Prefabricated multi-well plate for culturing single microorganism in liquid medium

Abstract

The purpose of the present disclosure is to provide a multi-well plate for culturing a single microorganism, capable of conveniently and rapidly dividing and culturing a culture medium containing a single microorganism in a plurality of multi-wells. The multi-well plate of the present disclosure is economical because a special device is not additionally used, and a method for culturing a single microorganism using same can efficiently culture a single microorganism in multi-wells without a process of diluting a culture medium in each well.

Description

Assembled multi-well plate for culturing single microorganisms in liquid medium

[0001] The present invention relates to an assembled multi-well plate for culturing a single microorganism in a liquid medium and a single microorganism culturing method using the same.

[0002] The continued emergence of antibiotic-resistant bacteria poses a major threat to global health, with warnings that by 2050, 10 million people will die annually from antibiotic-resistant infections. Therefore, developing new antibiotics is a critically important task. Most antibiotics developed to date have been derived from microorganisms. However, the search for new antibiotic-producing microorganisms faces limitations. Traditionally, agarose solid media has been used to isolate microorganisms, but this method, which cannot isolate many microorganisms that can only be cultured in liquid medium, poses a challenge for screening new microbial species.

[0003] Meanwhile, microalgae, with an estimated 200,000-800,000 known species, are a valuable resource on Earth, producing 15,000 natural substances, including various antibiotics and bioactive compounds. To utilize these microalgae, they must be isolated, but most isolated microalgae do not grow in solid-phase culture. Current isolation methods, such as those using capillary tubes, are extremely labor-intensive, resulting in only a few thousand individual microalgae currently held in microalgae banks.

[0004] Conventionally, the isolation and identification of microorganisms has generally involved smearing and culturing a Petri dish with agarose or other sieve medium using glass rods, glass beads, etc. to obtain a single colony. However, it has been reported that the conventional culture method using solid media can culture less than 1% of known microorganisms. On the other hand, the culture method using liquid media uses a solution in which microorganisms are dispersed to create droplets and isolate and identify the microorganisms through this process. However, there is a need for improvement due to the difficulty in sorting the droplets in which microorganisms are captured. In addition, a method for liquid culture of a single species of microorganism can be used by sequentially diluting the culture medium using a multiwell plate such as a 96-well plate, but this method requires a lot of effort when performed manually, which is problematic.

[0005] Accordingly, there is a need for the development of an efficient culture plate for efficiently isolating various microorganisms.

[0006] The present disclosure aims to provide a multi-well plate for culturing a single microorganism, which can efficiently form a plurality of isolated multi-wells and independently culture a single microorganism in a liquid phase.

[0007] The present disclosure aims to provide a method for culturing a single microorganism in a liquid phase without diluting the culture solution in each well of a multi-well plate, and in which the microorganisms grown in each well do not cross-contaminate each other.

[0008] The present disclosure provides a multi-well plate for culturing a single microorganism, comprising: a lower container including a bottom, a plurality of side walls, and a plurality of first connecting portions positioned on the side walls; a porous plate including a plurality of through holes and a plurality of second connecting portions formed at an edge and independently coupled to the first connecting portions; and a lid detachable from the top of the porous plate.

[0009] The lower container may include one or more partitions dividing the container across the container and one or more receiving grooves located in the partitions, and the porous plate may include one or more divided areas dividing the through holes across the plate and one or more protrusions located below the divided areas and engaging the receiving grooves.

[0010] The above lower container may further include an elastic body formed on the surface of the bottom, side walls and bulkhead.

[0011] The above elastic body may include polypropylene.

[0012] The hardness of the above elastic body may be 10 to 40.

[0013] The thickness of the above elastic body may be 1.0 to 2.5 mm.

[0014] The height (a) of the above through hole may satisfy the following relational expression 1.

[0015] [Relationship 1]

[0016] h o -h e <a< h o

[0017] (In the above relational expression 1,

[0018] a is the height of the through hole, h o is the height of the lower container, h e is the thickness of the elastic body.)

[0019] The diameter of the above through hole may be 1.0 to 3.0 mm.

[0020] The above lid may include a gas-permeable, breathable membrane.

[0021] The cross-sectional shape of the above through hole may be square, hexagonal or circular.

[0022] The present disclosure provides a method for culturing a single microorganism, comprising: a step of injecting a culture medium containing a single microorganism into a lower container including a bottom, a plurality of side walls, and a first connection portion positioned on the side walls; a step of connecting a porous plate including a plurality of through holes and a plurality of second connection portions formed at an edge and independently connected to the first connection portions to the lower container, thereby sealing the culture medium in each of the through holes; and a step of covering the porous plate with a lid from the top of the porous plate and culturing.

[0023] The lower container may include one or more partitions dividing the container across the container and one or more receiving grooves located in the partitions, and the porous plate may include one or more divided areas dividing the through holes across the plate and one or more protrusions located below the divided areas and engaging the receiving grooves.

[0024] The above lower container may further include an elastic body formed on the surface of the bottom, side walls and bulkhead.

[0025] The number (N) of single microorganisms injected into the above lower container may satisfy the following relationship 2.

[0026] [Relationship 2]

[0027] N≥0.1a (unit: CFU / (a*V)) (100 <a<50000)

[0028] (In the above relational expression 2, a is the number of wells, and V is the individual capacity of the well)

[0029] The multi-well plate for culturing a single microorganism of the present disclosure can easily and quickly divide a culture solution containing a single microorganism into multiple multi-wells and culture it, and is economical because it does not require additional special devices, and can efficiently culture a single microorganism in a multi-well without the process of diluting the culture solution in each well.

[0030] The single microorganism culture method of the present disclosure is such that the culture medium is divided into multiple wells in a single step and completely separated so that each well can independently culture a single microorganism.

[0031] Figure 1 shows a perspective view of a multiwell plate according to one embodiment of the present disclosure.

[0032] FIG. 2 illustrates a perspective view of a multiwell plate according to another embodiment of the present disclosure.

[0033] Figure 3 shows the front and back sides of the porous plate of the multiwell plate of the present disclosure.

[0034] Figure 4 shows the front surface of the lower container of the multiwell plate of the present disclosure.

[0035] Figure 5 shows the process of combining the lower container and the porous plate of the multiwell plate of the present disclosure.

[0036] Figure 6 shows the appearance of the elastic body (green) when the lower container and the porous plate of the multi-well plate of the present disclosure are combined. a is the height of the through hole, h o is the height of the lower container, h e is the thickness of the elastic body

[0037] Figure 7 shows a receiving groove located in a partition wall in the lower container of the multiwell plate of the present disclosure.

[0038] Hereinafter, with reference to the attached drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. The present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0039] Conventionally, to cultivate a single microorganism in a liquid medium, a multi-well plate was used to continuously dilute the culture medium, but this process required a lot of effort when performed manually, resulting in low efficiency. Accordingly, the present disclosure provides a multi-well plate for culturing a single microorganism, which can cultivate a single microorganism in a liquid phase without diluting the culture medium in each well, and in which the microorganisms growing in each well do not cross-contaminate each other.

[0040] The multi-well plate for culturing a single microorganism of the present disclosure may include a lower container (100) including a bottom (110), a plurality of side walls (120) and a plurality of first connecting portions (130) positioned on the side walls; a porous plate (200) including a plurality of through holes (210) and a plurality of second connecting portions (230) formed at an edge and independently connected to the first connecting portions; and a lid (300) detachable from the top of the porous plate.

[0041] The multi-well plate for culturing a single microorganism can form a multi-well for culturing microorganisms by tightly contacting the lower container (100) and the porous plate (200) through the connection of the first connecting portion (130) and the second connecting portion (230) so that each of the through holes (210) is strongly contacted with the bottom (110) of the lower container (100), thereby allowing single microorganisms to be cultured independently in each well without cross-contamination.

[0042] Specifically, the lower container (100) may include four side walls (120) and one to three first connecting portions (130) on each side wall, and the number of second connecting portions (230) of the porous plate (200) may be the same as the number of first connecting portions (130).

[0043] In addition, the thickness of the bottom (110) of the lower container (100) may be 1.0 to 4.0 mm, specifically 1.0 to 3.0 mm, and more specifically 1.5 to 2.5 mm. At this time, when the thickness of the bottom (110) is thinner than the above-mentioned range, there is a risk of deformation when the lower container (100) and the porous plate (200) are fastened.

[0044] Specifically, the porous plate (200) may have 100 to 50,000 through holes (210), but is not limited thereto as long as a large number of microorganisms can be separated. In addition, the diameter of the through holes (210) may be 0.5 to 5.0 mm, and more specifically, may be 0.5 to 4.0 mm or 1.0 to 3.0 mm. The size of the diameter of the through holes (210) is taken into consideration for recovering microorganisms from the wells in which the microorganisms have grown. If the diameter of the through holes (210) is smaller than the above-mentioned range, it is not easy to recover microorganisms cultured inside the wells, and if the diameter is larger than the above-mentioned range, it is difficult to purely culture many species of microorganisms at once.

[0045] According to one embodiment, the cross-sectional shape of the through hole (210) may be square, hexagonal or circular, but is not limited thereto.

[0046] A microhole can be formed at the top of the above through hole (210), and the microhole can reduce the outflow of gas generated inside the through hole (210) and the inflow of gas from the outside.

[0047] The above lid (300) is sterilized and provided while covering the top of the porous plate (200), thereby performing a contamination prevention function. In addition, it prevents contamination from the external environment, such as the worker's hands, gas, and pollutants, and prevents evaporation of liquid generated during culture.

[0048] According to one embodiment, the lid (300) may be composed of a transparent material. Due to the transparent material, it is possible to determine whether cells are growing inside the multiwell plate using light and turbidity, and the cell proliferation rate can be determined based on the turbidity of the solution.

[0049] According to another embodiment, the lid (300) may be composed of a gas-permeable, breathable membrane. The breathable membrane may facilitate gas exchange within the well, thereby assisting in microbial culture. Furthermore, when extracting cultured microorganisms from the well, the breathable membrane may be pierced with a syringe needle to extract the microbial culture solution. The breathable membrane may be secured to the upper surface of the porous plate (200) for use.

[0050] Meanwhile, in order for the culture medium to spread and be injected well into the lower container (100) and the plurality of through holes (210), the inner surface of the lower container, the surface of the porous plate (200), and the through holes (210) are subjected to hydrophilic surface treatment by plasma or chemical treatment, thereby allowing the microorganisms and the culture medium to be injected naturally and uniformly into the through holes (210) in the lower container.

[0051] According to one embodiment, the lower container (100) may include one or more partition walls (140) dividing the container across the container and one or more receiving grooves (150) positioned in the partition walls (140), and the porous plate (200) may include one or more divided areas (240) dividing the through holes (210) across the plate and one or more protrusions (250) positioned below the divided areas (240) and engaging the receiving grooves (150).

[0052] n (0) located on the above bulkhead (140) <n<10, n=1,2,3,4,5,쪋)의 수용홈(150)은 각각 상기 격벽(140) 길이의 1 / (n+1), 2 / (n+1), 3 / (n+1), ..., n / (n+1) 지점에 형성된 것일 수 있으며, 복수의 수용홈(150)의 위치에 대응하여 다공플레이트(200)의 분할영역(240) 하단부에 돌출부(250)가 형성된 것일 수 있다. 구체적으로, 도 7에서 나타난 것과 같이, 격벽(140)에 위치하는 붉은색 및 파란색 수용홈(150)은 노란색으로 표시되는 격벽의 길이의 1 / 2 지점에 형성된 것일 수 있다. 상기 수용홈(150) 및 돌출부(250)의 결합에 따라 다공플레이트(200)가 하부용기(100)에 더욱 안정적으로 밀착되어 결합될 수 있으며, 수용홈(150)의 웰과 웰 사이의 물질의 이동을 차단할 수 있다.

[0053] Specifically, the lower container (100) may include one to two bulkheads (140) and one to five receiving grooves (150) located in the bulkheads (140), and specifically, the lower container (100) may include two bulkheads (140) and two receiving grooves (150) located in one of the bulkheads (140). At this time, the porous plate (200) may include one or two divided areas (240), and the protrusion (250) may be located at the lower end of the divided area (240) in a portion corresponding to the receiving grooves (150) located in the bulkheads (140) of the lower container (100).

[0054] In addition, the material forming the multi-well plate is not particularly limited, and materials commonly used in microbial culture can be used. For example, transparent materials such as polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate resin, polypropylene resin, acrylic resin, polycarbonate resin, epoxy resin, and vinyl chloride resin can be used, and a resin material including at least one of the above that has undergone a surface hydrophilic treatment, a transparent inorganic material such as glass or quartz can be used, and a material capable of identifying the wells in which microorganisms grow can be used.

[0055] According to one embodiment, the lower container (100) may further include an elastic body (160) formed on the surfaces of the bottom (110), the side wall (120), and the partition wall (140). Specifically, the elastic body (160) may be formed on one surface where the bottom (110), the side wall (120), and the partition wall (140) of the lower container (100) are in contact with each other, and accordingly, when the porous plate (200) and the lower container (100) are coupled, each through hole (210) is locked by the elastic body (160) when it comes into contact with the bottom (110) of the lower container (100), thereby forming a plurality of wells. Liquid movement is blocked between the formed wells, thereby preventing contamination of microorganisms cultured inside the wells.

[0056] According to one embodiment, the elastomer may comprise a polypropylene copolymer. The polypropylene copolymer is a copolymer with an α-olefin monomer, and the olefin monomer may comprise at least one selected from the group consisting of ethylene, 1-butene, 1-pentene, and 1-hexene, and combinations thereof.

[0057] The polypropylene copolymer may include a homopolymer and a copolymer. Specifically, the polypropylene copolymer may include at least one selected from the group consisting of block polypropylene, random polypropylene, homopolypropylene, high-crystalline block polypropylene, high-crystalline homopolypropylene, metocene polypropylene, metallocene polypropylene, and combinations thereof.

[0058] The above polypropylene copolymer may have a weight average molecular weight (Mw) of 200,000 to 500,000 g / mol, specifically, the lower limit may be 250,000 or 300,000 g / mol, and the upper limit may be 500,000, 450,000 or 400,000 g / mol.

[0059] Specifically, the isotactic index (II) of the polypropylene copolymer may be 97.5 to 99.5%. In addition, the density of the polypropylene copolymer may be 0.80 to 0.95 g / cm. 3 It may be, specifically, 0.85 to 0.92 g / cm 3 may be, more specifically, 0.89 to 0.92 g / cm 3 It could be.

[0060] The above polypropylene copolymer may have a melt flow index measured at 230°C and a load of 2.16 kg of 0.1 g / 10 min to 1.0 g / 10 min, specifically 0.5 g / 10 min to 1.0 g / 10 min.

[0061] According to one embodiment, the hardness (A) of the elastic body may be 10 to 40, specifically 10 to 30, or 10 to 25.

[0062] According to one embodiment, the thickness of the elastic body may be 1.0 to 2.5 mm, and specifically, 1.5 to 2.5 mm. In this case, when the thickness is thinner than the aforementioned range, the locking action by the elastic body may be weak, and when the thickness is thick, the flatness may be reduced.

[0063] According to one implementation example, the height (a) of the through hole (210) of the porous plate may satisfy the following relational expression 1.

[0064] [Relationship 1]

[0065] h o -h e <a< h o

[0066] (In the above relational expression 1,

[0067] a is the height of the through hole (210), h o is the height of the lower container (100), h e is the thickness of the elastic body (160).

[0068] Specifically, the height (h) of the internal storage space of the lower container (100) o -h e ) is smaller than the height (a) of the through hole (210), the porous plate (200) is mounted on the lower container (100) and pressure can be applied to the elastic body (160), whereby the thickness of the elastic body (160) of the bottom (110) of the lower container (100) is reduced, so that the through hole (210) can be completely in contact with the bottom (110) of the lower container (100).

[0069] In addition, the present disclosure provides a method for culturing a single microorganism in a liquid phase without diluting the culture solution in each well of a multi-well plate, and in which the microorganisms grown in each well do not cross-contaminate each other.

[0070] Specifically, the single microorganism culture method of the present disclosure may include a step of injecting a culture medium containing a single microorganism into a lower container (100) including a bottom (110), a plurality of side walls (120) and a plurality of first connecting portions (130) positioned on the side walls (120); a step of connecting a porous plate (200) including a plurality of through holes (210) and a plurality of second connecting portions (230) formed at an edge (220) and independently connected to the first connecting portions (130) to the lower container (100) to seal the culture medium in each through hole (210); and a step of covering the porous plate (200) with a lid (300) from the top and culturing the porous plate.

[0071] The single microorganism culture method of the present disclosure can improve the convenience of the worker by quickly and simply injecting the microorganism and culture solution into the lower container (100) of the multi-well plate.

[0072] According to one embodiment, the lower container (100) may include one or more partition walls (140) dividing the container across the container and one or more receiving grooves (150) positioned in the partition walls (140), and the porous plate may include one or more divided areas (240) dividing the through holes (210) across the plate and one or more protrusions (250) positioned below the divided areas (240) and engaging the receiving grooves (150).

[0073] According to one embodiment, the lower container (100) may further include an elastic body (160) formed on the surface of the bottom (110), side wall (120), and bulkhead (140).

[0074] Specifically, the porous plate (200) including the plurality of through holes (210) is spaced apart from the lower container (100) before use and is hung on the hook of the connecting part, and when used, microorganisms and a culture solution are first injected into the lower container (100) on a horizontal work table, and then the porous plate (200) is brought into close contact with the lower container (100), the elastic body (160) between the lower container (100) and the porous plate (200) is pressed and brought into close contact, and then pressed by the close hook of the connecting means, so that each of the through holes (210) can be perfectly isolated to form a multi-well.

[0075] According to one embodiment, the number (N) of single microorganisms injected into the lower container (100) may satisfy the following relationship 2.

[0076] [Relationship 2]

[0077] N≥0.1a (unit: CFU / (a*V)) (100 <a<50000)

[0078] (In the above relational expression 2, a is the number of wells, and V is the individual capacity of the well)

[0079] Specifically, in the above relational expression 2, the lower limit of N may be 0.1a, 0.2a, 0.3a, 0.4a, 0.5a, 0.6a, 0.7a, 0.8a, 0.9a, or 1.0a, and the upper limit may be 1.5a, 1.4a, 1.3a, 1.2a, 1.1a, or 1.0a.

[0080] When injecting microorganisms satisfying the above concentration into the lower container (100), more than 1 CFU of microorganisms can be injected into each well after mounting the porous plate (200).

[0081] At this time, the number of microorganisms may be less than the number of wells because different microorganisms grow simultaneously in many wells if the number is greater than the number of multi-wells. Specifically, if the number of bacteria is 10% of the number of wells, it is effective for culturing a single microorganism.

[0082] In this way, the single microorganism cultivation method of the present disclosure has the advantage of allowing easy microbial cultivation by forming a large number of independent environments capable of culturing microorganisms at once simply by combining the porous plate (200) and the lower container (100).

[0083] Hereinafter, the present disclosure will be described in detail with reference to FIGS. 1 to 7.

[0084] Referring to FIG. 1, a multi-well plate for culturing a single microorganism according to one embodiment of the present disclosure can be used for culturing a single microorganism as an integrated body sequentially assembled with a lower container (100), a porous plate (200), and a lid (300). Specifically, the lower container (100) may include a bottom (110), a plurality of side walls (120), and a plurality of first connecting portions (130) positioned on the side walls (120), and the porous plate (200) may include a plurality of through holes (210) and a plurality of second connecting portions (230) formed on an edge (220).

[0085] When the porous plate (200) is connected from the top of the lower container (100), a plurality of first connecting portions (130) located on the side wall (120) of the lower container (100) may be independently connected to a plurality of second connecting portions (230) formed on the edge (220) of the porous plate (200) so that the porous plate (200) is mounted on the lower container (100).

[0086] Specifically, the first connecting portion (130) may be in the form of a protrusion, and the second connecting portion (230) may be in the form of a receiving groove, and may be coupled to each other so that the porous plate (200) is mounted and coupled to the lower container (100).

[0087] The above porous plate (200) is composed of a plurality of through holes (210). The cross-sectional shape of the through holes (210) can be any shape that is separated from the surroundings and can culture microorganisms. That is, although the drawing shows the well as having a uniform rectangular shape, it is not limited thereto and, for example, shapes such as pentagons, hexagons, and circles are possible, and the shape and size can be changed in consideration of the size and type of microorganisms to be cultured.

[0088] The lower container (100) can be injected with microorganisms and culture medium through an injection port located on one side or near a corner. Alternatively, microorganisms and culture medium can be injected directly into the center of the lower container. In this case, the operator can use a liquid handling device, such as a pipette or syringe, to inject microorganisms and culture medium into the injection port.

[0089] The above-mentioned inlet may be installed at an angle, and this angled inlet performs the function of facilitating the injection or discharge of the culture medium. In addition, one or more inlets may be provided on one side or near a corner of the lower container.

[0090] The lower container (100) above can have ribs formed at a certain height inside the side surface. More specifically, the ribs can be formed on the horizontal axis of the inner side surface of the lower container. After injecting the culture medium and microorganisms, the worker can apply physical force, such as shaking the lower container from side to side, so that the microorganisms can be uniformly distributed inside the lower container. At this time, the culture medium can be prevented from overflowing to the outside by the ribs installed at a certain height inside the lower container. In addition, the amount of culture medium injected can be confirmed by marking a rib or dotted line at a certain height inside the side surface of the lower container.

[0091] Referring to FIGS. 2 to 4, a multi-well plate for culturing a single microorganism according to another embodiment of the present disclosure may include a lower container (100) having at least one partition wall (140) dividing the container across the container and at least one receiving groove (150) positioned in the partition wall, and the porous plate may include at least one divided area (240) dividing a through hole across the plate and at least one protrusion (250) positioned below the divided area (240) and coupled with the receiving groove (150).

[0092] Referring to Fig. 5, when the porous plate (200) is mounted on the lower container (100), the protrusion (250) located at the bottom of the divided area (240) of the porous plate (200) may be received and coupled to the receiving groove (150) of the bulkhead (140) of the lower container (100). Accordingly, the porous plate (200) can be more stably and closely coupled to the lower container (100), and movement of materials between the wells of the receiving groove (150) can be blocked.

[0093] In addition, the lower container (100) may have an elastic body (160) attached in close contact to the surfaces of the bottom (110), side walls (120), and partition walls (140). Specifically, when the porous plate (200) is mounted on the lower container (100), the through hole (210) of the porous plate (200) is in close contact with the elastic body (160) formed on the bottom of the lower container (100), so that the space between the wells of the through hole (210) is completely sealed, and movement of the culture medium between the wells can be prevented.

[0094] Referring to Fig. 6, the height (a) of the through hole (210) of the porous plate (200) may satisfy the following relational expression 1.

[0095] [Relationship 1]

[0096] h o -h e <a< h o

[0097] (In the above relational expression 1,

[0098] a is the height of the through hole, h o is the height of the lower container, h e is the thickness of the elastic body.)

[0099] Height of the internal storage space (h) o -h e ) is smaller than the height (a) of the through hole, the porous plate (200) is mounted on the lower container (100) and pressure can be applied to the elastic body (160), whereby the thickness of the elastic body (160) of the bottom (110) of the lower container is reduced, so that the through hole (210) can be completely in contact with the bottom (110) of the lower container.

[0100] As described above, the present invention has been described through specific matters and limited examples, but these are provided only to help a more general understanding of the present invention, and the present invention is not limited to the above examples, and those skilled in the art to which the present invention pertains can make various modifications and variations based on this description.

[0101] Therefore, the idea of ​​the present invention should not be limited to the described embodiments, and all things that are equivalent or equivalent to the following claims as well as the claims are considered to fall within the scope of the idea of ​​the present invention.

[0102]

[0103] 100: Lower container

[0104] 110: Floor

[0105] 120: Side wall

[0106] 130: First connector

[0107] 140: Bulkhead

[0108] 150: Reception Home

[0109] 160: Elastic body

[0110] 200: Perforated plate

[0111] 210: Through hole

[0112] 220: Edge

[0113] 230: Second connector

[0114] 240: Partition area

[0115] 250: Protrusion

[0116] 300: Lid

Claims

1. A lower container comprising a bottom, a plurality of side walls and a plurality of first connecting portions positioned on the side walls; A porous plate comprising a plurality of through holes and a plurality of second connecting portions formed at the edges and independently coupled to the first connecting portion; A multi-well plate for culturing a single microorganism, comprising a lid that can be removed from the top of the above-mentioned porous plate.

2. In paragraph 1, The lower container comprises one or more bulkheads dividing the container across the container and one or more receiving grooves positioned in the bulkheads, A multi-well plate for culturing a single microorganism, wherein the porous plate comprises one or more partition areas dividing through holes across the plate and one or more protrusions positioned below the partition areas and coupled with the receiving grooves.

3. In paragraph 2, A multi-well plate for culturing a single microorganism, wherein the lower container further includes an elastic body formed on the surface of the bottom, side walls, and partition walls.

4. In paragraph 3, A multi-well plate for culturing a single microorganism, wherein the elastic body comprises polypropylene.

5. In paragraph 3, A multi-well plate for culturing a single microorganism, wherein the hardness of the elastic body is 10 to 40.

6. In paragraph 3, A multi-well plate for culturing a single microorganism, wherein the thickness of the elastic body is 1.0 to 2.5 mm.

7. In paragraph 1, A multi-well plate for culturing a single microorganism, wherein the height (a) of the above through hole satisfies the following relational expression 1. [Relationship 1] h o -h e <a< h o (In the above relational expression 1, a is the height of the through hole, h o is the height of the lower container, h e is the thickness of the elastic body.) 8. In paragraph 1, A multi-well plate for culturing a single microorganism, wherein the diameter of the above through hole is 1.0 to 3.0 mm.

9. In paragraph 1, A multi-well plate for culturing a single microorganism, wherein the lid includes a gas-permeable, breathable membrane.

10. In paragraph 1, A multi-well plate for culturing a single microorganism, wherein the cross-sectional shape of the above through hole is square, hexagonal or circular.

11. A step of injecting a culture medium containing a single microorganism into a lower vessel including a bottom, a plurality of side walls, and a first connecting portion located on the side walls; A step of sealing a culture medium in each of the through holes by connecting a porous plate including a plurality of second connecting portions formed at the edges and independently connecting with the first connecting portion to the lower container; and A single microorganism culture method, comprising a step of covering the lid from the top of the porous plate and culturing.

12. In paragraph 11, The lower container comprises one or more bulkheads dividing the container across the container and one or more receiving grooves positioned in the bulkheads, A single microorganism culture method, wherein the porous plate comprises one or more divided regions dividing through holes across the plate and one or more protrusions positioned below the divided regions and coupled with the receiving grooves.

13. In paragraph 11, A single microorganism culture method, wherein the lower vessel further comprises an elastic body formed on the surface of the bottom, side walls, and bulkhead.

14. In paragraph 11, A single microorganism cultivation method, wherein the number (N) of single microorganisms injected into the above lower container satisfies the following relationship 2. [Relationship 2] N≥0.1a (unit: CFU / (a*V)) (100 <50000)​ (In the above relational expression 2, a is the number of wells, and V is the individual capacity of the well)

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