JP2011220947A - Microbiological testing apparatus and microbiological testing chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microbiological testing chip which can reduce effect of reflection of excitation light even when an optical axis of the excitation light and a central axis of a condenser lens for fluorescence or an objective lens are coaxial, and provide a microbiological testing apparatus using the microbiological testing chip.SOLUTION: A microbiological testing chip 10 comprises a main body 15 and a bacteria detection part 17. The bacteria detection part 17 is formed by bonding a cover member 171 and a channel member 172 and comprises a bacteria detection channel 173. The bacteria detection part 17 is arranged to be inclined in the microbiological testing chip 10 so that a normal vector of the bacteria detection part 17 on an incident surface and the optical axis of the excitation light are not parallel.

Description

  The present invention relates to a microorganism testing apparatus and a microorganism testing chip that measure microorganisms.

  2. Description of the Related Art Conventionally, various bacterial count measuring devices for the purpose of speeding up and simplifying the viable count are known. Some bacterial count measuring apparatuses use a fluorescent flow cytometry method. The fluorescent flow cytometry method is a particle measurement method in which a fluid containing a specimen stained with a fluorescent dye directly measures the specimen one by one when passing through a small diameter channel.

  In the fluorescent flow cytometry method, as a technique for testing microorganisms more easily, a container for containing a sample liquid containing microorganisms and a reagent liquid (staining liquid) containing a fluorescent dye is provided in the microorganism test chip, The reagent solution is previously held in each container in the microorganism chip, and this microorganism test chip is placed or inserted in the detection device so that the sample liquid flows through the flow path in the microorganism test chip. Some detection parts are irradiated with excitation light to count microorganisms (Patent Documents 1 to 3).

JP 2008-157829 A JP 2009-178078 A JP 2009-281753 A

  In Patent Document 2, in order to avoid generation of stray light due to reflection of excitation light and avoid a decrease in the amount of received fluorescence, a microorganism detection unit is formed of a light-transmitting material, and at least around the microorganism detection unit The 90 ° range is exposed, the optical axis of the excitation light is made parallel to the main surface of the microbe inspection chip, and the central axis of the fluorescent light collecting lens for fluorescence detection is orthogonal to the main surface of the microbe inspection chip It is configured to be. Since the optical axis of the excitation light and the central axis of the fluorescent condensing lens are orthogonal, the light receiving optical system does not receive the reflected light of the excitation light as stray light.

  However, in order to simplify the optical system, it is desirable that the optical axis of the excitation light and the central axis of the fluorescent condensing lens (or objective lens) be coaxial. In addition, alignment is facilitated when the optical axis of the excitation light and the central axis of the fluorescent condensing lens are coaxial.

  The present invention relates to a microbe inspection chip capable of reducing the influence of reflection of excitation light even when the optical axis of excitation light and the central axis of a fluorescent condenser lens or objective lens are coaxial, and a microbe inspection using the same To provide an apparatus.

  The microorganism testing chip of the present invention is arranged in the microorganism testing chip by tilting the cell detection unit so that the normal vector on the incident surface of the cell detection unit and the optical axis of the excitation light are not parallel. It is characterized by that.

  If the angle formed by the surface of the fungus body detection unit and the optical axis of the excitation light is θ, the fungus body detection unit is inclined and provided in the microbe test chip so that θ is in the range of 80 to 30 ° (bacterial cells) If the angle between the normal vector on the incident surface of the detector and the optical axis of the excitation light is α, θ + α = 90 °, and θ = 80-30 °, in other words, α = 10-60 °. .)

  θ is more preferably 80 to 70 ° (in other words, α is more preferably 10 to 20 °).

  The microorganism testing apparatus of the present invention is characterized in that the microorganism testing chip configured as described above is attached at right angles to the optical axis of excitation light.

  According to the present invention, even if the optical axis of the excitation light and the central axis of the fluorescent condensing lens are coaxial, reflection of the excitation light is difficult to enter the light receiving optical system, so that the influence of reflection of the excitation light is reduced. Is possible.

  Further, in the present invention, since the microbial cell detection unit in the microbial test chip is tilted, the microbial test chip can be attached to the detection device as compared with the case where the entire microbial test chip is tilted and attached to the test device. It becomes easy.

It is a figure which shows the structural example of the microorganisms test chip | tip of this invention. It is a figure which shows the structural example of the microbial cell detection part of the microbe test chip | tip of this invention. It is a figure which shows the structural example of the microbe test | inspection apparatus of this invention. It is a schematic diagram for demonstrating the alignment of the X direction in the microbe test | inspection apparatus of this invention. It is a schematic diagram for demonstrating the alignment of the Y direction in the microorganisms testing apparatus of this invention. It is a figure which shows the procedure of the alignment of the microbe test | inspection chip | tip in the microbe test | inspection apparatus of this invention. It is a figure which shows the structural example of the detection apparatus in the microbe test | inspection apparatus of this invention. It is a figure which shows the procedure of the microbe test | inspection in the microbe test | inspection apparatus of this invention.

  Embodiments of the present invention will be described below with reference to the drawings.

  The microorganism testing chip used in the present embodiment has a main body and a fungus body detection unit attached to the main body. The main body has a detection window frame portion that is a through-hole or a through groove, and the fungus body detection portion is disposed so as to cover the detection window frame portion. The fungus body detection unit has a fungus body detection flow path connected to a flow path provided in the main body. The fungus body detection unit has a cover member and a flow path member, and is formed by bonding the two members together. The channel member has a groove, and the groove of the channel member forms a fungus body detection channel by bonding two members together. Regarding such a microorganism testing chip, the patent applicant has previously proposed as Japanese Patent Application No. 2008-263055. That is, the microbe test chip is required to be inexpensive in order to be disposable, and in order to improve the detection accuracy, generation of autofluorescence due to induced radiation or fluorescence from the microbe test chip itself is required. Is required to be suppressed. The microbe inspection chip proposed in Japanese Patent Application No. 2008-263055 satisfies these requirements.

  In the following description, the microorganism testing chip and the microorganism testing apparatus proposed in Japanese Patent Application No. 2008-263055 will be described first, and then embodiments of the microorganism testing chip of the present invention will be described in detail.

  FIG. 1 is a schematic view of a microorganism testing chip 10 used by the microorganism testing apparatus of the present invention. First, the configuration of the microorganism testing chip 10 will be described. The microbe inspection chip 10 includes a main body 15 that removes food residues contained in the specimen and stains the fungus body, and a fungus body detection unit 17 that detects the fungus body. The fungus body detection unit 17 includes a fungus body detection flow path 173 for irradiating excitation light from an external light source and observing the fluorescence of the microorganism. Details of the structure of the fungus body detection unit 17 will be described later with reference to FIGS. 2A and 2B. Here, the main body 15 will be described.

  The main body 15 includes a specimen container 151 for holding the specimen 1511, a dead bacteria staining liquid holding container 152 that is a reaction container for holding the dead bacteria staining dye 1521, and a reaction container that holds the whole bacteria staining dye 1531. A bacterial stain holding container 153, an alignment reagent holding container 154 for holding an alignment reagent 1541, a diluent holding container 155 for holding a diluent 1551, and a food residue contained in the sample It has a food residue removal unit 160 that is a filter for removing, and a detection liquid disposal container 156 for discarding various unnecessary waste liquids that have passed through the fungus body detection flow path 173. The waste liquid discarded in the detection liquid waste container 156 includes a sample 1511, a mixture of dead bacteria staining dye 1521, and a whole bacteria staining dye 1531, and an alignment reagent 1541.

  The bottom surfaces of these containers 151, 152, 153, 154, and 155 are formed in a funnel shape. As shown in the drawing, the microorganism testing chip 10 is held in a standing state with the bottom surfaces of these containers facing downward.

  The main body 15 further connects a specimen container 151, a food residue removal unit 160, a dead bacteria staining liquid holding container 152, a whole bacteria staining liquid holding container 153, and a bacterial cell detection flow path 173, and the specimen 1511 and the mixed liquid flow. Solution channels 1571 to 1576 for causing them to flow, vents 1591 to 1596 for causing the sample 1511 and the mixed liquid in each container to flow by atmospheric pressure, and vent channels for connecting the vents 1591 to 1596 and each container 1581 to 1586. The solution flow paths 1571 to 1576, the vents 1591 to 1596, and the flow paths 1581 to 1586 are named from the names of the containers to be connected, and the flow path 1571 between the specimen container and the dead bacteria staining liquid holding container, and the dead bacteria staining liquid holding container- Total bacterial stain holding container flow path 1572, Whole bacterial stain holding container-dilution holding container flow path 1573, Diluted liquid holding container-Bacterial cell detection flow path 1574, Alignment reagent holding container- Bacterial cell detection flow path 1575, Bacterial cell detection flow path-detection liquid waste container flow path 1576, specimen container vent 1591, dead bacterial stain holding container vent 1592, whole bacterial stain holding container vent Mouth 1593, diluent holding container vent 1594, alignment reagent holding container vent 1595, detection liquid disposal container vent 1596, specimen container vent flow path 1581, dead bacteria stain holding container vent flow path 1582, whole bacteria Color liquid holding container vent passage 1583, the diluent holding container vent passage 1584, positioning reagent holding container vent passage 1585, and detection liquid waste container vent passage 1586.

  The sample container 151, the food residue removing unit 160, the dead bacteria staining liquid holding container 152, the whole bacteria staining liquid holding container 153, the dilution liquid holding container 155, the bacterial cell detection flow path 173, and the detection liquid disposal container 156 are a solution. The flow paths 1571 to 1574 and 1576 are connected in series. The alignment reagent holding container-bacterial cell detection flow path channel 1575 joins the diluent holding container-bacterial cell detection flow path channel 1574.

  The depth and the channel width of the solution channel 1571 to 1576 are 10 μm to 3 mm, the depth and the channel width of the ventilation channel 1581 to 1586 are formed in the range of 10 μm to 3 mm, and the solution channel 1571 to 1576 is formed. Is formed so as to be larger than the cross-sectional area of the ventilation channels 1581 to 1586. The manufacturing method of the main body 15 is not specifically described here. As described in Journal of Biomolecular Techniques, Vol 14, Issue 2, pp. 119-127 and Japanese Patent Application Laid-Open No. 2005-245317 cited in Patent Documents 1 to 3, a method for producing a microbe test chip is known. .

  The material which comprises the main body 15 is demonstrated. Since the microorganism testing chip 10 is disposable, the main body 15 is formed of an inexpensive material. The main body 15 has a complicated structure inside. Therefore, the main body 15 is formed of a material that is easy to be finely processed and that has a low processing cost. Glass and quartz have less autofluorescence and excellent optical properties, but are not easily processed finely. That is, processing costs increase when microfabrication is performed. The main body 15 holds the specimen and staining solution before processing. Therefore, the main body 15 is formed of a chemical resistant material. From the above, materials used for the main body 15 include polypropylene, polyethylene terephthalate, polycarbonate, polystyrene, acrylonitrile butadiene styrene resin, polymethacrylic acid methyl ester, and the like. The main body 15 is formed of at least one material selected from these materials.

  The dead bacteria staining solution 1521, the whole bacteria staining solution 1531, and the alignment reagent 1541 are enclosed in advance in the microorganism testing chip 10. The specimen 1511 is injected into the specimen container 151 from the vent 1591 before the examination. The diluent 1551 is injected into the diluent holding container 155 from the vent 1594 before inspection.

  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 diluent 1551 is mainly a buffer solution such as physiological saline, pure water, or phosphate buffered saline. The dilution liquid 1551 is also used as a cleaning liquid.

  For example, PI (propidium iodide) (0.1 μg / ml to 1 mg / ml) is used for the dead bacteria staining solution 1521, and for example, DAPI (4 ′, 6- Diamidin-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), LDS751 ( 0.1 μg / ml to 1 mg / ml) or the like is used.

  As the alignment reagent 1541, a solution containing fluorescent dyes such as PI, DAPI, AO, EB, and LDS751 and fine particles that emit fluorescence of a specific wavelength is used. In order to make the optical system for detection common, the wavelength peak of the alignment reagent 1541 is preferably close to the wavelength peak of the dead bacteria staining solution 1521 or the whole bacteria staining solution 1531. For example, PI (peak wavelength 532 nm) may be used as the alignment reagent 1541 and dead bacteria staining solution 1521, and LDS751 (peak wavelength 710 nm) may be used as the whole bacteria staining solution 1531.

  Next, a specimen and solution flow method in the microorganism testing chip 10 will be described. In the viable cell count measurement using the microbe inspection chip 10, first, the microbe inspection chip 10 is aligned, that is, the fungus body detection flow path 173 is aligned, and then the viable cell count is measured. First, the flow of the alignment reagent 1541 in the alignment process will be described. As shown in the figure, the microorganism testing chip 10 is held in a standing state with the funnel-shaped bottom surface of the container facing downward. The alignment reagent 1541 is caused to flow to the detection liquid waste liquid container 156 via the fungus body detection flow path 173. The highest point of the alignment reagent holding container-bacteria cell detection flow path 1575 is formed to be higher than the water level of the alignment reagent 1541. Therefore, as long as the alignment reagent holding container vent 1695 and the detection liquid disposal container ventilation hole 1596 are closed, the alignment reagent 1541 held in the alignment reagent holding container 154 is not detected. 1596 does not flow. Therefore, pressure from the pressure supply device 14 (FIG. 3) is applied via the alignment reagent holding container vent 1695 to increase the pressure in the alignment reagent holding container 154. At the same time, the detection liquid waste container 156 is opened to the atmosphere through the detection liquid waste container vent 1596. Due to the pressure difference, the alignment reagent 1541 enters the detection liquid disposal container 156 via the alignment reagent holding container-bacteria cell detection channel 1575 and the cell detection channel 173. The fungus body detection flow path 173 is irradiated with excitation light from the detection device 11 (FIG. 3). Therefore, the alignment reagent 1541 emits fluorescence when passing through the fungus body detection flow path 173. This fluorescence is detected by the detection device 11 (FIG. 3). The position of the microorganism testing chip 10 is adjusted so that the fluorescence detected by the detection device 11 (FIG. 3) is maximized. After alignment, a part of the diluent 1551 is caused to flow into the fungus body detection flow path 173. Thereby, the fungus body detection flow path 173 is washed, and the alignment reagent 1541 remaining in the fungus body detection flow path 173 is removed.

  Next, the flow of each liquid in the viable count measurement step will be described.

  1. A step of causing the specimen 1511 to flow into the dead bacteria staining liquid holding container 152. The highest point of the flow path 1571 between the specimen container and dead bacteria staining liquid holding container is formed to be higher than the water level of the specimen 1511 held in the specimen container 151. Therefore, the specimen 1511 held in the specimen container 151 does not flow into the dead bacteria staining liquid holding container 152 as long as the specimen container ventilation hole 1591 and the dead bacteria staining liquid holding container ventilation hole 1592 are closed. The pressure from the pressure supply device 14 (FIG. 3) is applied through the vent 1591 to increase the atmospheric pressure in the sample container 151. At the same time, the dead germ stain holding container 152 is opened to the atmosphere through the dead germ stain holding container vent 1592. Due to the pressure difference, the specimen 1511 enters the dead bacteria staining liquid holding container 152 from the specimen container 151 via the food residue removal unit 160 and is mixed with the dead bacteria staining liquid 1531. When the sample 1511 passes through the food residue removal unit 160, the food residue in the sample 1511 is removed by the food residue removal unit 160. Dead bacteria in the specimen 1511 are stained with the dead bacteria staining solution 1521. On the other hand, the dead bacteria staining solution 1531 does not pass through the cell membrane of live bacteria. Therefore, viable bacteria in the specimen 1511 are not stained.

  The volume of the dead bacteria staining liquid holding container 152 is larger than the total volume of the specimen 1511 and the dead bacteria staining liquid 1521. The highest point of the flow path 1572 between the dead bacteria staining liquid holding container and the whole bacteria staining liquid holding container is formed to be higher than the water level of the mixed liquid of the two liquids held in the dead bacteria staining liquid holding container 152. The water level of the mixed liquid of the two liquids does not exceed the highest point of the flow path 1572 between the dead bacteria staining liquid holding container and the whole bacteria staining liquid holding container, and the air contained in the dead bacteria staining liquid holding container 152 is dead. It is discharged to the outside through the bacterial stain holding container vent 1592. Since the atmospheric pressure of the dead bacteria staining liquid holding container 152 is equal to the atmospheric pressure, the mixed liquid of the two liquids is not pushed out to the whole bacterial staining liquid holding container 153 and the dead liquid staining liquid holding container 152 is used for the time required for the reaction. Can be held in. Similarly, the whole bacteria staining solution 1531 is held in the whole bacteria staining solution holding container 153. That is, the whole bacteria staining solution 1531 is not pushed out to the diluent holding container 155 and does not flow back to the dead bacteria staining liquid holding container 152.

  At this time, in order to prevent the mixed solution from flowing into the whole bacteria staining solution holding container 153, the pressure from the pressure supply device is applied through each of the vents 1593 to 1596, and the whole is reduced to a range lower than the atmospheric pressure of the sample container 151. The pressure in the bacteria staining solution holding container 153, the diluent holding container 154, the alignment reagent holding container 155, and the detection liquid waste container 156 may be increased.

  During the staining, it is preferable to reduce the influence of the staining due to the temperature change by keeping the temperature of the microorganism testing chip 10 constant.

  2. A step of flowing a mixed liquid of the specimen 1511 and the dead bacteria staining liquid 1521 to the whole bacteria staining liquid holding container 153. Pressure from the pressure supply device 14 (FIG. 3) is applied through the vent 1592, and the atmospheric pressure in the dead bacteria staining liquid holding container 152 is increased. At the same time, the whole bacteria staining liquid holding container 153 is opened to the atmosphere via the whole bacteria staining liquid holding container vent 1593. Due to the difference in atmospheric pressure, the mixed liquid of the specimen 1511 and the dead bacteria staining liquid 1521 enters the whole bacteria staining liquid holding container 153 from the dead bacteria staining liquid holding container 152 and is mixed with the whole bacteria staining liquid 1531. Dead and live bacteria in the specimen 1511 are stained with the whole bacteria staining solution 1531.

  The volume of the whole bacteria staining liquid holding container 153 is larger than the total volume of the specimen 1511, the dead bacteria staining liquid 1521, and the whole bacteria staining liquid 1531. The highest point of the whole bacteria staining liquid holding container-dilution liquid holding container flow path 1573 is formed to be higher than the water level of the mixed liquid of three liquids held in the whole bacteria staining liquid holding container 153. Since the total bacterial stain holding container 153 has an atmospheric pressure equal to the atmospheric pressure, the three liquid mixture is not pushed out to the diluent holding container 154, and the mixed liquid is held in the whole bacterial stain holding container 153 for the time required for the reaction. can do.

  3. A step of flowing a mixed liquid of the specimen 1511, dead bacteria staining liquid 1521, and whole bacteria staining liquid 1531 to the diluent holding container 154. Pressure from the pressure supply device 14 (FIG. 3) is applied through the vent 1593, and the atmospheric pressure in the whole bacteria staining solution holding container 153 is increased. At the same time, the diluent holding container 154 is opened to the atmosphere via the diluent holding container vent 1594. Due to the atmospheric pressure difference, the mixed solution of the specimen 1511, the dead bacteria staining solution 1521, and the whole bacteria staining solution 1531 enters the diluent holding container 154 from the whole bacteria staining solution holding container 153 and mixes with the diluent 1541. Thereby, the density | concentration of the unbound pigment | dye (dead bacteria dyeing liquid 1521 which does not dye | stain microorganisms, and all the bacteria dyeing | staining liquid 1531) contained in a liquid mixture falls. By reducing the concentration of the unbound dye, the amount of fluorescence emitted by the unbound dye that causes noise during detection can be reduced.

  The volume of the diluent holding container 155 is larger than the total volume of the specimen 1511, the dead bacteria staining liquid 1521, the whole bacteria staining liquid 1531 and the dilution liquid 1551. Further, the highest point of the dilution liquid holding container-bacteria cell detection flow path 1574 is formed to be higher than the water level of the mixed liquid of the four liquids. Since the atmospheric pressure of the diluent holding container 155 is equal to the atmospheric pressure, the four liquid mixture is not pushed out to the detection liquid disposal container 156 and can be held in the diluent holding container 155.

  4). A step of causing a mixed liquid of the specimen 1511, the dead bacteria staining liquid 1521, the whole bacteria staining liquid 1531, and the dilution liquid 1541 to flow into the fungus body detection flow path 173. Pressure from the pressure supply device 14 (FIG. 3) is applied through the vent 1594, and the atmospheric pressure in the diluent holding container 154 is increased. At the same time, the detection liquid waste container 156 is opened to the atmosphere via the detection liquid waste container vent 1596. The liquid mixture diluted due to the pressure difference enters the detection liquid disposal container 156 from the dilution liquid holding container 154 via the cell detection flow path 173. Excitation light is applied to the fungus body detection channel 173 in the vertical direction, preferably at an angle that is deviated from the vertical direction. Thereby, the stained microorganisms fluoresce. The detection device 11 detects fluorescence. The fluorescence peak wavelength by the whole bacteria staining solution 1531 is different from the fluorescence peak wavelength by the dead bacteria staining solution 1521. By detecting the fluorescence from the total bacterial stain 1531, the total number of viable and dead bacteria can be detected. The number of dead bacteria can be detected by detecting fluorescence from the dead bacteria staining solution 1521. From the difference between the two, the viable cell count can be obtained.

  Next, details of the microbial cell detection unit 17 of the microbial test chip will be described with reference to FIG. FIG. 2A shows a cross section along the normal vector on the excitation light incident surface of the fungus body detection unit. As shown in FIG. 7, the fungus body detection unit is provided at an inclination on the main body of the microbe inspection chip. This inclination will be described in detail in the description of FIG.

  FIG. 2A is a cross-sectional view of the joint between the main body 15 and the fungus body detection unit 17. FIG. 2B is an exploded perspective view of the fungus body detection unit 17. The main body 15 and the fungus body detection unit 17 are produced in separate steps, and both are joined. First, the manufacturing method of the microbial cell detection part 17 is demonstrated. The fungus body detection unit 17 includes a cover member 171 and a flow path member 172, both of which are formed of a thin flat plate. A groove 1731 is formed in the flow path member 172, and through holes 1741 and 1751 are formed at both ends of the groove 1731. The cover member 171 and the flow path member 172 are bonded together so that the surface on which the groove 1731 is formed becomes the bonded surface. Thereby, the fungus body detection part 17 is formed. A fungus body detection flow path 173 is configured by the groove 1731 of the flow path member 172 and the cover member 171. The cell body detection channel inlet 174 and the cell body detection channel outlet 175 are configured by the through holes 1741 and 1751 of the channel member 172.

  On the other hand, the dilution liquid holding container-bacteria cell detection flow path channel 1574 formed in the main body 15 changes the flow path direction at the lower end to form an opening on the surface of the main body 15. Similarly, the fungus body detection flow path-detection liquid disposal container flow path 1576 has an opening on the surface of the main body 15 by changing the flow path direction at the upper end. The opening of the dilution liquid holding container-bacteria cell detection channel 1574 is connected to the cell detection channel inlet 174, and the opening of the cell detection channel-detection liquid waste container channel 1576 is: It is connected to the fungus body detection flow path outlet 175.

  A detection window frame 161 is formed on the main body 15. The detection window frame portion 161 is a through hole or a through groove. The detection window frame 161 is formed between the opening of the dilution liquid holding container-bacteria cell detection flow path channel 1574 and the opening of the fungus cell detection flow path-detection liquid waste container flow path 1576. . When the fungus body detection unit 17 is manufactured, it is attached to the main body 15. As shown in the figure, the fungus body detection unit 17 is mounted so that the fungus body detection flow path 173 is disposed on the detection window frame 161 of the main body 15.

  Stained bacterial cells 162 are a diluent holding container-bacteria cell detection channel 1574, a cell detection channel inlet 174, a cell detection channel 173, and a cell detection channel outlet 175. It flows in the order of the flow path 1576 between the flow path for detecting bacterial cells and the waste container for detection liquid. The excitation light 113 incident on the fungus body detection flow path 173 irradiates the fungus body 162 flowing in the fungus body detection flow path 173. As a result, the bacterial cell 162 emits fluorescence 121 and is detected by the detection device 11 (FIG. 3).

  According to this example, the detection window frame portion 161 that is a through hole or a through groove of the main body 15 is provided behind the fungus body detection flow path 173. Therefore, the excitation light 113 irradiates only the fungus body detection unit 17 and does not irradiate the main body 15. Reflected light from the main body 15 or autofluorescence that causes an increase in background light does not occur. In order for the excitation light 113 that has passed through the fungus body detection flow path 173 not to be irradiated to the main body 15, the cross-section of the through-hole constituting the detection window frame portion 161 increases along the radiation direction of the excitation light 113. It is preferable.

  In the example shown in FIG. 2A, the central axis of the fungus body detection flow path 173 is the flow path 1574 between the diluent holding container and the fungus body detection flow path and the flow path between the fungus body detection flow path and the detection liquid waste container 1576. It is eccentric with respect to the central axis. That is, the fungus body detection flow path 173 is not arranged on the same line as the diluent holding container-bacterial cell detection flow path 1574 and the fungus body detection flow path-detection liquid disposal container flow path 1576. . However, the fungus body detection flow path 173 is arranged on the same line as the diluent holding container-bacterial body detection flow path flow path 1574 and the fungus body detection flow path-detection liquid disposal container flow path 1576. May be configured. In this case, the fungus body detection flow path 173 is directly connected to the diluent holding container-bacteria body detection flow path path 1574 and the fungus body detection flow path-detection liquid waste container flow path 1576.

  The cover member 171 has a thickness of 0.01 μm to 1 mm. The thickness of the flow path member 172 is 0.01 μm to 1 mm. The cross-sectional shape of the fungus body detection flow path 173 is a square, a rectangle, or a trapezoid. The larger the cross-sectional dimension of the fungus body detection flow path 173, the smaller the pressure loss. However, the smaller the cross-sectional dimension, the better for flowing microorganisms one by one. One side of the cross section of the fungus body detection flow path 173 is preferably 1 μm to 1 mm, and the length is preferably 0.01 mm to 10 mm. The optical axis of the excitation light applied to the fungus body detection flow path 173 is perpendicular to the direction vector of the fungus body detection flow path 173.

  The material which comprises the microbial cell detection part 17 is demonstrated. The microorganism testing chip 10 is disposable. That is, after use, the fungus body detection unit 17 is discarded together with the main body 15. Therefore, the material used for the microbial cell detection part 17 must be cheap. The material used for the fungus body detection unit 17 needs to have excellent optical characteristics so as to be suitable for fluorescence measurement. That is, it is desirable that the autofluorescence is low and the light transmittance, surface accuracy, refractive index and the like are excellent. In order not to inhibit the detection of fluorescence from the microbial cells, it is preferable that the amount of autofluorescence generated by the microbial cell detection unit 17 itself be sufficiently smaller than the amount of fluorescence from the microbial cells.

  If a curved surface, unevenness, or the like exists on the surface of the fungus body detection unit 17, the light amount of the excitation light 113 irradiated to the fungus body measurement flow path 173 varies due to light refraction or irregular reflection on the surface. For this reason, the amount of fluorescence detected also fluctuates, and the measurement accuracy decreases. Therefore, the surface of the fungus body detection unit 17 needs to have a desired flatness. The fungus body detection unit 17 preferably has a flatness with an unevenness of 0.1 mm or less.

  In consideration of such conditions, the materials used for the cell detector 17 include glass, quartz, polymethacrylic acid methyl ester (PMMA), polydimethylsiloxane (PDMS), cycloolefin polymer (COP), polyethylene terephthalate, Polycarbonate etc. can be considered. The fungus body detection unit 17 is formed of one or more kinds of substances selected from these substances. The fungus body detection unit 17 is preferably formed of the same material as that of the main body 15. In particular, the flow path member 172 is formed of the same material as the main body 15.

  The cover member 171 is a simple flat plate, but the flow path member 172 is formed by forming a groove and a through hole in the flat plate. Therefore, the flow path member 172 is formed of a material that is easy to be finely processed and that has a low processing cost. Glass and quartz have excellent optical properties but are not easily processed finely. That is, processing costs increase when microfabrication is performed.

  Therefore, the cover member 171 may be made of glass or quartz, and the flow path member 172 may be made of polymethacrylic acid methyl ester, polydimethylsiloxane, cycloolefin polymer, polyethylene terephthalate, or polycarbonate. Preferably, the flow path member 172 may be made of polydimethylsiloxane. In this case, the cover member 171 and the flow path member 172 are joined using the self-adhesive property of polydimethylsiloxane.

  The amount of autofluorescence of the fungus body detection unit 17 depends not only on the material but also on the thickness dimension of the fungus body detection unit. In order to reduce the amount of autofluorescence, the thickness dimension of the fungus body detection unit may be reduced. As the thickness of the fungus body detection unit 17 is smaller, the amount of autofluorescence generated from the fungus body detection unit 17 is reduced. However, if the thickness dimensions of the cover member 171 and the flow path member 172 are reduced, manufacturing becomes difficult and flatness deteriorates. In order to maintain the necessary flatness and suppress the amount of autofluorescence so as not to inhibit the detection of fluorescence of the bacterial cells, it is necessary to limit the thickness dimensions of these parts to a predetermined range.

  The autofluorescence amounts of glass, quartz, and polydimethylsiloxane are almost the same. When the cover member 171 is made of glass or quartz, the thickness is preferably 0.05 mm or greater and 1 mm or less. When the flow path member 172 is made of polydimethylsiloxane, the thickness is preferably 0.1 mm or greater and 1 mm or less.

  Further, the cover member 171 and the flow path member 172 may be made of cycloolefin polymer, polymethacrylic acid methyl ester, polyethylene terephthalate, or polycarbonate. In this case, the amount of autofluorescence per unit volume is increased by about three times or more than when the cover member 171 is manufactured from glass or quartz and the flow path member 172 is manufactured from polydimethylsiloxane. Therefore, the thickness of the cover member 171 and the flow path member 172 is preferably 0.01 mm or more and 0.3 mm or less.

  FIG. 3 is a configuration diagram of the microorganism testing apparatus 1 of the present invention. The microorganism testing apparatus 1 includes a microorganism testing chip 10 having a cell detection unit 17, an XY stage 125 for moving the microorganism testing chip 10 in the XY direction, and excitation light to the cell detection unit 17 of the microorganism testing chip 10. A detection device 11 that irradiates and detects fluorescence therefrom, and a pressure supply device 14 that supplies a gas of a predetermined pressure to the microorganism testing chip 10 are provided. A system device 18 and an output device 19 are connected to the microorganism testing apparatus 1. The microorganism testing chip 10 has been described with reference to FIG. The XY stage 125 holds the microorganism testing chip 10 (held by mounting or insertion), moves the microorganism testing chip 10 in the XY direction by an instruction signal from the system device 18, and performs alignment thereof. As shown in the figure, the X axis and the Y axis are set on the horizontal plane. The X axis is perpendicular to the excitation light applied to the fungus body detection flow path 173 of the fungus body detection unit 17, and the Y axis is parallel to the excitation light.

  The pressure supply device 14 includes a cylinder 141 with a pressure adjusting device. The cylinder 141 is filled with high-pressure air, inert gas, or the like. The air vents 1591 to 1596 (FIG. 1) of the cylinder 141 and the microorganism testing chip 10 are connected by chip connecting pipes 1441 to 1446. Valves 1421 to 1426 are provided in the chip connecting pipes 1441 to 1446, respectively. By opening and closing the valves 1421 to 1426, a gas having a predetermined pressure is supplied to the container of the microorganism testing chip 10 or the container of the microorganism testing chip 10 is opened to the atmosphere. Thereby, as described with reference to FIG. 1, the specimen and the reagent are transported in the microorganism testing chip 10.

  The detection device 11 includes an excitation light source 111, an excitation light / fluorescence separation dichroic mirror 112, an objective lens 114, a band pass filter 117, a condenser lens 118, a pinhole 119, and a photodetector 120. The excitation light-fluorescence separation dichroic mirror 112 has a function of reflecting the excitation light 113 and transmitting fluorescence. That is, light in the wavelength band near the wavelength of the excitation light 113 is reflected, and light in the wavelength band near the wavelength of fluorescence is transmitted. The excitation light source 111, the excitation light-fluorescence separation dichroic mirror 112, and the objective lens 114 are arranged so that the optical axis of the excitation light 113 and the central axis of the objective lens 114 coincide. As a result, the excitation light 113 is condensed at the focal point of the lens system including the objective lens 114 and the condenser lens 118.

  When alignment is performed, the alignment reagent 1541 is caused to flow through the fungus body detection flow path 173. When measuring the number of cells, the sample is caused to flow in the cell detection channel 173.

  Excitation light 113 output from the excitation light source 111 is reflected by the excitation light-fluorescence separation dichroic mirror 112, collected by the objective lens 114, and irradiated to the fungus body detection flow path 173. The fluorescence 121 from the microorganisms or the alignment reagent passing through the fungus body detection flow path 173 of the fungus body detection unit 17 is converted into parallel rays by the objective lens 114 and transmitted through the excitation light-fluorescence separation dichroic mirror 112. The light passes through the bandpass filter 117, is collected by the condenser lens 118, and reaches the photodetector 120 via the pinhole 119. The pinhole 119 functions as a spatial filter for cutting stray light. An electrical signal from the detection device 11 is sent to the system device 18. The system device 18 processes the electrical signal and outputs the obtained measurement result to the output device 19.

  With reference to FIG.4 and FIG.5, the alignment method of the flow path 173 for a microbial cell detection is demonstrated. The Y axis is taken in parallel to the optical axis of the excitation light 113 irradiated to the fungus body detection flow path 173, and the X axis is taken perpendicular to the optical axis of the excitation light 113. 4 and 5 schematically illustrate the positions of the fungus body detection flow path 173 and the excitation light 113 when the fungus body detection unit 17 is cut along a plane parallel to the XY plane.

  A method for positioning the fungus body detection flow path 173 in the X direction will be described with reference to FIG. The alignment reagent 1541 is caused to flow in the fungus body detection flow path 173, and the microbial test chip 10 is moved in the X direction by the XY stage 125. FIG. 4A shows a position 173a in the X direction of the fungus body detection flow path 173 before movement and a position 173b in the X direction of the fungus body detection flow path 173 after movement. FIG. 4B shows an example of a graph 128 showing the relationship between the position in the X direction of the fungus body detection flow path 173 and the fluorescence amount I detected by the detection device 11 (FIG. 3). When the fungus body detection flow path 173 passes on the optical axis of the excitation light 113, the fluorescence amount I becomes maximum. Therefore, the position in the X direction may be set by the XY stage 125 when the fluorescence amount I is maximized.

  A method for positioning the fungus body detection flow path 173 in the Y direction will be described with reference to FIG. The alignment reagent 1541 is caused to flow in the fungus body detection flow path 173, and the microorganism testing chip 10 is moved in the Y direction by the XY stage 125. FIG. 5A shows a position 173c in the Y direction of the fungus body detection flow path 173 before movement and a position 173d in the Y direction of the fungus body detection flow path 173 after movement. FIG. 5B shows an example of a graph 129 showing the relationship between the position in the Y direction of the fungus body detection flow path 173 and the fluorescence amount I detected by the detection device 11 (FIG. 3). When the fungus body detection flow path 173 passes on the optical axis of the excitation light 113, the fluorescence amount I becomes maximum. Therefore, the position in the Y direction may be set by the XY stage 125 when the fluorescence amount I is maximized.

With reference to FIG. 6, the procedure of the alignment of the microorganism test chip 10, that is, the alignment of the fungus body detection flow path 173 will be described. In step S 101, the microorganism testing chip 10 is mounted on the XY stage 125, and the alignment reagent 1541 is caused to flow in the fungus body detection flow path 173. For example, PI (wavelength peak 532 nm) may be used as the alignment reagent 1541. In step S <b> 102, the microorganism testing chip 10 is moved in the direction perpendicular to the optical axis of the excitation light 113 by the XY stage 125. That is, as shown in FIG. 4A, the microorganism testing chip 10 is moved in the X direction. At the same time, as shown in FIG. 4B, the relationship between the position in the X direction and the profile of the fluorescence amount I detected by the detection device 11 is acquired. This profile is stored in the system device 18 (FIG. 3). In step S103, a position where the fluorescence amount I is maximized or a position where the first derivative of the fluorescence amount I with respect to the X direction is 0 (X = _Xc ) is obtained. Thus, a position in the X direction is obtained. The microbe inspection chip 10 is moved to that position.

Next, in step S <b> 104, the microorganism testing chip 10 is moved in the direction parallel to the optical axis of the excitation light 113 by the XY stage 125. That is, as shown in FIG. 5A, the microorganism testing chip 10 is moved in the Y direction. At the same time, as shown in FIG. 5B, the relationship between the position in the Y direction and the profile of the fluorescence amount I detected by the detection device 11 is acquired. This profile is stored in the system device 18 (FIG. 3). In step S105, a position where the fluorescence amount I is maximized or a position where the first derivative of the fluorescence amount I with respect to the Y direction is 0 (Y = Y _c ) is obtained. Steps S102 and S103 are positioning in the X direction, and steps S104 and S105 are positioning in the Y direction. By repeating the positioning in the X direction and the positioning in the Y direction, high-precision positioning is possible.

  In addition to the structure and method described above, the structure and method described in Japanese Patent Application No. 2009-109153 previously proposed by the patent applicant can be used for the alignment method.

  With reference to FIG. 7, the structural example of the detection apparatus of the microorganisms test | inspection apparatus by this invention is demonstrated. The detection device 11 of this example is suitable for measuring the number of viable bacteria in a food-derived specimen. That is, in the detection apparatus of this example, the number of viable bacteria and the number of dead bacteria are discriminated. The optical system of the detection apparatus may differ depending on the excitation spectrum and fluorescence spectrum of the fluorescent dye. Here, a case will be described in which PI (excitation wavelength peak 532 nm, fluorescence wavelength peak 615 nm) is used as a dead bacteria staining liquid, and LDS751 (excitation wavelength peak 541 nm, fluorescence wavelength peak 710 nm) is used as a whole bacteria staining liquid. The optical system of the detection apparatus of this example is preferably configured when two types of fluorescent dyes are used.

  In this example, in order to simplify the optical system, the optical axis of the excitation light and the central axis of the fluorescent condensing lens are coaxial. Further, by making the optical axis of the excitation light and the central axis of the fluorescent condensing lens coaxial, alignment can be easily performed at a time. The detection device 11 includes an excitation light source 111 (wavelength 532 nm), an excitation light-fluorescence separation dichroic mirror 112 that reflects the excitation light 113 and transmits the fluorescence of the fungus body, and a microorganism that passes through the fungus body detection flow path 173. The objective lens 114 that collects the fluorescence from the light and makes it parallel light, the dichroic mirror 115 for fluorescence separation that reflects light having a wavelength of 610 nm or less, and passes light having a wavelength of 610 nm or more, a mirror 116, and a wavelength near 610 nm A short-wavelength bandpass filter 1171 that passes only light having a wavelength, a long-wavelength bandpass filter 1172 that passes light having a wavelength near 710 nm, and a short-wavelength condensing lens 1181 that collects parallel light. A long wavelength condenser lens 1182 and a short wavelength pinhole 1191 used as a spatial filter for cutting stray light. Wavelength pinhole 1192, short wavelength photodetector 1201 that detects light that has passed through short wavelength bandpass filter 1171, and long wavelength photodetector that detects light that has passed through long wavelength bandpass filter 1172 1202.

  The excitation light source 111 uses a laser, and the short wavelength photodetector 1201 and the long wavelength photodetector 1202 use a photomultiplier. As described above, it is assumed that the alignment of the fungus body detection flow path 173 of the microorganism testing chip 10 has been completed. A fungus body detection flow path 173 of the microorganism testing chip 10 is disposed at the focal position of the objective lens 114.

  Excitation light (wavelength 532 nm) output from the excitation light source 111 reflects the excitation light-fluorescence separation dichroic mirror 112 and is irradiated to the fungus body detection flow path 173. Thereby, PI and LDS751 which dye | stained the microorganisms which flow through the microbe detection channel 173 are excited. Fluorescence 1211 (PI is center wavelength 610 nm) from dead bacteria staining liquid PI and fluorescence 1212 (center wavelength 710 nm) from whole bacteria staining liquid LDS751 are incident on objective lens 114. The fluorescence 1211 from the dead bacteria staining liquid PI is reflected by the fluorescence separation dichroic mirror 115, and the fluorescence 1212 from the whole bacteria staining liquid LDS751 passes through the fluorescence separation dichroic mirror 115. Thus, the fluorescence from the two dyes can be separated by the difference in wavelength. The fluorescence 1211 from the killed bacterial dye PI passes through the short wavelength band-pass filter 1171, is collected by the short wavelength condensing lens 1181, passes through the short wavelength pinhole 1191, and is short wavelength photodetector 1201. Is incident on. The fluorescence 1212 from the whole bacterial stain LDS751 passes through the long-wavelength bandpass filter 1172, is collected by the long-wavelength condensing lens 1182, passes through the long-wavelength pinhole 1192, and is detected by the long-wavelength photodetector 1202. Is incident on.

  The fluorescence detected by the short wavelength photodetector 1201 and the long wavelength photodetector 1202 is converted into an electrical signal, and the electrical signal is sent to the system device 18 (FIG. 3). The system device 18 processes the electrical signals sent from the short wavelength photodetector 1201 and the long wavelength photodetector 1202 and outputs information on the number of microorganisms to the output device 19 (FIG. 3) as a test result. The number of dead bacteria is obtained from the output of the short wavelength photodetector 1201, and the total number of bacteria is obtained from the output of the long wavelength photodetector 1202. The viable count is obtained from the difference between the two.

  In Japanese Patent Application No. 2008-263055, a part of the excitation light of the excitation light source may be surface-reflected by the fungus body detection unit and return to the detection device, so the normal vector of the fungus body detection unit and the light of the excitation light The whole microbe inspection chip is inclined with respect to the inspection device so that the axes are not parallel, so that the reflected light does not return to the detection device. On the other hand, in this embodiment, the fungus body detection unit 17 is tilted by θ with respect to the plane of the main body 15 and overlapped.

  That is, a part of the excitation light 113 from the excitation light source 111 may be reflected on the surface of the fungus body detection unit 17 and return to the detection device 11. In order to prevent this, the normal vector (not shown) of the fungus body detection unit 17 and the optical axis of the excitation light 113 are not parallel. That is, when the angle formed by the surface of the fungus body detection unit 17 and the optical axis of the excitation light 113 is θ, as shown in FIG. The main body 15) is provided with an inclined fungus body detection unit 17 (assuming that the angle between the normal vector on the incident surface of the fungus body detection unit 17 and the optical axis of the excitation light is α, θ + α = 90 °, θ = 80-30 °, in other words, α = 10-60 °). The minimum value of α is approximately ½ of the solid angle δ of the objective lens 114. Therefore, the optimum value of α varies depending on the objective lens used. In this embodiment, the objective lens 114 having a solid angle of 34 ° is used in consideration of the light collection efficiency and the depth of focus. In this case, α is optimally around 17 ° (in terms of θ, around 73 °). When δ is reduced, α is also reduced. However, since a lens having a small δ is difficult to collect light, the minimum value of α is about 10 ° (about 80 ° in terms of θ). The error range from the optimum value of α is determined in consideration of the incident angle of the excitation light (laser light). The incident angle β of the excitation light (laser light) is obtained by β = arctan ((d / 2) / L) where d is the diameter of the laser light incident on the objective lens and L is the distance from the lens to the focal point. . Considering these, θ is more preferably 80 to 70 ° (in other words, α is more preferably 10 to 20 °). The larger α is, the more difficult the reflected light of the excitation light enters the light receiving optical system, but the critical angle of total reflection from the glass layer (refractive index 1.5) to the channel layer (water: refractive index 1.3) is about Since it is 60 °, α is set to 60 °. In order to reduce the influence of the reflected light, α should be 10 ° or more, and in order to make the effect more effective, 10 to 20 ° is most desirable.

  The fungus body detection unit 17 is tilted so that the normal vector of the fungus body detection unit 17 and the optical axis of the excitation light 113 are not parallel to each other, but the excitation light is transmitted through the fungus body detection channel 173 (flow direction). Irradiate from the vertical direction.

  When the detection device (such as the XY stage 125) is attached to the microbe inspection chip 10 that is configured by tilting and superimposing the fungus body detection unit 17 on the main body 15 by θ, it is perpendicular to the optical axis of the excitation light 113. Compared with the case where the microorganism detection chip is not inclined and the entire microorganism detection chip is inclined with respect to the optical axis of the excitation light 113 and attached to the detection device. It becomes easy to attach to the detection device.

  With reference to FIG.8 and FIG.1, the procedure which measures the viable count in the sample derived from food using the microbe test chip | tip 10 of this invention is demonstrated. The dead bacteria staining solution 1521, the whole bacteria staining solution 1531, and the alignment reagent 1541 are enclosed in advance in the microorganism testing chip 10. Diluent 1551 is injected into diluent holding container 155 before inspection. In step S201, the specimen 1511 is injected into the specimen container 151 through the vent 1591. 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. In step S202, the microorganism testing chip 10 is mounted on the XY stage 125 of the microorganism testing apparatus. In step S203, alignment of the microorganism testing chip, that is, alignment of the fungus body detection flow path 173 is performed. As described with reference to FIG. 6, the alignment is performed by causing the alignment reagent 1541 to flow in the fungus body detection flow path 173. In step S204, a diluent is passed through the fungus body detection flow path 173, and the alignment reagent 1541 attached to the flow path is washed.

  Steps S205 to S211 are processing for measuring the number of viable bacteria in the sample. In step S <b> 205, the specimen 1511 is caused to flow from the specimen container 151 to the dead bacteria staining liquid holding container 152 via the food residue removing unit 160. In step S206, the specimen 1511 and the dead bacteria staining solution 1531 are mixed by stirring. Although dead bacteria in the specimen 1511 are stained with the dead bacteria staining solution 1521, live bacteria in the specimen 1511 are not stained. In step S207, the mixed liquid of the specimen 1511 and the dead bacteria staining liquid 1521 is caused to flow into the whole bacteria staining liquid holding container 153. In step S208, the mixed solution of the specimen 1511 and the dead bacteria staining solution 1521 is mixed with the whole bacteria staining solution 1531 by stirring. In step S209, the mixed liquid of the specimen 1511, the dead bacteria staining liquid 1521, and the whole bacteria staining liquid 1531 is caused to flow into the diluent holding container 154. In step S210, the mixed solution of the specimen 1511, the dead bacteria staining solution 1521, and the whole bacteria staining solution 1531 is mixed with the diluent 1541 by stirring. By adding the diluted solution 1541, the concentration of the unbound dye contained in the mixed solution is lowered. By reducing the concentration of the unbound dye, the amount of fluorescence emitted by the unbound dye that causes noise during detection can be reduced. In step S <b> 211, a mixed solution of the specimen 1511, the dead bacteria staining solution 1521, the whole bacteria staining solution 1531, and the diluent 1541 is caused to flow into the fungus body detection flow path 173. Excitation light is irradiated from the vertical direction to the fungus body detection flow path 173. However, it is preferable that the normal vector of the fungus body detection unit 17 and the optical axis of the excitation light 113 are not parallel. Thereby, the dyed microorganisms flowing through the fungus body detection flow path 173 emit fluorescence. The detection device 11 detects fluorescence. By detecting the fluorescence from the total bacterial stain 1531, the total number of viable and dead bacteria can be detected. The number of dead bacteria can be detected by detecting fluorescence from the dead bacteria staining solution 1521. From the difference between the two, the viable cell count can be obtained.

  Although the examples of the present invention have been described above, the present invention is not limited to the above-described examples, and it is easy for those skilled in the art to make various modifications within the scope of the invention described in the claims. Will be understood.

DESCRIPTION OF SYMBOLS 1 Microorganism test | inspection apparatus 10 Microorganism test | inspection chip 11 Detection apparatus 14 Pressure supply apparatus 15 Main body 17 Fungus body detection part 18 System apparatus 19 Output apparatus 111 Excitation light source 112 Excitation light-fluorescence separation dichroic mirror 113 Excitation light 114 Objective lens 115 For fluorescence separation Dichroic mirror 116 Mirror 117 Band pass filter 118 Condensing lens 119 Pinhole 120 Photo detector 121 Fluorescence 125 XY stage 127 Relationship between X direction position and output value 128 Relationship between Y direction position and output value 151 Sample container 152 Dead bacteria Staining solution holding container 153 Whole bacteria staining solution holding container 154 Alignment reagent holding container 155 Dilution solution holding container 156 Detection solution discarding container 157 Solution channel 158 Ventilation channel 159 Vent 160 160 Food residue removal unit 161 Detection window Frame 162 Bacteria 171 Hippo Member 172 channel member 173 fungus body detection flow path 174 fungus body detection flow path inlet 175 fungus body detection flow passage outlet

Claims (11)

A specimen container for holding a specimen, a reagent holding container for holding a staining reagent, a bacteria detection unit, a detection liquid disposal container for holding waste liquid, the specimen container, A flow path connecting between the reagent holding containers, a flow path connecting between the reagent holding containers and the fungus body detection unit, and a flow path connecting between the fungus body detection unit and the detection liquid disposal container A microbe inspection chip having
The fungus body detection unit is inclined and disposed in the microbe inspection chip body so that the normal vector on the incident surface of the excitation light of the fungus body detection unit and the optical axis of the excitation light are not parallel to each other. Microbe inspection chip. A main body and a fungus body detection unit mounted on the main body,
The main body connects a specimen container for holding a specimen, a reagent holding container for holding a staining reagent, a detection liquid waste container for holding waste liquid, and the specimen container and the reagent holding container. And a flow path connecting the reagent holding container and the fungus body detection unit, and a flow path connecting the fungus body detection unit and the detection liquid waste container. And
The fungus body detection unit has a fungus body detection flow path connected to the flow path, and forms the fungus body detection flow path by bonding a cover member and a flow path member having a groove. Is composed of
The main body has a detection window frame that is a through-hole or a through-groove,
The fungus body detection unit is disposed on the detection window frame portion so as to be inclined so that the normal vector on the excitation light incident surface of the fungus body detection unit and the optical axis of the excitation light are not parallel to each other. Microorganism testing chip characterized by   3. The microorganism test chip main body according to claim 1, wherein an angle formed between the surface of the fungus body detection unit and the optical axis of the excitation light is θ, and the fungus is placed in the microbe inspection chip body so that the angle is in a range of θ = 80 to 30 °. A microorganism testing chip, wherein the body detection unit is inclined.   3. The microorganism test chip main body according to claim 1, wherein an angle formed between the surface of the fungus body detection unit and the optical axis of the excitation light is θ, and the fungus is placed in the microbe inspection chip body such that θ is in a range of 80 to 70 °. A microorganism testing chip, wherein the body detection unit is inclined. The microorganism testing chip according to claim 1 or 2,
The cover member is formed of a glass or quartz plate having a thickness of 0.05 mm to 1 mm, and the flow path member is formed of a polydimethylsiloxane plate having a thickness of 0.1 mm to 1 mm. Microbiological testing chip that is characterized. The microorganism testing chip according to claim 1 or 2,
The microbe inspection chip, wherein the cover member and the flow path member are formed of a plate material of cycloolefin polymer, polymethacrylic acid methyl ester or polycarbonate having a thickness of 0.01 mm or more and 0.3 mm or less. The microorganism testing chip according to claim 1 or 2,
The microorganism testing chip, wherein the main body is made of one or more kinds of materials selected from polypropylene, polystyrene, polyethylene terephthalate, polycarbonate, acrylonitrile butadiene styrene resin, and polymethacrylic acid methyl ester. The microorganism testing chip according to claim 1 or 2,
The main body and the flow path member are made of the same substance, and the microorganism testing chip is characterized in that: The microbial test chip according to any one of claims 1 to 9, the biological test chip being detachably attached, an XY stage for moving the biological test chip in an XY direction, and the fungus body detection unit A detection device that irradiates the excitation light to detect fluorescence from the fungus body detection unit,
The optical axis of the excitation light in the detection device and the optical axis of the lens system for detecting the fluorescence are coaxial,
The microorganism testing apparatus, wherein the microorganism testing chip is attached to the XY stage so that the plane of the microorganism testing chip body is perpendicular to the optical axis of the excitation light.   10. The microorganism testing apparatus according to claim 9, wherein the excitation light is perpendicularly incident on a fungus body detection flow path of the fungus body detection unit. In claim 9, when the solid angle of the objective lens in the lens system for detecting the fluorescence is δ, and the error range obtained from the incident angle of the excitation light is β,
The microbe inspection apparatus characterized in that an angle θ between the surface of the fungus body detection unit and the optical axis of the excitation light is θ = 90 ° − (1/2) δ ± β.

Images