Soil microorganism observation device and observation method

Abstract

To provide an observation device of soil microorganisms which is capable of simulating time changes of interaction among soil microorganisms without replacing a flow channel connecting between a plurality of culture parts.SOLUTION: In an embodiment, an observation device of soil microorganisms comprises a plurality of culture parts, a flow channel and a control part. Soil microorganisms are cultured in each of the plurality of culture parts, and the plurality of culture parts are isolated from each other. The flow channel connects between the plurality of culture parts, and is capable of flowing medium through the flow channel. The control part, by controlling a diffusion state of a substance through the flow channel from each of the plurality of culture parts, is capable of changing the diffusion state of a substance through the flow channel from each of the plurality of culture parts with lapse of time.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] An embodiment of the present invention relates to a device and method for observing soil microorganisms. [Background technology]

[0002] Because soil is opaque to visible light, it is difficult to directly observe the interactions between soil microorganisms. This makes it difficult to understand the effects of soil manipulations, such as fertilization, on microbial activity and plant growth. For these reasons, observation devices that can simulate the interactions between soil microorganisms have been developed.

[0003] In an observation device that simulates interactions between soil microorganisms, multiple culture sections (chambers) that are isolated from one another are connected via flow paths. By using multiple observation devices with different separation distances between the multiple culture sections through the flow paths, the state of substance exchange between the multiple culture sections, i.e., the degree of interaction between the soil microorganisms between the multiple culture sections, can be observed with different observation devices.

[0004] Here, the soil condition changes over time because it becomes wet or dry due to rain, etc. Therefore, the degree of interaction between soil microorganisms changes over time in response to the change in soil condition over time. There is a demand for an observation device that can simulate the time-dependent changes in the interaction between soil microorganisms without replacing the flow paths connecting multiple culture sections. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Eyal Karzbrun, Alexandra M. Tayar, Vincent Noireaux, and Roy H. Bar‐Ziv. 2014. “Synthetic Biology. Programmable on‐chip DNA compartments as artificial cells.” Science 345 (6198): 829‐832 [Non-patent document 2] Felix JH Hol, Or Rotem, Edouard Jurkevitch, Cees Dekker, and Daniel A. Koster. 2016. “Bacterial Predator‐prey dynamics in microscale patchy landscapes.” Proceedings of the Royal Society B: Biological Sciences 283 (1824): 20152154 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a device and method for observing soil microorganisms that can simulate the changes over time in the interactions between soil microorganisms without replacing the flow paths connecting multiple culture sections. [Means for solving the problem]

[0007] In one embodiment of the present invention, a soil microorganism observation device includes a plurality of culture units, a flow path, and a control unit. Soil microorganisms are cultured in each of the plurality of culture units, and the plurality of culture units are isolated from one another. The flow path connects the plurality of culture units, and a medium can be flowed through the flow path. The control unit controls the diffusion state of a substance through the flow path from each of the plurality of culture units, thereby changing the diffusion state of the substance through the flow path from each of the plurality of culture units over time. The flow paths can be used to flow both a solution and air as media. The control unit changes the diffusion state of the substance through the flow paths from each of the multiple culture units by switching between a state in which the solution flows through the flow paths and a state in which air flows through the flow paths, and by changing the flow rate of the solution when the solution flows through the flow paths. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a device and method for observing soil microorganisms that can simulate the changes in interactions between soil microorganisms over time without replacing the flow paths connecting multiple culture sections. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram showing an observation device according to the first embodiment. [Figure 2] FIG. 2 is a schematic diagram showing an observation device according to the second embodiment. [Figure 3] FIG. 3 is a cross-sectional view that schematically shows a plurality of culture sections and their vicinity in an observation device according to a second embodiment, with a medium flowing through a flow channel. [Figure 4] FIG. 4 is a schematic diagram illustrating an example of image processing of image data including periodically captured images, which is performed by the control unit according to the second embodiment. [Figure 5] FIG. 5 is a cross-sectional view that schematically shows one of a plurality of culture sections and its vicinity in an observation device according to a modified example, with a solution flowing through a flow path. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0011] (First embodiment) First, a first embodiment will be described as an example of an embodiment. Fig. 1 is a schematic diagram showing an observation device 1 according to the first embodiment. The observation device 1 includes a plurality of culture sections 2 and flow channels 3. In the observation device 1, the culture sections 2 and flow channels 3 are formed inside a culture plate 5. The culture plate 5 is transparent to visible light, and the culture sections 2 and flow channels 3 can be observed (visually recognized) from outside the culture plate 5. Examples of materials for forming the culture plate 5 include polydimethylsiloxane (PDMS) and glass.

[0012] Each of the multiple culture sections 2 is formed from a chamber inside the culture plate 5, and each culture section 2 contains a suspension containing soil microorganisms 6 and a culture solution, and the soil microorganisms are cultured using the culture solution. The culture solution is a liquid containing a nutrient source for the soil microorganisms. Inside the culture plate 5, the multiple culture sections 2 are isolated from each other. Note that, although the example in FIG. 1 shows a configuration in which two culture sections 2 are provided, any configuration may be used as long as multiple culture sections 2 are provided. In one example, three or more culture sections 2 may be provided.

[0013] A medium such as a fluid can be passed through the flow path 3. The culture sections 2 are connected to one another via the flow paths 3. Here, in the culture plate 5, the culture sections 2 and the flow paths 3 are each formed using microfabrication technology. The dimensions and shapes of the culture sections 2 and the flow paths 3 are not particularly limited. In one example, the width of the flow path 3 is formed to be about 20 μm.

[0014] The culture plate 5 is also formed with a solution inlet 11, an air inlet 12, and an outlet 13, and the flow path 3 opens to the outside of the culture plate 5 at the solution inlet 11, the air inlet 12, and the outlet 13, respectively. The flow path 3 is formed with a main flow path section 15, supply flow path sections 16 and 17, and a junction 18. In the flow path 3, the supply flow path section 16 extends from the solution inlet 11 to the junction 18, and the supply flow path section 17 extends from the air inlet 12 to the junction 18. The supply flow path sections 16 and 17 merge at the junction 18. The main flow path section 15 extends from the junction 18 to the outlet 13. Inside the culture plate 5, the main flow path section 15 is connected to each of the multiple culture sections 2. In the flow path 3, the side where the solution inlet 11 and the air inlet 12 are located is the upstream side, and the side where the outlet 13 is located is the downstream side.

[0015] The observation device 1 is also provided with a solution supply unit 21 and an air supply unit 22. The solution supply unit 21 is connected to the solution inlet 11 of the flow path 3 via one or more tube members (not shown). The solution supply unit 21 is composed of, for example, a tank that stores a solution containing a culture medium, and a supply actuator such as a micropump. In the solution supply unit 21, the supply actuator such as a micropump is operated to supply the solution stored in the tank to the flow path 3. The solution supplied from the solution supply unit 21 flows from the solution inlet 11 into the flow path 3, and flows from upstream to downstream, passing through the supply flow path section 16, the junction section 18, and the main flow path section 15 in this order.

[0016] The air supply unit 22 is connected to the air inlet 12 of the flow path 3 via one or more tube members (not shown). The air supply unit 22 is composed of, for example, a tank for storing air, a compressor, an on-off valve, etc. In the air supply unit 22, the air stored in the tank is compressed by the compressor. Then, by opening the on-off valve, the compressed air is supplied to the flow path. The air supplied from the air supply unit 22 flows into the flow path 3 from the solution inlet 11, and flows from upstream to downstream, passing through the supply flow path section 16, the junction section 18, and the main flow path section 15 in this order.

[0017] A tube member (not shown) is connected to the outlet 13. The solution and air that have flowed through the flow path 3 as described above are each discharged from the outlet 13. At this time, the solution and air are each discharged through the inside of the tube member connected to the outlet 13. Due to the configuration as described above, it is possible to flow a solution containing a culture medium and air as media (fluids) through the flow path 3. In FIG. 1, the flow of the solution supplied from the solution supply unit 21 to the flow path 3, the flow of air supplied from the air supply unit 22 to the flow path 3, and the flow of the solution and air discharged from the flow path 3 are each indicated by dashed arrows.

[0018] Furthermore, the culture plate 5 is provided with the same number of filter units 2 as the culture units 2, with one filter unit 23 provided for each of the multiple culture units 2. Each filter unit 23 is disposed between a corresponding one of the culture units 2 and the main channel unit 15 of the channel 3. Each filter unit 23 has a large number of gaps 25 formed therein, which serve as permeable portions. Therefore, each filter unit 23 allows fluids, such as a solution containing a culture medium and air, to pass through only through the permeable portions. Therefore, in each culture unit 2, fluid can flow in from the channel 3 and out of the channel 3 only through the permeable portions of the corresponding filter unit 23. In one example, each filter unit 23 is formed from polydimethylsiloxane (PDMS), and in each filter unit 23, each gap 25, which serves as a permeable portion, has a width (diameter) of approximately 0.6 μm.

[0019] The observation device 1 also includes a photographing unit 27. The photographing unit 27 is composed of a camera or a device equipped with a photographing function, and photographs the photographing range T. At this time, the photographing unit 27 photographs a microscopic image using a microscope. The photographing range T includes all of the multiple culture units 2 and the connection parts of the culture units 2 to their respective flow channels 3. Because the culture plate 5 is transparent to visible light, it is possible to photograph the inside of the culture plate 5, such as the culture units 2. The photographing unit 27 photographs the photographing range T periodically at predetermined time intervals.

[0020] The observation device 1 includes a control unit 31 and a user interface 32. The control unit 31 controls the entire system in which the observation device 1 is installed. The control unit 31 is composed of a processing device such as a computer, and the processing device constituting the control unit 31 includes a processor or integrated circuit and a storage medium such as a memory. The processor or integrated circuit includes any of a CPU (Central Processing Unit), an ASIC (Application Specific Integrated Circuit), a microcomputer, an FPGA (Field Programmable Gate Array), and a DSP (Digital Signal Processor). The processing device constituting the control unit 31 may include only one integrated circuit or the like, or may include multiple integrated circuits or the like. The processing device constituting the control unit 31 performs processing by executing a program or the like stored in a storage medium or the like.

[0021] In one example, the control unit 31 may be composed of multiple computers, i.e., multiple processing devices. In this case, the multiple processing devices cooperate to perform processing by the control unit 31. In another example, the control unit 31 may be composed of a server in a cloud environment. In this case, processing by the control unit 31 is performed by a virtual processor such as a virtual CPU. Various operations are performed in the user interface 32 by the user of the observation device 1, etc. Furthermore, the user interface 32 can notify the user of the observation device 1, etc. of various information. The user interface 32 notifies information by either a screen display, a voice, etc.

[0022] The control unit 31 acquires image data including images periodically captured by the photographing unit 27 at predetermined time intervals. The control unit 31 detects changes in the state of the photographing range T over time by performing image processing on the image data. For example, the control unit 31 can detect the type of medium flowing through the flow path 3 and the flow rate of the solution flowing through the flow path based on the images captured by the photographing unit 27. Operation commands are input to the control unit 31 based on operations performed on the user interface 32 by a user of the observation device 1. The control unit 31 controls the operation of supply operating units such as a micropump in the solution supply unit 21 and the operation of a compressor, an on-off valve, and the like in the air supply unit 22 based on the results of image processing of the image data from the photographing unit 27 and the operation commands from the user interface 32. In FIG. 1 , data input to the control unit 31 and transmission of control commands from the control unit 31 are indicated by solid arrows.

[0023] The control unit 31, for example, operates the supply operating unit of the solution supply unit 21 to supply the solution to the flow path 3, and stops the supply of the solution to the flow path 3 by stopping the operation of the supply operating unit of the solution supply unit 21. Therefore, the control unit 31 can switch between supplying and not supplying the solution to the flow path 3 by controlling the operation of the solution supply unit 21. Furthermore, the control unit 31 can supply air to the flow path 3 by opening the on-off valve of the air supply unit 22, and stops the supply of air to the flow path 3 by closing the on-off valve of the air supply unit 22. Therefore, the control unit 31 can switch between supplying and not supplying air to the flow path 3 by controlling the operation of the air supply unit 22.

[0024] As described above, since the operation of each of the solution supply unit 21 and the air supply unit 22 is controlled, the control unit 31 can control and adjust the type of medium (fluid) flowing through the flow path 3, and can change the type of medium flowing through the flow path 3. That is, the control unit 31 can switch between a state in which a solution flows through the flow path 3 and a state in which air flows through the flow path 3. Furthermore, when a solution is being supplied to the flow path 3, the control unit 31 controls the operation of the supply actuation unit of the solution supply unit 21, thereby controlling the flow rate of the solution flowing through the flow path 3. At this time, the flow rate of the solution in the flow path 3 is adjusted by adjusting the discharge pressure and discharge amount of the solution from the micropump in the solution supply unit 21. Therefore, the control unit 31 can change the flow rate of the solution in the flow path 3.

[0025] In the observation device 1, the diffusion state of a substance through the flow path 3 from each of the culture units 2 changes when at least one of the type of medium flowing through the flow path 3 and the flow rate of the solution in the flow path 3 changes. Therefore, the control unit 31 controls the type of medium flowing through the flow path 3 and the flow rate of the solution in the flow path 3 as described above, thereby controlling the diffusion state of a substance through the flow path 3 from each of the culture units 2. Then, when the control unit 31 adjusts the diffusion state of a substance through the flow path 3 from each of the culture units 2, the degree of interaction between the soil microorganisms 6 among the multiple culture units 2 is adjusted.

[0026] For example, when a solution flows through the flow path 3 and the flow rate of the solution in the flow path 3 is zero or nearly zero, the degree of diffusion of substances from each of the culture units 2 through the flow path 3 is similar to simple diffusion. When a flow rate of the solution is generated in the flow path 3, substances are more likely to diffuse from each of the culture units 2 through the flow path 3, particularly downstream, compared to when the flow rate of the solution is zero. The faster the flow rate of the solution in the flow path 3, the more easily substances are likely to diffuse from each of the culture units 2 through the flow path 3. Therefore, when a solution is flowing through the flow path 3, the faster the flow rate of the solution in the flow path 3, the greater the degree of interaction between soil microorganisms 6 among multiple culture units 2.

[0027] Furthermore, when air is flowing through the flow paths 3, substances do not diffuse, or hardly diffuse, from each of the culture sections 2 through the flow paths 3. Therefore, when air is flowing through the flow paths 3, the degree of interaction between the soil microorganisms 6 among the multiple culture sections 2 is zero or nearly zero.

[0028] As described above, in this embodiment, the control unit 31 changes over time at least one of the flow rate of the solution in the flow path 3 and the type of medium flowing through the flow path 3, thereby making it possible to change over time the diffusion state of a substance through the flow path 3 from each of the multiple culture units 2. This makes it possible to change over time the degree of interaction between the soil microorganisms 6 among the multiple culture units 2, making it possible to simulate changes over time in the interaction between the soil microorganisms 6 using a single observation device 1. Therefore, it becomes possible to observe, using the observation device 1, changes over time in the interaction between the soil microorganisms 6 that correspond to changes over time in the soil state.

[0029] Furthermore, in this embodiment, the diffusion state of substances through the flow channels 3 from each of the culture units 2 is changed by changing at least one of the flow rate of the solution in the flow channels 3 and the type of medium flowing through the flow channels 3. Therefore, when observing changes over time in the interactions between soil microorganisms 6, there is no need to replace the flow channels 3 connecting multiple culture units 2, nor is there a need to use multiple observation devices with different separation distances through the flow channels 3 between the culture units 2. Therefore, the effort required to observe changes over time in the interactions between soil microorganisms 6 is reduced.

[0030] (Second embodiment) Next, a second embodiment will be described as another example of an embodiment. The second embodiment is a modified example of the first embodiment, which is modified as follows. Therefore, in the following description of the second embodiment, components and processes that are changed from the first embodiment will be described, and descriptions of components and processes that are the same as those of the first embodiment will be omitted.

[0031] 2 is a schematic diagram showing an observation device 1 according to a second embodiment. As shown in Fig. 2, in this embodiment, an inlet 41 and an outlet 42 are formed in a culture plate 5, and the flow path 3 opens to the outside of the culture plate 5 at each of the inlet 41 and the outlet 42. In this embodiment as well, the flow path 3 is connected to each of the multiple culture sections 2 inside the culture plate 5. In this embodiment, the side of the flow path 3 where the inlet 41 is located is the upstream side, and the side where the outlet 42 is located is the downstream side.

[0032] The observation device 1 is also provided with a supply unit 43. The supply unit 43 is connected to the inlet 41 of the flow path 3 via one or more tube members (not shown). The supply unit 43 is composed of, for example, a medium generating unit that generates a medium, which will be described later, and a supply operating unit such as a micropump. In the supply unit 43, the supply operating unit such as a micropump is operated, whereby the medium generated by the medium generating unit is supplied to the flow path 3. The medium (fluid) supplied from the supply unit 43 flows from the inlet 41 into the flow path 3 and flows through the flow path 3 from the upstream side to the downstream side.

[0033] A tube member (not shown) is connected to the outlet 42. The medium that has flowed through the flow path 3 as described above is discharged from the outlet 42. At this time, the medium is discharged through the inside of the tube member connected to the outlet 42. In FIG. 2, the flow of the medium supplied from the supply unit 43 to the flow path 3 and the flow of the medium discharged from the flow path 3 are indicated by dashed arrows.

[0034] The culture plate 5 of this embodiment is provided with the same number of electric field generators 45 as the culture sections 2, one electric field generator 45 for each of the plurality of culture sections 2. Each electric field generator 45 includes a pair of electrodes 46, 47. In each electric field generator 45, the pair of electrodes 46, 47 is arranged with a corresponding one of the culture sections 2 and the flow path 3 sandwiched therebetween. Therefore, in each electric field generator 45, the connection portion between a corresponding one of the plurality of culture sections 2 and the flow path 3 is located between the pair of electrodes 46, 47.

[0035] The observation device 1 is provided with the same number of power supply units 48 as the number of culture units 2 and electric field generators 45, one power supply unit 48 for each electric field generator 45. In each electric field generator 45, an electrode 46 is electrically connected to a power supply unit 48, and an electrode 47 is grounded. In each electric field generator 45, power is supplied from a corresponding one of the power supply units 48, and a voltage is applied between the pair of electrodes 46, 47. As a result, an electric field is generated in the region between the pair of electrodes 46, 47 in each electric field generator 45. Therefore, when power is supplied to each electric field generator 45, an electric field is generated in the corresponding one of the culture units 2 and in the connection between the corresponding one of the culture units 2 and the flow path 3.

[0036] 3 is a cross-sectional view schematically showing multiple culture sections 2 and their vicinity in the observation device 1 of this embodiment, with a medium flowing through the flow path 3. As shown in FIG. 3, in this embodiment, oil 51 and water-in-oil droplets 52 present in the oil 51 are supplied as a medium from a supply unit 43 to the flow path 3. In one example, with a medium being supplied from the supply unit 43 to the flow path 3, multiple water-in-oil droplets 52 flow through the flow path 3, and the multiple water-in-oil droplets 52 are spaced apart from one another at predetermined intervals in the direction of flow through the flow path 3. Each of the water-in-oil droplets 52 is a solution containing a culture medium, and is surrounded by a lipid membrane 53 formed by a surfactant.

[0037] Furthermore, in this embodiment, oil 54 is stored in each of the culture sections 2, and water-in-oil droplets 55 exist in the stored oil 54 in each of the culture sections 2. In each of the water-in-oil droplets 55, similar to the water-in-oil droplets 52, a solution containing the culture medium is enveloped by a lipid membrane 56 formed by a surfactant. In each of the culture sections 2, soil microorganisms 6 are cultured inside the water-in-oil droplets 55, i.e., in the solution portion of the water-in-oil droplets 55.

[0038] When no electric field is generated by a corresponding one of the electric field generators 45, each water-in-oil droplet 55 in the culture unit 2 does not fuse with the water-in-oil droplets 52 flowing in the flow path 3. On the other hand, when an electric field is generated by a corresponding one of the electric field generators 45, each water-in-oil droplet 55 in the culture unit 2 becomes able to fuse with the water-in-oil droplets 52 flowing in the flow path 3. Therefore, at the connection portion between each of the culture units 2 and the flow path 3, the fusion state of the water-in-oil droplets 55 with the water-in-oil droplets 52 flowing in the flow path 3 changes depending on the operating state of the corresponding one of the electric field generators 45, i.e., the state of generation of the electric field by the corresponding one of the electric field generators 45.

[0039] 2, in this embodiment, the control unit 31 controls the operation of a supply operating unit such as a micropump in the supply unit 43 and the supply of power from each of the multiple power supply units 48 to a corresponding one of the electric field generators 45, based on the results of image processing of image data from the imaging unit 27 and operation commands from the user interface 32. The control unit 31 controls the supply of power to each of the electric field generators 45, thereby controlling the operation of each of the electric field generators 45 and the state of electric field generation by each of the electric field generators 45. In FIG. 2, data input to the control unit 31 and transmission of control commands and the like from the control unit 31 are indicated by solid arrows.

[0040] In this embodiment, the control unit 31 controls the operation of each electric field generator 45 as described above, thereby controlling the fusion state of the water-in-oil droplets 52 flowing through the flow path 3 relative to each water-in-oil droplet 55 in the culture unit 2. Therefore, by changing the state of electric field generation by each electric field generator 45 under control of the control unit 31, the fusion state of the water-in-oil droplets 52 flowing through the flow path 3 relative to the water-in-oil droplet 55 present in the corresponding one of the culture units 2 changes.

[0041] In each of the culture units 2 of the observation device 1, the fusion state of the water-in-oil droplets 52 flowing through the flow path 3 relative to the water-in-oil droplets 55 changes, thereby changing the diffusion state of substances from the culture unit 2 through the flow path 3. For this reason, the control unit 31 controls the fusion state of the water-in-oil droplets 52 flowing through the flow path 3 relative to each of the water-in-oil droplets 55 in the culture unit 2 as described above, thereby controlling the diffusion state of substances from each of the culture units 2 through the flow path 3. Also in this embodiment, the control unit 31 adjusts the diffusion state of substances from each of the culture units 2 through the flow path 3, thereby adjusting the degree of interaction between the soil microorganisms 6 among the multiple culture units 2.

[0042] Here, for each electric field generator 45, the state in which it generates an electric field is referred to as the ON state, and the state in which it does not generate an electric field is referred to as the OFF state. For example, in each culture unit 2, when the frequency at which one of the electric field generators 45 is turned ON and the time per unit time that one of the electric field generators 45 is turned ON are at reference levels, the degree of diffusion of a substance from that culture unit 2 through the flow path is comparable to simple diffusion. In each culture unit 2, the more frequently one of the electric field generators 45 is turned ON, the more easily the substance from that culture unit 2 diffuses through the flow path 3. In addition, in each culture unit 2, the longer the time per unit time that one of the electric field generators 45 is turned ON, the more easily the substance from that culture unit 2 diffuses through the flow path 3.

[0043] Therefore, when a medium is flowing through the flow path 3, the degree of interaction between the soil microorganisms 6 among the multiple culture units 2 changes depending on the frequency with which each electric field generator 45 is turned on and the time per unit time that each electric field generator 45 is turned on. Furthermore, even when a medium is flowing through the flow path 3, if each electric field generator 45 is continuously turned off over time, no or almost no substances diffuse from each culture unit 2 through the flow path 3. Therefore, while the electric field generator 45 is continuously maintained in the off state, the degree of interaction between the soil microorganisms 6 among the multiple culture units 2 is zero or approximately zero.

[0044] The control unit 31 also detects each of the water-in-oil droplets 52 and 55 in the captured image by processing the image data from the imaging unit 27. Here, in the captured image of the imaging range T, the lipid membrane 53 surrounding the water-in-oil droplet 52 and the lipid membrane 56 surrounding the water-in-oil droplet 55 appear darker than other parts due to the refractive index of light. In one example, the control unit 31 performs binarization processing on the captured image of the imaging range T to generate a binary image of the captured image. Then, based on the positions of pixels darker than a threshold in the binary image, the control unit 31 detects each of the lipid membranes 53 and 56, i.e., the boundaries between each of the water-in-oil droplets 52 and the oil 51, and the boundaries between each of the water-in-oil droplets 55 and the oil 54, in the captured image.

[0045] By detecting the lipid membranes 53, 56 from the captured image as described above, the control unit 31 detects the portions enclosed by each of the lipid membranes 53 detected in the captured image as water-in-oil droplets 52. The control unit 31 then detects the portions enclosed by each of the lipid membranes 56 detected in the captured image as water-in-oil droplets 55. The control unit 31 also calculates position information for each of the water-in-oil droplets 52 detected in the captured image, for example, calculating the positions of the upstream end and downstream end of each of the detected water-in-oil droplets 52. The image capturing unit 27 also captures images periodically at predetermined time intervals. Therefore, the control unit 31 calculates the position information for each of the water-in-oil droplets 52 detected in each of the periodically captured images, thereby calculating the change over time in the position information for each of the water-in-oil droplets 52.

[0046] The control unit 31 controls the operation of each electric field generator 45 based on the position information for each water-in-oil droplet 52 and the change in the position information over time. Here, one of the water-in-oil droplets 52 flowing through the flow path 3 is assumed to be water-in-oil droplet 52A. When the water-in-oil droplet 52A is to fuse with each water-in-oil droplet 55 in the culture unit 2, the control unit 31 determines whether the water-in-oil droplet 52A is located at a position where it can fuse with each water-in-oil droplet 55 in the culture unit 2, based on the position information for the water-in-oil droplet 52A and the change in the position information over time. Then, the control unit 31 turns on one of the electric field generators 45 at the timing when the water-in-oil droplet 52A is located at a position where it can fuse with each water-in-oil droplet 55 in the culture unit 2.

[0047] Furthermore, among the multiple culture sections 2, one is designated as culture section 2A, and the other positioned adjacent to culture section 2A on the downstream side of flow path 3 is designated as culture section 2B. By performing image processing on image data including periodically captured images, control section 31 tracks water-in-oil droplet 52A flowing downstream through flow path 3 after water-in-oil droplet 52A has passed a position in culture section 2A where it can fuse with water-in-oil droplet 55. By tracking water-in-oil droplet 52A, control section 31 determines whether water-in-oil droplet 52A has reached a position in culture section 2B where it can fuse with water-in-oil droplet 55 after passing the position in culture section 2A where it can fuse with water-in-oil droplet 55.

[0048] 4 is a schematic diagram illustrating an example of image processing of image data including periodically captured images, performed by the control unit 31 of this embodiment. Here, assuming that time t is defined as a variable, in the example of FIG. 4, the imaging range T is imaged by the imaging unit 27 at each of multiple time points including time t1 and time t2, which is later than time t1. Furthermore, the control unit 31 sets a fusion region E of the water-in-oil droplets 55 relative to the water-in-oil droplets 52 flowing through the flow path 3 for each of the multiple culture units 2. For example, a fusion region Ea is set for culture unit 2A, and a fusion region Eb is set for culture unit 2B.

[0049] The control unit 31 acquires the pixel values ​​of the constituent pixels of each of the binarized images obtained by binarizing the images captured by the imaging unit 27. In each of the binarized images, pixels that are brighter than the threshold value have a pixel value of 0, and pixels that are darker than the threshold value have a pixel value of 1. The water-in-oil droplet 52 that is just one upstream of the water-in-oil droplet 52A is designated as water-in-oil droplet 52B.

[0050] The control unit 31 determines whether the sum of pixel values ​​in each of the fusion regions Ea and EB in each of the periodically acquired binarized images is equal to or greater than a reference value. Then, when the water-in-oil droplet 52B passes through the fusion region Ea of the culture unit 2A, the control unit 31 detects the timing at which the sum of pixel values ​​in the fusion region Ea in the binarized image changes from less than the reference value to equal to or greater than the reference value. Then, based on the fact that the sum of pixel values ​​in the fusion region Ea has changed to equal to or greater than the reference value, the control unit 31 determines that the upstream end Ua of the water-in-oil droplet 52A has reached the fusion region Ea of the culture unit 2A.

[0051] Then, the control unit 31 turns on one of the electric field generators 45 corresponding to the culture unit 2A at the timing when the upstream end Ua of the water-in-oil droplet 52A reaches the fusion region Ea or immediately thereafter. This allows the water-in-oil droplet 52A to properly fuse with the water-in-oil droplet 55 in the culture unit 2A. In the example of FIG. 4, in the binarized image I1 at time t1, it is determined that the sum of pixel values ​​in the fusion region Ea has changed to or exceeded a reference value. Then, based on the binarized image I1, it is determined that the upstream end Ua of the water-in-oil droplet 52A has reached the fusion region Ea for the culture unit 2A. Note that in each of the binarized images I1 and I2 in FIG. 4, areas brighter than the threshold are shown in white, and areas darker than the threshold are shown in black.

[0052] In tracking the water-in-oil droplet 52A after the upstream end Ua of the water-in-oil droplet 52A reaches the fusion region Ea, the control unit 31 calculates, for each pixel, the difference in pixel value between the binarized image I1 at time t1 and the binarized image I2 at time t2. At this time, the difference in pixel value is calculated as an absolute value. The control unit 31 then generates a difference value image I3 in which the difference in pixel value between the binarized images I1 and I2 is shown for each pixel. In the difference value image I3 in FIG. 4, parts where the difference in pixel value is smaller than a reference value are shown in white, and parts where the difference in pixel value is equal to or greater than the reference value are shown in black.

[0053] The control unit 31 calculates the position Ua(t=t2) of the upstream end Ua of the water-in-oil droplet 52A at time t2 based on the position Ua(t=t1) of the upstream end Ua of the water-in-oil droplet 52A at time t1 (binarized image I1) and the difference value image I3. At this time, the control unit 31 detects a portion of the flow path 3 downstream of the position Ua(t=t1) where the difference value in the difference value image I3 is equal to or greater than a reference value. Then, the control unit 31 identifies the portion closest to the position Ua(t=t1) from among the portions downstream of the position Ua(t=t1) where the difference value is equal to or greater than the reference value as the position Ua(t=t2) of the upstream end Ua of the water-in-oil droplet 52A at time t2.

[0054] In the example of FIG. 4, the control unit 31 sets a virtual line C along the flow path 3. Then, the control unit 31 gradually moves a frame enclosing the same area as each of the fusion regions E in the difference value image I3 from the position Ua(t=t1) of the upstream end Ua at time t1 downstream along the virtual line C. At this time, each time the control unit 31 moves the frame, it determines whether the sum of the difference values ​​in the range enclosed by the frame is equal to or greater than a reference value. By making such a determination, the control unit 31 can detect a portion of the flow path 3 downstream of the position Ua(t=t1) where the difference value is equal to or greater than the reference value, and can identify the position Ua(t=t2) of the upstream end Ua of the water-in-oil droplet 52A at time t2.

[0055] After time t2, the control unit 31 identifies the position of the upstream end Ua of the water-in-oil droplet 52A in the same way as it identifies the position Ua (t=t2) of the upstream end Ua of the water-in-oil droplet 52A at time t2. Therefore, after the upstream end Ua of the water-in-oil droplet 52A reaches the merging region Ea, the upstream end Ua of the water-in-oil droplet 52A is properly tracked, and the water-in-oil droplet 52A is properly tracked.

[0056] After the upstream end Ua of the water-in-oil droplet 52A reaches the fusion region Ea, the control unit 31 tracks the water-in-oil droplet 52A as described above to determine whether the upstream end Ua of the water-in-oil droplet 52A has reached the fusion region Eb of the culture unit 2B. Then, at the timing when the upstream end Ua of the water-in-oil droplet 52A reaches the fusion region Eb or immediately thereafter, the control unit 31 turns on one of the electric field generators 45 corresponding to the culture unit 2B. This allows the water-in-oil droplet 52A that has fused with the water-in-oil droplet 55 in the culture unit 2A to properly fuse with the water-in-oil droplet 55 in the culture unit 2B. Furthermore, the control unit 31 performs the same control as described above on the water-in-oil droplets 52 other than the water-in-oil droplet 52A, allowing them to fuse with the water-in-oil droplet 55 in the culture unit 2B after fusing with the water-in-oil droplet 55 in the culture unit 2A.

[0057] As described above, in this embodiment, the control unit 31 controls the operation of each of the multiple electric field generators 45, thereby controlling the fusion state of the water-in-oil droplets 52 flowing through the flow path 3 for each of the water-in-oil droplets 55 in the multiple culture units 2. This makes it possible to change the diffusion state of substances through the flow path 3 from each of the multiple culture units 2 over time. Therefore, in this embodiment, it is also possible to change the degree of interaction between the soil microorganisms 6 among the multiple culture units 2 over time, making it possible to simulate changes over time in the interaction between the soil microorganisms 6 using a single observation device 1. Therefore, in this embodiment, it is also possible to observe changes over time in the interaction between the soil microorganisms 6 corresponding to changes over time in the soil condition using the observation device 1.

[0058] Furthermore, in this embodiment, the diffusion state of substances through the flow paths 3 from each culture unit 2 is changed by changing the operating state of each electric field generator 45. Therefore, when observing changes over time in the interactions between soil microorganisms 6, there is no need to replace the flow paths 3 connecting multiple culture units 2, nor is there a need to use multiple observation devices with different separation distances between the culture units 2 through the flow paths 3. Therefore, in this embodiment as well, the effort required to observe changes over time in the interactions between soil microorganisms 6 is reduced.

[0059] (Variation) In one modified example, one or more of the plurality of filter sections 23 are formed as follows. Fig. 5 is a cross-sectional view schematically showing one of the plurality of culture sections 2 and its vicinity in an observation device 1 of one modified example, with the above-mentioned solution flowing through the flow path 3. As shown in Fig. 5, in this modified example, at least one of the filter sections 23 is formed to have a multi-layer structure (multi-stage structure). The multi-layer filter section 23 includes a first filter layer 61 and a second filter layer 62. The second filter layer 62 is stacked on the side of the first filter layer 61 opposite to the side where the flow path 3 is located.

[0060] In this modification, a number of gaps 25 serving as permeation portions are formed in each of the first filter layer 61 and the second filter layer 62 in the multi-layer filter unit 23. The gaps 25 in the second filter layer 62 are formed offset from the gaps 25 in the first filter layer 61. However, each of the gaps 25 in the second filter layer 62 communicates with one or more of the gaps 25 in the first filter layer 61. Therefore, fluid can flow from the flow path 3 into the corresponding culture unit 2 through the multi-layer filter unit 23, and can flow from the corresponding culture unit 2 to the flow path through the multi-layer filter unit 23. When a fluid flows through the multi-layer filter unit 23 between the corresponding culture unit 2 and the flow path 3, the fluid passes through the gaps 25 serving as permeation portions in each of the first filter layer 61 and the second filter layer 62.

[0061] In this modification, even if the width (diameter) of each of the gaps 25, which are permeable portions, in each of the first filter layer 61 and the second filter layer 62 of the filter unit 23 having a multi-layer structure is increased to some extent, excessive inflow of fluid from one flow path 3 to the corresponding flow path 3 is appropriately suppressed. By increasing the width of each of the gaps 25 in each of the first filter layer 61 and the second filter layer 62 to some extent, it is possible to reduce the effort and cost involved in manufacturing the filter unit 23 having a multi-layer structure.

[0062] The present invention is not limited to the above-described embodiments, and various modifications can be made in the implementation stage without departing from the spirit of the invention. Furthermore, the embodiments may be implemented in appropriate combinations, in which case the combined effects can be obtained. Furthermore, the above-described embodiments include various inventions, and various inventions can be extracted by combining selected elements from the disclosed elements. For example, if the problem can be solved and the desired effect can be obtained even if some elements are deleted from all elements shown in the embodiments, the configuration from which these elements are deleted can be extracted as an invention. [Explanation of symbols]

[0063] 1...Observation device 2(2A,2B)...Culture section 3...Flow path 5. Culture plate 6. Soil microorganisms 11...Solution inlet 12...Air inlet 13…Discharge port 15...Main flow path section 16, 17...Supply channel section 18...Confluence 21...Solution supply unit 22...Air supply section 23...Filter section 25...Gap 27...Photography Department 31...Control unit 32...User Interface 41...Inlet 42…Discharge port 43...Supply section 45...Electric field generating section 46,47...electrode 48...Power supply section 51,54…oil 52(52A,52B),55…Water droplets in oil 53,56…lipid membrane

Claims

1. a plurality of culture sections in which soil microorganisms are cultured and isolated from each other; a flow path that connects the plurality of culture units and allows a medium to flow therethrough; a control unit capable of changing the diffusion state of the substance through the flow path from each of the plurality of culture units over time by controlling the diffusion state of the substance through the flow path from each of the plurality of culture units; Equipped with The flow path can allow a solution and air to flow as the medium, the control unit changes the diffusion state of the substance through the flow path from each of the plurality of culture units by switching between a state in which the solution flows through the flow path and a state in which the air flows through the flow path, and by changing a flow rate of the solution when the solution flows through the flow path. A device for observing soil microorganisms.

2. A plurality of culture sections in which soil microorganisms are cultured and isolated from each other; a flow path that connects the plurality of culture units and allows a medium to flow therethrough; a control unit capable of changing the diffusion state of the substance through the flow path from each of the plurality of culture units over time by controlling the diffusion state of the substance through the flow path from each of the plurality of culture units; a plurality of electric field generators, each of which is provided for each of the plurality of culture units and which, when activated, generates an electric field at a connection portion between a corresponding one of the plurality of culture units and the flow path; Equipped with The flow channel can be configured to allow water-in-oil droplets, in which a solution is enclosed in a lipid membrane, to flow as the medium; Each of the plurality of culture sections contains water-in-oil droplets in which a solution is enclosed in a lipid membrane, The control unit controls the operation of each of the plurality of electric field generators to control the fusion state of the water-in-oil droplets flowing through the flow path with respect to the water-in-oil droplets of each of the plurality of culture units, and controls the diffusion state of the substance through the flow path from each of the plurality of culture units. A device for observing soil microorganisms.

3. A plurality of culture sections in which soil microorganisms are cultured and isolated from each other; a flow path that connects the plurality of culture units and allows a medium to flow therethrough; a control unit capable of changing the diffusion state of the substance through the flow path from each of the plurality of culture units over time by controlling the diffusion state of the substance through the flow path from each of the plurality of culture units; a plurality of filter units, each provided for each of the plurality of culture units, and each disposed between a corresponding one of the plurality of culture units and the flow path; Equipped with at least one of the plurality of filter sections includes a first filter layer and a second filter layer that is stacked on the first filter layer on a side opposite to a side on which the flow path is located, and that has a transmission portion that is shifted relative to the first filter layer; each of the transmissive portions of the second filter layer communicates with one or more of the transmissive portions of the first filter layer; A device for observing soil microorganisms.

4. Cultivating soil microorganisms in each of a plurality of culture sections isolated from one another; allowing a medium to flow through a flow path connecting the plurality of culture units; By controlling a diffusion state of the substance through the flow path from each of the plurality of culture units, the diffusion state of the substance through the flow path from each of the plurality of culture units is changed over time; Equipped with The flow path can allow a solution and air to flow as the medium, In controlling the diffusion state of the substance, the diffusion state of the substance through the flow path from each of the plurality of culture units is changed by switching between a state in which the solution flows through the flow path and a state in which the air flows through the flow path, and by changing the flow rate of the solution when the solution flows through the flow path. How to observe soil microorganisms.

5. Cultivating soil microorganisms in each of a plurality of culture sections isolated from each other; allowing a medium to flow through a flow path connecting the plurality of culture units; a plurality of electric field generators provided for each of the plurality of culture sections, each of the plurality of electric field generators being operated, thereby generating an electric field at a connection portion between a corresponding one of the plurality of culture sections and the flow path by each of the plurality of electric field generators; By controlling a diffusion state of the substance through the flow path from each of the plurality of culture units, the diffusion state of the substance through the flow path from each of the plurality of culture units is changed over time; Equipped with The flow channel can be configured to allow water-in-oil droplets, in which a solution is enclosed in a lipid membrane, to flow as the medium; In controlling the diffusion state of the substance, the operation of each of the plurality of electric field generating units is controlled to control the fusion state of the water-in-oil droplets flowing through the flow path with respect to the water-in-oil droplets of each of the plurality of culture units, thereby controlling the diffusion state of the substance through the flow path from each of the plurality of culture units. How to observe soil microorganisms.

6. Cultivating soil microorganisms in each of a plurality of culture sections isolated from each other; allowing a medium to flow through a flow path connecting the plurality of culture units; By controlling a diffusion state of the substance through the flow path from each of the plurality of culture units, the diffusion state of the substance through the flow path from each of the plurality of culture units is changed over time; Equipped with In culturing the soil microorganisms, a plurality of filter units are provided for each of the plurality of culture units, and each of the plurality of filter units is disposed between a corresponding one of the plurality of culture units and the flow path; In the culturing of the soil microorganisms, at least one of the plurality of filter units is provided with a first filter layer and a second filter layer laminated on the first filter layer on the side opposite to the side where the flow path is located, and a permeable portion of the second filter layer is formed to be offset from the permeable portion of the first filter layer, In the cultivation of the soil microorganisms, in at least one of the plurality of filter units in which the first filter layer and the second filter layer are provided, each of the permeation portions of the second filter layer is connected to one or more of the permeation portions of the first filter layer. How to observe soil microorganisms.

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