CN112852614A

CN112852614A - Anaerobic microorganism culture medium oxygen removal device capable of adjusting gas composition and oxygen removal method

Info

Publication number: CN112852614A
Application number: CN202110202711.7A
Authority: CN
Inventors: 王海松
Original assignee: Changshu Institute of Technology
Current assignee: Changshu Institute of Technology
Priority date: 2021-02-23
Filing date: 2021-02-23
Publication date: 2021-05-28

Abstract

The invention belongs to the technical field of anaerobic microorganism culture medium deoxygenation devices, and particularly provides an anaerobic microorganism culture medium deoxygenation device capable of adjusting gas composition, which comprises a controller, a main gas path and branch gas paths, and is characterized in that: the main gas path is sequentially connected with a vacuum system, a valve system I, an air pressure detection system I, a valve system II, a valve system III, an air pressure detection system II and a gas storage system from a gas outlet communicated with the outside; simultaneously, an automatic anaerobic microorganism culture medium preparation facilities is disclosed, including main gas circuit, box and controller, the upper surface of box can be dismantled and is connected with the support ring, the inside fixed surface of box is connected with the bottom plate, the inside of bottom plate has been placed main gas circuit, the right side of main gas circuit has vacuum meter, solenoid valve one and vacuum pump from the front to the back intercommunication in proper order, has deposited gas in the memory, can utilize the memory to provide the required special gas of microorganism, does benefit to and carries out microbial preparation.

Description

Anaerobic microorganism culture medium oxygen removal device capable of adjusting gas composition and oxygen removal method

Technical Field

The invention belongs to the technical field of microbial culture. The invention particularly relates to an anaerobic microorganism culture medium deoxygenation device capable of adjusting gas composition and an automatic culture medium deoxygenation method.

Background

Microorganisms have been implicated in various aspects of human productive life, such as microorganisms required for our fermented food processing, microorganisms required for biopharmaceuticals, microorganisms required for maintaining physical health, and microorganisms required for sewage treatment. According to the requirement of the microorganism on the oxygen content in the growing environment, the microorganism can be classified into aerobic microorganism, facultative anaerobic microorganism and anaerobic microorganism. Most of the microorganisms are aerobic or facultative anaerobic microorganisms because the surface air contains oxygen. However, microorganisms in the intestinal tract of humans or animals, deep-sea microorganisms, microorganisms in the bottom layer of swamp, and the like belong to anaerobic microorganisms because the living environment does not contact oxygen. The anaerobic microorganism has important significance for human or animal health, biological resource development, sewage treatment and the like.

Unlike aerobic microorganisms and facultative anaerobic microorganisms, anaerobic microorganisms have stringent requirements for the content of oxygen in the culture environment. For strictly anaerobic microorganisms, very small amounts of oxygen can inhibit their growth and even kill the microorganisms. At present, anaerobic jar method, anaerobic bag method, anaerobic glove box, anaerobic box, biological aerobic method, plate pyrogallol method, high-level agar culture method, Hungart anaerobic roller tube technology and the like are generally adopted for culturing anaerobic microorganisms. The methods have the defects of high technical requirement, complex operation, incapability of monitoring the growth condition of microorganisms in real time and the like, for example, the common anaerobic jar method adopts a common drying jar, and then uses a physical and chemical method to cause an anaerobic environment in the jar, so that the observation, sampling and detection of the growth process of the anaerobic microorganisms are inconvenient; the anaerobic bag method is characterized in that a gas generating tube (an ampoule of sodium bicarbonate solid with sodium borohydride and 5% citric acid), a methylene blue indicator tube, a palladium catalyst tube, a drying agent and the like are arranged in a plastic bag, and an anaerobic environment in the plastic bag is caused by complex operation, so that the method is high in cost, long in time for establishing the anaerobic environment, and inconvenient to observe, sample and detect in the growth process of anaerobic microorganisms like the anaerobic jar method; the anaerobic glove box is a closed large-scale metal box body, and before operation, the experimental material about to enter the box body needs to be subjected to long-time air extraction and nitrogen gas filling deoxidation treatment in a channel connected with the anaerobic glove box; the anaerobic box also cannot realize observation and operation in the growth process of microorganisms, and in addition, the anaerobic environment of the anaerobic box is unstable; the biological aerobic method is mainly characterized in that plants are placed in a closed container, oxygen is consumed, carbon dioxide is generated at the same time, the experimental operation difficulty is high, the consumed time is long, and observation, sampling, qualitative and quantitative detection in the growth process of anaerobic microorganisms are not facilitated; although the flat pyrogallic acid method and the high agar culture method have low cost and relatively simple operation, the culture effect of the anaerobic microorganisms is not ideal, in addition, the application range of the method is narrow, and the sampling and separating effect of the anaerobic microorganisms is not easy to control; the Hencatel anaerobic roller tube technology which is the most similar to the Hencatel anaerobic roller tube technology is the most effective technology for culturing strict and obligate anaerobic microorganisms at present, but the process is complex, the operation is complicated, the anaerobic roller tube cannot be manufactured in batches, the anaerobic roller tube can only be manufactured at present, and the effect of taking and using the anaerobic roller tube immediately cannot be achieved. Therefore, there is an urgent need to establish a method for culturing anaerobic microorganisms which is easy to operate, efficient and free from time and space limitations.

Disclosure of Invention

In view of the above circumstances, an object of the present invention is to provide a microbial anaerobic culture medium gas conditioning apparatus and an oxygen-removed gas conditioning method, which can adjust gas composition, provide a strict anaerobic environment, observe and sample at any time, and quantitatively detect the growth status of microorganisms.

The invention provides an anaerobic microorganism culture medium deoxygenation device capable of adjusting gas composition, which comprises a controller, a main gas path and branch gas paths, wherein the main gas path is sequentially connected with a vacuum system, a valve system I, an air pressure detection system I, a valve system II, a valve system III, an air pressure detection system II and a gas storage system from a gas outlet communicated with the outside;

a plurality of branch gas paths I are arranged between the air pressure detection system 1 and the valve system II, a valve I is arranged at the bottom end of each branch gas path I, and the other end of each valve I is hermetically connected with a culture container needing to establish an anaerobic environment;

and a branch gas circuit II is arranged between the valve system II and the gas storage system, a valve II is arranged at the tail end of the branch gas circuit II, and a gas needle is connected to the bottom of the valve II and communicated with the external environment.

The controller is used for receiving data of the air pressure detection system I and the air pressure detection system II and controlling the opening and closing of the valve system I, the valve system II and the valve system III.

The valve system II comprises an electromagnetic valve 2 and a main valve, the electromagnetic valve 2 is opened and closed by a controller, and the main valve is manually opened and closed.

The gas storage system comprises an argon gas storage system, a nitrogen gas storage system and a carbon dioxide storage system.

The sealed container comprises a sealed penicillin bottle and a sealed Hungate tube.

An oxygen removal method of an anaerobic microorganism culture medium oxygen removal device based on adjustable gas composition comprises the following steps:

putting a culture medium required by the growth of anaerobic microorganisms into an open container, and inserting the air needle into the bottom of the container; opening a gas storage system, and adjusting the gas pressure to be between 0MPa and 1.5 MPa; opening the valve II, and aerating the culture medium in the container for 0-30 min;

after the aeration is finished, closing the valve II, hermetically connecting the aerated container with the valve I, opening the valve system III, and adjusting the air pressure of an air path to be between 1.5 and 2.0 MPa; sequentially opening a valve system II and a valve I on the main air path;

and thirdly, opening the valve system I through the controller, closing the valve system II, opening the vacuum system, when the air pressure detection system I detects that the air pressure is-0.15-0.05 MPa, closing the valve system I through the controller, closing the valve system II, increasing the air pressure on the main air path and the branch air path, when the air pressure detection system I detects that the air pressure is 0.08-0.15 MPa, opening the valve system I through the controller, closing the valve system II, repeating the steps for 10-50 times, and finally, when the air pressure detection system I is 0.08-0.15 MPa, closing the valve system I through the controller, closing the valve system II, taking down the container, thereby obtaining the anaerobic microorganism culture medium after the gas composition is adjusted.

An automatic anaerobic microorganism culture medium preparation device comprises a main gas path, a box body and a controller, wherein a support ring is detachably connected to the upper surface of the box body, a bottom plate is fixedly connected to the lower surface inside the box body, the main gas path is placed inside the bottom plate, a vacuum meter, a first electromagnetic valve and a vacuum pump are sequentially communicated with the right side of the main gas path from front to back, the vacuum pump is placed on the right side of the upper surface of the bottom plate, a first branch gas path, a second valve, a third branch gas path and a fourth branch gas path are communicated with the bottom of the main gas path close to the left side of the vacuum meter, a fourth valve is communicated between the fourth branch gas paths, a third valve is communicated between the third branch gas paths, a second branch gas path is communicated between the second valves, a first valve is communicated between the first branch gas paths, and penicillin bottles are arranged below the first branch gas path and the second, a Hunter tube is arranged below the third branch gas path and the fourth branch gas path, the Hunter tube and the penicillin bottle are placed in front of the upper surface of the bottom plate, the left side of the main gas path is communicated with a main valve and a second electromagnetic valve, the left side of the main gas path, which is close to the lower part of the second electromagnetic valve, is communicated with a fifth branch gas path, a fifth valve is communicated between the fifth branch gas paths, an open bottle is arranged below the fifth valve, the open bottle is placed on the left side of the upper surface of the bottom plate, one side, far away from the vacuum gauge, of the main gas path is divided into three branches, a first flow valve and a first pressure gauge are communicated between one of the branches, an argon gas storage device is inserted at the bottom of the branch, a second flow valve and a second pressure gauge are communicated between the other branches, a nitrogen storage device is inserted at the bottom of the other branch, and a third flow valve and a third pressure gauge are communicated between the last branch, and the bottom of the last branch is inserted with a carbon dioxide storage, the nitrogen storage and the argon storage are respectively arranged behind the upper surface of the bottom plate, the controller is arranged above the inside of the box body, and the flow valve I, the flow valve II, the flow valve III, the electromagnetic valve I, the electromagnetic valve II, the vacuum pump and the vacuum meter are respectively and electrically connected with the controller through data lines.

The argon gas storage device, the nitrogen gas storage device and the carbon dioxide storage device form a gas storage device, the first branch gas circuit, the second branch gas circuit, the third branch gas circuit, the fourth branch gas circuit and the fifth branch gas circuit form a gas distribution branch, one end of the gas distribution branch is communicated with the main gas circuit, and the other end of the gas distribution branch is communicated with the penicillin bottle, the Henkete tube and the open bottle.

The box body is connected with a box door in a rotating mode in front of the box body, and a visual window is arranged inside the box door.

Advantageous effects

The invention has the following advantages:

1. according to the anaerobic microorganism culture medium deoxygenation device capable of adjusting gas composition, the device operation only needs to input device operation parameters through a human-computer exchange interface of a controller, the flow valve is controlled by a computer to adjust the gas flow, the computer automatically controls the starting and closing of the vacuum pump and the opening and closing of the electromagnetic valve.

2. By adopting the anaerobic microorganism culture medium deoxygenation device capable of adjusting gas composition, the culture medium without oxygen can be rapidly prepared in batches, and the culture environment of microorganisms is anaerobic.

3. The anaerobic sealed penicillin bottle or the Hencaut tube filled with the culture medium and prepared by the anaerobic microorganism culture medium deoxygenating device capable of adjusting gas composition can be stored for a long time at room temperature after being sterilized, and can be directly inoculated with anaerobic microorganisms to be cultured without secondary sterilization during use, so that the experimental operation flow is greatly simplified.

4. When the anaerobic microorganism is cultured, the real-time qualitative and quantitative monitoring of the growth of the anaerobic microorganism can be realized.

5. The invention relates to an anaerobic microorganism culture medium deoxygenation device capable of adjusting gas composition, which comprises a controller, a plurality of branch gas circuits, a plurality of pressure meters, a plurality of flow valves, a plurality of electromagnetic valves, a vacuum meter, a vacuum pump, a main gas circuit, a plurality of valves and the like, wherein the branch gas circuits are used for connecting sealed penicillin bottles, sealed Henkel tubes and open bottles, gas is stored in a memory, special gas required by microorganisms can be provided by using the memory, microbial preparation is facilitated, the pressure meters are used for detecting the pressure condition of an air outlet of the memory, the flow valves are used for detecting the current gas flow condition, the electromagnetic valves are used for controlling the opening and closing of the pipelines, the flow valves, the electromagnetic valves, the vacuum meters and the vacuum pump are all connected with the controller, a worker can know the preparation condition at any time, the improved device enables the microorganisms to be in an anaerobic environment, and a box, this chamber door has transparent visual window, and the staff can see through visual window and observe or open the chamber door and take a sample, and easy operation is convenient.

6. The invention has low preparation cost and ingenious structural design, can easily obtain a sterile environment which completely meets the requirements in a laboratory, saves a large amount of manpower and material resources, improves the working efficiency, simplifies the experiment and the working process, can ensure that vast scientific research personnel can culture microorganisms more conveniently and improve the scientific research efficiency.

Drawings

FIG. 1 is a diagram of the gas path for implementing the anaerobic microorganism culture medium oxygen removal device capable of adjusting the gas composition.

Figure 2 the pressure change of the gas in the hunter tube stored for different lengths of time.

Figure 3 growth curves of bifidobacterium infantis in anaerobic RCM medium at room temperature for different periods of time.

Fig. 4 is a schematic view of the entire structure of embodiment 2.

Fig. 5 is a schematic partial top view structure of embodiment 2.

Fig. 6 is a diagram showing the piping developed connection in example 2.

Fig. 7 is an external structural view of embodiment 2.

Detailed Description

Example 1

The embodiment provides an anaerobic microorganism culture medium oxygen removal device capable of adjusting gas composition, and the gas path structure of the device is shown in fig. 1.

Anaerobic microorganism culture medium deaerating plant that adjustable gas constitutes includes system controller, manometer, gas flow valve, solenoid valve, vacuometer, vacuum pump, gas circuit system and valve, and its structure is as follows:

the gas circuit system comprises a main gas circuit and a plurality of branch gas circuits, wherein one end of the main gas circuit is provided with a vacuum pump, the other end of the vacuum pump is connected with the outside atmosphere, and the vacuum pump is used for pumping out gas in the gas circuit. The other end of the vacuum pump is connected with an electromagnetic valve 1 through a gas circuit pipeline, and the other end of the electromagnetic valve 1 is provided with a vacuum meter for detecting the vacuum degree of the gas circuit system. The electromagnetic valve 1, the vacuum pump and the vacuum meter are all connected with the controller through data lines, and the controller can control the electromagnetic valve 1, the vacuum pump and the vacuum meter to be opened and closed.

The other end of main gas circuit is equipped with gas storage ware, gas storage ware includes the argon gas bomb, nitrogen gas bomb and carbon dioxide bomb, and manometer 1 is connected to the gas outlet of argon gas bomb, and manometer 2 is connected to the gas outlet of nitrogen gas bomb, and manometer 3 is connected to the gas outlet of carbon dioxide bomb, manometer 1, manometer 2, manometer 3's front end is equipped with flow valve 1, flow valve 2, flow valve 3 respectively, and flow valve 1, flow valve 2, flow valve 3's the other end all inserts in the main gas circuit, and flow valve 1, flow valve 2, flow valve 3 pass through the data line and link to each other with the controller, and the switching of flow valve can be controlled to the controller.

The main gas circuit comprises a vacuum pump, an electromagnetic valve 1, a vacuum meter, an electromagnetic valve 2, a main valve and a gas storage from left to right.

The electromagnetic valve 1 is used for controlling air inlet and outlet of the air path system and the outside, and the electromagnetic valve 2 is used for controlling on-off of an air path in the air path system.

A plurality of

branch gas circuits

1, 2, 3 and 4 are arranged between the vacuum meter and the electromagnetic valve 2, the bottom end of each gas circuit is provided with a valve, the other end of each valve is connected with a container needing to establish an anaerobic environment, and the container can be a sealed penicillin bottle, a sealed Hencauter tube or other closed containers; the number of the

branch gas circuits

1, 2, 3 and 4 is not limited to four, and any number of the branch gas circuits can be used, so that a plurality of anaerobic culture containers can be conveniently treated at the same time.

A branch gas circuit 5 is arranged between the main valve and the gas storage device, a valve 5 is arranged at the tail end of the branch gas circuit 5, and the valve 5 is communicated with the external environment through a gas needle with the length being more than 30 cm.

Based on the gas path, the anaerobic microorganism culture medium deoxygenation method capable of adjusting the gas composition is provided, and the method comprises the following specific steps:

and opening the controller, setting the running state of the vacuum pump on the controller to be normally open, setting the vacuum degree of the vacuum meter to be-0.15-0.05 MPa, and setting the pressure value to be 0.08-0.15 MPa. According to the gas demand when the microorganism is cultured, the argon, nitrogen or carbon dioxide flow valve is adjusted through the controller, thereby controlling the gas composition and the gas proportion in the main gas circuit. Such as: argon, nitrogen and carbon dioxide in a ratio of 1:2: 7.

Firstly, filling a culture medium required by the growth of anaerobic microorganisms into a Hungat tube or a penicillin bottle, wherein the volume of the liquid is not more than 1/3 of the volume of the Hungat tube or the penicillin bottle; then inserting a needle head of a stainless steel air needle at the tail end of the valve 5 into the bottom of the open Hencaut tube or the penicillin bottle; opening a valve of the gas storage cylinder, adjusting the opening of a valve, increasing the air pressure in the main pipeline, and controlling the pressure value of a pressure gauge to be between 0MPa and 1.5 MPa; opening the valve 5, adjusting the flow valve through a controller, and aerating the culture medium in the Hencatel tube or the penicillin bottle for 0-30 min; after aeration is finished, the valve 5 is closed, then the just aerated Hencai tube or Xilin bottle containing the culture medium is sealed by a rubber plug, and the aluminum cover is pressed tightly by a gland clamp for fixation. Then, one end of a needle head of 8-10cm is respectively connected with a branch gas circuit 1, a branch gas circuit 2, a branch gas circuit 3 and a branch gas circuit 4, and the other end of the needle head penetrates through a rubber plug and is connected with the upper space of the Hencott tube or the penicillin bottle; opening a gas storage cylinder valve, and adjusting the opening of a valve to enable the pressure value of a pressure gauge 1, a pressure gauge 2 or a pressure gauge 3 to be respectively indicated between 1.5MPa and 2.0 MPa; and sequentially opening a main valve on the main gas circuit, a valve 1 on the branch gas circuit 1, a valve 2 on the branch gas circuit 2, a valve 3 on the branch gas circuit 3 and a valve 4 on the branch gas circuit 4. Opening an electromagnetic valve 1 through a controller, closing an electromagnetic valve 2, starting a vacuum pump, connecting the vacuum pump with a main gas circuit between the electromagnetic valve 1 and the electromagnetic valve 2, a branch gas circuit 1, a branch gas circuit 2, a branch gas circuit 3 and a branch gas circuit 4, when the pressure of the vacuum meter is-0.15 to-0.05 MPa, feeding back a signal to a controller through a data line by the vacuum meter, closing the electromagnetic valve 1 through the controller, opening the electromagnetic valve 2, increasing the gas pressure on the main gas circuit and the branch gas circuit, when the pressure of the vacuum meter reaches 0.08 to 0.15MPa, feeding back a signal to the controller through the data line by the vacuum meter, closing the electromagnetic valve 2 through the controller, opening the electromagnetic valve 1, vacuumizing a penicillin bottle or a Hungart tube through the vacuum pump, repeating the steps for 10 to 50 times according to the size of the penicillin bottle or the Hungart tube, and finally when the indicated pressure of the vacuum meter reaches 0.08 to 0.15MPa, the vacuum meter feeds back signals to the controller through a data line, the electromagnetic valve 1 and the electromagnetic valve 2 are closed, and at the moment, the upper space of liquid in the penicillin bottle or the Hungate tube is argon, nitrogen, carbon dioxide or the combination gas of the three gases according to different gas component ratios. Taking down the penicillin bottle or the Hencauter tube from the branch gas circuit 1, the branch gas circuit 2, the branch gas circuit 3 or the branch gas circuit 4 respectively to obtain a culture medium, wherein the space of the top layer of the penicillin bottle or the Hencauter tube except the culture medium is an anaerobic environment.

And sterilizing an anaerobic penicillin bottle or a Hencatel tube filled with the culture medium at 121 ℃ for 15 minutes to obtain the anaerobic culture medium without the viable bacteria. In the case of good sealing, the medium can be stored at room temperature for a long time.

When the anaerobic microorganism culture is carried out, the Hencauter tube or the Hencauter bottle filled with the sterile anaerobic culture medium is taken, the anaerobic microorganism to be cultured is inoculated by a sterile injector in a biological safety cabinet or an ultra-clean workbench, and then the inoculated Hencauter tube or the Hencauter tube is placed in a constant temperature incubator or a shaking table for the time required by the growth of the microorganism. During the culturing process, the growth of the microorganisms can be monitored by sampling to detect or directly detecting the absorbance of the microorganism culture medium in a Hencatel anaerobic tube.

Example 2

As shown in fig. 4 to 7, the air passage system according to embodiment 1 provides an automatic anaerobic microorganism preparation device, which includes a main air passage 211, a box body 230 and a controller 232, wherein the upper surface of the box body 230 is detachably connected with a support ring 231, the lower surface inside the box body 230 is fixedly connected with a bottom plate 222, the main air passage 211 is placed inside the bottom plate 222, the right side of the main air passage 211 is sequentially communicated with a vacuum gauge 212, a first electromagnetic valve 214 and a vacuum pump 216 from front to back, the vacuum pump 216 is placed on the right side of the upper surface of the bottom plate 222, the left side, close to the vacuum gauge 212, of the bottom of the main air passage 211 is communicated with a first branch air passage 227, a second valve 204, a third branch air passage 208 and a fourth branch air passage 213, the fourth valve 215 is communicated between the fourth branch air passages 213, the third valve 205 is communicated between the third branch air passages 208, the second valve 204 is communicated with a second branch air passage, a penicillin bottle 221 is arranged below the first branch gas path 227 and the second valve 204, a hunter tube 218 is arranged below the third branch gas path 208 and the fourth branch gas path 213, the hunter tube 218 and the penicillin bottle 221 are arranged in front of the upper surface of the bottom plate 222, the left side of the main gas path 211 is communicated with a main valve 229 and a second solenoid valve 228, the left side of the main gas path 211 close to the second solenoid valve 228 is communicated with a fifth branch gas path 225, a fifth valve 224 is communicated between the fifth branch gas paths 225, an open bottle 223 is arranged below the fifth valve 224 and is arranged on the left side of the upper surface of the bottom plate 222, one side of the main gas path 211 far away from the vacuum gauge 212 is divided into three branches, a first flow valve 201 and a first pressure gauge 202 are communicated between one branch, an argon gas storage 220 is inserted at the bottom of the branch, a second flow valve 207 and a second pressure gauge 206 are communicated between the other branches, and a nitrogen storage 219 is inserted at the bottom of the other branch, a flow valve III 209 and a pressure gauge III 210 are communicated between the last branch, a carbon dioxide storage 217 is inserted into the bottom of the last branch, the carbon dioxide storage 217, the nitrogen storage 219 and the argon storage 220 are respectively arranged behind the upper surface of the bottom plate 222, a controller 232 is arranged above the inner part of the box body 230, and the flow valve I201, the flow valve II 207, the flow valve III 209, the electromagnetic valve I214, the electromagnetic valve II 228, the vacuum pump 216 and the vacuum gauge 212 are respectively and electrically connected with the controller 232 through data lines.

Further, the argon gas storage device 220, the nitrogen gas storage device 219 and the carbon dioxide storage device 217 form a gas storage device, the first branch gas path 227, the second branch gas path 203, the third branch gas path 208, the fourth branch gas path 213 and the fifth branch gas path 225 form a gas branch path, one end of the gas branch path is communicated with the main gas path 11, the other end of the gas branch path is communicated with the penicillin bottle 221, the hunter tube 218 and the open bottle 223, argon gas is stored in the argon gas storage device 220, nitrogen gas is stored in the nitrogen gas storage device 219, and similarly, carbon dioxide is stored in the carbon dioxide storage device 217, and the gas in the storage device provides some substances which cannot be synthesized by the carbon dioxide for microbial preparation.

Furthermore, the number of the gas distribution branches can be any, and the number of the gas distribution branches can be one, two, three or more according to the use requirement of workers.

Further, the branch gas path five 225 is communicated with the external environment.

Further, the front of the box body 30 is rotatably connected with a box door 233, and the inside of the box door 233 is provided with a visual window which is transparent glass, so that a worker can see through the visual window to observe the inside of the box body 230, and open the box door 233, thereby facilitating sampling.

In conclusion, the working process of the invention is as follows: the improved preparation device comprises a controller 232, a plurality of branch gas circuits, a plurality of pressure meters, a plurality of flow valves, a plurality of electromagnetic valves, a vacuum meter 212, a vacuum pump 216, a main gas circuit 211, a plurality of valves and the like, wherein the pressure meters are a first pressure meter 202 connected with an air outlet of an argon gas storage 220, a second pressure meter 206 connected with an air outlet of a nitrogen gas storage 219, a third pressure meter 210 connected with an air outlet of a carbon dioxide storage 217, the flow valves are a first flow valve 201 between the argon gas storage 220 and the main gas circuit 211, a second flow valve 207 between the nitrogen gas storage 219 and the main gas circuit 211, a third flow valve 209 between the carbon dioxide storage 217 and the main gas circuit 211, the plurality of flow valves are connected with the controller 232 through data lines, the electromagnetic valves are a first electromagnetic valve 214 for controlling an air inlet end in the main gas circuit 211 and a second electromagnetic valve 228 for controlling an air outlet end in the main, the vacuum meter 212 is connected with the controller 232 through a data line, the vacuum pump 216 is connected with the controller 232 through a data line, one end of each of the gas distributing branches is connected with the main gas path 211, the other end of each of the gas distributing branches is connected with a container needing to establish an anaerobic environment, the container can be a sealed penicillin bottle 221, a sealed Henkete tube 218 or other closed containers, and the gas distributing branch five 225 is communicated with the external environment.

Example 4

Bifidobacteria are strict anaerobes, and even if trace oxygen remains in the culture environment, the bifidobacteria cannot grow. In this embodiment, an oxygen-free environment for bifidobacterium growth is created by high-purity nitrogen, and with reference to fig. 1 to 3, the specific operation process is as follows:

38g of Clostridium thermocellum culture medium (RCM) (available from Merck, Germany) was accurately weighed, heated and dissolved in 1000mL of distilled water with stirring, after the RCM culture medium is completely dissolved, the RCM culture medium is subpackaged into Henkett tubes with the volume of 30mL, each Henkett tube is filled with 10mL of RCM culture medium solution, then an anaerobic microorganism culture medium oxygen removing device capable of adjusting the gas composition is used, aerating the culture medium in the Hencauter tube, respectively inserting 30cm long needles connected with the branch gas path 5 into the bottom of the Hencauter tube during aeration, then the nitrogen regulating valve and the valve 5 are opened, the gas flow passing through the flow valve 2 is controlled by the controller, aerating the culture medium in the Hencauter tube for 10-30min, closing the nitrogen regulating valve and the valve 5 after the aeration is finished, plugging a T-shaped butadiene-acrylonitrile rubber plug into the Hencauter tube, and locking the rubber plug by an aluminum cover to form a seal for the Hencauter tube.

The sealed Hencott tube is respectively connected with a branch gas circuit 1 or a branch gas circuit 2 or a branch gas circuit 3 or a branch gas circuit 4 through a sterile needle head with the length of 8-10cm, a main valve and a valve 1 or a valve 2 or a valve 3 or a valve 4 connected with the Hencott tube are opened, a controller sets a vacuum pump to be in a normally open mode, the controller sets the gas flow through a flow valve 2 to ensure that the nitrogen pressure displayed by a pressure gauge 2 is 1MPa, the pressure measurement range of a vacuum gauge is set to be negative 0.1MPa to positive 0.1MPa, namely, when the vacuum gauge displays that the nitrogen pressure of a main gas circuit is positive 0.1MPa, the vacuum gauge feeds back a signal to the controller through a data line to close the solenoid valve 2, the solenoid valve 1 is opened, the vacuum pump vacuumizes the Hencott tube connected with the main gas circuit and the branch gas circuit, and when the vacuum gauge displays that the nitrogen pressure of the main gas circuit is reduced from positive 0.1MPa to negative 0.1MPa, the vacuum gauge feeds back a signal to, opening the electromagnetic valve 2, filling nitrogen into the main gas path and the Henkel tube connected with the branch gas path by a high-pressure nitrogen bottle, when the vacuum meter shows that the nitrogen pressure of the main gas path is increased from 0.1MPa to positive 0.1MPa, feeding back a signal to the controller by the vacuum meter through a data line to close the electromagnetic valve 2, opening the electromagnetic valve 1, vacuumizing the Henkel tube connected with the main gas path and the branch gas path by the vacuum pump, repeating vacuumizing and filling for 25 times, replacing the air on the upper layer of the Henkel tube liquid into high-purity nitrogen without oxygen, wherein the air pressure on the upper layer of the Henkel tube liquid is between 0 and 0.1 MPa.

The prepared Hencatel tube containing RCM medium is sterilized at 121 deg.C for 15min, cooled, and stored at room temperature for culturing Bacillus bifidus.

In this example, Bifidobacterium infantis LMG8811(B.infantis LMG8811) was selected as an anaerobic environment for detecting Hencott tubes prepared by an anaerobic microorganism culture medium oxygen removal device capable of adjusting gas composition. Taking a hunter tube containing RCM medium and used for culturing Bifidobacterium which has been left at room temperature for 1 day, 1 week, 1 month, 3 months, 6 months and 12 months respectively, measuring the air pressure in the hunter tube stored for different lengths of time by an air pressure gauge, and detecting the sealing property of the hunter tube according to the change of the air pressure value. Then, 0.1mL of the Bifidobacterium stock solution was inoculated into the above-mentioned Hencatel tube by using a sterile syringe and cultured in a 37 ℃ incubator for 70 hours. During the culture process of the bifidobacterium, the absorbance (OD value) of the bacterial liquid in the Hungart tube is detected in real time by an ultraviolet-visible spectrophotometer, and the growth curve of the bifidobacterium infantis in the RCM culture medium under different standing times is drawn. As shown in fig. 3.

Claims

1. Anaerobic microorganism culture medium deoxidization device that adjustable gas constitutes, including controller, main gas circuit and branch gas circuit, its characterized in that: the main gas path is sequentially connected with a vacuum system, a valve system I, an air pressure detection system I, a valve system II, a valve system III, an air pressure detection system II and a gas storage system from a gas outlet communicated with the outside;

2. Anaerobic microorganism culture medium oxygen removal device that adjustable gas is constituteed, its characterized in that: the valve system II comprises an electromagnetic valve 2 and a main valve, the electromagnetic valve 2 is opened and closed by a controller, and the main valve is manually opened and closed.

3. Anaerobic microorganism culture medium oxygen removal device that adjustable gas is constituteed, its characterized in that: the gas storage system comprises an argon gas storage system, a nitrogen gas storage system and a carbon dioxide storage system.

4. Anaerobic microorganism culture medium oxygen removal device that adjustable gas is constituteed, its characterized in that: the sealed container comprises a sealed penicillin bottle and a sealed Hungate tube.

5. A deoxygenation method of an anaerobic microorganism culture medium deoxygenation device based on adjustable gas composition is characterized by comprising the following steps of: the method comprises the following steps:

6. An automatic anaerobic microorganism culture medium preparation facilities which characterized in that: including main gas circuit (211), box (230) and controller (232), the upper surface of box (230) can be dismantled and is connected with support ring (231), the inside lower fixed surface of box (230) is connected with bottom plate (222), place inside of bottom plate (222) main gas circuit (211), the right side of main gas circuit (211) is from preceding to back in proper order the intercommunication have vacuum gauge (212), solenoid valve (214) and vacuum pump (216), just vacuum pump (216) are placed the upper surface right side of bottom plate (222), the bottom of main gas circuit (211) is close to the left side intercommunication of vacuum gauge (212) has branch gas circuit (227), valve two (204), branch gas circuit three (208) and branch gas circuit four (213), just the intercommunication has valve four (215) between branch gas circuit four (213), the intercommunication has valve three (205) between branch gas circuit three (208), a branch air passage II (203) is communicated between the valve II (204), a valve I (226) is communicated between the branch air passages I (227), penicillin bottles (221) are arranged below the branch air passages I (227) and the valve II (204), Hungate tubes (218) are arranged below the branch air passages III (208) and the branch air passages IV (213), the Hungate tubes (218) and the penicillin bottles (221) are placed in front of the upper surface of the bottom plate (222), a main valve (229) and a solenoid valve II (228) are communicated with the left side of the main air passage (211), a branch air passage fifth (225) is communicated with the left side of the main air passage (211) close to the lower part of the solenoid valve II (228), a valve fifth (224) is communicated between the branch air passages fifth (225), an open bottle (223) is arranged below the valve fifth (224), and the open bottle (223) is placed on the left side of the upper surface of the bottom plate (222), one side of the main gas path (211) far away from the vacuum meter (212) is divided into three branches, a first flow valve (221) and a first pressure gauge (202) are communicated between one branch, an argon storage (220) is connected to the bottom of the branch in an inserted mode, a second flow valve (207) and a second pressure gauge (206) are communicated between the other branches, a nitrogen storage (219) is connected to the bottom of the other branch in an inserted mode, a third flow valve (209) and a third pressure gauge (210) are communicated between the last branch in an inserted mode, a carbon dioxide storage (217) is connected to the bottom of the last branch in an inserted mode, the carbon dioxide storage (217), the nitrogen storage (219) and the argon storage (220) are placed behind the upper surface of the bottom plate (222) respectively, and the controller (232) is installed above the interior of the box body (230), the flow valve I (221), the flow valve II (207), the flow valve III (209), the electromagnetic valve I (214), the electromagnetic valve II (228), the vacuum pump (216) and the vacuum meter (212) are respectively electrically connected with the controller (232) through data lines.

7. The automatic anaerobic microorganism culture medium preparation device according to claim 6, wherein: argon gas storage ware (220), nitrogen gas storage ware (219) with carbon dioxide storage ware (217) constitute the gas storage ware, branch gas circuit one (227), branch gas circuit two (203), branch gas circuit three (208), branch gas circuit four (213) and branch gas circuit five (225) constitute and divide the gas branch road, divide the one end of gas branch road with main gas circuit (211) are linked together, divide the other end of gas branch road with xiLin bottle (221), hunter's pipe (218), uncovered bottle (223) are linked together.

8. The automatic anaerobic microorganism culture medium preparation device according to claim 6, wherein: a box door (233) is rotatably connected to the front of the box body (230), and a visible window is arranged inside the box door (233).