Microorganism monitoring device, microorganism monitoring system and method

Abstract

The invention provides a microorganism monitoring device, a microorganism monitoring system and a method, and the microorganism monitoring device comprises a microcavity array chip which is provided with a plurality of culture cavities; the gas-permeable membrane is arranged at the bottom of the microcavity array chip, covers the bottom ends of all the culture cavities and is used for allowing gas in the culture cavities to enter and exit and preventing the liquid to be detected from flowing out of the bottom ends of the culture cavities; the first frame is connected with the top of the microcavity array chip, and a sample inlet and a containing space communicated between the sample inlet and the culture cavity are formed in the first frame; and the flexible film covers the top of the accommodating space. On the basis of improving the detection accuracy and reliability, the device is simple in structure, low in cost, simple to operate and high in applicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial monitoring, and in particular relates to a microbial monitoring device, a microbial monitoring system and a method. Background Art

[0002] At present, in order to achieve effective detection of bacteria or fungi in samples, microfluidics technology was born and plays an important role in microbial detection. It can miniaturize the detection process, manipulate samples through microchannels, quickly separate and enrich target microorganisms in pathogen detection, and improve detection sensitivity. In the existing technology, microfluidics chips used for microbial detection have a large number of microvalves and precision structures, which are complex in structure, high in cost, and difficult to mass produce. When the above-mentioned equipment is used for microbial detection, the external control system is complicated, and the professional knowledge and practical experience of the operator are required to be high. The interpretation of the results relies on expensive and environmentally demanding equipment. The operation is complicated and the efficiency is low. It is difficult to apply in resource-limited scenarios such as primary medical care and remote areas, and its applicability is low. Summary of the Invention

[0003] The present invention addresses the technical problems in the prior art such as the complex structure and high cost of microbial monitoring devices, as well as the complex operation and low applicability of microbial monitoring, and provides a microbial monitoring device, a microbial monitoring system and a method.

[0004] In view of the above technical problems, an embodiment of the present invention provides a microbial monitoring device, comprising: A microcavity array chip is provided with multiple culture chambers; a gas-permeable membrane, disposed at the bottom of the microcavity array chip and covering the bottom ends of all the culture cavities, for allowing gas in the culture cavities to enter and exit, and preventing the test liquid from flowing out through the bottom ends of the culture cavities; a first frame connected to the top of the microcavity array chip, the first frame being provided with an injection port and a receiving space communicating between the injection port and the culture chamber; A flexible film covers the top of the accommodating space.

[0005] The present invention also provides a microbial monitoring system, comprising a controller, an extrusion piece, and at least one of the microbial monitoring devices; the controller is communicatively connected to the microbial monitoring device; the extrusion piece is provided with an extrusion portion adapted to the accommodating space; the extrusion piece is used to extrude a flexible membrane through the extrusion portion and push the test liquid in the accommodating space to be evenly filled into each of the culture chambers after the test liquid enters the accommodating space through the sampling port.

[0006] The present invention also provides a microbial monitoring method, which is applied to the microbial monitoring system. The microbial monitoring method includes: receiving a sample loading signal and controlling the test liquid to enter the containing space from the sample inlet of the first frame; Controlling the extrusion part of the extrusion member to squeeze the flexible membrane and push the test liquid in the accommodation space of the microcavity array chip to fill each of the culture chambers, so that the flexible membrane is attached to the top of the culture chamber; The microbial monitoring device with the first frame removed is placed under preset culture conditions to culture the test liquid in the culture chamber, and the culture process is monitored to obtain microbial monitoring results.

[0007] The microbial monitoring device provided by the present invention includes: a microcavity array chip, which is provided with multiple culture chambers; a breathable membrane, which is arranged at the bottom of the microcavity array chip and covers the bottom ends of all culture chambers, is used to allow gas in the culture chamber to enter and exit, and prevent the test liquid from flowing out through the bottom ends of the culture chambers; a first frame, which is connected to the top of the microcavity array chip, and is provided with an injection port and a storage space connected between the injection port and the culture chamber; and a flexible membrane, which covers the top of the storage space.

[0008] The microbial monitoring device provided by the present invention can significantly shorten the incubation time required for signal detection by providing multiple culture chambers on the microcavity array chip, thereby enabling rapid detection and analysis. When microorganisms are present in the test liquid, the migration of microorganisms between different culture chambers can be effectively prevented, thus avoiding resource competition between microorganisms. This ensures that the test liquid in each culture chamber can be cultured in a relatively independent and stable environment, which is conducive to the enrichment of the microorganisms themselves and their metabolites, provides good conditions for the growth and color development of microorganisms, and significantly improves the accuracy and reliability of detection. At the same time, the microbial monitoring device of the present invention can quickly and evenly spread and disperse the test liquid into multiple culture chambers, improving the distribution efficiency of the test liquid while ensuring the consistency and stability of the test liquid. In addition, the gas permeable membrane is used to allow gas to enter and exit the culture chamber and prevent the test liquid from flowing out through the bottom of the culture chamber. This not only provides a stable gas exchange channel but also prevents the test liquid from being lost due to leakage of the culture chamber. When microorganisms are present in the test liquid, the normal growth of the microorganisms in the test liquid and the accuracy of detection can be ensured. The present invention has a simple structure while improving detection accuracy and reliability. In addition, the microfluidic device with a microcavity array used for sample discretization only requires a small amount of test liquid, which not only reduces material and detection costs but also reduces the impact on the environment. It is also simple to operate and has high applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention will be further described below with reference to the accompanying drawings and examples.

[0010] Figure 1 It is a schematic diagram of the assembly structure of a microbial monitoring device provided by one embodiment of the present invention.

[0011] Figure 2 It is a schematic diagram of the explosion structure of a microorganism monitoring device provided by another embodiment of the present invention.

[0012] Figure 3 It is a perspective structural diagram of a microorganism monitoring device provided by one embodiment of the present invention.

[0013] Figure 4 It is a perspective structural diagram of a microorganism monitoring device provided by another embodiment of the present invention.

[0014] Figure 5 This is a flow chart of a microbial monitoring method provided by one embodiment of the present invention.

[0015] Figure 6 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the first embodiment of the present invention.

[0016] Figure 7 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the second embodiment of the present invention.

[0017] Figure 8 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the third embodiment of the present invention.

[0018] Figure 9 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the fourth embodiment of the present invention.

[0019] Figure 10 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the fifth embodiment of the present invention.

[0020] Figure 11 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the sixth embodiment of the present invention.

[0021] Figure 12 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the seventh embodiment of the present invention.

[0022] Figure 13 This is an image of the test liquid in the culture chamber collected at intervals of 0.25 hours according to the eighth embodiment of the present invention.

[0023] The reference numerals in the specification are as follows: 10. Microcavity array chip; 11. Culture chamber; 20. Breathable membrane; 30. First frame; 31. Inlet; 32. Accommodation space; 40. Flexible membrane; 50. Extrusion piece; 51. Extrusion part; 60. Second frame; 61. Breathable channel. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] like Figures 1 to 4 As shown, an embodiment of the present invention provides a microbial monitoring device, comprising: The microcavity array chip 10 is provided with a plurality of culture chambers 11 ; wherein the microcavity array chip 10 can be made of materials such as polymethyl methacrylate, polycarbonate, cycloolefin copolymer or cycloolefin polymer.

[0026] A breathable membrane 20 is disposed at the bottom of the microcavity array chip 10 and covers the bottom ends of all the culture cavities 11. It allows gas to enter and exit the culture cavities 11 and prevents the test liquid from escaping through the bottom ends of the culture cavities 11. The breathable membrane 20 can be a waterproof or hydrophobic breathable membrane, or other air-permeable but water-impermeable thin film. The test liquid is a product of a drop or a small amount of sample liquid sampled from a large volume and mixed with an indicator. It is understood that the sample liquid (or test liquid) of the present invention may or may not contain microorganisms. The microbial monitoring results determined by the microbial monitoring method and system of the present invention can indicate the presence of microorganisms in the test liquid. Specifically, the waterproof breathable membrane 20 can be made of materials such as polytetrafluoroethylene, thermoplastic polyurethane rubber, or polyurethane. The waterproof breathable membrane 20 allows gaseous water molecules (water vapor) to pass through, but blocks liquid water from penetrating, achieving a "breathable but water-impermeable" effect. The hydrophobic breathable membrane 20 may be made of polyvinylidene fluoride, polypropylene or a hydrophobically modified material. The hydrophobic breathable membrane 20 can repel liquid water through the hydrophobicity of the material surface while maintaining gas permeability.

[0027] The first frame 30 is connected to the top of the microcavity array chip 10. The first frame 30 is provided with an injection port 31 and a receiving space 32 communicating between the injection port 31 and the culture chamber 11. The first frame 30 can be made of materials such as polymethyl methacrylate.

[0028] The flexible film 40 covers the top of the accommodating space 32 .

[0029] It is understood that the culture chamber 11 is a through-hole structure, and its bottom end is covered with the breathable membrane 20, which is used to allow gas in the culture chamber 11 to enter and exit, and prevent the test liquid from flowing out through the bottom end of the culture chamber 11. This not only provides a stable gas exchange channel, but also prevents the test liquid from being lost due to leakage from the culture chamber 11. When microorganisms are present in the test liquid, the normal growth of the microorganisms in the test liquid and the accuracy of the detection can be ensured. The breathable membrane 20 can be adhered to the bottom of the microcavity array chip 10 using double-sided tape to prevent the test liquid from flowing out through the bottom end of the culture chamber 11. The flexible membrane 40 is made of a non-permeable material to prevent the test liquid from leaking out of the flexible membrane 40 when the flexible membrane 40 squeezes and pushes the test liquid in the holding space 32. The flexible membrane 40 can also maintain the cleanliness and sterility of the holding space 32. The number of culture chambers 11 can be set according to actual conditions. The shape of the culture chamber 11 can be set according to actual conditions. For example, the shape, size and spacing of the culture chamber 11 can be adjusted according to the volume requirements of different samples to be tested, thereby improving the adaptability to various volumes of test liquids, so that the microbial monitoring device can be widely used in a variety of microbial culture scenarios. The first frame 30 and the microcavity array chip 10 match each other, so that the microcavity array chip 10 can be placed in the first frame 30, and the accommodating space 32 is formed between the first frame 30 and the microcavity array chip 10. The first frame 30 and the microcavity array chip 10 can be bonded to each other by double-sided tape to form a seal between the first frame 30 and the microcavity array chip 10 to prevent the test liquid in the accommodating space 32 from leaking in the gap that may be formed between the first frame 30 and the microcavity array chip 10.

[0030] In one embodiment, the plurality of culture chambers 11 are distributed in an array and have equal volumes. The number of the culture chambers 11 can be 100, that is, distributed in a 10 by 10 array. The volume of the culture chamber 11 can be 10 μL. In the microcavity array chip 10 of the present invention, a series of culture chambers 11 with highly consistent sizes and shapes are regularly arranged to form a microcavity array. When the flexible membrane 40 is squeezed or the microcavity array chip 10 is vibrated, a uniform and continuous force can be applied to the test liquid, thereby accurately distributing the sample to each independent microreaction unit (culture chamber 11). The discretized microreaction units are highly uniform in volume and shape, ensuring the consistency and stability of the detection.

[0031] In one embodiment, the culture chamber 11 may be hexagonal, circular, or square.

[0032] In one embodiment, the microcavity array chip 10 has a length of 15-30 mm and a width of 15-30 mm; the first frame 30 has a length of 25-50 mm and a width of 25-50 mm. The microcavity array chip 10 of the present invention can be flexibly designed based on actual needs. By varying the size and number of microchambers, it can adapt to different testing requirements and adjust the amount of test fluid, demonstrating excellent scalability. Furthermore, its compact size and portability allow it to be used in resource-limited environments such as on-site or grassroots laboratories, further reducing testing costs and improving testing convenience.

[0033] The thickness of the breathable membrane 20 can be set according to actual conditions. In one embodiment, the thickness of the breathable membrane 20 is 0.2 μm. The flexible membrane 40 is waterproof or hydrophobic. Under the action of an external force, such as squeezing the flexible membrane 40 on top of the flexible membrane 40 by an external component (including rolling or translational squeezing of the flexible membrane 40, or vibrating the entire microbial monitoring device by an external component), the test liquid can be evenly spread and distributed to each culture chamber 11 without leaking out of the flexible membrane 40. This improves the distribution efficiency of the test liquid, allowing the test liquid to quickly and evenly enter the culture chamber 11, providing a good sample distribution foundation for subsequent microbial detection.

[0034] In the above-described embodiment of the present invention, the multiple culture chambers 11 provided on the microcavity array chip 10 can significantly shorten the incubation time required for signal detection, thereby enabling rapid detection and analysis. When microorganisms are present in the test fluid, the migration of microorganisms between different culture chambers 11 can be effectively prevented, avoiding resource competition between microorganisms. This ensures that the test fluid in each culture chamber 11 can be cultured in a relatively independent and stable environment, facilitating the enrichment of the microorganisms themselves and their metabolites, providing favorable conditions for microbial growth and color development, and significantly improving the accuracy and reliability of detection. Furthermore, the microbial monitoring device of the present invention can quickly and evenly spread and disperse the test fluid into the multiple culture chambers 11, improving the distribution efficiency of the test fluid while ensuring the consistency and stability of the test fluid. Furthermore, the gas permeable membrane 20 allows gas to enter and exit the culture chambers 11 and prevents the test fluid from flowing out through the bottom of the culture chambers 11. This provides a stable gas exchange channel and prevents the test fluid from being lost due to leakage from the culture chambers 11. When microorganisms are present in the test fluid, this ensures the normal growth of the microorganisms in the test fluid and the accuracy of detection. The present invention has a simple structure while improving detection accuracy and reliability. In addition, the microfluidic device with a microcavity array used for sample discretization only requires a small amount of test liquid, which not only reduces material and detection costs but also reduces the impact on the environment. It is also simple to operate and has high applicability.

[0035] In one embodiment, a flow guide device (not shown) may be provided at one end of the injection port 31 facing the storage space 32 to guide the test liquid to be evenly distributed in the storage space 32, further improving the distribution uniformity of the test liquid. It is understood that the flow guide device may be a tapered channel provided between the injection port 31 and the storage space 32, with the larger end of the tapered channel connected to the storage space 32 and the smaller end connected to the injection port 31. In this way, when the test liquid enters the tapered channel from the injection port 31, the cross-sectional area of the tapered channel from the tip to the bottom increases, and the flow rate in the channel decreases, which is beneficial to the distribution and diffusion of the test liquid after entering the storage space 32, so that the test liquid is further evenly distributed in each culture chamber 11.

[0036] like Figures 1 to 4 As shown, in one embodiment, the microbial monitoring device further includes a second frame 60; the breathable membrane 20 is clamped between the second frame 60 and the microcavity array chip 10, and the second frame 60 is provided with a breathable channel 61 arranged opposite to the culture chamber 11. It can be understood that the second frame 60 further strengthens the connection stability between the breathable membrane 20 and the microcavity array chip 10, and prevents the test liquid from flowing out through the gap that may exist between the breathable membrane 20 and the microcavity array chip 10. The second frame 60 can also form an area for accommodating the microcavity array chip 10 together with the first frame 30 to support the microcavity array chip 10, improve the stability of the microcavity array chip 10, and facilitate the operator to perform operations such as loading and pushing the test liquid.

[0037] An embodiment of the present invention also provides a microbial monitoring system, including a controller, an extrusion piece 50, and at least one of the microbial monitoring devices; the controller is communicatively connected to the microbial monitoring device; the extrusion piece 50 is provided with an extrusion portion 51 adapted to the accommodating space 32; the extrusion piece 50 is used to squeeze the flexible membrane 40 through the extrusion portion 51 and push the test liquid in the accommodating space 32 to be evenly filled into each of the culture chambers 11 after the test liquid enters the accommodating space 32 through the sampling port 31.

[0038] It can be understood that the microbial monitoring system of the present invention is used to perform the following microbial monitoring method. The various devices used in the following microbial monitoring method, such as a pipette or syringe, a robotic arm or a slide, an image monitoring device, etc., can all belong to the microbial monitoring system and will not be described in detail here. The various modules in the above-mentioned controller can be implemented in whole or in part through software, hardware, or a combination thereof. The above-mentioned modules can be embedded in or independent of the controller in hardware form, or can be stored in the controller in software form, so that the controller can call and execute the operations corresponding to the above modules.

[0039] The extrusion member 50 is configured to cooperate with the flexible membrane 40. The extrusion portion 51 of the extrusion member 50 can squeeze the flexible membrane 40 on top of the flexible membrane 40 (including rolling or translating the flexible membrane 40), thereby pushing the test liquid in the holding space to move through the flexible membrane 40, thereby quickly and evenly spreading and distributing the sample to each culture chamber 11. This improves the distribution efficiency of the test liquid, allowing the test liquid to quickly and evenly enter the culture chamber 11, providing a good sample distribution foundation for subsequent microbial testing. In this embodiment, through the cooperation of the extrusion member 50 and the flexible membrane 40, the test liquid can be quickly and evenly spread and dispersed to multiple culture chambers through the flexible membrane 40 without the use of complex microfluidic pumps and valves, thereby improving the distribution efficiency of the test liquid while ensuring the consistency and stability of the test liquid.

[0040] In some embodiments, the extrusion member 50 is a roller. When the roller is needed, the roller is placed on the top of the flexible membrane 40 and driven to roll, thereby squeezing the top of the flexible membrane 40. The flexible membrane 40 pushes the test liquid in the holding space to move, thereby quickly and evenly spreading and distributing the sample to each culture chamber 11, thereby improving the distribution efficiency of the test liquid and allowing the test liquid to enter the culture chamber 11 quickly and evenly.

[0041] like Figure 5 As shown, an embodiment of the present invention further provides a microbial monitoring method, which is applied to the microbial monitoring system. The microbial monitoring method can be directly executed by the controller of the microbial monitoring system, or can be jointly executed by the controller in the microbial monitoring system and / or the control modules corresponding to other modules.

[0042] The microbial monitoring method comprises: S100: Receive a sample loading signal and control the test liquid to enter the storage space 32 from the injection port 31 of the first frame 30. Controlling the test liquid to enter the storage space 32 from the injection port 31 can be performed by a device such as a pipette or syringe. The pipette or syringe can be controlled by a controller, or a separate control module can be provided, as long as the control module is communicatively connected to and controllable by the controller. It is understood that the test liquid is a product obtained by sampling a drop or a small amount of sample liquid from a large volume of sample liquid and mixing it with an indicator. S200: Control the extrusion portion 51 of the extrusion member 50 to squeeze the flexible membrane 40 and push the test liquid in the storage space 32 of the microcavity array chip 10 into each of the culture chambers 11, so that the flexible membrane 40 is attached to the top of the culture chamber 11. The extrusion member 50 can be controlled by a device capable of performing linear motion, such as a robotic arm or a slide block, to move on top of the flexible membrane 40, thereby causing the extrusion portion 51 of the extrusion member 50 to squeeze the flexible membrane 40. The above-mentioned robotic arms or slide rails and sliders and other devices can be controlled by a controller, or a control module can be set separately, as long as the control module is connected to the controller for communication and can be controlled by the controller.

[0043] It will be appreciated that as the extruding member 50 moves on top of the flexible membrane 40, it squeezes the flexible membrane 40 below, thereby evenly pushing the test liquid in the lower holding space 32 into each culture chamber 11 through the flexible membrane 40. After the extruding member 50 completes one movement (i.e., the extruding portion 51 moves across all areas of the flexible membrane 40), the test liquid is completely discretized into stable droplets within all culture chambers 11. Furthermore, the extruding member 50 can squeeze the lower flexible membrane 40 with a uniform force and speed, with the force being 1-5 N and the speed being 1-5 mm / s.

[0044] S300: The microbial monitoring device, with the first frame 30 removed, is placed under preset culture conditions to culture the microorganisms in the culture chamber 11 and monitor the microorganisms in the culture chamber 11 to obtain microbial monitoring results. The test fluid in the culture chamber 11 can be monitored using an image monitoring device or visually. It is understood that the sample fluid (or test fluid) in the present invention may or may not contain microorganisms, and the microbial monitoring results indicate whether microorganisms are present in the test fluid.

[0045] In the present invention, the microcavity array in the microcavity array chip 10 can discretize samples, thereby dispersing microorganisms such as bacteria or fungi into microreaction units for growth. This facilitates the enrichment of the microorganisms themselves and their metabolites, significantly shortening the incubation time required for signal detection, and thus enabling rapid detection and analysis. Furthermore, the microarray discretization technology, through physical isolation, effectively reduces the impact of interfering substances that may exist in the microchannels and reaction chambers of the microfluidic chip, significantly improving the accuracy and reliability of detection.

[0046] In the above-described embodiment of the present invention, the multiple culture chambers 11 provided on the microcavity array chip 10 can significantly shorten the incubation time required for signal detection, thereby enabling rapid detection and analysis. When microorganisms are present in the test liquid, the migration of microorganisms in the test liquid between different culture chambers 11 can be effectively prevented, thereby avoiding resource competition between microorganisms and ensuring that the test liquid in each culture chamber 11 can be cultured in a relatively independent and stable environment, which is conducive to the enrichment of the microorganisms themselves and their metabolites, providing good conditions for the growth and color development reaction of the microorganisms, and significantly improving the accuracy and reliability of the detection. At the same time, through the cooperation of the extrusion member 50 and the flexible membrane 40, the test liquid can be quickly and evenly spread and dispersed into the multiple culture chambers 11 through the flexible membrane 40 without the need for complex microfluidic pumps and valves, thereby improving the distribution efficiency of the test liquid and ensuring the consistency and stability of the test liquid. Furthermore, the gas in the culture chamber 11 is allowed to enter and exit through the gas permeable membrane 20, and the test liquid is prevented from flowing out through the bottom of the culture chamber 11. This not only provides a stable gas exchange channel, but also prevents the test liquid from being lost due to leakage from the culture chamber 11. When microorganisms are present in the test liquid, the normal growth of the microorganisms in the test liquid and the accuracy of the test can be ensured. While improving the accuracy and reliability of detection, the present invention has a simple structure. Furthermore, the microfluidic device with a microcavity array used for sample discretization only requires a small amount of test liquid, which reduces material and testing costs and minimizes environmental impact. Furthermore, the device is simple to operate and highly applicable.

[0047] In one embodiment, in step S100, before receiving the sample loading signal, the method further includes: S400: Attach the breathable membrane 20 to the bottom of the microcavity array chip 10 to seal the bottom of the culture chamber 11 through the breathable membrane 20. The breathable membrane 20 can be attached to the bottom of the microcavity array chip 10 using double-sided tape to form a seal between the microcavity array chip 10 and the breathable membrane 20, thereby preventing the test solution from leaking from any gap between the microcavity array chip 10 and the breathable membrane 20.

[0048] S500: Cover the top of the receiving space 32 of the first frame 30 with the flexible film 40. The flexible film 40 may be attached to the top of the receiving space 32 of the first frame 30 by using double-sided tape.

[0049] S600 , connecting the microcavity array chip 10 with the gas permeable membrane 20 attached thereto to the bottom of the first frame 30 , so that the injection port 31 is connected to the culture chamber 11 through the accommodating space 32 .

[0050] It is understandable that the attaching operations in steps S400 and S500, as well as the operation of connecting the microcavity array chip 10 to the bottom of the first frame 30 in step S600, can all be performed by a device such as a robotic arm. The above-mentioned device can be controlled by a controller, or a separate control module can be provided, as long as the control module is in communication with the controller and can be controlled by the controller. In step S600, after connecting the microcavity array chip 10 to which the breathable membrane 20 is attached to the bottom of the first frame 30 so that the sample inlet 31 is connected to the culture chamber 11 through the accommodating space 32, the following steps may also be performed: placing the microbial monitoring device horizontally to prevent abnormal flow of the test liquid due to tilting.

[0051] In one embodiment, in step S200, after the test liquid in the receiving space 32 of the microcavity array chip 10 is pushed to be uniformly filled into each of the culture chambers 11 so that the flexible membrane 40 is attached to the top of the culture chamber 11, the process further includes: S700 , removing the first frame 30 in the microorganism monitoring device to obtain the microcavity array chip 10 with upper and lower ends respectively covered with the flexible membrane 40 and the breathable membrane 20 .

[0052] S800: A first sealing film is placed on top of the flexible film 40, and a second sealing film is placed on the lower surface of the breathable film 20 to seal the culture chamber 11. It will be appreciated that the first and second sealing films can prevent external impurities from entering the culture chamber 11, ensuring a stable reaction environment within the culture chamber 11. The first and second sealing films can be made of transparent materials to facilitate monitoring of microorganisms in the culture chamber 11.

[0053] In one embodiment, the microorganism monitoring system further includes an image monitoring device, and the controller is in communication with the image monitoring device.

[0054] In step S300, monitoring the microorganisms in the culture chamber 11 to obtain microorganism monitoring results includes: S310: Capture an image of the test liquid in the culture chamber 11 through an image monitoring device, and determine a microbial monitoring result based on the image of the test liquid. The image monitoring device may be a high-definition imaging device, such as a camera or a mobile phone with a camera function.

[0055] In one embodiment, the test liquid contains an indicator. The indicator may be a resazurin indicator. Understandably, if there are microorganisms in the test liquid, then when the test liquid comes into contact with resazurin, the color of resazurin will undergo significant and easily discernible changes due to the metabolic activities of bacteria or fungi. In the oxidized state, it appears clear blue or purple, and once reduced, the color gradually transitions to pink and may eventually become colorless, and the entire color change process is intuitive and clear. Whether it is a professional experimenter or an operator who has only received basic training, they can directly observe the color change with the naked eye, or use a mobile phone or camera to take pictures for analysis. This simple and intuitive reading method greatly expands the application scenarios of the present invention, and can efficiently complete detection tasks even in grassroots laboratories and on-site testing points with limited resources. For example, Figure 6 After incubation for 8.25 hours in the presence of the test liquid, the color of the droplets in some of the culture chambers 11 of the microcavity array chip 10 changed significantly, specifically manifesting as a transformation from purple resazurin to pink resorufin. In this way, by performing color recognition on the image of the test liquid in each culture chamber 11, color feature information from the image of the test liquid can be extracted, and the color change corresponding to the image of the test liquid can be determined to determine the presence of microorganisms (fungi, etc.) in the test liquid. Understandably, if no microorganisms are present in the test liquid, the test liquid will not undergo a color change under the influence of the indicator. Understandably, the resazurin indicator is sensitive to microbial metabolism, resulting in a higher contrast, more significant change in the color reaction, and high detection sensitivity. The color development results can be observed with the naked eye or by taking a photo with a mobile phone, directly determining the presence of microorganisms. This eliminates the need for specialized testing equipment and professional operators, reducing testing costs.

[0056] In step S310, the image monitoring device is used to collect an image of the liquid to be tested in the culture chamber 11, and the microorganism monitoring result is determined based on the image of the liquid to be tested, including: S311, using an image monitoring device to collect the image of the test liquid in the culture chamber 11 in a regular or real-time manner, and extracting color feature information from the image of the test liquid; specifically, using a preset image recognition model to perform color recognition on the image of the test liquid to extract color feature information, and the extracted color feature information may include the color features of each culture chamber 11. For example, Figure 6In the image of the test liquid corresponding to 8.25 hours, the identifiable color feature information is that all the culture chambers 11 are purple, while the identifiable color feature information in the image of the test liquid corresponding to 9.25 hours is that part of the culture chambers 11 are pink and the remaining culture chambers 11 are purple.

[0057] Understandably, the preset image recognition model can be trained based on a neural network model. For example, the model can be trained based on a historical data set that includes multiple historical monitoring samples, each of which is associated with a set of identification features (corresponding to color feature information). The historical monitoring samples are then input into the neural network model, and the neural network model is trained to enable the neural network model to recognize color feature information. The loss generated by the neural network model during training is calculated to assess whether the neural network model has converged. When the model converges, the neural network model is determined as the preset image recognition model. This preset image recognition model can then be used to extract color feature information from the image of the test liquid.

[0058] S312, obtaining color change information associated with the indicator and microorganisms in the test liquid; the color change information refers to the color change information displayed by the microorganism when it is mixed with the indicator at a preset culture stage; that is, the color change information can represent the color change of different microorganisms when they are mixed with the indicator at a preset culture stage during their incubation process. For example, for Figure 6 For the test liquid containing microorganisms (Escherichia coli), the corresponding color change information is: since the test liquid in the culture chamber 11 contains microorganisms (Escherichia coli), after incubation for 8-10 hours (corresponding to the preset culture stage), the color of at least a portion of the droplets in the culture chamber 11 will change from purple (resazurin) to pink (resorufin).

[0059] S313, determine the microbial monitoring result based on the color characteristic information and the color change information. In the culture chamber 11, microbial metabolites rapidly accumulate and act on resazurin. Compared with traditional methods, the color reaction has a higher contrast and more significant changes, which improves the detection sensitivity and achieves accurate detection of trace or low metabolic activity microorganisms. Figure 6Taking the cultivation process of the test liquid containing microorganisms (Escherichia coli) as an example, the preset cultivation stage of incubation in the culture chamber 11 is 8-10 hours. During this preset cultivation stage, part of the culture chambers 11 in the microcavity array chip 10 contain microorganisms (Escherichia coli), so the color of part of the droplets changes from purple resazurin to pink resorufin. At this time (within 8-10 hours), color feature information containing purple and pink can be extracted from the image of the test liquid according to the above step S311. Therefore, the microbial monitoring result can be determined as the presence of bacteria (microorganisms) in the test liquid based on the color change in this part of the culture chamber 11. If the sample discretization is not achieved through the microbial monitoring device in the present invention, it will take about 20 hours to be recognized that the color change (such as Figure 6 As shown in , it will take about 20.25 hours for most of the culture chambers 11 to turn pink. If the test liquid is not dispersed in multiple culture chambers 11, it will be around this time that the test liquid that was initially purple will turn pink and be identified), and it will take more than ten hours to obtain the microbial monitoring results. Therefore, the above scheme in the present invention can achieve rapid detection and analysis.

[0060] In this embodiment, the microcavity array in the microcavity array chip 10 can discretize samples, thereby miniaturizing microbial samples. Each microcavity serves as an independent reaction unit, dispersing microorganisms such as bacteria or fungi into the microreactor unit for growth. This facilitates the enrichment of the microorganisms themselves and their metabolites, significantly shortening the incubation time required for signal detection, thereby enabling rapid detection and analysis. Resazurin is sensitive to microbial metabolism and is reduced and discolored during microbial growth and metabolism.

[0061] In one embodiment, step S313, i.e., determining the microbial monitoring result based on the color characteristic information and the color change information, includes: When it is determined based on the first color characteristic information that a color change corresponding to the color change information occurs in the preset culture stage, the microbial monitoring result is determined to be that bacteria are present in the test liquid; the first color characteristic information refers to the color characteristic information corresponding to the image of the test liquid collected within the culture time range corresponding to the preset culture stage.

[0062] That is, in this embodiment, the calculation can be started from the start time of the culture until the culture duration range corresponding to the preset culture stage (for example, Figure 6The image of the test liquid is not collected until the incubation time period (corresponding to 8-10 hours) has passed. The images of the test liquid collected within the incubation time range are then used as a basis for determining the microbial monitoring result. That is, the color characteristic information corresponding to the images of the test liquid collected within the incubation time range is set as the first color characteristic information, and a determination is made based on the first color characteristic information whether a color change corresponding to the color change information occurs within the preset incubation stage (for example, if all the culture cavities 11 in the initially collected image of the test liquid are purple, and some pink culture cavities 11 appear in the subsequently collected image of the test liquid, indicating that some of the culture cavities 11 have changed from purple to pink). If a color change corresponding to the color change information occurs, the microbial monitoring result indicates that bacteria are present in the test liquid. If it is determined based on the first color characteristic information that no color change corresponding to the color change information occurs during the preset incubation stage (for example, if all the culture cavities in all the images of the test liquid collected within the incubation time range remain purple), the microbial monitoring result can be determined as sterility in the test liquid, thereby concluding that the sample liquid corresponding to the test liquid contains no microorganisms and is a qualified sample liquid.

[0063] In a further embodiment, after determining that the microbial monitoring result indicates that bacteria are present in the test liquid, the method includes: When it is determined based on the second color characteristic information that the color change corresponding to the color change information is continuing, it is determined that the verification of the microbial monitoring result is correct; the second color characteristic information refers to the color characteristic information corresponding to the image of the liquid to be tested collected within the preset time period after the preset culture stage. That is, in order to ensure the accuracy of the result, the microbial monitoring result can be further verified. At this time, it is necessary to count whether the number of culture cavities 11 with color changes in the image of the liquid to be tested within the preset time period has increased. When the number of culture cavities 11 with color changes increases, it means that the color change is continuing, and it can be confirmed that the verification of the microbial monitoring result is correct. If it is found that the number of culture cavities 11 with color changes remains unchanged, it means that the color change has stopped. At this time, it is necessary to indicate that the microbial monitoring result is abnormal and to conduct a manual inspection on it.

[0064] Similarly, after determining that the test fluid is sterile as a result of the microbial monitoring, the microbial monitoring result can be further verified. In this case, the second color characteristic information can be used to determine whether a color change corresponding to the color change information occurs within a preset time period after the preset incubation stage. If a color change corresponding to the color change information occurs, the microbial monitoring result can be similarly determined to indicate the presence of bacteria in the test fluid. If a color change corresponding to the color change information does not occur within a preset time period after the preset incubation stage, the microbial monitoring result is verified to be correct (the test fluid is sterile), thereby concluding that the sample fluid corresponding to the test fluid is free of microorganisms and is qualified.

[0065] In one embodiment, in step S100, injecting the test liquid from the injection port 31 into the containing space 32 includes: S110: Inject a preset volume of the test liquid from the injection port 31 into the receiving space 32 at a preset flow rate. The preset flow rate can be set according to actual conditions to ensure that the test liquid can enter the receiving space 32 evenly and completely.

[0066] In one embodiment, in step S300, after monitoring the microorganisms in the culture chamber 11 to obtain microorganism monitoring results, the following steps are performed: S900: Discard the microbial monitoring device into a pre-set biochemical waste bin. It is understandable that disposing the microbial monitoring device into a pre-set biochemical waste bin ensures that the waste is disposed of safely and in compliance with regulations.

[0067] In one embodiment, the test solution contains an Escherichia coli test solution with a bacterial count of 10 CFU (Colony-Forming Units) (the culture medium is Tryptone Soy Broth (TSB)) and a resazurin indicator at a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0068] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 6 It can be seen that when the incubation time reaches 8.25 hours, it can be determined based on the color feature information extracted from the image of the test liquid that the color of the droplets in some culture chambers 11 of the microcavity array chip 10 has changed significantly, specifically manifested as the transformation of purple resazurin into pink resorufin. As the incubation process continues to advance, when the time is extended to 20.25 hours, the droplets in more culture chambers 11 in the microcavity array chip 10 turn pink. However, the color of the droplets in the culture chambers 11 that do not contain Escherichia coli remains in its initial state throughout the incubation period and does not change.

[0069] In one embodiment, the test solution contains 11 CFU of Staphylococcus aureus (culture medium is Tryptone Soy Broth TSB) and 10 μg / mL of resazurin indicator. The preset culture condition in step S300 is 33 degrees Celsius.

[0070] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 7It can be seen that when S. aureus was incubated in the droplets for 19.25 hours, the color of the droplets in several culture chambers 11 of the microcavity array chip 10 changed from purple resazurin to pink resorufin, as determined by color feature information extracted from the test liquid image. As the incubation time extended to 27.25 hours, the number of culture chambers 11 in the microcavity array chip 10 that changed to pink gradually increased. Throughout the experiment, the color of the droplets in culture chambers 11 that did not contain S. aureus remained purple, without any change.

[0071] In one embodiment, the test solution contains a Pseudomonas aeruginosa test solution with a bacterial count of 10 CFU (the culture medium is Tryptone Soy Broth (TSB)) and a resazurin indicator at a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0072] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 8 It can be seen that when Pseudomonas aeruginosa is incubated in the droplets for 15 hours, the color of the droplets in some culture chambers 11 of the microcavity array chip 10 can be determined based on the color feature information extracted from the image of the test liquid. The color changes significantly from the initial purple to pink, for example, in the color change area indicated by the small red arrow in the figure. As the incubation time is extended to 20.25 hours, the number of culture chambers 11 in the microcavity array chip 10 that turn pink shows a gradually increasing trend. In the culture chambers 11 that are not allocated to Pseudomonas aeruginosa, the color of the droplets remains purple and does not change.

[0073] In one embodiment, the test solution contains a Bacillus subtilis test solution with a bacterial count of 14 CFU (the culture medium is Tryptone Soy Broth TSB) and a resazurin indicator with a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0074] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 9 It can be seen that after Bacillus subtilis was incubated in the droplets for 9.25 hours, the color characteristics extracted from the image of the test liquid could be used to determine that the color of the droplets in some culture chambers 11 of the microcavity array chip 10 changed from purple resazurin to pink resorufin. As the incubation time was further extended to 20.25 hours, the number of culture chambers 11 that appeared pink gradually increased. During this process, the color of the droplets that had previously turned pink gradually changed back to colorless resorufin. In contrast, the color of the droplets in the culture chambers 11 that did not contain Bacillus subtilis remained unchanged, purple, throughout.

[0075] In one embodiment, the test solution contains a Streptococcus pyogenes test solution with a bacterial count of 17 CFU (the culture medium is Tryptone Soy Broth TSB) and a resazurin indicator with a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0076] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 10 It can be seen that after 29.25 hours of incubation of S. pyogenes in the droplets, the color of the droplets in several culture chambers 11 changed from purple (resazurin) to pink (resorufin) based on the color feature information extracted from the test liquid image. As the incubation process progressed to 45.25 hours, the number of culture chambers 11 on the chip that turned pink gradually increased. Throughout the incubation period, the droplets in culture chambers 11 that did not contain S. pyogenes remained stably purple, without any change.

[0077] In one embodiment, the test solution contains a micrococcal test solution with a bacterial count of 86 CFU (the culture medium is tryptone soy broth TSB) and a resazurin indicator with a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0078] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 11 It can be seen that when the micrococcus is incubated in the droplet system for 28.25 hours, the color of the droplets in the culture chambers 11 of the microcavity array chip 10 changes from purple to pink based on the color feature information extracted from the image of the test liquid. Figure 11 The color change area is indicated by the arrow in the figure. As the incubation time was further extended to 40.25 hours, the number of pink culture chambers 11 on the microcavity array chip 10 gradually increased. Throughout the experiment, the color of the droplets in the culture chambers 11 without micrococci remained stable at purple, without any change.

[0079] In one embodiment, the test solution contains a Candida albicans test solution with a bacterial count of 15 CFU (the culture medium is Tryptone Soy Broth (TSB)) and a resazurin indicator with a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0080] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 12It can be seen that after C. albicans was incubated in the droplets for 17.25 hours, the color characteristics extracted from the test liquid image confirmed that the color of the droplets in several culture chambers 11 had changed from purple resazurin to pink resorufin. When the incubation time reached 25.25 hours, the number of culture chambers 11 that had turned pink gradually increased, while the droplets in culture chambers 11 that did not contain C. albicans remained purple.

[0081] In one embodiment, the test solution contains a test solution of Aspergillus niger with a bacterial count of 59 CFU (the culture medium is Tryptone Soy Broth TSB) and a resazurin indicator with a concentration of 10 μg / mL. The preset culture condition in step S300 is 33 degrees Celsius.

[0082] In step S311, an image monitoring device is used to collect images of the test liquid in the culture chamber 11 at intervals of 0.25 hours. Figure 13 It can be seen that after the Aspergillus niger was incubated in the droplets for 21.25 hours, the color characteristics extracted from the image of the test liquid confirmed that the color of several droplets in the culture chamber 11 changed from purple (resazurin) to pink (resorufin). When the incubation time reached 30.25 hours, the droplets in the culture chamber 11 turned pink over a large area.

[0083] It should be understood that the size of the serial numbers of the steps in the above embodiments does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0084] The above are merely embodiments of the microbial monitoring device, microbial monitoring system, and method of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A microbial monitoring device, characterized in that: include: A microcavity array chip is provided with multiple culture chambers; a gas-permeable membrane, disposed at the bottom of the microcavity array chip and covering the bottom ends of all the culture cavities, for allowing gas in the culture cavities to enter and exit, and preventing the test liquid from flowing out through the bottom ends of the culture cavities; a first frame connected to the top of the microcavity array chip, the first frame being provided with an injection port and a receiving space communicating between the injection port and the culture chamber; A flexible film covers the top of the accommodating space.

2. The microorganism monitoring device according to claim 1, wherein The microorganism monitoring device further includes a second frame; the air permeable membrane is clamped between the second frame and the microcavity array chip; and the second frame is provided with an air permeable channel arranged opposite to the culture cavity.

3. The microorganism monitoring device according to claim 1, wherein The plurality of culture chambers are distributed in an array and have equal volumes; or / and The culture chamber is hexagonal, circular or square; or / and The length of the microcavity array chip is 15-30 mm, and the width is 15-30 mm; the length of the first frame is 25-50 mm, and the width is 25-50 mm.

4. A microbial monitoring system, characterized in that: It comprises a controller, an extrusion piece, and at least one microbial monitoring device according to any one of claims 1 to 3; the controller is communicatively connected to the microbial monitoring device; the extrusion piece is provided with an extrusion portion adapted to the accommodating space; the extrusion piece is used to extrude a flexible membrane through the extrusion portion and push the test liquid in the accommodating space to be uniformly filled into each of the culture chambers after the test liquid enters the accommodating space through the sampling port.

5. A microbial monitoring method, characterized in that: Applied to the microbial monitoring system according to claim 4, the microbial monitoring method comprises: receiving a sample loading signal and controlling the test liquid to enter the containing space from the sample inlet of the first frame; Controlling the extrusion part of the extrusion member to squeeze the flexible membrane and push the test liquid in the accommodation space of the microcavity array chip to fill each of the culture chambers, so that the flexible membrane is attached to the top of the culture chamber; The microbial monitoring device with the first frame removed is placed under preset culture conditions to culture the test liquid in the culture chamber, and the culture process is monitored to obtain microbial monitoring results.

6. The microbial monitoring method according to claim 5, characterized in that: Before receiving the sample loading signal, the method further includes: Attaching the breathable membrane to the bottom of the microcavity array chip to block the bottom end of the culture cavity through the breathable membrane; covering the flexible film on the top of the receiving space of the first frame; The microcavity array chip with the air permeable membrane attached thereto is connected to the bottom of the first frame so that the sample inlet is connected to the culture chamber through the accommodation space.

7. The microbial monitoring method according to claim 5, characterized in that: After the test liquid in the accommodation space of the microcavity array chip is pushed to be filled into each culture cavity so that the flexible membrane is attached to the top of the culture cavity, the method further includes: Removing the first frame from the microbial monitoring device to obtain the microcavity array chip with upper and lower ends respectively covered with the flexible membrane and the breathable membrane; A first sealing film is covered on the top of the flexible film, and a second sealing film is covered on the lower surface of the breathable film to seal the culture chamber.

8. The microbial monitoring method according to claim 5, characterized in that: The microbial monitoring system further includes an image monitoring device, and the controller is in communication with the image monitoring device; The monitoring of the test liquid in the culture chamber to obtain microbial monitoring results includes: An image monitoring device is used to collect an image of the liquid to be tested in the culture chamber, and a microorganism monitoring result is determined based on the image of the liquid to be tested.

9. The microbial monitoring method according to claim 5, characterized in that: The liquid to be tested contains an indicator; The collecting of the image of the liquid to be tested in the culture chamber by the image monitoring device and determining the microorganism monitoring result according to the image of the liquid to be tested include: collecting an image of the test liquid in the culture chamber periodically or in real time by an image monitoring device, and extracting color feature information from the image of the test liquid; Acquiring color change information associated with the indicator in the test liquid; the color change information refers to the color change information displayed when the microorganism is mixed with the indicator during a preset culture stage; The microbial monitoring result is determined based on the color characteristic information and the color change information.

10. The microbial monitoring method according to claim 9, characterized in that: Determining the microbial monitoring result according to the color characteristic information and the color change information includes: When it is determined based on the first color characteristic information that a color change corresponding to the color change information occurs in a preset culture stage, the microbial monitoring result is determined to be that microorganisms are present in the liquid to be tested; the first color characteristic information refers to the color characteristic information corresponding to the image of the liquid to be tested collected within the culture time range corresponding to the preset culture stage.

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