JP2013044585A - Microorganism inspection device and inspection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism inspection device and inspection method capable of confirming a viable cell count is correctly measured by fluorescent flow cytometry.SOLUTION: For a flow state of a detection liquid or a position state of optical detection, information is obtained by various sensors even while measuring the viable cell count to recognize a measurement state. For instance, normal flow and abnormal flow (a cause of abnormality such as leakage of fluid and clogging of a flow path) are recognized by obtaining information from a flow rate sensor (differential pressure meter) and information on a bacteria count frequency distribution from a photodetector as the flow state of the detection liquid, a normal detection position and an abnormal detection position (positional deviation) are recognized by obtaining information from the photodetector as the position state of the optical detection, and the measurement state (whether it is normal or abnormal, and the cause when it is abnormal) is recognized from the information of the flow state and the position state.

Description

  The present invention relates to a microorganism testing apparatus and a testing method, and more particularly to a microorganism testing apparatus and a testing method for measuring the number of viable bacteria by a fluorescence cytometry method using a microorganism testing chip.

  Some microbiological testing devices that measure the number of viable bacteria use a fluorescent flow cytometry method. The fluorescence flow cytometry method is a particle measuring 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 microbiological testing equipment using the fluorescence flow cytometry method, a disposable chip (testing channel) that can be used to measure the number of specimens (detection channel) in order to reduce costs and eliminate the hassle of washing. It has been proposed to form by chip. In the microbe inspection apparatus using the inspection chip, every time the inspection chip is mounted on the inspection apparatus body, the detection channel is aligned, that is, the detection channel is aligned with the excitation light and the focus of the detector. Will do. As such a microbe inspection apparatus, there exist a thing of patent documents 1 and 2, for example.

  In addition, although it is not a fluorescence flow cytometry method, there exists a thing of patent document 3 as a technique which detects clogging of the fine flow path in a biochemical reaction cartridge. Patent Document 3 also describes that the flow path is inspected as a test immediately before the biochemical reaction is performed using the biochemical reaction cartridge in addition to the pre-shipment inspection at the time of manufacturing the biochemical reaction. Has been.

JP 2010-256278 A JP 2009-281753 A JP 2008-139096 A

  In order to perform viable counts without failure using the fluorescence flow cytometry method, it is essential that the detection channel is located in a location suitable for optical detection, and that the sample fluid flows in the detection channel. is there. In particular, in the case of a microbiological test apparatus that detaches and measures a test chip including a detection channel, every time the test chip is attached to the test apparatus, the position state of the test chip (detection channel) is recognized and the measurement state is changed. It is important to understand.

  Patent Documents 1 and 2 describe a method of aligning with high accuracy when an analysis chip (inspection chip) is attached to an inspection apparatus. However, after the measurement is started, the position state of the analysis chip and the optical detection device is not continuously grasped.

  Further, Patent Document 3 describes that a flow path is inspected as an inspection of a biochemical reaction cartridge immediately before a biochemical reaction is performed using the biochemical reaction cartridge. It is not possible to grasp the current state when

  That is, as described in Patent Document 1 and the like, conventionally, a microorganism testing apparatus or the like only recognizes that the position state and the flow path state are normal before the start of the testing. That is, conventionally, after starting the inspection, the position state and the flow state are not continuously grasped.

  However, even if the alignment operation or the like is performed with high accuracy before the start of the inspection, there may be a case where the position state or the flow state being measured is not in a normal state for some reason. If the number of viable bacteria is detected to some extent, it can be estimated that the detection flow path is in a normal position and the flow state is also normal. However, when viable bacteria are not detected (or the viable count is only slightly measured), it is due to the fact that there are no (or few) viable bacteria (microorganisms), or It is important to determine whether there is any problem on the microbe inspection device including the test chip in order to make the result of viable cell count measurement in the microbe test device more reliable. It can be said that there is.

  An object of the present invention is to provide a microorganism testing apparatus and a testing method capable of confirming that the viable count is correctly performed by the fluorescence flow cytometry method.

  Another object of the present invention is to provide a microorganism testing apparatus and a testing method capable of estimating the cause when it is determined that measurement is not correctly performed in viable count by fluorescent flow cytometry. Is to provide.

  In order to achieve the above object, the present invention obtains information from each sensor (detector) during measurement of the number of viable bacteria about the flow state of detection liquid or the position state of optical detection, and grasps the measurement state. Is.

  For example, in the present invention, the flow state of the detection liquid is obtained from a flow sensor (differential pressure gauge) and information on the bacterial count frequency distribution, and normal flow, abnormal flow (fluid leakage, flow path clogging, etc.) Cause), obtain information from the optical detector as the optical detection position status, recognize the normal detection position and abnormal detection position (positional deviation), and measure the measurement status (normal or not) from the flow status and position status information. It is possible to grasp the abnormality or the cause if it is abnormal).

  According to the present invention, it is possible to confirm that the viable count is correctly performed by the fluorescence flow cytometry method.

  In addition, according to the present invention, it is possible to estimate the cause when it is determined that the measurement is not performed correctly in the viable cell count measurement by the fluorescence flow cytometry method.

It is a figure which shows schematic structure of the microorganisms testing apparatus which concerns on one Example of this invention. It is a figure which shows schematic structure of the microbe inspection chip | tip in the microbe inspection apparatus shown in FIG. It is sectional drawing of the junction part of the main body and microbe detection part in the microbe detection chip | tip of the microbe inspection apparatus shown in FIG. It is a perspective view which shows the decomposition | disassembly structure of the microorganisms detection part shown to FIG. 3A. It is a figure which shows the other structural example of the pressure supply apparatus of the microbe test | inspection apparatus shown in FIG. It is a figure explaining one Example of the microorganisms testing method using the microorganisms testing apparatus shown in FIG. It is a figure which shows the flow state and position state when a measurement state is normal in the microbe inspection apparatus shown in FIG. It is a figure which shows the flow state and position state in case the measurement state has shifted | deviated in the microorganisms testing apparatus shown in FIG. FIG. 2 is a diagram showing a flow state and a position state when a fluid leakage occurs in the measurement state in the microorganism testing apparatus shown in FIG. 1. FIG. 2 is a diagram showing a flow state and a position state when a measurement state is misaligned and a fluid leak occurs in the microorganism testing apparatus shown in FIG. 1. It is a figure which shows the flow state and position state when the measurement state has clogged the flow path in the microbe inspection apparatus shown in FIG. It is a figure which shows description about the bacteria count histogram in case a measurement state is normal. It is a figure which shows description about a microbe count histogram in case a measurement state is clogged. It is a figure explaining the relationship between a flow state and a position state, and a measurement state (it is normal or abnormal, or the cause when abnormal).

  Embodiments of the present invention will be described below with reference to the drawings. Note that the embodiments described later are merely examples, and combinations of the embodiments, combinations with known or well-known techniques, and other modes by replacement are possible.

In the present specification, the microorganism testing apparatus and the testing method mean a testing apparatus and testing method for cells and microorganisms, and are not limited to a testing apparatus and testing method using only microorganisms. Further, in the present specification and claims, “cells or microorganisms” are not expressed, but “microorganisms” are simply described, but “cells” are also included.
(A) Overall Configuration Example of Microorganism Testing Apparatus FIG. 1 is a configuration diagram of a microorganism testing apparatus 1 according to an embodiment of the present invention. The microorganism testing apparatus 1 includes a microorganism testing chip 10, a detection device 11, a pressure supply device 14, a system device 18, an output device 19, and an XY movable stage 125.

The microbe inspection chip 10 has a mechanism for holding a specimen and a reagent therein and performing a process necessary for microbe measurement. The XY movable stage 125 holds the microorganism testing chip 10 and adjusts the position of the microorganism testing chip 10. The microbe inspection chip 10 is basically used only once, and is attached to the XY movable stage 125 for each inspection, position adjustment is performed, and is removed from the XY movable stage 125 after the inspection is completed. The detection device 11 irradiates microorganisms in the microorganism test chip 10 with excitation light, and converts scattered light and fluorescence from the microorganisms into electrical signals. The pressure supply device 14 controls the transport of the specimen and the reagent in the microbe test chip 10 via the chip connection tubes 1441 to 1444 connected to the microbe test chip 10 in order to perform the steps necessary for microbe measurement. The system device (control device) 18 is connected to each component (detection device 11, pressure supply device 14, output device 19, XY movable stage 125) of the microorganism testing device 1, and outputs a control signal to the pressure supply device 14. And the signal processing with respect to the electric signal input from the detection apparatus 11 is performed. The output device 19 displays the measurement result obtained by processing the electrical signal in the system device 18.
(B) Example of Microbe Test Chip Configuration Details of the microbe test chip 10 will be described. FIG. 2 is a plan view showing a configuration example of the microorganism testing chip 10. The microbe inspection chip 10 includes a specimen container 151 for holding a liquid specimen 1511, a microorganism staining liquid container 152 for holding a staining liquid (reagent liquid) 1521 for staining microorganisms in the specimen, a specimen and a staining liquid. A detection liquid container 155 for holding a detection liquid mixed and reacted, and a microorganism detection flow path 173 for irradiating the excitation light 113 from the excitation light source 111 of the detection apparatus 11 and observing the fluorescence of the microorganism are provided inside. And a detection liquid disposal container 156 for discarding a detection liquid that is a mixed liquid of the specimen 1511 and the staining liquid 1521 that have passed through the microorganism detection flow path 173. In this specification, the specimen container 151 side is defined as the upstream side and the microorganism detection flow path 173 side is defined as the downstream side along the specimen flow.

  Furthermore, the microorganism testing chip 10 connects the sample container 151, the microorganism staining liquid container 152, the detection liquid container 155, and the microorganism detection flow path 173, and the solution flow paths 1571 to 1574 through which the sample 1511 and the mixed liquid flow, Ventilation holes 1591 to 1594 connected to the chip connecting pipes 1441 to 1444 of the pressure supply device 14, and ventilation channels 1581 to 1584 for connecting the ventilation holes 1591 to 1594 and the containers (151, 152, 155, 156). I have.

  The solution flow paths 1571 to 1574, the vents 1591 to 1594, and the ventilation flow paths 1581 to 1584 are derived from the names of the containers to be connected, and the flow path 1571 between the specimen container and the microorganism staining solution container, and between the microorganism staining solution container and the detection solution container. A flow path 1572, a flow path 1573 between the detection liquid container and the microorganism detection flow path, a flow path 1574 between the flow path for microorganism detection and the detection liquid waste container, the specimen container vent 1591, the vent hole 1592 of the microorganism staining liquid container, and the detection liquid container A vent 1593, a detection liquid waste container vent 1594, a specimen container vent flow path 1581, a microorganism staining liquid container vent flow path 1582, a detection liquid container vent flow path 1583, and a detection liquid waste container vent flow path 1584.

  Further, a food residue removal unit 160 that is a filter for removing food residues contained in the sample is provided on the sample container 151 side of the flow channel 1571 between the sample container and the microorganism staining solution container.

  The sample container 151, the food residue removal unit 160, the microorganism staining liquid container 152, the detection liquid container 155, the microorganism detection flow path 173, and the detection liquid waste container 156 are connected in series by the solution flow paths 1571 to 1574. It is connected.

  In the case of FIG. 2, the depth and the channel width of the solution channels 1571 to 1574 are, for example, 10 μm to 1 mm, and the depth and the channel width of the ventilation channels 1581 to 1584 are, for example, in the range of 10 μm to 1 mm. The cross-sectional area of the solution flow paths 1571 to 1574 is formed to be larger than the cross-sectional area of the ventilation flow paths 1581 to 1584.

  The volume of the sample container 151 is larger than the volume of the sample 1511. The volume of the microorganism staining solution container 152 is larger than the total volume of the specimen 1511 and the microorganism staining solution 1521. The volume of the detection liquid container 155 is larger than the total volume of the specimen 1511 and the microorganism staining liquid 1521. Further, the highest point of the flow path 1571 between the specimen container and the microorganism staining solution container is formed to be higher than the water level of the specimen 1511 in the specimen container 151. Similarly, the highest point of the channel 1572 between the microorganism staining solution container and the detection solution container is formed so as to be higher than the water level of the mixed solution of the specimen 1511 and the microorganism staining solution 1521. Further, the highest point of the detection liquid container-microorganism detection flow path 1573 is formed to be higher than the water level of the detection liquid (mixed liquid) composed of the specimen 1511 and the microorganism staining liquid 1521.

  The microorganism staining liquid 1521 is enclosed in the microorganism testing chip 10 in advance. The specimen 1511 is injected into the specimen container 151 from the vent 1591 before the examination.

As a configuration example of the microbe inspection chip, another configuration example as described in JP 2008-157829 A may be used. For example, the detection liquid container 155 may be omitted by mixing and reacting the specimen and the staining liquid in the microbial staining liquid container 152 so as to hold the detection liquid as it is. In addition, when using a plurality of staining solutions, a plurality of microorganism staining solution containers are provided without holding the plurality of staining solutions in the same microorganism staining solution container, and after the staining with the first staining solution is completed, the second staining solution The liquid mixture may be flowed into a microorganism staining liquid container that holds the liquid, and mixed and reacted with the second staining liquid to form a final detection liquid.
(C) Structural Example of Microbe Detection Unit of Microbe Test Chip The structure of the microbe detection unit 17 in the microbe test chip 10 will be described with reference to FIGS. 3A and 3B. FIG. 3A shows a cross-sectional view of a joint portion between the main body 15 of the microbe inspection chip 10 and the microbe detection unit 17. FIG. 3B shows an exploded perspective view of the microorganism detection unit 17. The microorganism detection unit used in this embodiment is described in detail in Patent Document 1.

  The main body 15 and the microorganism detection unit 17 are produced in separate steps, and are joined together. First, the manufacturing method of the microorganism detection part 17 is demonstrated. The microorganism 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. The microorganism detection part 17 is formed by this bonding. A microbe detection flow path 173 is configured by the groove 1731 of the flow path member 172 and the cover member 171. The through holes 1741 and 1751 of the flow path member 172 constitute a microorganism detection flow path inlet 174 and a microorganism detection flow path outlet 175.

  On the other hand, the flow path 1573 between the detection liquid container and the microorganism detection flow path 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 flow path 1574 between the microorganism detection flow path and the detection liquid disposal container changes the flow path direction at the upper end to form an opening on the surface of the main body 15. The opening of the detection liquid container-microorganism detection flow path 1573 is connected to the microorganism detection flow path inlet 174, and the opening of the microbe detection flow path-detection liquid waste container flow path 1574 is the flow for detecting microorganisms. It is connected to the road exit 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 flow path 1573 between the detection liquid container and the microorganism detection flow path and the opening of the flow path 1574 between the flow path for microorganism detection and the detection liquid disposal container. The manufactured microorganism detection unit 17 is attached to the main body 15 as described above. As shown in FIG. 3A, the microorganism detection unit 17 is mounted so that the microorganism detection flow path 173 is disposed on the detection window frame 161 of the main body 15.

  In the case of the present embodiment, a detection window frame portion 161 that is a through hole or a through groove of the main body 15 is provided behind the microorganism detection flow path 173. Therefore, the excitation light 113 irradiates only the microorganism detection unit 17 and does not irradiate the main body 15. For this reason, the reflected light from the main body 15 and autofluorescence that cause an increase in background light are not generated. In order that the excitation light 113 that has passed through the microorganism detection flow path 173 is not 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. Is preferred.

  The thickness of the cover member 171 is, for example, 0.01 μm to 1 mm. The thickness of the flow path member 172 is, for example, 0.01 μm to 1 mm. The cross-sectional shape of the microorganism detection flow path 173 is formed in, for example, a square, a rectangle, or a trapezoid. The larger the cross-sectional dimension of the microorganism detection channel 173 is, the smaller the pressure loss is. However, it is preferable that the microorganism is flowed one by one. One side of the cross section of the microorganism detection flow path 173 is preferably 1 μm to 1 mm, for example, and the length is preferably 0.01 mm to 10 mm, for example. The optical axis of the excitation light 113 applied to the microorganism detection channel 173 is perpendicular to the direction vector of the microorganism detection channel 173.

  The material which comprises the microorganisms detection part 17 is demonstrated. The microorganism testing chip 10 is disposable. That is, after use, the microorganism detection unit 17 is discarded together with the main body 15. Therefore, the material used for the microorganism detection unit 17 must be inexpensive. The material used for the microorganism detection unit 17 needs to be excellent in 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 detection of fluorescence from microorganisms, it is preferable that the amount of autofluorescence generated by the microorganism detection unit 17 itself be sufficiently smaller than the amount of fluorescence from microorganisms.

  If a curved surface, unevenness, or the like exists on the surface of the microorganism detection unit 17, the light amount of the excitation light 113 irradiated on the microorganism 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 microorganism detection unit 17 needs to have a desired flatness. The microorganism detecting unit 17 preferably has a flatness with an unevenness of 0.1 mm or less.

  Considering such conditions, the materials used for the microorganism detection unit 17 include glass, quartz, polymethacrylic acid methyl ester (PMMA), polydimethylsiloxane (PDMS), cycloolefin polymer (COP), polyethylene terephthalate, and polycarbonate. Etc. are considered. The microorganism detection unit 17 is formed of one or more kinds of substances selected from these substances.

  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, in the case of this embodiment, the cover member 171 is made of glass or quartz, and the flow path member 172 is made of polymethacrylic acid methyl ester, polydimethylsiloxane, cycloolefin polymer, polyethylene terephthalate, and polycarbonate. Preferably, the flow path member 172 is made of polydimethylsiloxane. In this case, the self-adhesiveness of polydimethylsiloxane is used for joining the cover member 171 and the flow path member 172.

  The amount of autofluorescence of the microorganism detection unit 17 depends not only on the material but also on the thickness dimension of the microorganism detection unit. In order to reduce the amount of autofluorescence, the thickness dimension of the microorganism detection unit may be reduced. The smaller the thickness of the microorganism detection unit 17, the smaller the amount of autofluorescence generated from the microorganism detection unit 17. 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 hinder the detection of fluorescence of microorganisms, the thickness dimensions of these parts must be limited to a predetermined range.

  The autofluorescence amounts of glass, quartz and polydimethylsiloxane are almost the same. Therefore, when the cover member 171 is made of glass or quartz, the thickness is preferably 0.05 mm or more and 1 mm or less, for example. When the flow path member 172 is manufactured from polydimethylsiloxane, the thickness is preferably 0.1 mm or more and 1 mm or less, for example.

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 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, for example.
(D) Configuration Example of Pressure Supply Device Details of the pressure supply device 14 will be described. The pressure supply device 14 includes an air pump 141 with a regulator 1411. The air pump 141 is connected to the vent holes 1591 to 1594 of the microorganism testing chip 10 by chip connecting pipes 1441 to 1444. Valves 1421 to 1424 are provided in the chip connection tubes 1441 to 1444, respectively. By opening and closing the valves 1421 to 1424, a gas having a predetermined pressure is supplied to each container in the microorganism testing chip 10, or each container in the microorganism testing chip 10 is opened to the atmosphere. By controlling this pressure, the transport of the specimen and the reagent in the microorganism testing chip 10 is realized. The conveyance of the specimen and the reagent in the microorganism testing chip 10 by controlling the pressure is described in detail in Japanese Patent Application Laid-Open No. 2008-157829.

  A flow rate sensor 1412 is connected to the chip connection pipe 1444 connecting the valve 1424 and the detection liquid container 155, and the difference in the chip connection pipe 1444 generated by the flow of air flowing from the valve 1424 to the detection liquid container 155. The pressure is measured and used as flow state information.

Further, as shown in FIG. 4, the flow sensor may be a differential pressure gauge 1413. In this case, if a flow path resistance 1414 is arranged to facilitate the generation of a differential pressure and the differential pressure at both ends thereof is measured, a smaller amount of flow such as air leakage can be measured.
(E) Configuration Example of Detection Device The detection device 11 includes an excitation light source 111, a scattered light detection unit, and a fluorescence detection unit. Among these, the scattered light detection unit includes the scattered light detector 123 for detecting the scattered light 124 from the microorganisms passing through the microorganism detection flow path 173 and the excitation light 113 from the excitation light source 111. It is comprised from the light-shielding plate 122 for preventing that it injects directly. On the other hand, the fluorescence detection unit condenses the fluorescence 121 from the microorganisms passing through the microorganism detection flow path 173 and reflects the excitation light 113 in the direction of the microorganism detection unit 17 while collimating the objective lens 114. The fluorescent light 121 is transmitted through a dichroic mirror 112, a bandpass filter 117 that passes through the fluorescent light 121, a condensing lens 118 for condensing parallel light, a pinhole 119 used as a spatial filter for cutting stray light, It is comprised with the photodetector 120 which detects the light which passed the band pass filter 117. FIG. In addition, the irradiation part and the detection part are arrange | positioned so that a mutual focus may overlap.

  Before starting the measurement (before flowing the detection liquid into the microorganism detection channel), the microorganism detection channel 173 is adjusted to the position of the focal point of the excitation light. This positioning process will be described. Excitation light 113 output from the excitation light source 111 of the detection device 11 is irradiated to the microorganism detection channel 173 to detect the amount of scattered light generated from the microorganism detection channel 173 and the amount of fluorescence generated from the microorganism detection unit 17, respectively. By doing so, the relationship between the movable position of the XY stage 125 and each light quantity is acquired as a profile. In addition, the detection device 11 controls the XY movable stage 125 based on the acquired profile, and adjusts the microorganism testing chip 10 (specifically, the microorganism detection flow path 173) to a position suitable for detection. . That is, the position of the microorganism detection flow path where the amount of scattered light detected by the scattered light detection unit is maximized matches the optical axis of the excitation light, and the microorganism detection by which the fluorescence amount detected by the fluorescence detection unit is maximized The focus of the excitation light is made to coincide with the position of the flow path. This alignment is described in detail in Patent Document 1. For alignment, another method, for example, the method described in Patent Document 2 may be used. In this case, a holding container such as an alignment reagent, a solution flow path, a ventilation flow path, and the like are provided on the microorganism testing chip.

After the measurement is started, the photo-detector 120 detects fluorescence generated by the sample liquid consisting of the sample 1511 and the staining liquid 1521 flowing through the microorganism detection flow path, thereby obtaining the position state information.
(F) Measurement method (inspection method)
Hereinafter, a measurement method in the case of measuring the number of viable bacteria in a food-derived specimen using the microorganism testing apparatus 1 according to the present embodiment will be described. FIG. 5 is a flowchart showing the process of measuring the number of viable bacteria using the microorganism testing chip 10.

Prior to mounting the microorganism testing chip 10 on the XY movable stage 125, the sample 1511 is injected into the sample container 151 from the vent 1591 (S901). The specimen 1511 used here is obtained by adding a physiological saline having a mass ratio of 10 times to the food to be examined and performing a stomaching process.

  Next, the microbe inspection chip 10 is mounted on the XY movable stage 125 of the microbe inspection apparatus (S902).

  The measurement process after the microbe inspection chip 10 is mounted on the XY movable stage 125 includes a pretreatment process (S903 to S905) in which food residues are removed from the specimen and the microorganisms in the specimen are stained, and the microbe inspection chip 10 is aligned. The positioning step (S907) is performed, and the viable cell count measurement step (S908) that actually measures the viable cell count.

  Since the alignment step (S907) and the pretreatment steps (S903 to S905) are independent steps, they can be performed in parallel, and the viable cell count measurement step (S908) is performed when both steps are completed. Next, each step will be described.

  In the pretreatment step, first, the specimen 1511 is moved to the microorganism staining solution container 152 (S903). The pressure of the pressure supply device 14 is applied to the sample container 151 through the sample container vent 1591 to increase the pressure in the sample container 151. At the same time, the internal pressure of the microorganism staining solution container 152 is released to atmospheric pressure through the microorganism staining solution container vent 1592. Due to the pressure difference, the specimen 1511 enters the microorganism staining solution container 152 and is mixed with the microorganism staining solution 1521. When the specimen 1511 flows into the microorganism staining liquid container 152, the food residue contained in the specimen 1511 is removed by the food residue removing unit 160.

  The specimen 1511 from which the food residue has been removed is mixed with the staining solution 1521 in the microorganism staining solution container 152. The staining solution in the present example is a mixed solution of a dead bacteria staining solution for staining dead bacteria and a whole bacteria staining solution for staining whole bacteria. Examples of the dead bacteria staining solution include cyanine for dead microorganisms. Fluorescent dye (final concentration: 0.01 mM to 10 mM), PI (propidium iodide) (0.1 mg / ml to 20 mg / ml), EB (ethidium iodide) (0.1 mg / ml to 20 mg / ml) In the whole bacterial stain, for example, cyanine-based fluorescent dye (0.1 mM to 10 mM) for all microorganisms, LDS751 (0.1 mg / ml to 20 mg / ml), DAPI (4 ′, 6-diamidine) -2'-phenylindole) (0.1 mg / ml to 20 mg / ml), HOECHST33258 (0.1 mg / ml to 20 mg / ml), HOECHST34580 (0.1 mg / ml to 20 mg / ml). To use. DMSO (dimethyl sulfoxide), ethanols, water, etc. are used as the solvent.

  The mixing of the specimen 1511 and the staining solution 1521 in the microorganism staining solution container 152 is promoted by bubbling (S904). In the bubbling, for example, the pressure of the pressure supply device 14 is applied to the detection liquid container 155 via the detection liquid container vent 1593, and the microorganism staining liquid container 152 is opened to the atmosphere via the microorganism staining liquid container ventilation hole 1592. Air flows from the detection liquid container 155 through the microorganism staining liquid container-detection liquid container flow path 1572 and flows from below the microorganism staining liquid container 152 holding the mixed liquid of the specimen 1511 and the staining liquid 1521 to become bubbles. When the mixture rises from the bottom to the top, the mixture is stirred to promote mixing.

  Dead bacteria in the specimen 1511 are stained with a dead bacteria stain (here, PI (peak wavelength: 532 nm) is used) and a whole bacteria stain (here, LDS751 (peak wavelength: 710 nm) is used), while The living bacteria in the specimen 1511 are stained only with the whole bacteria staining solution.

  The water level of the liquid mixture of the two liquids does not exceed the highest point of the flow path 1572 between the microorganism staining liquid container and the detection liquid container that connects the microorganism staining liquid container 152 and the detection liquid container 155, and further enters the microorganism staining liquid container 152. The discharged air is discharged to the outside through the microorganism staining liquid container vent 1592. Since the atmospheric pressure of the microorganism staining liquid container 152 is equal to the atmospheric pressure, the two-liquid mixture is not pushed out to the detection liquid container 155, and the microorganism staining liquid is used during the time required for the reaction (for example, about 30 minutes to 1 hour). It can be held in the container 152.

  At this time, in order to prevent the mixed liquid from flowing into the detection liquid container 155, the pressure from the pressure supply device 14 is applied to each of the vents 1593 to 1594 to detect the detection liquid container 155 to a range lower than the pressure of the specimen container 151. The pressure of the detection liquid waste liquid container 156 may be increased.

  During dyeing, it is desirable to keep the temperature of the microorganism testing chip 10 constant so as to reduce the influence of staining due to temperature changes.

  Next, the detection liquid (mixed liquid) composed of the specimen 1511 and the staining liquid 1521 is moved to the detection liquid container 155 (S905).

  As described above, the alignment of the microorganism testing chip 10 executed in parallel with these pretreatment steps is to adjust the microorganism detection flow path 173 to the position of the focal point of the excitation light.

When the above operation is completed, the detection liquid composed of the specimen 1511 and the microorganism staining liquid 1521 is moved to the microorganism detection flow path 173, and viable bacteria in the specimen are measured by the fluorescence flow cytometry method. (S908). As shown in FIG. 1, the excitation light 113 is irradiated from the direction perpendicular to the paper surface. For this reason, the fluorescence from the pigment | dye which dye | stained microorganisms and the scattered light by microorganisms arise. The detection device 11 detects only the fluorescence of the whole bacterial stain for live bacteria, and detects the fluorescence of the whole bacterial stain and the dead bacterial stain for dead bacteria. For this reason, it becomes possible to distinguish between live bacteria and dead bacteria. In addition, since the amount of scattered light varies depending on the size of the microbial cell, the size of the microbial cell can also be determined. In FIG. 1, one photodetector 120 is shown. However, when a plurality of staining solutions (dead bacteria staining solution and whole bacteria staining solution) are used, it corresponds to the peak wavelength of fluorescence from each staining solution. Thus, a photodetector or the like is provided. That is, a dichroic mirror for fluorescence separation, a short wavelength bandpass filter, a long wavelength bandpass filter, a short wavelength photodetector, a long wavelength photodetector, and the like are provided. These configurations are described in detail in, for example, Japanese Patent Application Laid-Open No. 2008-157829, Patent Document 1, Patent Document 2, and the like.
(G) Grasp of measurement state Information recognition of a flow state and a position state in order to grasp a measurement state will be described with reference to FIGS. In this embodiment, the differential pressure gauge 1413 shown in FIG. 4 is used as the flow rate sensor.

  FIG. 6 shows a case where the measurement state is normal. That is, the detection liquid flows through the microorganism detection flow path 173 at a predetermined flow rate (represented by a long arrow in the figure), and the microorganism detection flow path 173 is irradiated with the excitation light 113. At this time, since the flow rate of the detection liquid flowing through the microorganism detection flow path 173 is very small, the detection liquid container 155, the detection liquid container vent flow path 1583, and the detection liquid are added to the flow path 1573 between the detection liquid container and the microorganism detection flow path. The differential pressure gauge 1413 connected through the container vent 1593 shows a low value. Further, the detection liquid is irradiated with the excitation light 113 to emit fluorescence, and the photodetector 120 detects the fluorescence, and the fluorescence intensity shows a high value. Thus, when the measurement state is normal, the differential pressure value is low and the fluorescence intensity is high.

  FIG. 7 shows a measurement state in which a positional deviation has occurred. That is, the detection liquid 1551 flows through the microorganism detection flow path 173 at a predetermined flow rate (represented by a long arrow in the figure), and the excitation light 113 is not irradiated to the microorganism detection flow path 173. At this time, since the flow rate of the detection liquid 1551 flowing through the microorganism detection flow path 173 is very small, the differential pressure gauge 1413 connected to the flow path 1573 between the detection liquid container and the microorganism detection flow path shows a low value. Further, since the detection liquid 1551 is not irradiated with the excitation light 113, fluorescence is not emitted, and the photodetector 120 does not detect fluorescence. For this reason, the fluorescence intensity shows a low value. Thus, when the measurement state is misalignment, the differential pressure value is low and the fluorescence intensity is weak.

  FIG. 8 shows a measurement state in which fluid leakage has occurred. That is, the detection liquid 1551 does not flow through the microorganism detection flow path 173 at a predetermined flow rate (expressed by a short arrow in the figure), and the microorganism detection flow path 173 is irradiated with the excitation light 113. At this time, since air leaks at the connection portion between the chip connecting tube 1444 and the detection liquid container vent 1593, the differential pressure gauge 1413 connected to the flow path 1573 between the detection liquid container and the microorganism detection flow path has a high value. Show. Further, the detection liquid 1551 is irradiated with the excitation light 113 to emit fluorescence, and the photodetector 120 detects the fluorescence, and the fluorescence intensity shows a high value. Thus, in the measurement state in which fluid leakage occurs, the differential pressure value is high and the fluorescence intensity is strong.

  FIG. 9 shows a measurement state in which misalignment and fluid leakage occur. That is, the detection liquid 1551 does not flow through the microorganism detection flow path 173 at a predetermined flow rate (represented by a short arrow in the figure), and the excitation light 113 is not irradiated to the microorganism detection flow path 173. At this time, since air leaks at the connection portion between the chip connecting pipe 1444 and the detection container vent 1593, the differential pressure gauge 1413 connected to the detection liquid container-microorganism detection flow path 1573 shows a high value. . Further, since the detection liquid 1551 is not irradiated with the excitation light 113, fluorescence is not emitted. Since the photodetector 120 does not detect fluorescence, the fluorescence intensity shows a low value. As described above, in the measurement state in which positional deviation and fluid leakage occur, the differential pressure value is high and the fluorescence intensity is weak.

  FIG. 10 shows a measurement state in which the flow path is clogged. That is, since the clogging 1552 is generated in the microorganism detection flow path 173, the detection liquid 1551 stays (represented by a non-shown arrow in the drawing), and the microorganism detection flow path 173 is irradiated with the excitation light 113. State. At this time, since the detection liquid 1551 in the microorganism detection flow path 173 is stagnant, the differential pressure gauge 1413 connected to the detection liquid container-microorganism detection flow path 1573 shows a low value. Further, the detection liquid 1551 is irradiated with the excitation light 113 to emit fluorescence, and the photodetector 120 detects the fluorescence, and the fluorescence intensity shows a high value. In this way, in the measurement state where the excitation light 113 irradiates the microorganism detection flow path 173, although the clogging has occurred in the flow path, the differential pressure value is low as in the case where the measurement state is normal, and , The fluorescence intensity increases.

  Here, both when the measurement state of FIG. 6 is normal and when the measurement state of FIG. 10 is a state in which the flow path is clogged, the differential pressure value is low and the fluorescence intensity is strong. Become. In order to distinguish between these measurement states and specify the measurement state, the determination is made from the distribution of the number of bacteria detected by the photodetector 120 (the number of detection frequencies). When the measurement state is normal, the frequency distribution of the number of bacteria is uniform as shown in FIG. 11A, and when the measurement state is clogged in the flow path, the frequency distribution of the number of bacteria is uneven as shown in FIG. 11B. become. That is, when clogging occurs and the detection liquid 1551 stays, the number of bacteria is not measured after a certain amount of time has passed.

  FIG. 12 shows a summary of the relationship between each measurement state, the position state, and the flow state. As described above, even after the microorganism test chip is aligned, information from the flow sensor (differential pressure gauge), the flow path for detecting the microorganism, and the excitation light as the flow state of the detection solution during the measurement of the number of viable bacteria. It is possible to grasp the measurement state by obtaining information from the photodetector as the optical position state. In addition, abnormalities other than air leakage can be recognized by obtaining information on the bacterial count frequency distribution for the fluorescence intensity from the photodetector.

  The measurement state, the position state, and the flow state may be displayed on the output device 19. That is, when outputting the measurement result of the viable count, information on the flow state obtained from the flow sensor (differential pressure gauge) and information on the intensity of the fluorescence intensity obtained from the photodetector are output together. Alternatively, when outputting the measurement result of the viable count, measurement is performed based on the relationship shown in FIG. 12 from information on the flow state obtained from the flow sensor (differential pressure gauge) and information on the intensity of the fluorescence intensity obtained from the photodetector. It is determined whether the result is based on normal operation, that is, there is no abnormality, and whether the measurement state is normal or abnormal is also output. If it is determined that there is an abnormality, the cause of the abnormality of the microorganism testing apparatus is estimated based on the relationship shown in FIG. 12, and the cause is output. Or, if there is no microorganism detection signal or smaller than the predetermined value, is there any abnormality in the microorganism testing device based on flow information obtained from the flow sensor (differential pressure gauge) or information on the intensity of the fluorescence intensity obtained from the photodetector It is also possible to judge and output the result.

  According to the above-described embodiment, based on the flow state information obtained from the flow rate sensor (differential pressure gauge) and the bacterial count frequency distribution information obtained from the photodetector, normal flow and abnormal as the flow state of the detection liquid It can recognize the flow (fluid leakage, clogging of the flow path), and the normal detection position and abnormal detection position (positional deviation) as the optical detection position status based on the intensity of fluorescence intensity obtained from the photodetector ) And can determine whether the measurement state is normal or abnormal from the flow state and the position state. In particular, when there are few microorganisms in the detection solution, it is possible to grasp whether the result is a measurement result based on the presence of some abnormality on the microorganism testing apparatus side or a measurement result in a normal measurement state.

  In the above-described embodiment, both the flow state and the position state are determined. However, the present invention can be applied to the case where either one is determined.

  DESCRIPTION OF SYMBOLS 1 ... Microorganism test apparatus, 10 ... Microorganism test chip, 11 ... Detection apparatus, 14 ... Pressure supply apparatus, 17 ... Microbe detection part, 18 ... System apparatus, 19 ... Output apparatus, 111 ... Excitation light source, 112 ... Dichroic mirror, 113 ... excitation light, 114 ... objective lens, 119 ... pinhole, 120 ... photodetector, 121 ... fluorescence, 124 ... scattered light, 125 ... XY movable stage, 141 ... air pump, 1411 ... regulator, 1412 ... flow rate sensor, 1413 ... Differential pressure gauge, 1414 ... Flow path resistance, 151 ... Sample container, 1511 ... Sample, 152 ... Microbe staining solution container, 1521 ... Staining solution, 155 ... Detection solution container, 1551 ... Detection solution, 156 ... Detection solution waste container, 1571-1574 ... Solution channel, 1581-1584 ... Ventilation channel, 1591-1594 ... Vent, 161 ... Detection Window portion, 173 ... microorganism detection flow path.

Claims (10)

Sample container for holding sample, reagent liquid container for holding reagent liquid, flow path for microorganism detection, flow path for connecting the sample container and the reagent liquid container, and the reagent liquid container and the flow path for microorganism detection, respectively A microbe testing chip comprising:
A stage for holding the microorganism testing chip and moving the microorganism testing chip;
A pressure connected to the microorganism testing chip, for transporting the specimen from the specimen container to the reagent liquid container, and transporting a detection liquid comprising the specimen and the reagent liquid from the reagent liquid container to the microorganism detection flow path; A pressure supply device for supplying,
A light source for irradiating the microorganism detection flow path with excitation light, a detection device having a photodetector for detecting fluorescence from the detection liquid flowing in the microorganism detection flow path and converting it into an electrical signal;
A flow sensor disposed between the pressure supply device and the microorganism testing chip;
A control device that is connected to the pressure supply device, the detection device, and the flow rate sensor, outputs a control signal to the pressure supply device, and processes an electrical signal from the detection device and the flow rate sensor;
An output device for displaying a detection result processed by the control device;
The control device determines the presence or absence of leakage between the microorganism testing chip and the pressure supply device based on an electrical signal from the flow sensor, and determines the intensity of fluorescence based on the electrical signal from the photodetector. And determining the position state of the microbe detection flow path, and outputting the presence / absence of the leak and the position state to the output device. Sample container for holding sample, reagent liquid container for holding reagent liquid, flow path for microorganism detection, flow path for connecting the sample container and the reagent liquid container, and the reagent liquid container and the flow path for microorganism detection, respectively A microbe testing chip comprising:
A stage for holding the microorganism testing chip and moving the microorganism testing chip;
A pressure connected to the microorganism testing chip, for transporting the specimen from the specimen container to the reagent liquid container, and transporting a detection liquid comprising the specimen and the reagent liquid from the reagent liquid container to the microorganism detection flow path; A pressure supply device for supplying,
A light source for irradiating the microorganism detection flow path with excitation light, a detection device having a photodetector for detecting fluorescence from the detection liquid flowing in the microorganism detection flow path and converting it into an electrical signal;
A control device connected to the pressure supply device and the detection device, and processing the output of a control signal to the pressure supply device and an electric signal from the detection device;
An output device for displaying a detection result processed by the control device;
The control device determines the position state of the microbe detection flow path by determining the intensity of fluorescence intensity based on an electric signal from the photodetector, and outputs the position state to the output device. Microbiological testing device. Sample container for holding sample, reagent liquid container for holding reagent liquid, flow path for microorganism detection, flow path for connecting the sample container and the reagent liquid container, and the reagent liquid container and the flow path for microorganism detection, respectively A microbe testing chip comprising:
A stage for holding the microorganism testing chip and moving the microorganism testing chip;
A pressure connected to the microorganism testing chip, for transporting the specimen from the specimen container to the reagent liquid container, and transporting a detection liquid comprising the specimen and the reagent liquid from the reagent liquid container to the microorganism detection flow path; A pressure supply device for supplying,
A light source for irradiating the microorganism detection flow path with excitation light, a detection device having a photodetector for detecting fluorescence from the detection liquid flowing in the microorganism detection flow path and converting it into an electrical signal;
A flow sensor disposed between the pressure supply device and the microorganism testing chip;
A control device that is connected to the pressure supply device, the detection device, and the flow rate sensor, outputs a control signal to the pressure supply device, and processes an electrical signal from the detection device and the flow rate sensor;
An output device for displaying a detection result processed by the control device;
The control device determines whether or not there is a leak between the microorganism testing chip and the pressure supply device based on an electrical signal from the flow sensor, and outputs the presence or absence of the leak to the output device. Microbiological testing device.   4. The microorganism testing apparatus according to claim 1, wherein the flow sensor is a differential pressure gauge.   5. The microorganism testing apparatus according to claim 4, wherein the differential pressure gauge measures a differential pressure at both ends of a channel resistance provided between the pressure supply device and the microorganism testing chip.   4. The control device according to claim 1, wherein the control device determines a flow state in the microbe test chip based on information on a detection frequency distribution from the photodetector, and outputs the flow state to the output device. A microorganism testing apparatus characterized by: Sample container for holding sample, reagent liquid container for holding reagent liquid, flow path for microorganism detection, flow path for connecting the sample container and the reagent liquid container, and the reagent liquid container and the flow path for microorganism detection, respectively A microbe test chip comprising: a stage for holding the microbe test chip and moving the microbe test chip; connected to the microbe test chip; transporting the sample from the sample container to the reagent solution container; A pressure supply device for supplying a pressure for conveying a detection liquid comprising the reagent liquid from the reagent liquid container to the microorganism detection flow path; a light source for irradiating the microorganism detection flow path with excitation light; and the microorganism detection A detection device having a photodetector for detecting fluorescence from the detection liquid flowing through the flow path and converting it into an electrical signal; and the pressure supply device and the detection device, and the pressure Microbiological testing using a microbiological testing device having a control device for processing a control signal output and an electrical signal from the detection device, and a flow rate sensor disposed between the pressure supply device and the microbiological testing chip. A method,
Attaching the microbe testing chip to a predetermined portion of the stage;
Aligning the microorganism testing chip and the detection device;
Pressure is supplied from the pressure supply device, the detection liquid is caused to flow in the microorganism testing chip, and when the detection liquid flows in the microorganism detection flow path, excitation light is emitted from the light source, and the light detection is performed. Detecting fluorescence with a vessel, converting it into an electrical signal and outputting it,
When the fluorescence is detected and converted into an electrical signal and output, the flow rate information obtained from the flow sensor and / or the intensity information of the fluorescence intensity obtained from the photodetector are output together. Microbial testing method. Sample container for holding sample, reagent liquid container for holding reagent liquid, flow path for microorganism detection, flow path for connecting the sample container and the reagent liquid container, and the reagent liquid container and the flow path for microorganism detection A microbe test chip comprising: a stage for holding the microbe test chip and moving the microbe test chip; connected to the microbe test chip; transporting the sample from the sample container to the reagent solution container; A pressure supply device for supplying a pressure for conveying a detection liquid composed of a specimen and the reagent liquid from the reagent liquid container to the microorganism detection flow path; a light source for irradiating the microorganism detection flow path with excitation light; and the microorganism detection A detection device having a photodetector for detecting fluorescence from the detection liquid flowing through the flow path and converting it into an electrical signal; and the pressure supply device and the detection device, and the pressure Microbiological testing using a microbiological testing device having a control device for processing a control signal output and an electrical signal from the detection device, and a flow rate sensor disposed between the pressure supply device and the microbiological testing chip. A method,
Attaching the microbe testing chip to a predetermined portion of the stage;
Aligning the microorganism testing chip and the detection device;
Pressure is supplied from the pressure supply device, the detection liquid is caused to flow in the microorganism testing chip, and when the detection liquid flows in the microorganism detection flow path, excitation light is emitted from the light source, and the light detection is performed. Detecting fluorescence with a vessel, converting it into an electrical signal and outputting it,
When detecting the fluorescence and converting it into an electrical signal and outputting it, it is determined whether there is an abnormality in the microbiological examination apparatus based on the flow rate information obtained from the flow sensor or the intensity information of the fluorescence intensity obtained from the photodetector. A method for testing microorganisms, comprising:   9. The microorganism testing method according to claim 8, further comprising the step of estimating an abnormality cause of the microorganism testing apparatus based on flow information obtained from the flow sensor and information on fluorescence intensity obtained from the photodetector. .   In Claim 8, when there is no detection signal of microorganisms or when it is smaller than a predetermined value, there is no abnormality in the microorganism inspection apparatus based on flow information obtained from the flow sensor or information on the intensity of fluorescence intensity obtained from the photodetector. A method for testing microorganisms, characterized in that

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