CN112210481B - Microorganism in-situ culture chip suitable for different temperatures and method thereof - Google Patents
Microorganism in-situ culture chip suitable for different temperatures and method thereof Download PDFInfo
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- CN112210481B CN112210481B CN202011145277.5A CN202011145277A CN112210481B CN 112210481 B CN112210481 B CN 112210481B CN 202011145277 A CN202011145277 A CN 202011145277A CN 112210481 B CN112210481 B CN 112210481B
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- 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
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Abstract
The invention discloses a microorganism in-situ culture chip and a method thereof, which are applicable to different temperatures. The chip comprises a first fixed plate, a culture plate, a contact plate and a second fixed plate; the first fixing plate is provided with a plurality of first through holes penetrating through the thickness direction of the plate body, and the culture plate is provided with a plurality of second through holes penetrating through the thickness direction of the plate body; the contact plate is provided with a contact chamber which penetrates through the thickness direction of the plate body; the upper surface of the second fixing plate is fixed with a plurality of cylindrical culture chambers for holding solid culture medium or liquid culture medium. According to the invention, through the optimization of the culture plate structure, the liquid and solid culture medium is switched according to the requirements of high temperature, low temperature and normal temperature habitat, the applicable temperature of the in-situ culture chip is expanded, the in-situ culture of microorganisms at different temperatures is realized, the temperature limitation of the traditional in-situ culture chip is broken, and a foundation is provided for the in-situ culture of functional microorganisms under high temperature media such as aerobic composting, anaerobic fermentation and the like.
Description
Technical Field
The invention belongs to the field of microorganism in-situ culture, and particularly relates to a microorganism in-situ culture chip and a method thereof, which are applicable to different temperatures.
Background
Most microorganisms cannot grow on a standard agar medium, so that only about 1% of microorganisms in the natural environment can be cultured by the existing culture methods. There are generally three explanations for 1% of the culturable microorganisms: the first explanation refers to that only 1% of the cells in a population can be cultured; the second explanation refers to that only 1% of the clusters in a community can be cultivated; the third explanation refers to 1% cell growth in a colony on standard agar medium. The different theoretical explanations all indicate that only a small number of microorganisms can be cultivated, which is limited by the conventional cultivation methods. Therefore, optimization of the culture method is required to reduce the limitation of the conventional methods such as plate streaking and gradient dilution method on the culture of microorganisms.
In 2002 Kaeberlein et al, the recovery rate of inoculated strains is over 40% by developing a microorganism diffusion chamber, but the strain still needs further culture after the diffusion chamber is cultured, so that the strain cannot be popularized on a large scale; the concept of in-situ culture chips is first proposed by Nichols in 2010, so that the quick enrichment and culture of marine microorganisms are realized, and the culture chips are initially perfected by Brittany in 2017, but the in-situ culture of microorganisms still cannot be realized at high temperature by the existing in-situ culture chips. The softened or liquefied agar medium increases the gap between the medium and the semipermeable membrane, resulting in the entry/exit of external/internal microorganisms into/from the culture chip, rendering the in situ culture chip ineffective. The high temperature environment is an important stage of typical waste biological treatment processes such as aerobic composting, anaerobic fermentation and the like, and the highest temperature in the composting and digestion processes can be over 60 ℃ and even 70-80 ℃, so that the defect limits the exploration of functional microorganisms and the utilization of functional strains in high temperature environment media. Therefore, the design of an in-situ culture chip applicable to different temperatures plays an important role in further exploring the high Wen Shengjing microorganism group and exploring the germplasm resources of various habitats.
Disclosure of Invention
The invention overcomes the defect that the traditional microorganism culture chip can not realize in-situ culture of microorganisms at different temperatures, realizes the switching of liquid and solid culture mediums by changing the culture plate structure and the assembly mode of the culture plate and the contact plate, and can realize in-situ culture of microorganisms at different temperatures; by improving the microorganism fixing method, the residual culture medium and microorganism cells on the culture plate in the agar culture medium solidification process are avoided, and the microorganism in-situ culture chip and the method suitable for different temperatures are provided.
The specific technical scheme adopted by the invention is as follows:
the first object of the invention is to provide a microorganism in-situ culture chip suitable for different temperatures, which comprises a first fixing plate, a culture plate and a contact plate, wherein the first fixing plate, the culture plate and the contact plate are all flat plates;
the first fixing plate is provided with a plurality of first through holes penetrating through the thickness direction of the plate body, and the culture plate is provided with a plurality of second through holes penetrating through the thickness direction of the plate body;
the contact plate is provided with a contact chamber which penetrates through the thickness direction of the plate body; the contact chamber comprises a first chamber and a second chamber which are mutually communicated along the thickness direction of the contact plate, and the vertical projection of the first chamber in the surface direction of the contact plate completely covers the vertical projection of the second chamber in the surface direction of the contact plate; the second chamber is a pore plate provided with a plurality of third through holes penetrating through the thickness direction of the plate body;
the first fixing plate, the culture plate and the contact plate are sequentially overlapped in a detachable mode from top to bottom, a first permeable membrane is clamped between the first fixing plate and the culture plate, and a second permeable membrane is clamped between the culture plate and the plate surface where the second chamber of the contact plate is located; the sizes of the first through hole, the second through hole and the third through hole are the same, and a plurality of straight channels are formed in one-to-one correspondence; the second through holes in each straight channel are used for containing the solid culture medium, and two ends of each second through hole are sealed by the first permeable membrane and the second permeable membrane; the first permeable membrane and the second permeable membrane are membrane bodies that allow nutrients and growth factors to pass through but do not allow microorganisms to pass through.
The second object of the present invention is to provide a microorganism in-situ culture chip suitable for different temperatures, which comprises a first fixing plate, a culture plate, a contact plate and a second fixing plate which are all flat plates;
the first fixing plate is provided with a plurality of first through holes penetrating through the thickness direction of the plate body, and the culture plate is provided with a plurality of second through holes penetrating through the thickness direction of the plate body;
the contact plate is provided with a contact chamber which penetrates through the thickness direction of the plate body; the contact chamber comprises a first chamber and a second chamber which are mutually communicated along the thickness direction of the contact plate, and the vertical projection of the first chamber in the surface direction of the contact plate completely covers the vertical projection of the second chamber in the surface direction of the contact plate; the second chamber is a pore plate provided with a plurality of third through holes penetrating through the thickness direction of the plate body;
a plurality of cylindrical culture chambers are fixed on the upper plate surface of the second fixed plate, and the joints of the culture chambers and the second fixed plate are closed; the height of the culture chamber is the same as the thickness of the contact plate;
the first fixing plate, the culture plate, the contact plate and the second fixing plate are sequentially overlapped in a detachable mode from top to bottom, a first permeable membrane is clamped between the first fixing plate and the culture plate, and the culture plate is connected with the plate surface where the first chamber of the contact plate is located; the culture chamber passes through a third through hole corresponding to the contact plate and is connected with the second through hole in a matching way, and the second through hole and the culture chamber jointly form a communicated pore canal for holding a solid culture medium or a liquid culture medium; the top of the culture chamber is provided with a hollow cylindrical leakage-proof rubber, and the joint of the second through hole and the culture chamber is kept closed by the leakage-proof rubber; the sizes of the first through holes and the second through holes are the same, and a plurality of straight channels are formed in one-to-one correspondence; the first through hole and the second through hole are sealed by a first permeable membrane, and the first permeable membrane is a membrane body which can pass through nutrients and growth factors but cannot pass through microorganisms.
As a preferred first object of the present invention, the first fixing plate, the culture plate and the contact plate are all made of stainless steel or polypropylene resin material.
As a preferable second object of the present invention, the first fixing plate, the culture plate, the contact plate and the second fixing plate are all made of stainless steel or polypropylene resin material.
As a preferable aspect of the first or second aspect of the present invention, the first and/or second permeable membrane is a semipermeable membrane having a pore size of 0.03 μm or less.
As a preferable aspect of the first or second aspect of the present invention, each of the laminate bodies in the microorganism in-situ culture chip is provided with a fixing hole penetrating through the thickness direction of the plate surface and corresponding to each other, and the fixing screw penetrates through the fixing hole and connects and fixes each laminate body through a fixing nut.
Preferably, the first or second object of the present invention is that the contact chamber is provided in plural.
The third object of the present invention is to provide a microorganism enrichment culture method based on the microorganism in-situ culture chip of the first object of the present invention, which comprises the following steps:
firstly, pretreating a sample of a target habitat, then enabling the concentration of microorganisms in a sample solution to reach target dilution by a gradient dilution method, and dripping the diluted sample solution into a solid culture medium to obtain a culture medium sample;
adding a culture medium sample into the cavity of the second through hole of the culture plate and filling the cavity with the culture medium sample in an environment below a target temperature; sequentially fixing a first permeable membrane and a first fixing plate above the culture plate, and sequentially fixing a second permeable membrane and a contact plate below the culture plate to obtain a microorganism in-situ culture chip;
the in-situ microorganism culture chip is placed in a target habitat, an environmental medium in the target habitat enters the first through hole and the inside of the contact chamber of the in-situ microorganism culture chip, so that a culture medium sample is exchanged with nutrient substances and growth factors of the medium in the environment through the first permeable membrane and the second permeable membrane, a growth environment of the target habitat is provided for enrichment culture of microorganisms in the culture medium sample, and meanwhile, the exchange of the culture medium sample with the microorganisms in the environmental medium is avoided.
The fourth object of the present invention is to provide a microorganism enrichment culture method based on the microorganism in-situ culture chip of the second object of the present invention, which comprises the following steps:
firstly, pretreating a sample of a target habitat, then enabling the concentration of microorganisms in a sample solution to reach target dilution by a gradient dilution method, and dripping the diluted sample solution into a solid culture medium or a liquid culture medium to obtain a culture medium sample;
adding a culture medium sample into the inner cavity of the culture chambers and filling the culture chambers with the culture medium sample in an environment with the temperature being greater than or equal to the target temperature, and connecting the second fixing plate and the contact plate by penetrating each culture chamber through a corresponding third through hole; a culture plate, a first permeable membrane and a first fixed plate are sequentially attached and fixed above the contact plate, and meanwhile, anti-leakage rubber is distributed at the top of the culture chamber so as to seal the joint of the culture plate and the culture chamber, thus obtaining a microorganism in-situ culture chip;
the in-situ microorganism culture chip is placed in a target habitat, an environmental medium in the target habitat enters the first through hole of the in-situ microorganism culture chip, so that a culture medium sample exchanges with nutrient substances in the environmental medium through a first permeable membrane, a growth environment of the target habitat is provided for enrichment culture of microorganisms in the culture medium sample, and meanwhile, the exchange of the culture medium sample and the microorganisms in the environmental medium is avoided.
As a preference of the third or fourth object of the present invention, the microorganism in-situ culture chip is sterilized with 75% ethanol after use for the next microorganism enrichment culture.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, through the structural optimization of the contact plate and the culture plate, the in-situ culture of microorganisms under the high-temperature condition is realized, the temperature applicable condition of the traditional in-situ chip is effectively expanded, and technical support is provided for the analysis of microorganism 'dark matters' and the development of germplasm resources under the high-temperature environment;
(2) The invention avoids the residues of the culture medium and the microbial cells on the culture plate in the agar culture medium solidification process through the optimization of the culture plate and the contact plate;
(3) According to the invention, through the optimization of the culture plate structure, the liquid and solid culture medium is switched according to the requirements of high temperature, low temperature and normal temperature habitat, the applicable temperature of the in-situ culture chip is expanded, the in-situ culture of microorganisms at different temperatures is realized, the temperature limitation of the traditional in-situ culture chip is broken, and a foundation is provided for the in-situ culture of functional microorganisms under high temperature media such as aerobic composting, anaerobic fermentation and the like;
(4) The invention optimizes the culture medium adding method, improves the problems of easy residue generation on the culture plate in the agar culture medium solidification process, and overcomes the defect of easy pollution of culture of the culture chip.
Drawings
FIG. 1 is a schematic view of the structure of the upper fixing plate, wherein (a) is a top view and (b) is a sectional view;
FIG. 2 is a schematic view of a culture plate, wherein (a) is a top view and (b) is a cross-sectional view;
fig. 3 is a schematic structural view of the contact plate, wherein (a) is a top view and (b) is a cross-sectional view;
fig. 4 is a schematic structural view of the lower fixing plate, wherein (a) is a top view and (b) is a sectional view;
FIG. 5 is a schematic structural diagram of a microorganism in-situ culture chip under low or normal temperature conditions;
FIG. 6 is a schematic diagram showing the structure of an in-situ microorganism culture chip under high temperature conditions;
in the figure: the culture plate comprises a first fixing plate 1, a culture plate 2, a contact plate 3, a second fixing plate 4, a fixing hole 5, a first through hole 61, a second through hole 62, a third through hole 63, a contact chamber 7, a culture chamber 8, a leakage-proof rubber 9, a fixing nut 10, a fixing screw 11, a first permeable membrane 12 and a second permeable membrane 13.
Detailed Description
The invention is further illustrated and described below with reference to the drawings and detailed description. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.
The existing in-situ culture method is to place the agar culture medium as a base in an in-situ habitat to realize the culture of microorganisms, but the characteristics of softening and liquefying the agar culture medium at high temperature limit the in-situ culture of microorganisms at high temperature. The softened or liquefied agar medium increases the gap between the medium and the semipermeable membrane, causing external or internal microorganisms to enter or leave the culture chip, rendering the in situ culture chip ineffective. The high temperature is an important process of waste treatment such as aerobic composting and anaerobic digestion, and the highest temperature can be over 60 ℃ and even can reach 70-80 ℃ in the composting and digestion treatment process, so that the limitation of the high temperature is one of important bottlenecks of in-situ cultivation of functional microorganisms in the waste treatment.
The invention provides a microorganism in-situ culture chip suitable for different temperatures, which comprises four components which are flat plates, namely a first fixing plate 1, a culture plate 2, a contact plate 3 and a second fixing plate 4, wherein the specific structures of the four components are as follows:
as shown in fig. 1, the first fixing plate 1 is provided with a plurality of first through holes 61 penetrating through the thickness direction of the plate body, and in practical application, the number of the arrays of the first through holes 61 can be set according to the requirements of different culturing and identifying devices, and the number of the first through holes 61 in each array can be switched according to different requirements.
As shown in fig. 2, the culture plate 2 has the same structure as the first fixing plate 1, and a plurality of second through holes 62 penetrating the plate body in the thickness direction are formed in the culture plate 2. In practical application, the number of the arrays of the second through holes 62 can be set according to the requirements of different culturing and identifying devices, and the number of the second through holes 62 in each array can be switched according to different requirements. However, it is necessary to ensure that the second through holes 62 and the first through holes 61 are the same in size, and that the positions of all the second through holes 62 and the first through holes 61 can be communicated in one-to-one correspondence, respectively.
As shown in fig. 3, the contact plate 3 is provided with a contact chamber 7 penetrating the plate body in the thickness direction. The contact chamber 7 includes a first chamber and a second chamber which are communicated with each other in the thickness direction of the contact plate 3, and the perpendicular projection of the first chamber in the plate direction of the contact plate 3 completely covers the perpendicular projection of the second chamber in the plate direction of the contact plate 3. The second chamber is an orifice plate provided with a plurality of third through holes 63 penetrating through the thickness direction of the plate body, and the thickness of the second chamber should be set thinner, generally not exceeding the thickness of the first fixing plate 1.
As shown in fig. 4, the upper plate surface of the second fixing plate 4 is fixed with a plurality of hollow cylindrical culture chambers 8 for holding solid culture medium or liquid culture medium, and the connection of the culture chambers 8 and the second fixing plate 4 is closed to prevent leakage of the culture medium held therein. The height of the culture chamber 8 is the same as the thickness of the contact plate 3, and the height of the culture chamber 8 may be made to protrude into the second through-hole 62 of the culture plate 2 slightly beyond the thickness of the contact plate 3, but is not preferably too high beyond the tip of the second through-hole 62.
In practical application, the materials adopted by the first fixing plate 1, the culture plate 2, the contact plate 3 and the second fixing plate 4 are diversified, and can be processed and manufactured by stainless steel metal materials, and also can be manufactured by adopting materials such as polypropylene resin and the like through 3D printing technology. The first permeable membrane 12 and the second permeable membrane 13 may each employ a semi-permeable membrane having a pore size of 0.03 μm or less, because the pore size of 0.03 μm or less allows diffusion of growth factors and nutrients while being small enough to prevent microorganisms from entering or/and leaving the culture chip, thereby forming an isolated growth environment and forming colonies.
The number of the contact chambers 7 may be plural as required.
The four components can be in different assembly forms so as to be suitable for different temperature environments, the temperature limitation of the traditional in-situ culture chip is broken through, and a foundation is provided for in-situ culture of functional microorganisms under high-temperature media such as aerobic composting and anaerobic fermentation. The following is a specific description by way of examples.
Example 1
As shown in fig. 5, the first fixing plate 1, the culture plate 2 and the contact plate 3 are sequentially stacked in a detachable manner from top to bottom in an environment lower than a target temperature (i.e., normal temperature or low temperature, which may be set according to actual conditions). In this embodiment, the first fixing plate 1, the culture plate 2 and the contact plate 3 may be in a detachable manner in the process of assembling, that is, the first fixing plate 1, the culture plate 2 and the contact plate 3 are provided with fixing holes 5 which are through and correspond to each other one by one along the thickness direction of the plate body, and the fixing screws 11 penetrate through the fixing holes 5 and fix the plate bodies through the fixing nuts 10.
A first permeable membrane 12 is sandwiched between the first fixing plate 1 and the culture plate 2, and the first permeable membrane 12 can completely cover the plate surfaces of the first fixing plate 1 and the culture plate 2 which are in contact with the first permeable membrane.
A second permeable membrane 13 is clamped between the culture plate 2 and the plate surface of the second chamber of the contact plate 3, and the second permeable membrane 13 can completely cover the culture plate 2 contacted with the second permeable membrane and the plate surface of the contact plate 3. This is because the larger first chamber is facing the ambient medium, the contact area of the second permeable membrane 13 with the ambient medium can be increased, thereby enhancing the exchange of microorganisms with nutrients and growth factors in the environment.
The first through holes 61, the second through holes 62 and the third through holes 63 have the same size, and a plurality of straight channels are formed in one-to-one correspondence. The second through-hole 62 in each straight passage is for receiving the solid medium, and both ends of the second through-hole 62 are sealed by the first permeable membrane 12 and the second permeable membrane 13, respectively. The first permeable membrane 12 and the second permeable membrane 13 are each a membrane body that allows nutrients and growth factors to pass therethrough but does not allow microorganisms to pass therethrough.
The method for enrichment culture of the microorganisms in the environment below the target temperature by utilizing the microorganism in-situ culture chip comprises the following steps:
before preparing the culture chip, the sample of the target habitat needs to be pretreated. Among other things, aquatic sediment or soil can be treated by ultrasound to remove microbial cells from the particles, and mud, powdered crushed stone and water samples can be used directly as cell sources.
The concentration of microorganisms in the sample solution is then brought to a target dilution by a gradient dilution method, and in particular the cells can be counted to determine the target dilution in the agar medium, so as to equally divide the dilution into culture chips, ensuring that each through-hole contains a small number of cells. The target dilution may be selected according to the actual situation. And then dripping the diluted sample solution into the agar solid culture medium to obtain a culture medium sample.
The sample of the non-solidified medium after the gradient dilution is added by a pipette and fills the inner cavity of the second through hole 62 of the culture plate 2. A first permeable membrane 12 and a first fixing plate 1 are sequentially fixed above the culture plate 2, a second permeable membrane 13 and a contact plate 3 are sequentially fixed below the culture plate 2, and the microorganism in-situ culture chip is obtained through assembly.
The in-situ microorganism culture chip is placed in a target habitat, and an environmental medium in the target habitat enters the first through hole 61 of the in-situ microorganism culture chip and the inside of the contact chamber 7, so that a culture medium sample exchanges with nutrient substances and growth factors of the medium in the environment through the first permeable membrane 12 and the second permeable membrane 13, a growth environment of the target habitat is provided for enrichment culture of microorganisms in the culture medium sample, and meanwhile, the exchange of the culture medium sample and the microorganisms in the environmental medium is avoided.
Example 2
As shown in fig. 6, the first fixing plate 1, the culture plate 2, the contact plate 3 and the second fixing plate 4 are sequentially stacked in a detachable manner from top to bottom in an environment equal to or higher than a target temperature (i.e., a high temperature, which may be set according to actual conditions). In this embodiment, the first fixing plate 1, the culture plate 2, the contact plate 3 and the second fixing plate 4 may be in a detachable manner in the assembly process, that is, fixing holes 5 which are through and in one-to-one correspondence with each other are formed on the first fixing plate 1, the culture plate 2, the contact plate 3 and the second fixing plate 4 along the thickness direction of the plate body, and fixing screws 11 penetrate through the fixing holes 5 and fix the plate bodies through fixing nuts 10.
A first permeable membrane 12 is sandwiched between the first fixing plate 1 and the culture plate 2, and the first permeable membrane 12 can completely cover the plate surfaces of the first fixing plate 1 and the culture plate 2 which are in contact with the first permeable membrane.
The culture plate 2 is connected with the plate surface of the first chamber of the contact plate 3, the culture chamber 8 passes through the third through hole 63 corresponding to the culture chamber 8 on the contact plate 3, and the top of the culture chamber 8 is level with the top of the contact plate 3. By adopting the connection mode, the contact plate 3 plays a role in supporting and fixing the culture chamber 8, so that the assembled microorganism in-situ culture chip has stronger stability. The top of cultivate room 8 is equipped with hollow tubular leak protection rubber 9, and the junction of cultivate board 2 and cultivate room 8 keeps sealedly through leak protection rubber 9 to phenomenon such as the softening that solid medium took place under the high temperature environment, liquefaction, make the culture medium reveal the problem such as culture chip inefficacy that leads to from the junction of cultivate board 2 and cultivate room 8.
The first through hole 61, the second through hole 62, the third through hole 63 and the culture chamber 8 have the same cross-sectional dimensions and form a plurality of straight passages in one-to-one correspondence. The first through hole 61 and the second through hole 62 in each straight passage are sealed by the first permeable membrane 12, and the first permeable membrane 12 is a membrane body that allows nutrients and growth factors to pass therethrough but does not allow microorganisms to pass therethrough.
The method for enriching and culturing the microorganisms in the environment with the temperature being more than or equal to the target temperature by utilizing the microorganism in-situ culture chip comprises the following steps:
before preparing the culture chip, the sample of the target habitat is pretreated firstly, then the microorganism concentration in the sample solution reaches the target dilution by a gradient dilution method, and the diluted sample solution is dripped into an agar solid culture medium or a liquid culture medium to obtain a culture medium sample.
The culture medium sample is added by a pipette and filled in the inner cavity of the culture chamber 8, or the liquid level of the culture medium sample is made to be level with the culture plate, and the second fixing plate 4 and the contact plate 3 are connected by passing each culture chamber 8 through the corresponding third through hole 63. The culture plate 2, the first permeable membrane 12 and the first fixed plate 1 are sequentially attached and fixed above the contact plate 3, meanwhile, the leakproof rubber 9 is distributed at the top of the culture chamber 8 so that the joint of the culture plate 2 and the culture chamber 8 is closed, and the microorganism in-situ culture chip is obtained through assembly.
The microorganism in-situ culture chip is placed in a target habitat, an environmental medium in the target habitat enters the first through hole 61 of the microorganism in-situ culture chip, so that a culture medium sample exchanges with nutrient substances in the environmental medium through the first permeable membrane 12, a growth environment of the target habitat is provided for enrichment culture of microorganisms in the culture medium sample, and meanwhile, the culture medium sample is prevented from exchanging with microorganisms in the environmental medium.
That is, when in-situ cultivation of microorganisms in high temperature environments such as composting and anaerobic digestion is needed, the contact chamber is changed into a closed space through the combination of the second fixing plate and the contact plate, so that the solid culture medium or the liquid culture medium can be conveniently switched, and the problems of liquefaction, softening and the like of the agar culture medium caused by the high temperature environments are avoided.
According to the invention, through the optimization of the culture plate structure, the liquid and solid culture medium is switched according to the requirements of high temperature, low temperature and normal temperature habitat, the applicable temperature of the in-situ culture chip is expanded, the in-situ culture of microorganisms at different temperatures is realized, the temperature limitation of the traditional in-situ culture chip is broken, and a foundation is provided for the in-situ culture of functional microorganisms under high temperature media such as aerobic composting, anaerobic fermentation and the like.
In addition, the microorganism in-situ culture chip is sterilized by 75% ethanol after being used for the next microorganism enrichment culture.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (5)
1. The microorganism in-situ culture chip suitable for different temperatures is characterized by comprising a first fixing plate (1), a culture plate (2), a contact plate (3) and a second fixing plate (4), wherein the first fixing plate, the culture plate (2), the contact plate and the second fixing plate are all flat plates;
a plurality of first through holes (61) penetrating through the thickness direction of the plate body are formed in the first fixed plate (1), and a plurality of second through holes (62) penetrating through the thickness direction of the plate body are formed in the culture plate (2);
the contact plate (3) is provided with a contact chamber (7) which penetrates through the thickness direction of the plate body; the contact chamber (7) comprises a first chamber and a second chamber which are mutually communicated along the thickness direction of the contact plate (3), and the vertical projection of the first chamber in the plate surface direction of the contact plate (3) completely covers the vertical projection of the second chamber in the plate surface direction of the contact plate (3); the second chamber is a pore plate provided with a plurality of third through holes (63) penetrating through the thickness direction of the plate body;
a plurality of cylindrical culture chambers (8) are fixed on the upper plate surface of the second fixed plate (4), and the joints of the culture chambers (8) and the second fixed plate (4) are closed; the height of the culture chamber (8) is the same as the thickness of the contact plate (3);
the first fixing plate (1), the culture plate (2), the contact plate (3) and the second fixing plate (4) are sequentially overlapped in a detachable mode from top to bottom, a first permeable membrane (12) is clamped between the first fixing plate (1) and the culture plate (2), and the culture plate (2) is connected with the plate surface where the first chamber of the contact plate (3) is located; the culture chamber (8) passes through a third through hole (63) corresponding to the contact plate (3) and is connected with a second through hole (62) in a matching way, and the second through hole (62) and the culture chamber (8) jointly form a communicated pore canal for containing a solid culture medium or a liquid culture medium; the top of the culture chamber (8) is provided with a hollow cylindrical leakage-proof rubber (9), and the joint of the second through hole (62) and the culture chamber (8) is kept closed by the leakage-proof rubber (9); the first through holes (61) and the second through holes (62) have the same size and form a plurality of straight channels in one-to-one correspondence; the first through hole (61) and the second through hole (62) are sealed by a first permeable membrane (12), and the first permeable membrane (12) is a membrane body which can pass through nutrient substances and growth factors but can not pass through microorganisms;
the first permeable membrane (12) is a semi-permeable membrane with the pore diameter less than or equal to 0.03 mu m;
the number of the contact chambers (7) is plural.
2. The microorganism in-situ culture chip according to claim 1, wherein the first fixing plate (1), the culture plate (2), the contact plate (3) and the second fixing plate (4) are all made of stainless steel or polypropylene resin materials.
3. The microorganism in-situ culture chip according to claim 1, wherein each plate body in the microorganism in-situ culture chip is provided with fixing holes (5) which penetrate through the thickness direction of the plate surface and are in one-to-one correspondence, and fixing screws (11) penetrate through the fixing holes (5) and connect and fix each plate body through fixing nuts (10).
4. A microorganism enrichment culture method based on the microorganism in-situ culture chip of claim 1, which comprises the following steps:
firstly, pretreating a sample of a target habitat, then enabling the concentration of microorganisms in a sample solution to reach target dilution by a gradient dilution method, and dripping the diluted sample solution into a solid culture medium or a liquid culture medium to obtain a culture medium sample;
adding a culture medium sample into and filling the inner cavity of the culture chambers (8) in an environment with the temperature being greater than or equal to a target temperature, and connecting the second fixing plate (4) and the contact plate (3) by passing each culture chamber (8) through a corresponding third through hole (63); the culture plate (2), the first permeable membrane (12) and the first fixed plate (1) are sequentially attached and fixed above the contact plate (3), and meanwhile, the leakproof rubber (9) is distributed at the top of the culture chamber (8) so as to seal the joint of the culture plate (2) and the culture chamber (8) to obtain a microorganism in-situ culture chip;
the in-situ microorganism culture chip is placed in a target habitat, an environmental medium in the target habitat enters the first through hole (61) of the in-situ microorganism culture chip, so that a culture medium sample exchanges with nutrient substances in the environmental medium through the first permeable membrane (12), a growth environment of the target habitat is provided for enrichment culture of microorganisms in the culture medium sample, and meanwhile, the exchange of the culture medium sample and the microorganisms in the environmental medium is avoided.
5. The method according to claim 4, wherein the in-situ microorganism culture chip is sterilized with 75% ethanol after use for the next microorganism enrichment culture.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865751A (en) * | 2012-12-17 | 2014-06-18 | 中国科学院上海生命科学研究院 | Array-type high-throughput microbe separating culturing apparatus |
| CN104498322A (en) * | 2014-11-18 | 2015-04-08 | 中国海洋大学 | In-situ high flux culture apparatus for sea microbe |
| CN105255712A (en) * | 2015-10-12 | 2016-01-20 | 国家海洋局第一海洋研究所 | High-throughput microorganism in-situ culture device |
| WO2016187622A1 (en) * | 2015-05-21 | 2016-11-24 | Northeastern University | Method and device for cultivation and analysis of novel microbial species with unknown growth requirements |
| RU182023U1 (en) * | 2018-02-27 | 2018-07-31 | Войсковая Часть 41598 | GROWTH CAMERA FOR CULTIVATION OF MICRO-ORGANISMS |
| WO2019058390A1 (en) * | 2017-09-20 | 2019-03-28 | Fertis India Pvt. Ltd. | Process for isolation and cultivation of uncultivable endophytes |
| CN214327723U (en) * | 2020-10-23 | 2021-10-01 | 浙江大学 | Microorganism in-situ culture chip suitable for different temperatures |
-
2020
- 2020-10-23 CN CN202011145277.5A patent/CN112210481B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865751A (en) * | 2012-12-17 | 2014-06-18 | 中国科学院上海生命科学研究院 | Array-type high-throughput microbe separating culturing apparatus |
| CN104498322A (en) * | 2014-11-18 | 2015-04-08 | 中国海洋大学 | In-situ high flux culture apparatus for sea microbe |
| WO2016187622A1 (en) * | 2015-05-21 | 2016-11-24 | Northeastern University | Method and device for cultivation and analysis of novel microbial species with unknown growth requirements |
| CN105255712A (en) * | 2015-10-12 | 2016-01-20 | 国家海洋局第一海洋研究所 | High-throughput microorganism in-situ culture device |
| WO2019058390A1 (en) * | 2017-09-20 | 2019-03-28 | Fertis India Pvt. Ltd. | Process for isolation and cultivation of uncultivable endophytes |
| RU182023U1 (en) * | 2018-02-27 | 2018-07-31 | Войсковая Часть 41598 | GROWTH CAMERA FOR CULTIVATION OF MICRO-ORGANISMS |
| CN214327723U (en) * | 2020-10-23 | 2021-10-01 | 浙江大学 | Microorganism in-situ culture chip suitable for different temperatures |
Non-Patent Citations (2)
| Title |
|---|
| Use of Ichip for High-Throughput In Situ Cultivation of "Uncultivable" Microbial Species;D. Nichols等;APPLIED AND ENVIRONMENTAL MICROBIOLOGY;第76卷(第8期);2445–2450 * |
| 未培养微生物原位培养技术研究进展;张作艳等;天然产物研究与开发;第30卷(第05期);907-913 * |
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