CN101624567A - Microorganism online observation device - Google Patents
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- CN101624567A CN101624567A CN200910102130A CN200910102130A CN101624567A CN 101624567 A CN101624567 A CN 101624567A CN 200910102130 A CN200910102130 A CN 200910102130A CN 200910102130 A CN200910102130 A CN 200910102130A CN 101624567 A CN101624567 A CN 101624567A
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- 244000005700 microbiome Species 0.000 title claims abstract description 44
- 238000012258 culturing Methods 0.000 claims description 37
- 238000007789 sealing Methods 0.000 claims description 17
- 230000008602 contraction Effects 0.000 claims description 5
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- 235000015097 nutrients Nutrition 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
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- 238000002474 experimental method Methods 0.000 description 7
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- 238000000034 method Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 4
- 230000002906 microbiologic effect Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 229910052593 corundum Inorganic materials 0.000 description 3
- 239000010431 corundum Substances 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
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- 238000005070 sampling Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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Abstract
The invention discloses a microorganism online observation device which can simulate high-pressure extreme environment and mainly comprises a sealed observer, wherein the observer is internally provided with a culture chamber and an observation chamber, one end of the culture chamber is connected with a transition joint in a sealed way, the other end of the culture chamber is communicated with the observation chamber, a position on the wall of the observer, which corresponds to the observation chamber, is provided with an observation hole, the observation hole is communicated with the observation chamber and is internally provided with a first transparent device, and the first transparent device and the observation chamber are arranged together in a sealed way. The invention has convenient connection, simple operation and wider application, not only can be used for singly carrying out high-pressure anaerobe culture and online observing high-pressure microorganism in real time, but also can be connected with a high-pressure flow system via a pressure pipe and a pressure switch to realize long-time continuous online culture and online micro observation.
Description
Technical field
The present invention relates to the microscopic observation device of microorganism under the ultra-high pressure environment, particularly the online microscopic observation device of marine organisms under abiotic ecological factor conditions such as high hydrostatic pressure.
Background technology
The research and development of marine microorganism resource are one of the themes of biotechnology in this century, are in microbe research wherein the focus especially under high pressure and the cryogenic deep-sea extreme environment.Notion (after this also claiming piezophile) that 1949, Zobell and Johnson at first proposed " have a liking for press biological, barophile ", and in nineteen fifty-seven the microorganism mixed culture by abyssal sediment, obtained bacterium and had a liking for the evidence of pressing growth.1979, the Yayano reported first have a liking for and press biological pure culture.Since the eighties in 20th century, separate and culture technique separation and purification and train bacterium from abyssal sediment with high pressure, the result shows that they all have to have a liking for usually and presses or the characteristic of withstand voltage growth pressure that confirmation pressure is the main abiotic factors of marine organisms.In addition, the abiotic factor of ocean environment also comprises illumination, temperature, salinity, various gas dissolved and suspended matter etc., and they have significant effects to aspects such as halobiontic distribution, growth, breeding and productivity.
In view of the deep-sea Sampling techniques have been mature on the whole in gordian techniquies such as high pressure seal, negative-pressure sealing, pollution control, protection against corrosion, sampling observations, the detection or the sampling gained information of carrying out can guarantee accurate arriving that predetermined depth is returned land laboratory again for existing instrument or equipment.The device or the equipment of the simulated high-pressure extreme environment of latest domestic, can realize testing lab simulation high pressure extreme environmental conditions as the disclosed a kind of tandem extreme environment simulated flow reaction unit of Chinese patent CN1559659A, microbial growth, growth, atomization are (for meiobenthos but these equipment can't directly detect by an unaided eye, its size is at 80～200 mu m ranges), unless finish experiment, after the pressure release, the extraction sample segment is placed on microscopically and observes.Obviously, this cultivation in continuous hyperbaric environment and real-time online that can't satisfy the high pressure microorganism is observed.Therefore, in conjunction with micro-imaging technique, the online microorganism observation device that research one cover can match with extreme environment simulated flow reaction unit in the laboratory, both can use separately, also can connect and insert the extreme environment flow system, thereby make device both can keep extreme environmental conditions such as high pressure, realize synchronized culture and the real-time monitored of microorganism simultaneously, metabolic processes such as online record microbial growth, growth, breeding, significant to the research marine organisms.At present, in the relevant equipment, the device that carries out the online microscopic observation of microorganism under condition of high voltage does not also occur.
Summary of the invention
The objective of the invention is to overcome the unobservable problem of microorganisms cultures in the analogous pole terminal pressure condition experiment process of prior art, and a kind of online microscopic observation device of simulating the microorganism under the high hydrostatic pressure condition of deep-sea is provided.
Inventive concept of the present invention is: in conjunction with microscopic examination technology and the negative packing technique of high pressure, make the material and the thickness of the wall of observer of the present invention, the spy hole of opening on the wall, the size of light hole and in establish the material and the thickness of transparent unit, guaranteeing to bear the safe pressure scope on the one hand is 0～60MPa, on the other hand, can make distance between observation room and micro objective in the operating distance of micro objective (as PlanN 10 *, its object lens operating distance is 10.6mm), thereby make the present invention meet the microscopic examination principle, reach the growth of real-time microscopic observation microorganism under multiple envrionment conditions, grow, metabolic purposes such as breeding.Observation device of the present invention is inserted extreme environment simulated flow reaction unit, or adopt high pressure syringe pump to suppress the injection nutrient solution, and make high hydrostatic pressure and the various abiotic ecological factors such as hypoxemia, high salt condition in the nutrient solution simulation marine microorganism nature environment in the system, by the mobile renewal of each composition in the observation device of the present invention in the valve control realization system.Microbiological specimens can be lived in the culturing room of observer of the present invention, because observation room and culturing room are communicated with, when microorganism enters observation room, can observe or microimaging microbiological specimens by observation room.
The present invention realizes that the technical scheme that its goal of the invention adopts is: this microorganism online observation device mainly comprises the observer of sealing, be provided with culturing room and observation room in the described observer, one end of described culturing room and crossover sub sealing are installed together, the other end of described culturing room is communicated with observation room, position relative with observation room on the wall of described observer has spy hole, described spy hole is communicated with observation room, be provided with first transparent unit in the described spy hole, described first transparent unit and observation room sealing are installed together.
Further, opposite side relative with spy hole on the wall of observer of the present invention also has light hole, and described light hole is communicated with observation room, is provided with second transparent unit in the described light hole, and described second transparent unit and observation room sealing are installed together.
Further, there is a contraction mouth junction of culturing room of the present invention and observation room.
Further, culturing room of the present invention has more than two.
Further, the present invention also comprises pressure switch, and described pressure switch is connected with crossover sub by pressure piping.
Compared with prior art, the invention has the beneficial effects as follows:
(1) apparatus of the present invention can be born the safe pressure of the highest 60Mpa, in 0～60MPa scope, can carry out the control that adds fast or at a slow speed, reduces pressure by high pressure syringe pump, but, also can under pressure constant state, carry out the microorganism microscopic examination by the microbial activities under the observation room real-time monitored different pressures condition.
(2) design of apparatus of the present invention observation room meets the microscopic observation principle, and whole device can directly be placed on the micro-Stage microscope and observe, and also can be used for fluorescence microscopy observation, realizes the online microscopic observation of living microorganism.If microscope configuration microscope camera system can be taken pictures in real time and make a video recording research object, behind micro-imaging, magnification is 1 times～100 times.
(3) apparatus of the present invention can be carried out cultivation of high pressure anaerobion and microscopic observation separately, also can be connected high-pressure flow system (in 0～60Mpa scope) with pressure switch by pressure piping, by upgrading nutrient solution, realize long-time continuous cultivation and online microscopic observation.Complete assembly is easy to connect, and is simple to operate, widely applicable.
Description of drawings
Fig. 1 is the external structure synoptic diagram of microorganism online observation device of the present invention;
Fig. 2 is the internal structure cross-sectional schematic of microorganism online observation device of the present invention;
Fig. 3 is the A-A sectional view of Fig. 2;
Fig. 4 is linked into structural representation in the microorganism culturing high-pressure flow system with microorganism online observation device of the present invention;
Among the figure: 1. pressure switch, 2. pressure piping, 3. crossover sub, 4. culturing room, 5. observation room, 6. gland, 7. tightness system, 81. first transparent units, 82. second transparent unit, the 9. wall of observer, 10. observer, 11. the nutrient solution case, 12. high pressure syringe pumps, 13. pressure transmitters, 14. reducing valve, 15. waste liquid bottles, 16. twist switches, 17. the contraction mouth of junction between culturing room and the observation room, 18. spy holes, 19. light holes.
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing.
As Fig. 1, Fig. 2 and shown in Figure 3, microorganism online observation device of the present invention includes observer 10 and pressure switch 1, is provided with culturing room 4 and observation room 5 in the observer 10.One end of culturing room 4 and crossover sub 3 sealings are installed together, and the other end of culturing room 4 is communicated with observation room 5.Position relative with observation room 5 on the wall 9 of observer 10 has spy hole 18, and spy hole 18 is communicated with observation room 5.First transparent unit 81 also is installed in the spy hole 18, and first transparent unit 81 of the present invention can adopt, and corundum corrosion-resistant, high pressure resistant material is made.Can use hollow gland 6 to fix first transparent unit 81 also is installed together first transparent unit 81 and observation room 5 sealings as tightness system 7 with hollow/○Xing Mifengquan.Stopping property and light transmission through sealing and fixed observation room 5 are good, and the safe pressure scope that can bear is 0～60MPa.Because observation room 5 and culturing room 4 are communicated with, microorganism can enter observation room 5, and the viewer uses stereoscope or microscope, makes object lens aim at spy hole 18, and the microorganism that sees through in 81 pairs of observation rooms of first transparent unit 5 is observed.
For making the viewer can observe microorganism in the observation room 5, can on the wall 9 of observer 10, have light hole 19 as shown in Figure 1 to Figure 3, this light hole 19 is positioned on another sidewall relative with spy hole 18.The same with spy hole 18, can in light hole 19, also be provided with second transparent unit, 82, the second transparent units 82 and be installed together with observation room 5 sealings.Second transparent unit 82 can be also the same with first transparent unit 81, adopts corundum corrosion-resistant, high pressure resistant material to make.Because light hole 19 is relative in the both sides and the position of observation room 5 with spy hole 18, make stereoscope or microscopically light source to enter in the observation room 5, and between light hole 19, observation room 5 and spy hole 18, form a light path so that there is enough light can observe the microorganism of observation room 5 by light hole 19 and spy hole 18.Certainly, if in observation room 5 or at spy hole 18, be provided with means of illumination, then light hole 19 can be set.
As shown in Figures 1 to 3, the wherein end that the present invention can respectively be provided with 4, two culturing room 4 an of culturing room symmetrically at the two ends of observation room 5 all is communicated with observation room 5, makes the microorganism of each culturing room 4 can enter in the observation room 5.The other end of two culturing room 4 is then by crossover sub 3 sealings.Because culturing room 4, observation room 5 all seal by tightness system 7, thereby make whole observer 10 form the structure of a sealing.Culturing room 4 of the present invention can have more than two, they can be distributed in observation room 5 around, and be communicated with observation room 5 respectively.
Need to prove, there is a contraction mouth 17 junction between culturing room 4 and the observation room 5, the purpose that this contraction mouth 17 is set is in order to prevent that the suspended particles except microorganism such as bait from also entering into observation room 5, avoiding these suspended particles to disturb observation, thereby improve the accuracy of observing to microorganism.
Apparatus of the present invention can be carried out anaerobion cultivation and observation separately; Also can insert microorganism culturing high-pressure flow system, supply with new nutritive medium, same sample is carried out long-term online microscopic observation by this high-pressure flow system.
If apparatus of the present invention will be linked in the microorganism culturing high-pressure flow system, then as shown in Figures 1 to 4, the crossover sub in the culturing room 4 of apparatus of the present invention 3 can be connected with pressure switch 1 by pressure piping 2.Pressure switch 1 can the control observation device pressure in 10, after high pressure syringe pump 12 pressurizations, pressure switch 1 is closed the whole apparatus of the present invention in back and is in the high pressure seal state, then can carry out cultivation and the observation experiment of anaerobion under pressure constant state separately.If 5 of the observation rooms of apparatus of the present invention are connected with a culturing room 4, for apparatus of the present invention being linked in the microorganism culturing high-pressure flow system, then can on observation room 5, set up a nutrient solution outlet, this nutrient solution outlet is installed together with the crossover sub sealing, and this crossover sub is connected with pressure switch by pressure piping, makes nutrient solution can dynamically flow through by culturing room 4 and observation room 5.
As shown in Figure 4, after observation device of the present invention inserted external microbial and cultivate the high-pressure flow system, total system comprised nutrient solution case 11, high pressure syringe pump 12, pressure transmitter 13, reducing valve 14 and waste liquid bottle 15, observer 10 of the present invention, the pressure switch 1 at the left and right two ends of observer 10.Wherein, the pressure switch 1 of observer 10 1 ends of the present invention is connected with high pressure syringe pump 12, and the pressure switch 1 of observer 10 the other ends is connected with reducing valve 14.High pressure syringe pump 12 can be manual quick high-pressure syringe pump or high pressure constant flow pump, in order to realize the pressurization or the pressure release of total system, pressure in the pressure switch 1 control observation device 10 at observer 10 two ends, the pressure switch 1 on observer 10 the right also can connect second pressure switch to be used to connect other peripheral equipments.
Below introduce the use of apparatus of the present invention.
Carry out applied voltage test in the laboratory continuously: open crossover sub 3, with an amount of sample pack into two culturing room 4 and the observation room 5 of observer 10, the whole device of the present invention connects and guarantees good seal.Apparatus of the present invention are inserted in the microorganism culturing high-pressure flow system, the inside of nutrient solution case 11 is cleaned up in advance, and the required nutrient solution of test of packing into, open twist switch 16, high pressure syringe pump 12 adds nutrient solution, close twist switch 16 and reducing valve 14, open the pressure switch 1 at pressure transmitter 13 and observer 10 two ends, adopt pressurization fast or slowly be pressurized to the needed pressure of experiment according to testing program.Then, observer 10 is placed on stereoscope or the microscopical Stage microscope, can see through the physiological activity of same microbiological specimens under the different pressures condition that first transparent unit 81 in the spy hole 18 is observed in the observation room 5, if microscope is equipped with camera system, then can take pictures in real time and make a video recording.
In order to reach the long observation needs of sample under high pressure conditions, nutrient solution need upgrade in time, at this moment, slowly pressurize by high pressure syringe pump 12 when need slowly open reducing valve 14, also can directly use constant flow pump, setting per minute flow, as 0.1ml/min, this flow can not be disturbed substantially with the assurance sample and add new nutrient solution simultaneously, thereby reaches the day-night observation to same sample, and holdout device internal pressure level remains unchanged substantially simultaneously.
Decompression experiment in the laboratory: pressure process adopts to be forced into the required pressure of experiment fast with above continuous pressurization experiment, opens reducing valve 14 then fast or slowly and realizes decompression.According to the size of observed sample, the flow size of control reducing valve 14 guarantees that sample can not be disturbed substantially and reaches day-night observation to same sample, reaches device internal pressure level simultaneously and slowly is reduced to desired level.Can take pictures in real time and make a video recording at microscopically by the microorganism in 18 pairs of observation rooms of spy hole 5 in the experimentation.
By the pressure-controlling that apparatus of the present invention realized and the change of environmental parameters such as the PH in the nutrient solution, salinity and dissolved oxygen, microscopic examination in real time and shooting, the adaptation mechanism of the different biologies of research microorganism physiological activity under different conditions such as high pressure, high salt, hypoxemia, low nutrition.
As mentioned above, the parts such as wall 9, first transparent unit 81, second transparent unit 82, pressure piping 2, pressure switch 1 and crossover sub 3 of observer 10 can adopt corrosion-resistant high pressure resistant material to make.Can throw in the essential suspended particles of biological long-term life such as bait in the culturing room 4.The sealing property of the observer 10 after the assembling can reach the safe pressure of maximum 60MPa, the light transmission 90% of observation room 5 more than.The withstand voltage properties of observer 10 is relevant with the size and the thickness of thickness, first transparent unit 81 and second transparent unit 82 of the wall 9 of selected material and observer 10, those skilled in the art can select by the required concrete pressure that bears of observation device accordingly according to the present invention: if the good pressure-resistant performance of the selected material of observer 10, then the thickness of the wall 9 of observer 10 can approach; If the withstand voltage properties of the material that observer 10 is selected is low, then the thickness of the wall 9 of observer 10 can be thicker, for example, when observer 10 of the present invention uses high pressure resistant, during corrosion resistant stainless material, the thickness of the wall 9 of observer 10 is about 9mm, culturing room 4 highly is 20mm, observation room 5 highly is 3mm, first transparent unit 81 and second transparent unit 82 can adopt the corundum of Φ 16 * 8mm, the height of whole observer 10 is about 38mm, be fit to be placed on the Stage microscope of microscope (the object lens operating distance is 10.6mm) of stereoscope or long reach, object lens are about 10.4mm apart from the minor increment of observation room 5 bottoms, can carry out microscopic observation by observation room 5.Certainly, the whole height of observer 10 is not thick more good more, and the whole height of observer 10 should be less than the ultimate range between micro objective and Stage microscope, otherwise observer 10 can't be put into Stage microscope.If microscope is equipped with camera system, then microimaging in real time.The whole device of the present invention inserts microorganism culturing high-pressure flow system by pressure switch 1, constitutes the mobile high pressure online observation system of cultivating of microorganism.The application of the invention device can be implemented in the laboratory the long-term online microscopic observation to same high pressure microbiological specimens.
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. 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. 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 microorganism online observation device in the claim 1 to 3, it is characterized in that: described culturing room (4) has more than two.
5. 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893549A (en) * | 2010-07-05 | 2010-11-24 | 浙江大学 | Method for acquiring transmitted spectrum of bio-culture solution and reactor adopting same |
| CN101982761A (en) * | 2010-09-27 | 2011-03-02 | 浙江大学 | On-line fluorescence reaction kettle |
| FR3025802A1 (en) * | 2014-09-16 | 2016-03-18 | Ubo Cnrs | ANAEROBIC BIOREACTOR DEVICE WITH REMOVABLE OBSERVATION MODULE |
| CN110118771A (en) * | 2019-03-29 | 2019-08-13 | 亳州学院 | A kind of intelligent microscopic examination device and microscopy method for active sludge microorganism |
| CN111440711A (en) * | 2020-04-20 | 2020-07-24 | 佛山科学技术学院 | Temperature and potential-based microbial environment simulation test system |
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| JP2006000052A (en) * | 2004-06-17 | 2006-01-05 | Olympus Corp | Biological sample observation system |
| CN1896222A (en) * | 2006-06-02 | 2007-01-17 | 西北农林科技大学 | Microbe culturing dish made from adjusting gas |
| CN201007773Y (en) * | 2007-02-08 | 2008-01-16 | 范飞 | Bacteria colonies and bacteriostatic circle dark background imaging system |
| CN201495225U (en) * | 2009-08-13 | 2010-06-02 | 浙江大学 | Online microbe observation device |
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2009
- 2009-08-13 CN CN2009101021305A patent/CN101624567B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893549A (en) * | 2010-07-05 | 2010-11-24 | 浙江大学 | Method for acquiring transmitted spectrum of bio-culture solution and reactor adopting same |
| CN101982761A (en) * | 2010-09-27 | 2011-03-02 | 浙江大学 | On-line fluorescence reaction kettle |
| CN101982761B (en) * | 2010-09-27 | 2012-07-04 | 浙江大学 | On-line fluorescence reaction kettle |
| FR3025802A1 (en) * | 2014-09-16 | 2016-03-18 | Ubo Cnrs | ANAEROBIC BIOREACTOR DEVICE WITH REMOVABLE OBSERVATION MODULE |
| CN110118771A (en) * | 2019-03-29 | 2019-08-13 | 亳州学院 | A kind of intelligent microscopic examination device and microscopy method for active sludge microorganism |
| CN111440711A (en) * | 2020-04-20 | 2020-07-24 | 佛山科学技术学院 | Temperature and potential-based microbial environment simulation test system |
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