High-pressure environment single colony multi-stage coating separation device and control method

Abstract

The present invention discloses a high-pressure environment single colony multi-stage coating and separation device, comprising an incubator and a liquid injection port installed at the top of the inner wall of the incubator. The interior of the incubator is stacked with at least two culture dishes spaced apart along its own axial direction. An avoidance hole is provided in the center of the culture dish, and the avoidance hole radially expands toward the edge of the culture dish to form a fan-shaped separation port. Adjacent separation ports are staggered from each other in the vertical direction. A stirring rod is provided through the axis of the incubator, and there is no motion interference between the stirring rod and the avoidance hole. A coating unit is sleeved on the lower end of the stirring rod, and the projection of the coating unit on the horizontal plane is located inside the projection of the separation port. The beneficial effect of the present invention is that the stirring rod is used to drive the coating unit to rotate. When the coating unit moves to the separation port area of ​​the current layer, the coating unit will lose the support provided by the culture dish and automatically fall to the culture dish of the next layer under the action of gravity.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial cultivation, and in particular to a high-pressure environment single colony multi-stage coating separation device and a control method. Background Art

[0002] For the isolation of microorganisms in special marine habitats, existing technologies are mainly carried out under normal pressure, and single colonies are rarely isolated and cultured under high pressure. Even when single colonies are isolated under high pressure, the separation efficiency is low and the operation process is relatively cumbersome. As a result, the number of culturable microorganisms is less than 1% of the deep-sea environment, which brings certain difficulties to our correct understanding and utilization of marine resources. Rapid, large-scale and effective isolation technologies and methods for deep-sea microorganisms are important means to restrict the efficient utilization of deep-sea microbial resources.

[0003] CN114456909A discloses a deep-sea microbial separation and cultivation device. An internal thread is provided on the inner wall surface of the base, and a sampling pipe is used to put a culture liquid sample into the internal thread of the separation and cultivation device. The sample slides downward along the internal thread under the action of gravity. During the movement, the microbial liquid it carries will gradually decrease, realizing the separation of microorganisms. It can realize the automatic streaking separation of the microbial liquid enriched by environmental sampling under the in-situ environmental conditions of the ocean, effectively improving the efficiency of microbial solid separation and cultivation, and providing an important basic means for the separation and cultivation of efficient special marine microorganisms.

[0004] However, the above-mentioned device completely relies on gravity to separate samples and can only perform streak separation once, which greatly reduces the probability of isolating a single colony. Summary of the Invention

[0005] In response to the above problems, the present invention proposes a high-pressure environment single colony multi-stage coating separation device and control method, which mainly solves the problems that existing microbial high-pressure separation devices are not suitable for uniformly coating samples over a large area and single colonies are difficult to separate.

[0006] In order to solve the above technical problems, the first aspect of the present invention proposes a high-pressure environment single colony multi-stage coating and separation device, including a culture vessel and an injection port installed on the top of the inner wall of the culture vessel. The interior of the culture vessel is stacked with at least two culture dishes along its own axial direction. An avoidance hole is provided in the center of the culture dish. The avoidance hole radially expands toward the edge of the culture dish to form a fan-shaped separation port. Adjacent separation ports are staggered from each other in the vertical direction. A stirring rod is provided passing through the axis of the culture vessel, and there is no motion interference between the stirring rod and the avoidance hole. The lower end of the stirring rod is sleeved with a coating unit, and the projection of the coating unit on the horizontal plane is located inside the projection of the separation port.

[0007] In some embodiments, a baffle is further included, and the baffle is installed on the left side of the separation port.

[0008] In some embodiments, the coating unit includes a sleeve slidably connected to the stirring rod, a connecting rod fixed to the outer wall of the sleeve, and a scratching plate is installed at the other end of the connecting rod.

[0009] In some embodiments, the cross-section of the stirring rod is rectangular, the sleeve is loosely fitted with the stirring rod, the avoidance hole is circular, and the diameter of the avoidance hole is greater than or equal to the diagonal length of the sleeve.

[0010] In some embodiments, a detachable cover is provided on the top of the incubator, and supporting feet are provided on the bottom of the incubator.

[0011] In some embodiments, the culture dish is detachable.

[0012] In some embodiments, a first buckle is provided at the edge of the culture dish, and a second buckle that cooperates with the first buckle is provided on the inner wall of the culture vessel.

[0013] In some embodiments, the angle between two adjacent separation openings is less than or equal to the angle at which the separation openings are opened.

[0014] In some embodiments, the distance between two adjacent culture dishes is greater than the height of the coating unit.

[0015] The second aspect of the present invention proposes a control method for the above-mentioned high-pressure environment single colony multi-stage coating and separation device, wherein the culture dish is loaded with a solid culture medium, the height of the solid culture medium is level with the upper end surface of the culture dish, the coating unit is placed on the surface of the topmost culture dish, and the stirring rod is driven according to a preset rotation speed and a preset time.

[0016] The beneficial effects of the present invention are as follows: by vertically arranging multiple levels of culture dishes in the incubator, a separation port is provided in each culture dish, and a stirring rod is used to drive the coating unit to rotate. When the coating unit moves to the separation port area of ​​the current layer, the coating unit will lose the support provided by the culture dish, and under the action of gravity, the coating unit will automatically fall to the culture dish of the next layer, and continue to be coated by the stirring rod. During the rotation of the stirring rod, since an avoidance hole is provided in the center of the culture dish, the stirring rod will not drive the culture dish to rotate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic structural diagram of the high-pressure environment single bacterial colony multi-stage coating and separation device disclosed in Example 1 of the present invention;

[0018] Figure 2This is a schematic diagram of the three-dimensional structure of the culture dish disclosed in Example 1 of the present invention;

[0019] Figure 3 This is a schematic diagram of the top view of the culture dish disclosed in Example 1 of the present invention;

[0020] Figure 4 This is a schematic diagram of the three-dimensional structure of the coating unit disclosed in the first embodiment of the present invention;

[0021] Figure 5 This is a schematic diagram of the cross-sectional structure of the coating unit disclosed in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the present invention more clear and distinct, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. It should also be noted that, for ease of description, the accompanying drawings only illustrate portions relevant to the present invention, not all of the present invention.

[0023] Example 1

[0024] This embodiment proposes a high-pressure environment single colony multi-stage coating separation device, which improves the cultivability of marine microorganisms by reshaping their in-situ environment for automatic multi-stage coating separation and cultivation of microorganisms, providing an important basic means for the development and utilization of high-pressure environment microbial resources.

[0025] like Figure 1-5 As shown, the device mainly includes a culture vessel 2 and a liquid injection port 55 installed on the top of the inner wall of the culture vessel 2. At least two culture dishes 24 are stacked along the axial direction of the culture vessel 2. A avoidance hole 242 is provided in the center of the culture dish 24. The avoidance hole 242 radially expands toward the edge of the culture dish 24 to form a fan-shaped separation port 241. Adjacent separation ports 241 are staggered from each other in the vertical direction. A stirring rod 232 is provided through the axis of the culture vessel 2, and there is no motion interference between the stirring rod 232 and the avoidance hole 242. The lower end of the stirring rod 232 is sleeved with a coating unit, and the projection of the coating unit on the horizontal plane is located inside the projection of the separation port 241.

[0026] In this embodiment, a multi-level culture dish 24 is vertically arranged in the culture vessel 2, a separation port 241 is provided in each culture dish 24, and a stirring rod 232 is used to drive the coating unit to rotate on the culture medium surface of the culture dish 24. When the coating unit moves to the separation port 241 area of ​​the current layer of culture dish 24, the coating unit will lose the support provided by the culture dish 24, and under the action of gravity, the coating unit will automatically fall to the culture medium surface of the next layer of culture dish 24, and continue to be driven by the stirring rod 232 for coating. During the rotation of the stirring rod 232, since an avoidance hole 242 is provided in the center of the culture dish 24, the stirring rod 232 will not drive the culture dish 24 to rotate.

[0027] In a preferred embodiment, a baffle 243 is also included, mounted to the left of the separation port 241. In this embodiment, the baffle 243 primarily blocks bacterial solution dripping from the injection port 55, preventing it from spreading into the next layer. This allows the coating unit to smoothly land on the surface of the culture medium in the next culture dish 24, where it can continue coating via the stirring rod 232, ensuring a successful coating. Specifically, the coating unit should be located to the left of the baffle 243, with the injection port 55 vertically centered on the left side of the baffle 243. This allows the bacterial solution to drip onto the culture medium in the culture dish 24, allowing the coating unit to immediately begin coating. As can be expected, bacterial solution dripping from the injection port 55 will converge on the left side of the baffle 243. Due to the blocking effect of the baffle 243, the bacterial solution can only pass through the coating unit, circling the culture dish 24, before passing through the separation port 241 of the current layer and into the next layer.

[0028] The coating unit can be any mechanical device that can be driven to rotate by the stirring rod 232 and can slide along the axial direction of the stirring rod 232. In one example, the coating unit includes a sleeve 253 that is slidably connected to the stirring rod 232, a connecting rod 252 fixed to the outer wall of the sleeve 253, and a scratching plate 251 installed at the other end of the connecting rod 252. The cross-section of the stirring rod 232 is rectangular, the sleeve 253 and the stirring rod 232 are clearance-matched, the avoidance hole 242 is circular, and the diameter of the avoidance hole 242 is greater than or equal to the diagonal length of the sleeve 253, so that there is no motion interference between the stirring rod 232 and the avoidance hole 242. In the above scheme, the stirring rod 232 and the sleeve 253 are set to be square, so that the torque of the stirring rod 232 can be transmitted to the sleeve 253, and then the torque is transmitted to the connecting rod 252 and the paddle 251 through the sleeve 253. At the same time, the square fit can also enable the sleeve 253 to slide along the outer wall of the stirring rod 232. Therefore, under the action of gravity, the sleeve 253 can carry the connecting rod 252 and the paddle 251 vertically down, serving as the mechanical basis for the coating unit to automatically fall to the next layer of culture dish 24.

[0029] Optionally, a detachable cover 23 is provided on the top of the incubator 2, and a support leg 21 is provided on the bottom of the incubator 2. In this embodiment, the cover 23 and the incubator 2 can be connected by a snap fastener, and the liquid injection port 55, temperature sensor 31, pressure sensor 47 and stirring rod 232 can be fixed to the cover 23. The support leg 21 is used to raise the placement height of the incubator 2 to leave space for the bottom end of the stirring rod 232.

[0030] Furthermore, the culture dish 24 is of a detachable design, which facilitates the removal of the culture dish 24 from the inner wall of the incubator 2. In one example, the edge of the culture dish 24 is provided with a first buckle, and the inner wall of the incubator 2 is provided with a second buckle that cooperates with the first buckle.

[0031] The culture dish 24 is filled with a solid culture medium with different nutrient ratios for the culture matrix. The present invention involves three culture dishes 24, namely the first culture dish, the second culture dish, and the third culture dish. The number of culture dishes 24 can be determined according to the experimental requirements and is not limited here. The angle between two adjacent separation ports 241 is less than or equal to the angle at which the separation ports 241 are expanded, that is, in the vertical projection perspective, the separation ports 241 of the culture dishes 24 of the next layer should be staggered with the separation ports 241 of the culture dishes 24 of the previous layer, and the staggered angle is less than or equal to the angle at which the separation ports 241 are expanded, so as to avoid a reduction in the coating area of ​​the culture dishes 24 of the next layer.

[0032] In order to enable the coating unit to rotate smoothly, the distance between two adjacent culture dishes 24 should be greater than the height of the coating unit, and the height of the solid culture medium should be level with the culture dishes 24 .

[0033] The following is a further description of the auxiliary device of this device. Figure 1 , also includes a pressure control unit 4, which is mainly used to inject gas into the incubator 2 to increase the pressure, so that the pressure environment in the incubator 2 is consistent with the pressure value of the microorganisms in situ in the ocean, and at the same time monitor the pressure changes in the incubator 2. The pressure control unit 4 includes an air compressor 41, a booster pump 42, a gas storage tank 43, a pressure regulating valve 44 and an air supply pipe 46 connected in sequence. A valve 45 is provided in the middle of the air supply pipe 46, and the end of the air supply pipe 46 is directly connected to the cover 23 or the side of the incubator 2. It also includes an enrichment system 5, which includes a microorganism enrichment kettle 51, a microinjection pump 52 and a liquid outlet pipe 54 connected in sequence. In order to ensure the pressure stability of the entire device, a valve 53 is provided in the middle of the liquid outlet pipe 54, and the end of the liquid outlet pipe 54 enters the interior of the incubator 2 from the cover 23 and forms the above-mentioned liquid injection port 55. The above-mentioned temperature sensor 31, pressure sensor 47, pressure control unit 4, and stirring rod 232 are all acquired and operated by the control unit 1.

[0034] Example 2

[0035] This embodiment provides a control method for the aforementioned high-pressure environment single colony multi-stage coating and separation device. A solid culture medium is loaded into a culture dish 24, the solid culture medium being flush with the upper end surface of the culture dish 24. A coating unit is placed on the surface of the topmost culture dish 24, and a stirring rod 232 is driven according to a preset rotation speed and for a preset time. In this embodiment, referring to the arrangement of baffle 243 described in Example 1, the motor is intended to drive stirring rod 232 to rotate counterclockwise.

[0036] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the essence of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A high-pressure environment single colony multi-stage coating separation device, comprising an incubator and a liquid injection port installed on the top of the inner wall of the incubator, characterized in that: At least two culture dishes are stacked at intervals along the axial direction of the culture vessel, an avoidance hole is provided in the center of the culture dish, the avoidance hole radially expands toward the edge of the culture dish to form a fan-shaped separation opening, adjacent separation openings are staggered from each other in the vertical direction, a stirring rod is provided passing through the axis of the culture vessel, and the stirring rod has no motion interference with the avoidance hole, a coating unit is sleeved on the lower end of the stirring rod, and the projection of the coating unit on the horizontal plane is located inside the projection of the separation opening; It also includes a baffle, which is installed on the left side of the separation port; The coating unit includes a sleeve slidably connected to the stirring rod, a connecting rod fixed to the outer wall of the sleeve, and a scratching plate installed at the other end of the connecting rod; The distance between two adjacent culture dishes is greater than the height of the coating unit.

2. The high-pressure environment single colony multi-stage coating and separation device according to claim 1, characterized in that: The cross section of the stirring rod is rectangular, the sleeve is loosely fitted with the stirring rod, the avoidance hole is circular, and the diameter of the avoidance hole is greater than or equal to the diagonal length of the sleeve.

3. The high-pressure environment single colony multi-stage coating and separation device according to claim 1, characterized in that: The top of the incubator is provided with a detachable cover, and the bottom of the incubator is provided with supporting feet.

4. The high-pressure environment single colony multi-stage coating separation device according to claim 1, characterized in that: The culture dish is detachable.

5. The high-pressure environment single colony multi-stage coating and separation device according to claim 4, characterized in that: The edge of the culture dish is provided with a first buckle, and the inner wall of the culture container is provided with a second buckle that cooperates with the first buckle.

6. The high-pressure environment single colony multi-stage coating and separation device according to claim 1, characterized in that: The included angle between two adjacent separation openings is less than or equal to the expanded angle of the separation openings.

7. A control method for the high-pressure environment single colony multi-stage coating separation device according to any one of claims 1 to 6, characterized in that: The culture dish is loaded with solid culture medium, the height of the solid culture medium is flush with the upper end surface of the culture dish, the coating unit is placed on the surface of the uppermost culture dish, and the stirring rod is driven according to a preset rotation speed and a preset time.

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