JP2006333812A - Method for metering microorganism and apparatus for metering microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting microbial cells in a specimen containing or possibly containing microbial cells by using a staining reagent and effective for reducing the under-estimation of the detection of live cells and improving the accuracy compared with conventional method and provide a microorganism metering apparatus. <P>SOLUTION: The invention provides a microorganism metering method having reduced under-estimation of live cells caused by fluorescent cross talk and a microorganism metering apparatus 1 by making a live and dead cell reagent to the long-wavelength side relative to a dead cell reagent. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention is a method for detecting a microorganism using a fluorescent dye with respect to a specimen containing or possibly containing a microorganism, and reduces the noise and improves the accuracy as compared with a conventionally known method. It relates to a method and a microbial metering device that can be increased.

  Conventionally, as a means for detecting a microorganism using a fluorescent dye, the luminescence obtained by staining the microorganism with a fluorescent dye for staining live bacteria and a fluorescent dye for staining dead bacteria as disclosed in Patent Document 1 There has been proposed a method for detecting viable and dead bacteria from the ratio of the fluorescence intensities in two kinds of fluorescence wavelength ranges emitted by an object. This is because the fluorescent dye that stains live bacteria is green fluorescence derived from a dye that stains microorganisms with esterase activity (for example, calcein AM), and the fluorescent dye that stains dead bacteria is red fluorescence derived from propidium iodide. It is detected by flow cytometry, and the intensity of green fluorescence and the intensity of red fluorescence of the luminescent material are detected, respectively, and it is determined from the fluorescence intensity ratio whether the luminescent material is live or dead.

  Further, as a means for detecting a microorganism using double staining of a fluorescent dye, a microorganism detection method using a nucleic acid staining dye that selectively prepares membrane permeability of a cell membrane as shown in Non-Patent Document 1. Has been proposed. This is due to a dye that is an example of a monomethine cross-linked asymmetric cyanine dye that is selectively permeable to the cell membranes of live and dead bacteria, and propidium iodide that is impermeable to the cell membrane of live bacteria. However, as shown in the texts on pages 626 and 627 and FIG. 15.39, a liquid obtained by sterilizing a cell membrane of a living Escherichia coli using isopropyl alcohol with a living Escherichia coli A sample obtained by mixing and staining a sample at an appropriate ratio is obtained by measuring a fluorescence spectrum with a fluorescence spectrophotometer.

According to this method, the green fluorescence due to the fluorescence wavelength of the monomethine-bridged asymmetric cyanine dye decreases with the proportion of viable bacteria contained, while the red fluorescence due to propidium iodide increases. By this method, live bacteria and dead bacteria can be labeled.
JP-A-11-146798 Richard P. Haugland "Handbook Of Fluorescent Probes And Research Products Ninth Edition", published by Molecular Probes, 2002, 626-627

  However, the microbiological weighing method described in Patent Document 1 and Non-Patent Document 1 does not always provide a satisfactory result. In both cases, since the fluorescence wavelength of live or dead bacteria is green and the fluorescence wavelength of dead bacteria is red, a part of the fluorescence spectrum of green fluorescence is red on the long wavelength side due to the characteristics of the fluorescent dye. A phenomenon that overlaps with the wavelength range of fluorescence, so-called fluorescence crosstalk, is seen, and even with only green fluorescence, red fluorescence may be detected by mistake, resulting in underestimating the number of viable bacteria, This is because the number of viable bacteria may not be accurately measured. In addition, depending on the difference in sensitivity to the staining reagent for each microorganism, when a certain type of microorganism has a strong coloration on one staining reagent, such a reflection is likely to be seen, and in unknown samples, When the types are mixed, it is difficult to obtain the measurement accuracy of viable bacteria, and there is a possibility that a stable viable cell detection result cannot be obtained.

  The present invention solves such a conventional problem, and is a method for detecting a microorganism using a fluorescent dye from a specimen containing or possibly containing a living bacterium. It is an object of the present invention to provide a method and a microorganism detection apparatus capable of accurately detecting viable bacteria while eliminating the adverse effects caused by reflection, such as the existing methods.

  In order to achieve the above object, the microorganism detection method of the present invention stains a microorganism with a staining reagent as described in claim 1 and detects the stained microorganism, and reacts with any of live and dead bacteria. A first staining reagent that develops color of both living and dead bacteria and a second staining reagent that reacts only with dead bacteria and develops color of only dead bacteria at a wavelength different from the color development are simultaneously brought into contact with the microorganism. The staining reagent is characterized in that it develops color at an excitation wavelength longer than that of the second staining reagent, and simultaneously detects both live and dead bacteria from the difference in wavelength and the amount of color development. By making the long wavelength side affected by the reflection of the living organism a dye for viable and dead bacteria, underestimation of the viable count can be prevented, the viable count detection accuracy can be improved, and a stable measurement result can be obtained.

  The microorganism weighing method according to claim 2 is characterized in that in the microorganism weighing method according to claim 1, a specimen containing microorganisms is captured on a filter, and the microorganisms on the filter are detected. By using a method called filtration, not only can the measurement range be constant regardless of the amount of sample, but also the effects of staining-inhibiting components contained in the sample can be removed, enabling stable measurement. it can.

  The microorganism weighing method according to claim 3 is the microorganism weighing method according to claim 1 or 2, wherein the first staining reagent and the second staining reagent are dropped on a filter capturing bacteria or microorganisms. It is characterized by filtering, and the staining process can be carried out simply and quickly.

  The microorganism weighing method according to claim 4 is the microorganism weighing method according to any one of claims 1 to 3, wherein a drying inhibitor is mixed in the first staining reagent and the second staining reagent. Therefore, it is possible to reduce the measurement instability due to the fading of the dye and to maintain high detection accuracy.

  Further, the microorganism measuring device according to claim 5 is different from the first coloring reagent that reacts with any of the living and dead bacteria to develop color of both the living and dead bacteria and only the dead bacteria that react with only the dead bacteria. A second staining reagent that develops color at the same time is brought into contact with a microorganism at the same time, and the first staining reagent develops color at an excitation wavelength longer than that of the second staining reagent. It is a device for detecting both germs and dead bacteria at the same time. It is equipped with a light source and a light receiving means. By detecting the wavelength and color development amount of the staining reagent using a configuration including the above, a highly sensitive metering device can be provided.

  The microorganism weighing device according to claim 6 is characterized in that, in the microorganism weighing device according to claim 5, the second staining reagent is colored by blue excitation light, and has an SN ratio with autofluorescence. By using a blue excitation wavelength region having a high value, microorganisms can be weighed with high sensitivity.

  A microorganism measuring device according to claim 7 is the microorganism measuring device according to claim 5 or 6, wherein the first staining reagent and the second staining reagent are nucleic acid binding compounds. In addition, since there is little difference in sensitivity due to microbial species such as enzyme activity, stable measurement can be provided without underestimating unknown samples.

  Further, the microorganism weighing device according to claim 8 is the microorganism weighing device according to any one of claims 5 to 7, which is excited in a predetermined wavelength range in a predetermined area on the filter surface on which the stained microorganism is captured. One or a plurality of light sources for irradiating light, light receiving means for receiving light in a predetermined wavelength range that is emitted by the excitation light at a part or all of a certain area of the filter surface, and the light source A microorganism judging means for receiving the emitted and emitted light within a set time, and judging that the received light quantity is within a set threshold value range and within a set area range; From the microorganism judging means, the moving means for moving the filter, the light source or the light receiving means continuously or intermittently to move the area necessary for the measurement of the filter surface. It is characterized by having means for accumulating the number of microorganisms in the specimen contacting means by judging that the size is determined to be 2 to 7 μm as one microorganism and sequentially accumulating the judged signals. It is possible to simultaneously and quickly grasp the life and death state and / or number or presence or absence of microorganisms contained on the filter, and minimize the irradiation area and time of the excitation light to minimize the state of the compound by the excitation light. It can suppress fading. Furthermore, by providing a threshold value of fluorescence intensity, it is possible to distinguish between microorganisms and foreign substances, and a microorganism measuring device capable of quickly and simultaneously measuring the number of microorganisms contained in a certain area is obtained.

  The microorganism measuring device according to claim 9 is the microorganism measuring device according to any one of claims 5 to 8, wherein the color is developed by the staining reagent and is set out of the coloring points determined to be microorganisms by the microorganism judging means. It is characterized by judging the size based on the threshold value, and classifying the type of microorganisms by size, and by counting for each type of bacteria, such as bacteria and yeast, It becomes possible to obtain more detailed information at a time, and the time required for the inspection required for each bacterial species can be shortened and the cost can be reduced.

  Further, the microorganism weighing device according to claim 10 is the microorganism weighing device according to any one of claims 5 to 9, wherein among the one or more coloring points determined to be microorganisms by the microorganism determining means, The light emitting point colored by the staining reagent has a judging means that uses the light emitting point as viable bacteria when not colored by the second staining reagent. By collating the light emission points, the instability of the number due to the addition of noise included in each wavelength region can be reduced, and the detection accuracy can be increased.

  The microorganism measuring device according to claim 11 is the microorganism measuring device according to any one of claims 5 to 10, wherein the first color among the one or more coloring points determined to be microorganisms by the microorganism determining means. In the apparatus for judging the luminescent point as a viable cell when the luminescent point colored by the staining reagent is not colored by the second staining reagent, the difference between the coordinates of the chromogenic point by the first and second staining reagents Are made to coincide with each other by giving a correction value, and a mechanical error existing between two images including the light emission points of the first and second staining reagents, respectively. As a means for correcting the error in coordinates derived from the above, after detecting the light emitting points of the image, by comparing them, the optimum correction value can be calculated and the light emitting points can be collated. High precision with a simple device configuration It is possible to realize a Do measurement.

  Further, the microorganism weighing device according to claim 12 is the microorganism weighing device according to any one of claims 5 to 11, wherein in the microorganism weighing device, a difference in coordinates of the coloring point by the first and second staining reagents is determined. When the correction value is given to either one, the correction value is calculated first, and then the image is formed after giving this value, and only the minimum processing time required for collation of the light emission point is used. Thus, high-precision measurement can be performed, and quick measurement can be realized.

  Further, the microorganism weighing device according to claim 13 is the microorganism weighing device according to any one of claims 5 to 10, wherein the first of the coloring points determined to be microorganisms by the microorganism determining means is the first color. In an apparatus for determining a color development point developed by a staining reagent as a living microbe when the color development point is not colored by the second staining reagent, a difference in coordinates of the color development point by the first and second staining reagents It has optical means to reduce the image, and by providing a mechanism to reduce the error mechanically, the image processing can be simplified, and the time required for image processing and the time for developing image processing software is shortened. Can be speeded up and developed at low cost.

  Further, the microorganism measuring device according to claim 14 is the microorganism measuring device according to any of claims 5 to 10 or 13, wherein the first of the coloring points determined to be microorganisms by the microorganism determining means. In the apparatus for judging the luminescent point as a living microbe when the luminescent point developed with one staining reagent is not colored with the second staining reagent, means for expanding the luminescent point on the filter surface and a parallel light beam The optical means for obtaining is provided, and the spectroscopic means for splitting light in a predetermined wavelength range is provided in the parallel light flux region, thereby reducing the difference in the coordinates of the coloring point due to the first and second staining reagents. It does not require time for image processing for collation of the light emission points derived from the first staining reagent and the second staining reagent, and can be measured in a shorter time and with higher accuracy. To realize It can be.

  According to the microorganism weighing method and the microorganism weighing device of the present invention, the first staining reagent is the second staining reagent in the first staining reagent for staining live and dead bacteria and the second staining reagent for staining dead bacteria. By using the longer wavelength side of the staining reagent, it is possible to reduce the detection accuracy due to reflection of the fluorescence intensity of the short wavelength side to the long wavelength side, without underestimating the number of viable bacteria. A microorganism measuring method and a microorganism measuring apparatus capable of obtaining a stable measurement result by measurement can be provided.

  According to the first aspect of the present invention, in the method of staining a microorganism using a staining reagent and detecting the stained microorganism, the first method of reacting with any of the live and dead bacteria to develop any of the live and dead bacteria A staining reagent and a second staining reagent that reacts only with dead bacteria and causes only the dead bacteria to develop color at a wavelength different from that of the color development are simultaneously brought into contact with the microorganism, and the first staining reagent is obtained from the second staining reagent. Is characterized by the fact that it produces color at a long excitation wavelength, and simultaneously detects both live and dead bacteria from the difference in wavelength and amount of color development. In consideration of the effect of reflection on the side, a dead-bacteria staining reagent is used on the short wavelength side and a viable-bacteria staining reagent is used on the long wavelength side. In this way, by directing the fluorescence emission of dead bacteria to the live and dead bacteria side, it is possible to give accuracy to distinguish between live and dead bacteria, and fundamentally eliminate the influence on the count of bacteria. , Has the effect that the weighing is stabilized.

  The invention described in claim 2 is characterized in that a specimen containing microorganisms is captured on a filter and the microorganisms on the filter are detected, and the range of the amount that can be filtered by the microorganism capturing filter Can be measured without change, and the influence of staining inhibition components contained in the sample can be removed, so that stable staining can be performed, and stable sampling and staining methods can be obtained. , It has the effect that stable measurement can be performed.

  The invention according to claim 3 is characterized in that the first staining reagent and the second staining reagent are dropped on a filter capturing bacteria or microorganisms and then filtered, and the staining step By providing a general-purpose method that anyone can easily handle, this method can be widely used.

  The invention according to claim 4 is characterized in that the first staining reagent and the second staining reagent are mixed with an anti-drying agent to prevent discoloration due to drying of the dye. It is possible to reduce the fluctuation of the luminescence intensity caused by the microorganism, reduce the instability of detection and measurement of the microorganism, and obtain high detection accuracy.

  The invention according to claim 5 is the first staining reagent that reacts with any of the live and dead bacteria to develop color of both the live and dead bacteria, and only the dead bacteria that react only with the dead bacteria and have a wavelength different from that of the color development. The second staining reagent to be colored is brought into contact with the microorganism at the same time, and the first staining reagent develops a color with an excitation wavelength longer than that of the second staining reagent. It is a device for simultaneous detection of both bacteria, and it is characterized by providing a light source and a light receiving means, and measuring the microorganisms from the wavelength difference of the colored microorganisms and the amount of fluorescent coloration, distinguishing the microorganisms with high accuracy, It can measure and has the effect | action that a small and low-cost apparatus can be provided by setting it as the minimum required structure.

  The invention according to claim 6 is characterized in that the second staining reagent is colored by blue excitation light, and has a high S / N ratio with autofluorescence compared to ultraviolet excitation or the like. By using the wavelength band, it has an effect that highly sensitive weighing can be performed. Even if the first staining reagent is set on the long wavelength side, it can be in the visible light range of orange to red, and it is easy to give a wavelength difference from the staining reagent for blue excitation, It has the effect | action that discrimination | determination can be performed with sufficient precision.

  The invention according to claim 7 is characterized in that the first and second staining reagents are nucleic acid binding compounds, and uses an enzyme retained in a cell as an index. As described above, since there is little difference in susceptibility due to microbial species and the environment, there is an effect that staining can be performed stably without underestimating unknown samples.

  Further, the invention according to claim 8 is characterized in that one or a plurality of light sources for irradiating excitation light in a predetermined wavelength range in a predetermined area on a filter surface on which stained microorganisms are captured, and a fixed surface of the filter surface. A light receiving means for receiving light in a predetermined wavelength range emitted by the excitation light in part or all of the area, and receiving the light emitted by the light source within a set time, Microorganism judging means for judging that the received light quantity is within a set threshold value range and within a set area range, and the filter or light source or light receiving means for the fixed area continuously or intermittently. A moving means that moves the area necessary for the measurement of the filter surface, and a microorganism that has been determined by the microorganism judging means to be 0.2 to 7 μm in size is determined as one microorganism, Is characterized by having means for integrating the number of microorganisms of the specimen contact means by sequentially integrating the signals determined in the above, and at the same time the state of life, death or number of microorganisms contained on the filter, And it has the effect | action that it can measure rapidly. In addition, by suppressing the light fading of the staining reagent due to the excitation light by minimizing the irradiation area and time of the excitation light, the light emission intensity can be detected stably. Furthermore, by providing a threshold value of the fluorescence intensity, a method for distinguishing microorganisms from foreign substances is provided, and an apparatus for accurately and quickly measuring microorganisms contained in a certain area can be provided.

  In the invention according to claim 9, the color is developed by the staining reagent, and the size is determined by a set threshold value among the coloring points determined to be microorganisms by the microorganism determination means, and the microorganisms are determined by the size. It is characterized by classifying types, and detailed information can be obtained at once by counting for each type of bacteria, such as bacteria and yeast, with more complicated information. It has the effect that it can be provided. In addition, since the inspection time required for each bacterial species can be shortened, the measurement time can be shortened and the cost of the measurement can be reduced.

  Further, in the invention described in claim 10, among the one or more coloring points determined to be microorganisms by the microorganism determining means, the luminescent point colored by the first staining reagent is colored by the second staining reagent. And a means for judging that the luminescence point is a living microbe when the luminescence point is not, and by the means for comparing the first and second luminescence points, what kind of luminescence each microorganism has Can be accurately detected, and the instability of the number due to the addition of noise included in each wavelength region can be reduced, and the detection accuracy can be increased.

  The invention according to claim 11 is such that, among the one or more coloring points determined to be microorganisms by the microorganism determining means, a light emitting point colored by the first staining reagent is colored by the second staining reagent. In the apparatus for judging the luminescent point as a living bacterium when not, the difference in coordinates of the coloring point by the first and second staining reagents is matched by giving a correction value to one of them. The measurement of the luminescence state of each microorganism by the deviation of coordinates derived from mechanical errors existing between two images including the luminescence points of the first and second staining reagents. Because it is a software process that can solve difficult factors, it is possible to accurately check the light emission point without using a large-scale weighing device, and it is highly accurate with a small, low-cost device configuration. Realizing measurement It has the effect of that.

  According to the twelfth aspect of the present invention, in the microorganism weighing apparatus, when a correction value is given to either one of the differences in the coordinates of the coloring point by the first and second staining reagents, the correction value is calculated first. However, it is characterized in that it is imaged after giving this value, and the processing time required for collation of light emission points is shortened by enlarging the image information by obtaining the coordinates of all the images. And has the effect that it can be performed quickly with high accuracy.

  Further, the invention according to claim 13 is that the coloring point developed with the first staining reagent among the one or more coloring spots determined as microorganisms by the microorganism judging means is colored with the second staining reagent. In the apparatus for judging the color development point as a living microbe when it is not, it has an optical means for reducing the difference between the coordinates of the color development point by the first and second staining reagents, and the error is reduced on the apparatus side. By providing such a mechanism, image processing by software can be simplified, the time required for image processing can be shortened, and measurement can be performed quickly. In addition, since the time for developing image processing software can be shortened, the development cost can be reduced.

  Further, in the invention described in claim 14, a light emitting point that is colored by the first staining reagent among the one or more coloring points determined to be microorganisms by the microorganism judging means is colored by the second staining reagent. An apparatus for judging the light emission point as a living microbe when it is not equipped with means for enlarging the light emission point on the filter surface and optical means for obtaining a parallel light beam, and splits light in a predetermined wavelength range And the first and second staining reagents are characterized in that the difference in the coordinates of the coloring points by the first and second staining reagents is reduced by providing a spectroscopic means in the parallel light flux region. Therefore, it does not require time for image processing for collating the light emission points included in each image according to the above, and it is possible to perform highly accurate measurement more quickly.

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

(Embodiment 1)
First, a liquid sample is sucked and filtered from above a microorganism collection filter that is a specimen contact means, and microorganism cells are captured two-dimensionally on the filter surface. In the present invention, the specimen containing or possibly containing the bacterium is a liquid specimen, but when the test object is a liquid sample such as drinking water, the specimen itself is a liquid specimen. If the test object is a solid sample such as vegetables or meat, homogenize it to make a liquid sample, or collect bacteria from the surface using a cotton swab, etc., and use it for physiological saline or phosphate buffer. Release into liquid etc. to make liquid specimen. When a cooking utensil such as a cutting board is an object to be examined, bacteria are collected from the surface using a cotton swab or the like, and are released into physiological saline or the like to form a liquid sample. Bacteria can be captured on the filter by suction-filtering such a liquid sample with a filter for collecting microorganisms.

  Next, an anti-drying component is mixed, and a fixed amount of about 0.1 to 1 ml of a staining reagent containing a constant concentration of either a living or dead bacteria staining reagent or a dead bacteria staining reagent or both is dropped onto the filter surface.

  The fluorescent staining reagent is preferably a nucleic acid-binding compound, and as a viable and dead bacteria reagent, emits orange to red fluorescence. Trimethine cross-linked asymmetric cyanine dye-based compounds can be used. In addition, as the killed bacteria reagent, those emitting blue to green fluorescence, for example, monomethine cross-linked asymmetric cyanine dye dimers such as acridine dimer, thiazole orange dimer, oxazole yellow dimer, SYTOX Green, TO Monomethine cross-linked asymmetric cyanine dye compounds such as -PRO-1 can be used.

  Two or more different staining reagents should be mixed in advance to a sample containing microorganisms so that the concentration is 0.1 to 100 μM and allowed to act simultaneously, or separately, without taking time or as appropriate. It is assumed that the action is performed with a long time interval.

  As a means for preventing the surface of the substance containing the bacteria captured on the filter for collecting microorganisms from drying out during measurement and changing the luminescence intensity, 10 to 60% w / v glycerol or 1 to 10% w / v polyvinyl alcohol, 10 to 90% v / v propylene glycol or ethylene glycol, or one or more components such as D (-)-mannitol and D-sorbitol are mixed. deep.

  As the microorganism collection filter, for example, a known filter having a pore size of 0.2 μm to 1 μm can be used.

  Using a condensing lens or the like, the filter on which the fluorescent bacteria are spread is irradiated with excitation light so that the irradiation range is a constant area with a diameter of about 3 mm, for example, to obtain a light emission point.

  For example, in the case of a trimethine cross-linked asymmetric cyanine dye, when an excitation light having a wavelength of about 540 nm to 610 nm is irradiated, fluorescence having a wavelength of about 560 nm to 630 nm is emitted. When excitation light having a wavelength of about 480 nm to about 510 nm is irradiated, fluorescence having a wavelength of about 510 nm to about 540 nm is emitted.

  Therefore, in the case of a light emitting diode as an excitation light source, a blue light source preferably emits a wavelength near 480 nm, a green light source preferably emits a wavelength near 535 nm, and a yellow light source Preferably, those capable of emitting a wavelength near 560 nm are used.

  In the case of using a solid-state laser, a blue laser beam that can emit a wavelength of preferably around 475 nm is used, and a green laser beam that can emit a wavelength of preferably around 535 nm is used.

  When a halogen lamp or a mercury lamp is used, an optimum bandpass filter or the like according to the excitation wavelength of the staining reagent can be used as an appropriate spectral filter. In addition, the reflection angle or transmission type diffraction grating having a wavelength resolution of 0.1 to 10 nm can give an optimum angle and extract excitation light including an arbitrary wavelength.

  As the fluorescent filter, a green filter preferably has a transmission performance in the range from 510 nm to 540 nm, an orange filter preferably has a transmission performance in the range from 580 nm to 610 nm, and a red filter has a transmission performance in the range from 590 nm to 630 nm. Use ones with transmission performance in the range up to. The fluorescence is obtained by photographing an image with an exposure time of about 0.1 to 10 seconds using a CCD camera as a photoelectric conversion element through a magnifying lens system.

  The fluorescence images acquired by these operations include emission points derived from the fluorescence of viable bacteria. These emission points have appropriate emission intensity according to a threshold value set in advance by the microorganism judging means, and bacteria When setting based on the size of a microorganism such as a fungus or a fungus, a means that the diameter is determined to be 0.2 to 7 μm is extracted as one microorganism, and the number is added and measured To determine the number of microorganisms. Impurities other than microorganisms do not emit fluorescence but emit scattered light of excitation light and the like, and may be affected during light reception. Therefore, a case where the amount of received light shows a certain value or more is determined as a microorganism. Also, depending on the type of impurities, it may be assumed that the fluorescent light is emitted more than the microorganism, so that it is determined that the microorganism is within the set light amount range, and basically one light is received within the range. Weigh as microorganisms.

  It should be noted that the coloring of the staining reagent is made fluorescent so that it can be detected with high sensitivity, and even a minute sample can be detected with high sensitivity.

  These image acquisition operations are performed for each staining reagent, and acquire live and dead bacteria images and dead bacteria images, respectively, and are performed by continuously or intermittently moving the minute area irradiated with excitation light. . These movements are performed, for example, using moving means such as a combination of a motor, a belt, a gear, and a position sensor.

  Further, by arranging a plurality of photoelectric conversion elements included in the light receiving means in a linear shape, movement in the X-axis direction (arrangement direction) becomes unnecessary, and a certain range is achieved by movement only in the Y-axis direction (movement direction). Detection can be performed, and weighing can be performed quickly. The light receiving means is only required to receive light having a target wavelength, and includes a light receiving filter and a photoelectric conversion element.

  In addition, by comparing the luminescent spots included in the killed bacteria image at the same position as the obtained live and dead bacteria image, it is judged whether the luminescent spots are live bacteria or dead bacteria, and the respective numbers are obtained, integrated, and weighed. It will be.

  Thereby, the number of living microorganisms and the number of dead bacteria contained in the original microorganism sample can be measured.

  The compounds contained in the first and second staining reagents are selected as harmless as possible. This microbiological weighing device is often used mainly in food factories, etc. When the target of the sample is a cooking utensil or the like, even if it is harmless, contact with the sample contact means containing the reagent is a sanitary matter. It is not preferable. In addition, depending on the characteristics of the reagent, it may be desirable to react with the specimen immediately before irradiation with the excitation light. The compounds contained in the first and second staining reagents are separated from the cotton swab and the specimen contacting means. In some cases, it may be better to add to the filter immediately before measurement. Based on this viewpoint, for example, SYTO82, which is one of reagents that penetrate nonspecifically from the cell membrane surface of live and dead bacteria and specifically bind to nucleic acids present in the cells, is used as a stain for live and dead bacteria. Then, a method may be considered in which the microorganisms collected on the filter are infiltrated from a specimen reagent containing part provided separately, in contact with the filter part. The same applies to the case of double staining using SYTOX Green, which is one of the reagents that simultaneously penetrate nonspecifically from the cell membrane surface of dead bacteria and specifically bind to nucleic acids present in the cells. The life and death of the collected microorganisms can be determined by irradiating each of them with excitation light having a specific wavelength to cause color development.

  In addition, the signal obtained from the microorganisms contained on the filter is recognized as binarized point coordinates, and the point coordinates obtained by an external operation terminal used in connection with a microcomputer as a microorganism judging means are used. By detecting and accumulating the number, the microorganisms contained in the specimen contact means can be easily and quickly measured. Further, by recognizing adjacent signals as one signal, the correlation with the culture method can be enhanced. Furthermore, the shape of the adhering matter adhering to the specimen contact means can be grasped from the adjacent signal, and the shape other than the microorganism can be removed from the measurement, and the accuracy can be improved.

  FIG. 1 is a conceptual diagram showing an embodiment of a microorganism weighing apparatus for suitably carrying out the microorganism weighing method of the present invention. The microorganism weighing device 1 includes a light source 3, a condensing lens 5 as a light source condensing unit, and a light receiving unit 10. In order to extract the target wavelength from the excitation light emitted from the light source 3, the light is spectrally separated by the excitation light spectral filter 4. The split excitation light is condensed through a condenser lens 5 onto a microorganism collection filter 2 set on an examination table 6 (which captures bacteria stained on the filter by a separate operation). The excitation light emitted from the light source 3 is condensed by the condensing lens 5, and at this time, the range irradiated with the excitation light by the condensing lens 5 is condensed on a small fixed area. Here, the minute fixed area indicates a range of about 1 mm in diameter when set based on a microorganism having a size of 0.2 to 7 μm. The range in which the excitation light is irradiated (a small fixed area) can be moved continuously or intermittently on the filter by moving means including the motor 12 and the belt 13. The fluorescence emitted by the living bacteria by the excitation light reaches the light receiving unit 10. Further, the light reaches the photoelectric conversion element 9 built in the light receiving unit 10 through the fluorescence spectral filter 8 and the lens 7 for extracting only the target fluorescence, and is converted into a signal. The signal thus obtained is imaged and subjected to image processing by the computing unit 11 which is a microorganism judging means. The computing unit 11, that is, the microorganism judging means is a microcomputer programmed with image processing according to the procedure shown in detail in FIG. 2, and is used in communication with software operated by an externally connected terminal or the like. Also applicable.

  FIG. 2A is a live and dead bacteria image, FIG. 2B is a dead bacteria image, and FIG. 2C is a conceptual diagram showing the live and dead bacteria determination results. 14, 15, and 16 from the coloring points a to c included in the live and dead bacteria image, and 17, 18, and 19 from the coloring points A to C included in the dead bacteria image are shown in the figure. At this time, it is assumed that 14 and 16, 15 and 18, and 16 and 19 each have a coordinate deviation (x, y) as a value. At this time, the correction value is calculated as (−x, −y), and calculated to the coordinate values of 17, 18, and 19. As a result, it can be seen that 14, 15, 16 and 17, 18, 19 each show the same light emission point, and in the killed bacteria image, 18 light emission points with low emission luminance are viable bacteria, both of which emit light. 17 and 19 are detected as dead bacteria, and the number is obtained, integrated, and weighed. Thereby, the number of living bacteria and the number of dead bacteria can be obtained.

(Embodiment 2)
FIG. 3 is a conceptual diagram showing another aspect of the microorganism weighing apparatus for favorably implementing the microorganism weighing method of the present invention. The microorganism weighing device 20 includes a light source 3, a condensing lens 5 as a light source condensing unit, and a light receiving unit 10. In order to extract a target wavelength from the excitation light emitted from the light source 3, the light is split by the excitation light spectral filter 4. The excitation light is condensed through a condenser lens 5 onto a microorganism collection filter 2 set on an examination table 6 (which captures bacteria stained on the filter by a separate operation). The fluorescence emitted by the microorganisms by the excitation light is magnified through the lens unit 21 that is a magnification means, and becomes a parallel light flux while retaining the two-dimensional position information. Here, the fluorescence is imaged by the collimator lens 23 through the fluorescence spectroscopic filter unit 22 in order to extract only the target wavelength, and reaches the photoelectric conversion element 9 installed at the place to be signaled. The signal thus obtained is converted into an image, and the calculation unit 11 performs image processing to detect a light emitting point indicating light emission of live bacteria and dead bacteria, and measures the number. Thereby, the number of viable bacteria can be obtained.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is limited to the following description and is not interpreted at all.

Example 1: Microbial weighing method of the present invention One pre-cultured Escherichia coli was suspended in filter sterilized distilled water or the like to prepare a liquid sample. The liquid specimen was suction filtered using a manifold into a microorganism collecting membrane filter having a pore diameter of 0.45 μm, and Escherichia coli was captured on the filter.

  There is a first staining reagent containing SYTO82 at a concentration of 5 μM and a second staining reagent containing 5 μM SYTOX Green. Both of these were prepared in a mixed aqueous solution of 50% w / v glycerol. First, the first staining reagent is dropped on the membrane filter and stained at room temperature for 5 minutes. The excess reagent on the membrane filter is again filtered by suction to remove the liquid component on the filter, and then the second staining reagent is dropped on the membrane filter and stained at room temperature for 5 minutes. The remaining reagent on the membrane filter completely removes liquid components by suction filtration staining. The microorganism collection filter 2 is set on the inspection table 6 of the microorganism weighing device 1. When the operation command is given from the computing unit to the microorganism weighing device 1, the inspection table 6 on which the microorganism collection filter 2 is set is moved below the condenser lens 5, and the filter is irradiated with excitation light and the image is displayed. Taken.

  FIG. 4 shows the fluorescence spectrum 24 of the killing reagent of SYTOX Green, which is the killing reagent, and the fluorescence spectral filter transmittance 27 of the killing reagent, and the fluorescence spectrum 25 of the living, killing reagent of SYTO82, which is the killing reagent. The fluorescence spectrum filter transmittance 28 and the fluorescence intensity addition 26 to the living and dead bacteria reagent due to the fluorescence crosstalk of the death bacteria reagent, which is the addition of the fluorescence intensity of SYTOX Green reflected in the fluorescence wavelength of SYTO 82, are shown. In this way, it is possible to prevent the reflection of live and dead bacteria in the dead bacteria image, while in the live and dead bacteria image, the brightness of the reflected of the dead bacteria reagent is added to the brightness of the dead and dead bacteria reagent due to the reflection of the dead bacteria reagent. Accumulated and brightness increases.

  The present invention is a method for measuring viable and dead bacteria from a specimen containing microorganisms by using a staining reagent, and is a method for measuring microorganisms that is more accurate and stable as compared with conventionally known methods. The present invention has industrial applicability in that it can provide a method and a microorganism weighing device capable of performing the above.

The conceptual diagram which shows the microorganisms measuring device of Embodiment 1 of this invention Schematic diagram showing live / dead bacteria image, dead bacteria image, live bacteria, dead bacteria discrimination result ((a) live / dead bacteria image (b) same dead bacteria image (c) acquired by the microorganism weighing device of Embodiment 1 of the present invention ) Results of distinguishing between same and dead bacteria) The conceptual diagram which shows the microorganisms measuring device of Embodiment 2 of this invention The figure which shows the wavelength of the dyeing reagent of Example 1 of this invention, and a fluorescence spectroscopy filter

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microorganism measuring apparatus 2 Microorganism collection filter 3 Light source 4 Excitation light spectral filter 5 Condensing lens 6 Inspection table 7 Lens 8 Fluorescence spectral filter 9 Photoelectric conversion element 10 Light receiving part 11 Calculation part 12 Motor 13 Belt 14 Coloring point a
15 Coloring point b
16 Coloring point c
17 Coloring point A
18 Coloring point B
19 Coloring point C
DESCRIPTION OF SYMBOLS 20 Microorganism measuring device 21 Lens unit 22 Fluorescence spectral filter unit 23 Collimator lens 24 Fluorescence spectrum of dead bacteria reagent 25 Fluorescence spectrum of dead bacteria reagent 26 Fluorescence intensity addition to dead bacteria reagent by fluorescence crosstalk of dead bacteria reagent 27 Dead bacteria Fluorescence spectral filter transmittance of reagent 28 Fluorescent spectral filter transmittance of living and dead bacteria reagent

Claims (14)

In a method of staining a microorganism with a staining reagent and detecting the stained microorganism, the first staining reagent that reacts with any of the living and dead bacteria to develop a color of any of the living and dead bacteria and only reacts with the dead bacteria. A second staining reagent that causes only the dead bacteria to develop color at a wavelength different from that of the color development is simultaneously brought into contact with the microorganism, and the first staining reagent develops color at an excitation wavelength longer than that of the second staining reagent, and the wavelength difference therebetween. And a method for measuring microorganisms, wherein both viable and dead bacteria are simultaneously detected from the difference in color development amount. The method for measuring microorganisms according to claim 1, wherein a specimen containing microorganisms is captured on a filter, and the microorganisms on the filter are detected. 3. The method for measuring microorganisms according to claim 1, wherein the first staining reagent and the second staining reagent are dropped on a filter capturing bacteria or microorganisms and then filtered. The method for measuring microorganisms according to any one of claims 1 to 3, wherein an anti-drying agent is mixed in the first staining reagent and the second staining reagent. A first staining reagent that reacts with any of the living and dead bacteria and develops both the living and dead bacteria and a second staining reagent that reacts only with the dead bacteria and develops only the dead bacteria with a wavelength different from the above-described coloring An apparatus for bringing a first staining reagent into color at an excitation wavelength longer than that of the second staining reagent, and simultaneously detecting both viable and dead bacteria from the difference in wavelength and the amount of color development, in contact with a microorganism A microorganism measuring apparatus that includes a light source and a light receiving means, and measures microorganisms from the wavelength difference of the colored microorganisms and the amount of fluorescent coloration. 6. The microorganism measuring apparatus according to claim 5, wherein the second staining reagent is colored by blue excitation light. The microorganism weighing device according to claim 5 or 6, wherein the first staining reagent and the second staining reagent are nucleic acid binding compounds. One or a plurality of light sources that irradiate excitation light in a predetermined wavelength range with a predetermined area on a filter surface on which stained microorganisms are captured; and a part or all of the constant area of the filter surface A light receiving means for receiving light in a predetermined wavelength range that is emitted by excitation light, and a light that is emitted by the light source to emit light and is received within a set time, and the received light quantity is a set threshold value And a microbe judgment means for judging that the microbe is within a set area, and the filter, the light source or the light receiving means is moved continuously or intermittently over the certain area, and the measurement of the filter surface is performed. A moving means for moving the area necessary for the measurement and a microorganism judged by the microorganism judging means to be 0.2-7 μm are judged as one microorganism, and the judged signals are integrated. Microorganisms measuring device according to any of claims 5-7 having integrating means for integrating the quantity of microorganisms of the specimen contact means in Rukoto. 6. The color is determined by a staining reagent, the size is determined by a set threshold value among the coloring points determined to be microorganisms by the microorganism determination means, and the type of microorganism is classified by the size. The microbial measuring apparatus in any one of -8. Among the coloring points developed by one or more staining reagents determined to be microorganisms by the microorganism judging means, the coloring point when the coloring point developed by the first staining reagent is not colored by the second staining reagent. The microorganism weighing device according to any one of claims 5 to 9, wherein the microorganism is determined to be a living bacterium. Among the one or more coloring points determined to be microorganisms by the microorganism judging means, when the coloring point developed by the first staining reagent is not colored by the second staining reagent, the coloring point is judged as a living microbe. 11. The apparatus according to claim 5, wherein the difference in coordinates of the coloring point between the first staining reagent and the second staining reagent is made coincident by giving a correction value to one of them. The microorganism measuring device according to 1. In the microorganism weighing apparatus, when a correction value is given to either one of the differences in the coordinates of the coloring point between the first staining reagent and the second staining reagent, the correction value is calculated first, and the image is obtained after giving this value. The microorganism weighing device according to any one of claims 5 to 11, wherein Among the one or more coloring points determined to be microorganisms by the microorganism judging means, when the coloring point developed by the first staining reagent is not colored by the second staining reagent, the coloring point is judged as a living microbe. The microorganism weighing device according to any one of claims 5 to 10, further comprising an optical unit that reduces a difference in coordinates of the coloring point between the first staining reagent and the second staining reagent. Among the one or more coloring points determined to be microorganisms by the microorganism judging means, when the coloring point developed by the first staining reagent is not colored by the second staining reagent, the coloring point is judged as a living microbe. In the apparatus to be provided with means for enlarging the color development point on the filter surface and optical means for obtaining a parallel light beam, and provided with a spectroscopic means for dispersing light in a predetermined wavelength region in the parallel light beam region, 14. The microorganism weighing apparatus according to claim 5, wherein a difference in coordinates of the coloring point between the first staining reagent and the second staining reagent is reduced.

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