Microbial culture kit

Abstract

To provide a microbe culture kit that, only by itself, allows for easily constituting a microorganism culture device having a simple design.SOLUTION: A microorganism culture kit disclosed herein includes three frames 1 each including an internal space 11, which are a top layer, a middle layer and a bottom layer. The three frames 1 are mutually stacked and connected to form a three-layer-stacked structure. The top-layer frame 1 allows nutrient-containing liquid or nutrient-containing gas to circulate in the internal space 11. The middle-layer frame 1 can hold a microorganism-containing medium in the internal space 11. The bottom-layer frame 1 allows environmental component-containing liquid or environmental component-containing gas to circulate in the internal space 11.SELECTED DRAWING: Figure 24

Description

Technical Field

[0001] The present invention relates to a microorganism culture kit that can be composed only of a microorganism culture device.

Background Art

[0002] In the conventional agar plate surface smearing method, it is said that only about 1% of the microorganisms that can be cultured are the microorganisms in the environment. The reasons are considered as follows. (a) Since the culture environment is closed, excessive product substances from microorganisms cannot be discharged outside the system. As a result, metabolites and environmental components of microorganisms accumulate and inhibit the growth of microorganisms. (b) In a solid medium, it is difficult to maintain the nutrient concentration required for the growth of the target microorganism.

[0003] Therefore, culture techniques as shown in Patent Documents 1 and 2, for example, have been proposed. In Patent Document 1, culture is performed while continuously supplying a liquid medium. In Patent Document 2, a solid medium containing microorganisms is placed in the natural environment for culture.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in the method of Patent Document 1, since only the medium components are controlled, various culture conditions cannot be realized. Further, in the method of Patent Document 2, since environmental components in the natural environment are supplied, stable culture conditions cannot be realized.

[0006] Moreover, the culture apparatuses described in Patent Documents 1 and 2 could hardly be described as having a simple configuration. Therefore, the culture process was not easy using the culture apparatuses described in Patent Documents 1 and 2.

[0007] The present invention aims to provide a microbial culture kit that allows for the easy construction of a microbial culture apparatus with a simple configuration, which enables the acquisition of a variety of difficult-to-culture microorganisms by realizing diverse culture conditions. [Means for solving the problem]

[0008] The microbial culture kit of the present invention It comprises three frame structures: an upper, middle, and lower layer, each with an internal space. The three frames described above constitute a three-layer laminated structure that is stacked and connected to one another. The upper frame allows for the circulation of a nutrient-containing liquid or nutrient-containing gas within its internal space. The intermediate frame is capable of holding a microorganism-containing culture medium in its internal space. The lower frame allows for the circulation of a liquid or gas containing environmental components within its internal space. It is characterized by the following. [Effects of the Invention]

[0009] According to the present invention, a microbial culture apparatus with a simple configuration can be constructed. Furthermore, since the constructed microbial culture apparatus allows for the cultivation of microorganisms with simple operations, it is possible to realize diverse culture conditions and obtain a variety of difficult-to-culture microorganisms. [Brief explanation of the drawing]

[0010] [Figure 1] This is a perspective view showing a microbial culture kit according to the first embodiment of the present invention. [Figure 2] This is a plan view showing the first mold of the microbial culture kit. [Figure 3] This is a cross-sectional view taken along line III-III in Figure 2. [Figure 4]It is a cross-sectional view showing the connection work between the first type of frame bodies. [Figure 5] It is a partial cross-sectional side view showing a vibrator. [Figure 6] It is a view seen from the direction of arrow VI in FIG. 5. [Figure 7] It is a schematic cross-sectional view showing a microorganism culture device constituted by a microorganism culture kit of the first embodiment. [Figure 8] It is a perspective view of a base. [Figure 9] It is a view seen from the direction of arrow IX in FIG. 8. [Figure 10] It is a cross-sectional view taken along the line X-X in FIG. 9.

[0011] [Figure 11] It is a bottom view of a lid body. [Figure 12] It is a cross-sectional view taken along the line XII-XII in FIG. 11. [Figure 13] It is a schematic cross-sectional view showing a first modification example of a microorganism culture device constituted by a microorganism culture kit of the first embodiment. [Figure 14] It is a schematic cross-sectional view showing a second modification example of a microorganism culture device constituted by a microorganism culture kit of the first embodiment. [Figure 15] It is a schematic cross-sectional view showing a third modification example of a microorganism culture device constituted by a microorganism culture kit of the first embodiment. [Figure 16] It is a perspective view showing a microorganism culture kit of the second embodiment of the present invention. [Figure 17] It is a plan view showing a second type of frame body of a microorganism culture kit. [Figure 18] It is a cross-sectional view taken along the line XVIII-XVIII in FIG. 17. [Figure 19] It is a cross-sectional view showing the connection work between the first type of frame body and the second type of frame body. [Figure 20] It is a schematic cross-sectional view showing a first modification example of a microorganism culture device constituted by a microorganism culture kit of the second embodiment.

[0012] [Figure 21]This is a schematic cross-sectional view showing a second modified example of a microbial culture apparatus configured with the microbial culture kit of the second embodiment. [Figure 22] This is a schematic cross-sectional view showing a third modified example of a microbial culture apparatus configured with the microbial culture kit of the second embodiment. [Figure 23] This is a schematic cross-sectional view showing a fourth modified example of a microbial culture apparatus configured with the microbial culture kit of the second embodiment. [Figure 24] This is a perspective view showing an example of a microbial culture kit according to a third embodiment of the present invention. [Figure 25] This is a perspective view showing another example of a microbial culture kit according to a third embodiment of the present invention. [Figure 26] Figure 24 is a schematic cross-sectional view showing a microbial culture apparatus composed of the microbial culture kit. [Figure 27] This is a schematic cross-sectional view showing a first modified example of a microbial culture apparatus configured with the microbial culture kit shown in Figure 24. [Figure 28] Figure 25 is a schematic cross-sectional view showing a microbial culture apparatus configured using a microbial culture kit. [Figure 29] Figure 25 is a schematic cross-sectional view showing a first modified example of a microbial culture apparatus configured with the microbial culture kit. [Figure 30] This is a perspective view showing an example of a microbial culture kit according to a fifth embodiment of the present invention.

[0013] [Figure 31] This is a view from arrow XXXX in Figure 30. [Figure 32] This is a view (plan view) from the direction of arrow XXXII in Figure 30. [Figure 33] This is a cross-sectional view taken along the line XXXIII-XXXIII in Figure 32. [Figure 34] Figure 32 is a perspective view of the cross-section shown. [Figure 35] This is a schematic cross-sectional view showing a microbial culture apparatus configured with the microbial culture kit of the fifth embodiment. [Figure 36] This is a perspective view showing a modified example of the second formwork. [Figure 37]This is a view taken along arrow XXXVII in Figure 36. [Figure 38] This is a cross-sectional view taken along the line XXXVIII-XXXVIII in Figure 37. [Figure 39] Figure 36 is a cross-sectional perspective view enlargement of the second formwork. [Figure 40] This is a schematic cross-sectional view showing the microbial culture apparatus used in the example. [Figure 41] This figure shows the results of the example. [Modes for carrying out the invention]

[0014] Embodiments of the microbial culture kit of the present invention will be described with reference to the figures.

[0015] [First Embodiment] The microbial culture kit of this embodiment comprises two frames that can be stacked on top of each other. As shown in Figure 1, in the microbial culture kit 10A of this embodiment, both frames are first type frame 1.

[0016] Figure 2 is a plan view of the first formwork 1. Figure 3 is a cross-sectional view taken along line III-III in Figure 2. The first formwork 1 comprises an annular first frame body 12 surrounding a first internal space 11. As shown in Figure 3, the first frame body 12 has an annular outer fitting portion 122 with an internal thread 121 at its lower end, and an annular inner fitting portion 124 with an external thread 123 at its upper end. The outer fitting portion 122 has dimensions that allow it to fit onto the inner fitting portion 124. The inner fitting portion 124 has dimensions that allow it to fit onto the outer fitting portion 122. The first internal space 11 comprises an internal space 111 surrounded by the outer fitting portion 122, and the remaining internal space 112. The inner fitting portion 124 has an O-ring 125 on its upper surface.

[0017] As shown in Figure 4, the two first mold bodies 1 are stacked and connected by screwing the internal thread 121 of the upper first mold body 1 into the external thread 123 of the lower first mold body 1. The two connected first mold bodies 1 are sealed by an O-ring 125.

[0018] Furthermore, the first frame body 12 has an inlet passage 13 for introducing fluid into the internal space 11 and an outlet passage 14 for releasing fluid from the internal space 11. Preferably, pipes 15 and 16, as shown in Figure 5, are connected to the inlet passage 13 and the outlet passage 14, respectively, and protrude radially outward. Figure 5 is a partial cross-sectional side view of pipe 15, and Figure 6 is a view taken along arrow VI in Figure 5. The inlet passage 13 and the outlet passage 14 can be closed by inserting a plug 17 (Figure 7) in place of the pipes 15 and 16. In this way, the inlet passage 13 and the outlet passage 14 can be opened and closed.

[0019] The first mold 1 is designed to allow fluid to flow through the first internal space 11 when the inlet passage 13 and outlet passage 14 are open. The fluid is a nutrient-containing gas or nutrient-containing liquid or an environmental component-containing gas or environmental component-containing liquid. Furthermore, when the inlet passage 13 and outlet passage 14 are closed, the first mold 1 is designed to hold a microbial culture medium, a nutrient-containing material, or an environmental component-containing material in the first internal space 11.

[0020] The microbial culture kit 10A of this embodiment can be used as follows. That is, as shown in Figure 7, a two-layer stacked structure can be constructed by stacking two first mold bodies 1, and this two-layer stacked structure can be used as a microbial culture device 100A. In this two-layer stacked structure, the first mold body 1 with the inlet passage 13 and outlet passage 14 closed is placed in the lower layer, and the first mold body 1 with the inlet passage 13 and outlet passage 14 open is placed in the upper layer. It is preferable to place a membrane filter (not shown) between the upper first mold body 1 and the lower first mold body 1. In the microbial culture device 100A, the lower first mold body 1 holds the microbial culture medium in the first internal space 11, and the upper first mold body 1 allows nutrient-containing liquid to flow through the first internal space 11. In other words, the microbial culture apparatus 100A has a two-layered structure consisting of a layered culture section A, which is made up of a lower first mold body 1 that holds a microbial culture medium, and a layered nutrient supply section B, which is made up of an upper first mold body 1 positioned on the first surface A11 of the culture section A and supplies nutrients to the culture section A.

[0021] Furthermore, it is preferable that the lower first formwork body 1 is stacked on the base 8. Figures 8 to 10 show the base 8. Figure 8 is a perspective view of the base 8. Figure 9 is a view taken along arrow IX in Figure 8. Figure 10 is a cross-sectional view taken along arrow XX in Figure 9. The base 8 is a circular plate in plan view and has an internal fitting portion 82 with external threads 81 at its upper part. The internal fitting portion 82 has dimensions that allow it to be fitted into the external fitting portion 122 of the first frame body 1.

[0022] Furthermore, it is preferable that the upper first mold body 1 is covered with a lid 7 so as to cover the first internal space 11. Figures 11 and 12 show the lid 7. Figure 11 is a bottom view of the lid 7. Figure 12 is a cross-sectional view taken along line XII-XII in Figure 11. The lid 7 is a circular plate in plan view and has an annular outer fitting portion 72 with internal threads 71 ​​at its lower end. The outer fitting portion 72 has dimensions that allow it to be fitted onto the inner fitting portion 124 of the first frame body 12.

[0023] According to the microbial culture apparatus 100A with the above configuration, by circulating a nutrient-containing liquid through the first internal space 11 of the upper first mold body 1, nutrients can be supplied from above to the microorganisms in the first internal space 11 of the lower first mold body 1, thereby enabling the culture of microorganisms.

[0024] Furthermore, instead of nutrient-containing liquid, nutrient-containing gas, environmental component-containing gas, or environmental component-containing liquid may be circulated in the first internal space 11 of the upper first mold frame 1. When environmental component-containing gas or environmental component-containing liquid is circulated, environmental components can be supplied from above to the microorganisms in the first internal space 11 of the lower first mold frame 1, thereby enabling the cultivation of microorganisms.

[0025] Thus, with the microbial culture apparatus 100A, microorganisms can be cultured with simple operations, and the type or concentration of nutrients or environmental components supplied can be easily changed. Therefore, it is possible to achieve diverse culture conditions and obtain a variety of difficult-to-culture microorganisms.

[0026] The microbial culture kit 10A of this embodiment can exhibit the following effects. (a) A microbial culture apparatus 100A with a two-layer stacked structure can be constructed as shown in Figure 7. (b) Since it only requires two first mold bodies 1, a microbial culture apparatus with a simple configuration can be realized. (c) By using a screw mechanism with internal and external threads, the microbial culture apparatus 100A can be constructed simply by connecting two first mold bodies 1, thereby improving the productivity of the apparatus and allowing for easy commencement of microbial culture operations. (d) The first mold body 1 consists of an annular first frame body 12 surrounding the first internal space 11, and therefore has a simple structure. In other words, the microbial culture kit 10A of this embodiment consists of simple components.

[0027] [Modified version of the first embodiment] (1) As shown in Figure 13, the upper and lower layers may be reversed compared to the apparatus in Figure 7. In this case, nutrients or environmental components can be supplied from below to the microorganisms in the first internal space 11 of the first mold body 1 of the upper layer, so that the microorganisms can be cultured.

[0028] (2) Preferably, one of the first mold bodies 1 is equipped with one or more sensors for detecting the culture state. The sensors are selected from a temperature sensor, a pH sensor, and a gas concentration sensor. Preferably, the same first mold body 1 is also equipped with one or more stimulating parts for applying physical stimulation to the microbial culture medium from the outside. The "one of the first mold bodies 1" is used to hold the microbial culture medium.

[0029] For example, in the microbial culture apparatus 100A shown in Figure 14, the lower first mold body 1 is equipped with a temperature sensor 51, a pH sensor 52, and an ultrasonic oscillator 53. In this case, the temperature sensor 51 and pH sensor 52 can detect the culture state of the microorganisms, and this can be monitored by an external device (not shown), allowing for a quick and accurate determination of the culture state. Moreover, based on the monitoring results, at least one of the type and concentration of the nutrient-containing liquid or nutrient-containing gas or environmental component-containing gas or environmental component-containing liquid circulating in the upper first mold body 1 can be changed, making it easy to achieve culture conditions suitable for the microorganisms even during the culture process.

[0030] (3) Two first mold bodies 1 may be used with both the inlet passage 13 and the outlet passage 14 closed. In the microbial culture apparatus 100A shown in Figure 15, the first mold bodies 1 with the inlet passage 13 and the outlet passage 14 closed are arranged in the upper and lower layers. In this case, one first mold body 1 (for example, the lower layer) holds the microbial culture medium, and the other first mold body 1 (for example, the upper layer) holds the nutrient-containing material or the environmental component-containing material. This also allows the microorganisms in the first internal space 1 of one first mold body 1 to be supplied with nutrients or environmental components from the other first mold body 1, thereby enabling the culture of microorganisms.

[0031] [Second Embodiment] The microbial culture kit of this embodiment comprises two frames that can be stacked on top of each other. As shown in Figure 16, in the microbial culture kit 10B of this embodiment, the two frames are the first frame 1 and the second frame 2.

[0032] Figure 17 is a plan view of the second formwork 2. Figure 18 is a cross-sectional view taken along line XVIII-XVIII in Figure 17. The second formwork 2 comprises an annular second frame body 22 surrounding the second internal space 21. As shown in Figure 18, the second frame body 22 has an annular outer fitting portion 222 with internal threads 221 at its lower end, and an annular inner fitting portion 224 with external threads 223 at its upper end. The outer fitting portion 222 has dimensions that allow it to fit onto the inner fitting portion 224 and the inner fitting portion 124 of the first formwork 1. The inner fitting portion 224 has dimensions that allow it to fit onto the outer fitting portion 222 and the outer fitting portion 122 of the first formwork 1. The second internal space 21 comprises an internal space 211 surrounded by the outer fitting portion 222 and the remaining internal space 212. The inner fitting portion 224 has an O-ring 225 on its upper surface.

[0033] The first formwork body 1 is the same as the first formwork body 1 of the first embodiment. The outer fitting portion 122 of the first formwork body 1 has dimensions that allow it to be fitted onto the inner fitting portion 224 of the second formwork body 2, and the inner fitting portion 124 of the first formwork body 1 has dimensions that allow it to be fitted onto the outer fitting portion 222 of the second formwork body 2.

[0034] The first formwork 1 and the second formwork are stacked and connected, for example, as shown in Figure 19, by screwing the internal thread 121 of the upper first formwork 1 into the external thread 223 of the lower second formwork 2. In this case, the connected first formwork 1 and second formwork 2 are sealed by an O-ring 225.

[0035] Furthermore, the second mold frame 2 is designed to hold a microbial culture medium, a nutrient-containing material, or an environmental component-containing material in the second internal space 21.

[0036] The microbial culture kit 10B of this embodiment can be used as follows. That is, as shown in Figure 20, a two-layer laminated structure can be constructed by stacking the first mold body 1 on the second mold body 2, and this two-layer laminated structure can be used as a microbial culture device 100B. In this two-layer laminated structure, the second mold body 2 is located in the lower layer, and the first mold body 1, with the inlet passage 13 and outlet passage 14 open, is located in the upper layer. It is preferable to place a membrane filter (not shown) between the upper first mold body 1 and the lower second mold body 2. In the microbial culture device 100B, the lower second mold body 2 holds the microbial culture medium in the second internal space 21, and the upper first mold body 1 allows nutrient-containing liquid to flow through the first internal space 11. In other words, the microbial culture apparatus 100B has a two-layered structure consisting of a layered culture section A, which is made up of a lower second mold body 2 that holds a microbial culture medium, and a layered nutrient supply section B, which is made up of an upper first mold body 1 positioned on the first surface A11 of the culture section A and supplies nutrients to the culture section A.

[0037] Furthermore, it is preferable that the lower second mold frame 2 is stacked on the base 8. Also, it is preferable that the upper first mold frame 1 is closed with a lid 7. The lid 7 and base 8 are the same as the lid 7 and base 8 used in the first embodiment. The outer fitting portion 72 of the lid 7 has dimensions that allow it to fit onto the inner fitting portion 224 of the second mold frame 2, and the inner fitting portion 82 of the base 8 has dimensions that allow it to fit onto the outer fitting portion 222 of the second mold frame 2.

[0038] According to the microbial culture apparatus 100B with the above configuration, by circulating a nutrient-containing liquid through the first internal space 11 of the upper first mold body 1, nutrients can be supplied from above to the microorganisms in the second internal space 21 of the lower second mold body 2, thereby enabling the culture of microorganisms.

[0039] Furthermore, instead of a nutrient-containing liquid, a nutrient-containing gas, an environmental component-containing gas, or an environmental component-containing liquid may be circulated in the first internal space 11 of the upper first mold frame 1. When an environmental component-containing gas or an environmental component-containing liquid is circulated, environmental components can be supplied from above to the microorganisms in the second internal space 21 of the lower second mold frame 2, thereby enabling the cultivation of microorganisms.

[0040] Thus, with the microbial culture apparatus 100B, microorganisms can be cultured with simple operations, and the type or concentration of nutrients or environmental components supplied can be easily changed. Therefore, it is possible to achieve diverse culture conditions and obtain a variety of difficult-to-culture microorganisms.

[0041] The microbial culture kit 10B of this embodiment can exhibit the following effects. (a) A microbial culture apparatus 100B with a two-layer stacked structure can be constructed as shown in Figure 20. (b) A microbial culture apparatus with a simple configuration can be realized by simply comprising a first mold body 1 and a second mold body 2. (c) The microbial culture apparatus 100B can be constructed simply by connecting the first mold body 1 and the second mold body 2 using a screw mechanism with internal and external threads, thereby improving the productivity of the apparatus and allowing for easy commencement of microbial culture work. (d) The first mold body 1 and the second mold body 2 consist of an annular frame body surrounding the internal space, and therefore have a simple structure. In other words, the microbial culture kit 10B of this embodiment consists of simple components.

[0042] [Modified version of the second embodiment] (1) As shown in Figure 21, the upper and lower layers may be reversed compared to the apparatus in Figure 20. In this case, nutrients or environmental components can be supplied from below to the microorganisms in the second internal space 21 of the upper second mold body 2, so that the microorganisms can be cultured.

[0043] (2) Preferably, the second mold body 2 is equipped with one or more sensors for detecting the culture state. The sensors are selected from a temperature sensor, a pH sensor, and a gas concentration sensor. Preferably, the second mold body 2 is also equipped with one or more stimulating parts for applying physical stimuli to the microbial culture medium from the outside. The second mold body 2 is used to hold the microbial culture medium.

[0044] For example, in the microbial culture apparatus 100B shown in Figure 22, the lower second mold body 2 is equipped with a temperature sensor 51, a pH sensor 52, and an ultrasonic oscillator 53. In this case, the temperature sensor 51 and pH sensor 52 can detect the culture state of the microorganisms, and this can be monitored by an external device (not shown), allowing for a quick and accurate determination of the culture state. Moreover, based on the monitoring results, at least one of the type and concentration of the nutrient-containing liquid or nutrient-containing gas or environmental component-containing gas or environmental component-containing liquid circulating in the upper first mold body 1 can be changed, making it easy to achieve culture conditions suitable for the microorganisms even during the culture process.

[0045] As shown in Figure 23, the first mold body 1 is used with the inlet passage 13 and outlet passage 14 closed, and either the first mold body 1 or the second mold body 2 holds the microorganism-containing culture medium, while the other of the first mold body 1 or the second mold body 2 holds the nutrient-containing material or the environmental component-containing material. When the first mold body 1 holds the microorganism-containing culture medium, the first mold body 1 may be equipped with a temperature sensor 51, a pH sensor 52, and an ultrasonic oscillator 53.

[0046] [Third Embodiment] The microbial culture kit of this embodiment is the microbial culture kit 10A of the first embodiment with one additional frame added. The additional frame is either a first mold frame 1 or a second mold frame 2. That is, the microbial culture kit of this embodiment includes a microbial culture kit 10C equipped with three first mold frames 1, as shown in Figure 24, and a microbial culture kit 10D equipped with two first mold frames 1 and one second mold frame 2, as shown in Figure 25. Note that the first mold frame 1 is the same as the first mold frame 1 of the first embodiment, and the second mold frame 2 is the same as the second mold frame 2 of the second embodiment.

[0047] (Microbial Culture Kit 10C) The microbial culture kit 10C of this embodiment can be used as follows. That is, as shown in Figure 26, a three-layer stacked structure can be constructed by stacking three first mold bodies 1, and this three-layer stacked structure can be used as a microbial culture device 100C. In this three-layer stacked structure, first mold bodies 1 with the inlet passage 13 and outlet passage 14 open are arranged in the upper and lower layers, and first mold bodies 1 with the inlet passage 13 and outlet passage 14 closed are arranged in the middle layer. It is preferable to place a membrane filter (not shown) between each layer. In the microbial culture device 100C, the middle layer first mold body 1 holds the microbial culture medium in the first internal space 11, the upper layer first mold body 1 allows nutrient-containing liquid to flow through the first internal space 11, and the lower layer first mold body 1 allows environmental component-containing liquid to flow through the first internal space 11. In other words, the microbial culture apparatus 100C has a three-layer laminated structure consisting of a layered culture section A made of a middle layer first mold body 1 holding a microbial culture medium, a layered nutrient supply section B made of an upper layer first mold body 1 placed on the first surface A11 of the culture section A and supplying nutrients to the culture section A, and a layered environmental component supply section C made of a lower layer first mold body 1 placed on the second surface A12 of the culture section A and supplying environmental components to the culture section A.

[0048] The stacking and connection of the three first formwork bodies 1 can be performed by screwing the internal thread 121 of the upper first formwork body 1 into the external thread 123 of the lower first formwork body 1. The connected first formwork bodies 1 are sealed together by an O-ring 125.

[0049] Furthermore, it is preferable that the lower first formwork 1 is stacked on the base 8. Also, it is preferable that the upper first formwork 1 is closed with a lid 7. The lid 7 and base 8 are the same as those used in the first and second embodiments.

[0050] According to the microbial culture apparatus 100C with the above configuration, by circulating a nutrient-containing liquid through the first internal space 11 of the upper first mold body 1 and circulating an environmental component-containing liquid through the first internal space 11 of the lower first mold body 1, nutrients can be supplied to the microorganisms in the first internal space 11 of the middle first mold body 1 from above and environmental components from below, thereby enabling the cultivation of microorganisms.

[0051] Furthermore, instead of nutrient-containing liquid, nutrient-containing gas, environmental component-containing gas, or environmental component-containing liquid may be circulated in the first internal space 11 of the upper first mold frame 1. When environmental component-containing gas or environmental component-containing liquid is circulated, environmental components can be supplied to the microorganisms in the first internal space 11 of the middle first mold frame 1 from above, thus enabling the cultivation of microorganisms.

[0052] Furthermore, instead of a liquid containing environmental components, a gas containing environmental components, a gas containing nutrients, or a liquid containing nutrients may be circulated in the first internal space 11 of the lower first mold frame 1. When a gas containing nutrients or a liquid containing nutrients is circulated, nutrients can be supplied to the microorganisms in the first internal space 11 of the middle first mold frame 1 from below, thus enabling the cultivation of microorganisms.

[0053] The microbial culture kit 10C of this embodiment can exhibit the following effects. (a) A microbial culture apparatus 100C with a three-layer stacked structure can be constructed as shown in Figure 26. (b) A microbial culture apparatus with a simple configuration can be realized by providing only three first mold bodies 1. (c) By using a screw mechanism with internal and external threads, the microbial culture apparatus 100C can be constructed simply by connecting three first mold bodies 1, thereby improving the productivity of the apparatus and allowing for easy commencement of microbial culture operations. (d) The first mold body 1 is an annular frame body surrounding the internal space, and therefore has a simple structure. In other words, the microbial culture kit 10C of this embodiment consists of simple components.

[0054] In the microbial culture apparatus 100C, not only the first mold body 1 located in the middle layer, but also the first mold body 1 located in at least one of the upper and lower layers may be used with the inlet passage 13 and outlet passage 14 closed. In the microbial culture apparatus 100C shown in Figure 27, the first mold body 1 in the lower layer is also used with the inlet passage 13 and outlet passage 14 closed. The first mold body 1 in the middle layer holds the microbial culture medium, the first mold body 1 in the upper layer allows nutrient-containing liquid or nutrient-containing gas to flow through, and the first mold body 1 in the lower layer holds environmental component-containing material. This also allows nutrients to be supplied to the microorganisms in the first internal space 11 of the first mold body 1 in the middle layer from above and environmental components from below, so that the microorganisms can be cultured. In the case of Figure 27, the first mold body 1 in the upper layer allows environmental component-containing liquid or environmental component-containing gas to flow through, and the first mold body 1 in the lower layer holds nutrient-containing material.

[0055] (Microbial culture kit 10D) The microbial culture kit 10D of this embodiment can be used as follows. That is, as shown in Figure 28, a three-layer laminated structure can be constructed by stacking the first mold body 1 above and below the second mold body 2, and this three-layer laminated structure can be used as a microbial culture device 100D. In this three-layer laminated structure, the first mold body 1, with the inlet passage 13 and outlet passage 14 open, is arranged in the upper and lower layers, and the second mold body 2 is arranged in the middle layer. It is preferable to place a membrane filter (not shown) between each layer. In the microbial culture device 100D, the second mold body 2 in the middle layer holds a microbial culture medium in the second internal space 21, the first mold body 1 in the upper layer allows nutrient-containing liquid to flow through the first internal space 11, and the first mold body 1 in the lower layer allows environmental component-containing liquid to flow through the first internal space 11. In other words, the microbial culture apparatus 100D has a three-layer laminated structure consisting of a layered culture section A made of a middle layer second mold body 2 that holds a microbial culture medium, a layered nutrient supply section B made of an upper layer first mold body 1 placed on the first surface A11 of the culture section A and supplying nutrients to the culture section A, and a layered environmental component supply section C made of a lower layer first mold body 1 placed on the second surface A12 of the culture section A and supplying environmental components to the culture section A.

[0056] The stacking and connection of the first formwork 1 and the second formwork 2 can be performed by screwing the internal thread 121 of the upper first formwork 1 onto the external thread 223 of the lower second formwork 2, and both are sealed by an O-ring 225. The stacking and connection of the second formwork 2 and the first formwork 1 can also be performed by screwing the internal thread 221 of the upper second formwork 2 onto the external thread 123 of the lower first formwork 1, and both are sealed by an O-ring 125.

[0057] Furthermore, it is preferable that the lower first formwork 1 is stacked on the base 8. Also, it is preferable that the upper first formwork 1 is closed with a lid 7. The lid 7 and base 8 are the same as those used in the first and second embodiments.

[0058] According to the microbial culture apparatus 100D with the above configuration, by circulating a nutrient-containing liquid through the first internal space 11 of the upper first mold body 1 and circulating an environmental component-containing liquid through the first internal space 11 of the lower first mold body 1, nutrients can be supplied to the microorganisms in the second internal space 21 of the middle second mold body 2 from above, and environmental components can be supplied from below, thereby enabling the cultivation of microorganisms.

[0059] Furthermore, instead of nutrient-containing liquid, nutrient-containing gas, environmental component-containing gas, or environmental component-containing liquid may be circulated in the first internal space 11 of the upper first mold frame 1. If environmental component-containing gas or environmental component-containing liquid is circulated, environmental components can be supplied to the microorganisms in the second internal space 21 of the middle second mold frame 2 from above, thus enabling the cultivation of microorganisms.

[0060] Furthermore, instead of a liquid containing environmental components, a gas containing environmental components, a gas containing nutrients, or a liquid containing nutrients may be circulated in the first internal space 11 of the lower first mold frame 1. When a gas containing nutrients or a liquid containing nutrients is circulated, nutrients can be supplied to the microorganisms in the second internal space 21 of the middle second mold frame 2 from below, thus enabling the cultivation of microorganisms.

[0061] The microbial culture kit 10D of this embodiment can exhibit the following effects. (a) A microbial culture apparatus 100D with a three-layer stacked structure can be constructed as shown in Figure 28. (b) A microbial culture apparatus with a simple configuration can be realized by providing only two first mold bodies 1 and one second mold body 2. (c) By using a screw mechanism with internal and external threads, the microbial culture apparatus 100D can be constructed simply by connecting two first mold bodies 1 and one second mold body 2, thereby improving the productivity of the apparatus and allowing for easy commencement of microbial culture work. (d) The first mold body 1 and the second mold body 2 are annular frame bodies that surround the internal space, and therefore have a simple structure. In other words, the microbial culture kit 10D of this embodiment consists of simple components.

[0062] In the microbial culture apparatus 100D, the first mold body 1, which is placed in at least one of the upper and lower layers, may be used with the inlet passage 13 and outlet passage 14 closed. In the microbial culture apparatus 100D shown in Figure 29, the first mold bodies 1 of the upper and lower layers are used with the inlet passage 13 and outlet passage 14 closed. The second mold body 2 of the middle layer holds the microbial culture medium, the first mold body 1 of the upper layer holds the nutrient-containing material, and the first mold body 1 of the lower layer holds the environmental component-containing material. This also allows nutrients to be supplied from above and environmental components from below to the microorganisms in the second internal space 21 of the second mold body 2 of the middle layer, so that the microorganisms can be cultured. In the case of Figure 29, the first mold body 1 of the upper layer may hold the environmental component-containing material, and the first mold body 1 of the lower layer may hold the nutrient-containing material.

[0063] [Fourth Embodiment] The microbial culture kit of this embodiment is the microbial culture kit 10B of the second embodiment with one additional frame added. This additional frame is the first mold frame 1. In other words, the microbial culture kit of this embodiment comprises two first mold frames 1 and one second mold frame 2. Therefore, the microbial culture kit of this embodiment is the same as the microbial culture kit 10D of the third embodiment.

[0064] [Fifth Embodiment] The microbial culture kit of this embodiment includes any number of microbial culture kits 10A to 10D from the first to fourth embodiments. According to this, a multilayer laminated structure can be constructed by stacking four or more frames, and this multilayer laminated structure can be used as a microbial culture device. Figure 30 is a perspective view showing a microbial culture device 100E consisting of a seven-layer laminated structure with seven frames stacked. Figure 31 is a view taken along arrow XXXI in Figure 30. Figure 32 is a view taken along arrow XXXII in Figure 30 (plan view). Figure 33 is a cross-sectional view taken along line XXXIII-XXXIII in Figure 32. Figure 34 is a perspective view of the cross-section shown in Figure 32.

[0065] In this seven-layer laminated structure, the bottom layer (1st layer) is the first formwork 1, the 2nd to 6th layers are the second formwork 2, and the top layer (7th layer) is the first formwork 1. The first formwork 1 of the 1st layer is laminated on a base 8, and the first formwork 1 of the 7th layer is sealed by a lid 7.

[0066] In the microbial culture apparatus 100E, the first mold 1 of the first layer is configured to circulate a liquid or gas containing environmental components, the second mold 2 of the second to fourth layers and the sixth layer hold a culture medium containing microorganisms, the second mold 2 of the fifth layer holds a nutrient-containing material, and the first mold 1 of the seventh layer is configured to circulate a liquid or gas containing nutrients.

[0067] In other words, as shown in Figure 35, a schematic cross-sectional view, the microbial culture apparatus 100E has a seven-layer laminated structure consisting of four culture sections A made up of second mold bodies 2 of layers 2 to 4 and 6 that hold microbial culture media, two nutrient supply sections B made up of layers 5 and 7 that supply nutrients to the culture sections A, and one environmental component supply section C made up of layer 1 that supplies environmental components to the culture sections A. A temperature sensor 51, a pH sensor 52, and an ultrasonic oscillator 53 are provided in the second mold bodies 2 of layers 2 to 4 and 6, respectively. A membrane filter 55 is also placed between each layer.

[0068] This type of microbial culture apparatus 100E can exhibit the following effects: (a) By circulating a liquid or gas containing environmental components through the first mold 1 of the first layer, the microorganisms in the second mold 2 of the second to fourth layers can be supplied with environmental components from below and nutrients from above, so that microorganisms can be cultured in the second mold 2 of the second to fourth layers. Furthermore, by circulating a liquid or gas containing nutrients through the first mold 1 of the seventh layer, the microorganisms in the second mold 2 of the sixth layer can be supplied with nutrients from both below and above, so that microorganisms can be cultured in the second mold 2 of the sixth layer.

[0069] (b) Since the culture conditions for the layered structure of layers 1-5 and the layered structure of layers 5-7 are different, two different culture conditions can be implemented. Therefore, the efficiency of the culture condition selection process can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0070] (c) In layers 2 to 4, since culture section A has a three-layer structure, the culture conditions differ in each layer. For example, the concentration of supplied environmental components is highest in layer 2 and lowest in layer 4. Similarly, the concentration of supplied nutrients is highest in layer 4 and lowest in layer 2. Therefore, the efficiency of selecting culture conditions can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0071] (d) Microorganisms can be cultured simply by circulating a liquid or gas containing environmental components in the first layer and a liquid or gas containing nutrients in the seventh layer, thus simplifying the cultivation of microorganisms. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0072] (e) The first formwork 1 and the second formwork 2 can be assembled simply by connecting them to each other, thus improving the productivity of the device.

[0073] (f) The first mold frame 1 and the second mold frame 2 can be easily removed by releasing the connection, and a different first mold frame 1 or second mold frame 2 can be newly connected in their place. In other words, each layer can be easily replaced. Therefore, the culture conditions can be easily changed, the efficiency of the culture condition selection process can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved. For example, the fifth layer and / or the seventh layer, which are nutrient supply units, can be replaced with another first mold frame 1 or second mold frame 2, which are nutrient supply units, or with a first mold frame 1 or second mold frame 2, which are environmental component supply units. Also, the first layer, which is an environmental component supply unit, can be replaced with another first mold frame 1 or second mold frame 2, which are environmental component supply units, or with a first mold frame 1 or second mold frame 2, which are nutrient supply units.

[0074] (g) By increasing the number of layers, the number of three-layered structures having nutrient supply sections B and / or environmental component supply sections C on both sides of the culture section A can be increased. Furthermore, the culture conditions can be made different for each of these three-layered structures. Therefore, the efficiency of the culture condition selection process can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0075] (h) At least one of the type and concentration of the nutrient-containing liquid or nutrient-containing gas circulating in the fifth and / or seventh layer can be changed. Also, at least one of the type and concentration of the environmental component-containing liquid or environmental component-containing gas circulating in the first layer can be changed. Therefore, a variety of culture conditions can be easily realized, and the process of selecting culture conditions suitable for microorganisms can be easily performed.

[0076] (i) The temperature sensor 41 and / or pH sensor 42 can detect and monitor the culture state of microorganisms in layers 2 to 4 and layer 6. Therefore, the culture state in each layer can be determined quickly and accurately.

[0077] (j) Based on the monitoring results, at least one of the type and concentration of the nutrient-containing liquid or nutrient-containing gas flowing through the fifth and / or seventh layer can be changed, and at least one of the type and concentration of the environmental component-containing liquid or environmental component-containing gas flowing through the first layer can be changed. Therefore, suitable culture conditions for microorganisms can be easily achieved even during the culture process.

[0078] (k) By applying vibrations to the microorganisms in layers 2 to 4 and 6 using the ultrasonic oscillator 43, the culture can be activated. Therefore, the culture efficiency can be improved.

[0079] [Another embodiment] (1) The first formwork 1 and the second formwork 2 are not limited to annular shapes, and may have external shapes such as triangles, quadrilaterals, other polygons, or ellipses in plan view.

[0080] (2) The connection between the first formwork bodies 1 and between the first formwork body 1 and the second formwork body 2 is not limited to a screw mechanism using internal and external threads, but can be carried out by, for example, a sliding fitting mechanism, a recessed fitting mechanism, or an external connecting member.

[0081] (3) Figures 36 to 38 show another second mold 2A. In this second mold 2A, the second internal space 21 is composed of a large number of through holes 210. Figure 36 is a perspective view of the second mold 2A. Figure 37 is a view taken along arrow XXXVII in Figure 36. Figure 38 is a cross-sectional view taken along line XXXVIII-XXXVIII in Figure 37. The second mold 2A has an annular outer fitting portion 222 with internal threads 221 at the bottom and an inner fitting portion 224A with external threads 223 at the top. The second internal space 21 has an internal space 211 surrounded by the outer fitting portion 222 and the remaining internal space, the "remaining internal space" consisting of a large number of through holes 210. In this second mold 2A, all of the through holes 210 are filled with a culture medium containing microorganisms.

[0082] (4) The second mold body 2A may include a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43, as shown in Figure 39. These are provided in each of the through-holes 210. The temperature sensor 41 and the pH sensor 42 are located inside the second mold body 2A to detect the temperature and pH of the microbial culture medium filled in the through-holes 210 and are connected to an external device (not shown). The external device is capable of monitoring the temperature and pH of the microbial culture medium via both sensors 41 and 42. The ultrasonic oscillator 43 is located inside the second mold body 2A to impart vibration to the microbial culture medium filled in the through-holes 210.

[0083] (5) The internal space 212 (Figure 18) of the second mold body 2 that constitutes the culture section A may be a space arbitrarily partitioned in the horizontal direction, or a space arbitrarily partitioned in the vertical direction.

[0084] (6) When the first mold body 1 is used as a culture section A with the inlet passage 13 and outlet passage 14 closed, the first internal space 11 may be a space arbitrarily partitioned in the lateral direction, or a space arbitrarily partitioned in the vertical direction.

[0085] (7) The opening and closing mechanisms for the inlet passage 13 and outlet passage 14 are not limited to a mechanism using a plug 17, but may also be a mechanism using an opening / closing cover, a mechanism using a check valve, etc.

[0086] [Examples] A microbial culture apparatus 100E configured using the microbial culture kit of the fifth embodiment was used. However, as shown in Figure 40, the first mold 1 was used as the fifth layer instead of the second mold 2.

[0087] (Composition of each part) • Nutrient supply section (7th layer) ·Substrate A solution • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.32g / 100mL Pure water 100mL

[0088] • Nutrient supply section (5th layer) ·Substrate B solution • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.032g / 100mL Pure water 100mL

[0089] ·Environmental component supply department (1st layer) • Soil extract • Prepared by mixing 5g of soil with 15g of pure water.

[0090] • Culture section (Layers 2-4 and Layer 6) • Agar powder (manufactured by Nakalai Tesque Co., Ltd.) 1.5g / 100mL Pure water 95mL The above agar aqueous solution was autoclaved (121°C / 20 minutes), and when it reached approximately 60°C, 5 mL of soil extract dilution was added and stirred before filling the internal space of the jacket, which served as the culture section. The soil extract dilution was prepared by adding 15 mL of pure water to 5 g of soil, stirring, letting it stand for 1 hour, and then sequentially diluting the supernatant 10,000 times. • Membrane filters VCWP (Merck Millipore K.K., 0.1 μm) were placed between each section.

[0091] (Culture work) Substrate A solution was continuously supplied to the nutrient supply section (layer 7), substrate B solution to the nutrient supply section (layer 5), and soil extract to the environmental component supply section (layer 1) for one week during cultivation.

[0092] (Analysis work) After culturing, the colonies generated in the culture area were collected and genetic analysis was performed at Techno Suruga Labs Co., Ltd. Homology analysis was conducted on approximately 600 base pairs in the V1-V4 region of 16S rDNA, a simplified molecular phylogenetic tree was constructed, and the species was identified. The homology rate indicates the degree of sequence agreement; a homology rate lower than 98% was considered a new species. • DNA extraction using achromopeptidase (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) • PCR amplification using PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) • Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) • Base sequencing: ChromasPro1.7 (Technelysium) • Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.) International DNA sequencing database Search date: March 15, 2018

[0093] (Comparative example) Colonies were generated using the agar plate surface smear method, and similar genetic analysis was performed.

[0094] (Analysis results) Figure 41 shows the results of the gene analysis. In Figure 41, A represents 100-98% known species, B represents 98-94% new species, C represents 94-91% new genera, and D represents less than 91% new orders. According to this example, approximately 40% of the acquired microorganisms were equivalent to new species, and new microorganisms at the new order and new genus levels were also acquired. Therefore, it was confirmed that this method is very effective in acquiring microorganisms that are difficult to culture. [Industrial applicability]

[0095] The microbial culture kit of the present invention has great industrial value because it allows for the construction of a microbial culture apparatus with a simple configuration. [Explanation of symbols]

[0096] 1. First formwork 11 1st interior space 12. Main body of the first frame 121 Internal thread 123 External thread 13 Inflow channel 14 Outflow channel 15, 16 pipes 2. Second formwork 21 Second interior space 22. Main body of the second frame 221 Internal thread 223 External thread 7 Lid 8 bases

Claims

1. It comprises three frame structures: an upper, middle, and lower layer, each having an internal space. The three frames described above constitute a three-layer laminated structure that is stacked and connected to one another. The layers are separated by filters that allow for the flow of nutrients / environmental components but prevent the flow of microorganisms. The upper frame allows for the circulation of a nutrient-containing liquid or nutrient-containing gas within its internal space. The intermediate frame is capable of holding a microorganism-containing culture medium in its internal space. The lower frame allows for the circulation of a liquid or gas containing environmental components within its internal space. Each of the three frames has a first fitting portion and a second fitting portion. The first fitting portion and the second fitting portion are mutually fittingable. The three frames mentioned above are either all first-type frames, or a first-type frame and a second-type frame. The first formwork comprises a first frame body surrounding a first internal space, and the first frame body is provided with an inlet passage for introducing fluid into the first internal space and an outlet passage for releasing fluid from the first internal space, both of which are openable and closable. The second formwork comprises a second frame body that surrounds the second internal space, The upper and lower frames are the first formwork, The intermediate frame is either the first formwork or the second formwork. The first mold body arranged in the upper layer is configured such that, when the inlet and outlet passages are open, the nutrient-containing liquid or nutrient-containing gas can be circulated through the first internal space, and when the inlet and outlet passages are closed, the nutrient-containing material can be held in the first internal space. The first mold body arranged in the lower layer is configured such that when the inlet and outlet passages are open, the liquid or gas containing environmental components can be circulated through the first internal space, and when the inlet and outlet passages are closed, the material containing environmental components can be held in the first internal space. It further comprises a base and a lid, The base supports the lower frame so as to close the first internal space of the lower first formwork, The cover closes the upper frame so as to cover the first internal space of the upper first formwork. Microbial culture kit.

2. The first frame body has an annular shape, The second frame body has an annular shape, The first fitting portion is an annular outer fitting portion having an internal thread, The second fitting portion is an annular inner fitting portion having an external thread, The outer fitting portion of the first frame body can be fitted onto the inner fitting portion of the first frame body and the inner fitting portion of the second frame body by the internal threads being screwed onto the external threads. The inner fitting portion of the first frame body can be fitted into the outer fitting portion of the first frame body and the outer fitting portion of the second frame body by the external screw being screwed into the internal screw, and the outer fitting portion of the second frame body can be fitted into the inner fitting portion of the first frame body and the inner fitting portion of the second frame body by the internal screw being screwed into the external screw, The inner fitting portion of the second frame body can be fitted into the outer fitting portion of the first frame body and the outer fitting portion of the second frame body by the external screw being screwed into the internal screw. The base has an annular inner fitting portion with an external thread at its upper part, and the inner fitting portion is capable of being fitted into the outer fitting portion of the first formwork body arranged in the lower layer. The lid has an annular outer fitting portion with internal threads at its lower part, and the outer fitting portion is externally fitted onto the internal fitting portion of the first mold body arranged in the upper layer. A microbial culture kit according to claim 1.

3. The first formwork has pipes extending to the outside from the inlet and / or outlet. A microbial culture kit according to claim 1.

4. The first fitting portion and the second fitting portion are, (i) A screwing mechanism using internal and external threads, (ii) Slide fitting mechanism, (iii) uneven fitting mechanism; Constituting at least one of the following: A microbial culture kit according to claim 1.

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