CN103865751A - Array-type high-throughput microbe separating culturing apparatus - Google Patents
Array-type high-throughput microbe separating culturing apparatus Download PDFInfo
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- CN103865751A CN103865751A CN201210548502.9A CN201210548502A CN103865751A CN 103865751 A CN103865751 A CN 103865751A CN 201210548502 A CN201210548502 A CN 201210548502A CN 103865751 A CN103865751 A CN 103865751A
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
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- C—CHEMISTRY; METALLURGY
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
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- C—CHEMISTRY; METALLURGY
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
Abstract
The invention relates to an array-type high-throughput microbe separating culturing apparatus, and discloses a novel microbe separating culturing apparatus. The apparatus is capable of realizing high-throughput separation and culture of cultured and/or uncultured microbes in a laboratory, operation is simplified, and the apparatus has good compatibility with conventional laboratory equipment. The separating culturing apparatus is capable of simulating in-situ culturing of microbe by natural environment, and is capable of efficiently separating difficult-to cultured microbe ( or named uncultured microbe or unculturable microbe). The microbe separating culturing apparatus is applicable to separate and culture difficult-to cultured microbe from environment, also is applicable to research interacting relationship between microbe and microbe, between microbe and environment factors, and between microbe and nutrition factors, and also is applicable to fields such as microbe source investigation and research, environment and ecology relationship, natural medicine discovery, biological technology research and the like.
Description
Technical field
The invention belongs to microorganism culturing field; More specifically, the present invention relates to a kind of array microorganism seperation culture device.
Background technology
In physical environment, existing abundant microbial species resource, is the resource treasure-house of the great sustainable developments such as drug discovery, the functional microorganism resource excavating (as bioenergy, biochemical industry, biomaterial produce bacterium) and solution mankind nowadays society and face, environment, Population and Health.The educable microbe species of the known mankind is only the 0.01-0.1% of marine microorganism, 1% left and right of land microorganism, in many microorganism classification doors (Phylum) or guiding principle (Class), be not also separated to so far a representative bacterial classification (Rapp é, M.S, et al.The uncultured microbial majority.Annual Review of Microbiology, 2003,57:369 – 394).But 100 for many years adopt and separate not culturing micro-organisms (Uncultured Microbes) for the normal isolation culture method of culturing micro-organisms (Cultured Microbes) and encountered the technology barrier that is difficult to go beyond.Conventional microorganism separating and culturing method adopts dull and stereotyped dilution to separate, effort, consuming time, and nutrient agar has obvious selectivity and the Preference to certain quasi-microorganism growth, make the mankind be difficult to approach those rich and varied not culturing micro-organisms (Karsten, Z., et al.Cultivating the uncultured.Proceedings of the National Academy of Sciences of the United States of America, 2002,99 (24): 15681-15686).Microbiology and pharmacology screening study are usually absorbed in repeated isolation, repeat to find in the cycle operation of known microorganisms and known compound.Therefore, the not new device of the separation and Culture of culturing micro-organisms, novel method, new industrial research, is that microbiology fundamental research in the world and Applied research fields are wide concerned and face a Task of very large technological challenge.
It is a kind of effective novel method that simulating natural environment culture method separates not culturing micro-organisms, the method separates the microorganism novel species in seawater, fresh water or soil by a kind of diffuser casing of design (Diffusion Growth Chamber), obtain good effect (Kaeberlein, T., et al.Isolating " uncultivable " microorganisms in pure culture in a simulated natural environment.Science, 2002,296:1127-1129).Diffuser casing is by two membranes and a principle structure that stainless steel packing ring forms, (aperture is that 0.03 μ m) first cements with packing ring to lower membrane, mix with the warm agar after thawing taking from after microorganism on hemipelagic sediment is diluted, pour diffuser casing into, then upper layer film is adhered to the top of packing ring.Diffuser casing is put back in the environment of original sampling spot and is cultivated, or be put in simulation these microorganism Natural Survival environment water in cultivate.This training strategy allows the nutrition species in environment can free in and out diffuser casing, and microorganism is restricted to diffuser casing inside.Cultivating after one week has the microcolony coming in every shape to be in a large number monitored to.The bacterium colony rate of recovery of the method maximum has reached 40 ± 13% of microbe inoculation number.Compared with the plate isolation method of standard, utilizing the stable microorganism being separated to of diffuser casing is about 300 times of flat band method.When this method being applied to the multiple-microorganism sample in other environment sources, prove that this is the strategy of a kind of extremely successful separation and Culture microorganism.The labor capacity of considering this method is very large, limit its application in drug discovery, diffuser casing is developed into massive parallel culturing micro-organisms device in a kind of high-throughout never culturing micro-organisms by Nichols, be called separating chips (Isolation Chip, or iChip), they have built two matrixes in the scope of 72 × 19 × 1mm size, each matrix has 192 micropores, each micro-pore diameter is 1mm, and totally 384 micropores, are equivalent to 384 little diffuser casings.1 cell of evenly distribute in each cell when use, so just realize and cultivated and separate a step and complete (Nichols, D, et al.Use of ichip forhigh-throughput in situ cultivation of " uncultivable " microbial species.Applied andEnvironmental Microbiology, 2010,76 (8): 2445-2450).Gavrish has slightly changed over diffuser casing again a grabber (Trap), is used for specially separating filamentous microorganism, as actinomycetes, mould.Different from diffuser casing: lower membrane changes the polycarbonate membrane of 0.2-0.6 μ m into, lower membrane and packing ring fastening good after, by sterilizing not containing in the agar flood chamber of bacterium, cover upper layer film.Grabber is placed in to sample surfaces to be separated (as the pedotheque that will separate), close contact.The method has utilized the mycelia of filamentous microorganism in the time spreading growth, can penetrate filter membrane and enter into the indoor agar of grabber, actinomycetes generally can penetrate the film of 0.2 μ m, mould can penetrate the film of 0.4-0.6 μ m, nutritive ingredient can free diffusing in agar.By controlling the aperture of lower membrane, just optionally separating payingoff bacteria or mould.These two kinds of separation and Culture modes all belong to Culture in situ (the in situ Cultivation) strategy (Gavrish of simulating natural environment, E., et al.A trap in situ cultivation of filamentous actinobacteria.Journal ofMicrobiological Methods, 2008,72:257-262).
But still there are many technical problems in the method for Nichols, because the hole on iChip is very little, by iChip is immersed in the nutrient agar containing bacterium completely toward the way of distributing substratum in hole and being dispersed in the microorganism in substratum, the substratum that is melting state is diffused in aperture, Deng taking out from culture medium solution after iChip, substratum is solidificated in aperture, reach the object (reference (Nichols, 2010 in diagram) that distributes substratum and microorganism.But this mode inevitably can be at whole iChip remained on surface substratum and microorganism cells, be difficult to like this avoid microbial growth and the interference to experiment outside these holes, so the mode of this distribution substratum can not overcome crossed contamination between hole, this is the problem that this iChip structure itself is brought.Secondly, cultivating is only a link in whole microorganism mask work flow process, iChip is only to provide a culture environment, microorganism after cultivation also needs to carry out the processes such as sample analysis separates with microorganism, purifying, sample after iChip cultivates must be shifted out to Yi Ge hole, a hole, and can think about it that on so little chip, sample in so many apertures being shifted out is a very difficult job, if only with manual operations, working efficiency reduces greatly, and very easily makes mistakes.This like this script is wished the high-throughput design concept of increasing work efficiency, and finally also can not embody due to the speed limit bottleneck of follow-up work flow process.High-throughput techniques is to need the high-throughput between each step of a whole set of flow process to mate to realize, otherwise will run into " short-board effect " problem, is decided by that the committed step of flux magnitude is just the step that flux is minimum.For this nonstandard design of iChip, having no bibliographical information has special automatization or its use of high-throughput matched.These defects make said apparatus finally not form commercial prod.
Therefore, the device that improves diffuser casing culturing micro-organisms, in the urgent need to further studying, is developed in this area, overcomes above technological deficiency.
Summary of the invention
The object of the present invention is to provide a kind of array microorganism seperation culture device.
In a first aspect of the present invention, a kind of microorganism seperation culture device is provided, described separation and Culture device comprises: culture apparatus (2) and environment simulator (1);
Described culture apparatus (2) comprising:
Middle culture plate (24), is distributed with 6-1536 through hole on it;
Upper filtering membrane (23), it is covered in the top of middle culture plate (24);
Lower filtering membrane (25), it is covered in the below of middle culture plate (24);
Upper mounted plate (21), it is covered in the top of filtering membrane (23), is distributed with 6-1536 through hole on it, and specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24);
Bottom plate (27), it is covered in the below of lower filtering membrane (25), is distributed with 6-1536 through hole on it, and specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24);
Described upper mounted plate (21), upper filtering membrane (23), middle culture plate (24), lower filtering membrane (25), bottom plate (27) stacked arrangement from top to bottom, fixing with fastening piece (28);
Described environment simulator (1) comprising:
Liquid storage container (11), it can hold described culture apparatus (2);
Bracket (12), it is positioned at liquid storage container (11) (preferably, described bracket is level), for carrying described culture apparatus (2);
Upper cover (13), the opening of itself and liquid storage container (11) matches.
In a preference, described upper mounted plate, upper filtering membrane, middle culture plate, lower filtering membrane, bottom plate are after fastener is fastening, the environment thing that in each through hole, culture can see through in filtering membrane and environment simulator carries out material (not comprising cell) exchange, but direct material (comprising cell) exchange does not occur between each through hole.
In another preference, described liquid storage container is opening.
In another preference, between described upper mounted plate (21) and upper filtering membrane (23), also comprise upper sealing panel (22); On it, be distributed with 6-1536 through hole, specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24); And/or
Between described bottom plate (27) and lower filtering membrane (25), also comprise lower seal plate (26); On it, be distributed with 6-1536 through hole, specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24).
In another preference, the effect of culture in the each through hole of culture plate in the middle of described upper sealing panel and lower seal plate performance isolation, make the environment thing that in each through hole, culture sees through in filtering membrane and environment simulator carry out material (not comprising cell) exchange, but direct material (comprising cell) exchange does not occur between each through hole.
In another preference, on fixed plate (21), upper sealing panel (22), middle culture plate (24), lower seal plate (26), bottom plate (27), specification, quantity and the distributing position of through hole are consistent, and number of openings is 6-1536; And/or the matrix form that is arranged as of through hole is arranged.
In another preference, the peripheral shape of the shape of cross section of described liquid storage container (11) and described culture apparatus (2) matches, and described culture apparatus (2) is arranged on the bracket in described reservoir.
In another preference, the thickness of described middle culture plate is not less than 0.5mm; And/or the diameter of described through hole is at 1-35mm.
In another preference, the diameter of described through hole is at 2-22mm; More preferably, the diameter of described through hole is: 2-9mm.
In another preference, the thickness of described middle culture plate is not less than 1mm; Preferred thickness is 1-5mm.
In another preference, described fastening piece (28) is selected from (but being not limited to): screw fastener, magnetic-type fastening piece, clamping type fastening piece.
In another preference, the inside top surface of described upper cover (13) is provided with gasket (14); And/or filtering membrane.
In another preference, the end face of described upper cover (13) can arrange one or more through holes;
On described gasket, one or more through holes can be set, this through hole is consistent with specification, quantity and the distributing position of the through hole of the end face of upper cover; With
Between described upper cover top surface and gasket, also comprise one deck filtering membrane.
In another preference, the material of described upper mounted plate (21), upper filtering membrane (23), middle culture plate (24), lower filtering membrane (25), bottom plate (27), liquid storage container (11), upper cover (13) and bracket (12) is selected from (but being not limited to): stainless steel, aluminium, polypropylene, polycarbonate; And/or
Described upper sealing panel (22) and the material of lower seal plate (26) are selected from (but being not limited to): silica gel, rubber, tetrafluoroethylene etc.
In another preference, the withstand high temperatures of described material; Preferably, tolerate the temperature of 121 DEG C.
In another preference, described upper filtering membrane (23) and the filter membrane aperture of lower filtering membrane (25): 0.01-10.0um.
In another preference, filter membrane material can be: Mierocrystalline cellulose acid esters class, polyamide-based, polysulfones, polyvinylidene difluoride (PVDF), tetrafluoroethylene, polycarbonate, polyolefins.
In another preference, the form of arranging in the described upper hole of middle culture plate (24) is matrix structure, through-hole spacing 4-25mm, and through-hole diameter 2-25mm, the middle thickness of slab of cultivating is not less than 1mm (preferably, 1-5mm).
In another preference, described middle culture plate is consistent with general 384 hole microtest plates, long 128 ± 1mm, wide 86 ± 1mm; Through hole number is 384, and through-hole spacing and distribution are consistent with general 384 orifice plates, and through-hole diameter 2-4mm cultivates thickness of slab 1-3mm.
In another preference, described middle culture plate is consistent with general 96 hole microtest plates, long 128 ± 1mm, wide 86 ± 1mm; Through hole number is 96, and through-hole spacing and distribution are consistent with general 96 orifice plates, and through-hole diameter 4-8mm cultivates thickness of slab 1-3mm.
In another preference, described middle culture plate is consistent with general 48 hole microtest plates, long 128 ± 1mm, wide 86 ± 1mm; Through hole number is 48, and through-hole spacing and distribution are consistent with general 48 orifice plates, and through-hole diameter 6-11mm cultivates thickness of slab 2-4mm.
In another preference, described middle culture plate is consistent with general 24 hole microtest plates, long 128 ± 1mm, wide 86 ± 1mm; Through hole number is 24, and through-hole spacing and distribution are consistent with general 24 orifice plates, and through-hole diameter 9-19mm cultivates thickness of slab 3-5mm.
In another preference, described middle culture plate is consistent with general 12 hole microtest plates, long 128 ± 1mm, wide 86 ± 1mm; Through hole number is 12, and through-hole spacing and distribution are consistent with general 12 orifice plates, and through-hole diameter 12-22mm cultivates thickness of slab 3-5mm.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Brief description of the drawings
The one-piece construction schematic diagram of Fig. 1, microorganism seperation culture device of the present invention.
The structural representation of Fig. 2, culture apparatus (culturing room).
The structural representation of Fig. 3, environment simulator (simulating chamber).
The assembling schematic diagram of Fig. 4, environment simulator (simulating chamber).
The principle of work schematic diagram of Fig. 5, microorganism seperation culture device of the present invention.
Mark in figure is respectively:
1. environment simulator (environmental chamber); 11. liquid storage containers (reservoir);
12. brackets; 13. upper covers;
14. gaskets; 15. ambient water or mixture;
2. culture apparatus (culturing room); 21. upper mounted plates;
22. upper sealing panels; Filtering membrane on 23.;
Culture plate in the middle of 24.; 25. times filtering membranes;
26. lower seal plates; 27. bottom plates;
28. screw fasteners; 29. semisolids or liquid nutrient medium;
210. diffuser casing; 211. diffuser casing.
Embodiment
In prior art, lack the not experimental installation of culturing micro-organisms of special separation and Culture in order to solve, the present invention is based on diffuser casing and cultivate principle, manufacture and design a kind of novel microorganism high-throughput isolation culture apparatus, device of the present invention can be realized high-flux microorganism diffusion type cultural method, simplified control, and good with existing laboratory equipment compatibility, diffuser casing is interior except can quantitatively add the solid medium that contains cell under high-throughput mode, outside semisolid medium, can also quantitatively add the liquid nutrient medium that contains cell, overcome and in iChip technology, be difficult to realize the defect that high-throughput quantification adds liquid nutrient medium, also having overcome in iChip technology distributes nutrient agar operation to be easy to bring cross-contamination issue between Kong foreign minister bacterium and hole, after cultivating, in sample analysis and treating processes, operation is easy and make the defects such as working efficiency reduces greatly.Separation and Culture device of the present invention can simulating natural environment carry out Culture in situ to microorganism, and not culturing micro-organisms of energy high efficiency separation; Also can be applicable to the interaction relationship between microorganisms and microorganism, microorganism and environmental factors, microorganism and nutritional factor; Also can be applicable to the fields such as Microbial resources investigation and research, environment and ecology relation, natural drug discovery, biotechnology research.
Microorganism seperation culture device
The invention provides a kind of microbial cultivation device that can simulating natural environment, this device comprises two main functional modules: one is microbial cultivation device (microwell plate diffuser casing), and microorganism growth space is provided; Another is environment simulator (environmental chamber), simulates microorganism spontaneous growth environment or builds specific microorganism ecological environment, carries out exchange of substance with microbial cultivation device, the needed nutritive ingredient of supply microorganism growth.
Culture apparatus
Described culture apparatus is a kind of " sandwich " formula structure, comprises culture plate and lays respectively at filtering membrane and the retaining plate of the upper and lower both sides of culture plate.Specifically comprise from top to bottom: upper mounted plate, upper filtering membrane, middle culture plate, lower filtering membrane, bottom plate.Above-mentioned each laminate and film are fixedly clamped by screw or other fastening means.Except two-layer filtering membrane, on retaining plate and culture plate, be all provided with the through hole of quantity, distributing position, specification corresponding (consistent), the through hole on middle culture plate is as microorganism culturing container, and each hole is a diffuser casing.In situation about fastening by upper and lower fixing plate clamp, each diffuser casing on culture plate is two-layerly close to filtering membrane on it microorganism or other cell and external environment is separated by upper and lower, cell is limited in microwell plate diffuser casing, and nutritive substance in external environment can free diffusing turnover diffuser casing.Each through hole of middle culture plate is separately as culturing room independently, direct exchange of substance and cell exchange can not occur between mutually; But, the filtering membrane covering up and down by through hole, each through hole can with filtering membrane outside environment generation exchange of substance (containing cell exchange).
As optimal way of the present invention, between described upper mounted plate and upper filtering membrane, also comprise upper sealing panel; Between described bottom plate and lower filtering membrane, also comprise lower seal plate.On sealing plate, be also distributed with through hole, specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate.Sealing plate application can more effectively bring into play sealing function, culture in the each through hole of culture plate in the middle of isolation, make the environment thing that in each through hole, culture sees through in filtering membrane and environment simulator carry out material (not comprising cell) exchange, but direct material (comprising cell) exchange does not occur between each through hole.In the time clamping between by upper and lower two-layer retaining plate by screw or alternate manner, sealing plate just will separate between the Kong Yukong on middle culture plate completely, make to spread culturing room's inner cell by upper and lower two-layer filtering membrane again and external environment is separated, microorganism cells is completely restricted in diffuser casing like this, and in external environment nutritive substance can free diffusing turnover diffuser casing, and in diffuser casing, carry out material transfer and exchange.Sealing plate major function be to seal, therefore this two-layer sealing plate also can use sealing-ring (when being circular when through hole, adopt O type circle) substitute, can adopt the sealing-ring that quantitatively, size identical with diffuser casing hole count mated with diffuser casing aperture to seal, upper and lower two ports of diffuser casing in the middle of sealing-ring can be separately positioned on culture plate, clamp by upper and lower two-layer retaining plate; More preferably can locate each sealing-ring at the through hole periphery processing sealing-ring locating slot (groove identical with sealing-ring size) of upper and lower two-layer retaining plate.Described sealing-ring also can be arranged on the retaining plate of upper and lower both sides; Sealing-ring locating slot also can be arranged on middle culture plate alternatively.
On fixed plate, upper sealing panel, middle culture plate, lower seal plate, bottom plate, specification, quantity and the distributing position of through hole are consistent, and number of openings is 6-1536; It is more preferably 12-384; As 384 holes, 96 holes, 48 holes, 24 holes, 12 holes etc.Through hole arrange and hole count can be set as required; Preferably, each through hole is arranged; Preferred, the pitch of holes on the microwell plate of through-hole spacing and identical hole count general on present market is identical, is convenient to and automatic fluid workstation compatibility.The shape of through hole is optional, and such as cross section is circle, square, ellipse etc.As optimal way of the present invention, the cross section of described through hole is circular.
At present, microwell plate (Mciro-Well Plate) is for microorganism culturing widespread use, after deliberation very detailed of the comprehensive parameters such as pore structure to microwell plate, volume of culture, oscillation frequency, surface tension, gas transfer efficiency, mixing of materials.Microwell plate has the advantage of array arrangement, stdn, collimation, high-throughput and self-reacting device compatibility.Therefore, as optimal way of the present invention, on middle culture plate of the present invention, specification, quantity and the distributing position of each through hole and existing known microwell plate are (as general 384 hole microtest plates, 96 hole microtest plates, 48 hole microtest plates, 24 hole microtest plates, 12 hole microtest plates) consistent; Thereby, make the not principle of culturing micro-organisms of cells culture in situ high efficiency separation that microorganism seperation culture device of the present invention can simulating natural environment, can realize again the diffuser casing culture apparatus with the microwell plate form of the high-throughput isolation of automatic fluid treatment unit highly compatible, formation can commercially be installed, and meets those skilled in the art's research needs.
Environment simulator
In the present invention, described environment simulator is made up of liquid storage container, upper cover and bracket.Described liquid storage container can hold described culture apparatus; Described bracket is positioned at liquid storage container (preferably, described bracket is level), for carrying described culture apparatus; Described upper cover, the opening of itself and liquid storage container matches.Preferably, described culture apparatus and environment simulator can closely be fixed together, can realize tight fixing by limiting the size of culture apparatus and environment simulator inside, also can realize closely fixing by the element such as bayonet socket, draw-in groove is set on liquid storage container inside or bracket.
As optimal way of the present invention, described liquid storage container shape is rectangular parallelepiped or square, and it is completely unlimited that upper end is, and sidewall is perpendicular to bottom surface.
As optimal way of the present invention, liquid storage container bottom external length and width size and existing known microwell plate are (as general 384 hole microtest plates, 96 hole microtest plates, 48 hole microtest plates, 24 hole microtest plates, 12 hole microtest plates) length and width are measure-alike or smaller, are convenient to the equipment and instrument for microwell plate on compatible market, in the situation that not needing with miscellaneous equipment instrument compatibility, also can adopt other different shape and size; The internal depth of liquid storage container is greater than total height and the bracket total height sum of microwell plate diffuser casing.
Described bracket can directly be arranged in liquid storage container, for example, be arranged on the wall of liquid storage container; Or described bracket and liquid storage container are detachable.As long as should be understood that the effect that can bring into play support culture apparatus, shape, the structure of described bracket are not particularly limited.As optimal way of the present invention, described bracket can form a horizontal plane, and liquid storage container is can horizontality placed on it.Certainly,, for the needs of cultivating, bracket also can be arranged to non-level state; For example, this bracket can be the bracket of capable of regulating height.As optimal way of the present invention, it (can be also the leg of other quantity that bracket is made up of a horizontal pane and four legs equal in length, but more than at least three, and on same straight line, do not distribute), bracket is put in liquid storage container, and bracket is used for placing described culture apparatus.Reach the liquid that has certain space to hold between culture apparatus lower end and liquid storage container bottom to take from environment or the mixture of liquid and solid by bracket support.
Described upper cover and the opening of liquid storage container match, and it can directly be arranged on liquid storage container, for example, can come to be connected with liquid storage container by hinge arrangement; Or described upper cover and liquid storage container are detachable.As long as can bring into play the sealing process of lid, the upper cover of any shape and structure is all available.
As optimal way of the present invention, described upper cover is surrounded by end face and side, and lateral vertical is in end face; Inside the horizontal section of upper cover side, shape is identical with the outer shape of liquid storage container side horizontal section, and its horizontal section interior dimensions is slightly larger than the outside dimension of liquid storage container side horizontal section.More preferably, there are a filtering membrane and a gasket in upper cover top surface inner side, upper cover top surface atresia or punching, punching can be made a call to one or several through hole, if upper cover top surface punching, also magnitude setting, shape, the consistent through hole of distributing position of correspondence position on the gasket of upper cover top surface inner side, is clipped in the middle of lid and gasket with filtering membrane.
More preferably, the gasket of upper cover top surface midsole layer filtering membrane and one deck silica gel material or other material, on lid, do not punch or make a call to one or several through hole, on the gasket of lid inner side, correspondence position is provided with the through hole of equal amts and shape, and filtering membrane is just clipped in the middle of lid and gasket.
Material
The parts such as the upper bottom plate of apparatus of the present invention, middle culture plate, microenvironment simulator liquid storage container, upper cover, bracket can adopt materials processing or the castings such as stainless steel, aluminium, polypropylene material, polycarbonate, as long as they have some strength.Preferably, these materials can be high temperature-resistant disinfected; More preferably can tolerate 121 DEG C or above high temperature.The each several part of device of the present invention can pass through the various ways manufactures such as mechanical workout, casting, injection moulding, drawing-die.Each accessory adopts above-mentioned material processing and manufacturing, belongs to durable products, does not have the problem such as resource consumption and environmental pollution that on market, the disposable use of microwell plate brings that is similar to.
Described upper-lower seal plate, be alternatively arranged in the middle of sealing-ring on culture plate or upper bottom plate can adopt the silica-gel plate of various materials or other to have certain elastic material, as long as it can realize the effect of sealing.Preferably, it can be high temperature-resistant disinfected; More preferably can tolerate 121 DEG C or above high temperature.
Because apparatus of the present invention have adopted filtering membrane fixed form more flexibly, the replacement of film, the screening of membrane material are very easy to, by changing the mould material of different pore size, microwell plate diffuser casing just can become high-throughput Trap, for the microorganism (as actinomycetes or fungi) of special sample separation.
Application
When use, described culture apparatus is placed on the bracket in environment simulator, covers after upper cover, form a set of complete separation and Culture device.This device can be used for leaving standstill to be cultivated, and is also applicable to concussion and cultivates.
Described culture apparatus and environment simulator are closely fixed together, between the microwell plate diffuser casing of culture apparatus and environment simulator, can exchange nutritive substance by filtration medium (film), microorganism is limited in the through hole of diffuser casing, microbe population in each through hole can be according to research and design manual control, in through hole, substratum can be selected different forms or composition according to research purpose, and the filling of substratum can be mated multichannel pipettor or automatization liquor removing workstation.In lower floor's environment simulator, can be from the seawater in sampling environment, fresh water, soil etc., can be also according to these environmental qualities manual simulation's solution or solid dielectric.Lower floor's environment simulator can be an ameristic simulating chamber, can be to be also divided into the simulating chamber that the band of more environment is cut apart.Described culture apparatus also can not coordinate with environment simulator, is placed directly in sampling spot or other environment and cultivates.
Under working order, described culture apparatus is positioned on the bracket in liquid storage container (reservoir), between microwell plate diffuser casing bottom and liquid storage container inner bottom part, form a cavity, as environment simulator, can filling liquid in environment simulator or semi-solid solution (from seawater, river, lake water in microorganism to be separated physical environment of living in; Or the mixture of water and mud; Or the nutrient solution of artificial preparation etc., represent with " ambient water or mixture " in the drawings).Because each diffuser casing on culture plate is separated microorganism (or other cell) and external environment by the upper and lower two-layer filtering membrane of being close on it, cell is limited in (in diffuser casing, being semisolid medium or liquid nutrient medium) in microwell plate diffuser casing, and nutritive substance in external environment (be injected into contain in ambient water in liquid storage container or mixture various small-molecule substances etc.) can see through filtering membrane free diffusing turnover diffuser casing; And cell metabolite in diffuser casing also can free diffusing turnover diffuser casing.Like this, by diffusion, especially under wave and culture mode, can be by same environmental chamber generation exchange of substance between each diffuser casing, therefore, this device can be used between microorganisms and environment, interaction relationship between microorganism and microorganism, between microorganism and nutritional factor, can microorganism separately under pure culture state microorganisms the relation such as cultivate altogether.
Owing to being high-throughput training method, the microorganism cells quantity (as 1 cell in average every hole) in the middle of can controlling as required in each hole of culture plate, can realize microorganism separation and purifying and synchronously carry out.
Known microorganism that at present can artificial culture is only the 0.01-0.1% of marine microorganism, 1% left and right of land microorganism, in physical environment, exceeding 99% not educable microorganism, is the resource treasure-house that drug discovery, functional microorganism excavated and solved great sustainable development.But adopt conventional flat board dilution separation to wait microorganism separating and culturing method, not only effort, consuming time, and nutrient agar has obvious selectivity and the Preference to the growth of certain quasi-microorganism, make the mankind be difficult to approach those rich and varied not culturing micro-organisms.Therefore, exploitation microorganism separation and Culture new technology and new clothes are set to and break this deadlock a kind of technique means is provided.Device of the present invention is to realize this goal becomes possibility.The application of this device can reach and improve microorganism separation efficiency, obtain more multifarious microorganism novel species and the new compound of not cultivating, for the researchs such as drug discovery, the separation of difficult culturing micro-organisms, synthetic biology Component screening provide a kind of technique means, and be applicable to study the Eco-physiological mechanism between these difficult culturing micro-organisms and environment, biology, chemical molecular.Therefore, this device use aspect life science fundamental research, applied research is suitable wide.The application of this novel method, new technology, new device can promote the technical progress of relevant industries, and brings indirectly economic benefit.
The invention has the beneficial effects as follows:
(1) compared with traditional plate isolation method, apparatus of the present invention have designed special structure and have realized simulating natural environment and separation and Culture microorganism, not aspect culturing micro-organisms, have clear superiority especially at separation and Culture occurring in nature.
(2) culture apparatus of the present invention has the feature of high-throughput, microminiaturization, has improved the scientific effort efficiency of separate microorganism, has reduced the input of scientific research effort intensity and minimizing experimental site, equipment and material reagent.
(3) interaction mechanism that the present invention can be used between microorganisms and environment, microorganism and microorganism, microorganism and nutritional factor is studied, as symbiotic relationship, sensitive factor discovery etc.;
(4) device of the present invention is preferably standard microwell plate specification, can with some automatic equipment compatibilities of existing supporting conventional microwell plate, fully increase work efficiency and the utilization ratio of existing installation; And as long as change the hole knockout on microwell plate diffuser casing, just can be on market switch between 384,96,48,24,12,6 holes of standard, versatility is very high.
(5) the present invention can adopt the metal material processing and manufacturings such as stainless steel, and device belongs to durable products, does not have the problem such as resource consumption and environmental pollution that on market, the disposable use of microwell plate brings that is similar to.
(6) apparatus structure is small and exquisite, and highly versatile is applicable to mass production manufacture.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, the condition of conventionally advising according to normal condition or according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Referring to Fig. 1, provide a kind of matrix type separation and Culture device comprise environment simulator 1 and be arranged on the culture apparatus 2 (culturing room 2) in environment simulator 1, culturing room 2 provides microorganism growth space; Environment simulator 1 is for supplying the environment composition that microorganism growth is relevant.When use, culturing room 2 is placed in environment simulator 1, after cover lid, forms a set of complete device.
Referring to Fig. 2, culturing room 2 is a kind of " sandwich " formula structures, by two-layer retaining plate, two-layer sealing plate, one deck culture plate and two-layer filtering membrane form; Be followed successively by from top to bottom upper mounted plate 21, upper sealing panel 22, upper filtering membrane 23, middle culture plate 24, lower filtering membrane 25, lower seal plate 26, bottom plate 27.Above-mentioned each laminate and film are fixedly clamped by screw fastener 28.Except the two-layer filtering membrane of culturing room 2, on above-mentioned each layer of retaining plate, sealing plate and culture plate, be all provided with the through hole communicating up and down, through hole is set to be 96 holes (12 × 8) of arranged; Aperture is 6mm; Pitch of holes is 9mm (distance between the central point in two holes).Each through hole on middle culture plate 24 is a diffuser casing.Each diffuser casing on culture plate is separated microorganism or other cell by the upper and lower two-layer filtering membrane of being close on it with external environment, cell is limited in diffuser casing, and nutritive substance in external environment can pass in and out diffuser casing by free diffusing.Filtering membrane is polycarbonate membrane, aperture 0.1um, and thickness 3-5um, can high-temperature sterilization.
Two-layer sealing plate (upper sealing panel, lower seal plate) employing medical silica-gel plate, the more than 121 DEG C high temperature of its ability, can tolerate steam moist heat sterilization, in the time clamping between by upper and lower two-layer retaining plate by screw or alternate manner, silica gel sealing plate just will separate (separating by diffuser casing and diffuser casing) completely between the Kong Yukong on middle culture plate, by upper and lower two-layer filtering membrane, diffuser casing and external environment are separated again, microorganism cells is completely restricted in diffuser casing like this, and nutritive substance can pass in and out diffuser casing by free diffusing in external environment, with in diffuser casing, carry out material transfer and exchange.
Referring to Fig. 3-4, environment simulator 1 is made up of bottom liquid storage container 11 (reservoir 11), top upper cover 13 and middle part bracket 12.Reservoir 11 shapes are rectangular parallelepiped, and it is completely unlimited that upper end is, and lateral vertical is in bottom surface; On the inner length and width size in reservoir 11 bottoms and market, the microwell plate length and width of standard are measure-alike, and figure peripheral length and width are of a size of 130mm × 90mm.The internal depth of reservoir 11 is greater than the total height of culturing room and the total height sum of bracket 12; In figure, the internal depth of reservoir 11 is 45mm; Culturing room's total height is 11mm; Bracket 12 is highly 15mm.
The upper mounted plate 21 and the bottom plate 27 that in figure, install adopt stainless steel; Middle culture plate 24 adopts stainless steel; The reservoir 11 of environment simulator 1 adopts stainless steel; Upper cover 13 adopts stainless steel, bracket 12 to adopt stainless steel.Filtering membrane adopts polycarbonate material.
The combination of embodiment 2, the each parts of matrix type diffusion type microbial cultivation device
Referring to Fig. 1, culture apparatus 2 is fixed together with environment simulator 1, between two Room, can exchange nutritive substance by filtration medium (upper and lower filtering membrane), microorganism is limited in the hole of diffuser casing, microbe population in each hole can need manual control according to research, in hole, substratum can be selected different forms or composition (as semi-solid state or liquid state) according to research purpose, and the filling of substratum can be mated multichannel pipettor or automatization liquor removing workstation.
Under another kind of mode, culture apparatus 2 can be departed to environment simulator, be placed directly in sampling spot or other environment and cultivate.
The mode of operation of embodiment 3, matrix type diffusion type microbial cultivation device
Referring to Fig. 5, the working process of device of the present invention is, culturing room 2 is positioned on the bracket 12 in reservoir 11, between the bottom of culturing room 2 and reservoir 11 inner bottom parts, form a cavity, in reservoir 11, be full of liquid or semi-solid solution, be ambient water or mixture 15, it is from seawater, river, lake water in microorganism to be separated physical environment of living in; Or the mixture of water and mud; Or the nutrient solution of artificial preparation etc.Because each diffuser casing on middle culture plate 24 is separated microorganism (or other cell) and external environment by the upper and lower two-layer filtering membrane of being close on it, cell is limited in diffuser casing 210,211, in diffuser casing 210,211, be semisolid medium 29 or liquid nutrient medium, and nutritive substance in external environment (is injected into various small-molecule substances that contain in ambient water in reservoir 11 or mixture 15 etc., as c, d, e in Fig. 5 ... Deng) can see through filtering membrane free diffusing turnover diffuser casing 210,211 etc.; And cell metabolite in diffuser casing 210,211 also can free diffusing turnover diffuser casing 210,211 (the metabolite b that the cell B in metabolite a, the diffuser casing 211 producing as the cell A in diffuser casing 210 produces etc. can be spread in reservoir 11, and can again be diffused in other diffuser casings).Like this, by diffusion, especially under wave and culture mode, between each diffuser casing, can there is exchange of substance, therefore, this device can be used between microorganisms and environment, interaction relationship between microorganism and microorganism, between microorganism and nutritional factor, can microorganism separately under pure culture state microorganisms the relation such as cultivate altogether.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a microorganism seperation culture device, is characterized in that, described separation and Culture device comprises: culture apparatus (2) and environment simulator (1);
Described culture apparatus (2) comprising:
Middle culture plate (24), is distributed with 6-1536 through hole on it;
Upper filtering membrane (23), it is covered in the top of middle culture plate (24);
Lower filtering membrane (25), it is covered in the below of middle culture plate (24);
Upper mounted plate (21), it is covered in the top of filtering membrane (23), is distributed with 6-1536 through hole on it, and specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24);
Bottom plate (27), it is covered in the below of lower filtering membrane (25), is distributed with 6-1536 through hole on it, and specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24);
Described upper mounted plate (21), upper filtering membrane (23), middle culture plate (24), lower filtering membrane (25), bottom plate (27) stacked arrangement from top to bottom, fixing with fastening piece (28);
Described environment simulator (1) comprising:
Liquid storage container (11), it can hold described culture apparatus (2);
Bracket (12), it is positioned at liquid storage container (11), for carrying described culture apparatus (2);
Upper cover (13), the opening of itself and liquid storage container (11) matches.
2. microorganism seperation culture device as claimed in claim 1, is characterized in that, between described upper mounted plate (21) and upper filtering membrane (23), also comprises upper sealing panel (22); On it, be distributed with 6-1536 through hole, specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24); And/or
Between described bottom plate (27) and lower filtering membrane (25), also comprise lower seal plate (26); On it, be distributed with 6-1536 through hole, specification, quantity and the distributing position of each through hole is consistent with the through hole on middle culture plate (24).
3. microorganism seperation culture device as claimed in claim 1, it is characterized in that, on fixed plate (21), upper sealing panel (22), middle culture plate (24), lower seal plate (26), bottom plate (27), specification, quantity and the distributing position of through hole are consistent, and number of openings is 6-1536; And/or
The matrix form that is arranged as of through hole is arranged.
4. spreading culture apparatus as claimed in claim 1, is characterized in that, the thickness of described middle culture plate is not less than 0.5mm; And/or
The diameter of described through hole is at 1-35mm.
5. microorganism seperation culture device as claimed in claim 1, is characterized in that, described fastening piece (28) is selected from: screw fastener, magnetic-type fastening piece, clamping type fastening piece.
6. microorganism seperation culture device as claimed in claim 1, is characterized in that, the inside top surface of described upper cover (13) is provided with gasket (14); And/or
Filtering membrane.
7. microorganism seperation culture device as claimed in claim 1, is characterized in that, the end face of described upper cover (13) can arrange one or more through holes;
On described gasket, one or more through holes can be set, this through hole is consistent with specification, quantity and the distributing position of the through hole of the end face of upper cover; With
Between described upper cover top surface and gasket, also comprise one deck filtering membrane.
8. microorganism seperation culture device as claimed in claim 1, it is characterized in that, the material of described upper mounted plate (21), upper filtering membrane (23), middle culture plate (24), lower filtering membrane (25), bottom plate (27), liquid storage container (11), upper cover (13) and bracket (12) is selected from: stainless steel, aluminium, polypropylene, polycarbonate; And/or
Described upper sealing panel (22) and the material of lower seal plate (26) are selected from: silica gel, rubber, tetrafluoroethylene etc.
9. microorganism seperation culture device as claimed in claim 1, is characterized in that, described upper filtering membrane (23) and the filter membrane aperture of lower filtering membrane (25): 0.01-10.0um.
10. microorganism seperation culture device as claimed in claim 1, is characterized in that, the form of arranging in the described upper hole of middle culture plate (24) is matrix structure, through-hole spacing 4-25mm, and through-hole diameter 2-25mm, the middle thickness of slab of cultivating is not less than 1mm.
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