Gas replacement device for anaerobic microorganism culture

Abstract

The invention relates to the technical field of anaerobic microorganism culture, and discloses a gas replacement device for anaerobic microorganism culture, which comprises a plurality of groups of gas supply gas circuit components and at least one gas replacement station, the gas replacement station comprises a secondary pressure reducing valve, a plurality of groups of gas charging / exhausting channels and a vacuum pump, the plurality of groups of gas supply gas circuit assemblies are connected with the input end of the secondary pressure reducing valve, the output end of the secondary pressure reducing valve is connected with one ends of the plurality of groups of gas charging / exhausting channels through a flow divider, and the other end of each group of gas charging / exhausting channel is used for being connected with a replaceable sterile needle head; the sterile needle head is connected with a culture bottle, and the output end of the vacuum pump is connected with the output end of the secondary pressure reducing valve. A plurality of channels can be operated at the same time, the requirement of a plurality of students for carrying out anaerobic operation at the same time is met, in the inflation and deflation process, a vacuum needle or an inflation needle does not need to be pulled and inserted, gas pollution is avoided, meanwhile, gas of different proportions can be accurately regulated and controlled, and the culture requirements of different types of anaerobic microorganisms are met.

Description

Technical Field

[0001] The invention relates to the technical field of anaerobic microorganism cultivation, in particular to a gas replacement device for anaerobic microorganism cultivation. Background Art

[0002] Microorganisms are divided into two major categories: aerobic and anaerobic. The cultivation of anaerobic microorganisms requires strict control of oxygen concentration, and the cultivation process is generally completed in an anaerobic chamber. Anaerobic microorganisms are widely present in the natural environment and participate in the geobiochemical cycles of elements such as carbon, nitrogen, and sulfur. Their research is of great significance in many fields such as medicine, industry, and ecological protection. Research on anaerobic bacteria often combines omics sequencing analysis and anaerobic culture technology, but the latter is often limited by harsh culture conditions. Due to their high sensitivity to oxygen, the cultivation process has high requirements for equipment and operation, and the cultivation is more difficult, which makes operations such as experimental verification and production application relatively difficult. Therefore, the technological upgrade of anaerobic microbial culture equipment is of great value to their research.

[0003] Another limitation of anaerobic microbial culture is the lack of proficiency of experimental members in anaerobic operations. At present, anaerobic microbial culture experiments are rarely carried out in college classroom teaching, mainly due to the limitation of teaching equipment. Traditional anaerobic culture devices Existing anaerobic culture equipment generally consists of a nitrogen bottle connected to a pressure valve and a gas needle at the end. One gas needle is used for each microbial culture bottle at a time. This method has the following main problems: 1. Only one culture bottle can be operated at a time, which makes it difficult to meet the needs of dozens of students to carry out standardized anaerobic operations. 2. During the filling and deflation process, it is necessary to perform vacuum-inflation-vacuum-inflation-vacuum-inflation, and repeat the cycle three times. Each operation will pull out and insert a vacuum needle or inflation needle. Each plugging and unplugging will cause a large amount of air to enter the bottle. Especially when the vacuum needle is pulled out and inserted, the entry of oxygen will cause the death of anaerobic microorganisms. 3. The precision is low, and it is difficult to achieve precise control of different proportions of gas. Summary of the Invention

[0004] The purpose of the present invention is to provide a gas replacement device for anaerobic microbial culture to solve the problems that the existing anaerobic microbial culture process is difficult for multiple people to operate, the inflation and deflation process in the operation process is easy to mix with gas, and it is difficult to achieve precise control of different proportions of gas.

[0005] The technical solution of the present invention is: A gas replacement device for anaerobic microbial culture, comprising multiple groups of gas supply circuit components and at least one gas replacement station; each group of gas supply circuit components is used to transport different types of non-oxygen gases; the gas replacement station comprises a secondary pressure reducing valve, multiple groups of charging / exhaust channels and a vacuum pump, the output ends of the multiple groups of gas supply circuit components are connected to the input end of the secondary pressure reducing valve, the output end of the secondary pressure reducing valve is connected to one end of the multiple groups of charging / exhaust channels via a diverter, the other end of each group of charging / exhaust channels is used to be connected to a replaceable sterile needle, the sterile needle is used to be connected to a culture bottle, and the output end of the vacuum pump is connected to the output end of the secondary pressure reducing valve.

[0006] Preferably, as a further improvement of the present invention, it further includes a switching valve, one connecting end of the switching valve is connected to the output end of the vacuum pump, and the other connecting end of the switching valve is connected to the output end of the secondary pressure reducing valve.

[0007] Preferably, as a further improvement of the present invention, there are two gas exchange stations, and the switching valve is a three-way two-way switching ball valve. The two connecting ends of the three-way two-way switching ball valve are respectively connected to the output ends of the secondary pressure reducing valves in the two gas exchange stations, and the other connecting end of the three-way two-way switching ball valve is connected to the output end of the vacuum pump.

[0008] The output end of the secondary pressure reducing valve is connected to a pipeline emptying ball valve, and the pipeline emptying ball valve is used to release air to relieve pressure.

[0009] Preferably, as a further improvement of the present invention, the gas supply circuit assembly includes a gas cylinder and a gas pipeline connected to the bottle mouth of the gas cylinder, the gas pipeline is connected to a first-level pressure reducing valve near the bottle mouth of the gas cylinder, and the end of each gas pipeline away from the gas cylinder is connected to the input end of the second-level pressure reducing valve through a merger.

[0010] Preferably, as a further improvement of the present invention, a one-way fire-stop valve is connected to the gas pipeline.

[0011] Preferably, as a further improvement of the present invention, a gas leakage protection component is provided on the gas transmission pipeline, and the gas leakage protection component includes a solenoid valve, a gas detection alarm unit and an exhaust fan; the solenoid valve is arranged on the gas transmission pipeline; the gas detection alarm unit is connected to the solenoid valve, and is used to control the solenoid valve to automatically cut off the gas transmission when a gas leakage is detected and a threshold is reached; the exhaust fan is connected to the gas detection alarm unit, and is used to open and discharge the leaked gas when an alarm is sounded.

[0012] Preferably, as a further improvement of the present application, the gas detection alarm unit comprises an oxygen alarm, a combustible gas alarm and a smoke alarm, which are respectively electrically connected with the electromagnetic valve and the exhaust fan.

[0013] Preferably, as a further improvement of the present application, each group of filling / air suction channels is connected with the sterile needle through a filter sterilization device.

[0014] Preferably, as a further improvement of the present application, the filter sterilization device comprises a needle tube, a filter sterilization structure and a luer joint; a rubber plug is sealingly connected inside the inlet end of the needle tube, and an interface connected with the filling / air suction channel is arranged on the rubber plug; the filter sterilization structure comprises a sterile absorbent cotton layer, a glass fiber layer and a nylon layer arranged in the inside of the needle tube from top to bottom; and the luer joint is connected between the outlet end of the needle tube and the sterile needle.

[0015] Compared with the prior art, the present application has the following beneficial effects: 1. Multiple channels can be operated simultaneously to complete the replacement of culture medium oxygen or anaerobic culture bottle gas with high throughput, meeting the needs of multiple students to simultaneously carry out anaerobic operation.

[0016] 2. Directly connected with a vacuum pump, without pulling out the vacuum needle or the air filling needle during the filling and air suction process, gas pollution is avoided.

[0017] 3. Rapid and efficient gas switching can be performed, more gas combinations can be achieved through simple operation, and the culture needs of more anaerobic microorganisms can be met.

[0018] 4. Continuous culture can be achieved to ensure that the culture process is continuously supplemented with gas without contamination by bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a structural schematic diagram of an anaerobic microbial culture gas replacement device according to the present application.

[0020] Figure 2 Figure 2 is a structural schematic diagram of a filter sterilization device in an anaerobic microbial culture gas replacement device according to the present application. DETAILED DESCRIPTION

[0021] The following will be described in detail Figure 1~Figure 2, a detailed description of the specific embodiments of the present invention is provided. In the description of the invention, it should be understood that the terms "center," "upper," "lower," "front," "back," "left," "right," "vertical," "horizontal," "top," "bottom," "inside," "outside," and the like, indicating directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings and are intended only to facilitate the description of the present invention and simplify the description. They do not indicate or imply that the devices or components referred to must have a specific direction, be constructed, or operate in a specific direction. Therefore, they should not be construed as limiting the present invention.

[0022] The terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the quantity of the technical features indicated. Therefore, features specified as "first" or "second" may explicitly or implicitly include one or more of such features; and in the description of the invention, unless otherwise specified, "plurality" means two or more.

[0023] Example like Figure 1~Figure 2 As shown, an embodiment of the present invention provides a gas replacement device for anaerobic microbial culture, comprising multiple groups of gas supply path components and at least one gas replacement station; each group of gas supply path components is used to transport different types of non-oxygen gases; the gas replacement station comprises a secondary pressure reducing valve 1, multiple groups of charging / exhaust channels 2 and a vacuum pump 31, the output ends of the multiple groups of gas supply path components are connected to the input end of the secondary pressure reducing valve 1, the output end of the secondary pressure reducing valve 1 is connected to one end of the multiple groups of charging / exhaust channels 2 through a diverter, the other end of each group of charging / exhaust channels 2 is used to be connected to a replaceable sterile needle, the sterile needle is used to be connected to a culture bottle, and the output end of the vacuum pump 31 is connected to the output end of the secondary pressure reducing valve 1.

[0024] In this embodiment, the gas replacement station is provided to allow multiple groups of charging / exhausting channels 2 to operate simultaneously, thereby completing the deoxygenation of the culture medium or the replacement of the gas in the anaerobic culture bottle with high throughput, meeting the needs of multiple people to operate, and the secondary pressure reducing valve is provided to achieve precise regulation of different proportions of gas to meet the cultivation needs of different types of anaerobic microorganisms. The pressure in the anaerobic culture bottle can be controlled to achieve pressurized culture and cultivation of high-pressure microorganisms. Since the vacuum pump 31 is connected to the output end pipeline of the secondary pressure reducing valve 1, the vacuum pump 31 can be directly controlled to perform the charging and discharging process without plugging and unplugging the vacuum needle or the charging needle, avoiding gas pollution, and preventing the entry of gas to cause pollution and the death of anaerobic microorganisms.

[0025] In order to be able to switch between inflation / vacuuming modes with one click, a switching valve 3 is also included. One connection end of the switching valve 3 is connected to the output end of the vacuum pump 31, and the other connection end of the switching valve 3 is connected to the output end of the secondary pressure reducing valve 1. The setting of the switching valve 3 can switch between inflation / vacuuming modes with one click, and the operation is simple and quick.

[0026] Among them, the gas supply path assembly has seven routes, and the gas supply path assembly includes a gas cylinder 51 and a gas supply pipeline 52 connected to the bottle mouth of the gas cylinder 51. The gas supply pipeline 52 is connected to a first-level pressure reducing valve near the bottle mouth of the gas cylinder 51, and the end of each gas supply pipeline 52 away from the gas cylinder 51 is connected to the input end of the second-level pressure reducing valve 1 through a merger.

[0027] like Figure 1 As shown, the gases stored in the seven gas cylinders 51 from left to right are CO2, CO2 / N2 mixed gas, N2, N2 / H2 mixed gas, CH4, H2, and high-purity air.

[0028] In another embodiment of the present invention, in order to facilitate operation by more people, there are two gas exchange stations, and the switching valve 3 is a three-way two-way switching ball valve. The two connecting ends of the three-way two-way switching ball valve are respectively connected to the output ends of the secondary pressure reducing valve 1 in the two gas exchange stations, and the other connecting end of the three-way two-way switching ball valve is connected to the output end of the vacuum pump 31.

[0029] Among them, such as Figure 1 As shown, there are 10 groups of filling / exhausting channels 2 in each gas exchange station, so that up to 20 students can operate at the same time.

[0030] In another embodiment of the present invention, the output end of the secondary pressure reducing valve 1 is connected to a pipeline emptying ball valve 4, which is used to deflate and relieve pressure. The pipeline emptying ball valve 4 can be quickly deflated to quickly reduce the pressure on the inflation station and ensure inflation safety; at the same time, other types of gases remaining in the pipeline can be quickly emptied.

[0031] The following is a further explanation of the principle of precise control of gases in different proportions achieved by this application with reference to an application example: The cultivation of anaerobic methanogenic archaea requires a composition of 2% CO2, 15% H2, 6% CH4, and 67% N2. High accuracy is required, with an error of no more than 1%. Converted to a pressure gauge (typically, anaerobic culture equipment can withstand pressures of 15-45 psi), using a conventional anaerobic bottle at 15 psi as an example, the appropriate gas levels are 1.8 psi of CO2, 2.2 psi of H2, 1 psi of CH4, and 10 psi of N2. Manual mixing is used.

[0032] The deployment steps are as follows: S1. After connecting the anaerobic bottle to the sterile needle, switch the switching valve 3 to the vacuum pump 31 to perform vacuuming (0 psi); S2. Open the CO2 cylinder, switch the switching valve 3 to the secondary pressure reducing valve 1, and manually adjust the secondary pressure reducing valve 1 to 1.8 psi. After the secondary pressure reducing valve 1 reading stabilizes for one minute, close the CO2 cylinder and open the drain ball valve 3 to release the gas. After the release is complete, close the pipeline drain ball valve 4. S3. Open the H2 gas cylinder and manually adjust the secondary pressure reducing valve to 1 to 4 psi (1.8 psi + 2.2 psi). After the reading stabilizes for one minute, close the H2 gas cylinder and open the drain ball valve 3 to release the gas. After the release is complete, close the pipeline drain ball valve 4. S4. Open the CH4 gas cylinder and manually adjust the secondary pressure reducing valve to 1 to 5 psi (4 psi + 1 psi). After the reading stabilizes for one minute, close the CH4 gas cylinder and open the drain ball valve 3 to release the gas. After the release is complete, close the pipeline drain ball valve 4. S5. Open the N2 gas cylinder and manually adjust the secondary pressure reducing valve from 1 to 15 psi (5 psi + 10 psi). After the reading stabilizes for one minute, close the N2 gas cylinder and open the drain ball valve 3 to release the gas. After the release is completed, close the pipeline drain ball valve 4 to complete the gas filling.

[0033] In the past, it was almost impossible to complete this operation because one bottle of gas corresponded to one needle. The reasons are as follows:

[0034] 1) The pressure gauge corresponding to each gas cylinder displays the internal pressure, generally 5-15 MPa (approximately 725-2175 psi). Usually, the gauge does not display psi units, only MPa. Even if psi units are displayed, the value on the gauge may be too large, resulting in inaccurate gas output. This application uses a two-stage pressure reducing valve 1 to reduce the pressure in the filling / exhaust channel and display it on the pressure gauge of the pressure reducing valve. By adjusting the pressure reducing valve, the internal pressure of the cylinder can be precisely controlled.

[0035] 2) Previous devices required repeated plugging and unplugging, each of which would cause gas leakage and change the gas ratio. In the present application, switching between different gases is accomplished by opening and closing different inflation gas paths. Residual gas in the path can be released by opening the pipeline evacuation ball valve 4, avoiding the problem of gas ratio changes due to leakage caused by plugging and unplugging the needle.

[0036] 3) In the past, the device's inflation and deflation process involved three cycles of vacuuming, inflation, vacuuming, inflation, vacuuming, and inflation. Each cycle involved inserting and removing the vacuum or inflation needle. This process caused a large amount of air to enter the bottle, especially when the vacuum needle was inserted. The oxygen introduced into the bottle killed anaerobic microorganisms.

[0037] However, the present application does not involve the plugging and unplugging process. The three-way two-way switching ball valve 3 can be switched between the inflation / vacuuming mode with one click. The two sets of gas replacement stations can provide 20 channels for simultaneous operation, completing the deoxygenation of the culture medium or the replacement of the gas in the anaerobic culture bottle with high throughput, and realizing the precise control of different proportions of gas to meet the cultivation requirements of different types of anaerobic microorganisms.

[0038] In another embodiment of the present invention, a one-way fire damper 53 is connected to the gas pipeline 52 to ensure one-way flow of gas, thereby protecting the pipeline system and preventing flammable and explosive gases from being ignited at the end and causing accidents.

[0039] In another embodiment of the present invention, a gas leakage protection component is provided on the gas transmission pipeline 52, and the gas leakage protection component includes a solenoid valve 61, a gas detection alarm unit 62 and an exhaust fan 63; the solenoid valve 61 is arranged on the gas transmission pipeline 52; the gas detection alarm unit 62 is connected to the solenoid valve 61, and is used to control the solenoid valve 61 to automatically cut off the gas transmission when a gas leakage is detected and the threshold is reached; the exhaust fan 63 is connected to the gas detection alarm unit 62, and is used to open and discharge the leaked gas when an alarm is sounded. Through the above arrangement, the problem of gas leakage causing harm during the gas distribution process can be effectively avoided.

[0040] Specifically, the gas detection alarm unit 62 includes an oxygen alarm, a combustible gas alarm and a smoke alarm. The oxygen alarm, the combustible gas alarm and the smoke alarm are electrically connected to the solenoid valve 61 and the exhaust fan 63 respectively. When oxygen, combustible gas or smoke is detected, the solenoid valve 61 is controlled to cut off the gas supply and open at the same time to discharge the leaked gas.

[0041] In another embodiment of the present invention, each group of filling / exhaust channels 2 is connected to a sterile needle through a filter sterilization device 7, and a HEPA filter, ULPA filter or other size membrane filter can be replaced to meet the requirements of accurately intercepting particles of a specific size and ensuring that the anaerobic bottle is continuously inflated without bacterial contamination.

[0042] Specifically, the filtration and sterilization device 7 includes a needle tube 71, a filtration and sterilization structure and a Luer connector 75; the inlet end of the needle tube 71 is sealed internally connected to a rubber plug 76, and the rubber plug 76 is provided with an interface connected to the filling / exhaust channel 2; the filtration and sterilization structure includes a sterile cotton wool layer 72, a glass fiber layer 73 and a nylon layer 74 arranged inside the needle tube 71 and arranged in sequence from top to bottom, wherein the sterile cotton wool is used to filter dust, large particles of dust, bacteria, and fungal spores in the gas and the pipeline; the glass fiber layer is used to filter particles of dust, bacteria, and some bacteriophages in the pipeline; the nylon layer is used to filter medium particles of dust, volatile organic compounds and aerosols; the Luer connector 75 is connected to the lower end of the needle tube 71; the Luer connector 75 is connected between the outlet end of the needle tube 71 and the sterile needle.

[0043] Through the above-mentioned setting, terminal sterilization can be achieved; at the same time, it can be sterilized and reused repeatedly; inflation and vacuuming can be completed in one step to avoid bacterial contamination, and the sterile absorbent cotton layer 72 can absorb liquid, thereby preventing liquid from being drawn back into the pipeline and vacuum pump during vacuuming, and can also improve the adaptability of consumables, and all links can be completed using one type of sterile needle.

[0044] Among them, three groups of gas pipelines 52 in the seven groups of gas supply gas circuit components are connected to the anaerobic glove box and the anaerobic culture equipment respectively. Figure 1 The gas circuits here primarily supply two types of equipment: the off-board culture equipment described in this article, and the in-cabin culture equipment (such as an anaerobic glove box). These two devices, when used together, can accommodate most anaerobic microorganisms. The two gas circuits on the upper left side connect to the three-gas incubator.

[0045] The above disclosures are only several preferred specific embodiments of the present invention. However, the embodiments of the present invention are not limited thereto. Any changes that can be conceived by those skilled in the art should fall within the scope of protection of the present invention.

Claims

1. A gas replacement device for anaerobic microbial culture, characterized in that: include: Multiple groups of gas supply circuit components, each group of gas supply circuit components is used to transport different types of non-oxygen gases; At least one gas exchange station, the gas exchange station comprising a secondary pressure reducing valve (1), multiple groups of filling / exhaust channels (2) and a vacuum pump (31), the output ends of the multiple groups of gas supply path components are connected to the input end of the secondary pressure reducing valve (1), the output end of the secondary pressure reducing valve (1) is connected to one end of the multiple groups of filling / exhaust channels (2) through a diverter, the other end of each group of filling / exhaust channels (2) is used to be connected to a replaceable sterile needle, the sterile needle is used to be connected to a culture bottle, and the output end of the vacuum pump (31) is connected to the output end of the secondary pressure reducing valve (1).

2. The gas replacement device for anaerobic microorganism cultivation according to claim 1, characterized in that: It also includes a switching valve (3), one connection end of the switching valve (3) is connected to the output end of the vacuum pump (31), and the other connection end of the switching valve (3) is connected to the output end of the secondary pressure reducing valve (1).

3. The gas replacement device for anaerobic microorganism cultivation according to claim 2, characterized in that: There are two gas exchange stations, and the switching valve (3) is a three-way two-way switching ball valve. Two connection ends of the three-way two-way switching ball valve are respectively connected to the output ends of the secondary pressure reducing valves (1) in the two gas exchange stations, and the other connection end of the three-way two-way switching ball valve is connected to the output end of the vacuum pump (31).

4. The gas replacement device for anaerobic microorganism cultivation according to claim 1, characterized in that: The output end of the secondary pressure reducing valve (1) is connected to a pipeline drain ball valve (4), and the pipeline drain ball valve (4) is used to release air for pressure relief.

5. The gas replacement device for anaerobic microorganism cultivation according to claim 1, characterized in that: The gas supply circuit assembly comprises a gas cylinder (51) and a gas delivery pipeline (52) connected to the bottle mouth of the gas cylinder (51); the gas delivery pipeline (52) is connected to a primary pressure reducing valve near the bottle mouth of the gas cylinder (51); and one end of each gas delivery pipeline (52) away from the gas cylinder (51) is connected to the input end of the secondary pressure reducing valve (1) through a confluence.

6. The gas replacement device for anaerobic microorganism cultivation according to claim 5, characterized in that: A one-way fire-stop valve (53) is connected to the gas transmission pipeline (52).

7. The gas replacement device for anaerobic microorganism cultivation according to claim 5, characterized in that: The gas transmission pipeline (52) is provided with a gas leakage protection component, and the gas leakage protection component comprises: A solenoid valve (61) is provided on the gas transmission pipeline (52); A gas detection alarm unit (62) is connected to the solenoid valve (61) and is used to control the solenoid valve (61) to automatically cut off gas transmission when gas leakage is detected and the threshold is reached; An exhaust fan (63) is connected to the gas detection alarm unit (62) and is used to open and exhaust the leaked gas when an alarm is sounded.

8. The gas replacement device for anaerobic microorganism cultivation according to claim 7, characterized in that: The gas detection alarm unit (62) comprises an oxygen alarm, a combustible gas alarm and a smoke alarm, and the oxygen alarm, combustible gas alarm and smoke alarm are electrically connected to the solenoid valve (61) and the exhaust fan (63) respectively.

9. The gas replacement device for anaerobic microorganism cultivation according to claim 1, characterized in that: Each set of filling / exhausting channels (2) is connected to the sterile needle via a filter sterilization device (7).

10. The gas replacement device for anaerobic microorganism cultivation according to claim 9, characterized in that: The filtration and sterilization device (7) comprises: A needle tube (71) having an inlet end sealedly connected to a rubber plug (76), wherein the rubber plug (76) is provided with an interface connected to the inflation / evacuation channel (2); A filtration and sterilization structure, comprising a sterile absorbent cotton layer (72), a glass fiber layer (73), and a nylon layer (74) arranged inside the needle tube (71) and arranged in sequence from top to bottom; A Luer connector (75) is connected between the outlet end of the needle tube (71) and the sterile needle.

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