JP2009178078A - Microbiological testing chip and microbiological testing instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microbiological testing chip, testing instrument and testing method to prevent the generation of stray light caused by the reflection of excitation light, thereby preventing the decrease in the received light quantity of fluorescent light. <P>SOLUTION: The microbiological testing chip 10 has a substrate and a microorganism detection part 18 attached to the substrate and containing a microorganism detection flow channel 181, the microorganism detection part 18 is formed of a light-transmitting material, and at least 90° of the circumference of the microorganism detection part is exposed in the chip 10. Also provided are a microbiological testing instrument and a testing method to use the microbiological testing chip 10. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microorganism testing apparatus for measuring microorganisms contained in a sample and a microorganism testing chip used therefor.

  In recent years, food poisoning caused by microorganisms such as enterohemorrhagic Escherichia coli O157 has become a major social problem. For this reason, demands on food safety of general consumers are increasing. Food suppliers include many businesses ranging from general restaurants to large companies with food manufacturing plants. For these businesses, food hygiene management is essential to ensure the safety of the food provided. In general, food hygiene managers manage food hygiene.

  As an objective index for determining whether or not a food is “hygienic”, the number of viable bacteria such as general viable bacteria and coliform bacteria contained in the food is used.

  Conventionally, culture methods have been used as means for measuring the number of viable bacteria. In the culture method, first, a suspension of a food sample is applied on a medium to culture microorganisms. Next, the number of viable bacteria in the food is measured by measuring colonies formed by the grown microorganisms. In the culture method, it takes one to several days for the microorganisms to form colonies. Therefore, the culture method requires a long test time. Furthermore, the culture method includes steps requiring specialized knowledge and skill such as suspension, dilution, application, colony counting. Therefore, there are problems that human error occurs and inspection costs are high. Therefore, for food suppliers, the culture method is not a test for ensuring the safety of shipped food, but is a means of investigating the cause after the occurrence of food poisoning.

  Conventionally, various methods and apparatuses for the purpose of speeding up and simplifying the viable count have been tried. As one of them, a bacterial count measuring apparatus using a fluorescent flow cytometry method has attracted attention. In the fluorescence flow cytometry method, each specimen stained with a fluorescent dye is caused to flow along an extremely thin path. In this method, the number of samples is directly measured one by one, but the measurement can be completed in a short time.

  Improvements have been made to the bacterial count measuring apparatus using the fluorescent flow cytometry method. For example, in order to prevent components in the specimen from adhering to the channel wall surface, the specimen and sheath fluid flows are laminarized and the pressure difference between the two flows is used to narrow the specimen laminar flow diameter. Things have been done.

  Further, Non-Patent Document 1 describes that a flow path portion for performing measurement by the fluorescent flow cytometry method is formed by a disposable chip in order to realize a reduction in price and to save the labor of washing. The measurement is performed in the disposable chip and discarded after the measurement.

Journal of Biomolecular Techniques, Vol14, Issue2, pp.119-127

  Generally, a disposable microbial test chip is produced by attaching a transparent flat substrate such as glass or PMMA (methyl methacrylate resin) on a substrate having large and small grooves formed by fine processing. Therefore, the detection flow path is configured on the substrate. Therefore, in order to detect the fluorescent substance flowing in the detection flow path in the chip, the optical axis of the excitation light and the central axis of the detection condensing lens for detecting fluorescence are installed on the same surface side with respect to the chip. The Therefore, when the central axis of the detection condensing lens is arranged perpendicular to the chip surface including the detection flow path, the optical axis of the excitation light is coaxial with the central axis of the detection condensing lens, Or, it becomes an inclined direction.

  When the optical axis of the excitation light is coaxial with the central axis of the detection condensing lens, the reflected light of the excitation light from the flat substrate or the reflected light of the excitation light from the bottom surface of the detection flow path becomes the detection condensing lens. enter. Therefore, the reflected light is detected as stray light, making correct fluorescence measurement difficult.

  When the optical axis of the excitation light is arranged in a direction inclined with respect to the central axis of the detection condensing lens, a part of the excitation light is reflected on the flat substrate, so that the excitation incident on the detection flow path The amount of light decreases. Therefore, the amount of fluorescence obtained is also reduced, and the detection accuracy is reduced.

  An object of the present invention is to avoid generation of stray light due to reflection of excitation light and avoid a decrease in the amount of fluorescence received in a microorganism testing chip.

  The microbe inspection chip of the present invention has a substrate and a microbe detection unit attached thereto. The microorganism detection unit has a microorganism detection flow path and is formed of a light-transmitting material. A range of at least 90 degrees around the microbe detection part is exposed.

  According to the present invention, generation of stray light due to reflection of excitation light can be avoided and a decrease in the amount of received fluorescence can be avoided in the microorganism testing chip.

  With reference to FIG. 1, the structure of the 1st example of the microbe test | inspection chip | tip used with the microbe test | inspection apparatus of this invention is demonstrated. The microbe test chip 10 of this example includes a sample container 151 for holding a sample 1511, first and second reaction containers 153 and 154 for mixing and reacting a reagent with the sample, and unnecessary components contained in the sample. The unnecessary component removing unit 16 that is a filter for removing, the excitation light from the external light source is irradiated, the microorganism detecting unit 18 having the microorganism detecting channel 181, and the solution containing the specimen that has passed through the microorganism detecting unit 18 is discarded. A waste liquid container 163. The arrow A in the figure indicates the optical axis of the excitation light that irradiates the microorganism detection flow path 181 of the microorganism detection unit 18.

  As shown in the figure, the microorganism testing chip 10 of this example is used in an upright state with the waste liquid container 163 facing down.

  Here, a case where the microorganisms in the food are inspected by the microorganism inspection apparatus of the present invention will be described. In this case, the specimen 1511 is a food to be examined. In addition, the first reaction vessel 153 holds a killed bacteria dye 1531 that stains only killed bacteria. Therefore, the first reaction container 153 is hereinafter referred to as a dead bacteria staining reagent holding container 153. The second reaction vessel 154 holds a whole cell staining dye 1541 that stains all cells. Therefore, the second reaction container 154 is hereinafter referred to as a whole bacteria staining reagent holding container 154. The unnecessary component removal unit 16 removes food residues. A mixed liquid of the specimen 1511, dead bacteria staining dye 1531, and whole bacteria staining dye 1541 passes through the microorganism detection unit 18 and is then held in the waste liquid container 163.

  The lower end of the sample container 151 is connected to the unnecessary component removal unit 16 through a flow path or directly. The lower end of the unnecessary component removal unit 16 is connected to the upper end of the killed bacteria staining reagent holding container 153 by a flow path 1571. The lower end of the dead bacteria staining reagent holding container 153 is connected to the upper end of the whole bacteria staining reagent holding container 154 by a flow path 1572. The lower end of the whole bacteria staining reagent holding container 154 is connected to the upper end of the microorganism detection unit 18 by a flow path 1573. The lower end of the microorganism detection unit 18 is connected to the upper end of the waste liquid container 163 by a flow path 1574. By the flow paths 1571, 1572, 1573, and 1574, the sample container 151, the unnecessary component removal unit 16, the dead bacteria staining reagent holding container 153, the whole bacteria staining reagent holding container 154, the microorganism detection unit 18, and the waste liquid container 163 are connected in series. It is connected to.

  An air flow path 1584 is connected to the upper end of the waste liquid container 163. The upper end of the air flow path 1584 is a vent hole 1634. An air flow path 1581 is connected to the upper end of the sample container 151. The upper end of the air flow path 1581 is a vent 1631. An air flow path 1582 is connected to the upper end of the killed bacteria staining reagent holding container 153. The upper end of the air flow path 1582 is a vent 1632. An air flow path 1583 is connected to the upper end of the whole bacteria staining reagent holding container 154. The upper end of the air flow path 1583 is a vent 1633. Is the specimen container 151, the dead bacteria staining reagent holding container 153, the whole bacteria staining reagent holding container 154, and the waste liquid container 163 connected to a predetermined pressure source through the vents 1631, 1632, 1633, and 1634, respectively? Or open to the atmosphere.

  The solution channels 1571 to 1574 are pipes having a rectangular or circular cross section with an inner diameter of 10 μm to 1 mm. The air flow paths 1581 to 1584 are pipes having a rectangular cross section or a circular cross section with an inner diameter of 10 μm to 1 mm. The cross-sectional area of the solution flow paths 1571 to 1574 is larger than the cross-sectional area of the air flow paths 1581 to 1584. The inner diameter of the microorganism detection flow path of the microorganism detection unit 18 is 1 μm to 1 mm.

  The volume of the sample container 151 is larger than the volume of the sample 1511. The volume of the dead bacteria staining reagent holding container 153 is larger than the total volume of the specimen 1511 and the dead bacteria staining dye 1531. The volume of the whole bacteria staining reagent holding container 154 and the waste liquid container 163 is larger than the total volume of the specimen 1511, dead bacteria staining dye 1531, and whole bacteria staining dye 1541, respectively.

  The highest point of the solution flow channel 1571 that connects the sample container 151 and the dead bacteria staining reagent holding container 153 is higher than the liquid level of the sample 1511 in the sample container 151. The highest point of the solution flow path 1572 that connects the killed bacteria staining reagent holding container 153 and the whole bacteria staining reagent holding container 154 is the liquid mixture of the specimen 1511 and the killed bacteria staining dye 1531 in the killed bacteria staining reagent holding container 153. Higher than the surface. The highest point of the solution flow path 1573 connecting the whole bacteria staining reagent holding container 154 and the microorganism detecting unit 18 is the specimen 1511 in the whole bacteria staining reagent holding container 154, the dead bacteria staining dye 1531, and the whole bacteria staining dye 1541. It is higher than the liquid level of the mixed liquid. Thus, by setting the height of the highest point of the flow path higher than the height of the liquid level of the upstream container, the solution flows downstream from the upstream container via the flow path due to the liquid level difference. Moving to the side container is prevented.

  The dead bacteria staining dye 1531 and the whole bacteria staining dye 1541 are enclosed in advance in the dead bacteria staining reagent holding container 153 and the whole bacteria staining reagent holding container 154 of the microorganism test chip 10.

  For example, PI (propidium iodide) (1 μg / ml to 1 mg / ml) may be used as the dead bacteria staining dye 1531. For example, DAPI (4 ′, 6-diamidine-2′-phenylindole) (1 μg / ml to 1 mg / ml), AO (acridine orange) (1 μg / ml to 1 mg / ml), EB (Ethidium bromide) (1 μg / ml to 1 mg / ml), SYTO® (1 μg / ml to 1 mg / ml), and the like may be used.

  A specimen 1511 is obtained by adding a physiological saline having a mass ratio of 10 times to a food to be examined and performing a stomaching process. The specimen 1511 is injected into the specimen container 151 from the vent 1633 before the examination.

  Next, a method for measuring microorganisms using the microorganism testing chip 10 and movement of each liquid by the method will be described.

  As shown in the figure, the microorganism testing chip 10 is used in an upright state so that the vents 1631 to 1634 are arranged on the upper side and the waste liquid container 163 is arranged on the lower side. First, the sample is introduced into the sample container 151. The vent 1631 is opened to the atmosphere, and the specimen is introduced into the specimen container 151 from the vent 1631. When the sample is introduced into the sample container 151, the same volume of gas is discharged from the sample container 151 through the vent 1631.

  Next, the specimen 1511 is moved to the dead bacteria staining reagent holding container 153. A pressure source is connected to the vent 1631 and the vents 1632 to 1634 are opened to the atmosphere. Pressurized air is introduced into the specimen container 151 through the vent 1631. The pressure of the air above the sample in the sample container 151 increases. As a result, the pressure in the specimen container 151 becomes larger than the pressure in the dead bacteria staining reagent holding container 153. The sample in the sample container 151 moves to the dead bacteria staining reagent holding container 153 via the unnecessary component removing unit 16 and the solution flow channel 1571. When the specimen is introduced into the killed bacteria staining reagent holding container 153, the same volume of gas is discharged out of the killed bacteria staining reagent holding container 153 through the vent 1632.

  Further, when the sample 1511 passes through the unnecessary component removing unit 16, unnecessary components in the sample 1511 are removed.

  The specimen is mixed with the dead bacteria staining dye 1531 in the dead bacteria staining reagent holding container 153. Although dead bacteria in the specimen 1511 are stained with the dead bacteria staining dye 1531, live bacteria in the specimen 1511 are not stained with the dead bacteria staining dye 1531.

  The liquid level of the mixed liquid of the specimen and the dead bacteria staining dye 1531 does not exceed the highest point of the solution flow path 1572 that connects the dead bacteria staining liquid holding container 153 and the whole bacteria staining liquid holding container 154. Since the atmospheric pressure in the dead bacteria staining liquid holding container 153 and the whole bacteria staining liquid holding container 154 is equal to the atmospheric pressure, the mixed liquid is held in the dead bacteria staining liquid holding container 153 and moves to the whole bacteria staining liquid holding container 154. There is nothing. The mixed solution can be held in the dead bacteria staining solution holding container 153 for a time required for the staining reaction. During the staining, the temperature of the microorganism testing chip 10 is kept constant. Thereby, the influence of dyeing due to temperature change can be reduced.

  Note that the atmospheric pressure in the waste liquid container 163 is also equal to the atmospheric pressure. Therefore, the whole bacteria staining dye 154 in the whole bacteria staining reagent holding container 154 does not move to the microorganism detection unit 18 and does not flow back to the dead bacteria staining liquid holding container 153. That is, the whole bacteria staining reagent holding container 154 is held.

  In order to more reliably prevent the mixed liquid from moving from the dead bacteria staining liquid holding container 153 to the whole bacteria staining liquid holding container 154, a pressure source may be connected to the vent holes 1633 and 1634. That is, the atmospheric pressure in the whole bacteria staining liquid holding container 154 and the detection liquid waste liquid container 163 is made larger than the atmospheric pressure and smaller than the atmospheric pressure in the sample container 151. Thereby, the mixed liquid is reliably held in the dead bacteria staining liquid holding container 153.

  Next, the mixed solution is moved to the whole bacteria staining reagent holding container 154. A pressure source is connected to the vents 1631 and 1632 to open the vents 1633 and 1634 to the atmosphere. Pressurized air is introduced into the dead bacteria staining liquid holding container 153 through the vent 1632. The pressure of the air on the upper side of the mixed liquid in the dead bacteria staining liquid holding container 153 increases. Thereby, the pressure in the dead bacteria staining liquid holding container 153 becomes larger than the pressure in the whole bacteria staining reagent holding container 154. The mixed liquid in the dead bacteria staining liquid holding container 153 moves to the whole bacteria staining reagent holding container 154 via the solution flow path 1572. When the mixed solution is introduced into the whole bacteria staining reagent holding container 154, the same volume of gas is discharged from the whole bacteria staining reagent holding container 154 through the vent 1633.

  The specimen is mixed with the whole bacteria staining dye 1541 in the whole bacteria staining reagent holding container 154. Dead and live bacteria in the specimen 1511, that is, all the bacteria are stained with the whole bacteria staining dye 1541.

  Next, a mixed liquid of the specimen 1511, dead bacteria staining dye 1531 and whole bacteria staining dye 1541 is passed through the microorganism detection unit 18. A pressure source is connected to the vents 1631, 1632 and 1633, and the vent 1634 is opened to the atmosphere. Pressurized air is introduced into the whole bacteria staining reagent holding container 154 through the vent 1633. The pressure of the air above the mixed liquid in the whole bacteria staining reagent holding container 154 increases. Thereby, the pressure in the whole bacteria staining reagent holding container 154 becomes larger than the pressure in the waste liquid container 163. The mixed liquid in the whole bacteria staining reagent holding container 154 moves to the waste liquid container 163 via the solution flow path 1573 and the microorganism detection unit 18.

  When the liquid mixture is discharged into the waste liquid container 163, the same volume of gas is discharged from the waste liquid container 163 through the vent 1634.

  When the mixed solution passes through the microorganism detection flow path 181 of the microorganism detection unit 18, excitation light is irradiated from the direction of arrow A in FIG. When a microorganism is irradiated with excitation light, it emits fluorescence. The number of dead bacteria can be obtained by counting the number of passing fluorescence of the dead bacteria staining dye 1531. By counting the number of fluorescent passages of the whole bacteria staining dye 1541, the total number of bacteria can be obtained. The number of viable bacteria can be obtained by subtracting the number of dead bacteria from the total number of bacteria.

  The microorganism testing chip 10 of the present invention shown in FIG. 1 has two reaction containers, that is, a dead bacteria staining reagent holding container 153 and a whole bacteria staining reagent holding container 154, but is configured to have one reaction container. Alternatively, it may be configured to have three or more reaction vessels. The reaction in the reaction vessel is staining of microorganisms with a staining reagent, but other operations may be performed.

  With reference to FIG. 2, the microorganisms detection part 18 of the microorganisms test chip | tip 10 of this invention is demonstrated. FIG. 2 is a perspective view of the microorganism detecting unit 18 as viewed from the side of the microorganism testing chip 10. A microorganism detecting unit 18 is mounted in the concave portion of the microorganism testing chip 10. The microorganism detection unit 18 may not be an integral structure with the microorganism testing chip 10 but may be a separately manufactured member. Therefore, the microorganism detecting unit 18 may be made of a material different from the material constituting the microorganism testing chip 10.

  The microorganism detection unit 18 is formed of a light-transmitting material that has excellent optical characteristics and a small change in shape. By using such a material, optimal measurement conditions such as a physical quantity such as an incident amount of excitation light and a focal length can be stably maintained. The microorganism detection unit 18 is preferably formed of quartz or optical glass. Quartz and optical glass are preferable because they are excellent in optical performance and have less autofluorescence than resin.

  The microorganism detection unit 18 has a through hole. This hole is the microorganism detection flow path 181. The cross section of the microorganism detection flow path 181 is rectangular or circular, but is preferably square. The larger the inner diameter of the microorganism detection channel 181 is, the smaller the pressure loss is. However, in order to flow the microorganisms one by one, it is better to have a smaller one.

  The inner diameter of the microorganism detection flow path 181 is 1 μm to 1 mm. That is, the cross section of the microorganism detection flow path 181 is a square having a side of 1 μm to 1 mm, or a circle having a diameter of 1 μm to 1 mm.

  The cross section of the microorganism detection unit 18 is rectangular or circular, but is preferably square. The outer diameter of the microorganism detection unit 18 is 0.1 mm to 5 mm. That is, the cross section of the microorganism detection unit 18 is a square having a side of 0.1 mm to 5 mm, or a circle having a diameter of 0.1 mm to 5 mm.

  The upper and lower ends of the microorganism detection unit 18 are embedded in the microorganism test chip 10. The length of the part exposed from the microorganism test chip 10 in the microorganism detection unit 18 is preferably 0.01 mm to 10 mm.

  The microorganism detecting channel 181 is connected to the upper solution channel 1573 and the lower solution channel 1574. The inner diameters of the solution flow channels 1573 and 1574 are 10 μm to 1 mm. Therefore, the cross-sectional area of the microorganism detection flow path 181 is sufficiently smaller than the cross-sectional areas of the solution flow paths 1573 and 1574.

  The detection optical system will be described. The detection optical system includes an irradiation optical system having a light source that emits excitation light, and a light receiving optical system having a light receiving element that detects fluorescence from microorganisms. Arrow A in the figure indicates the optical axis of the excitation light. An arrow B in the figure indicates the central axis of the objective lens 120 provided in the light receiving optical system. In this example, the optical axis of the excitation light and the central axis of the objective lens 120 are configured to be orthogonal. The optical axis of the excitation light is parallel to the main surface of the microorganism testing chip 10, but the central axis of the objective lens 120 is orthogonal to the main surface of the microorganism testing chip 10.

  The microorganism detection flow path 181 is irradiated with excitation light. The excitation light is applied to the irradiation range 183 of the microorganism detection flow path 181. The microorganism 175 flows in the microorganism detection flow path 181 from the top to the bottom of the figure. The microorganism 175 is irradiated with excitation light when passing through the irradiation range 183. The microorganism 175 emits fluorescence of the dead bacteria staining dye 1531 or the whole bacteria staining dye 1541. This fluorescence is detected by a light receiving optical system including the objective lens 120.

  In this example, the problems of stray light generation due to reflection of excitation light and a decrease in the amount of received fluorescence can be solved. Since the optical axis of the excitation light and the central axis of the objective lens are orthogonal to each other, the light receiving optical system does not detect the reflected light of the excitation light as stray light. Further, the optical axis of the excitation light is orthogonal to the main surface of the microorganism testing chip 10. Therefore, the amount of excitation light reflected on the main surface of the microorganism testing chip 10 is small. Therefore, in this example, the excitation light can be efficiently applied to the microorganism detection flow path 181.

  When a part of the microorganism testing chip 10 is on the optical axis of the excitation light or on the central axis of the objective lens 120, the amount of the excitation light irradiated to the microorganism detection flow path 181 is reduced. Further, when a part of the microbe inspection chip 10 is present on the central axis of the objective lens 120, the amount of fluorescence received by the light receiving element is reduced.

  According to the present invention, the microorganism detecting unit 18 is not surrounded by or surrounded by a part of the microorganism testing chip 10. In this example, the optical axis of the excitation light and the central axis of the objective lens 120 are configured to be orthogonal. Therefore, the surroundings of the microorganism detection unit 18 may be exposed in a range of at least 90 degrees. It is sufficient that there are no obstacles that interfere with the irradiation of excitation light and the detection of fluorescence within a range of at least 90 degrees around the microorganism detection unit 18. Preferably, the surroundings of the microorganism detection unit 18 may be exposed in a range of at least 180 degrees. It is sufficient that there is nothing that interferes with irradiation of excitation light and detection of fluorescence within a range of at least 180 degrees around the microorganism detection unit 18.

  Therefore, the excitation light from the light source is directly applied to the microorganism detection flow path 181 without being blocked by a part of the microorganism test chip 10. Further, the fluorescence from the microorganism detection channel 181 is directly received by the light receiving optical system without being blocked by a part of the microorganism testing chip 10. Therefore, there is no reduction in the amount of excitation light irradiated and no reduction in the amount of fluorescence received.

  An example of the configuration of the microorganism testing apparatus 1 according to the present invention will be described with reference to FIG. The microorganism testing apparatus 1 of this example includes an analysis apparatus 11, a system apparatus 111, an output apparatus 112, and a communication device 114. When the microorganisms are measured by the microorganism testing apparatus 1, the microorganism testing chip 10 is loaded inside the analyzer 11. By changing the type of the microorganism testing chip 10, it is possible to carry out an optimal microorganism count measurement process for various specimens. The system apparatus 111 performs control of the microorganism testing apparatus 1 and processing of electrical signals output from the microorganism testing apparatus 1. The output device 112 outputs the analysis result obtained by the analysis device 11.

  In addition, the system device 111 exchanges data with the server 113 through the communication device 114. The server 113 collects output results from the plurality of microorganism testing apparatuses 1 via the communication device 114 and performs analysis. Further, the server 113 transmits an optimal control method of the microorganism testing apparatus 1 to the plurality of microorganism testing apparatuses 1 via the communication device 114 in order to perform the microorganism count measurement process corresponding to the type of specimen.

  A detailed example of the configuration of the microorganism testing apparatus 1 according to the present invention will be described with reference to FIG. The microorganism testing apparatus 1 includes an analysis device 11, a system device 111, and an output device 112. The analysis device 11 includes a holding unit that holds the microorganism testing chip 10, a detection device 12 that optically detects microorganisms flowing through the microorganism detection unit of the microorganism testing chip 10, and a pressure source that provides a differential pressure to the microorganism testing chip 10. It has a device 14. The microbe inspection chip 10 held in the holding unit and the pressure source device 14 are connected by a chip connecting pipe 144. The detection device 12 and the pressure source device 14 are connected to the system device 111. The system device 111 is connected to the output device 112.

  As shown in FIG. 1, the microbe inspection chip 10 includes a mechanism for holding a specimen and a staining reagent therein and performing a process necessary for microbe measurement. The pressure source device 14 opens the vent holes 1631, 1632, 1633, and 1634 (FIG. 1) of the microorganism testing chip 10 to the atmosphere via the chip connection pipe 144, or supplies air of a predetermined pressure thereto. Thereby, steps necessary for microbial measurement are executed. The detection device 12 has a detection optical system. That is, the detection device 12 includes an irradiation optical system having a light source that emits excitation light, and a light receiving optical system having a light receiving element that detects fluorescence from microorganisms. The detection optical system will be described later with reference to FIG. The detection device 12 generates an electrical signal indicating the detection result of the microorganism.

  The system device 111 outputs a control signal to the pressure source device 14, receives an electrical signal indicating a detection result from the detection device 12, and processes it. A measurement result obtained by processing the electrical signal is displayed on the output device 112.

  An example of the detection optical system included in the detection device 12 will be described with reference to FIG. The configuration of the detection optical system may change depending on the excitation spectrum and fluorescence spectrum of the dye used for staining the microorganism. Here, the case where DAPI (4 ′, 6-diamidine-2′-phenylindole) is used as the dead bacteria staining dye 1531 and PI (propidium iodide) as the whole bacteria staining dye 1541 will be described. The excitation wavelength peak of PI is 532 nm, and the fluorescence wavelength peak is 615 nm. The excitation wavelength peak of DAPI is 365 nm, and the fluorescence wavelength peak is 460 nm.

  The detection device 12 of this example has an irradiation optical system and a light receiving optical system. The irradiation optical system includes a short wavelength (wavelength 365 nm) laser light source 135, a long wavelength (wavelength 532 nm) laser light source 136, a light source dichroic mirror 137 that reflects light having a wavelength of 450 nm or more, and a laser beam luminous flux. Cylindrical lenses 133 and 134 for condensing the light. The light receiving optical system includes an objective lens 120 that condenses fluorescent light into parallel rays, a detection dichroic mirror 123 that reflects light having a wavelength of 450 nm or less, a mirror 124, and a short-wavelength optical device that passes only light having a wavelength near 460 nm. A filter 125, a long-wavelength optical filter 126 that allows light having a wavelength in the vicinity of 615 nm to pass through, condensing lenses 127 and 128 for condensing parallel light, and pinholes 129 and 130 used as spatial filters for cutting stray light A short wavelength photo 131 for detecting light that has passed through the short wavelength optical filter 125, and a long wavelength photo 132 for detecting light that has passed through the long wavelength optical filter 126.

  The microorganism detection flow path 181 is disposed at the focal points of the cylindrical lens 144 and the objective lens 120, and the pinholes 129 and 130 are disposed at the focal positions of the condenser lenses 127 and 128.

  The short wavelength laser light source 135 is a short wavelength excitation light source for DAPI, and generates excitation light having a wavelength of 365 nm. The long wavelength laser light source 136 is a long wavelength excitation light source for PI, and generates excitation light having a wavelength of 532 nm. Laser light from the short wavelength laser light source 135 passes through the light source dichroic mirror 137, is transformed into a light beam having an elliptical cross section by the cylindrical lenses 133 and 134, and is irradiated to the microorganism detection flow path 181. The laser light from the long wavelength laser light source 136 is reflected by the light source dichroic mirror 137, changed in its path by 90 degrees, progressed by the cylindrical lenses 133 and 134, and transformed into an elliptical light beam, and the microorganism detection flow path 181. Is irradiated. When laser light is irradiated to the microorganism 175 that passes through the microorganism detection channel 181, the microorganism emits fluorescence.

  Excitation light (wavelength 532 nm) from the long wavelength laser light source 136 excites the PI that stains the microorganism 175. Fluorescence with a wavelength peak of 615 nm is generated from PI. Excitation light (wavelength 360 nm) from the short wavelength laser light source 135 excites the DAPI that stains the microorganism 175. From DAPI, fluorescence having a wavelength peak of 460 nm is generated.

  This fluorescence is collected by the objective lens 120 and converted into parallel rays. The parallel light is guided to the detection dichroic mirror 123.

  Fluorescence from the PI (fluorescence wavelength peak 615 nm) passes through the detection dichroic mirror 123. The fluorescence from PI further passes through the long wavelength optical filter 126 and is collected by the condenser lens 128. The condensed fluorescence passes through the pinhole 130, whereby stray light is cut and reaches the long wavelength photomultiplier 132.

  Fluorescence (fluorescence wavelength peak 460 nm) from DAPI reflects the dichroic mirror for detection 123, travels by changing the course by 90 degrees, reflects by the mirror 124, and travels again by changing the course by 90 degrees. The fluorescence from the DAPI further passes through the short wavelength optical filter 125 and is collected by the condenser lens 127. The condensed fluorescence passes through the pinhole 129, whereby stray light is cut and reaches the short wavelength photomultiplier 131.

  In this way, in this example, since the detection dichroic mirror 123 that reflects light having a wavelength of 450 nm or less is used in the light receiving optical system, the fluorescence derived from the two dyes can be separated by the difference in wavelength.

  The fluorescence that has entered the short wavelength photomultiplier 131 and the long wavelength photomal 132 is converted into an electrical signal, and the electrical signal is sent to the system device 111. The system apparatus 111 processes the electrical signals sent from the short wavelength photomultiplier 131 and the long wavelength photomal 132 to obtain information on the number of microorganisms. That is, the number of dead bacteria is detected based on the fluorescence signal from the PI output from the long wavelength photo 132. Based on the fluorescence signal from DAPI output from the short wavelength photomultiplier 131, the total number of bacteria is detected. The inspection result is output to the output device 112.

  The assembly method of the first example of the microorganism testing chip 10 according to the present invention will be described with reference to FIG. The microorganism testing chip 10 of this example is created by bonding an upper member 101 and a lower member 102 together. The upper member 101 and the lower member 102 may be manufactured by injection molding resin. The upper member 101 is formed of a plate material whose both surfaces are flat. Ventilation holes 1631, 1632, 1633, and 1634 are formed in the upper member 101. These vents are through holes. Dashed lines 151, 153, 154, 161 shown in the upper member 101 indicate the positions of the sample container 151, the dead bacteria staining reagent holding container 153, the whole bacteria staining reagent holding container 154, and the unnecessary component removing unit 16. A broken line 180 shown on the upper member 101 indicates the position of the microorganism detection unit support 180 formed on the lower member 102.

  Solid lines 1631, 1632, 1633, and 1634 shown in the lower member 102 indicate holes that constitute the vent holes. Solid lines 151, 153, 154, and 161 shown in the lower member 102 represent grooves that constitute the sample container 151, the dead bacteria staining reagent holding container 153, the whole bacteria staining reagent holding container 154, and the unnecessary component removing unit 16. In addition, solid lines 1581 to 1584 shown in the lower member 102 indicate grooves constituting the air flow paths 1581 to 1584. Solid lines 1571 to 1574 shown in the lower member 102 indicate grooves constituting the solution flow paths 1571 to 1574. By attaching the upper member 101 and the lower member 102 together, the grooves formed in the lower member 102 form a specimen container 151, a dead bacteria staining reagent holding container 153, a whole bacteria staining reagent holding container 154, and a solution flow path 1571 to 1574. , And air flow paths 1581 to 1584 are formed. In order to prevent liquid leakage, a viscous material such as an adhesive may be applied to the contact surface between the upper member 101 and the lower member 102 for sealing.

  The unnecessary component removal unit 16 includes a filter 160. That is, the filter 160 is fixed by inserting the filter 160 into a groove formed in the lower member 102 and bonding the upper member 101 and the lower member 102 together. Filter 160 is preferably configured to remove particles having a diameter of 10 microns or greater. In this case, a groove for engaging the filter 160 may also be provided in the upper member 101.

  In this example, the microorganism detection unit 18 is formed of a columnar member. The lower member 102 is formed with a microorganism detection unit support unit 180. The microorganism detection unit support unit 180 may be a recess. The microorganism detection unit 18 is fixed by engaging the microorganism detection unit 18 with the microorganism detection unit support unit 180 and bonding the upper member 101 and the lower member 102 together. In this case, the upper member 101 may also be provided with a groove for engaging the microorganism detection unit 18.

  The structure of the second example of the microorganism testing chip according to the present invention will be described with reference to FIG. Here, a case where the microorganisms in the food are inspected by the microorganism inspection apparatus of the present invention will be described. In this case, the specimen 1511 is a food to be examined. The microbe inspection chip 20 of this example includes a specimen container 151 for holding a specimen 1511, a dead bacteria staining reagent holding container 153 for holding a dead bacteria staining dye 1531 for staining only dead bacteria, and a whole container for staining all bacteria. The whole bacteria staining reagent holding container 154 that holds the bacteria staining dye 1541, the unnecessary component removal unit 16 that is a filter for removing food residues contained in the sample, and excitation light from an external light source are irradiated into the sample. A microorganism detection unit 28 for observing fluorescence from contained microorganisms and a waste liquid container 163 for discarding a solution containing the specimen that has passed through the microorganism detection unit 28 are provided. Similar to the first example of FIG. 1, the microorganism testing chip 20 of this example includes a solution flow channel 1571 to 1574, an air flow channel and vent holes 1631 to 1634. As shown in the figure, the microorganism testing chip 20 of this example is used in an upright state so that the waste liquid container 163 is at the bottom.

  In the first example shown in FIG. 1, the microorganism detection flow path included in the microorganism detection unit 18 is arranged in parallel to the main surface of the microorganism testing chip. However, in the microorganism testing chip 20 of the present example, the microorganism detection flow path included in the microorganism detection unit 28 is perpendicular to the main surface of the microorganism testing chip 20. That is, the microorganism detection flow path included in the microorganism detection unit 28 is arranged along the thickness direction of the microorganism testing chip 20.

  Arrow B in the figure indicates the optical axis of the excitation light. An arrow D in the figure indicates the central axis of the objective lens provided in the light receiving optical system. In this example, the optical axis of the excitation light and the central axis of the objective lens are configured to be orthogonal. Both the optical axis of the excitation light and the central axis of the objective lens are parallel to the main surface of the microorganism testing chip 20.

  Details of the microorganism detection unit 28 will be described with reference to FIG. FIG. 8 shows a cross-sectional configuration cut along the line a-a ′ of FIG. 7. The microorganism testing chip 20 of this example includes an upper member 201, a lower member 202, an upper flat plate 203, and a lower flat plate 204. The upper member 201 and the lower member 202 are bonded to each other, and the upper flat plate 203 and the lower flat plate 204 are bonded to both sides thereof. The microorganism detection unit 28 is mounted between the upper member 201 and the lower member 202. Packings 191 and 192 are attached to both ends of the microorganism detection unit 28. The microorganism detection unit 28 is elastically supported by the two packings 191 and 192. The microorganism detection unit 28 of this example may have the same structure as the microorganism detection unit 18 of the first example. As illustrated, the microorganism detection unit 28 includes a microorganism detection flow path 281. The microorganism detection flow path 281 is a through hole formed in the microorganism detection unit 28. In FIG. 8, in order to explain the microorganism 175 passing through the microorganism detection flow path 281, the inner diameter of the microorganism detection flow path 281 is exaggerated. Actually, as shown in FIG. 9, the inner diameter of the microorganism detection channel 281 is sufficiently smaller than the inner diameters of the solution channels 1573 and 1574.

  An arrow D in the figure indicates the central axis of the objective lens provided in the light receiving optical system. The optical axis of the excitation light is perpendicular to the paper surface. The excitation light is applied to the irradiation range 183 of the microorganism detection flow path 181. The microorganism 175 flows in the microorganism detection channel 181 from the left to the right in the figure. The microorganism 175 is irradiated with excitation light when passing through the irradiation range 183. The microorganism 175 emits fluorescence of the dead bacteria staining dye 1531 or the whole bacteria staining dye 1541. This fluorescence is detected by a light receiving optical system including the objective lens 120.

  The members constituting the microorganism testing chip 20 are manufactured by resin injection molding. The microorganism detection unit 28 is manufactured as a separate member from the microorganism testing chip 20. Therefore, the installation position of the microorganism detecting unit 28 in the microorganism testing chip 20 is not always accurate. The installation position of the microorganism detection unit 28 varies for each microorganism testing chip. The position of the microorganism testing chip loaded in the analyzer 11 also varies for each microorganism testing chip. Therefore, it is necessary to align the microbe detection unit 28 of the microbe inspection chip loaded in the analyzer 11 and the objective lens 120 of the detection optical system.

  According to the present invention, as described below, the microorganism detection unit 28 and the objective lens 120 of the detection optical system can be easily positioned.

  The structure of the detection optical system according to the present invention will be described with reference to FIG. As shown in the figure, an objective lens 120 is provided in the vicinity of the microorganism detection unit 28. The objective lens 120 is held by a lens holder 121. The lens holder 121 is supported on the microorganism detection unit 28 by two blades 122. The microorganism detection unit 28 is attached to the upper member 101 and the lower member 102 via packings 191 and 192, respectively. The packings 191 and 192 are made of an elastically deformable material.

  The positioning in the detection optical system according to the present invention will be described with reference to FIG. The objective lens 120 is held by a pair of lens holders 121 so as to be sandwiched from both sides. Each lens holder 121 is provided with a blade 122. Each of the two blades 122 has a tapered portion 122a. The two taper portions 122a face each other, and a V-shape having a predetermined sandwich angle is formed between them. Preferably, the inclination angles of the two tapered portions 122a are symmetric with respect to a line connecting the central axis of the objective lens 120 and the center of the microorganism measurement flow path 281. In this example, the microorganism detection unit 28 is formed of a columnar member. The V-shape formed by the two taper portions 122a is in contact with the cylindrical surface of the columnar member of the microorganism detection unit 28.

  As shown in the figure, the Z axis is taken along a line connecting the center of the microorganism measurement flow path 281 and the center of the objective lens 120, and the Y axis is taken in the direction of the microorganism measurement flow path 281 (perpendicular to the paper surface). The X axis is taken in a direction orthogonal to

  The tapered portions 122a of the two blades 22 define the distance between the microorganism measurement flow path 281 and the objective lens 120, and at the same time arrange the center of the microorganism measurement flow path 281 and the center of the objective lens 120 in a straight line. .

  The distance between the microbe measurement channel 281 and the objective lens 120 is set so that the focal point of the objective lens 120 is arranged at the center of the microbe measurement channel 281. Next, the position of the objective lens 120 in the X direction with respect to the microorganism measurement flow path 281 is set so that the center axis of the objective lens 120 and the center of the microorganism measurement flow path 281 are aligned. Thus, when the position of the objective lens 120 with respect to the microorganism measurement flow path 281 is set, the length of the blade 122 and the inclination angle of the tapered portion 122a can be determined.

  Thus, when the size and shape of the two blades 22 are determined, the objective lens 120 is positioned. The objective lens 120 and the lens holder 121 are arranged so that the two tapered portions 122a are in contact with the cylindrical surface of the columnar member of the microorganism detecting unit. The tapered portions 122a of the two blades 22 are pressed against the cylindrical surface of the columnar member of the microorganism detecting unit 28 by a slight force. At this time, due to elastic deformation of the packings 191 and 192, the position or posture of the microorganism detection unit 28 can be slightly changed.

  The relative position between the microorganism detection unit 28 and the objective lens 120 is determined by the contact between the tapered portion 122a of the two blades 122 and the microorganism detection unit 28. That is, the focal position of the objective lens 120 can be arranged on the central axis of the irradiation range 183 of the microorganism detection flow path 181 of the microorganism detection unit 28. When the outer diameter of the columnar member of the microorganism detection unit 28 changes, the distance between the center of the objective lens 120 and the center of the microorganism measurement flow path 281 is slightly changed. However, the irradiation range 283 of the microorganism detection flow path 281 can be arranged on the central axis of the objective lens 120.

  According to this example, even if the accuracy of the installation position of the microorganism measurement channel 281 in the microorganism test chip is not high, or even if the accuracy of the installation position of the microorganism test chip in the microorganism test device 1 is not high, The objective lens 120 can be easily positioned with respect to the flow path 281.

  The objective lens support mechanism described with reference to FIG. 10 is also applicable to the first example of the microorganism testing chip of the present invention shown in FIG.

  A method for assembling the second example of the microorganism testing chip according to the present invention will be described with reference to FIG. The microorganism testing chip 20 of this example is created by bonding the upper member 201 and the lower member 202 together. An upper flat plate 203 is bonded to the outer surface of the upper member 201, and a lower flat plate 204 is bonded to the outer surface of the lower member 202. The upper member 201 and the lower member 202 may be manufactured by injection molding a resin. The upper member 201, the upper flat plate 203, and the lower flat plate 204 are formed of plate materials having flat surfaces.

  When the microorganism testing chip 20 of this example is compared with the first example shown in FIG. 6, the structure of the microorganism detecting unit 28 is different. Further, the structure of the solution flow path connected to the microorganism detection unit 28 is different. In the microorganism testing chip 20 of this example, an upper flat plate 203 and a lower flat plate 204 are used.

  Here, the structure of the microorganism detecting unit 28 of the microorganism testing chip 20 of this example and the solution flow path connected thereto will be described. As described with reference to FIG. 7, the solution flow channels 1573 and 1574 are connected to the microorganism detection unit 28. The solution channel 1573 is a channel that connects the whole bacteria staining reagent holding container 154 and the microorganism detection unit 28. The solution channel 1574 is a channel that connects the microorganism detection unit 28 and the waste liquid container 163.

  As shown in FIG. 11, a solid line 1573 a shown in the lower member 202 and a solid line 1573 b shown in the upper flat plate 203 indicate grooves constituting the solution flow path 1573. A broken line 1573c shown in the upper flat plate 203 indicates a flow path connecting the two. The flow path 1573 c extends in a direction orthogonal to the main surface of the microorganism testing chip 20. A solid line 1574b and a broken line 1574a shown in the lower member 202 indicate grooves constituting the solution flow path 1574. In addition, the groove | channel represented by the broken line 1574a is formed in the surface on the opposite side to the surface in which the groove | channel represented by the continuous line 1574b is formed. A broken line 1574c shown in the lower member 202 indicates a flow path connecting the two.

  By attaching the upper member 201 and the lower member 202 together, the groove 1573 a formed in the lower member 202 forms a part of the solution flow path 1573. By bonding the upper member 201 and the upper flat plate 203, the groove 1573 b formed in the upper member 201 forms a part of the solution flow path 1573. The two flow paths are connected by a hole 1573 c that penetrates the upper member 201.

  By bonding the upper member 201 and the lower member 202, the groove 1574 b formed in the lower member 202 forms a part of the solution flow path 1574. By attaching the lower member 202 and the lower flat plate 204 together, the groove 1574 a formed in the lower member 202 forms a part of the solution flow path 1574. The two flow paths are connected by a hole 1574 c that penetrates the lower member 202.

  In this example, the microorganism detection unit 28 is formed of a columnar member. The microorganism detection unit 28 is mounted such that the microorganism measurement flow path 28 is arranged along a direction orthogonal to the main surface of the microorganism testing chip 20. The microorganism detection unit 28 is mounted between the upper member 201 and the lower member 202 while being sandwiched between the two packings 191 and 192.

  The inlet of the microorganism detection unit 28 is connected to the flow channel 1574 b of the solution flow channel 1573, and the outlet of the microorganism detection unit 28 is connected to the flow channel 1574 a of the solution flow channel 1574.

  As shown in FIG. 6, in the first example of the microorganism testing chip, both ends of the microorganism detecting unit 18 are supported by the members of the microorganism testing chip. Therefore, the dimension of the microorganism measurement flow path 18 becomes long. On the other hand, in the microorganism testing chip 20 of this example, both ends of the microorganism detecting unit 28 are supported by packings 191 and 192. Therefore, the length of the microorganism measurement flow path 281 can be shortened. Since the length of the microorganism measurement flow path is shortened, the pressure loss of the fluid flowing inside can be further reduced. In addition, there is no defect due to the protruding adhesive.

  The example of the present invention has been described above. However, the present invention is not limited to the above-described example, and various modifications can be easily made by those skilled in the art within the scope of the invention described in the claims. Will be understood.

It is a figure which shows the 1st example of the microbe test chip | tip of this invention. It is a perspective view of the principal part of the 1st example of the microbe test chip | tip of this invention. It is a figure which shows the example of a structure for the microorganisms test | inspection apparatus of this invention. It is a figure which shows the state which also loaded the microbe test | inspection chip | tip of the microbe test device of this invention. It is a figure which shows the example of a structure of the detection optical system of the microbe test | inspection apparatus of this invention. It is a figure explaining the assembly method of the 1st example of the microorganisms test chip | tip of this invention. It is a figure which shows the 2nd example of the microbe test chip | tip of this invention. It is a figure which shows the cross-sectional structure of the principal part of the 2nd example of the microbe test chip | tip of this invention. It is a figure which shows the example of the support mechanism of the objective lens of the detection optical system of the microbe test | inspection apparatus of this invention. It is a figure which shows the example of the support mechanism of the objective lens of the detection optical system of the microbe test | inspection apparatus of this invention. It is a figure explaining the assembly method of the 2nd example of the microbe test chip | tip of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Microorganism test apparatus, 10 ... Microorganism test chip, 11 ... Analysis apparatus, 12 ... Detection apparatus, 14 ... Pressure source apparatus, 16 ... Unnecessary component removal part, 18 ... Microbe detection part, 20 ... Microorganism test chip, 28 ... Microorganism Detecting unit, 101 ... upper member, 102 ... lower member, 111 ... system device, 112 ... output device, 113 ... server, 114 ... communication device, 120 ... objective lens, 121 ... lens holder, 122 ... blade, 123 dichroic for detection Mirror, 124 ... Mirror, 125 ... Short wavelength optical filter, 126 ... Long wavelength optical filter, 127, 128 ... Condensing lens, 129 ... Pinhole, 131 ... Short wavelength photomultiplier, 132 ... Long wavelength photomultiplier, 133 134 ... Cylindrical lens, 135 ... Short wavelength laser, 136 Long wavelength laser, 137 ... Dichroic mirror for light source DESCRIPTION OF SYMBOLS 144 ... Chip | tip connection pipe | tube, 151 ... Sample container, 153 ... Dead bacteria dye container, 154 ... Whole bacteria dye container, 155 ... Eluent container, 156 ... Detection liquid container, 157 ... Solution flow path, 158 ... Air 160, unnecessary component removing member, 161 ... unnecessary component removing unit holding unit, 163 ... waste liquid container, 175 ... microorganism, 180 ... microorganism detecting channel holding unit, 181 ... microorganism detecting channel, 182 ... excitation Light, 183 ... excitation light irradiation range, 184 ... fluorescence, 191, 192 ... packing, 201 ... upper member, 202 ... lower member, 203 ... upper flat plate, 204 ... lower flat plate, 281 ... channel for detecting microorganisms

Claims (20)

In a microorganism testing chip having a substrate, and a microorganism detecting unit attached to the substrate and having a microorganism detecting channel inside,
The substrate is
A sample container for holding a sample containing microorganisms, a reaction container for holding a reagent that reacts with the sample, a waste liquid container for storing a mixed solution of the sample and the reagent, and one end connected to the sample container A first air channel having the other end connected to the vent, a second air channel having one end connected to the reaction vessel and the other end connected to the vent, and one end of the waste vessel And a third air flow path having the other end connected to the vent, and
The substrate further includes the sample container, the reaction container, a microorganism detection flow path of the microorganism detection unit, and a solution flow path connected in series with the waste liquid container,
The microbe detection chip, wherein the microbe detection unit is formed of a light-transmitting material, and a range of at least 90 degrees around the microbe detection unit is exposed.   2. The microbe inspection chip according to claim 1, wherein a range of at least 180 degrees around the microbe detection unit is exposed.   2. The microbe inspection chip according to claim 1, wherein the microbe detection flow path is arranged in a direction parallel to the main surface of the substrate.   2. The microbe inspection chip according to claim 1, wherein the microbe detection flow path is arranged in a direction perpendicular to the main surface of the substrate.   The microbe inspection chip according to claim 1, wherein both ends of the microbe detection part are supported by elastic members.   2. The microorganism testing chip according to claim 1, wherein the microorganism detection flow path has a square cross section with a side of 1 μm to 1 mm or a circular cross section with a diameter of 1 μm to 1 mm.   The microbe inspection chip according to claim 1, wherein the microbe detection unit has a square cross section with a side of 0.1 mm to 5 mm or a circular cross section with a diameter of 0.1 mm to 5 mm. Chip.   2. The microorganism testing chip according to claim 1, wherein the solution flow path has a square cross section with a side of 10 μm to 1 mm or a circular cross section with a diameter of 10 μm to 1 mm.   2. The microorganism testing chip according to claim 1, wherein the substrate is constituted by adhering two or more members.   2. The microorganism testing chip according to claim 1, wherein the microorganism detecting unit is made of quartz or glass.   2. The microorganism testing chip according to claim 1, wherein a filter is provided on the outlet side of the specimen container.   The thing of Claim 1 WHEREIN: The said reaction container is the whole bacteria dyeing | staining which hold | maintains the dead bacteria dyeing | staining reagent holding | maintenance container which hold | maintains the dead bacteria dyeing pigment | dye which dye | stains only dead bacteria, A microorganism testing chip comprising a reagent holding container. In a microorganism testing apparatus comprising: a holding unit that holds a microorganism testing chip; a pressure source device that provides a differential pressure to the microorganism testing chip held in the holding unit; and a detection device that generates excitation light and receives fluorescence. ,
The microorganism testing chip includes a substrate, and a microorganism detecting unit that is attached to the substrate and has a microorganism detecting channel therein.
The substrate includes a vent that can be connected to the pressure source device, a sample container for holding a specimen containing microorganisms, a reaction container for holding a reagent that reacts with the specimen, and a mixture of the specimen and the reagent A waste liquid container for storing liquid; a first air flow path having one end connected to the sample container and the other end connected to the vent; and one end connected to the reaction container and the other end to the vent A second air flow path connected, and a third air flow path having one end connected to the waste liquid container and the other end connected to the vent;
The substrate further includes the sample container, the reaction container, a microorganism detection flow path of the microorganism detection unit, and a solution flow path connected in series with the waste liquid container,
The detection device receives a light source that irradiates excitation light to the microorganism detection flow path, an objective lens that collects fluorescence from the microorganism detection flow path, and fluorescence collected by the objective lens, A light receiving element that converts it into an electrical signal,
The microorganism testing apparatus, wherein an optical axis of the excitation light is orthogonal to a central axis of the objective lens.   14. The microorganism testing apparatus according to claim 13, wherein the microorganism detection unit is formed of a light transmissive material, and a range of at least 90 degrees around the microorganism detection unit is exposed. apparatus.   14. The microorganism testing apparatus according to claim 13, wherein the microorganism detecting unit is formed of a light transmissive material, and a range of at least 180 degrees around the microorganism detecting unit is exposed. apparatus.   14. The microorganism testing apparatus according to claim 13, wherein the microorganism detection flow path is arranged in a direction perpendicular to the main surface of the substrate or in a direction parallel to the main surface of the substrate. Microbiological testing device.   14. The microorganism testing apparatus according to claim 13, wherein the detection device includes a pair of lens holders for holding the objective lens, and blades attached to the lens holders, and the blades have a tapered portion. The microbe inspection apparatus is configured such that the relative position of the objective lens with respect to the microbe detection part is determined by the taper part of the blade contacting the outer surface of the microbe detection part.   14. The microorganism testing apparatus according to claim 13, wherein the focal point of the objective lens is disposed in the microorganism detection flow path. In a microorganism testing method using a microorganism testing chip having a substrate, and a microorganism detecting unit attached to the substrate and formed of a light-transmitting material having a microorganism detecting channel inside,
Holding the microorganism testing chip upright;
Irradiating the microorganism detection unit with excitation light from a direction orthogonal to the microorganism detection flow path;
Receiving the fluorescence from the microorganism detection flow path by a light receiving optical system;
And the optical axis of the objective lens included in the light receiving optical system is orthogonal to the optical axis of the excitation light. The microorganism testing method according to claim 19,
The substrate is
A sample container for holding a sample containing microorganisms, a reaction container for holding a reagent that reacts with the sample, a waste liquid container for storing a mixed solution of the sample and the reagent, and one end connected to the sample container A first air channel having the other end connected to the vent, a second air channel having one end connected to the reaction vessel and the other end connected to the vent, and one end of the waste vessel And a third air flow path having the other end connected to the vent, and
The substrate further includes the sample container, the reaction container, a microorganism detection channel of the microorganism detection unit, and a solution channel connected in series with the waste liquid container. Method.

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