CN101624567B - Microorganism online observation device - Google Patents
Microorganism online observation device Download PDFInfo
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- CN101624567B CN101624567B CN2009101021305A CN200910102130A CN101624567B CN 101624567 B CN101624567 B CN 101624567B CN 2009101021305 A CN2009101021305 A CN 2009101021305A CN 200910102130 A CN200910102130 A CN 200910102130A CN 101624567 B CN101624567 B CN 101624567B
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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
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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
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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
- 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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- 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 mikrobe under the ultra-high pressure environment, particularly the online microscopic observation device of sea life 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 mikrobe mixed culture through 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 sea life.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 yield-power.
Be mature on the whole in gordian techniquies such as high pressure seal, negative-pressure sealing, pollution control, protection against corrosion, sampling observations in view of the deep-sea Sampling techniques, 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 like the disclosed a kind of tandem extreme environment simulated flow reaction unit of Chinese patent CN1559659A; Microbial growth, growth, atomization (as far as meiobenthos, its size is at 80～200 mu m ranges) but these equipment can't directly detect by an unaided eye are only if 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 mikrobe is observed.Therefore, in conjunction with micro-imaging technique, the online mikrobe 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 mikrobe simultaneously; Metabolic processes such as online record microbial growth, growth, breeding, significant to the research sea life.At present, in the relevant equipment, the device that under condition of high voltage, carries out the online microscopic observation of mikrobe 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 mikrobe under the high hydrostatic pressure condition of deep-sea is provided.
Inventive concept of the present invention is: combine the negative packing technique of microscopic examination technology and high pressure; Make the spy hole opened on material and thickness, the wall of the wall of observer of the present invention, light hole size 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 metabolic purposes such as the growth of real-time microscopic observation mikrobe under multiple envrionment conditions, growth, 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, through 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 mikrobe gets into observation room, can observe or microimaging microbiological specimens through 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 said observer; One end of said culturing room and crossover sub sealing are installed together; The other end of said culturing room is communicated with observation room, and position relative with observation room on the wall of said observer has spy hole, and said spy hole is communicated with observation room; Be provided with first transparent unit in the said spy hole, said first transparent unit and observation room sealing are installed together.
Further, opposite side relative with spy hole on the wall of observer according to the invention also has light hole, and said light hole is communicated with observation room, is provided with second transparent unit in the said light hole, and said second transparent unit and observation room sealing are installed together.
Further, there is a contraction mouth junction of culturing room according to the invention and observation room.
Further, culturing room according to the invention has more than two.
Further, the present invention also comprises PS, and said PS is connected with crossover sub through 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 through high pressure syringe pump; But, also can under pressure constant state, carry out the mikrobe microscopic examination through 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 and make a video recording research object in real time, 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 PS through pressure piping; Through 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 the structural representation in the microorganism culturing high-pressure flow system with microorganism online observation device of the present invention;
Among the figure: 1. PS, 2. pressure piping, 3. crossover sub, 4. culturing room, 5. observation room; 6. gland, 7. tightness system, 81. first transparent units, 82. second transparent units, the 9. wall of observer; 10. observer, 11. nutrient solution casees, 12. high pressure syringe pumps, 13. pressure transmitters, 14. reducing valve; 15. waste liquid bottle, 16. twist switches, the contraction mouth of junction between 17. culturing room and the observation room, 18. spy holes, 19. light holes.
Embodiment
Below in conjunction with accompanying drawing the present invention is further described.
Like Fig. 1, Fig. 2 and shown in Figure 3, microorganism online observation device of the present invention includes observer 10 and PS 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.On the wall 9 of observer 10, have spy hole 18 with observation room 5 relative positions, 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 fixing first transparent units 81 also as tightness system 7 first transparent unit 81 and observation room 5 sealings to be installed together 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 is communicated with culturing room 4, mikrobe can get into observation room 5, and the viewer uses stereoscope or microscope, makes object lens aim at spy hole 18, and the mikrobe that sees through in 81 pairs of observation rooms of first transparent unit 5 is observed.
For making the viewer can observe the mikrobe in the observation room 5, can on the wall 9 of observer 10, have light hole 19 like Fig. 1 to shown in 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 get 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 mikrobe of observation room 5 through 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.
Shown in Fig. 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 mikrobe 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 through 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 mikrobe such as bait from also entering into observation room 5; Avoiding these suspended particles to disturb observation, thereby improve the accuracy of observing to mikrobe.
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 will apparatus of the present invention be linked in the microorganism culturing high-pressure flow system, then extremely shown in Figure 4 like Fig. 1, can the crossover sub in the culturing room 4 of apparatus of the present invention 3 be connected with PS 1 through pressure piping 2.PS 1 can the control observation device pressure in 10, after high pressure syringe pump 12 pressurizations, PS 1 closes afterwards that whole apparatus of the present invention are 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; The outlet of this nutrient solution is installed together with the crossover sub sealing, and this crossover sub is connected with PS through 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 PS 1 at the left and right two ends of observer 10.Wherein, the PS 1 of observer 10 1 ends of the present invention is connected with high pressure syringe pump 12, and the PS 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 PS 1 control observation device 10 at observer 10 two ends, the PS 1 on observer 10 the right also can connect second PS 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 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, twist switch 16 opened; High pressure syringe pump 12 adds nutrient solution; Close twist switch 16 and reducing valve 14, open the PS 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, the nutrient solution that need upgrade in time, at this moment; Slowly pressurize through high pressure syringe pump 12 when need slowly open reducing valve 14, also can directly use constant flow pump, setting PM flow; Like 0.1ml/min; This flow can not be disturbed basically 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 basically 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, the assurance sample can not be disturbed basically and reach the 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 through the mikrobe in 18 pairs of observation rooms of spy hole 5 in the experimentation.
Through 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 mikrobe physiological activity under different conditions such as high pressure, high salt, hypoxemia, low nutrition.
As stated, parts such as the wall 9 of observer 10, first transparent unit 81, second transparent unit 82, pressure piping 2, PS 1 and crossover sub 3 can adopt corrosion-resistant high pressure resistant material to process.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 material of being selected for use and observer 10; Those skilled in the art can select by the required concrete pressure that bears of observation device according to the present invention accordingly: if the good pressure-resistant performance of the material that observer 10 is selected for use, 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 for use is low; Then the thickness of the wall 9 of observer 10 can be thicker, for example, and when observer of the present invention 10 uses high pressure resistant, corrosion resistant stainless material; The thickness of the wall 9 of observer 10 is about 9mm; Culturing room 4 highly is 20mm, and observation room 5 highly is 3mm, and 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 through 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 through PS 1, constitutes the mobile high pressure online observation system of cultivating of mikrobe.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 said observer (10); The sealing of one end of said culturing room (4) and crossover sub (3) is installed together, and the other end of said culturing room (4) is communicated with observation room (5), on the wall (9) of said observer (10) and the relative position of observation room (5) have spy hole (18); Be provided with first transparent unit (81) in the said spy hole (18), said first transparent unit (81) is installed together with observation room (5) sealing; Said observation room (5) is provided with the nutrient solution outlet, and this nutrient solution exit seal is equipped with second crossover sub.
2. microorganism online observation device according to claim 1; It is characterized in that: the wall (9) of said observer (10) is gone up the opposite side relative with spy hole (18) and is also had light hole (19); Be provided with second transparent unit (82) in the said light hole (19), said 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 said 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: said culturing room (4) has more than two.
5. microorganism online observation device according to claim 1 is characterized in that: it also comprises PS (1), and said PS (1) is connected with crossover sub (3) through pressure piping (2).
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| 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 |
| CN101982761B (en) * | 2010-09-27 | 2012-07-04 | 浙江大学 | On-line fluorescence reaction kettle |
| FR3025802B1 (en) * | 2014-09-16 | 2019-12-27 | Cnrs | ANAEROBIC BIOREACTOR DEVICE WITH RETRACTABLE 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 |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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