JP2006284604A - Filter for collecting microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To microorganisms from for efficiently collecting an object to be inspected, and accurately detect and weigh the collected microorganisms. <P>SOLUTION: A microorganism collecting chip comprises a filter for removing foreign substances and a filter for collecting microorganisms. A microorganism collecting kit comprises the microorganism-collecting chip and a suction filtration means. The suction filtration means is a negative pressure tube provided with a rubber plug at an opening. The microorganism-collecting chip has a liquid specimen injected vessel into which a liquid specimen is injected, and a hollow needle for penetrating the rubber plug provided at the opening of the negative pressure tube. The liquid specimen is injected into the liquid specimen injected vessel, and sucked and filtrated by a pressure in the negative pressure tube. The foreign substance is removed by the foreign substance removing filter. The microorganisms are collected on the microorganism collecting filter, detected and weighed by using a unit including the microorganism collecting filter. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention is a means for collecting microorganisms adhering to a test object, and these are colored, luminescent and fluorescent, and efficiently collect and detect live and dead cells or specific microorganisms present in the test object. The present invention relates to a filter for collecting microorganisms for measurement.

  Conventionally, as this kind of microorganism collecting means, microorganisms are filtered from a liquid sample that can contain microorganisms by pressure change due to operation of a pump and collected on a filter, and then the unit is disassembled to be fine like tweezers. There was one that collected and measured the filter to which the collected microorganisms were attached using a tool capable of performing work.

  Such a conventional microorganism collecting means requires a large device such as a pump for collecting microorganisms. Further, in the case of a sample that is directly affected by foreign matter, a foreign matter removal process is required separately. Furthermore, it is necessary to collect only the filter in order to inspect the microorganisms collected on the filter. Since this work requires tools for performing fine work such as tweezers, the efficiency is lowered and a large amount of samples cannot be processed. Furthermore, since a pump is used, the equipment becomes large, and this is not possible in a place with a small work space such as a food inspection process. These require a certain amount of testing time and technique in microbial testing that requires rapidity. Therefore, there is a problem that not everyone can easily and quickly carry out.

The present invention solves such a conventional problem, and by using a dark filter as a microorganism collection filter, it is possible to accurately measure microorganisms while suppressing light emission in the background (background). The object is to provide a microbe collection chip.

  It is another object of the present invention to provide a microorganism collection chip capable of accurately measuring microorganisms by suppressing the light emission of the background (background) by forming a thin film made of certain components on the microorganism collection filter. It is said.

  Moreover, it aims at providing the microbe collection chip | tip which makes the effect of forming a thin film more reliable by making the film thickness of a thin film into an appropriate value.

In order to achieve the above object, the present invention is characterized in that in a microorganism collection filter for collecting microorganisms, the spectral reflectance of the filter is lowered and background light emission is suppressed .

Further, the microorganism collection filter is a dark filter. And according to this invention, it can suppress effectively that a background (background) light-emits and inhibits accurate microorganism measurement.

In addition, a thin film containing at least one metal component selected from gold, copper, chromium, platinum and palladium is formed on the filter. And according to this invention, it can suppress effectively that a background (background) light-emits and inhibits accurate microorganism measurement.

The thin film has a thickness of 10 to 50 nm. And according to this invention, the effect of forming a thin film can be made more reliable.

In addition, a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on a dark filter. And according to this invention, it can suppress more effectively that a background (background) light-emits and inhibits accurate microorganism measurement .

ADVANTAGE OF THE INVENTION According to this invention, the filter for microorganisms collection in which it was suppressed effectively that the background (background) light-emits and obstructs accurate microorganisms measurement is provided.

The present invention will be described below with reference to the drawings. However, the present invention is not limited to the following description.

1 (a) is a cross-sectional view of one embodiment of a micro-organism collection kit. The microorganism collection kit A includes a microorganism collection chip B and suction filtration means C. Microorganism-collecting chip B was formed a dark (eg black) carbon film microorganism-collecting filter 2 (e.g. on a white filter of the present invention in a filter 1 and the rear for removing foreign substances as a pre-filter in the previous stage as a basic structure Stuff). The pore diameter of the foreign matter removing filter 1 is set to 5 to 20 μm in order to remove foreign matters as much as possible, and the pore size of the microorganism collecting filter 2 is set to 0.2 to 0.8 μm in order to reliably collect microorganisms. Here, the hole diameter means the minimum hole diameter. The microorganism collection chip B has a liquid sample injection container 3, and the foreign matter removing filter 1 is the bottom of the liquid sample injection container 3. The microorganism collection filter 2 is disposed on a pedestal 4, and the pedestal 4 is held by a pedestal holding member 5. A hollow needle 6 is integrally formed on the pedestal 4 held by the pedestal holding member 5. The liquid specimen injection container 3 and the pedestal holding member 5 are detachably fitted. The suction filtration means C is a negative pressure tube 7, and the mouth of the negative pressure tube 7 is sealed with a rubber stopper 8.

By using the filter 2 for collecting microorganisms of the present invention as a dark filter, it is possible to effectively prevent the background (background) from emitting light and inhibiting accurate microbial measurement. A thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium may be formed on the filter. Since these components have a property of low spectral reflectance with respect to excitation light having a wavelength of about 300 to 550 nm, the effect can be obtained if the microorganism collection filter 2 is a dark filter and the above-described thin film is formed on the filter. (Aluminum and silver cannot be employed because they have high spectral reflectance with respect to the excitation light having the above wavelength). The thin film may be made of an alloy, a metal oxide, a metal carbide, a metal nitride, a metal carbonitride, or the like in addition to a kind of metal component. Moreover, a laminated thin film may be sufficient. As a method for forming the thin film, a known vapor phase growth method such as a vacuum deposition method, an ion sputtering method, or an ion plating method is preferably employed. The thickness of the thin film is desirably 10 to 50 nm. If the thickness is less than 10 nm, the effect of forming a thin film may not be sufficiently exhibited. On the other hand, if the thickness exceeds 50 nm, the pores of the filter may be clogged, which may affect the original function of the filter.

  The liquid sample itself is a liquid sample when the test object is a liquid sample such as drinking water. If the object to be inspected is a solid sample such as vegetables or meat, homogenize it to prepare a liquid specimen, or collect microorganisms from the surface using a cotton swab, etc., and use this as a physiological saline solution. Release to make liquid specimen. When a cooking utensil such as a cutting board is an object to be inspected, microorganisms are collected from the surface using a cotton swab or the like and released into physiological saline or the like to obtain a liquid sample. The liquid specimen thus prepared is injected into the liquid specimen injection container 3 of the microorganism collection chip B. Thereafter, the hollow needle 6 is pierced into the rubber stopper 8 at the mouth of the negative pressure tube 7 in the negative pressure state, and the liquid specimen is suction filtered by passing the rubber stopper 8 through the hollow needle 6 as shown in FIG. Is done. The lower part of the pedestal holding member 5 is extended so as to provide a function as a guide for reliably piercing the hollow needle 6 into the rubber plug 8 of the negative pressure tube 7 and so that the operator does not injure the hollow needle 6. Yes.

When the liquid specimen passes through the foreign matter removal filter 1, foreign matters larger than the microorganisms are collected and removed by the foreign matter removal filter 1. A filter for collecting large foreign matters, such as vegetable scraps and fibers, may be provided in the preceding stage of the foreign matter removing filter 1, and a filter for removing foreign matters may be formed in a laminated form. The liquid specimen that has passed through the foreign matter removing filter 1 reaches the microorganism collecting filter 2 of the present invention, and when the liquid specimen contains microorganisms, the microorganisms are collected in the microorganism collecting filter 2. A groove is provided on the boundary surface between the microorganism collection filter 2 and the pedestal 4 so that the liquid sample can be distributed evenly over the entire surface of the microorganism collection filter 2. As a result, it is possible to suppress a phenomenon in which microorganisms are concentrated and collected in the local area of the microorganism collecting filter 2.

  Since the liquid sample injection container 3 and the pedestal holding member 5 are detachably fitted, after collecting the microorganisms on the microorganism collection filter 2, the liquid sample injection container 3 is removed, and FIG. As shown in (), only the part including the filter 2 for collecting microorganisms can be moved to the weighing table of the microorganism weighing device.

  In addition, a liquid specimen injection container may be separately provided in the front stage of the foreign matter removing filter.

  The central part of the rubber stopper at the mouth of the negative pressure tube may be a thin layer.

  The liquid sample may be prepared separately using a test tube or the like. However, physiological saline or the like is injected into the liquid sample injection container 3 in advance, and the microorganisms collected using a cotton swab or the like are released. It is good also as a liquid specimen.

  The liquid sample injection container 3 and the pedestal holding member 5 are detachably fitted to each other, but may be joined using screws. In addition, both of them may be integrally formed at the beginning of manufacture, and a separation groove for facilitating the separation of both is provided at an appropriate position over the outer periphery, and the two are made using this separation groove. You may make it isolate | separate.

  The hollow needle is not limited in length as long as it penetrates the rubber stopper and reaches the inside of the negative pressure tube.

2 (a) is a sectional view of another embodiment of the micro-organism collection kit. The microorganism collection kit D includes a microorganism collection chip E and suction filtration means F. The difference between the microorganism collection chip E and the microorganism collection chip B shown in FIG. 1A is that a lid 52 with a cotton swab 51 covering the opening is provided on the liquid sample injection container 13. By attaching a cotton swab to the microorganism collection kit, it is possible to easily and easily sample from inspection objects with complicated shapes such as inspection objects having many scratches on the surface and corners of the kitchen. Further, the shape can be simplified by integrating the cotton swab 51 with the lid 52 like the microorganism collecting chip E. As shown in FIG. 2A, if the physiological saline 53 is injected into the liquid specimen injection container 13 in advance and the cotton swab 51 has a length soaked in the physiological saline 53, a cotton swab 52 is provided. After the microorganisms are collected from the test object using, the microorganisms attached to the cotton swab 51 are shaken into the physiological saline 53 by covering the liquid sample injection container 13 with the lid 52 and shaking the microorganism collection chip E up and down and left and right. It is easy to prepare a liquid specimen by liberation.

After releasing the microorganisms into the physiological saline 53, as shown in FIG. 2B, the liquid specimen is sucked and filtered by passing the rubber plug 18 through the hollow needle 16, and on the microorganism collection filter 12 of the present invention. Microbes are collected. Thereafter, in the same manner as shown in FIG. 1C, the liquid specimen injection container 13 is removed, and only the part including the microorganism collecting filter 12 is moved to the measuring table of the microorganism measuring apparatus for measurement.

  Note that the cotton swab need not be integrated with the lid.

  Note that the swab does not necessarily have a length soaked in physiological saline, and any length can be used as long as the microorganism attached to the cotton swab can be released into the physiological saline by shaking. It may be the same.

  The cotton swab may be immersed in physiological saline from the beginning of the microorganism collection kit. In that case, since the cotton swab is wet with physiological saline, it is possible to effectively collect microorganisms from a particularly dry specimen.

  Alternatively, the liquid specimen may be prepared by separately releasing the microorganisms adhering to the cotton swab into the physiological saline using a test tube or the like, and the prepared liquid specimen may be injected into the liquid specimen injection container.

The microorganisms collected on the microorganism collection filter of the present invention as described above can be detected and weighed by various methods, and in the following, both live and dead cells or either A method for simultaneously detecting and weighing a specific microorganism will be described.

The first microorganism measuring method includes: a first compound that develops viable and dead cells; a second compound that develops dead cells with a wavelength different from the color development; and a third compound that develops live cells with a wavelength different from the color development; Among them, at least one or more types of the fourth compound that develops color at a wavelength different from the color developed by reacting with one or a plurality of species and a specific microorganism-derived substance is brought into contact with the liquid specimen, and the microorganism is contained in the liquid specimen. In the case where the microorganism is stained, the microorganism is collected on the microorganism collection filter of the present invention using the microorganism collection chip, and then, from the wavelength difference and the color development amount, either live cells and / or dead cells are collected. This is a method of simultaneously detecting one and a specific microorganism. According to this method, viable and dead cells, live cells, dead cells, and specific microorganisms develop colors at different wavelengths, so that the specific microorganisms are detected simultaneously with either or both of live and dead cells from the wavelength difference and color development amount. be able to.

  Examples of color development include fluorescence. In this case, by using the first compound as a nucleic acid binding compound, it is possible to measure the living and dead cells with high accuracy at the single cell level. In addition, by using the second compound as a nucleic acid-binding compound, dead cells can be accurately measured at a single cell level. Moreover, by making the third compound a compound that develops color by reacting with a microorganism-derived substance, only living microorganisms can be measured due to the difference in the amount of color development. At this time, by making the microorganism-derived substance an enzyme protein, the viability of the microorganism can be discriminated from its reactivity. Moreover, the life and death of a specific microorganism can be discriminate | determined from the reactivity by making a specific microorganism origin substance into an enzyme protein.

  Examples of the first compound that develops viable and dead cells used for fluorescent staining of microorganisms include life and death such as 4 ′, 6-diamidino-2-phenylindole dihydrochloride (which may be a derivative). A compound that penetrates non-specifically from the cell membrane surface and develops color by specifically binding to a nucleic acid present in the cell. Examples of the second compound that develops color of dead cells at a wavelength different from that of the color development include, for example, nonspecific penetration from the membrane surface of dead cells such as propidium iodide (which may be a derivative) Examples thereof include compounds that develop color by specifically binding to nucleic acids present therein. Examples of the third compound that develops viable cells at a wavelength different from that of the color development include 6-carboxyfluorescein diacetate, 2 ′, 7 ′ dichlorofluorescein diacetate, 6- (N-succinimidyloxycarbonyl)- Penetration into cells such as 3 ′, 6′-0,0′-diacetylfluorescein, dihydrorhodamine, fluorescein diacetate, 4-azidofluorescein diacetate (these may be derivatives) and in living cells And compounds that develop color when decomposed by an esterase. Examples of the fourth compound that develops color at a wavelength different from the color development by reacting with a specific microorganism-derived substance include 4-methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl-β-D- Glucuronides (these may be derivatives). These are decomposed by specifically reacting with β-glucuronidase and β-galactosidase which are enzyme proteins produced by Escherichia coli and coliforms to produce 4-methylumbelliferone. 4-Methylumbelliferone is excited by ultraviolet light and emits fluorescence.

  The first to fourth compounds are used, for example, in a state dissolved in physiological saline, and can be added to a liquid sample to stain the microorganism when the microorganism is contained in the liquid sample. An appropriate amount of the first to fourth compounds dissolved in physiological saline may be stored in an eye drop container or the like so that they can be added to a liquid specimen at the time of use. It should be noted that the staining time should be appropriately set according to the type of test object and the volume of the liquid sample.

By a liquid specimen containing the stained microorganisms suction filtration using a micro-organism collection kit, collecting microorganisms that stained microorganisms for collecting on the filter of the present invention in the microorganism-collecting chip. At this time, for example, if a surfactant such as polysorbate 80 is added to a liquid specimen containing stained microorganisms, it adheres to the foreign matter collected by the foreign matter removal filter and is captured by the microorganism collection filter. Microorganisms that are not collected can be efficiently passed through the foreign matter removing filter and collected in the microorganism collecting filter. Further, if a medium component such as polypeptone is added to the liquid specimen, the activity of the microorganism during the operation can be maintained. A liquid agent containing a medium component together with a surfactant, for example, LP diluent “DAIGO” (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) is preferably used to achieve such an object. After the suction filtration, the measurement is performed by moving only the portion including the microorganism collection filter 2 as shown in FIG. If a polyhydric alcohol such as glycerin is added to a liquid specimen containing stained microorganisms, it is possible to prevent inactivation of microorganisms and decrease in light emission due to drying of the filter surface for collecting microorganisms.

In the microorganism collection chip, it is possible to install a filter carrying the first to fourth compounds in the preceding stage of the microorganism collection filter of the present invention .

FIG. 3 is a conceptual diagram showing one embodiment of the microorganism weighing device. This microorganism weighing device includes a light source 104, a lens 110 as a light source condensing unit, and a light receiving unit 111. In order to extract a target wavelength from the excitation light emitted from the light source 104, the light is split by the excitation light spectral filter 112. The split excitation light passes through the prism 113 and the optical path is changed. The excitation light whose optical path has been changed includes the microorganism collecting filter 2 of the present invention installed on the examination table 116 through the lens 110, that is, the microorganism collecting filter 2, the pedestal 4, the pedestal holding member 5, and the hollow needle. 6 is condensed on the surface of the filter 2 for collecting microorganisms. Therefore, the fluorescence of the microorganisms excited by the excitation light passes through the prism 113 again. At this time, the fluorescence passes through the prism 113 as it is and reaches the light receiving unit 111. The fluorescence that reaches the light receiving unit 111 passes through the fluorescence spectroscopic filter 114 to extract only the target fluorescence, reaches the photoelectric conversion element 115 built in the light receiving unit 111, is converted into a signal, and is recognized. Although not shown in the drawing, the microorganism weighing device includes means for moving the examination table 116, and can receive all or part of the fluorescence emitted from the surface of the microorganism collecting filter 2.

  The fluorescence that has reached the photoelectric conversion element 115 is determined to be a microorganism or a foreign substance by the microorganism determination means 105, and the fluorescence determined to be a microorganism is integrated and the quantity thereof is measured.

  The excitation light generated from the light source 104 is condensed by the lens 110. At this time, the range irradiated with the excitation light by the lens 110 is condensed on a small fixed area. In this case, the small fixed area indicates a range of about 0.2 μm to 7.0 μm on a side when set based on the size of the microorganism. In addition, when based on a comparison with the agar medium diffusion method, which is one of the most utilized microorganism detection means, colonies formed by a population of microorganisms cultured and grown by the agar medium diffusion method have a distance of When they are close to each other, colonies may overlap with each other, and when they are finally visually confirmed, there may be cases where the colonies are recognized as one colony. Therefore, the minute fixed area in this case indicates a range of about 100 μm to 500 μm on a side when based on a distance where colonies do not overlap each other.

  The irradiation time of the excitation light collected by the lens 110 depends on the quenching time and excitation light intensity of the fluorescent compound. Depending on the type of compound, it may be decomposed by ultraviolet light existing in nature, and it is desirable to irradiate the excitation light within a range of about 2 seconds to 300 seconds.

  When detecting light emission, if the wavelength range of the light source 104 is wide, the excitation light spectral filter 112 can adjust the excitation wavelength and perform spectroscopy. Since the excitation light spectral filter 112 can be changed according to the target detection target, the excitation light spectral filter 112 can cope with various fluorescent compounds. At the same time, when the emitted fluorescence wavelength is wide, it is possible to cope with various fluorescent compounds by changing the fluorescence spectral filter 114 according to the target detection target in order to detect the target emission. .

  Examples of the light source 104 include various diodes, halogen lamps, xenon lamps, cold cathode tubes, lasers, black lights, mercury lamps, and the like. Among these light sources, a diode, a cold cathode tube, a black light, etc., whose maximum excitation wavelength is relatively limited may be implemented without using the excitation light spectral filter 112 and the fluorescent spectral filter 114 in some cases. For halogen lamps, mercury lamps, and the like, it may be necessary to use the excitation light spectral filter 112 and the fluorescent spectral filter 114.

  The prism 113 and the lens 110 each have a property of transmitting ultraviolet light as necessary. Quartz glass etc. are mentioned as what has the property which permeate | transmits ultraviolet light. Thus, it can be applied to a compound excited by ultraviolet light. The inspection table 116 on which the part including the microorganism collecting filter 2 is installed has a rotating ability. The excitation light collected by the lens 110 moves a distance corresponding to the radius from the outer periphery of the microorganism collection filter 2 to the center or from the center to the outer periphery. At that time, the excitation light collected by the lens 110 is changed by changing the rotation speed of the examination table 116 between the position of the excitation light collected by the lens 110 and the center of the excitation light. Can be prevented from occurring in the fluorescence emitted from the excited compound between the time when the light is present at the outer peripheral portion and the time when the light is present at the central portion, afterimage and afterglow.

As shown in FIG. 3, the inspection table 116 has a depressed portion (apparatus groove) for fitting a portion including the microorganism collection filter 2 of the present invention, and the portion including the microorganism collection filter 2 is left as it is. The shape can be incorporated. At this time, for example, the inspection table 116 is provided with a metal flat plate so that the microorganism collecting filter 2 is positioned thereon, and the microorganism collecting filter 2 is incorporated in such a state that it is pressed against the metal flat plate. Thus, if the microorganism collecting filter 2 is held smoothly on the inspection table 116 without unevenness, the quantification of the microorganisms collected in the microorganism collecting filter 2 can be made more reliable.

  It should be noted that by providing a means for recognizing the condensed position, the position of the excitation light collected by the lens 110 is recognized, so that the condensed light does not deviate from the orbit, and when it deviates, it returns to the orbit again. Is set to

  Note that the minute fixed area to which the excitation light is irradiated is not limited to a polygon including a square, but may be a circle, an ellipse, or the like as long as the sample can be irradiated.

  It is also possible to use a diffraction grating or the like as means for separating excitation light or fluorescence.

  Although the fluorescence afterimage and afterglow are prevented by adjusting the rotation speed of the examination table 116, it is also possible to prevent afterimage and afterglow by adjusting the moving speed of the lens 110 that irradiates the excitation light. It is.

  The means for recognizing the condensed position does not necessarily need to directly recognize the condensing position of the excitation light, and may be any means that can grasp the trajectory on the microorganism collection filter 2.

The second microorganism measuring method is a first microorganism collection method in which microorganisms are collected from a liquid specimen that may contain microorganisms on a microorganism collection filter of the present invention using a microorganism collection chip, and then the captured microorganisms develop viable and dead cells. Reaction with one or more of a second compound that develops a color of a compound and dead cells at a wavelength different from the color development and a third compound that produces a color of a living cell at a wavelength different from the color development, and a specific microorganism-derived substance By contacting at least one or more types of the fourth compound that develops color at a wavelength different from that of the color development and staining the microorganism, it is determined from the wavelength difference and the color development amount that both live cells and dead cells are identified. This is a method for simultaneously detecting microorganisms. According to this method, even if the volume of the liquid specimen is large, the microorganism is stained after collecting the microorganism on the microorganism collection filter. Therefore, the amount of the first to fourth compounds used is small, Live cells, dead cells and specific microorganisms can be accurately and easily measured.

By adding black ink to a liquid sample, the microorganisms are collected on the filter for collecting microorganisms of the present invention, so that the background of the background and foreign matter are suppressed and the microorganisms are accurately measured. Can be done well. The ink is not necessarily added to the liquid specimen, but may be added from above the microorganism collecting filter on which the microorganisms are collected.

Figure 4 shows an embodiment of a perspective view of a normal state confirmation test sample of microorganisms metering device (partly transparent view). The specimen 151 for normal state confirmation test has a substrate (for example, a substrate on which a predetermined number of inclination height confirmation marks 155 are printed and a light emitter 154 that emits light with excitation light having a specific number of specific wavelengths is fixed on the surface thereof (for example, Resin film) 152 and its support frame 153.

  Examples of the illuminant to be fixed on the surface of the substrate include polymer fluorescent particles and stained microorganisms. Polymer fluorescent particles maintain stable and uniform light emission over a long period of time, making it possible to check the status of the device repeatedly and repeatedly, and in terms of ensuring safety to the human body. Is expensive. Polymer fluorescent particles are particles made of polystyrene, styrene-divinylbenzene, etc., and are manufactured by adding fluorescent dyes that emit light by excitation light of a specific wavelength when the particles are polymerized. Are commercially available. The polymeric fluorescent particles preferably have a particle diameter of 0.1 to 1.0 μm. If it is too small, it will be difficult to accurately measure the number, or if a filter for collecting microorganisms is used as the base material as will be described later, the polymer fluorescent particles will fall from the hole in the filter. This is because, while the workability is deteriorated, if it is too large, the emission intensity is too strong, which may affect the measurement accuracy. The polymer fluorescent particle may be a single type of polymer fluorescent particle fixed. For example, the polymer fluorescent particle emits blue light by excitation light having a wavelength of 350 to 400 nm, and excitation light having a wavelength of 500 to 550 nm. A plurality of types of polymer fluorescent particles that emit light in different colors by excitation light having different wavelengths may be fixed, such as polymer fluorescent particles that emit red light.

  When immobilizing dyed microorganisms on the surface of a substrate, the microorganisms to be immobilized depend on whether the microorganisms to be tested are viable or dead or both, viable, dead, or dead / live mixed bacteria (For example, if the microorganism to be tested is dead, it is necessary to confirm the normal state of the light source for measuring dead bacteria, so the dead bacteria must be fixed to the substrate) . It should be noted that when fixing live or dead / live mixed bacteria, safety to the human body in operation must be ensured.

  The substrate may be glass, paper, metal, etc. in addition to the resin film. Moreover, you may use the filter for microorganism collection as a base material. If the filter for collecting microorganisms is used as a base material, the light emitting body that emits light by excitation light having a specific wavelength is filtered, so that the light emitting body is easily collected on the filter, that is, fixed to the surface of the base material. be able to. The substrate is preferably dark (for example, black). This is because it is possible to accurately check the state of the apparatus by suppressing the light emission of the background (background). In addition, if a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on a base material on which a light emitter that emits light with excitation light having a specific wavelength is fixed, these components are used. Has a low spectral reflectance with respect to excitation light having a wavelength of about 300 to 550 nm, so that the base material is dark and the above-mentioned light emitting body that emits light by excitation light having a specific wavelength is fixed on the surface. If a thin film is formed, the effect is further improved (aluminum and silver cannot be employed because they have a high spectral reflectance with respect to the excitation light having the above wavelength). In addition, when such a thin film is formed, it is possible to adjust the light emission intensity of the light emitter, for example, to reduce the light emission intensity when the light emission intensity is too strong, and to remove the light emitter fixed to the surface of the substrate. It is also possible to prevent such a situation. The thin film may be made of an alloy, a metal oxide, a metal carbide, a metal nitride, a metal carbonitride, or the like in addition to a kind of metal component. Moreover, a laminated thin film may be sufficient. As a method for forming the thin film, a known vapor phase growth method such as a vacuum deposition method, an ion sputtering method, or an ion plating method is preferably employed. The thickness of the thin film is preferably 10 to 1000 nm, and more preferably 20 to 100 nm. If the thickness is less than 10 nm, the effect of forming a thin film may not be sufficiently exhibited. On the other hand, if the thickness exceeds 1000 nm, the number of light emitters fixed on the surface of the substrate can be accurately measured and the light emission intensity of the light emitter can be adjusted. This is because it becomes difficult.

  FIG. 5 is a front view of the resin film 152 on which the light emitters 154 that emit light by the excitation light having a predetermined number of specific wavelengths are fixed on the surface shown in FIG. 4 and the predetermined number of inclination height confirmation marks 155 are printed. is there. When the luminescent material 154 is used as a stained microorganism, for example, the staining of the microorganism may be performed by, for example, using the first compound that develops viable cells, the second compound that develops dead cells, or the third compound that develops viable cells. What is necessary is just to use it suitably. The illuminant 154 can be detected by a microorganism weighing device as shown in FIG. After confirming that a predetermined number of illuminants can be detected using the normal state confirmation test specimen, the microorganisms of the liquid specimen are measured. If a predetermined number of light emitters can be detected, it means that the apparatus is in a normal state. On the other hand, if the predetermined number of light emitters cannot be detected, it means that the apparatus is not in a normal state, and it is necessary to adjust the apparatus.

  On the surface of the resin film 152 shown in FIG. 5, a light emitting body 154 that emits light with excitation light having a specific number of specific wavelengths is fixed, and four inclination height confirmation marks 155 are printed. By irradiating the four tilt height confirmation marks 155 with light and checking whether or not the output level of the reflected light is uniform, for example, the presence or absence of the tilt of the examination table 116 in the microorganism weighing apparatus shown in FIG. Can be inspected. Further, by checking whether or not the output level is as specified, it is possible to inspect whether or not the inspection table 116 has a specified height.

Incidentally, although not have particular limitation on the shape of the normal state checking test sample of microorganisms metering device, for example, a recessed portion (device groove) having the inspection table 116 in a microorganism weighing apparatus shown in FIG. 3 It is desirable to have a shape that can be incorporated in the same manner as the part including the filter 2 for collecting microorganisms. Further, a metal flat plate is provided on the inspection table 116 so that the base material 152 of the normal state confirmation test specimen 151 is positioned thereon, and the base material 152 is incorporated in a state of being pressed against the metal flat plate. Thus, the normal state of the apparatus may be more surely confirmed by holding the base material 152 smoothly on the inspection table 116 without unevenness.

INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide a filter for collecting microorganisms in which the background (background) emits light and effectively inhibits microbial weighing from being inhibited .

It is a cross-sectional view of one embodiment of the micro-organism collection kit. It is a cross-sectional view of another embodiment of the micro-organism collection kit. It is a schematic diagram of one embodiment of an apparatus for performing the microorganism metered using microbial collection kit. Which is one embodiment of a perspective view of a normal state confirmation test sample of microorganisms metering device (partly transparent view). It is a front view of the resin film in the test specimen shown in FIG.

Explanation of symbols

A, D Microorganism collection kit B, E Microorganism collection chip C, F Suction filtration means 1 Foreign matter removal filter 2, 12 Microorganism collection filter of the present invention 3, 13 Liquid specimen injection container 4 Base 5 Base holding member 6, 16 Hollow Needle 7, 17 Negative pressure tube 8, 18 Rubber stopper 51 Cotton swab 52 Lid 53 Saline 151 Specimen for normal state confirmation test of microorganism weighing device 152 Base material (resin) that emits light emitted by excitation light of specific wavelength the film)
153 Support frame 154 Luminescent body that emits light by excitation light having a specific wavelength 155 Tilt height confirmation mark

Claims (26)

  A microbe collection chip that combines a foreign matter removal filter as a pre-filter in the front stage and a microbe collection filter in the back stage, filters the microbe from a liquid sample that may contain microbes, and collects the microbe on the microbe collection filter.   The microbe collection chip according to claim 1, wherein the foreign matter removing filter has a pore size of 5 to 20 µm, and the microbe collection filter has a pore size of 0.2 to 0.8 µm.   The microorganism collecting chip according to claim 1 or 2, wherein the foreign matter removing filter serves as a bottom portion of the liquid specimen injection container.   The microorganism collection chip according to claim 1 or 2, wherein a liquid sample injection container is provided further upstream of the filter for removing foreign substances.   The microorganism collection chip according to claim 3 or 4, further comprising a lid with a cotton swab covering the liquid sample injection container opening.   The microorganism collection chip according to any one of claims 1 to 5, wherein a part including a filter for collecting microorganisms can be detached independently.   The microorganism collection chip according to any one of claims 1 to 6, wherein the microorganism collection filter is a dark filter.   The microorganism collection chip according to any one of claims 1 to 7, wherein a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on the filter.   The microbe collection chip according to claim 8, wherein the thickness of the thin film is 10 to 50 nm.   A filter for collecting microorganisms and aspiration for collecting microorganisms on a filter for collecting microorganisms by filtering microorganisms from a liquid sample that can contain microorganisms, by combining a filter for removing foreign substances as a pre-filter in the first stage and a filter for collecting microorganisms in the second stage. Microorganism collection kit comprising filtration means.   The microorganism collection kit according to claim 10, wherein the suction filtration means is a negative pressure tube.   The microorganism collection kit according to claim 11, wherein a rubber stopper is provided at the mouth of the negative pressure tube.   The microorganism collection kit according to claim 12, wherein a central portion of the rubber plug is a thin layer.   The microorganism collection kit according to any one of claims 11 to 13, wherein a hollow needle that reaches the inside of the negative pressure tube is provided below the microorganism collection filter of the microorganism collection chip.   One or more of a first compound that develops viable cells, a second compound that develops dead cells with a wavelength different from the color development, and a third compound that develops viable cells with a wavelength different from the color development And at least one fourth compound that develops color at a wavelength different from that of the color developed by reacting with a specific microorganism-derived substance is brought into contact with the liquid specimen, and if the microorganism is contained in the liquid specimen, the microorganism is stained. Then, after collecting the microorganisms on the filter for collecting microorganisms using the microorganism collecting chip according to claim 1, the living cells and / or dead cells and the specific microorganism are simultaneously used from the wavelength difference and the color development amount. Microbial weighing method to detect.   The first compound and the dead cells that cause the captured microorganisms to develop viable and dead cells after collecting the microorganisms from the liquid specimen that can contain the microorganisms using the microorganism collecting chip according to claim 1, By reacting with one or more kinds of a second compound that develops color at a wavelength different from the color development and a third compound that produces viable cells at a wavelength different from the color development, the color development occurs by reacting with a specific microorganism-derived substance. A microorganism that contacts at least one or more types of fourth compounds that develop color at different wavelengths, stains the microorganism, and then simultaneously detects a specific microorganism and / or both living and / or dead cells from the difference in wavelength and the amount of color development Weighing method.   The method for measuring microorganisms according to claim 15 or 16, wherein ink is added to the liquid specimen.   17. The microorganism measuring method according to claim 15 or 16, wherein after collecting microorganisms on a microorganism collecting filter, black ink is added on the microorganism collecting filter to blacken the microorganism collecting filter.   Using a specimen for normal state confirmation inspection of a microbial measuring device including a base material on which a luminescent material that emits light of excitation light of a specific wavelength is fixed, and detecting the luminescent material in advance, and the microbial measuring device is in a normal state The microorganism weighing method according to claim 15 or 16, wherein the specific microorganism is detected simultaneously with live cells and / or dead cells after the confirmation.   The method for measuring microorganisms according to claim 19, wherein the illuminant is a polymeric fluorescent particle.   The method for measuring microorganisms according to claim 19, wherein the illuminant is a stained microorganism.   Specimen for normal state confirmation inspection of a microbial measuring device for confirming that the microbial measuring device is in a normal state before performing microbial weighing, including a base material on which a light emitting body that emits light by excitation light of a specific wavelength is fixed. .   The specimen for normal state confirmation inspection of the microorganism weighing device according to claim 22, wherein the base material is dark.   The thin film containing at least one kind of metal component selected from gold, copper, chromium, platinum, and palladium is formed on a base material on which a light emitting body that emits light with excitation light having a specific wavelength is fixed. Sample for normal state confirmation test of microbial measuring device.   The specimen for normal state confirmation test of the microorganism weighing device according to claim 24, wherein the thickness of the thin film is 10 to 1000 nm.   2. A light source for irradiating excitation light in a predetermined wavelength range in a minute fixed area of the microorganism collection filter in the microorganism collection chip according to claim 1, and receiving light in a predetermined wavelength range emitted by the excitation light. A light receiving means that receives the light emitted from the light source and emits light within a set time, and determines the microorganism when the received light quantity is within a set threshold range; and A microorganism measuring apparatus comprising: a moving unit that continuously or intermittently moves the minute fixed area; and an integrating unit that integrates the number of microorganisms from a signal determined to be a microorganism by the microorganism determining unit, the excitation light having a specific wavelength A device that includes a substrate on which a luminescent material that emits light is fixed on the surface, so that the microorganism weighing device can be confirmed to be in a normal state before weighing microorganisms. .

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