Cell detection device and cell detection method

Abstract

To provide a technique of detecting a cell-derived substance highly sensitively without breaking a cell.SOLUTION: A cell detection device comprises: a culture medium adding device which adds a partial culture medium of prepared culture medium to a culture vessel for holding a cell to be detected; a culture medium recovery device which recovers the culture medium from the culture vessel; and a detector which detects light from a luminescent reagent mixed with the recovered culture medium.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 a very small number 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 and then detecting them, but a major problem is that it takes a long time to culture for more than a day to obtain a sufficient number of bacteria for detection. Therefore, several rapid test methods have been developed in recent years.

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

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

[0005] Patent Document 2 discloses a method in which a sample is filtered to collect and concentrate bacteria on the filter, and then ATP outside the bacteria is eliminated to remove background ATP before extracting ATP inside the bacteria. 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] JP 2013-116083 A Summary of the Invention [Problem to be solved by the invention]

[0007] On the other hand, if bacteria are detected in the test for the presence or absence of bacteria, 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 that have contaminated the sample, estimate the route of contamination, and reflect the results in quality control. However, in the method of measuring intracellular ATP as described above, it is necessary to destroy the bacteria when extracting ATP from the bacteria, so it is not possible to subsequently culture the bacteria and perform identification tests.

[0008] In order to further enrich and culture the bacteria after detecting their presence, it is possible to use a method in which the bacteria are partially destroyed and tested using the ATP method. In other words, the bacteria are grown in a liquid medium, and a portion of the liquid medium is taken at regular intervals to measure the ATP in the bacteria. If an increase in ATP is observed, it is considered that the bacteria have grown. In this method, live bacteria remain in the original liquid medium, so it is possible to enrich and culture the bacteria and proceed to other tests. However, since the liquid medium is repeatedly taken, it is necessary to start the culture with a medium volume that takes into account the maximum number of times it is taken, and the bacteria are diluted in the medium, resulting in a low bacterial concentration, so there is room for improvement in the detection sensitivity of the bacteria.

[0009] The above problems are not limited to bacteria, but also apply to the detection of cells in general, for the purpose of detecting small numbers of cells, such as cultured cells, with high sensitivity and rapidity without destroying them.

[0010] Therefore, the present disclosure provides a technique 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 of the present specification and the accompanying drawings. Also, aspects of the present disclosure may be realized and realized by the elements and combinations of various elements and aspects of the following detailed description and the appended claims. The descriptions in this specification are exemplary and illustrative only and are not intended to limit the scope or application of the present disclosure in any way. Effect 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 description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic cross-sectional view showing a cell detection device according to a first embodiment. [Diagram 2] FIG. 1 is a functional block diagram of a cell detection device according to a first embodiment. [Diagram 3] 3 is a flowchart showing a cell detection method according to the first embodiment. [Figure 4A] FIG. 13 is a schematic diagram showing a measurement screen. [Figure 4B] FIG. 13 is a schematic diagram showing a measurement method setting screen. [Figure 4C] FIG. 13 is a schematic diagram showing a screen for displaying a graph of the measurement results. [Diagram 5] FIG. 11 is a schematic cross-sectional view showing a cell detection device according to a second embodiment. [Figure 6] FIG. 11 is a functional block diagram of a cell detection device according to a second embodiment. [Figure 7] 13 is a graph showing the results of bacteria detection by the cell detection device according to the second embodiment. [Figure 8] FIG. 11 is a schematic diagram showing a cell detection device according to a third embodiment. [Figure 9] FIG. 11 is a schematic cross-sectional view showing a cell detection device according to a fourth embodiment. [Figure 10] FIG. 13 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. 23 is a schematic diagram showing a configuration of a portion of a cell detection device according to a tenth embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] [First embodiment] <Configuration of cell detection device> Fig. 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 using 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 for holding a filter 102, a collection container 105, a lid 106, and a luminescence measuring device 109.

[0016] The syringe 101 (culture medium container, culture medium addition device) contains the culture medium 1, and is configured to be able to dispense a predetermined amount (a portion) of the total amount of the culture medium 1. Although not shown in the figure, the syringe 101 is attached to a drive device (culture medium addition device) that controls the vertical movement of the syringe 101 itself and the sliding of the plunger of the 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 a sample solution using the filter 102 and a filtering device (not shown). The filter 102 is placed on the mesh 104 of the filter holder 103 after filtering the sample solution to capture the bacteria 2.

[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, for example, 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, for example, 1 μm can be used.

[0019] The shape of the filter 102 may be a circular shape that is generally available on the market, 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 a circular filter. Even if the filter is circular, the filter area can be increased by forming a corrugated surface rather than a flat surface, and clogging of the filter by particles contained in the sample can be prevented. Furthermore, clogging of the filter by particles contained in the sample can also be prevented by stacking a filter with a large pore size on a filter with a small pore size.

[0020] The pore size of the mesh 104 may be large enough so that the culture medium 1 can 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 configured to be detachable from the filter holder 103, and covers the space above the filter 102, thereby preventing contamination from the outside. A septum 107 is provided on the upper 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 equal to the outer diameter of collection container 105.

[0023] The collection container 105 is a container such as a microtube, and contains a luminescence reagent 3 containing, for example, luciferin and luciferase. After the bacteria 2 are cultured on the filter 102, the medium 1 containing ATP, which is a secretion of the bacteria 2, is collected in the collection container 105 by, for example, a centrifuge (medium collection device) performing a centrifugation operation with the filter holder 103 and the collection container 105 connected. As a result, the ATP in the medium 1 collected in the collection container 105 is mixed with the luminescence reagent 3, and luminescence can be generated by a reaction. If the medium 1 on the filter 102 can be collected in the collection container 105, a medium collection device other than a centrifuge can also be used. For example, the medium 1 may be collected from the filter 102 to the collection container 105 by applying pressure such as air pressure from the side of the lid 106 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 luminescence from the luminescence reagent 3 in the collection container 105 can be detected. 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 in the figure, 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 regulator, and the centrifuge are connected to the above-mentioned arithmetic device, and the arithmetic device 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 device operates the driving device, the luminescence measuring device 109, the temperature regulator, 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 the bacteria 2 are cultured, and a storage location for the luminescence reagent 3 (recovery container) are arranged separately, and the culture medium 1 moves from the culture medium container to the culture container and then to the recovery container. Furthermore, the culture medium 1 is characterized in that it can be added to the filter 102 by a syringe 101 (culture medium adding device) in a fixed amount at a time, rather than the entire amount. With this configuration, it is possible to repeatedly perform the following operations: adding a small amount of the culture medium 1 from the syringe 101 to the filter 102, culturing the bacteria 2 on the filter 102, recovering the culture medium 1 by a centrifuge (culture medium recovering device) into a recovery container 105 to react with the luminescence reagent 3, and measuring the bacteria-derived ATP in the culture medium 1. The amount of the culture medium 1 added to the filter 102 at one time will be described later.

[0028] By culturing the bacteria 2 in a small amount of medium 1 on the filter 102, the concentration of ATP secreted by the bacteria 2 in the medium 1 increases, and extracellular ATP can be measured with high sensitivity. As a result, bacteria can be detected quickly. In addition, since the secretions outside the bacteria are measured, there is no need to destroy the bacteria 2, and it is possible to measure changes over time associated with the proliferation of the same bacterial sample. After detection of the bacteria, the bacteria can be further cultured and subjected to 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 luminescent reagent 3 is stored in advance in the collection container 105, and the culture medium 1 collected from the filter 102 is repeatedly mixed therein. In this way, since a mechanism for dispensing the luminescent reagent 3 is not required, the cost of the device can be reduced.

[0030] When the purpose is to detect anaerobic bacteria, the collection container 105, the filter holder 103 and the lid 106 are sealed to keep the inside airtight, the inside is filled with an oxygen-free gas, and the culture medium 1 is degassed in advance and filled into the syringe 101. This allows anaerobic culture. An anaerobic environment can also be created by adding a reducing agent to the culture 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 adding 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 measuring device 78 (detection device), a calculation device 79, an input device 80, an output device 81, and a temperature regulator 82.

[0032] Culture medium section 71 is a place 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, etc. Culture medium section 71 is connected to culture medium adding device 72. Culture medium adding device 72 is, for example, syringe 101 and its drive device, and the drive device is composed of a part for fixing syringe 101, a motor for moving the part, a motor for pushing the plunger of syringe 101 a certain distance to discharge culture medium 1, etc.

[0033] The culture section 73 holds the bacteria (cells) to be detected. The culture section 73 is, for example, the filter 102 and the filter holder 103 of the cell detection device 100. A part 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 so that the culture medium or components in the culture medium do not move from the culture section 73 to the culture medium section 71.

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

[0035] The medium recovery unit 75 stores the medium transferred from the culture unit 73. The 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 medium recovery unit 75 are connected to each other and are used for a medium recovery operation by the medium recovery unit 75.

[0036] The luminescence reagent section 76 is a place for holding a luminescence reagent for a luminescence reaction, and is, for example, a container or syringe containing the luminescence reagent. In the cell detection device 100, the luminescence reagent 3 is contained in the collection container 105, so the luminescence 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 section 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 section 76 to the calculation device 79.

[0038] The arithmetic device 79 is a computer terminal such as a personal computer, a smartphone, a tablet, or a 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 medium adding device 72 and the medium recovering device 74 are also connected to the arithmetic device 79, and the arithmetic device 79 controls the operations of these devices. An input device 80 and an output device 81 are also connected to the arithmetic device 79, and a user can determine measurement parameters via the input device 80. The arithmetic device 79 controls the operations of the medium adding device 72 and the medium recovering device 74 in accordance with the parameters. The output device 81 displays the results of calculation of the ATP amount by the arithmetic device 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, and thus it is possible to measure the bacteria 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 bacteria detection threshold, the culture time (measurement time interval), the maximum culture time, and the amount of medium 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 capturing surface side of the filter 102, pours the sample solution into the funnel, and filters the solution. This captures and concentrates the bacteria 2 on the filter 102, and removes the solvent. The 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 of the luminescence measurement originating from the sample solution.

[0046] Next, the user introduces the luminescence reagent 3 into the collection container 105, and connects the filter holder 103 to the upper part 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 by 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 medium adding device 72 based on the medium adding amount set in step S1 to cause the needle 108 of the syringe 101 to pierce the septum 107 of the lid 106, and add a portion of the medium 1 from the syringe 101 to the filter 102. The amount of medium 1 to be added can be the minimum amount necessary to wet the entire filter 102.

[0049] In step S5, the arithmetic device 79 judges 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 arithmetic unit 79 drives the culture medium adding device 72 to pull out the needle 108 of the syringe 101 from the septum 107. Thereafter, the arithmetic unit 79 moves the lid 106, the filter holder 103, and the collection container 105 in a connected state 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 by 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 luminescence reagent 3 (step S7).

[0051] In step S8, the luminescence measuring device 109 detects 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 the 0th hour of culture.

[0052] In step S9, the arithmetic device 79 judges whether or not 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. After that, in step S5, the arithmetic device 79 judges 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, for example, to about 30 to 40° C. if the detection target is bacteria, or to about 20 to 30° C. if the detection target is mold.

[0054] Thereafter, steps S6 to S9 are executed in the same manner as described above. When culturing is performed for one hour, the calculation device 79 stores the amount of luminescence calculated in step S8 for the second time in the storage device as the amount of luminescence after one hour of culturing.

[0055] In step S11, the arithmetic device 79 judges 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 judges that bacteria have been detected (positive) and outputs the judgment result to the output device 81. If the output device 81 is a monitor, it displays a screen showing the 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 culture is less than the threshold in step S11 (No), the process proceeds to step S13. In step S13, the calculation device 79 judges whether the culture time has reached the maximum culture time set in step S1. If the maximum culture time has been reached (Yes), in step S14, the calculation device 79 judges that the bacteria is not detected (negative) and outputs the judgment result to the output device 81.

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

[0058] As described above, when the amount of luminescence is equal to or greater than the threshold and the result is determined to be positive, or when the maximum culture time is reached and the result is determined to be negative, detection of bacteria is terminated. Note that in the determination in step S13, it may be determined 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 value 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 value on a GUI screen for setting conditions displayed on the output device 81 at the stage of step S11. This allows appropriate setting of detection conditions even for detection targets with different characteristics, and enables inspection using the same cell detection device 100 and cell detection method.

[0060] Furthermore, the determination of the 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 the 0th hour, 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 proliferation or growth rate of the bacteria, the amount of culture medium or the amount of luminescence reagent prepared initially, and the like. The amount of ATP secreted outside the bacteria is considered to be proportional to the number of bacteria. Generally, the number of bacteria doubles in about 20 minutes for the fastest dividing bacteria species (such as E. coli). Therefore, in order to detect bacterial proliferation by an increase in extracellular ATP, an incubation time of 20 minutes or more is considered appropriate, and the measurement interval can be set to be longer than 20 minutes, taking into account the measurement variability and measurement sensitivity. For example, the interval can be one hour or two hours.

[0062] The amount of culture medium and the amount of luminescence reagent prepared in step S2 are calculated from the amount of culture medium added at one time and the set value of the maximum number of measurements. For example, if the amount of culture medium added to the filter is 50 μL and the maximum number of culture medium additions is to be 20, the amount of culture medium in the syringe needs to be 1 mL. In addition, taking into consideration that the luminescence reagent becomes diluted each time the culture medium is mixed with the luminescence reagent and the luminescence efficiency decreases in proportion to the concentration, for example, if the dilution rate of the luminescence reagent is allowed up to 1.5 times, if the amount of culture medium recovered from the filter is 50 μL each time and the maximum number of measurements is 20, 2 mL of luminescence reagent needs to be stored in the recovery container in advance. The allowable dilution rate of the luminescence reagent can be determined by measuring the relationship between the dilution rate and the luminescence intensity using a standard ATP solution in advance.

[0063] The type of medium to be added can be selected according to the bacteria to be detected. For general bacteria, bouillon medium or soybean-casein digest medium can be used, but is not limited to these. A medium selected for the purpose of detecting only specific bacteria can also be used. For example, when the purpose is to detect Enterobacteriaceae, EE bouillon medium for Enterobacteriaceae detection can be used.

[0064] In step S4, the amount of medium added onto the filter 102 can be minimized to increase the extracellular ATP concentration. However, it is necessary to add an amount of medium that is sufficient to spread the medium evenly over the entire filter and provide sufficient nutrients to the bacteria captured on the filter. It is also necessary to take into account 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 was calculated as area × thickness, and was 25 μL. When 50 μL of medium was added to this filter, the entire filter was sufficiently moistened, and then the medium was removed by centrifugation, 5 μL of medium remained on the filter. In addition, 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, about 10 μL of water in the medium on the filter evaporated. Therefore, the amount of medium added to the filter can be 20 μL, which is 15 μL of the residual and evaporated parts combined, plus the minimum amount required for the luminescence reaction, for example, 5 μL. More preferably, by adding the residual and evaporated parts to the filter volume to make it 40 μL or more, the medium can be maintained throughout the entire filter even if evaporation occurs, and damage to the bacteria is thought to be reduced.

[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 x thickness of the filter, and preferably 1 to 2 times.

[0067] As a measurement target substance, in addition to ATP secreted outside the bacterial body, various secretions can be targeted. In addition to secretions, bacteria can also be detected by adding a substrate that emits fluorescence or light when decomposed by the target enzyme, targeting specific enzymes possessed by bacteria.

[0068] For example, a luciferin derivative that is decomposed by bacterial esterase to produce luciferin can be added to medium 1. The luciferin derivative is decomposed in proportion to the bacterial esterase activity, and luciferin is produced in medium 1. If a luminescence reagent containing ATP and luciferase is added to this, the amount of liberated luciferin can be quantified. This method makes it possible to detect bacterial growth based on esterase activity.

[0069] In addition, 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, instead of the luminescence measurement device 78, an excitation light source and a light measurement device are used.

[0071] In addition, the technology of this 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 the GUI screen displayed on the output device 81 in the case of 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 been obtained, the end time is displayed. If the set value of the most recent ATP measurement is outside the settable 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] Fig. 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 for setting a threshold value, which is a criterion for determining that a sample is positive for bacteria, a measurement time interval (culture time), an amount of medium to be added from the medium section to the culture section, and a maximum value of the culture time (test time). A calculation method for the threshold value and time interval can 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 the time course of the measurement value, the time and result of the positive / negative judgment, the threshold value used for the judgment, and the like for the selected specimen. By using the graph screen 4c, the progress of the measurement 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 contains 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 contains the luminescence reagent 3. The cell detection device 100 adds a part of the culture medium 1 in the syringe 101 to the filter 102 by the syringe 101, recovers the culture medium 1 that has come into contact with the bacteria 2 in the recovery container 105 by the culture medium recovery device, and measures the luminescence from the luminescence reagent 3 by the luminescence measuring device 109. In this way, since the culture medium that has come into contact with the bacteria is recovered from the filter 102, it is possible to add a part 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 a series of operations, namely, contact between the part of the culture medium 1 and the bacteria 2, movement of the culture medium 1 that has come into contact with the bacteria 2, and detection of light, multiple times for each culture time.

[0079] The cell detection device 100, having such a configuration, can recover a high concentration of the extracellular secretions of the bacteria 2 captured on the filter 102 and measure the luminescence at any culture time. Therefore, the presence or absence of bacterial proliferation can be detected quickly and with high sensitivity without destroying the bacteria 2. In addition, since the detected bacteria 2 are non-destructive, other tests can be performed 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 in 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 in different collection containers for each culture time. In this embodiment, the same components as in the first embodiment are denoted by 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 a state when a culture medium 1 is added to a filter 102 for the first time. As shown in FIG. 5(a), the cell detection device 200 includes five collection containers 105a to 105e (a plurality of collection containers). Furthermore, a luminescence reagent 3 has not been introduced in advance into the collection container 105. The other configurations are similar to those of the cell detection device 100 of the first embodiment. Furthermore, 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 is selected according to the number of luminescence measurements required.

[0082] The 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 FIG. 5(a), a filter holder 103 having a filter 102 is connected to the first collection container 105a, and a needle 108 of a syringe 101 is inserted into a septum of a lid 106 to add a portion of the medium 1, and then the needle 108 of the syringe 101 is removed. Thereafter, the collection container 105a and the filter holder 103 are set in a centrifuge in a connected state, and centrifuged to collect the medium 1 on the filter 102 into the collection container 105a.

[0083] 5(b) shows the state after the culture medium 1 has been collected in the first collection container 105a. The syringe 101 and filter holder 103 are withdrawn from the first collection container 105a in which the culture medium 1 has been collected, and 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 either the luminescence measuring device 109 or the collection containers 105a to 105e, luminescence measurement can be performed for each collection container 105.

[0084] Although not shown in the figure, 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] Fig. 6 is a functional block diagram of a cell detection device 200 according to the second embodiment. As shown in Fig. 6, in the cell detection device 200, a culture medium recovery section 75 and a luminescence reagent section 76 are arranged separately, and a luminescence reagent adding device 77 is connected to the luminescence reagent section 76. The other points are the same as those of 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, a storage section for the luminescent reagent 3 of 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, a motor that pushes the plunger of the syringe 201 a certain distance to eject the luminescent reagent 3, and the like.

[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. A 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 described above, and the culture medium 1 is collected in the first collection container 105a. After the completion of step S6, the calculation device 79 uses a mechanism (not shown) to move the filter holder 103 away from the first collection container 105a, and connects them to a new second collection container 105b.

[0090] In step S7, the arithmetic device 79 drives the luminescence reagent adding device 77 to add the luminescence 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 calculation 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 the filter holder 103 to move away from the second collection container 105b by a mechanism (not shown), and connects them to a new third collection container 105c.

[0094] In step S7, the arithmetic device 79 drives the luminescence reagent adding device 77 to add the luminescence 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 multiple collection containers, and for each repeated luminescence measurement, the medium 1 after culturing the bacteria on the filter 102 is collected into a different collection container and mixed with the luminescence reagent 3. This makes it possible to keep the mixture ratio of the collected medium 1 and the luminescence reagent 3 constant, so that luminescence measurement can be performed with the same sensitivity regardless of the culture time. This improves the detection accuracy of the bacteria.

[0097] <Experimental Example> An experiment was carried out 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, and the 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 respective components of the cell detection device 200. Filter: Area 0.2 cm 2 , pore size 0.45 μm Collection container: Microtube 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. As a result, the bacteria were collected and concentrated on the filter, and the solvent was removed.

[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 in 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 in 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) is the incubation time, and the vertical axis is the amount of luminescence proportional to the amount of ATP (CPS: counts per second). 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 by the bacteria into the medium 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 at 2 hours, but an increase was observed at 4 hours. When the number of staphylococci was 100 CFU, an increase in ATP was observed at 2 hours.

[0108] <<Comparison of extracellular ATP amount and intracellular ATP amount>> For a sample of 10 CFU, after 6 hours of culture, 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 side are the units of the measurement results of the amount of extracellular ATP at 10 CFU in Figure 7(a) converted to amol. In other words, the figures plot the amount of ATP secreted outside the cells between 4 and 6 hours after culturing Staphylococcus aureus with 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 (results of two measurements). In other words, the results show the amount of ATP contained within the cells of Staphylococcus aureus after culturing the cells for 6 hours with an initial cell count of 10 CFU.

[0111] The amount of ATP outside the cells was 15,000 amol on average, and the amount of ATP inside the cells was 120,000 amol on average, which indicates that the amount of ATP outside the cells was approximately 1 / 8 of the amount of ATP inside the cells.

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

[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 with 10 CFU / 1 mL, culturing the bacteria, and then taking 30 μL aliquots to measure intracellular ATP.

[0114] The initial bacterial concentration after adding the SCD medium is 10 CFU / 10 mL. After culturing this for 6 hours at 37°C, 30 μL of the culture medium is taken, and 10 μL of the free ATP elimination reagent and 10 μL of the intracellular ATP extraction reagent are added. 30 μL of the luminescence reagent is added, and the 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 the 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) Filter 1 mL of sample onto the filter, capture and concentrate 10 CFU of bacteria on the filter. Add 50 μL of SCD medium to the sample, culture at 37°C for 2 hours, and remove the medium. Repeat this process three times. Add 30 μL of luminescence reagent to the 50 μL of medium collected the third time, measure luminescence, and calculate the amount of ATP, which is 15,000 amol.

[0117] In this manner, in this embodiment, by culturing the bacteria concentrated on a filter in a small amount of culture medium and measuring the extracellular secretions, it was found that it is possible to improve sensitivity 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, thereby enabling more rapid bacterial detection.

[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 forming an upper surface and a disk 303 forming a 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] The disk 303 is formed with collection containers 305 in the form of a plurality of wells. The luminescence reagent 3 is not introduced in advance into the collection containers 305. At least one of the disks 302 and 303 is configured to be rotatable around its center as a rotation axis. This allows the filter 102 to be positioned above each collection container 305. In order not to interfere 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 opening diameter of the collection container 305 is larger than the size of the filter 102. The rotation of the disk 302 or 303 is controlled by, for example, 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 keeps the inside and outside airtight.

[0123] 8(b) shows the state after the culture medium 1 has been collected in the first collection container 305. By rotation of the disk 302 or 303, the syringe 101 and the 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. In addition, the syringe 201 containing the luminescence reagent 3 is used to pierce the septum 304 with the needle of the syringe 201 to add the luminescence reagent 3 to the first collection container 305, 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 side wall surface of the sealed container 301 is shown so that the internal configuration can be seen. As shown in FIG. 8(c), the septa 107 and 304 covering the openings provided in the disk 302 are located directly above the two collection containers 305 provided in the disk 303. When the position of the luminescence measuring device 109 is fixed, the disk 302 is fixed and the disk 303 is rotated, so that luminescence measurement can be performed using a plurality of collection containers 305 by simply rotating the disk 303. Of course, the disk 303 may be fixed and the disk 302 may be rotated, and the position of the luminescence measuring device 109 may be changed every time luminescence measurement is performed in 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 of 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 store the filtrate in one of the multiple collection containers 305.

[0127] The method of this embodiment also differs 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 and collection of the medium 1, as well as the luminescence measurement, are carried out in the sealed container 301. This reduces the risk of bacteria being mixed in from the outside when the filter 102 is moved above a different collection container 305 and transferred to the collection container. In addition, since the gas composition in the sealed container 301 can be controlled, the culture conditions can be kept anaerobic or aerobic depending on the type of bacteria to be detected.

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

[0130] <Configuration of cell detection device> Fig. 9(a) is a schematic cross-sectional view for explaining a method for filtering a sample solution using filter 102. Filter 102 is, for example, a filter with a volume of 100 µL. As shown in Fig. 9(a), filter 102 is held by an annular filter holder 403. A large amount of liquid sample can be filtered by connecting a funnel 5 to the cell capturing surface side of filter 102 and a filtration stage 6 to the opposite surface. By connecting a suction pump (not shown) to the filtration stage 6 and performing suction filtration, bacteria in the sample can be collected and concentrated on 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 medium container that contains the medium 1 and the culture container in which 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 stage 6 are removed from the filter holder 403, and a syringe 401 (culture medium container, culture medium adding device) is connected to the cell capturing surface side of the filter holder 403, and an adapter 404 (a member defining a space) is connected to the opposite surface. As a result, a space 402 (culture container) is formed 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 accommodated in the space 402.

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

[0134] The adaptor 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 luminescence 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 luminescence wavelength so that luminescence in adjacent wells 405 does not interfere with measurement, and a transparent window 415 is provided only near the surface where luminescence is measured by the luminescence measuring device 109. Each well 405 is covered with a seal 406 to maintain a tight seal. As the seal 406, a rubber seal that can maintain a tight seal even if a capillary 408 penetrates it, an aluminum material that prevents deterioration of the luminescence reagent 3, or the like can be used.

[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 a plurality of collection containers. In this method, the calculation device actually drives each drive device to drive the syringe 401, etc., but for the sake of simplicity, the following description may be given assuming that the subject of the operation is simply the calculation device.

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

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

[0141] In luminescence measurement at the 0th hour of culture, the calculation 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 calculation device. The calculation 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 performed, and keeps the filter 102 at, for example, 37° C. by a temperature regulator, and cultures the bacteria on the filter 102 .

[0143] After 2 hours of culture, the arithmetic device causes the capillary 408 to pierce the seal 406 and insert it into a well 405 different from that at 0 hours. Then, 200 μL of medium 1 is delivered by the syringe 401, and the medium containing the substance secreted by the cells on the filter 102 is dropped into the well 405 and mixed 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 arithmetic device. The arithmetic device calculates the amount of luminescence corresponding to the detection signal and regards this as the luminescence at the second hour.

[0144] The computing device compares the amount of luminescence at the 0th hour with the amount of luminescence at the 2nd hour, and if a significant increase in the amount of luminescence is observed at the 2nd hour, it 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 with the amount of luminescence at the 0th hour. As described above, the method is not limited to determining whether the result is positive or negative by comparing the measured amount of luminescence with the amount of luminescence at the 0th hour, and the determination may be made by comparing with a predetermined threshold value or the like.

[0145] <Technical effect> As described above, cell detection device 400 according to the fourth embodiment uses a combination of syringe 401 that contains 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 makes it unnecessary to move the syringe that contains the culture medium and filter holder 403 having filter 102 separately, simplifying the operation.

[0146] Furthermore, since the luminescence reagent 3 is dispensed in advance into the multiple wells 405, a dispensing mechanism for the luminescence 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 mixture ratio of the collected medium 1 and the luminescent reagent 3 can be kept constant, and luminescence measurement can be performed with the same sensitivity regardless of the culture time. Therefore, the detection accuracy of bacteria is improved.

[0148] [Fifth embodiment] In the first to fourth embodiments, the cell detection device is configured to add the culture medium from a direction substantially perpendicular to the filter surface. In contrast, 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> Fig. 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 paths 508 and 509, and before the culture medium is added, flow paths 508 and 509 are sealed with plugs 510 and 511, respectively. Flow path 508 is provided on the bacteria capture surface side of filter 102, and flow path 509 is provided on the surface side 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 a 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 (members defining a space) are attached to both sides of filter 102 so as to surround filter 102. Providing lids 506 and 507 on filter holder 503 forms filter cartridge 502 (culture vessel) in which filter 102 is sealed in a vessel with a small volume of approximately within twice 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), the plugs 510 and 511 sealing the two flow paths 508 and 509 are removed. The medium container 501 is connected to the flow path 508 via a check valve 513 and a liquid supply pump 512, and the 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 side 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. That is, the flow direction of the medium 1 is approximately parallel to the filter surface. By flowing the medium approximately parallel to the filter surface, the medium can be distributed over the entire filter surface even with a small amount of medium compared to the case of flowing it vertically.

[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 description may be given simply assuming that the calculation device is the subject of the operations.

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

[0157] <Technical effect> As described above, the cell detection device 500 according to the fifth embodiment includes the 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 makes it possible to efficiently collect a small amount of culture medium even in the case of a filter with a large area, without the culture medium remaining on the periphery of the filter.

[0158] [Sixth embodiment] In the first to fifth embodiments, a cell detection device that detects 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 colonies of detected bacteria is proposed.

[0159] <Configuration of cell detection device> Fig. 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 substantially 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 by 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 5 times the volume (area x thickness) of the filter 102.

[0160] The material of the fixing member 603 can be any material, such as the same material as the filter 102 or an agar medium (gel), 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 is detected using the cell detection device 600, the bacteria 2 are further cultured to form visible colonies, and the filter cartridge 602 can be removed and observed as is.

[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 live bacteria can be counted by forming visible colonies by culturing the bacteria.

[0162] [Seventh embodiment] In the above-mentioned fifth embodiment, a method of collecting the culture medium in different wells (collection containers) for each culture time by using a filter cartridge with a built-in filter and measuring the luminescence has been described. In order to collect the culture medium in different wells in the fifth embodiment, the filter cartridge 502 is moved and the capillary 515 is inserted into each well, but in the seventh embodiment, a method of collecting the culture medium in multiple wells without moving the filter cartridge 502 is proposed.

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

[0167] First, before performing luminescence measurement, the arithmetic device flushes and discharges a sufficient amount of medium 1 using the liquid delivery pump 512 to 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 delivers a predetermined amount of 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 medium 1 in a different well 705 each time, mixes it with a luminescence reagent to cause a luminescence 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 switching valve 702 for switching the flow path 703 so that the flow path 701 connected to the filter cartridge 502 communicates with one well 705, as a configuration for recovering the culture medium 1 into a different well 705 for each culture time. This makes it possible to perform luminescence measurement for each culture time while keeping the culture medium container 501, the filter cartridge 502, and the well 705 sealed, thereby preventing the intrusion of foreign matter from the outside during culture. In addition, since it is not necessary to move the filter cartridge 502 away from each well 705 or to insert the flow path 701 into each well 705, no space is required for moving the filter cartridge 502, and the device can be made smaller in size.

[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> Fig. 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 adaptors 806 and 807 (members that define a space). Recesses are formed on the surfaces of the adaptors 806 and 807 that face the filter 102. This forms a space in which the filter 102 is housed. The adaptor 806 is provided with two flow paths 804 that connect this space with the outside. A medium container 801 (multiple medium containers) that houses the medium 1 is connected to each of the two flow paths 804. A valve 805 is provided at the boundary between the flow paths 804 and the medium container 801, separating the space in which the filter 102 is housed from the 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 be stored in the culture medium containers 801. In this case, it is possible to detect bacteria that can grow according to the type of culture medium, so that the bacteria contained in the sample can be categorized according to the culture medium in which they could or could not grow.

[0174] For example, a device (culture medium adding 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 of a predetermined value or more to the culture medium container 801, 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 a space in which the filter 102 is accommodated. 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 described briefly 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 the capillary 808. Next, the 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 the medium container 801 is sent to the filter cartridge 802 for culture for a predetermined time. That is, a part 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 luminescence reagent to cause a luminescence 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 an 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 a small amount 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 method of each embodiment is non-destructive, and can be used for subsequent analysis. In the sixth embodiment, a cell detection device capable of counting the number of colonies of detected bacteria was described. In the ninth embodiment, another cell detection device capable of counting the number of colonies of detected bacteria is proposed.

[0180] <Configuration of cell detection device> Fig. 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 a 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 adaptors 906 and 907. Recesses are formed on the surfaces of the adaptors 906 and 907 facing the filter 102. This forms a space in which the filter 102 is housed. A valve 905 is provided in the recess of the adaptor 906, and a culture medium container (not shown in FIG. 14) is connected to the valve 905. This allows the space in which the filter 102 is housed to communicate with the culture medium container. A valve 909 is provided in the recess of the adaptor 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 cells. Bacteria 2 that enter each cell cannot move to another cell.

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

[0185] First, filter 102 is placed in filter holder 903, and in a state where partition wall 904 is 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 masses.

[0186] Thereafter, the filter holder 903 is sandwiched between the adaptors 906 and 907 to assemble the filter cartridge 902 .

[0187] Next, when culture medium is added via valve 905, the culture medium can 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 to generate 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 adaptor 907 is removed from the filter cartridge 902, the filter 102 is placed on an appropriate agar medium or liquid medium, and further culture is performed, whereby cell growth in each square can be confirmed by visual inspection or optical measurement means. 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 mass 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 after bacteria detection.

[0191] [Tenth embodiment] In the first to ninth embodiments, the medium added to the filter is centrifuged and collected in the 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 a filter cartridge 1002 and a collection container cartridge 1010 of a 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 provided perpendicular to the filter 102 at the radial end of the filter holder 1003 .

[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 housing the filter 102 by penetrating the septum 1007 with a capillary connected to a container for housing the culture medium or the needle of a syringe housing the culture medium. 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 luminescence reagent 3 is accommodated on the seal 1011. The space below the seal 1011 communicates with the flow path 1008 of the filter holder 1003. For example, the upper surface of the collection container 1005 on the side where the luminescence reagent 3 is accommodated can also be made breakable. This allows, for example, the upper surface of the collection container 1005 and the seal 1011 to be broken by a needle penetrating them, and the luminescence reagent 3 can be added to the space below the seal 1011.

[0196] When the flow passage 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 in the figure, the cell detection device 1000 has a rotation mechanism (culture medium recovery 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). By the rotation about the rotation axis 1009, centrifugal force is applied to the culture medium present in the space in which the filter 102 is housed, and the culture medium moves in a direction away from the rotation axis 1009.

[0198] Fig. 15(b) is a schematic top view showing the filter cartridge 1002 and the collection container cartridge 1010. As shown in Fig. 15(b), the flow path 1008 is provided on the 180° opposite side of the rotation axis 1009. Due to this positional relationship, the culture medium in the filter cartridge 1002 can 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 similar to 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, a collection container cartridge 1010 is attached around the filter cartridge 1002, and a flow path 1008 and a first collection container 1005 are connected to each other.

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

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

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

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

[0205] Thereafter, luminescence measurement is performed in the same manner as for the first collection container 1005. As described above, the addition of culture medium, incubation for a predetermined time, collection of the culture 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.

[0206] <Technical effect> As described above, in the cell detection device 1000 according to the tenth embodiment, the filter cartridge 1002 rotates about the rotation axis 1009 provided on the periphery of the filter cartridge 1002, with the space in which the filter 102 is housed 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, when collecting the culture medium in the collection container 1005, it is not necessary to move the filter cartridge 1002 and the collection container cartridge 307 to a separately prepared centrifuge. This eliminates the need for space for moving the filter cartridge 1002 and the collection container cartridge 1010, thereby realizing a compact device.

[0208] [Variations] The present disclosure is not limited to the above-described embodiments, and includes various modified examples. 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 configurations described. In addition, a part of an embodiment can be replaced with a configuration of another embodiment. In addition, a configuration of another embodiment can be added to a configuration of an embodiment. In addition, a part of the configuration of each embodiment can be added to, deleted from, or replaced with a part of the configuration of another embodiment.

[0209] [Note] <Specific matter 1> a culture medium adding device that adds a part of the culture medium from among 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 cell detection device comprising: 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, 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 as 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 greater than or equal to 1 / 2 and less than or equal to 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 described in 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, characterized in that the culture medium recovery device further includes a selection mechanism for connecting any of a plurality of recovery containers in which the portion of the culture medium is respectively recovered to the culture container.

[0214] <Specific matter 6> The cell detection device described in specific item 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 described in specific feature 2, wherein the culture vessel has a member covering the surface of the filter that retains the cells.

[0216] <Specific matter 8> The cell detection device described in specific item 1, 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 of which contains a portion of the culture medium, to the culture container.

[0217] <Specific matter 9> A cell detection device according to specific item 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> The cell detection device 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 described in specific item 1, further comprising a luminescent reagent adding device that adds the luminescent reagent to the culture medium recovered by the culture medium recovery device.

[0220] <Specific matter 12> A cell detection device as described in specific item 1, characterized in that the culture container and the collection container containing the collected culture medium are arranged at separate positions within the same enclosed 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 in which the filter is housed.

[0222] <Specific matter 14> The cell detection device described in specific item 5, wherein the selection mechanism is a valve that switches a flow path that connects the culture container with one of the multiple collection containers.

[0223] <Specific matter 15> The cell detection device described in specific feature 2, further comprising a partition 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, wherein 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 part of the prepared medium to a culture vessel holding the cells to be detected; Recovering the medium from the culture vessel; and detecting light from a luminescent reagent mixed with the recovered medium.

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

[0227] <Specific matter 19> The culture vessel has a filter having a pore size smaller than the size of the cells, The cell detection method includes: The cell detection method described in specific 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 during the multiple repetitions.

[0230] <Specific matter 22> The cell detection method described in specific item 19, characterized in that the culture medium is moved into the culture vessel approximately parallel to the filter surface.

[0231] <Specific matter 23> The cell detection method described in 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, 20. 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> The cell detection method according to specific item 18, characterized in that in the multiple repetitions, the entire amount of the medium is added to the culture vessel from a different medium vessel each time.

[0234] <Specific matter 26> The cell detection method described in specific item 18, 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.

[0235] <Specific matter 27> The cell detection method according to specific 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, comprising: a medium addition device that adds a part of a medium prepared in the medium to a culture vessel having a filter with a pore size smaller than the size of the cell and holding the detection target cell in the filter; a medium recovery device that recovers an amount of the medium added from the culture vessel, the amount being a difference between the amount remaining in the culture vessel and the amount evaporated, thereby recovering adenosine triphosphate produced in the medium from the cells; a detection device for 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; A cell detection device characterized in that the volume of the portion of culture medium added to the culture vessel by the culture medium addition device is greater than or equal to the sum of the amount remaining in the culture vessel and the amount that evaporates plus the area and thickness of the filter.

2. The cell detection device according to claim 1, characterized in that the volume of the portion of culture medium added to the culture vessel by the culture medium addition device is greater than or equal to two and less than or equal to five times the value obtained by multiplying the area and thickness of the filter.

3. 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, 2. The cell detection device according to claim 1, wherein the light is luminescence produced by a reaction between the adenosine triphosphate and the luciferin and the luciferase.

4. The cell detection device according to claim 1 , wherein the culture medium recovery device further comprises a selection mechanism for connecting any one of a plurality of recovery containers in which the portion of culture medium is respectively recovered to the culture vessel.

5. The cell detection device according to claim 1 , wherein the culture medium addition device adds the culture medium to the culture vessel parallel to a surface of the filter that holds the cells.

6. The cell detection device according to claim 1 , wherein the culture vessel has a member for covering a surface of the filter that retains the cells.

7. The cell detection device according to claim 1, 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 of which contains a portion of the culture medium, to the culture container.

8. 2. The cell detection device according to claim 1, wherein at least a portion of a collection vessel that contains the medium collected from the culture vessel transmits the light from the luminescent reagent.

9. The cell detection device according to claim 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.

10. 2. The cell detection device according to claim 1, wherein the medium recovery device is a centrifuge.

11. A cell detection method for non-destructively detecting cells, comprising: Adding a part of the prepared medium to a culture vessel having a filter with a pore size smaller than the size of the cell and holding the detection target cell on the filter; recovering an amount of the medium added from the culture vessel that is a difference between the amount remaining in the culture vessel and the amount evaporated, thereby recovering adenosine triphosphate produced in the medium from the cells; 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; A cell detection method, wherein the volume of the portion of culture medium is equal to or greater than the sum of the area and thickness of the filter multiplied by the amount of culture medium remaining in the culture vessel and the amount of culture medium that evaporates.

12. repeating the adding, withdrawing, and detecting the light a plurality of times; The cell detection method according to claim 11, further comprising comparing the results of the multiple detections.

13. The cell detection method described in claim 12, characterized in that in the multiple repetitions, the amount of culture medium minus the amount remaining in the culture vessel and the amount evaporating from the portion of the culture medium is recovered into a different recovery container each time.

14. The cell detection method according to claim 11, wherein the recovering is performed using a centrifuge.

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