Cell detection device and cell detection method

Abstract

To provide a technique for detecting a cell-derived substance with high sensitivity without destroying cells.SOLUTION: A cell detection device of the present disclosure includes: a medium adding device configured to add a portion of a prepared culture medium to a culture container holding cells to be detected; a medium collection device configured to collect an amount of the added portion of the culture medium from the culture container, excluding a portion remaining in the culture container and a portion evaporated, thereby collecting adenosine triphosphate generated in the culture medium from the cells; and a detection device configured to detect light from a luminescent reagent, which reacts with the adenosine triphosphate to emit light, having been mixed with the collected culture medium and the adenosine triphosphate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a cell detection device and a cell detection method. [Background technology]

[0002] In sterility testing of pharmaceuticals, etc., it is necessary to detect very small numbers of bacteria or fungi (hereinafter simply referred to as "bacteria"). Conventionally, the culture method used for sterility testing involves culturing bacteria in a medium to increase the number of cells for detection, but a major issue is that it takes time, requiring cultivation for more than a day to obtain a sufficient number of bacteria for detection. Therefore, several rapid testing methods have been developed in recent years.

[0003] Among these, the ATP (Adenosine Triphosphate) method is known as a highly sensitive method for detecting bacteria. The ATP method detects bacterial ATP through bioluminescence caused by the luciferin-luciferase reaction, and can generally detect approximately 100 CFU (Colony Forming Unit) of bacteria.

[0004] For example, Patent Document 1 discloses that bacterial growth and death can be detected with high sensitivity by dispensing a bacterial culture medium and a luminescence reagent onto a plate and measuring luminescence using the ATP method.

[0005] Patent Document 2 also discloses a method in which a sample is filtered to capture and concentrate bacteria on the filter, and then extracellular ATP is eliminated to remove background ATP before extracting intracellular ATP. The method of Patent Document 2 enables highly sensitive detection of bacteria, and can detect even a few bacteria. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 2696081 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-116083 Summary of the Invention [Problem to be solved by the invention]

[0007] On the other hand, if bacteria are detected in a bacterial presence test, it is desirable to perform other tests, such as bacterial species identification, using the same sample. This is to understand the details of the bacteria contaminating the sample, estimate the route of contamination, and reflect the results in quality control. However, the method of measuring intracellular ATP as described above requires the bacteria to be destroyed when extracting intracellular ATP, making it impossible to subsequently culture the bacteria and perform identification tests.

[0008] To further enrich and culture the bacteria after detecting their presence, one possible method is to partially destroy the bacteria and test them using the ATP method. This method involves growing the bacteria in a liquid medium, then periodically aliquoting a portion of the liquid medium to measure the intracellular ATP. An increase in ATP is considered to indicate bacterial growth. With this method, live bacteria remain in the original liquid medium, allowing for subsequent enrichment and further testing. However, because repeated aliquots are taken from the liquid medium, it is necessary to start the culture with a medium volume that takes into account the maximum number of aliquots. Furthermore, the bacteria are diluted in the medium, resulting in a low bacterial concentration, leaving room for improvement in the sensitivity of bacterial detection.

[0009] The above-mentioned problems are not limited to bacteria, but also apply to general cell detection, which involves detecting a small number of cells, such as cultured cells, with high sensitivity and speed without destroying them.

[0010] Therefore, the present disclosure provides a technology for detecting cells with high sensitivity without destroying the cells. [Means for solving the problem]

[0011] In order to solve the above problems, the cell detection device disclosed herein is characterized by comprising a culture medium addition device that adds a portion of prepared culture medium to a culture vessel holding the cells to be detected, a culture medium recovery device that recovers the culture medium from the culture vessel, and a detection device that detects light from a luminescent reagent mixed with the recovered culture medium.

[0012] Further features related to the present disclosure will become apparent from the description and accompanying drawings of this specification, and aspects of the present disclosure may be realized and realized by the elements and combinations of various elements and aspects set forth in the following detailed description and the appended claims. The descriptions herein are exemplary and illustrative only and are not intended to limit the scope or application of the present disclosure in any way. [Effects of the Invention]

[0013] According to the cell detection device of the present disclosure, cells can be detected with high sensitivity without destroying the cells. Problems, configurations, and effects other than those described above will become apparent from the following description of the embodiments. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic cross-sectional view showing a cell detection device according to a first embodiment. [Figure 2] FIG. 1 is a functional block diagram of a cell detection device according to a first embodiment. [Figure 3] 1 is a flowchart showing a cell detection method according to the first embodiment. [Figure 4A] FIG. 10 is a schematic diagram showing a measurement screen. [Figure 4B] FIG. 10 is a schematic diagram showing a measurement method setting screen. [Figure 4C] FIG. 10 is a schematic diagram showing a screen that displays a graph of the measurement results. [Figure 5] FIG. 10 is a schematic cross-sectional view showing a cell detection device according to a second embodiment. [Figure 6] FIG. 10 is a functional block diagram of a cell detection device according to a second embodiment. [Figure 7] 10 is a graph showing the results of bacteria detection by the cell detection device according to the second embodiment. [Figure 8] FIG. 10 is a schematic diagram showing a cell detection device according to a third embodiment. [Figure 9] FIG. 10 is a schematic cross-sectional view showing a cell detection device according to a fourth embodiment. [Figure 10] FIG. 10 is a schematic diagram showing the configuration of a portion of a cell detection device according to a fifth embodiment. [Figure 11] FIG. 13 is a schematic cross-sectional view showing the configuration of a portion of a cell detection device according to a sixth embodiment. [Figure 12] FIG. 13 is a schematic diagram showing a cell detection device according to a seventh embodiment. [Figure 13] FIG. 13 is a schematic cross-sectional view showing the configuration of a portion of a cell detection device according to an eighth embodiment. [Figure 14] FIG. 13 is a schematic cross-sectional view showing the configuration of a portion of a cell detection device according to a ninth embodiment. [Figure 15] FIG. 22 is a schematic diagram showing the configuration of a part of a cell detection device according to a tenth embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0015] [First embodiment] <Configuration of cell detection device> 1 is a schematic cross-sectional view showing a cell detection device 100 according to a first embodiment. The cell detection device 100 is a device for detecting bacteria or fungi (hereinafter referred to as "bacteria") contained in a sample solution with high sensitivity based on extracellular ATP secreted by the bacteria, without destroying the bacteria. As shown in FIG. 1, the cell detection device 100 includes a syringe 101, a filter holder 103 that holds a filter 102, a collection container 105, a lid 106, and a luminescence measuring device 109.

[0016] Syringe 101 (culture medium container, culture medium addition device) contains culture medium 1, and syringe 101 is configured to be able to dispense a predetermined amount (a portion) of the total amount of culture medium 1. Although not shown in the figure, syringe 101 is attached to a drive device (culture medium addition device) that controls the vertical movement of syringe 101 itself and the sliding of the plunger of syringe 101.

[0017] A mesh 104 is provided at the bottom of a filter holder 103 (culture vessel), and a filter 102 is placed on the mesh 104. The bacteria 2 are captured on the filter 102, for example, by filtering the sample solution using the filter 102 and a filtration device (not shown). After filtering the sample solution to capture the bacteria 2, the filter 102 is placed on the mesh 104 of the filter holder 103.

[0018] The pore size of the filter 102 is selected to be large enough to capture the bacteria 2 (cells) to be detected. For example, when detecting bacteria, a filter with a pore size of 0.5 μm or less is generally used, and when detecting particularly small bacteria, a filter with a pore size of 0.2 μm or 0.1 μm is used. When detecting large cells such as yeast or mold, a filter with a pore size of 1 μm, for example, can be used.

[0019] The shape of the filter 102 may be a circular shape, which is commonly available commercially, or may be another shape. For example, by using a cylindrical or roll-shaped filter, the diameter can be reduced while maintaining the filter area, and when multiple filters are used in a row, more filters can be arranged in the same space compared to circular filters. Even if the filter is circular, by forming a corrugated surface rather than a flat surface, the filter area can be increased and filter clogging by particles contained in the sample can be prevented. Furthermore, by using a filter with a larger pore size on top of a filter with a smaller pore size, filter clogging by particles contained in the sample can also be prevented.

[0020] The pore size of the mesh 104 may be large enough to allow the culture medium 1 to pass through when the culture medium 1 is recovered by a culture medium recovery device, which will be described later.

[0021] The lid 106 is detachably attached to the filter holder 103 and covers the space above the filter 102, thereby preventing contamination from outside. A septum 107 is provided on the top surface of the lid 106, and by piercing the needle 108 of the syringe 101 through the septum 107, a predetermined amount of culture medium 1 can be supplied onto the filter 102 while keeping the space above the filter 102 sealed. By supplying the culture medium 1 to the filter 102 that has filtered the sample solution, bacteria 2 captured and concentrated from the sample can be cultured on the filter 102.

[0022] 1, filter holder 103 is formed in a cylindrical shape with a flange on the side, with lid 106 supported on the upper surface of the flange and the bottom surface of the flange supported by collection container 105. The outer diameter of filter holder 103 is smaller than the inner diameter of collection container 105, and the outer diameter of the flange of filter holder 103 can be approximately the same as the outer diameter of collection container 105.

[0023] The collection container 105 is, for example, a microtube or the like, and contains a luminescence reagent 3 containing, for example, luciferin and luciferase. After the bacteria 2 are cultured on the filter 102, the filter holder 103 and the collection container 105 are connected and centrifuged, for example, using a centrifuge (culture medium collection device), to collect the medium 1 containing ATP, which is a secretion of the bacteria 2, into the collection container 105. This causes the ATP in the medium 1 collected in the collection container 105 to mix with the luminescence reagent 3, resulting in a reaction that generates luminescence. A culture medium collection device other than a centrifuge can also be used as long as it can collect the medium 1 on the filter 102 into the collection container 105. For example, the medium 1 may be collected from the filter 102 into the collection container 105 by applying pressure, such as air pressure, from the lid 106 side using a pressure pump or the like.

[0024] The luminescence measuring device 109 (detection device) is placed at a position (for example, near the bottom of the collection container 105) where it can detect luminescence from the luminescent reagent 3 in the collection container 105. The luminescence measuring device 109 outputs a detection signal to, for example, an external calculation device. The calculation device calculates the amount of luminescence from the detection signal of the luminescence measuring device 109, and calculates the amount of ATP corresponding to the amount of luminescence.

[0025] Although not shown, the cell detection device 100 may have a temperature regulator for regulating the temperature of at least the filter 102 from the outside.

[0026] The driving device of the syringe 101, the luminescence measuring device 109, the temperature controller, and the centrifuge are connected to the above-mentioned arithmetic unit, and the arithmetic unit is connected to input / output devices such as a monitor, a keyboard, a touch panel, etc. A user can set various parameters by operating the input / output devices, and the arithmetic unit operates the driving device, the luminescence measuring device 109, the temperature controller, and the centrifuge in accordance with the parameters.

[0027] As shown in FIG. 1 , in the cell detection device 100, a storage location for the culture medium 1 (culture medium container), a filter 102 (culture container) in which bacteria 2 are cultured, and a storage location for the luminescent reagent 3 (collection container) are arranged separately, and the culture medium 1 moves from the culture medium container to the culture container and then to the collection container. Furthermore, the cell detection device 100 is characterized in that the culture medium 1 can be added to the filter 102 in fixed amounts rather than all at once using a syringe 101 (culture medium addition device). This configuration allows repeated operations to be performed: adding a small amount of culture medium 1 from the syringe 101 to the filter 102, culturing the bacteria 2 on the filter 102, recovering the culture medium 1 into a collection container 105 using a centrifuge (culture medium recovery device) to react with the luminescent reagent 3, and measuring the bacteria-derived ATP in the culture medium 1. The amount of culture medium 1 added to the filter 102 at one time will be described later.

[0028] By culturing bacteria 2 in a small amount of medium 1 on filter 102, the concentration of ATP secreted by bacteria 2 in medium 1 increases, allowing for highly sensitive measurement of extracellular ATP. As a result, bacteria can be detected quickly. Furthermore, since the extracellular secretions are measured, there is no need to destroy bacteria 2, and it is possible to measure changes over time associated with the growth of the same bacterial sample. After bacteria detection, the bacteria can be further enriched and cultured for other tests (drug susceptibility tests, bacterial species identification tests, genetic tests, etc.), or the bacterial strain can be preserved.

[0029] In the cell detection device 100, the collection container 105 contains the luminescent reagent 3 in advance, and the culture medium 1 collected from the filter 102 is repeatedly mixed therein. In this way, there is no need for a mechanism for dispensing the luminescent reagent 3, and therefore the cost of the device can be reduced.

[0030] If the purpose is to detect anaerobic bacteria, the collection container 105, filter holder 103, and lid 106 are tightly attached to keep the interior airtight, the interior is filled with an oxygen-free gas, and the medium 1 is degassed in advance and filled into the syringe 101. This allows anaerobic cultivation. An anaerobic environment can also be created by adding a reducing agent to the medium 1. On the other hand, since the luminescent reagent in the ATP method requires oxygen, it is advisable to seal oxygen in the space on the luminescent reagent 3 side.

[0031] Fig. 2 is a functional block diagram of the cell detection device 100 according to the first embodiment. As shown in Fig. 2, the cell detection device 100 includes a culture medium section 71 (culture medium container), a culture medium addition device 72, a culture section 73 (culture container), a culture medium recovery device 74, a culture medium recovery section 75 (recovery container), a luminescence reagent section 76 (recovery container), a luminescence measurement device 78 (detection device), a calculation device 79, an input device 80, an output device 81, and a temperature controller 82.

[0032] Culture medium section 71 is a location where a culture medium suitable for cells to be detected is stored. Culture medium section 71 is, for example, a storage section for culture medium 1 of syringe 101 shown in FIG. 1. Culture medium section 71 is connected to culture medium addition device 72. Culture medium addition device 72 is, for example, syringe 101 and its drive device, and the drive device is composed of parts that fix syringe 101, a motor that moves the parts, and a motor that pushes the plunger of syringe 101 a certain distance to dispense culture medium 1.

[0033] The culture section 73 holds bacteria (cells) to be detected. The culture section 73 is, for example, the filter 102 and filter holder 103 of the cell detection device 100. A portion of the culture medium is supplied from the culture medium section 71 to the culture section 73. In the cell detection device 100, the culture medium held in the culture medium section 71 and the culture medium supplied from the culture medium section 71 to the culture section 73 are separated by being contained in different containers, and are configured to prevent the culture medium or components in the culture medium from moving from the culture section 73 to the culture medium section 71.

[0034] The culture medium recovery device 74 performs an operation of moving the culture medium added to the culture unit 73 to the culture medium recovery unit 75. For example, the culture medium recovery device 74 includes a device for transporting a liquid, such as a centrifuge, a suction pump, a pressure pump, or a tube pump.

[0035] The culture medium recovery unit 75 stores the culture medium transferred from the culture unit 73. The culture medium recovery unit 75 is, for example, the recovery container 105 shown in Fig. 1. In the cell detection device 100, the culture unit 73 and the culture medium recovery unit 75 are connected to each other and used for the culture medium recovery operation by the culture medium recovery unit 75.

[0036] The luminescent reagent section 76 is a place for holding a luminescent reagent for a luminescent reaction, and is, for example, a container or syringe containing the luminescent reagent. In the cell detection device 100, the luminescent reagent 3 is contained in the collection container 105, so the luminescent reagent section 76 and the culture medium collection section 75 are integrated.

[0037] The luminescence measuring device 78 is a device that detects light generated by a luminescence reaction in the culture medium recovery unit 75, and specifically includes sensors such as a CCD sensor, a CMOS sensor, a photomultiplier tube, etc. The luminescence measuring device 78 outputs a detection signal of luminescence from the luminescence reagent unit 76 to the calculation device 79.

[0038] The arithmetic device 79 is a computer terminal such as a personal computer, smartphone, tablet, or mobile phone, and processes the detection signal from the luminescence measuring device 78. Specifically, when the arithmetic device 79 receives an input of the detection signal from the luminescence measuring device 78, it calculates the amount of luminescence from the detection signal and calculates the amount of ATP corresponding to the amount of luminescence.

[0039] The arithmetic unit 79 is also connected to the medium addition device 72 and the medium recovery device 74, and controls their operations. The arithmetic unit 79 is also connected to an input device 80 and an output device 81, and the user can set measurement parameters via the input device 80. The arithmetic unit 79 controls the operations of the medium addition device 72 and the medium recovery device 74 in accordance with the parameters. The output device 81 displays the results of calculation of the ATP amount by the arithmetic unit 79, a GUI screen for the user to input measurement parameters, and the like.

[0040] The temperature regulator 82 is, for example, an incubator, and regulates the temperature of the culture section 73 to an appropriate temperature according to the type of bacteria.

[0041] In this way, by arranging the culture medium section 71, the culture section 73, and the culture medium recovery section 75 separately and performing luminescence measurement in the culture medium recovery section 75, it is possible to repeatedly perform the operation of intermittently adding a predetermined amount of culture medium from the culture medium section 71 to the culture section 73 and recovering the culture medium in the culture medium recovery section 75 after cultivation, thereby enabling bacteria to be measured non-destructively and with high sensitivity.

[0042] <Cell detection method> FIG. 3 is a flowchart showing a cell detection method using the cell detection device 100.

[0043] In step S1, the user sets the bacterial detection threshold, the culture time (measurement time interval), the maximum culture time, and the amount of medium to be added per culture by operating the condition setting GUI screen displayed on the output device 81 and the input device 80. The calculation device 79 stores these set parameters in a storage device (not shown in FIGS. 1 and 2).

[0044] In step S2, the user prepares a sample solution that may contain bacteria, a syringe 101 that contains a culture medium 1, a filter 102, a filter holder 103, a lid 106, a collection container 105, and a luminescence reagent 3.

[0045] In step S3, the user connects the filter holder 103 with the filter 102 set therein to a filtration device (not shown). Next, the user connects a funnel (not shown) to the bacteria capture surface of the filter 102, pours the sample solution into the funnel, and filters it. This captures and concentrates the bacteria 2 on the filter 102, and also removes the solvent. Filtration using the filter 102 can be performed by a method suitable for the sample, such as suction filtration, pressure filtration, or centrifugal filtration. After filtration, the filter 102 can be washed by adding a cleaning solution that does not affect the bacteria and filtering the solution, thereby reducing the background light emission measurement originating from the sample solution.

[0046] Next, the user introduces the luminescent reagent 3 into the collection container 105 and connects the filter holder 103 to the top of the collection container 105. After that, the user places the lid 106 on the filter holder 103 to prevent the filter 102 from being contaminated with bacteria from the outside.

[0047] After steps S1 to S3 are completed, the user operates the input device 80 to input an instruction to the arithmetic device 79 to start operation.

[0048] In step S4, the computing device 79 drives the culture medium adding device 72 based on the amount of culture medium to be added set in step S1, causing the needle 108 of the syringe 101 to pierce the septum 107 of the lid 106, and adding a portion of the culture medium 1 from the syringe 101 to the filter 102. The amount of culture medium 1 to be added can be the minimum amount necessary to wet the entire filter 102.

[0049] In step S5, the arithmetic unit 79 determines whether the measurement to be performed is the first luminescence measurement. If it is the first measurement (Yes), the process proceeds to step S6.

[0050] In step S6, the computing device 79 drives the culture medium adding device 72 to pull out the needle 108 of the syringe 101 from the septum 107. Thereafter, the computing device 79 moves the lid 106, the filter holder 103, and the collection container 105, while they are connected, to the culture medium collection device 74, and collects the culture medium in the filter holder 103 (on the filter 102) into the collection container 105. If the culture medium collection device 74 is a centrifuge, the culture medium held on the filter 102 can be dropped into the collection container 105 by centrifugal force. The collection operation in step S6 causes the collected culture medium to be mixed with the luminescent reagent 3 (step S7).

[0051] In step S8, the luminescence measuring device 109 detects the luminescence generated by the reaction between the ATP in the culture medium and the luminescence reagent 3, and outputs a detection signal to the arithmetic device 79. The arithmetic device 79 calculates the amount of luminescence based on the detection signal, and stores this in the storage device as the amount of luminescence at 0 hours into the culture.

[0052] In step S9, the arithmetic device 79 determines whether this is the first measurement. If this is the first measurement, the process returns to step S4. In step S4, the arithmetic device 79 adds the same amount of culture medium 1 as in the previous step S4 onto the filter 102. Thereafter, in step S5, the arithmetic device 79 determines that this is not the first measurement (No), and proceeds to step S10.

[0053] In step S10, the computing device 79 cultivates the bacteria on the filter 102 for a predetermined time while maintaining the temperature of the filter 102 at an appropriate temperature using the temperature regulator 82. The temperature may be set according to the bacteria to be detected, such as about 30 to 40°C if the detection target is bacteria, or about 20 to 30°C if the detection target is mold.

[0054] Thereafter, steps S6 to S9 are executed in the same manner as above. If the culture has been carried out for one hour, the calculation device 79 stores the luminescence amount calculated in step S8 for the second time in the storage device as the luminescence amount for one hour after the culture.

[0055] In step S11, the arithmetic device 79 determines whether the amount of luminescence after cultivation is equal to or greater than the threshold value set in step S1. If it is equal to or greater than the threshold value (Yes), the amount of luminescence increases due to ATP being secreted into the medium as the bacteria grow, and so in step S12, the arithmetic device 79 determines that bacteria have been detected (positive) and outputs the determination result to the output device 81. If the output device 81 is a monitor, it displays a screen indicating a positive result. If the output device 81 is a speaker, it emits an alarm sound or the like.

[0056] If the amount of luminescence after cultivation is less than the threshold value in step S11 (No), the process proceeds to step S13. In step S13, the calculation device 79 determines whether the cultivation time has reached the maximum cultivation time set in step S1. If the maximum cultivation time has been reached (Yes), in step S14, the calculation device 79 determines that bacteria have not been detected (negative) and outputs the determination result to the output device 81.

[0057] If the maximum incubation time has not been reached in step S13 (No), the process returns to step S4, and the addition of medium, incubation, medium recovery container, and luminescence measurement are repeated.

[0058] As described above, when the amount of luminescence exceeds the threshold and is determined to be positive, or when the maximum incubation time is reached and is determined to be negative, detection of bacteria is terminated. Note that the determination in step S13 may also be made as to whether the maximum number of measurements has been reached.

[0059] The increase in the amount of luminescence in step S11 may be determined using a threshold set by the user in step S1 as described above, or may be automatically calculated by the arithmetic device 79 from the measurement data. Alternatively, the user may set the threshold on a GUI screen for setting conditions displayed on the output device 81 at the stage of step S11. This allows the detection conditions to be set appropriately even for detection targets with different characteristics, and enables testing using the same cell detection device 100 and cell detection method.

[0060] Furthermore, the determination of an increase in the amount of light emission in step S11 is not limited to the method of comparing the measured amount of light emission with a threshold value as described above. For example, the difference between the measured amount of light emission and the amount of light emission at time 0, or the difference between the amount of light emission measured this time and the amount of light emission measured last time, may be compared with the corresponding threshold value.

[0061] The allowable range of the incubation time set in step S1 is determined based on the growth rate or growth rate of the bacteria, the amount of culture medium or the amount of luminescence reagent initially prepared, and other factors. The amount of ATP secreted outside the bacteria is thought to be proportional to the number of bacteria. In general, the number of bacteria doubles in about 20 minutes for the fastest dividing bacterial species (such as E. coli). Therefore, in order to detect bacterial growth by an increase in extracellular ATP, an incubation time of 20 minutes or more is considered appropriate, and the measurement interval can be set longer than 20 minutes, taking into account measurement variability and measurement sensitivity. For example, this could be one hour or two hours.

[0062] The required amounts of medium and luminescent reagent to be prepared in step S2 are calculated based on the volume of medium added per addition and the maximum number of measurements. For example, if 50 μL of medium is added to the filter and the maximum number of additions is 20, 1 mL of medium is required in the syringe. Furthermore, considering that the luminescent reagent becomes diluted each time the medium is mixed with the luminescent reagent, and the luminescence efficiency decreases in proportion to the concentration, for example, if a dilution rate of the luminescent reagent is allowed up to 1.5 times, and the volume of medium recovered from the filter is 50 μL each time and the maximum number of measurements is 20, 2 mL of luminescent reagent must be pre-stored in the recovery container. The allowable dilution rate of the luminescent reagent can be determined by measuring the relationship between dilution rate and luminescence intensity using a standard ATP solution.

[0063] The type of medium to be added can be selected depending on the bacteria to be detected. For general bacteria, broth medium or soybean-casein digest medium can be used, but there is no limitation. A medium selected for the purpose of detecting only specific bacteria can also be used. For example, if the purpose is to detect Enterobacteriaceae, EE broth medium for Enterobacteriaceae detection can be used.

[0064] In step S4, the amount of culture medium added to the filter 102 can be minimized to increase the extracellular ATP concentration. However, it is necessary to add an amount of culture medium that is sufficient to distribute the medium evenly over the entire filter and provide sufficient nutrients to the bacteria captured on the filter. It is also necessary to consider the amount of water that evaporates during incubation.

[0065] For example, an area of ​​0.2 cm 2The amount of medium added was examined using a PVDF filter with a thickness of 0.125 mm and a pore size of 0.45 μm. The volume of this filter, calculated as area x thickness, was 25 μL. When 50 μL of medium was added to this filter, the entire filter was thoroughly wetted, and then the filter was centrifuged to remove the medium, 5 μL of medium remained on the filter. Furthermore, when 50 μL of medium was added to the filter, it was placed in a sealed container, and left in an incubator at 37°C for 2 hours, approximately 10 μL of water from the medium on the filter evaporated. Therefore, the amount of medium added to the filter can be 20 μL, which is the sum of the residual and evaporated medium (15 μL) plus the minimum amount required for the luminescence reaction, e.g., 5 μL. More preferably, adding the residual and evaporated medium to the filter volume to a volume of 40 μL or more will ensure that the medium remains distributed throughout the filter even if evaporation occurs, thereby minimizing damage to the bacteria.

[0066] Thus, it is considered appropriate that the amount of culture medium added to the filter is between 1 / 2 and 5 times the area and thickness of the filter, and preferably 1 to 2 times.

[0067] In addition to ATP, which bacteria secrete outside the bacterial cell, various other secretions can be targeted as measurement targets. In addition to secretions, bacteria can also be detected by adding a substrate that generates fluorescence or luminescence when broken down by the target enzyme, targeting enzymes specific to bacteria.

[0068] For example, one could add a luciferin derivative to Medium 1, which is decomposed by bacterial esterase to produce luciferin. The luciferin derivative is decomposed in proportion to the bacterial esterase activity, and luciferin is produced in Medium 1. If a luminescent reagent containing ATP and luciferase is added to this mixture, the amount of liberated luciferin can be quantified. This method allows bacterial growth to be detected based on esterase activity.

[0069] Moreover, by using a luciferin derivative that is decomposed only by an enzyme specific to a bacterial species to produce luciferin, it is also possible to detect only a specific bacterial species. For example, D-luciferin-O-β-D-glucuronide derivative that is decomposed by β-glucuronidase specific to Escherichia coli, and luciferin derivative 6-O-β-Galactopyranosyl-Luciferin targeting β-galactosidase specific to coliform bacteria.

[0070] Here, the quantification in the bioluminescence method using luciferin has been described, but it is also possible to perform fluorescence detection using a derivative of a fluorescent substance. In addition, by using derivatives of a plurality of luminescent substances or fluorescent substances having different wavelengths, different enzyme activities can be detected simultaneously. When performing fluorescence detection, an excitation light source and a light measurement device are used instead of the luminescence measurement device 78.

[0071] In addition, the technology of the present embodiment can be applied not only to bacteria but also as a method for detecting the activity and growth of cultured animal cells and the like. By adding a substrate corresponding to various enzymes specific to the target cells, the growth of the target cells can be detected.

[0072] <Example of GUI screen> An example of a GUI screen displayed on the output device 81 when detecting bacteria for a plurality of sample solutions will be described.

[0073] FIG. 4A is a schematic diagram of a measurement screen 4a showing the inspection conditions and inspection status of a plurality of sample solutions. As shown in FIG. 4A, on the measurement screen 4a, a new row can be created by clicking the "Create new sample" button, and the measurement of a new sample can be set by inputting the sample number, name, and other information. By clicking the "Start" button, the measurement can be started, and by clicking the "Pause" button, the measurement can be stopped. In addition, the culture start time, culture elapsed time, detection positive / negative, or that the measurement is in progress of each sample is displayed. For the sample for which the inspection result has come out, the end time is displayed. If the set value of the most recent ATP measurement is outside the set possible range, an error is displayed.

[0074] When the user clicks the "Method" button on the measurement screen 4a, a measurement method setting screen opens, allowing the user to set the measurement method.

[0075] 4B is a schematic diagram showing the measurement method setting screen 4b. As shown in FIG. 4B, the measurement method setting screen 4b can be called up and a measurement method can be set for one or more samples selected on the measurement screen 4a. The measurement method setting screen 4b has fields that allow the user to set the threshold value, which is the criterion for determining whether a sample is positive for bacteria, the measurement time interval (culture time), the amount of medium to be added from the culture medium section to the culture section, and the maximum value of the culture time (test time). Calculation methods for the threshold value and time interval can also be set in advance in the arithmetic unit 79 and then called up and used.

[0076] When the user clicks the "Show graph" button on the measurement screen 4a in FIG. 4A, a graph screen can be called up for one or more samples selected on the measurement screen 4a.

[0077] Figure 4C is a schematic diagram showing the graph screen 4c. As shown in Figure 4C, the graph screen 4c displays, for a selected sample, the change in measurement value over time, the time and result of the determination if positive or negative, the threshold value used for the determination, and the like. By using the graph screen 4c, the progress of measurements for multiple samples can be visually confirmed.

[0078] <Technical effect> As described above, the cell detection device 100 according to the first embodiment includes a syringe (culture medium container, culture medium adding device) that stores a culture medium, a filter 102 (culture container) that holds the bacteria 2, a culture medium recovery device such as a centrifuge, and a recovery container 105 that stores the luminescent reagent 3. The cell detection device 100 adds a portion of the culture medium 1 in the syringe 101 to the filter 102 using the syringe 101, recovers the culture medium 1 that has come into contact with the bacteria 2 into the recovery container 105 using the culture medium recovery device, and measures the luminescence from the luminescent reagent 3 using the luminescence measuring device 109. In this way, the culture medium that has come into contact with the bacteria is recovered from the filter 102, so that it is possible to add a portion of the culture medium 1 again to the filter 102 that holds the bacteria 2 and culture the bacteria. Therefore, the cell detection device 100 can repeat the series of operations of bringing the portion of the culture medium 1 into contact with the bacteria 2, moving the culture medium 1 that has come into contact with the bacteria 2, and detecting light multiple times for each culture time.

[0079] With this configuration, the cell detection device 100 can recover a high concentration of extracellular secretions of the bacteria 2 captured on the filter 102 and measure the luminescence at any culture time. Therefore, it is possible to quickly and sensitively detect the presence or absence of bacterial growth without destroying the bacteria 2. Furthermore, because the detected bacteria 2 are not destroyed, it is possible to perform other tests on the bacteria 2.

[0080] [Second embodiment] In the first embodiment, a method was described in which a cell detection device having one collection container is used to collect culture medium into the same collection container for each culture time and measure luminescence. In contrast, in the second embodiment, a method is proposed in which multiple collection containers are used to collect culture medium into different collection containers for each culture time. In this embodiment, the same components as in the first embodiment are assigned the same reference numerals, and repeated explanations will be omitted.

[0081] <Configuration of cell detection device> FIG. 5(a) is a schematic cross-sectional view showing a cell detection device 200 according to the second embodiment, illustrating the state when the culture medium 1 is added to the filter 102 for the first time. As shown in FIG. 5(a), the cell detection device 200 includes five collection containers 105a to 105e (plurality of collection containers). Furthermore, the collection container 105 does not have a luminescence reagent 3 pre-filled therein. The other configurations are the same as those of the cell detection device 100 of the first embodiment. Note that the filter holder 103 and the lid 106 are illustrated in a simplified manner. The number of collection containers 105 is five in FIG. 5(a), but this is selected depending on the number of luminescence measurements required.

[0082] The multiple collection containers 105 are, for example, individual microtubes, and can be individually installed in a centrifuge, a luminescence measuring device, or a temperature controller. In the state shown in Figure 5(a), a filter holder 103 having a filter 102 is connected to the first collection container 105a, and the needle 108 of the syringe 101 is inserted into the septum of the lid 106 to add a portion of the culture medium 1, and then the needle 108 of the syringe 101 is removed. Thereafter, the collection container 105a and the filter holder 103 connected to each other are set in a centrifuge and centrifuged, and the culture medium 1 on the filter 102 is collected into the collection container 105a.

[0083] 5(b) shows the state after the culture medium 1 has been collected into the first collection container 105a. The syringe 101 and filter holder 103 are retracted from the first collection container 105a into which the culture medium 1 has been collected, and these are set in the second collection container 105b. Furthermore, the luminescence reagent 3 is added to the first collection container 105a using the syringe 201 containing the luminescence reagent 3, and luminescence measurement is performed by the luminescence measuring device 109. By moving the luminescence measuring device 109 or any of the collection containers 105a to 105e, luminescence measurement can be performed for each collection container 105.

[0084] Although not shown, the syringe 201 is attached to a drive device (luminescence reagent adding device) that controls the vertical movement of the syringe 201 itself and the sliding of the plunger of the syringe 201.

[0085] 6 is a functional block diagram of a cell detection device 200 according to the second embodiment. As shown in Fig. 6, the cell detection device 200 has a culture medium recovery section 75 and a luminescent reagent section 76 arranged separately, and a luminescent reagent adding device 77 is connected to the luminescent reagent section 76. In other respects, the cell detection device 200 is similar to the cell detection device 100 of the first embodiment shown in Fig. 2.

[0086] In the second embodiment, the luminescent reagent section 76 is, for example, the storage section for the luminescent reagent 3 in the syringe 201 shown in Fig. 5(b). The luminescent reagent adding device 77 is, for example, the syringe 201 and its drive device. The drive device for the syringe 201 is made up of a part that fixes the syringe 201, a motor that moves the part, and a motor that pushes the plunger of the syringe 201 a certain distance to dispense the luminescent reagent 3.

[0087] The arithmetic device 79 controls the operation of the culture medium adding device 72 and the culture medium recovering device 74, as well as the operation of the luminescent reagent adding device 77. The user can determine the amount of luminescent reagent 3 to be added via the input device 80, and the arithmetic device 79 controls the operation of the luminescent reagent adding device 77 in accordance with the parameters.

[0088] <Cell detection method> The cell detection method using the cell detection device 200 according to the second embodiment is almost the same as that of the first embodiment (FIG. 3), so only the differences from the first embodiment will be described below.

[0089] First, in the first measurement, steps S1 to S6 are executed in the same manner as above to collect the culture medium 1 into the first collection container 105a. After step S6 is completed, the computing device 79 uses a mechanism (not shown) to move the filter holder 103 away from the first collection container 105a and connect them to a new second collection container 105b.

[0090] In step S7, the arithmetic device 79 drives the luminescent reagent adding device 77 to add the luminescent reagent 3 from the syringe 201 to the first collection container 105a.

[0091] Thereafter, steps S8 and S9 are performed for the first collection container 105a in the same manner as in the first embodiment, and the process returns to step S4, where the computing device 79 adds culture medium from the syringe 101 to the filter 102 set in the second collection container 105b.

[0092] Next, in step S5, it is determined that this is the second measurement, and the process proceeds to step S10, where the cells are cultured for a predetermined period of time.

[0093] Next, in step S6, the culture medium 1 is collected into the second collection container 105b. After step S6 is completed, the computing device 79 causes a mechanism (not shown) to retract the filter holder 103 from the second collection container 105b and connect them to a new third collection container 105c.

[0094] In step S7, the arithmetic device 79 drives the luminescent reagent adding device 77 to add the luminescent reagent 3 from the syringe 201 to the second collection container 105b.

[0095] Thereafter, steps S8 to S14 are carried out for the second collection container 105b in the same manner as in the first embodiment. The above operations are repeated until a positive or negative detection result is obtained in the luminescence measurement for the second collection container 105b and thereafter.

[0096] <Technical effect> As described above, the cell detection device 200 according to the second embodiment has a plurality of collection containers, and for each repeated luminescence measurement, the medium 1 after culturing bacteria on the filter 102 is collected into a different collection container and mixed with the luminescence reagent 3. This makes it possible to maintain a constant mixing ratio between the collected medium 1 and the luminescence reagent 3, and therefore luminescence measurement can be performed with the same sensitivity regardless of the culture time. This improves the accuracy of bacterial detection.

[0097] <Experimental Example> An experiment was conducted to detect bacteria (cells) in a sample solution using the cell detection device 200 according to the second embodiment.

[0098] <<Preparation of Sample and Cell Detection Device>> The detection target was Staphylococcus aureus (S. aureus), and a bacterial solution of Staphylococcus aureus was used as the sample solution. Three types of bacterial counts, 0 CFU (Colony forming unit), 10 CFU, or 100 CFU, were prepared.

[0099] The following were used as the components of the cell detection device 200. Filter: Area 0.2 cm 2 and pore size 0.45 μm Collection container: Microtube Culture medium: Soybean casein digest medium (SCD medium) Luminescence reagent: Luminescence reagent containing luciferin and luciferase

[0100] <<Measurement of ATP Amount>> (1) First, the bacterial solution of Staphylococcus aureus was added onto the filter, placed in a microtube with the lid on, and centrifuged using a centrifuge. This collected and concentrated the bacteria on the filter and removed the solvent.

[0101] (2) Next, the microtube was replaced with a new one (the first collection container), and 50 μL of SCD medium was added to the filter. This was centrifuged, and the medium added to the filter was collected into the microtube.

[0102] (3) 30 μL of the luminescence reagent was added to the collected medium, and the luminescence amount was measured. The unit of luminescence measurement is CPS (Count per second).

[0103] (4) Next, the filter was transferred to a new microtube (the second collection container), 50 μL of SCD medium was added to the filter again, and the lid was put on. After incubating this at 37°C for 2 hours, it was centrifuged, and the medium was collected into the microtube (the second collection container). 30 μL of the luminescence reagent was added to the collected medium, and the luminescence amount was measured.

[0104] (5) The procedure in (4) was repeated twice more, and the luminescence intensity after 4 hours of culture and after 6 hours of culture was measured.

[0105] (6) Two samples were prepared for each of the 0 CFU, 10 CFU, and 100 CFU samples, and procedures (1) to (5) were performed for each sample. The amount of ATP corresponding to the measured luminescence level was calculated for each sample. The results are shown in Figure 7(a).

[0106] Figure 7(a) is a graph showing the results of two tests performed on three types of sample solutions. The horizontal axis of the graph in Figure 7(a) represents the incubation time, and the vertical axis represents the amount of luminescence (CPS: counts per second), which is proportional to the amount of ATP. The ATP value at each time corresponds to the amount of extracellular ATP secreted into the medium during the two-hour incubation. For example, the ATP value at the fourth hour is the total amount of ATP secreted into the medium by the bacteria from the second to fourth hours.

[0107] As shown in Figure 7(a), when the number of staphylococci was 0 CFU, no increase in ATP was observed between 0 and 6 hours. When the number of staphylococci was 10 CFU, no increase in ATP was observed after 2 hours, but an increase was observed after 4 hours. When the number of staphylococci was 100 CFU, an increase in ATP was observed after 2 hours.

[0108] <<Comparison of extracellular and intracellular ATP amounts>> After culturing the 10 CFU sample for 6 hours, the ATP in the cells remaining on the filter was extracted by a known method and the amount of ATP was measured.

[0109] Figure 7(b) is a graph comparing the amount of extracellular ATP and the amount of intracellular ATP after 6 hours of culture. The vertical axis of Figure 7(b) is the amount of ATP (amol). The two bar graphs on the left are the units of the extracellular ATP measurement results for 10 CFU in Figure 7(a), converted to amol. In other words, the graph plots the amount of ATP secreted extracellularly between 4 and 6 hours after culturing Staphylococcus aureus at an initial cell count of 10 CFU.

[0110] The two bar graphs on the right show the results of measuring the amount of intracellular ATP after measuring the amount of extracellular ATP using a 10 CFU sample (two measurements). In other words, the amount of ATP contained within the cells was measured after culturing Staphylococcus aureus with an initial cell count of 10 CFU for 6 hours.

[0111] The amount of extracellular ATP was 15,000 amol on average, and the amount of intracellular ATP was 120,000 amol. Therefore, the amount of extracellular ATP was approximately 1 / 8 of the amount of intracellular ATP.

[0112] <<Comparison of cell detection sensitivity>> Using a 1 mL sample containing 10 CFU of staphylococci, the samples were cultured for 6 hours using the three methods shown below, and the sensitivity of ATP measurement was calculated and compared using the ATP amount values ​​described above.

[0113] (Method 1) Add a sample to the culture medium, culture it, and measure the intracellular ATP (destruction of the bacteria) This method involves adding 9 mL of SCD medium to 1 mL of a sample of staphylococcus at 10 CFU / 1 mL, culturing the sample, and then measuring the intracellular ATP by aliquoting 30 μL of the medium.

[0114] The initial bacterial concentration after adding SCD medium is 10 CFU / 10 mL. After culturing this at 37°C for 6 hours, 30 μL of the culture medium is taken, and 10 μL of free ATP elimination reagent and 10 μL of intracellular ATP extraction reagent are added. 30 μL of luminescence reagent is added, and luminescence is measured. The amount of ATP is calculated to be 360 ​​amol (120,000 amol ÷ 10 mL × 30 μL).

[0115] (Method 2) Adding a sample to a culture medium and culturing it to measure extracellular ATP (non-destructive to bacteria) Cultivation was carried out in the same manner as in Method 1 above, and 30 μL of luminescence reagent was added to 50 μL of this culture medium. The amount of luminescence was measured, and the amount of ATP was calculated to be 75 amol (15,000 amol ÷ 10 mL × 50 μL).

[0116] (Method 3) Cultivating bacteria in a small amount of medium on a filter (Second embodiment) 1 mL of sample is filtered through a filter, capturing and concentrating 10 CFU of bacteria on the filter. 50 μL of SCD medium is added to this, and the mixture is incubated at 37°C for 2 hours. The medium is then removed, and this process is repeated three times. 30 μL of luminescence reagent is added to the 50 μL of medium recovered from the third run, and luminescence is measured. The amount of ATP is calculated to be 15,000 amol.

[0117] In this way, in this embodiment, by culturing bacteria concentrated on a filter in a small amount of culture medium and measuring the extracellular secretions, it was found that sensitivity can be improved by about 50 times, even without destroying the bacteria, compared to a normal method in which samples are taken from the culture medium at regular intervals and the ATP amount is measured by luminescence, making it possible to detect bacteria more quickly.

[0118] [Third embodiment] In the second embodiment, a cell detection device having a plurality of separate collection containers has been described, whereas in the third embodiment, a cell detection device having a plurality of integrally formed collection containers is proposed.

[0119] <Configuration of cell detection device> Fig. 8(a) is a schematic cross-sectional view showing a cell detection device 300 according to the third embodiment. As shown in Fig. 8, the cell detection device 300 includes a sealed container 301 whose interior is sealed. The sealed container 301 has a disk 302 that forms the top surface and a disk 303 that forms the bottom surface.

[0120] One filter holder 103 (culture vessel) that holds a filter 102 is attached to the disk 302 , and an opening for the filter holder 103 is formed in the disk 302 and is closed by a septum 107 .

[0121] A plurality of collection containers 305 in the form of wells are formed on the disk 303. No luminescent reagent 3 has been previously introduced into the collection containers 305. At least one of the disks 302 and 303 is configured to be rotatable around its center as the rotation axis. This allows the filter 102 to be positioned above each collection container 305. To avoid interfering with the rotation of the disk 302 or 303, there is a gap between the opening of the collection container 305 and the filter 102 that is large enough to prevent the culture medium from leaking out, and the diameter of the opening of the collection container 305 is larger than the size of the filter 102. The rotation of the disk 302 or 303 is controlled, for example, by a rotating electric machine that is driven based on instructions from a computing device.

[0122] The space between disks 302 and 303 is sealed. In this specification, "sealed" means that there are no gaps through which bacteria can enter from the outside. A gas with a different composition from the outside air can be sealed inside sealed container 301 for anaerobic cultivation, and the sealed structure maintains airtightness between the inside and outside.

[0123] 8(b) shows the state after the culture medium 1 has been collected into the first collection container 305. By rotating the disk 302 or 303, the syringe 101 and filter holder 103 are retracted from above the first collection container 305 in which the culture medium 1 has been collected, and are stopped above the second collection container 305. The disk 302 has an opening covered with a septum 304. Furthermore, the syringe 201 containing the luminescent reagent 3 is used to add the luminescent reagent 3 to the first collection container 305 by piercing the septum 304 with the needle of the syringe 201, and luminescence measurement is performed by the luminescence measuring device 109.

[0124] FIG. 8(c) is a perspective view showing the state of FIG. 8(b). In FIG. 8(c), the internal configuration is shown through the side wall of the sealed container 301. As shown in FIG. 8(c), the septa 107 and 304 covering the openings provided in the disk 302 are respectively positioned directly above the two collection containers 305 provided on the disk 303. When the position of the luminescence measuring device 109 is fixed, by fixing the disk 302 and rotating the disk 303, it is possible to perform luminescence measurement using multiple collection containers 305 simply by rotating the disk 303. Of course, it is also possible to fix the disk 303 and rotate the disk 302, and change the position of the luminescence measuring device 109 each time luminescence measurement is performed for each collection container 305.

[0125] <Cell detection method> The cell detection method using the cell detection device 300 according to the third embodiment is almost the same as that according to the second embodiment, so only the differences from the second embodiment will be described below.

[0126] In the method of this embodiment, in step S3, the user sets the filter 102, which has filtered the sample solution and captured the bacteria 2, in the filter holder 103 of the disk 302. Alternatively, the user may set the filter 102 on the disk 302, add the sample solution to the filter 102, centrifuge the sealed container 301, and collect the filtrate in one of the multiple collection containers 305.

[0127] The method of this embodiment is also different from the second embodiment in that the positional relationship between the filter 102 and each collection container 305 is changed by rotating the disk 302 or 303.

[0128] <Technical effect> As described above, in the cell detection device 300 according to the third embodiment, the filter 102 for culturing bacteria and the collection container 305 for luminescence measurement are disposed in the internal space of the sealed container 301, and the cultivation of bacteria, collection of the medium 1, and luminescence measurement are carried out within the sealed container 301. This reduces the risk of bacteria being mixed in from outside when the filter 102 is moved above a different collection container 305 and transferred to the collection container. Furthermore, since the gas composition within the sealed container 301 can be controlled, the culture conditions can be maintained as anaerobic or aerobic depending on the type of bacteria to be detected.

[0129] [Fourth embodiment] In the second and third embodiments, a method was described in which a luminescent reagent was added to each of a plurality of collection containers using a syringe each time luminescence measurement was performed, whereas in the fourth embodiment, a cell detection device is proposed in which a luminescent reagent is dispensed in advance into a plurality of collection containers.

[0130] <Configuration of cell detection device> FIG. 9(a) is a schematic cross-sectional view illustrating a method for filtering a sample solution using a filter 102. The filter 102 is, for example, a filter with a volume of 100 μL. As shown in FIG. 9(a), the filter 102 is held in a ring-shaped filter holder 403. A funnel 5 is connected to the cell-capturing surface of the filter 102, and a filtration base 6 is connected to the opposite surface, thereby enabling filtering of a large amount of liquid sample. A suction pump (not shown) is connected to the filtration base 6 to perform suction filtration, allowing bacteria in the sample to be captured and concentrated on the filter 102.

[0131] Fig. 9(b) is a schematic cross-sectional view showing a cell detection device 400 according to the fourth embodiment. As shown in Fig. 9(b), in the cell detection device 400, the structures of the culture medium container that contains the culture medium 1 and the culture container in which the culture is performed are different from those of the cell detection devices 100 to 300 of the first to third embodiments.

[0132] After the bacteria are captured on the filter 102, the funnel 5 and the filtration base 6 are removed from the filter holder 403, and a syringe 401 (culture medium container, culture medium addition device) is connected to the cell capturing surface side of the filter holder 403, and an adapter 404 (a member that defines a space) is connected to the opposite surface. This forms a space 402 (culture container) surrounded by the bottom surface of the syringe 401, the inner wall surface of the filter holder 403, and the adapter 404, and the filter 102 is housed in the space 402.

[0133] Syringe 401 is provided with a check valve 409 that controls dripping and sealing of culture medium 1. Check valve 409 allows culture medium 1 to flow only in the direction from syringe 401 to filter 102. During culture, check valve 409 prevents diffusion of cell-derived substances from filter 102 to culture medium 1.

[0134] The adapter 404 is provided with a valve 410 and a capillary 408 for allowing the culture medium 1 that has passed through the filter 102 to flow out. The valve 410 is configured to allow the culture medium to flow from the filter 102 to the capillary 408 only when a certain level of pressure or more is applied, and prevents the culture medium in the filter 102 from flowing toward the capillary 408 due to gravity while cells are being cultured on the filter 102.

[0135] Although not shown, the syringe 401 is attached to a drive device (culture medium addition device) that controls the vertical movement of the syringe 401 itself, the sliding of the plunger of the syringe 401, the driving of the check valve 409, and the driving of the valve 410.

[0136] The luminescent reagent 3 is dispensed, for example, in 200 μL portions into a plate-shaped container having multiple wells 405 (multiple collection containers). The wells 405 are formed from a material that does not transmit light of the luminescent wavelength so that luminescence in adjacent wells 405 does not interfere with measurement, and only the surface near which luminescence is measured by the luminescence measuring device 109 is provided with a transparent window 415. Each well 405 is covered with a seal 406 to maintain a tight seal. The seal 406 may be a rubber seal that can maintain a tight seal even when a capillary 408 penetrates it, or an aluminum material that prevents deterioration of the luminescent reagent 3.

[0137] The luminescence measuring device 109 is provided with a cover 416 that covers the periphery of one well 405, which makes it possible to prevent light from an adjacent well 405 from being detected.

[0138] <Cell detection method> The cell detection method according to the fourth embodiment is almost the same as the methods according to the second and third embodiments, which use multiple collection containers. In this method, the calculation device actually drives each drive device to drive syringe 401, etc., but for the sake of simplicity, the explanation may be given simply assuming that the calculation device is the subject of the operation.

[0139] 3, the computing device may operate the syringe 401 to flow the culture medium 1 and clean the flow path from the filter 102 to the capillary 408. At this time, the culture medium 1 discharged from the capillary 408 is discarded.

[0140] In addition, in this embodiment, the amount of culture medium 1 added onto the filter 102 at one time can be the total volume of the space 402 in which the filter 102 exists, the valve 410, and the capillary 408 (for example, 200 μL).

[0141] In measuring luminescence at the 0th hour of culturing, the computing device inserts the capillary 408 through the seal 406 into one well 405, drops 200 μL of medium 1 into the well 405, and mixes it with the luminescence reagent 3. The luminescence measuring device 109 detects the luminescence generated by the reaction through the transparent window 415 and transmits a detection signal to the computing device. The computing device calculates the amount of luminescence corresponding to the detection signal and regards this as the luminescence at the 0th hour.

[0142] Next, the computing device pulls out the capillary 408 from the well 405 where the luminescence measurement at 0 hours was carried out, and maintains the filter 102 at, for example, 37° C. using a temperature regulator, and cultivates the bacteria on the filter 102 .

[0143] After two hours of culture, the computing device causes capillary 408 to pierce seal 406 and insert it into a well 405 different from that used at time 0. Then, 200 μL of medium 1 is delivered using syringe 401, and the medium containing the substance secreted by the cells on filter 102 is dropped into well 405 and mixed with luminescent reagent 3. Luminescence measuring device 109 detects the luminescence generated by the reaction through transparent window 415 and transmits a detection signal to the computing device. The computing device calculates the amount of luminescence corresponding to the detection signal and regards this as the luminescence at the second hour.

[0144] The calculation device compares the amount of luminescence at hour 0 with the amount of luminescence at hour 2, and if a significant increase in the amount of luminescence is observed at hour 2, the result is determined to be positive for bacteria. In other cases, the same operation is repeated until an increase in the amount of luminescence is detected by comparing it with the amount of luminescence at hour 0. As described above, the method of determining whether the result is positive or negative is not limited to a method of comparing the measured amount of luminescence with the amount of luminescence at hour 0, and the determination may also be made by comparing it with a predetermined threshold value, etc.

[0145] <Technical effect> As described above, cell detection device 400 according to the fourth embodiment uses a combination of syringe 401 that stores and drips culture medium 1 and filter 102 that captures bacteria, and culture medium 1 in syringe 401 and culture medium 1 added to filter 102 are arranged separately. This eliminates the need to separately move the syringe that stores the culture medium and filter holder 403 that has filter 102, making operation simple.

[0146] Furthermore, since the luminescent reagent 3 is dispensed into the multiple wells 405 in advance, a dispensing mechanism for the luminescent reagent 3 is not required, and the overall configuration of the device can be simplified.

[0147] Furthermore, as in the second embodiment, multiple wells 405 into which a predetermined amount of luminescent reagent 3 is dispensed are used, so the mixing ratio of the collected culture medium 1 and luminescent reagent 3 can be kept constant, and luminescence measurement can be performed with the same sensitivity regardless of the culture time, thereby improving the accuracy of bacterial detection.

[0148] [Fifth embodiment] In the first to fourth embodiments, the cell detection device is described as being configured to add the culture medium from a direction substantially perpendicular to the filter surface, whereas in the fifth embodiment, a configuration is proposed in which the culture medium is added from a direction substantially horizontal to the filter surface.

[0149] <Configuration of cell detection device> 10(a) is a schematic cross-sectional view showing a filter cartridge 502 of a cell detection device 500 according to the fifth embodiment. As shown in FIG. 10(a), the filter cartridge 502 includes a filter holder 503, a mesh 504, lids 506 and 507, flow channels 508 and 509, and plugs 510 and 511.

[0150] The filter 102 is fixed on a mesh 504 held by an annular filter holder 503. The mesh 504 plays a role in supporting the filter 102 when the strength of the filter 102 is weak.

[0151] Filter holder 503 is provided with flow channels 508 and 509, which are sealed with plugs 510 and 511, respectively, before the culture medium is added. Flow channel 508 is provided on the bacteria capture surface side of filter 102, and flow channel 509 is provided on the surface of filter 102 opposite to the bacteria capture surface.

[0152] A funnel is attached to the bacteria capture surface side of filter 102, a filtration stand is attached to the mesh 504 side, and sample solution is supplied from the funnel, thereby filtering the sample and capturing cells on filter 102. After filtering the sample, disk-shaped lids 506 and 507 (components that define a space) are attached to both sides of filter 102 so as to surround filter 102. By providing lids 506 and 507 to filter holder 503, filter 102 becomes filter cartridge 502 (culture vessel) in which filter 102 is sealed in a vessel with a small volume of approximately two times or less the volume of the filter.

[0153] FIG. 10(b) is a top view showing a configuration for adding culture medium to the filter cartridge 502.

[0154] As shown in FIG. 10( b), plugs 510 and 511 sealing the two flow paths 508 and 509 are removed. A medium container 501 is connected to the flow path 508 via a check valve 513 and a liquid supply pump 512, and a capillary 515 is connected to the flow path 509 via a valve 514. The flow path 508 is connected to the bacteria capture surface of the filter 102, and the flow path 509 is connected to the opposite surface. The medium 1 is supplied to the bacteria capture surface of the filter 102 via the check valve 513 and the flow path 508 by the liquid supply pump 512, and flows through the filter 102 to the flow path 509. In other words, the flow direction of the medium 1 is approximately parallel to the filter surface. By flowing the medium approximately parallel to the filter surface, a smaller amount of medium can be distributed over the entire filter surface compared to when the medium is flowed perpendicularly.

[0155] <Cell detection method> The cell detection method using the cell detection device 500 of this embodiment is almost the same as the cell detection methods of the second to fourth embodiments described above, and will be briefly described below. In this method, the calculation device actually drives each drive device to drive the liquid feed pump 512 and the like, but for the sake of simplicity, the explanation may be given simply assuming that the calculation device is the subject of the operation.

[0156] First, before performing luminescence measurement, the arithmetic device uses the liquid feed pump 512 to feed a sufficient amount of medium 1 to wash the filter 102 and the flow paths 508 and 509, and then discharges the medium from the capillary 515. Thereafter, the cell detection operation can be performed using a plate having a plurality of wells (collection containers) into which a luminescence reagent has been dispensed in advance, similar to the fourth embodiment. Specifically, the arithmetic device drives the liquid feed pump 512 to feed a predetermined amount of medium 1 from the medium container 501 into the filter holder 503, collects the medium into the first well through the capillary 515, and performs luminescence measurement using the luminescence measurement device. Thereafter, the arithmetic device removes the filter cartridge 502 from the first well, again feeds a predetermined amount of medium 1 into the filter holder 503, and after culturing for a predetermined time, collects the medium into the second well through the capillary 515, and performs luminescence measurement using the luminescence measurement device. In this way, the arithmetic device repeats the operation of adding medium 1 to the filter 102, culturing for a predetermined time, and then performing luminescence measurement.

[0157] <Technical effect> As described above, the cell detection device 500 according to the fifth embodiment includes a filter cartridge 502 configured so that the culture medium 1 is added from a direction approximately parallel to the surface of the filter 102. This prevents the culture medium from accumulating on the periphery of the filter, even when a filter with a large area is used, and allows a small amount of culture medium to be efficiently collected.

[0158] [Sixth embodiment] In the first to fifth embodiments, a cell detection device that tests for the presence or absence of bacteria using a filter 102 that filters a sample solution has been described. Since the bacteria detected by the method of each embodiment are non-destructive, they can also be used for subsequent analysis. Therefore, in the sixth embodiment, a cell detection device that can count the number of detected bacterial colonies is proposed.

[0159] <Configuration of cell detection device> 11 is a schematic cross-sectional view showing a filter cartridge 602 of a cell detection device 600 according to the sixth embodiment. As shown in FIG. 11, the filter cartridge 602 (culture vessel) has a configuration similar to that of the filter cartridge of the fifth embodiment (FIG. 10), but differs in that a fixing member 603 is adhered to the back surface of the lid 506. The fixing member 603 contacts the bacteria capture surface of the filter 102. The bacteria 2 captured on the filter 102 are fixed by being sandwiched between the filter 102 and the fixing member 603. The internal volume of the filter cartridge 602 including the fixing member 603 can be set to within five times the volume (area × thickness) of the filter 102.

[0160] The material of the fixing member 603 is not particularly limited and may be, for example, the same material as the filter 102, an agar medium (gel), or any other material that can fix the position of the bacteria 2 and does not affect the bacteria 2. When a transparent gel is used as the fixing member 603, after the bacteria 2 are detected using the cell detection device 600, the bacteria 2 are further cultured to form visible colonies, which can then be observed by removing the filter cartridge 602.

[0161] <Technical effect> As described above, the cell detection device 600 according to the sixth embodiment includes a filter cartridge 602 configured so that the culture medium 1 is added from a direction approximately parallel to the surface of the filter 102, and a fixing member 603 for fixing the position of the bacteria 2 is provided on the back surface of the lid 506 of the filter cartridge 602. As a result, after detecting positive bacteria, the number of viable bacteria can be counted by forming visible colonies through cultivation.

[0162] [Seventh embodiment] In the above-described fifth embodiment, a method was described in which a filter cartridge with a built-in filter was used to collect the culture medium in different wells (collection containers) for each culture time and perform luminescence measurement. In order to collect the culture medium in different wells in the fifth embodiment, the filter cartridge 502 was moved and the capillary 515 was inserted into each well. However, in the seventh embodiment, a method is proposed in which the culture medium is collected in multiple wells without moving the filter cartridge 502.

[0163] <Configuration of cell detection device> Fig. 12 is a schematic diagram showing a cell detection device 700 according to the seventh embodiment. As shown in Fig. 12, the cell detection device 700 includes a culture medium container 501 (culture medium container) that contains a culture medium 1, a liquid delivery pump 512 (culture medium adding device), a filter cartridge 502 (culture container), a flow path 701, a switching valve 702 (selection mechanism, culture medium collecting device), and a plurality of wells 705 (a plurality of collecting containers) that contain a luminescent reagent 3.

[0164] In the filter cartridge 502 , the flow path from which the culture medium 1 is discharged is connected to a flow path 701 , and the flow path 701 is connected to a switching valve 702 .

[0165] The switching valve 702 has a flow path 703 that can be switched to communicate with each of a plurality of wells 705, and the switching of the flow path 703 is controlled by a valve controller (not shown in FIG. 12) connected to a computing device.

[0166] <Cell detection method> The cell detection method using the cell detection device 700 of this embodiment is almost the same as the cell detection methods of the second to fourth embodiments described above, and will therefore be briefly described below.

[0167] First, before performing luminescence measurement, the arithmetic device uses the liquid feed pump 512 to send a sufficient amount of culture medium 1 to wash and drain the filter and flow path 701. Next, the arithmetic device connects the flow path 701 to the switching valve 702. Next, as in the fifth embodiment, the arithmetic device sends a predetermined amount of culture medium 1 to the filter cartridge 502 for each culture time, and after culturing for a predetermined time, switches the switching valve 702 to recover the cultured culture medium 1 into a different well 705 each time, mixes it with a luminescent reagent to cause a luminescent reaction, and performs luminescence measurement using a luminescence measuring device.

[0168] <Technical effect> As described above, the cell detection device 700 according to the seventh embodiment has a configuration for recovering the culture medium 1 into different wells 705 for each culture time, and includes a switching valve 702 that switches the flow path 703 so that the flow path 701 connected to the filter cartridge 502 communicates with one well 705. This makes it possible to perform luminescence measurement for each culture time while maintaining the airtightness of the culture medium container 501, the filter cartridge 502, and the wells 705, thereby preventing the intrusion of foreign matter from outside during culture. Furthermore, since there is no need to move the filter cartridge 502 away from each well 705 or to insert the flow path 701 into each well 705, there is no need for space for moving the filter cartridge 502, and the device can be made more compact.

[0169] [Eighth embodiment] In the above-mentioned first to seventh embodiments, a cell detection device having only one container (culture medium container) for accommodating a culture medium and a predetermined amount of the culture medium is added to a filter has been described. In the eighth embodiment, a cell detection device having a plurality of containers for accommodating a culture medium is proposed.

[0170] <Configuration of cell detection device> 13 is a schematic cross-sectional view showing a culture medium container 801 and a filter cartridge 802 of a cell detection device 800 according to the eighth embodiment. As shown in Fig. 13, the filter cartridge 802 (culture container) includes an annular filter holder 803 that holds the filter 102, adapters 806 and 807, a valve 809, and a capillary 808.

[0171] The filter holder 803 is sandwiched between adapters 806 and 807 (members that define a space). Recesses are formed on the surfaces of the adapters 806 and 807 that face the filter 102. This forms a space in which the filter 102 is housed. The adapter 806 is provided with two flow paths 804 that connect this space with the outside. Culture medium containers 801 (multiple culture medium containers) that house the culture medium 1 are respectively connected to the two flow paths 804. A valve 805 is provided at the boundary between the flow paths 804 and the culture medium container 801, separating the space in which the filter 102 is housed from the culture medium 1.

[0172] The medium container 801 contains a sterilized amount of medium 1 required for one culture, which is calculated in advance.

[0173] Different types of culture media can also be stored in each culture medium container 801. In this case, it is possible to detect bacteria that can grow depending on the type of culture medium, and therefore it is possible to categorize the bacteria contained in the sample depending on which medium they could or could not grow in.

[0174] For example, a device (culture medium addition device) capable of compressing the culture medium container 801 (applying pressure to the culture medium container 801) is connected to the culture medium container 801, and by applying pressure to the culture medium container 801 that is greater than or equal to a predetermined value, the culture medium 1 in the culture medium container 801 passes through the valve 805 and is added onto the filter 102.

[0175] The adapter 227 is provided with a capillary 808 connected to the space in which the filter 102 is housed. A valve 809 is provided between the capillary 808 and the spaces in the adapters 806 and 807, and the drive of the valve 809 is controlled by a drive device (culture medium addition device) not shown.

[0176] <Cell detection method> The cell detection method using the cell detection device 800 of this embodiment is almost the same as the cell detection methods of the second to fourth embodiments described above, and will therefore be briefly described below.

[0177] First, before luminescence measurement, a sufficient amount of medium 1 is flowed through the flow path 804 to wash and discharge the filter 102 and capillary 808. Next, medium containers 801 are connected to each flow path 804. Next, as in the fifth embodiment, one medium container 801 is compressed for each culture time, and the entire amount of medium 1 in that medium container 801 is sent to the filter cartridge 802, where culture is performed for a predetermined time. In other words, a portion of the total amount of medium 1 in all medium containers 801 is sent to the filter cartridge 802. Thereafter, the medium 1 after culture is collected in a different well each time, mixed with a luminescent reagent to cause a luminescent reaction, and luminescence measurement is performed.

[0178] <Technical effect> As described above, the cell detection device 800 according to the eighth embodiment has a plurality of medium containers 801, each containing the amount of medium 1 required for one culture, and the plurality of medium containers 801 are connected to the filter cartridges 802. This makes it possible to easily add small amounts of medium to the filter 102 multiple times without requiring a precise mechanical mechanism.

[0179] [Ninth embodiment] As described above, the bacteria detected by the methods of each embodiment are non-destructive and can be used for subsequent analysis. In the sixth embodiment, a cell detection device capable of counting the number of detected bacterial colonies was described. Therefore, in the ninth embodiment, another cell detection device capable of counting the number of detected bacterial colonies is proposed.

[0180] <Configuration of cell detection device> 14 is a schematic cross-sectional view showing a filter cartridge 902 of a cell detection device 900 according to the ninth embodiment. As shown in Fig. 14, the filter cartridge 902 (culture vessel) includes an annular filter holder 903 that holds the filter 102, a partition wall 904, adapters 906 and 907, valves 905 and 909, and a capillary 908.

[0181] The filter holder 903 is sandwiched between adapters 906 and 907. Recesses are formed on the surfaces of the adapters 906 and 907 facing the filter 102. This forms a space for accommodating the filter 102. A valve 905 is provided in the recess of the adapter 906, and a culture medium container (not shown in FIG. 14) is connected to the valve 905. This allows the space for accommodating the filter 102 to communicate with the culture medium container. A valve 909 is provided in the recess of the adapter 907, and a capillary 908 is connected to the valve 909.

[0182] Partitions 904 are disposed on the cell trapping surface of filter 102. Partitions 904 divide the cell trapping surface of filter 102 into a plurality of squares. Bacteria 2 that enter each square cannot move to another square.

[0183] The shape of the upper surface of the partition wall 904 is not particularly limited and may be, for example, a lattice shape, a radial shape, a concentric circle shape, or any combination thereof. However, from the viewpoint of accurately counting the bacteria 2, the shape of the partition wall 904 may be selected so that the area of ​​each square is approximately equal. The partition wall 904 may be made of, for example, resin.

[0184] <Cell detection method> The cell detection method using the cell detection device 900 of this embodiment is almost the same as the cell detection methods of the second to fourth embodiments described above, and will therefore be briefly described below.

[0185] First, filter 102 is placed in filter holder 903, and with partition wall 904 placed on filter 102, a sample solution is added from above filter 102 and filtration is performed. At this time, bacteria 2 contained in the sample are captured in one of the cells.

[0186] Thereafter, the filter holder 903 is sandwiched between the adapters 906 and 907, and the filter cartridge 902 is assembled.

[0187] Next, when the culture medium is added via valve 905, the culture medium can be spread over the entire surface of filter 102 by capillary action. Alternatively, the culture medium can be spread over the entire surface of filter 102 by rotating filter cartridge 902 on central axis 910 of filter cartridge 902 and generating centrifugal force from the center of filter 102 toward the edge (radially outward).

[0188] Next, the added medium is collected through the valve 909 and the capillary 908, and luminescence measurement is performed. As explained above, the addition of medium, incubation for a predetermined time, collection of medium, and luminescence measurement are repeated, and when an increase in the amount of luminescence is observed, the result is determined to be positive for bacteria.

[0189] Thereafter, the adapter 907 is removed from the filter cartridge 902, and the filter 102 is placed on an appropriate agar medium or liquid medium for further cultivation, whereby cell growth in each square can be confirmed visually or by optical measurement. If the number of cells contained in the original sample is smaller than the total number of squares, the number of squares in which cell growth was observed can be considered to be the viable cell count in the original sample.

[0190] <Technical effect> As described above, in the cell detection device 900 according to the ninth embodiment, the partition 904 that forms a grid on the cell trapping surface is provided on the filter 102. This makes it possible to count the number of live bacteria by further culturing the bacteria after detection.

[0191] [Tenth embodiment] In the first to ninth embodiments, the culture medium added to the filter is centrifuged and collected in a collection container disposed below the filter. In the tenth embodiment, an example is proposed in which the collection container is disposed on approximately the same plane as the filter.

[0192] <Configuration of cell detection device> 15(a) is a schematic cross-sectional view showing the filter cartridge 1002 and collection container cartridge 1010 of the cell detection device 1000 according to the tenth embodiment. As shown in FIG. 15, the collection container cartridge 1010 is formed in an annular shape and is provided around the annular filter cartridge 1002.

[0193] The filter cartridge 1002 has an annular filter holder 1003 that holds the filter 102, upper and lower lids 1006 that seal the inside of the filter holder 1003, and a rotation shaft 1009 that is provided at the radial end of the filter holder 1003 and perpendicular to the filter 102.

[0194] The filter holder 1003 has a mesh 1004, and the filter 102 on which the bacteria 2 are captured is placed on the mesh 1004. A septum 1007 is provided in the center of the upper lid 1006. The culture medium can be supplied to the space containing the filter 102 by passing a capillary connected to a container for the culture medium or the needle of a syringe containing the culture medium through the septum 1007. The filter holder 1003 is provided with a flow path 1008 that is approximately parallel to the filter 102.

[0195] A plurality of collection containers 1005 are formed inside the collection container cartridge 1010 along the circumferential direction of the collection container cartridge 1010. One of the collection containers 1005 communicates with a flow path 1008 (selection mechanism) of the filter holder 1003. The internal space of the collection container 1005 is divided into upper and lower parts by a breakable seal 1011, and the luminescent reagent 3 is contained above the seal 1011. The space below the seal 1011 communicates with the flow path 1008 of the filter holder 1003. The collection container 1005 can also have, for example, an upper surface on the side where the luminescent reagent 3 is contained, breakable. This allows, for example, the upper surface of the collection container 1005 and the seal 1011 to be broken by a needle that pierces them, and the luminescent reagent 3 can be added to the space below the seal 1011.

[0196] When the flow path 1008 and the collection container 1005 are to be communicated with each other, the collection container cartridge 1010 is rotated relative to the filter cartridge 1002 to align them.

[0197] Although not shown, the cell detection device 1000 has a rotation mechanism (culture medium collection device) that rotates the filter cartridge 1002 about a rotation axis 1009 in a state in which the filter cartridge 1002 and the collection container cartridge 1010 are assembled (in a state in which the flow path 1008 and the collection container 1005 are in communication). The rotation about the rotation axis 1009 applies centrifugal force to the culture medium present in the space in which the filter 102 is housed, causing the culture medium to move in a direction away from the rotation axis 1009.

[0198] Figure 15(b) is a schematic top view showing the filter cartridge 1002 and the collection container cartridge 1010. As shown in Figure 15(b), the flow path 1008 is provided on the opposite side of the rotation axis 1009 by 180 degrees. This positional relationship allows the culture medium in the filter cartridge 1002 to be collected into the collection container 1005 with a high recovery rate and with little residual liquid.

[0199] <Cell detection method> The cell detection method using the cell detection device 1000 of this embodiment is almost the same as the cell detection methods of the second to fourth embodiments described above, and will therefore be briefly described below.

[0200] First, the sample solution is filtered by the filter 102 of the filter cartridge 1002 to capture the bacteria 2. Next, a lid 1006 and a rotating shaft 1009 are attached to the filter cartridge 1002, and a collection container cartridge 1010 is attached around the filter cartridge 1002, and the flow path 1008 and the first collection container 1005 are connected to each other.

[0201] Next, for example, using a syringe containing culture medium, culture medium is added into the filter cartridge 1002 through the septum 1007, and the filter cartridge 1002 is rotated around the rotation axis 1009 using a rotation mechanism, and the culture medium is collected in the space below the seal 1011 of the first collection container 1005.

[0202] Next, the collection container 1005 and the seal 1011 are broken, for example, using a syringe needle to break them, and the culture medium collected in the first collection container 1005 is reacted with the luminescence reagent. A luminescence measuring device is placed at a position where it can detect luminescence from the bottom surface of the collection container 1005, and luminescence measurement is performed.

[0203] Next, collection container cartridge 1010 is rotated relative to filter cartridge 1002 to connect flow path 1008 to second collection container 1005 .

[0204] Next, using a syringe, culture medium is added into the filter cartridge 1002 through the septum 1007, and after culturing for a predetermined period of time, the filter cartridge 1002 is rotated around the rotation axis 1009 using a rotation mechanism, and the culture medium is collected in the space below the seal 1011 of the second collection container 1005.

[0205] Thereafter, luminescence measurement is carried out in the same manner as for the first collection container 1005. As explained above, the addition of culture medium, incubation for a predetermined time, collection of culture medium, and luminescence measurement are repeated, and when an increase in the amount of luminescence is observed, the result is judged to be positive for bacteria.

[0206] <Technical effect> As described above, in the cell detection device 1000 according to the tenth embodiment, the filter cartridge 1002 rotates around the rotation axis 1009 provided on the periphery of the filter cartridge 1002, with the space accommodating the filter 102 communicating with one collection container 1005. This allows the culture medium to be collected from the filter 102 into the collection container 1005 by centrifugal force, thereby achieving a high collection rate.

[0207] Furthermore, there is no need to move filter cartridge 1002 and collection container cartridge 307 to a separately prepared centrifuge when collecting the culture medium in collection container 1005. This eliminates the need for space to move filter cartridge 1002 and collection container cartridge 1010, allowing for the miniaturization of the device.

[0208] [Variations] The present disclosure is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure, and it is not necessary to include all of the described configurations. Furthermore, a part of one embodiment can be replaced with a configuration of another embodiment. Furthermore, a configuration of another embodiment can be added to a configuration of one embodiment. Furthermore, a part of the configuration of each embodiment can be added to, deleted from, or substituted for a part of the configuration of another embodiment.

[0209] [Note] <Specific matter 1> a culture medium addition device that adds a portion of the prepared culture medium to a culture vessel that holds the cells to be detected; a medium recovery device that recovers the medium from the culture vessel; a detection device that detects light from a luminescent reagent mixed with the recovered culture medium.

[0210] <Specific matter 2> The cell detection device described in specific item 1 is characterized in that the culture vessel has a filter with a pore size smaller than the size of the cells, and the cells are retained on the filter.

[0211] <Specific matter 3> A cell detection device described in specific item 2, characterized in that the volume of the portion of culture medium added to the culture vessel by the culture medium addition device is between 1 / 2 and 5 times the value obtained by multiplying the area and thickness of the filter.

[0212] <Specific matter 4> the luminescent reagent contains luciferase, The cell detection device according to specific item 1, characterized in that the light is luminescence resulting from a reaction between adenosine triphosphate or luciferin produced in the culture medium from the cells and the luciferase.

[0213] <Specific matter 5> The cell detection device described in specific item 1 is characterized in that the culture medium recovery device further includes a selection mechanism for connecting any of a plurality of recovery containers from which the portion of the culture medium is recovered to the culture container.

[0214] <Specific matter 6> The cell detection device according to specific feature 2, wherein the culture medium addition device adds the culture medium to the culture vessel approximately parallel to the filter surface.

[0215] <Specific matter 7> The cell detection device according to specific feature 2, wherein the culture vessel has a member covering the surface of the filter that holds the cells.

[0216] <Specific matter 8> The cell detection device described in specific item 1 is characterized in that the culture medium addition device adds the entire amount of the culture medium in any one of a plurality of culture medium containers, each containing a portion of the culture medium, to the culture container.

[0217] <Specific matter 9> A cell detection device according to specific feature 1, characterized in that at least a portion of a collection container that contains the culture medium collected from the culture container is transparent to the light from the luminescent reagent.

[0218] <Specific matters 10> A cell detection device as described in specific item 1, characterized in that the culture medium container, the culture vessel, and the recovery container are arranged so that the culture medium moves in the following order: a culture medium container containing the prepared culture medium, the culture vessel, and a recovery container containing the culture medium recovered from the culture vessel.

[0219] <Specific matter 11> The cell detection device according to specific feature 1, further comprising a luminescent reagent adding device that adds the luminescent reagent to the culture medium collected by the culture medium collecting device.

[0220] <Specific matter 12> A cell detection device according to specific item 1, characterized in that the culture vessel and the collection vessel containing the collected culture medium are arranged at separate positions within the same sealed space.

[0221] <Specific matter 13> The cell detection device described in specific feature 2, wherein the culture vessel has a filter holder that holds the filter and a member that defines a space that accommodates the filter.

[0222] <Specific matter 14> The cell detection device according to specific feature 5, wherein the selection mechanism is a valve that switches a flow path that connects the culture vessel with one of the plurality of collection vessels.

[0223] <Specific matter 15> The cell detection device described in specific item 2 is characterized in that it further comprises a partition wall arranged on the surface of the filter that retains the cells and divides the surface into multiple squares.

[0224] <Specific matter 16> the plurality of collection vessels are arranged around the culture vessel; the selection mechanism is a flow path that connects the culture vessel with one of the plurality of collection vessels, The cell detection device described in specific item 5, characterized in that the culture medium recovery device rotates the culture vessel while the culture vessel is in communication with one of the multiple recovery vessels.

[0225] <Specific matter 17> Adding a portion of the prepared medium to a culture vessel holding cells to be detected; Recovering the medium from the culture vessel; detecting light from a luminescent reagent mixed with the recovered medium.

[0226] <Specific matter 18> repeating the adding of the medium, the withdrawing of the medium, and the detecting of the light a plurality of times; Item 18. The cell detection method according to item 17, further comprising comparing the results of the multiple detections.

[0227] <Specific matter 19> the culture vessel has a filter with a pore size smaller than the size of the cells, The cell detection method includes: Item 17. The cell detection method according to item 17, further comprising capturing the cells on the filter.

[0228] <Specific matters 20> A cell detection method described in specific item 19, characterized in that the volume of the portion of culture medium added to the culture vessel is between 1 / 2 and 5 times the value obtained by multiplying the area and thickness of the filter.

[0229] <Specific matter 21> Item 19. The cell detection method according to item 18, wherein the culture medium is collected in a different collection container each time the plurality of times are repeated.

[0230] <Specific matter 22> 20. The cell detection method according to item 19, wherein the culture medium is moved into the culture vessel substantially parallel to the filter surface.

[0231] <Specific matter 23> A cell detection method according to specific item 19, further comprising providing a member for covering the surface of the filter that retains the cells in the culture vessel.

[0232] <Specific matter 24> the luminescent reagent contains luciferase; Item 18. The cell detection method according to item 17, wherein the light is luminescence resulting from a reaction between adenosine triphosphate or luciferin produced in the medium from the cells and the luciferase.

[0233] <Specific matter 25> Item 19. The cell detection method according to item 18, wherein the entire amount of the medium is added to the culture vessel from a different medium vessel each time during the multiple repetitions.

[0234] <Specific matter 26> The cell detection method described in specific item 18, further comprising judging the cell to be positive if the comparison determines that the amount of a substance derived from the cell has increased, and judging the cell to be negative if the comparison determines that the amount of the substance has not increased.

[0235] <Specific matter 27> Item 27. The cell detection method according to item 26, further comprising culturing the cells in the culture vessel after the cells are determined to be positive. [Explanation of symbols]

[0236] 1. Culture medium 2. Bacteria 3...Luminescent reagent 100~1000...Cell detection device 101...Syringe 102...Filter 103...Filter holder 104...Mesh 105...Collection container 106…Lid 107...Septum 108...syringe needle 109...Luminescence measuring device

Claims

1. A cell detection device for non-destructively detecting cells, a culture medium addition device that adds a portion of the prepared culture medium to a culture vessel that holds the cells to be detected; a medium recovery device that recovers the adenosine triphosphate produced in the medium from the cells by recovering an amount of the medium that is the difference between the amount that remains in the culture vessel and the amount that evaporates from the culture vessel, from the portion of the medium added from the culture vessel; a detection device that detects light from a luminescent reagent that reacts with the adenosine triphosphate and emits light, the luminescent reagent being mixed with the recovered culture medium and the adenosine triphosphate.

2. 2. The cell detection device according to claim 1, further comprising a luminescent reagent adding device that adds the luminescent reagent to the culture medium collected by the culture medium collecting device.

3. 2. The cell detection device according to claim 1, wherein the culture vessel and the collection vessel containing the collected culture medium are arranged at separate positions within the same sealed space.

4. the culture vessel has a filter with a pore size smaller than the size of the cells, and the cells are retained by the filter; 2. The cell detection device according to claim 1, wherein the culture vessel comprises a filter holder that holds the filter, and a member that defines a space that accommodates the filter.

5. the culture medium recovery device includes a selection mechanism that connects any one of a plurality of recovery containers from which the portion of the culture medium is recovered to the culture vessel; 2. The cell detection device according to claim 1, wherein the selection mechanism is a valve that switches a flow path that connects the culture vessel with one of the plurality of collection vessels.

6. the culture vessel has a filter with a pore size smaller than the size of the cells, and the cells are retained by the filter; The cell detection device according to claim 1 , further comprising partitions arranged on a surface of the filter that holds the cells and that divide the surface into a plurality of cells.

7. the culture medium recovery device further includes a selection mechanism for connecting any one of a plurality of recovery containers into which the portion of the culture medium is recovered to the culture vessel; the plurality of collection vessels are arranged around the culture vessel; the selection mechanism is a flow path that connects the culture vessel with one of the plurality of collection vessels, 2. The cell detection device according to claim 1, wherein the culture medium recovery device rotates the culture vessel while the culture vessel is in communication with one of the plurality of recovery vessels.

8. A cell detection method for non-destructively detecting cells, comprising: Adding a portion of the prepared medium to a culture vessel holding cells to be detected; recovering the adenosine triphosphate produced in the medium from the cells by subtracting the amount remaining in the culture vessel and the amount evaporated from the portion of the medium added from the culture vessel; detecting light from a luminescent reagent that reacts with the adenosine triphosphate and emits light, the luminescent reagent being mixed with the collected medium and the adenosine triphosphate.

9. the culture vessel has a filter with a pore size smaller than the size of the cells, The cell detection method includes: further comprising capturing the cells on the filter; The cell detection method according to claim 8, wherein the culture medium is moved into the culture vessel parallel to the surface of the filter that holds the cells.

10. the culture vessel has a filter with a pore size smaller than the size of the cells, The cell detection method includes: further comprising capturing the cells on the filter; The cell detection method according to claim 8 , further comprising providing a member for covering the surface of the filter that holds the cells in the culture vessel.

11. the luminescent reagent contains luciferin, or the medium contains a luciferin derivative that reacts with an enzyme in the cells to produce luciferin; the luminescent reagent contains luciferase; 9. The cell detection method according to claim 8, wherein the light is luminescence produced by a reaction between the adenosine triphosphate and the luciferin and the luciferase.

12. repeating the adding of the medium, the withdrawing of the medium, and the detecting of the light a plurality of times; and comparing the results of the multiple detections; 9. The cell detection method according to claim 8, wherein the entire amount of the medium is added to the culture vessel from a different medium vessel each time during the multiple repetitions.

13. repeating the adding of the medium, the withdrawing of the medium, and the detecting of the light a plurality of times; and comparing the results of the multiple detections; The cell detection method described in claim 8, further comprising determining that the cell is positive if the comparison determines that the amount of a substance derived from the cell has increased, and determining that the cell is negative if the comparison determines that the amount of the substance has not increased.

14. The cell detection method according to claim 13 , further comprising culturing the cells in the culture vessel after the cells are determined to be positive.

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