image. Microbial detection method. The microorganism detection method according to any one of claims 1 to 11, further comprising hardness measurement means for measuring the hardness of the specimen. The microorganism detection method according to any one of claims 1 to 12, further comprising charge detection means for detecting the presence or absence or the strength of the charge of the specimen. The microorganism detection method according to claim 2, wherein the fluorescent staining reagent is a reagent for staining living microorganisms. The microorganism detection method according to claim 2, wherein the fluorescent staining reagent is a reagent for staining dead microorganisms. The microorganism detection method according to claim 2, wherein the fluorescent staining reagent is a reagent for staining all microorganisms. The microorganism detection method according to any one of claims 1 to 16, further comprising differential interference observation means for producing two lights using light deflection and observing the two lights using the interference.

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