CN201495225U - A microbial online observation device - Google Patents

Abstract

The utility model discloses of an online microbe observation device capable of stimulating high-pressure extreme environment conditions, which mainly comprises a sealed observer arranged with a cultivation chamber and an observation chamber, wherein one end of the cultivation chamber is mounted in a sealing way with a transition joint, the other end of the cultivation chamber is communicated with the observation chamber, an observation hole is formed on part of the wall of the observer corresponding with the observation chamber, and is communicated with the observation chamber; and a first transparent device arranged in the observation hole is mounted in a sealing way with the observation chamber. The utility model has the advantages of convenient connection, simple operation and extensive application, not only can realize cultivation of high-pressure anaerobic microbes as well real-time on-line observation of high-pressure anaerobic microbes in the laboratory, but also can be connected with a high-pressure flow system through a pressure pipe and a pressure switch, so as to realize long-term continuous cultivation and on-line micro-observation.

Description

A microbial online observation device

technical field

The utility model relates to a microscopic observation device for microorganisms in an extremely high-pressure environment, in particular to an online microscopic observation device for marine organisms under the conditions of high hydrostatic pressure and other non-biological ecological factors.

Background technique

The research and development of marine microbial resources is one of the themes of biotechnology in this century, and the research on microorganisms under the extreme environment of high pressure and low temperature in the deep sea is a hot spot among them. In 1949, Zobell and Johnson first proposed the concept of "barophile" (hereinafter also called piezophile), and in 1957, they obtained evidence of bacterial barophilic growth through the mixed culture of microorganisms in deep-sea sediments. In 1979, Yayano first reported the pure culture of barophiles. Since the 1980s, high-pressure separation and culture techniques have been used to isolate, purify and cultivate bacteria from deep-sea sediments. The results show that they usually have the characteristics of pressure-loving or pressure-resistant growth pressure, confirming that pressure is the main abiotic factor of marine organisms . In addition, the abiotic factors of the marine environment also include light, temperature, salinity, various dissolved gases and suspended substances, etc., which have an important impact on the distribution, growth, reproduction and productivity of marine organisms.

Given that deep-sea sampling technology has basically matured in key technologies such as high-pressure sealing, negative pressure sealing, pollution control, anti-corrosion, and sampling observation, the detection or sampling obtained by existing instruments or equipment after reaching the predetermined depth and then returning to the land laboratory Information has been guaranteed to be accurate. The latest domestic devices or equipment for simulating high-pressure extreme environments, such as a series-type extreme-environment simulating flow reaction device disclosed in Chinese patent CN1559659A, can simulate high-pressure and extreme environmental conditions in the laboratory, but these devices cannot directly observe the growth of microorganisms with the naked eye , development, and differentiation process (for small benthic organisms, the size is in the range of 80-200 μm), unless the experiment is over, after the pressure is released, take some samples and put them under the microscope for observation. Obviously, this cannot satisfy the cultivation and real-time online observation of high-pressure microorganisms in a continuous high-pressure environment. Therefore, combined with microscopic imaging technology, a set of online microbial observation device that can be matched with the extreme environment simulation flow reaction device in the laboratory can be used alone or connected in series to the extreme environment flow system, so that the device can be used Maintaining extreme environmental conditions such as high pressure, while realizing simultaneous cultivation and real-time observation of microorganisms, and online recording of metabolic processes such as growth, development, and reproduction of microorganisms are of great significance to the study of marine organisms. At present, among related equipment, there is no device for online microscopic observation of microorganisms under high pressure conditions.

Utility model content

The purpose of the utility model is to overcome the unobservable problem of microbial cultures in the experimental process of simulating extreme pressure conditions in the prior art, and provide an online microscopic observation device for simulating microorganisms under high hydrostatic pressure conditions in deep sea.

The inventive idea of the present utility model is: combine microscopic observation technique and high pressure negative sealing technique, make the material and thickness of the wall of the utility model observer, the observation hole that opens on the wall, the size of the light hole and the internal transparent device The material and thickness, on the one hand, ensure that the safe pressure range is 0-60MPa, on the other hand, the distance between the observation room and the microscope objective lens can be within the working distance of the microscope objective lens (such as PlanN10×, the objective lens working distance is 10.6mm ), so that the utility model conforms to the principle of microscopic observation, and achieves the purpose of real-time microscopic observation of the metabolism of microorganisms such as growth, development, and reproduction under various environmental conditions. Connect the observation device of the utility model to the extreme environment simulation flow reaction device, or use a high-pressure injection pump to pressurize and inject the culture solution, and make the culture solution in the system simulate the high hydrostatic pressure, low oxygen, and high salt conditions in the original living environment of marine microorganisms, etc. Various non-biological ecological factors realize the flow update of each component in the observation device of the utility model in the system through valve control. Microbial samples can live in the cultivation chamber of the observer of the utility model. Since the observation chamber and the cultivation chamber are connected, when microorganisms enter the observation chamber, the microbial samples can be observed or microscopically photographed through the observation chamber.

The technical solution adopted by the utility model to realize the purpose of the invention is: the microorganism on-line observation device mainly includes a sealed observer, the observer is provided with a cultivation room and an observation room, and one end of the cultivation chamber is sealed and installed with the transition joint. Together, the other end of the cultivation chamber communicates with the observation chamber, and an observation hole is opened at a position opposite to the observation chamber on the wall of the observer, and the observation hole communicates with the observation chamber, and the observation hole is provided with The first transparent device is sealed and installed together with the observation chamber.

Further, there is a light hole on the wall of the observer of the utility model opposite to the observation hole, and the light hole communicates with the observation room, and a second transparent device is arranged in the light hole , the second transparent device is sealed and installed together with the observation chamber.

Further, there is a constriction opening at the connection between the cultivation room and the observation room of the utility model.

Further, there are more than two cultivation chambers in the utility model.

Further, the utility model also includes a pressure switch, and the pressure switch is connected to the transition joint through a pressure-resistant tube.

Compared with the prior art, the beneficial effects of the utility model are:

(1) The device of this utility model can bear the highest safe pressure of 60Mpa. In the range of 0-60MPa, the high-pressure injection pump can be used for fast or slow acceleration and decompression control, and the observation room can be used for real-time observation of microorganisms under different pressure conditions. Activities, microscopic observation of microorganisms can also be carried out under constant pressure.

(2) The design of the observation room of the device of the utility model conforms to the principle of microscopic observation. The whole device can be directly placed on the microscopic stage for observation, and can also be used for fluorescence microscopic observation to realize online microscopic observation of living microorganisms. If the microscope is equipped with a microscopic camera system, it can take pictures and videos of the research objects in real time. After microscopic imaging, the magnification is 1 to 100 times.

(3) The device of the utility model can carry out high-pressure anaerobic microbial cultivation and microscopic observation alone, and can also connect a high-pressure flow system (within the range of 0 to 60Mpa) through a pressure-resistant tube and a pressure switch, and realize long-term continuous production by updating the culture solution. Culture and online microscopic observation. The whole set of device is convenient to connect, easy to operate and widely applicable.

Description of drawings

Fig. 1 is a schematic diagram of the external structure of the utility model microorganism online observation device;

Fig. 2 is a schematic cross-sectional view of the internal structure of the utility model microorganism online observation device;

Fig. 3 is A-A sectional view of Fig. 2;

Fig. 4 is a structural schematic diagram of connecting the microorganism online observation device of the present utility model into the high-pressure flow system for microorganism cultivation;

In the figure: 1. Pressure switch, 2. Pressure-resistant pipe, 3. Transition joint, 4. Cultivation room, 5. Observation room, 6. Pressure sleeve, 7. Sealing device, 81. First transparent device, 82. Second Transparent device, 9. Observer wall, 10. Observer, 11. Culture solution tank, 12. High-pressure syringe pump, 13. Pressure sensor, 14. Pressure reducing valve, 15. Waste liquid bottle, 16. Knob switch, 17 . The constriction port at the connection between the culture chamber and the observation chamber, 18. Observation hole, 19. Light-through hole.

Detailed ways

Below in conjunction with accompanying drawing, the utility model is further described.

As shown in Fig. 1, Fig. 2 and Fig. 3, the microorganism online observation device of the present utility model includes an observer 10 and a pressure switch 1, and the observer 10 is provided with a cultivation chamber 4 and an observation chamber 5. One end of the cultivation chamber 4 is hermetically installed with the transition joint 3 , and the other end of the cultivation chamber 4 communicates with the observation chamber 5 . An observation hole 18 is opened at a position opposite to the observation chamber 5 on the wall 9 of the observer 10 , and the observation hole 18 communicates with the observation chamber 5 . A first transparent device 81 is also installed in the observation hole 18, and the first transparent device 81 of the utility model can be made of corundum which is corrosion-resistant and high-pressure-resistant material. The first transparent device 81 can be fixed by using the hollow pressing sleeve 6 and the first transparent device 81 and the observation chamber 5 can be hermetically installed together by using the hollow/O-ring as the sealing device 7 . The sealed and fixed observation chamber 5 has good airtightness and light transmittance, and can withstand a safe pressure range of 0-60 MPa. Since the observation chamber 5 communicates with the cultivation chamber 4, microorganisms can enter the observation chamber 5, and the observer uses a stereoscope or a microscope to align the objective lens with the observation hole 18 and observe the microorganisms in the observation chamber 5 through the first transparent device 81. .

In order to enable the observer to observe the microorganisms in the observation chamber 5, as shown in FIGS. on the other side wall. Like the observation hole 18 , a second transparent device 82 may also be provided in the light-through hole 19 , and the second transparent device 82 is hermetically installed with the observation chamber 5 . The second transparent device 82 can also be made of corundum, which is a corrosion-resistant and high-pressure-resistant material, like the first transparent device 81 . Because the light through hole 19 and the observation hole 18 are on both sides of the observation chamber 5 and the positions are opposite, so that the light source under the stereoscopic mirror or the microscope can enter the observation chamber 5 through the light through hole 19 and the observation hole 18, and pass through the light through hole 19 1. An optical path is formed between the observation chamber 5 and the observation hole 18 so that there is enough light to observe the microorganisms in the observation chamber 5 . Of course, if an illuminating device is provided in the observation chamber 5 or in the observation hole 18, the light through hole 19 may not be provided.

As shown in Figures 1 to 3, the utility model can be respectively provided with a cultivation chamber 4 symmetrically at both ends of the observation chamber 5, and one of the ends of the two cultivation chambers 4 is all communicated with the observation chamber 5, so that each cultivation chamber 4 Microbes can enter the observation chamber 5 . The other ends of the two culture chambers 4 are sealed by a transition joint 3 . Since the cultivation chamber 4 and the observation chamber 5 are sealed by the sealing device 7 , the entire observer 10 forms a sealed structure. There can be more than two cultivation chambers 4 of the present utility model, and they can be distributed around the observation chamber 5 and communicate with the observation chamber 5 respectively.

It should be noted that there is a constriction port 17 at the connection between the cultivation chamber 4 and the observation chamber 5. The purpose of setting the constriction port 17 is to prevent bait and other suspended matter particles other than microorganisms from entering the observation chamber 5, so as to avoid These suspended particles interfere with the observation of microorganisms, thereby improving the accuracy of observation.

The device of the utility model can perform anaerobic microorganism cultivation and observation alone; it can also be connected to a microorganism cultivation high-pressure flow system, and the high-pressure flow system supplies new nutrient solution for long-term on-line microscopic observation of the same sample.

If the utility model device is connected to the microbial culture high-pressure flow system, then as shown in Fig. 1 connection. The pressure switch 1 can control the pressure in the observer 10. After pressurizing with the high-pressure injection pump 12, the entire utility model device is in a high-pressure sealed state after the pressure switch 1 is closed, and then the cultivation of anaerobic microorganisms under constant pressure can be carried out separately. and observational experiments. If the observation chamber 5 of the device of the present invention is only connected with a cultivation chamber 4, in order to connect the device of the present invention to the high-pressure flow system for microorganism cultivation, a culture solution outlet can be added on the observation chamber 5, and the culture solution outlet It is sealed and installed together with the transition joint, and the transition joint is connected to the pressure switch through the pressure-resistant tube, so that the culture fluid can flow through the culture chamber 4 and the observation chamber 5 dynamically.

As shown in Figure 4, after connecting the observation device of the present utility model to the high-pressure flow system for cultivating external microorganisms, the whole system includes a culture solution tank 11, a high-pressure injection pump 12, a pressure sensor 13, a pressure reducing valve 14 and a waste liquid bottle 15. The observer 10 of the utility model, the pressure switch 1 at the left and right ends of the observer 10. Wherein, the pressure switch 1 at one end of the observer 10 of the present invention is connected with a high-pressure injection pump 12 , and the pressure switch 1 at the other end of the observer 10 is connected with a pressure reducing valve 14 . The high-pressure injection pump 12 can be a manual rapid high-pressure injection pump or a high-pressure constant-flow pump to realize the pressurization or pressure relief of the entire system. The pressure switch 1 at both ends of the observer 10 controls the pressure in the observer 10, and the pressure switch 1 on the right side of the observer 10 Pressure switch 1 can also be connected to a second pressure switch for connection to other external devices.

Introduce the use process of the utility model device below.

The pressure test is carried out continuously in the laboratory: the transition joint 3 is opened, and an appropriate amount of samples are loaded into the two cultivation chambers 4 and the observation chamber 5 of the observer 10, and the entire device of the utility model is connected and sealed well. Connect the device of the present utility model into the high-pressure flow system for microbial cultivation, pre-clean the inside of the culture solution tank 11, and fill it with the culture solution required for the test, turn on the knob switch 16, add the culture solution to the high-pressure injection pump 12, and turn off the knob switch 16 and the decompression valve 14, open the pressure switch 1 at both ends of the pressure sensor 13 and the observer 10, and adopt rapid or slow pressurization to the pressure required for the experiment according to the test plan. Then, the observer 10 is placed on the stage of the stereoscope or the microscope, and the physiological activities of the same microbial sample in the observation chamber 5 under different pressure conditions can be observed through the first transparent device 81 in the observation hole 18, If the microscope is equipped with a camera system, it can take pictures and video in real time.

In order to meet the long-term observation needs of the sample under high pressure, it is necessary to update the culture medium in time. At this time, it is necessary to slowly open the pressure reducing valve 14 while slowly pressurizing through the high-pressure syringe pump 12. It is also possible to directly use a constant flow pump. Set the flow rate per minute, such as 0.1ml/min, to ensure that the sample will not be disturbed and at the same time add new culture medium, so as to achieve continuous observation of the same sample, while maintaining the pressure level in the device basically unchanged .

Decompression experiment in the laboratory: the pressurization process is the same as the above continuous pressurization experiment, using rapid pressurization to the required pressure for the experiment, and then quickly or slowly opening the pressure relief valve 14 to achieve decompression. According to the size of the sample to be observed, the flow rate of the pressure reducing valve 14 is controlled to ensure that the sample is basically not disturbed to achieve continuous observation of the same sample, and at the same time, the pressure level in the device is slowly reduced to the required level. During the experiment, the microorganisms in the observation chamber 5 can be photographed and videotaped in real time under a microscope through the observation hole 18 .

Through the pressure control realized by the device of the utility model and the change of environmental parameters such as PH, salinity and dissolved oxygen in the culture medium, real-time microscopic observation and photography, the study of different organisms under high pressure, high salt, low oxygen, low nutrition, etc. Adaptation mechanisms of microbial physiological activities under different conditions.

As mentioned above, the wall 9 of the observer 10, the first transparent device 81, the second transparent device 82, the pressure pipe 2, the pressure switch 1 and the transition joint 3 can be made of corrosion-resistant and high-pressure resistant materials. Suspended particles necessary for the long-term life of organisms such as bait can be put in the cultivation chamber 4 . The sealing performance of the assembled observer 10 can reach a maximum safety pressure of 60 MPa, and the light transmission of the observation chamber 5 is above 90%. The pressure resistance of the observer 10 is related to the material selected and the thickness of the wall 9 of the observer 10, the size and thickness of the first transparent device 81 and the second transparent device 82, those skilled in the art can observe the device according to the utility model The specific pressure that needs to bear is selected accordingly: if the pressure resistance of the selected material of the observer 10 is good, the thickness of the wall 9 of the observer 10 can be thinner; Lower, then the thickness of the wall 9 of observer 10 can be thicker, for example, when the utility model observer 10 uses high pressure resistant, corrosion-resistant stainless steel material, the thickness of the wall 9 of observer 10 can be about 9mm, cultivates The height of the chamber 4 is 20mm, the height of the observation chamber 5 is 3mm, the first transparent device 81 and the second transparent device 82 can use Ф16×8mm corundum, the height of the entire observer 10 is about 38mm, suitable for placing in a stereoscope or a long On the stage of the microscope of the working distance (the working distance of the objective lens is 10.6mm), the minimum distance between the objective lens and the bottom of the observation chamber 5 is about 10.4mm, and the microscopic observation can be carried out through the observation chamber 5 . Of course, the overall height of the observer 10 is not as thick as possible, and the overall height of the observer 10 should be smaller than the maximum distance between the microscope objective lens and the stage, otherwise the observer 10 cannot be put into the object stage. If the microscope is equipped with a camera system, real-time microscopic photography is possible. The whole device of the utility model is connected to the high-pressure flow system for microbial cultivation through the pressure switch 1 to form a high-pressure online observation system for microbial flow cultivation. By using the device of the utility model, long-term on-line microscopic observation of the same high-pressure microbial sample in a laboratory can be realized.

Claims (5)

1. microorganism online observation device, it is characterized in that: it comprises the observer (10) of sealing, be provided with culturing room (4) and observation room (5) in the described observer (10), one end of described culturing room (4) and crossover sub (3) sealing are installed together, the other end of described culturing room (4) is communicated with observation room (5), go up the position relative at the wall (9) of described observer (10) and have spy hole (18) with observation room (5), described spy hole (18) is communicated with observation room (5), be provided with first transparent unit (81) in the described spy hole (18), described first transparent unit (8) is installed together with observation room (5) sealing.

2. a kind of microorganism online observation device according to claim 1, it is characterized in that: the wall (9) of described observer (10) is gone up the opposite side relative with spy hole (18) and is also had light hole (19), described light hole (19) is communicated with observation room (5), be provided with second transparent unit (82) in the described light hole (19), described second transparent unit (82) is installed together with observation room (5) sealing.

3. a kind of microorganism online observation device according to claim 1 is characterized in that: there is a contraction mouth (17) in described culturing room (4) with the junction of observation room (5).

4. according to any one described a kind of microorganism online observation device in the claim 1 to 3, it is characterized in that: described culturing room (4) has more than two.

5. a kind of microorganism online observation device according to claim 1 is characterized in that: it also comprises pressure switch (1), and described pressure switch (1) is connected with crossover sub (3) by pressure piping (2).

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