CN104498322B - A kind of device cultivated for marine microorganism original position high-throughput - Google Patents

A kind of device cultivated for marine microorganism original position high-throughput Download PDF

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Publication number
CN104498322B
CN104498322B CN201410658474.5A CN201410658474A CN104498322B CN 104498322 B CN104498322 B CN 104498322B CN 201410658474 A CN201410658474 A CN 201410658474A CN 104498322 B CN104498322 B CN 104498322B
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culture
base plate
cell
division board
throughput
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CN201410658474.5A
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CN104498322A (en
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刘晨光
魏文浩
张晓华
李诚博
张增虎
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Ocean University of China
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Ocean University of China
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/02Form or structure of the vessel
    • C12M23/04Flat or tray type, drawers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/02Form or structure of the vessel
    • C12M23/12Well or multiwell plates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/34Internal compartments or partitions
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02Membranes; Filters
    • C12M25/04Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts

Abstract

The invention discloses a kind of device cultivated for marine microorganism original position high-throughput, comprise seawater storehouse and independently cultivate cell; Seawater storehouse comprises from the bottom to top by fixing base plate, reserve liquid layer, division board and the culture layer of bolt, offers reservoir, division board and culture layer offer consistent size, position circular hole opposing upper and lower in the middle of reserve liquid layer; Be provided with one deck filter membrane between division board and culture layer, to cultivate cell be the circular hole that is positioned in culture layer and be positioned on filter membrane.When device is used for laboratory simulation in situ environment, be placed in constant temperature oscillator, base plate is seal base, cultivates the cell separate cap sealing being connected with conduit; During for ocean environment Culture in situ, base plate is the base plate having sieve aperture, cultivates cell top seal translucent roof plate.This device can overall sterilizing, has the feature of high pressure resistant, the resistance to temperature difference, impact resistant, can maintain stable Culture in situ environment in the lab with in ocean environment simultaneously, reduces environmental change to the damage of microorganism.

Description

A kind of device cultivated for marine microorganism original position high-throughput
Technical field
The present invention relates to a kind of marine microorganism culture apparatus, be specifically related to a kind of device cultivated for marine microorganism original position high-throughput, belong to the field of microbial cultivation device.
Background technology
Very abundant Microbial resources are contained in ocean, more and more closer with contacting of the productive life of the mankind and environment protection.But owing to lacking effective cultural method, hinder us to the understanding of its diversity, physiological ecological function and the exploitation to its function.Up to now, the ratio of sum is accounted for less than 1% by the marine microorganism of traditional cultural method culture of isolated.
High-throughput (highthroughput) technology is a kind of new technique occurred the eighties in 20th century, is cultivate the experimental system of milligram ammonia on molecule or cell levels, qualification, screening.High-throughput techniques is at drug development, and in biological detection and microorganism culturing, Application comparison is extensive.Wherein high-throughput culture method (highthroughputculturing, HTC) was proposed by Connon in 2002, along with the application of high-throughput techniques, have developed many high-throughput culture environment method of microorganism and device:
As: the high-throughput based on culture plate is cultivated, and the method effectively improves the culture efficiency of microorganism, but effect is not remarkable.Diffusion chamber device, can the composition required for growth be provided for microorganism and remove meta-bolites, and the existence acted between microflora can be ensured, improve the Culturability of microorganism, but, the operation of this device is more loaded down with trivial details, and the high-throughput of microorganism is cultivated and species sorting can not efficiently be carried out simultaneously.Hollow fiber membrane device, its porous-film can carry out the exchanges such as nutritive substance, meta-bolites, signaling molecule, there is the interaction between the necessary kind of microorganism growth and in planting, but, this device is cultivated must depend on dilution to the hatching of microorganism cells, only has segregating population in a small amount to obtain cultivation in one apparatus.Separating chips device, by material diffusion acquisition in situ environment, parallelly cultivate can be carried out wherein, be suitable for microorganism growth, this device forms the microorganism cells quantity of bacterium colony apparently higher than classical culture protocols, is easy to observe, but, the effect of cultivating sorting also depends on the dilute strength of sample, and it is strict to make material requirements that is dull and stereotyped and film, can not produce harm to the existence of microorganism cells.
At present, Application comparison is that chromatography column combines with peristaltic pump widely, and peristaltic pump can maintain environment stable, and culture environment then can be carried out subregion by chromatography column, thus can carry out large-scale parallel isolate forster; The method principle is simple, but complex operation, higher to equipment requirements.There is shortcoming more or less in high-throughput culture method, how to improve the difficult point that its culture efficiency is microorganism culturing.
Original position (insitu) is cultivated, and refers to that microorganism grows in the physical environment of originally growth, can reduce the impact of extra outside atmosphere so to greatest extent, make to cultivate or microorganism that future carries out screening is grown normally; Compared with traditional method, quantitatively there is greater advantage cultivating of microorganism.Micro-embedded technology refers to and adopts natural or synthesized polymer material, by chemistry, physics or physico-chemical processes, another kind of material is wrapped up, a kind of technology with the minigel of semipermeability or sealing cyst membrane can be formed according to material difference, adopt micro-embedded technology can provide the space needed for independently surviving for microorganism; Compared with the slat chain conveyor method of traditional microbiological, on the producing level in space, micro-embedded technology has great advantage.
How the high-throughput that is applied to strong to prior art such as Culture in situ, micro-embedded technology is cultivated field, improving high-throughput culture efficiency is emphasis of the present invention.
Summary of the invention
The deficiency that technical problem to be solved by this invention exists for prior art, and provide a kind of for marine microorganism original position high-throughput cultivate, the device of high pressure resistant under can meeting ocean environment hydrologic condition, the resistance to temperature difference, impact resistant; By being combined of specific device and micro-embedded technology, high-throughout Culture in situ can being carried out to microorganism, significantly improve the Culturability of marine microorganism, and preliminary screening can be carried out to marine microorganism.
The present invention is achieved through the following technical solutions:
For the device that marine microorganism original position high-throughput is cultivated, comprising one provides the seawater storehouse of simulating ocean environment and several separate cultivation cells; Described seawater storehouse comprises the base plate, reserve liquid layer, division board and the culture layer that are fixedly linked with bolt from the bottom to top, rectangle or foursquare reservoir is offered in the middle of reserve liquid layer, division board and culture layer offer consistent size, with the circular hole cultivated cell and match, and division board is relative up and down with the circular hole position in culture layer; Be provided with one deck filter membrane between described division board and culture layer, cultivate cell and be arranged in the circular hole of culture layer and be placed on filter membrane; Described base plate is seal base.
Preferably, described seawater storehouse entirety is rectangular parallelepiped warehouse, and specification is high 5-7cm, long 10-20cm, wide 10-20cm; Cultivating cell is cylinder shape warehouse, and specification is diameter 1-2cm, height 2-4cm.
Preferably, described reserve liquid layer is provided with water-in for seawater circulation and water outlet.
Preferably, filter membrane used is tetrafluoroethylene hydrophilic film, and aperture is 0.22 μm.
Preferably, the application of installation that described marine microorganism original position high-throughput is cultivated is in laboratory simulation in situ environment, and device is positioned in constant temperature oscillator.
Preferably, the separate cap sealing being connected with conduit of described each cultivation cell, the end of every root conduit is connected with independently miniature two-port valve; Cultivate between end or incubation period, water inlet valve is opened, and outlet valve is closed, and opens the duct valve of the cultivation cell of required sampling, can carry out sample collecting separately to each culturing room.
Preferably, the described separate cap with conduit, the material of top cover and conduit is tetrafluoroethylene.
Described device can also be applied to ocean environment Culture in situ, and during cultivation, base plate is the opening base plate having sieve aperture, and described cultivation cell top is sealed light-transmitting top board.
Preferably, the material of described transparent roof panels is toughened glass.
Preferably, the sieve aperture of described opening base plate is the bar shaped screen hole of width 2-4mm, length 30-40mm.
Further, above-mentioned cultivation cell, base plate, reserve liquid layer, division board and culture layer form by the stainless steel of respective thickness, tetrafluoroethylene, toughened glass or the Vehicle Processing of epoxy resin sheet material
Apparatus of the present invention, when laboratory simulation in situ environment, device can be placed in constant temperature oscillator, and the seawater storehouse of bottom of device is seal base, carries out seawater circulation by means of only into and out of water hole; The separate cap sealing being connected with conduit of each cultivation cell, wherein the end of every root conduit is connected with independently miniature two-port valve, by the hydrostatic control of these micro valves and water-in, water outlet, realizes carrying out independent sampling to each cultivation cell at any time.
Apparatus of the present invention, under ocean on the spot in-situ condition, other floatation devices auxiliary under, the respective depth in required marine site can be suspended in; Device base plate is open sieve aperture base plate, seawater enter seawater storehouse by these sieve apertures and through filter membrane with cultivate cell UNICOM, the top of device with the good top plate seal of light transmission, by these, achieve each cultivation cell and temperature in ocean environment, pressure, illumination, substances content synchronous.
Apparatus of the present invention can maintain a stable Culture in situ environment, to reduce the damage of environmental change to microorganism and experimental result; Separate cultivation cell, makes to have in culturing process independently to cultivate isolating mechanisms, makes each cultivation cell each microballoon even wherein can be used as single reactor and carries out screening and culturing, thus better achieves high-throughput cultivation; High-throughput culture technique is combined with Culture in situ, can provide stable in situ environment for microorganism, does not need transfer, avoid the pollution of pilot process in microbial cultivation process.Apparatus of the present invention can adapt to complicated ocean condition simultaneously, and device adopts specific sheet material to carry out simple Vehicle Processing, is connected and fixed by bolt, has high pressure resistant under concrete hydrological environment, the resistance to temperature difference, the advantage of impact resistant.
Accompanying drawing explanation
Fig. 1: the present invention is used for the device that marine microorganism original position high-throughput is cultivated, and is applied to the broken away view of structure during laboratory simulation in situ environment;
Fig. 2: the present invention carries out structural representation when high-throughput is cultivated for laboratory simulation in situ environment;
Fig. 3: the present invention be used for marine microorganism original position high-throughput cultivate device, be applied to ocean on the spot in-situ condition time broken away view;
Fig. 4: the present invention be applied to ocean on the spot in-situ condition time, the structural representation of the inner and opening base plate of device;
Fig. 5: apparatus of the present invention cultivate rear microsphere optical microscope schematic diagram;
Fig. 6: apparatus of the present invention cultivate rear microsphere fluorescence microscope schematic diagram;
Fig. 7: apparatus of the present invention cultivate rear microballoon internal electrical mirror intention;
Fig. 8: apparatus of the present invention cultivate rear microsphere fluorescence microscope schematic diagram;
Wherein: 1 seawater storehouse, 2 cultivate cell, 3 base plates, 4 reserve liquid layers, 5 division boards, 6 culture layer, 7 reservoirs, 8 circular holes, 9 filter membranes, 10 water-ins, 11 water outlets, 12 conduits, 13 top boards, the keyhole of 14 bolts.
Embodiment
Below in conjunction with accompanying drawing, the device that the present invention is used for the cultivation of marine microorganism original position high-throughput is described in detail.
As depicted in figs. 1 and 2:
For the device that marine microorganism original position high-throughput is cultivated, be applied in laboratory simulation in situ environment, device is positioned in constant temperature oscillator, comprises seawater storehouse 1 and several separate cultivation cells 2 that one provides simulating ocean environment; Seawater storehouse comprises base plate 3, reserve liquid layer 4, division board 5 and culture layer 6 from the bottom to top, each structure has keyhole 14, is fixedly linked with bolt each other.Offers rectangle or foursquare reservoir 7 in the middle of reserve liquid layer, division board and culture layer offer consistent size, with the circular hole 8 cultivated cell and match, and division board is relative up and down with the circular hole position in culture layer.Be provided with one deck filter membrane 9 between division board and culture layer, cultivate cell and be arranged in the circular hole of culture layer and be placed on filter membrane; Base plate is seal base, and reserve liquid layer 4 is provided with water-in 10 for seawater circulation and water outlet 11.
As shown in Figure 2, the separate cap sealing being connected with conduit 12 of each cultivation cell 2, the end of every root conduit is connected with independently miniature two-port valve; Cultivate between end or incubation period, water-in 10 valve open, water outlet 11 valve closes, open the duct valve of the cultivation cell of required sampling, sample collecting can be carried out to each culturing room separately.The material of top cover and conduit is tetrafluoroethylene.
Seawater storehouse 1 entirety is rectangular parallelepiped warehouse, and specification is high 5-7cm, long 10-20cm, wide 10-20cm; Cultivating cell 2 is cylinder shape warehouse, and specification is diameter 1-2cm, height 2-4cm.Filter membrane 9 is tetrafluoroethylene hydrophilic film, and aperture is 0.22 μm.
Device of the present invention can also be applied to ocean environment Culture in situ, and as shown in Figure 3 and Figure 4, now, base plate 3, for having the opening base plate of sieve aperture, cultivates cell 2 top seal transparent roof panels 13.Translucent roof plate 13 material is toughened glass; The sieve aperture of opening base plate is the bar shaped screen hole of width 2-4mm, length 30-40mm.
In addition, cultivate cell (2), base plate (3), reserve liquid layer (4), division board (5) and culture layer (6) form by the stainless steel of respective thickness, tetrafluoroethylene, toughened glass or the Vehicle Processing of epoxy resin sheet material.
Under ocean on the spot in-situ condition, other floatation devices auxiliary under, the respective depth in required marine site can be placed in; Device base plate is open sieve aperture base plate, seawater enter seawater storehouse by these sieve apertures and through filter membrane with cultivate cell UNICOM, the top of device with the good top plate seal of light transmission, by these, achieve each cultivation cell and temperature in ocean environment, pressure, illumination, substances content synchronous.
The present invention also provides a kind of method utilizing said apparatus to carry out cultivating, screening marine microorganism, recycles the seawater obtained on the spot, coordinates light and temperature to control, simulation marine microorganism growth in situ environment.Cultivation cell and simulating ocean environment utilize tetrafluoroethylene hydrophilic film to isolate, to ensure the carrying out of exchange of substance, and infecting of isolation miscellaneous bacteria.
Below in conjunction with specific embodiment to utilizing said apparatus to cultivate, screen the method for marine microorganism and be described in detail:
Embodiment 1:
When being applied to laboratory simulation in situ environment, utilizing the device described in Fig. 1 and Fig. 2 to carry out cultivating, screening the method for marine microorganism, comprise the steps;
(1) marine microorganism microballoon is prepared: concentrated seawater (200:1) is prepared liquid with microballoon and mixes, prepared the calcium alginate microsphere of the particle diameter about 50 microns being embedded with marine microorganism by coaxial air-flow method, in prepared microballoon and microballoon, the ratio of institute's imbedded microbe is 10:1;
(2) after cultivating cell sterilising treatment, add the microballoon 80 milligrams of preparation in step 1 in each little indoor, cover the separate cap sealing being connected with conduit 12;
(3) in constant temperature oscillator, add the seawater recycling and obtain on the spot, device entirety is placed in the constant temperature oscillator that temperature is 8 DEG C, and the valve opening intake-outlet carries out seawater circulation;
(4) cultivate between end or incubation period, water inlet valve is opened, and outlet valve is closed, and opens the duct valve of the cultivation cell of required sampling, can carry out sample collecting separately to each culturing room; Sample carries out microscopic examination, and result such as Fig. 5 and Fig. 6 shows, Fig. 5 is photo under opticmicroscope, and Fig. 6 is fluorescent microscopy images, can observe microorganism embed by microballoon; And thalli growth is good in the short period of time, there is not breakage in microballoon.
Strain identification is carried out to the microorganism that step 4 obtains: extract DNA according to prior art disclosed in this area, and gained DNA is carried out the PCR of 16srRNA, check order subsequently, by gained sequence and database comparison, obtain a result, find wherein have the thalline of Pseudoalteromonas and Halomonas to be cultivated out.
Embodiment 2:
Be applied to ocean on the spot in-situ condition time, utilize the device described in Fig. 3 and Fig. 4 to carry out cultivating, screening the method for marine microorganism, comprise the steps;
(1) prepare marine microorganism microballoon: concentrated seawater (200:1) is prepared liquid with microballoon and mixes, prepared the calcium alginate microsphere of the particle diameter about 50 microns being embedded with marine microorganism by coaxial air-flow method;
(2), after cultivating cell sterilising treatment, in each hole, add the microballoon 80 milligrams of preparation in step 1, cover light transmission top plate seal;
(3) other floatation devices auxiliary under, be suspended in the lower 40 meters of degree of depth in South China Sea sea, seawater enters seawater storehouse by the sieve aperture of open base plate and through filter membrane and cultivation cell UNICOM;
(4) after cultivation terminates, open top board, sample collecting can be carried out to each culturing room separately; Sample carries out microscopic examination, and as shown in Figure 7 and Figure 8, Fig. 7 is photo under opticmicroscope to result, and Fig. 8 is fluorescent microscopy images, can observe microorganism embed by microballoon; And thalli growth is good in the short period of time, there is not breakage in microballoon.
Strain identification is carried out to the microorganism that step 4 obtains: extract DNA according to prior art disclosed in this area, and gained DNA is carried out the PCR of 16srRNA, check order subsequently, by gained sequence and database comparison, obtain a result, find wherein have the thalline of Pseudoalteromonas and Halomonas to be cultivated out.
Device of the present invention, cultivates cell and can be used as independent reaction vessel, can not produce mutual pollution each other, thus can carry out high-throughout Culture in situ.
Above-mentioned explanation is not the restriction to patent of the present invention; also be not limited in above-mentioned citing in patent of invention, the change of those skilled in the art done by essential scope of the present invention, remodeling, interpolation or replacement also should belong to protection scope of the present invention.

Claims (6)

1. the device cultivated for marine microorganism original position high-throughput, comprise one and the seawater storehouse (1) of simulating ocean environment and several separate cultivation cells (2) are provided, it is characterized in that, described seawater storehouse comprises the base plate (3) be fixedly linked with bolt (14) from the bottom to top, reserve liquid layer (4), division board (5) and culture layer (6), rectangle or foursquare reservoir (7) is offered in the middle of reserve liquid layer, division board and culture layer offer consistent size, the circular hole (8) matched with cultivation cell, and division board is relative up and down with the circular hole position in culture layer, be provided with one deck filter membrane (9) between described division board and culture layer, cultivate cell and be arranged in the circular hole of culture layer and be placed on filter membrane, described base plate is seal base, described reserve liquid layer (4) is provided with water-in (10) for seawater circulation and water outlet (11), the application of installation that described marine microorganism original position high-throughput is cultivated is in laboratory simulation in situ environment, and device is positioned in constant temperature oscillator, described each cultivation cell (2) seals with the separate cap being connected with conduit (12), and the end of every root conduit is connected with independently miniature two-port valve, cultivate between end or incubation period, water-in (10) valve open, water outlet (11) valve closes, open the duct valve of the cultivation cell of required sampling, sample collecting can be carried out to each culturing room separately, the described separate cap with conduit (12), the material of top cover and conduit is tetrafluoroethylene.
2. the device as requested described in 1, is characterized in that, described seawater storehouse (1) entirety is rectangular parallelepiped warehouse, and specification is high 5-7cm, long 10-20cm, wide 10-20cm; Cultivating cell (2) is cylinder shape warehouse, and specification is diameter 1-2cm, height 2-4cm.
3. the device as requested described in 1, is characterized in that, filter membrane (9) used is tetrafluoroethylene hydrophilic film, and aperture is 0.22 μm.
4. the device as requested described in any one of 1-3, it is characterized in that, described device can also be applied to ocean environment Culture in situ, now, base plate (3) is for having the opening base plate of sieve aperture, and described cultivation cell (2) top is sealed transparent top board (13).
5. the device as requested described in 4, is characterized in that, the material of described transparent roof panels (13) is toughened glass.
6. the device as requested described in 4, is characterized in that, the sieve aperture of described opening base plate is the bar shaped screen hole of width 2-4mm, length 30-40mm.
CN201410658474.5A 2014-11-18 2014-11-18 A kind of device cultivated for marine microorganism original position high-throughput Expired - Fee Related CN104498322B (en)

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CN104928148A (en) * 2015-06-03 2015-09-23 常州大学 Novel bacterial biofilm culture device suitable for atomic force microscope (AFM) to perform in-situ detection
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CN105969637A (en) * 2016-06-17 2016-09-28 国家海洋局第海洋研究所 Deep sea microorganism in-situ cultivation and enrichment device
CN107937249A (en) * 2017-12-30 2018-04-20 常州水精灵环保设备有限公司 A kind of Deep-Sea Microorganisms in-situ enrichment device
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