CN114456909A - Deep-sea microorganism separation culture device and culture method - Google Patents
Deep-sea microorganism separation culture device and culture method Download PDFInfo
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Abstract
The invention provides a deep-sea microorganism separation culture device, which comprises a separation culture device, a control acquisition system, a sample injection pipeline and a liquid injection unit, wherein under the condition that the internal pressure and temperature of the separation culture device are consistent with the deep-sea microorganism culture environment, microorganism liquid is injected into the sample injection pipeline through the liquid injection unit, a culture bacterial liquid sample is put into an internal thread of the separation culture device through the sample injection pipeline, the sample slides downwards along the internal thread under the action of gravity, and the microorganism liquid carried by the sample injection pipeline is gradually reduced in the moving process, so that the separation of microorganisms is realized; the invention also provides a deep-sea microorganism separation culture method, the sample slides downwards in the inner thread under the action of gravity, the automatic lineation separation of the microorganism liquid obtained by environmental sampling and enrichment can be realized under the condition of marine in-situ environment, the efficiency of microorganism solid separation culture is effectively improved, and an important basic means is provided for the separation culture of efficient marine special microorganism.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a deep-sea microbial separation culture device and a deep-sea microbial separation culture method.
Background
Huge microbial resources are stored in the ocean, and the investment of a large amount of financial resources and material resources in various countries in the world is attracted by the wide application prospect of the huge microbial resources. The marine environment is special, has the characteristics of oligotrophism, low temperature, low oxygen and high pressure, and the special environments cause the diversity and the particularity of marine microorganism species, such as stress resistance (salt resistance, high pressure resistance, cold resistance and the like), biodegradation characteristics (degradation on organic pollutants such as petroleum and the like and heavy metal ions) and other excellent biological characteristics (insect killing and sterilization).
The conventional microorganism solid plate separation method mainly comprises a coating plate method and an inoculation and marking method, and needs complicated processes of disinfection, inoculation and marking, is difficult to carry out automatic screening and needs professional operators. In addition, microorganisms in marine environment generally exist in high-pressure environment, and single colony streaking separation can be carried out only after pressure is released in the separation process, so that the development of marine special environment microorganism culture engineering is restricted by the technology, and the understanding of marine microorganisms is further limited.
In this regard, the prior art discloses a deep sea microbial cultivation tank comprising: the device comprises a linear bearing, a tension spring, a pressure compensation cavity, a fixed top plate, a deep sea motor assembly, a fixed bottom plate, a hose and a culture cabin body; according to the scheme, the deep sea motor assembly rotates to jack the end cover of the culture cabin body, microorganism enrichment culture is carried out in a completely open state, and in the process of distribution and recovery, the end cover of the culture cabin body is closed to seal the microorganism culture cabin body. Although the enrichment culture of microorganisms in a deep sea in-situ state can be realized, marine microorganisms are not subjected to separation culture, and the success rate of culture cannot be effectively improved.
Disclosure of Invention
In order to solve at least one technical defect, the invention provides a deep-sea microorganism separation culture device and a culture method, which realize an automatic streaking separation process under a high-pressure environment, effectively improve the pure culture efficiency and provide an important basic means for separating and culturing high-efficiency marine special microorganisms.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a deep sea microorganism separation culture device comprises a separation culture device, a control acquisition system, a pressure control system, a temperature control system, a sample introduction pipeline and a liquid injection unit; the separation incubator comprises a base, an upper cover and a screw rod; the base is in threaded connection with the upper cover; the surface of the inner wall of the base is provided with an internal thread, and the shape of the internal thread is U-shaped; the outer surface of the screw is provided with an external thread which is matched and connected with the internal thread, and the shape of the external thread is similar to an L shape; after the base and the screw rod are connected through the internal thread and the external thread, a certain gap is formed between the internal thread and the external thread and is used for filling a solid culture medium; the sample introduction pipeline is arranged on the upper cover and used for putting a culture bacteria liquid sample into the separation culture device; the liquid injection unit is connected with the sample injection pipeline and is used for injecting the microbial liquid into the sample injection pipeline; the pressure control system and the temperature control system are respectively connected with the separation culture device and are used for ensuring that the internal pressure and temperature of the separation culture device are consistent with the deep-sea microbial culture environment; the control end of the pressure control system, the acquisition end of the pressure control system, the control end of the temperature control system, the acquisition end of the temperature control system, the control end of the sample injection pipeline and the control end of the liquid injection unit are electrically connected with the control acquisition system.
After the solid culture medium is solidified, taking out the screw, closing the upper cover, and opening the pressure control system and the temperature control system to ensure that the internal temperature and pressure of the separation culture device are consistent with the environment for the growth of microorganisms; injecting microbial bacteria liquid into the sample injection pipeline by the liquid injection unit, putting a culture bacteria liquid sample into the internal thread of the separation culture device by the sample injection pipeline, enabling the sample to slide downwards along the internal thread under the action of gravity, and gradually reducing the amount of the microbial bacteria liquid carried by the sample in the moving process to realize the separation of microorganisms; the separated microorganisms grow on a solid culture medium, so that the separation culture of the microorganisms is realized.
In the scheme, the sample slides downwards in the inner thread under the action of gravity, so that the automatic streaking separation of the microbial liquid enriched by environmental sampling can be realized under the marine in-situ environmental condition, the difficulty that the pressure release is required in the conventional flat plate streaking separation culture is improved, the efficiency of the microbial solid separation culture is effectively improved, and an important basic means is provided for the separation culture of efficient marine special microbial bacteria.
The separation culture device is the core of the scheme, and can directly realize the separation process of the flora under the in-situ high-pressure environment; meanwhile, inoculation and lineation operations can be carried out by utilizing an inoculating loop without depending on professionals, and the problems that mixed bacteria are introduced nonstandard in the disinfection process of the inoculating loop or the temperature of the inoculating loop is not proper, so that the selected bacteria die and the lineation growth process fails are solved.
In the scheme, the control acquisition system comprises a central control system, a computer and the like, and the functions of changing various environmental data information, acquiring, processing, storing, outputting images and the like in real time in the separation and culture processes of the enriched marine microorganisms in the high-pressure environment are realized.
The gasket is arranged in the middle of one side of the internal thread and used for ensuring a certain gap between the internal thread and the external thread when the internal thread is matched with the external thread on the screw rod and used for filling a solid culture medium.
The pressure control system comprises an air storage tank, a pressure regulating valve, an air compressor, a booster pump, an air injection pipeline, a pressure sensor and an air injection valve; the air compressor, the booster pump, the air storage tank and the pressure regulating valve are sequentially connected through an air injection pipeline, and finally the air injection pipeline is connected with the upper cover through an air injection valve; the pressure sensor probe is arranged in the separation incubator, and the signal output end of the pressure sensor probe is electrically connected with the control acquisition system; the pressure regulating valve control end, the air compressor control end, the booster pump control end and the gas injection valve control end are electrically connected with the control acquisition system.
Wherein the temperature control system comprises a water bath device and a temperature sensor; the water bath device is wrapped on the outer wall of the separation incubator, and the control end of the water bath device is electrically connected with the control acquisition system; the temperature sensor probe is arranged in the separation incubator, and the signal output end of the temperature sensor probe is electrically connected with the control acquisition system.
In the above scheme, pressure sensor and temperature sensor's setting is arranged in the control and cultivates the in-process, and the environmental parameter of isolated culture ware changes, is convenient for adjust in real time, ensures the stability of cultivateing the environment.
The sample introduction pipeline comprises a sample throwing valve, a pipeline main body, a sample throwing valve and a sample carrying ball; the pipeline main body is fixedly arranged on the upper cover and is communicated with the internal thread; the sample throwing valve is fixedly arranged on the pipeline main body outside the upper cover; the sample throwing valve is fixedly arranged between the sample throwing valve and the pipeline main body outside the upper cover; the sample throwing valve control end and the sample throwing valve control end are electrically connected with the control acquisition system; wherein: opening a sample throwing valve, throwing sample carrying balls into the pipeline main body, and closing the sample throwing valve; starting the liquid injection unit to inject microbial bacteria liquid into the pipeline main body, so that the sample-carrying pellet is immersed in the microbial bacteria liquid; then open and throw a kind valve for take kind pellet to drop to the internal thread of isolated culture ware in, take kind pellet to slide down along the internal thread under the action of gravity, at the removal in-process, the microorganism fungus liquid that it carried will progressively decrement, realized the separation to the microorganism.
The liquid injection unit comprises a microorganism culture kettle, a micro injection pump and a liquid injection pipeline; the microorganism culture kettle is connected with the pipeline main body through a liquid injection pipeline, and the micro injection pump is arranged on the liquid injection pipeline; the control end of the micro-injection pump is electrically connected with the control acquisition system; the microorganism culture kettle is used for culturing deep-sea microorganisms and producing microorganism liquid, and the microorganism injection pump pumps the microorganism liquid into the pipeline main body.
The scheme also provides a deep-sea microorganism separation culture method, which is realized by applying the deep-sea microorganism separation culture device and specifically comprises the following steps:
s1: cleaning and sterilizing the deep-sea microorganism separation culture device, and filling a solid culture medium;
s2: determining the pressure value in the isolated culture device according to the environmental pressure value of deep-sea microorganism culture, opening a gas injection valve, and injecting gas or inert gas required by microorganism culture into the isolated culture device by using a pressure control system until the pressure value in the isolated culture device is consistent with the pressure value of microorganism culture;
s3: determining a temperature value in the isolated culture device according to an environment temperature value for deep-sea microorganism culture, and starting a temperature control system to enable the temperature in the isolated culture device to be consistent with the microorganism culture environment;
s4: injecting microbial bacteria liquid into the sample injection pipeline by the liquid injection unit, putting a culture bacteria liquid sample into the internal thread of the separation culture device by the sample injection pipeline, enabling the sample to slide downwards along the internal thread under the action of gravity, and gradually reducing the amount of the microbial bacteria liquid carried by the sample in the moving process to realize the separation of microorganisms;
s5: the separated microorganisms grow on a solid culture medium, so that the separation culture of the microorganisms is realized.
In step S1, the process of cleaning and sterilizing the deep-sea microbial isolation and culture device specifically includes: cleaning the isolated culture device, opening the upper cover, the base and the screw rod, and wiping the upper cover by using 75% alcohol; and filling 75% alcohol into the base, recovering 75% alcohol from the base after alcohol disinfection, and sterilizing for 15min under ultraviolet.
In step S1, the process of loading the solid medium specifically includes:
s11: after the sterilization is finished, rotating the screw rod to the inside of the base in the anticlockwise direction, adding the solid culture medium which is sterilized in advance, and then closing the upper cover;
s12: and when the solid culture medium is solidified, opening the upper cover, fixing the screw rod, rotating by holding the side wall of the base, taking out the screw rod in a rotating manner, and finally closing the upper cover to finish the process of filling the solid culture medium.
Wherein, step S4 specifically includes the following steps:
s41: opening a sample throwing valve, and closing the sample throwing valve after throwing sample carrying balls into the pipeline main body;
s42: starting a micro-injection pump to pump the microbial liquid in the microbial culture kettle into the pipeline main body, so that the sample-carrying pellet is immersed in the microbial liquid;
s43: open and throw a kind valve for take kind pellet to drop to the internal thread of isolated culture ware in, take kind pellet to slide down along the internal thread under the action of gravity, at the removal in-process, the microorganism fungus liquid that it carried will progressively decrement, realized the separation to the microorganism.
The scheme mainly relates to a deep-sea microorganism separation culture device and a culture method, and provides an automatic lineation separation culture device and an automatic separation culture process under the condition of high-pressure environment. The scheme overcomes the difficulty that marine special strains can be separated after pressure is released, the device can realize automatic operation scribing without professional operators, growth and separation of microorganisms are realized by moving and automatically scribing with the small balls carrying bacterial liquid, the labor cost is reduced, and the screening and utilization efficiency of engineering bacteria is improved.
Compared with the existing solid plate separation culture technology, the scheme does not need professionals to pick bacterial liquid for streak inoculation after sterilization by using an inoculating loop, can realize automatic streak by only injecting a small amount of bacterial liquid, completes separation work and improves the universality of operators; compared with the difficulty that the existing marine special strains can be separated only by pressure release in the screening process, the scheme realizes the separation work of single bacterial colony in the pressure maintaining process, improves the screening efficiency of the microbes difficult to culture and improves the screening and cultivating efficiency of the engineering bacteria with special functions.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
the invention provides a deep-sea microorganism separation culture device and a culture method, wherein a sample slides downwards in an internal thread under the action of gravity, so that the automatic streaking separation of an environment sampling enriched microorganism liquid can be realized under the condition of a marine in-situ environment, the difficulty that the pressure release is required in the conventional flat plate streaking separation culture is improved, the microorganism solid separation culture efficiency is effectively improved, and an important basic means is provided for the separation culture of efficient marine special microorganism bacteria.
Drawings
FIG. 1 is a schematic structural diagram of a deep-sea microorganism separation and culture apparatus according to the present invention;
FIG. 2 is a schematic diagram of a circuit module connection of the control acquisition system according to the present invention;
FIG. 3 is a schematic flow chart of the deep-sea microorganism isolation and culture method according to the present invention;
FIG. 4 is a schematic structural diagram of a screw according to an embodiment of the present invention;
wherein: 1. a separate incubator; 11. a base; 12. an upper cover; 13. a screw; 131. a handle; 2. controlling the acquisition system; 3. a pressure control system; 31. a gas storage tank; 32. a pressure regulating valve; 33. an air compressor; 34. a booster pump; 35. a gas injection pipe; 36. a pressure sensor; 37. a gas injection valve; 4. a temperature control system; 41. a water bath device; 42. a temperature sensor; 5. a sample introduction pipeline; 51. a sample throwing valve; 52. a pipe body; 53. a sample feeding valve; 54. carrying a sample small ball; 6. a liquid injection unit; 61. a microbial culture kettle; 62. a micro-injection pump; 63. and a liquid injection pipeline.
Detailed Description
The drawings are for illustrative purposes only and are not to be construed as limiting the patent;
the embodiment is a complete use example and has rich content
For the purpose of better illustrating the embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product;
it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.
The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
Example 1
As shown in fig. 1, fig. 2 and fig. 4, a deep-sea microorganism separation culture device is provided, which comprises a separation culture device 1, a control acquisition system 2, a pressure control system 3, a temperature control system 4, a sample injection pipeline 5 and a liquid injection unit 6; wherein: the separate incubator 1 comprises a base 11, an upper cover 12 and a screw 13; the base 11 is in threaded connection with the upper cover 12; the inner wall of the base 11 is provided with internal threads, and the shape of the internal threads is U-shaped; the outer surface of the screw rod 13 is provided with an external thread which is matched and connected with the internal thread 11, and the shape of the external thread is similar to an L shape; after the base 11 and the screw 13 are connected through the internal thread and the external thread, a certain gap is formed between the internal thread and the external thread and is used for filling a solid culture medium; the sample introduction pipeline 5 is arranged on the upper cover 12 and is used for feeding a culture bacteria liquid sample into the separation culture device 1; the liquid injection unit 6 is connected with the sample injection pipeline 5 and is used for injecting microbial liquid into the sample injection pipeline 5; the pressure control system 3 and the temperature control system 4 are respectively connected with the separation incubator 1 and are used for ensuring that the internal pressure and temperature of the separation incubator 1 are consistent with the deep-sea microbial culture environment;
the control end of the pressure control system 3, the acquisition end of the pressure control system 3, the control end of the temperature control system 4, the acquisition end of the temperature control system 4, the control end of the sample injection pipeline 5 and the control end of the liquid injection unit 6 are electrically connected with the control acquisition system 2;
after the solid culture medium is solidified, taking out the screw 13, closing the upper cover 12, and opening the pressure control system 3 and the temperature control system 4 to ensure that the internal temperature and pressure of the separation culture device 1 are consistent with the environment for the growth of microorganisms; injecting microbial bacteria liquid into the sample injection pipeline 5 by the liquid injection unit 6, putting a culture bacteria liquid sample into the internal thread of the separation culture device 1 by the sample injection pipeline 5, sliding the sample downwards along the internal thread under the action of gravity, and gradually decreasing the carried microbial bacteria liquid in the moving process to realize the separation of microorganisms; the separated microorganisms grow on a solid culture medium, so that the separation culture of the microorganisms is realized.
In the specific implementation process, the sample slides downwards in the internal thread under the action of gravity, so that the automatic streaking separation of the microbial liquid enriched by environmental sampling can be realized under the marine in-situ environmental condition, the difficulty that the pressure release is required in the conventional flat plate streaking separation culture is improved, the efficiency of microbial solid separation culture is effectively improved, and an important basic means is provided for the separation culture of efficient marine special microbial bacteria.
The separation culture device 1 is the core of the scheme, and can directly realize the separation process of flora under the in-situ high-pressure environment; meanwhile, inoculation and lineation operations can be carried out by utilizing an inoculating loop without depending on professionals, and the problems that mixed bacteria are introduced nonstandard in the disinfection process of the inoculating loop or the temperature of the inoculating loop is not proper, so that the selected bacteria die and the lineation growth process fails are solved.
In the specific implementation process, the control acquisition system 2 comprises a central control system, a computer and the like, and realizes the functions of various environmental data information changes, real-time acquisition, processing, storage, image output and the like of the enriched marine microorganisms in the high-pressure environment separation and culture processes.
More specifically, be provided with the gasket on the lateral wall of internal thread one side, the gasket is located the middle part of internal thread one side for when internal thread and the external screw thread on the screw rod mutually mate, guarantee to have certain space between internal thread and the external screw thread, be used for packing into solid culture medium.
More specifically, the pressure control system 3 includes an air tank 31, a pressure regulating valve 32, an air compressor 33, a booster pump 34, an air injection pipe 35, a pressure sensor 36, and an air injection valve 37; the air compressor 33, the booster pump 34, the air storage tank 31 and the pressure regulating valve 32 are sequentially connected through an air injection pipeline 35, and finally the air injection pipeline 35 is connected with the upper cover 12 through an air injection valve 37; the probe of the pressure sensor 36 is arranged in the separation incubator 1, and the signal output end of the probe is electrically connected with the control acquisition system 2; the control end of the pressure regulating valve 32, the control end of the air compressor 33, the control end of the booster pump 34 and the control end of the gas injection valve 37 are electrically connected with the control acquisition system 2.
More specifically, the temperature control system 4 includes a water bath device 41 and a temperature sensor 42; the water bath device 41 is wrapped on the outer wall of the separation incubator 1, and the control end of the water bath device is electrically connected with the control acquisition system 2; the temperature sensor 42 probe is arranged in the separation culture device 1, and the signal output end of the temperature sensor is electrically connected with the control acquisition system 2.
In the specific implementation process, the pressure sensor 36 and the temperature sensor 42 are arranged to monitor the change of the environmental parameters of the isolated culture device 1 during the culture process, so as to facilitate real-time adjustment and ensure the stability of the culture environment.
More specifically, the sample introduction pipeline 5 comprises a sample throwing valve 51, a pipeline main body 52, a sample throwing valve 53 and a sample carrying ball 54; the pipeline main body 52 is fixedly arranged on the upper cover 12; the sample throwing valve 51 is fixedly arranged on the pipeline main body 52 outside the upper cover 12, and the sample throwing valve 53 is fixedly arranged on the sample throwing valve 51 and the pipeline main body 52 outside the upper cover 12; the control end of the sample throwing valve 51 and the control end of the sample throwing valve 53 are electrically connected with the control acquisition system 2; wherein: firstly, opening a sample throwing valve 51, throwing a sample carrying ball 54 into a pipeline main body 52, and then closing the sample throwing valve 51; starting the liquid injection unit 6 to inject microbial bacteria liquid into the pipeline main body 52, so that the sample carrying pellet 54 is immersed in the microbial bacteria liquid; then the sample feeding valve 53 is opened, so that the sample carrying small balls 54 fall into the internal threads of the separation culture device 1, the sample carrying small balls 54 slide downwards along the internal threads under the action of gravity, and the microorganism liquid carried by the sample carrying small balls gradually decreases gradually in the moving process, thereby realizing the separation of microorganisms.
More specifically, the liquid injection unit 6 comprises a microorganism culture kettle 61, a micro injection pump 62 and a liquid injection pipeline 63; the microorganism culture kettle 61 is connected with the pipeline main body 52 through a liquid injection pipeline 63, and a micro injection pump 62 is arranged on the liquid injection pipeline 63; the control end of the micro-injection pump 62 is electrically connected with the control acquisition system 2; the microorganism culture kettle 61 is used for culturing deep-sea microorganisms and producing microorganism liquid, and the microorganism injection pump (62) pumps the microorganism liquid into the pipeline main body 52.
In the specific implementation process of the embodiment, the sample slides downwards in the inner thread under the action of gravity, so that the automatic streaking separation of the microbial liquid enriched by environmental sampling can be realized under the marine in-situ environmental condition, the difficulty that the pressure release is required in the conventional flat plate streaking separation culture is improved, the efficiency of microbial solid separation culture is effectively improved, and an important basic means is provided for the separation culture of efficient marine special microbial bacteria.
Example 2
More specifically, on the basis of embodiment 1, as shown in fig. 3, this embodiment proposes a deep-sea microorganism separation culture method implemented by using a deep-sea microorganism separation culture apparatus, which specifically includes the following steps:
s1: cleaning and sterilizing the deep-sea microorganism separation culture device, and filling a solid culture medium;
s2: determining the pressure value in the isolated culture device 1 according to the environmental pressure value of deep-sea microorganism culture, opening the gas injection valve 37, and injecting gas or inert gas required by microorganism culture into the isolated culture device 1 by using the pressure control system 3 until the pressure value in the isolated culture device 1 is consistent with the pressure value of microorganism culture;
s3: determining the temperature value in the isolated incubator 1 according to the environmental temperature value of deep-sea microorganism culture, and starting the temperature control system 4 to enable the temperature in the isolated incubator 1 to be consistent with the microorganism culture environment;
s4: injecting microbial bacteria liquid into the sample injection pipeline 5 by the liquid injection unit 6, putting a culture bacteria liquid sample into the internal thread of the separation culture device 1 by the sample injection pipeline 5, sliding the sample downwards along the internal thread under the action of gravity, and gradually decreasing the carried microbial bacteria liquid in the moving process to realize the separation of microorganisms;
s5: the separated microorganisms grow on a solid culture medium, so that the separation culture of the microorganisms is realized.
More specifically, in step S1, the process of cleaning and sterilizing the deep sea microbe separation and culture apparatus specifically includes: cleaning the isolated culture device 1, opening the upper cover 12, the base 11 and the screw 13, and wiping the upper cover 12 with 75% alcohol; and filling 75% alcohol into the base 11, recovering the 75% alcohol in the base after the alcohol disinfection is finished, and sterilizing for 15min under ultraviolet.
More specifically, in step S1, the process of loading the solid medium is specifically as follows:
s11: after the sterilization is finished, rotating the screw 13 into the base 11 in the anticlockwise direction, adding the sterilized solid culture medium, and closing the upper cover 12;
s12: when the solid culture medium is solidified, the upper cover 12 is opened, the screw 13 is fixed, the screw 13 is rotated by holding the side wall of the base 11, the screw 13 is taken out in a rotating way, and finally the upper cover 12 is closed, so that the process of filling the solid culture medium is completed.
More specifically, step S4 specifically includes the following steps:
s41: opening the sample throwing valve 51, throwing the sample carrying ball 54 into the pipeline main body 52, and closing the sample throwing valve 51;
s42: starting a micro-injection pump 62 to pump the microbial liquid in the microbial culture kettle 61 into the pipeline main body 52, so that the sample carrying pellet 54 is immersed in the microbial liquid;
s43: the sample feeding valve 53 is opened, so that the sample carrying small balls 54 fall into the internal threads of the separation culture device 1, the sample carrying small balls 54 slide downwards along the internal threads under the action of gravity, and the microorganism liquid carried by the sample carrying small balls gradually decreases gradually in the moving process, so that the separation of microorganisms is realized.
The embodiment mainly relates to a deep-sea microorganism separation culture device, and provides an automatic lineation separation culture device and an automatic separation culture process under a high-pressure environment condition. The method overcomes the difficulty that the marine special strains can be separated only after pressure is released, can realize automatic operation scribing without professional operators, realizes the growth and separation of microorganisms by moving and automatically scribing with the small balls carrying bacterial liquid, reduces the labor cost, and improves the screening and utilization efficiency of engineering bacteria.
Compared with the existing solid plate separation culture technology, the embodiment does not need to use a bacterium liquid picked by professionals after the inoculation ring is sterilized for streak inoculation, can realize automatic streak by injecting a small amount of bacterium liquid, completes separation work, and improves the universality of operators; compared with the difficulty that the existing marine special strains can be separated only by pressure release in the screening process, the separation work of single bacterial colonies is completed in the pressure maintaining process, the screening efficiency of the microorganisms difficult to culture is improved, and the screening and cultivating efficiency of the engineering bacteria with special functions is improved.
Example 3
In order to further explain the technical implementation process and technical effects of the present solution, the present embodiment provides a deep-sea methanotrophic bacterium separation culture apparatus and technique. The separation culture device 1 is the core of the embodiment, and can directly realize the separation process of flora under the in-situ high-pressure environment; meanwhile, inoculation and lineation operations can be carried out by utilizing an inoculating loop without depending on professionals, and the problems that mixed bacteria are introduced nonstandard in the disinfection process of the inoculating loop or the temperature of the inoculating loop is not proper, so that the selected bacteria die and the lineation growth process fails are solved.
The isolated culture apparatus 1 according to the present embodiment is composed of an upper cover 12 and a base 11, and the upper cover 12 and the base 11 are connected by a screw. The left side of upper cover 12 is provided with sample introduction pipeline 5, sets up on sample introduction pipeline 5 and throws a kind valve 51 and throw a kind valve 53, and the diameter of throwing a kind valve 51, throwing a kind valve 53 and pipeline main part 52 all is a bit more than the diameter of carrying a kind bobble 54, carries a kind bobble 54 to be located sample introduction pipeline 5, throws a kind valve 51, throws the position of a kind valve 53 control its in sample introduction pipeline 5. The bacterial liquid on the sample-carrying pellet 54 is derived from the microbial liquid injected by the micro-injection pump 62. The left side of the upper cover 12 is provided with an air inlet pipeline connected with the pressure control system 3, and methane gas is selected according to the specificity of the type of the cultured microorganisms. The front of the upper cover 12 is provided with a temperature sensor 42 and a pressure sensor 36 which are mainly used for monitoring the environmental parameter change of the separation incubator 1 in the culture process. The inside of base 11 is provided with the internal thread, and the internal thread is "U" type, links to each other with the inner wall welding of base 11, and the lateral wall of internal thread one side is provided with the gasket, and the gasket is located the middle part position of internal thread one side, and this gasket mainly acts on for when this internal thread and the external screw thread on the screw rod 13 when mutually supporting, has certain space between the external screw thread on internal thread and the screw rod, and this space is used for adorning solid medium. The pressure in the separate culture device 1 and each pipeline is controlled and supplemented by a pressure control system 3.
The pressure control system 3 is mainly used for pressurizing gas injected into the separation culture device 1 and comprises a gas storage tank 31, a pressure regulating valve 32, an air compressor 33 and a booster pump 34. The control acquisition system 2 related to the embodiment comprises a central control system, a computer and the like, and realizes the functions of information change of various environmental data, real-time acquisition, processing, storage, image output and the like of the enriched marine microorganisms in the separation and culture processes of the high-pressure environment.
The embodiment relates to a deep-sea methanotrophic bacterium separation culture device and technology, which mainly constructs a high-pressure environment in a separation culture device, wherein the high-pressure environment is the same as that of microorganisms living in a marine environment. Firstly cleaning the isolated culture device 1, opening the upper cover 12, the base 11 and the screw 13, wiping the upper cover 12 with 75% alcohol, filling the base 11 with 75% alcohol, recovering the 75% alcohol in the base 11 after the alcohol disinfection is finished, and then placing under ultraviolet for sterilization for 15 min. After the sterilization is finished, the screw 13 is rotated to the inside of the base 11 in the counterclockwise direction, the solid culture medium which is sterilized in advance is added, and finally the upper cover 12 is closed. When the solid culture medium is completely solidified, the upper cover 12 is opened, the handle 131 at the top of the screw 13 is held by one hand, the side wall of the base 11 is held by one hand, the screw 13 is rotated and taken out, and finally the upper cover 12 is closed. Then, the pressure value in the isolated culture vessel 1 is determined according to the pressure value in the microorganism enrichment kettle 61, and methane gas is injected into the isolated culture vessel 1 by the pressure control system 3 by opening the gas injection valve 37, so that the pressure value in the isolated culture vessel 1 is consistent with the microorganism enrichment kettle 61. Then, the temperature value in the isolation incubator 1 is determined based on the temperature value in the microorganism-enriching still 61, and the temperature in conformity with the temperature in the microorganism-enriching still 61 is obtained by placing the isolation incubator 1 in the water bath environment of the water bath apparatus 41. Then, the sample throwing valve 51 on the sample introduction pipe 5 is opened, the sterilized sample carrying small ball 54 is placed in the sample introduction pipe 5, the sample throwing valve 51 is closed, and the sample carrying small ball 54 enters the upper part of the sample throwing valve 53. Then, a small amount of bacteria liquid containing microorganisms is injected from the microorganism enrichment tank 61 into the upper part of the sample injection valve 53 through the micro-injection pump 62, so that the sample carrying pellet 54 is completely wrapped by the bacteria liquid. Through the control of opening of the sample feeding valve 53, the sample carrying small ball 54 falls on the solid culture medium on the inner thread, and is scribed on the surface of the solid culture medium according to a certain trajectory under the action of gravity, and finally separated microorganisms grow according to the scribed trajectory, so that the separation of single colonies is realized. After culturing for a certain period, the separation culture work of the single colony can be started. In the whole selection and culture process, the pressure and temperature in the separation culture device 1 are kept consistent with the pressure and temperature environment in the microorganism enrichment kettle 61 where the microorganism is originally located, so that the microorganism is separated in the in-situ high-pressure condition.
The embodiment provides a deep-sea microorganism separation culture device and a culture method, the sample carrying ball 54 slides downwards in the inner thread under the action of gravity, the automatic streak separation of the microorganism liquid of environment sampling enrichment can be realized under the condition of marine in-situ environment, the difficulty that the pressure release is required in the existing flat plate streak separation culture is improved, the microorganism solid separation culture efficiency is effectively improved, and an important basic means is provided for the separation culture of efficient marine special microorganism.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A deep sea microorganism separation culture device is characterized by comprising a separation culture device (1), a control acquisition system (2), a pressure control system (3), a temperature control system (4), a sample injection pipeline (5) and a liquid injection unit (6); wherein:
the separate incubator (1) comprises a base (11), an upper cover (12) and a screw (13); the base (11) is in threaded connection with the upper cover (12); an internal thread is arranged on the surface of the inner wall of the base (11), an external thread which is matched and connected with the internal thread (11) is arranged on the outer surface of the screw rod (13), and after the base (11) and the screw rod (13) are connected through the internal thread and the external thread, a certain gap is formed between the internal thread and the external thread and is used for filling a solid culture medium;
the sample introduction pipeline (5) is arranged on the upper cover (12) and is used for putting a culture bacteria liquid sample into the separation incubator (1); the liquid injection unit (6) is connected with the sample injection pipeline (5) and is used for injecting microbial liquid into the sample injection pipeline (5);
the pressure control system (3) and the temperature control system (4) are respectively connected with the separation culture device (1) and are used for ensuring that the internal pressure and temperature of the separation culture device (1) are consistent with the deep-sea microorganism culture environment;
the control end of the pressure control system (3), the acquisition end of the pressure control system (3), the control end of the temperature control system (4), the acquisition end of the temperature control system (4), the control end of the sample injection pipeline (5) and the control end of the liquid injection unit (6) are electrically connected with the control acquisition system (2);
after the solid culture medium is solidified, taking out the screw (13), closing the upper cover (12), and opening the pressure control system (3) and the temperature control system (4) to ensure that the internal temperature and pressure of the separation culture device (1) are consistent with the environment for the growth of microorganisms;
injecting microbial bacteria liquid into the sample injection pipeline (5) by the liquid injection unit (6), putting a culture bacteria liquid sample into the internal thread of the separation culture device (1) by the sample injection pipeline (5), sliding the sample downwards along the internal thread under the action of gravity, and gradually decreasing the carried microbial bacteria liquid in the moving process to realize the separation of microorganisms;
the separated microorganisms grow on a solid culture medium, so that the separation culture of the microorganisms is realized.
2. The deep-sea microbe separating and culturing device as claimed in claim 1, wherein a spacer is provided on the side wall of the side of the internal thread, the spacer is located in the middle of the side of the internal thread, and is used for ensuring a certain gap between the internal thread and the external thread when the internal thread and the external thread on the screw rod are matched with each other, and is used for filling a solid culture medium.
3. The deep sea microorganism separation and culture device according to claim 1, wherein the pressure control system (3) comprises an air storage tank (31), a pressure regulating valve (32), an air compressor (33), a booster pump (34), an air injection pipeline (35), a pressure sensor (36) and an air injection valve (37); the air compressor (33), the booster pump (34), the air storage tank (31) and the pressure regulating valve (32) are sequentially connected through an air injection pipeline (35), and finally the air injection pipeline (35) is connected with the upper cover (12) through an air injection valve (37); the probe of the pressure sensor (36) is arranged in the separation incubator (1), and the signal output end of the probe is electrically connected with the control acquisition system (2);
the control end of the pressure regulating valve (32), the control end of the air compressor (33), the control end of the booster pump (34), the control end of the gas injection valve (37) and the control acquisition system (2) are electrically connected.
4. The deep-sea microbe separating and culturing device as claimed in claim 1, wherein the temperature control system (4) comprises a water bath device (41) and a temperature sensor (42); the water bath device (41) is wrapped on the outer wall of the separation incubator (1), and the control end of the water bath device is electrically connected with the control acquisition system (2); the probe of the temperature sensor (42) is arranged in the separation incubator (1), and the signal output end of the probe is electrically connected with the control acquisition system (2).
5. The deep-sea microorganism separation and culture device according to claim 1, wherein the sample introduction pipeline (5) comprises a sample throwing valve (51), a pipeline main body (52), a sample throwing valve (53) and a sample carrying ball (54); the pipeline main body (52) is fixedly arranged on the upper cover (12) and is communicated with the internal thread; the sample throwing valve (51) is fixedly arranged on the pipeline main body (52) outside the upper cover (12), and the sample throwing valve (53) is fixedly arranged on the pipeline main body (52) inside the upper cover (12); the control end of the sample throwing valve (51) and the control end of the sample throwing valve (53) are electrically connected with the control acquisition system (2); wherein:
firstly, opening a sample throwing valve (51), throwing sample carrying small balls (54) into a pipeline main body (52), and then closing the sample throwing valve (51); starting the liquid injection unit (6) to inject microbial bacteria liquid into the pipeline main body (52) so that the sample-carrying small balls (54) are immersed in the microbial bacteria liquid; then the sample feeding valve (53) is opened, so that the sample carrying small balls (54) fall into the internal threads of the separation incubator (1), the sample carrying small balls (54) slide downwards along the internal threads under the action of gravity, and in the moving process, the carried microbial liquid is gradually reduced, and the separation of microorganisms is realized.
6. The deep sea microorganism separating and culturing device according to claim 5, wherein the liquid injection unit (6) comprises a microorganism culturing kettle (61), a micro injection pump (62) and a liquid injection pipeline (63); the microorganism culture kettle (61) is connected with the pipeline main body (52) through a liquid injection pipeline (63), and a micro injection pump (62) is arranged on the liquid injection pipeline (63); the control end of the micro-injection pump (62) is electrically connected with the control acquisition system (2);
the microorganism culture kettle (61) is used for culturing deep-sea microorganisms and producing microorganism liquid, and the microorganism injection pump (62) pumps the microorganism liquid into the pipeline main body (52).
7. A deep-sea microorganism separation culture method is realized by applying the deep-sea microorganism separation culture device as claimed in claims 1-6, and specifically comprises the following steps:
s1: cleaning and sterilizing the deep-sea microorganism separation culture device, and filling a solid culture medium;
s2: determining the pressure value in the isolated culture device (1) according to the environmental pressure value of deep-sea microorganism culture, opening a gas injection valve (37), and injecting gas or inert gas required by microorganism culture into the isolated culture device (1) by using a pressure control system (3) until the pressure value in the isolated culture device (1) is consistent with the pressure value of microorganism culture;
s3: determining a temperature value in the separation incubator (1) according to an environment temperature value for deep-sea microorganism culture, and starting a temperature control system (4) to enable the temperature in the separation incubator (1) to be consistent with the microorganism culture environment;
s4: injecting microbial bacteria liquid into the sample injection pipeline (5) by the liquid injection unit (6), putting a culture bacteria liquid sample into the internal thread of the separation culture device (1) by the sample injection pipeline (5), sliding the sample downwards along the internal thread under the action of gravity, and gradually decreasing the carried microbial bacteria liquid in the moving process to realize the separation of microorganisms;
s5: the separated microorganisms grow on a solid culture medium, so that the separation culture of the microorganisms is realized.
8. The method for isolated culture of microorganisms in deep sea according to claim 7, wherein the step S1 comprises the steps of:
cleaning the isolated culture device (1), opening the upper cover (12), the base (11) and the screw (13), and wiping the upper cover (12) with 75% alcohol; the base (11) is filled with 75% alcohol, 75% alcohol in the base is recovered after the alcohol disinfection is finished, and then the base is placed under ultraviolet for sterilization for 15 min.
9. The method for isolated culture of microorganisms in deep sea according to claim 7, wherein the step S1 comprises the steps of:
s11: after the sterilization is finished, rotating the screw (13) to the inside of the base (11) in the anticlockwise direction, adding the sterilized solid culture medium, and closing the upper cover (12);
s12: when the solid culture medium is solidified, the upper cover (12) is opened, the screw (13) is fixed, the screw (13) is rotated by holding the side wall of the base (11), the screw is taken out in a rotating way, and finally the upper cover (12) is closed, so that the process of filling the solid culture medium is completed.
10. The method for isolated culture of microorganisms in deep sea according to claim 7, wherein the step S4 comprises the following steps:
s41: opening a sample throwing valve (51), throwing a sample carrying small ball (54) into a pipeline main body (52), and then closing the sample throwing valve (51);
s42: starting a micro-injection pump (62) to pump the microbial liquid in the microbial culture kettle (61) into the pipeline main body (52) so that the sample carrying pellet (54) is immersed in the microbial liquid;
s43: open and throw a kind valve (53) for in taking a kind pellet (54) drops the internal thread of isolated culture ware (1), take a kind pellet (54) and slide down along the internal thread under the action of gravity, at the removal in-process, the microorganism fungus liquid that it carried will progressively decrement, has realized the separation to the microorganism.
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