Preformed multiwell plates for culturing individual microorganisms in liquid media

Abstract

The purpose of this disclosure is to provide a multi-well plate for culturing single microorganisms, which allows for convenient and rapid partitioning of a culture medium containing a single microorganism and culturing it in multiple wells. The multi-well plate of this disclosure is economical because it does not require additional special equipment, and a method for culturing single microorganisms using the microplate can efficiently culture single microorganisms in multiple wells without the need for diluting the culture medium in each well.

Description

Technical Field

[0001] The present invention relates to a prefabricated multiwell plate for culturing a single microorganism in a liquid culture medium and a method for culturing a single microorganism using the multiwell plate. Background Technology

[0002] The persistent emergence of antibiotic-resistant bacteria is one of the greatest threats to global health, and it is warned that by 2050, 10 million people will die annually from antibiotic-resistant bacterial infections. Therefore, developing new antibiotics is a critical and urgent priority. Most antibiotics developed to date have been derived from microorganisms. However, there are limitations in discovering new microorganisms to produce antibiotics. Traditionally, the isolation of microorganisms has been performed using agarose solid medium, and this method has problems in screening for new microbial species because many microorganisms that can only be cultured in liquid phase cannot be isolated individually.

[0003] Meanwhile, the number of known microalgae species, estimated to be between 200,000 and 800,000, is a valuable resource of Earth, producing 15,000 natural substances, including various antibiotics and physiologically active substances. For microalgae to be used, single-cell isolation should be performed; however, most single-celled microalgae do not grow in solid-phase culture. Because current single-cell isolation methods using capillaries are labor-intensive, microalgae libraries hold only a few thousand individual microalgae.

[0004] In routine microbial isolation and identification, methods are typically used to obtain single colonies by spreading and culturing on solid culture media (such as agarose in Petri dishes) using glass rods, glass beads, etc. However, conventional culturing methods using solid media have reportedly only been able to culture less than 1% of known microorganisms. Meanwhile, culturing methods using liquid culture media involve generating droplets from a solution containing dispersed microorganisms and purely isolating and identifying microorganisms in the process. However, these methods require improvement because it is difficult to individually classify the droplets containing microorganisms. Furthermore, methods can be used to liquid culture a single type of microorganism by continuously diluting the culture medium using multi-well plates such as 96-well plates, but this requires considerable effort when performed manually.

[0005] Therefore, there is a need to develop high-efficiency culture plates for the efficient single isolation of various microorganisms. Summary of the Invention

[0006] Technical issues One object of this disclosure is to provide a multi-well plate for culturing single microorganisms, wherein the multi-well plate can independently culture single microorganisms in a liquid phase by forming a plurality of separate pores.

[0007] Another object of this disclosure is to provide a method for culturing a single microorganism, which can culture a single microorganism in a liquid phase without the need for diluting the culture medium in each well of a multi-well plate and without allowing cross-contamination between microorganisms growing in each well.

[0008] Technical solution In one general aspect, a multi-well plate for culturing a single microorganism includes: a lower container including a bottom, a plurality of sidewalls, and a plurality of first connecting portions disposed on the sidewalls; a perforated plate including a plurality of through holes and a plurality of second connecting portions formed in an edge and independently connected to the first connecting portions; and a cap detachably coupled to the top of the perforated plate.

[0009] The lower container may include one or more partition walls that divide the container across the space; and one or more receiving grooves placed on the partition walls, and the perforated plate may include one or more dividing regions that divide the through holes across the plate; and one or more protrusions placed in the lower portion of the dividing regions and engaging with the receiving grooves.

[0010] The lower container may also include an elastomer formed on the surfaces of the bottom, the sidewalls, and the partition walls.

[0011] The elastomer may include polypropylene.

[0012] The elastomer may have a hardness of 10 to 40.

[0013] The elastomer may have a thickness of 1.0 mm to 2.5 mm.

[0014] The height (a) of the through hole can satisfy the following relationship 1: [Relation 1] h o -h e <a<h o in, a is the height of the through hole, h o h is the height of the lower container. e It is the thickness of the elastomer.

[0015] The through hole can have a diameter of 1.0 mm to 3.0 mm.

[0016] The cover may include a gas-permeable membrane.

[0017] The cross-sectional shape of the through hole can be quadrilateral, hexagonal, or circular.

[0018] In another general aspect, a method for culturing a single microorganism includes: injecting a culture medium comprising the single microorganism into a lower container, the lower container including a bottom, a plurality of sidewalls, and a first connecting portion disposed on the sidewalls; attaching a perforated plate to the lower container to seal the culture medium within each of the through-holes, the perforated plate including a plurality of through-holes and a plurality of second connecting portions formed in an edge and independently connected to the first connecting portion; and covering the top of the perforated plate with a cap and performing the culture.

[0019] The lower container may include one or more partition walls that divide the container across; and one or more receiving grooves placed on the partition walls, and the perforated plate includes one or more dividing regions that divide the through holes across the plate; and one or more protrusions placed in the lower portion of the dividing regions and connected to the receiving grooves.

[0020] The lower container may also include an elastomer formed on the surfaces of the bottom, the sidewalls, and the partition walls.

[0021] The number (N) of individual microorganisms injected into the lower container can satisfy the following relationship 2: [Relation 2] N≥0.1a (unit: CFU / (a)) V))(100 <a<50000) Where a is the number of holes, and V is the individual capacity of each hole.

[0022] Beneficial effects The multi-well plate disclosed herein for culturing single microorganisms allows for convenient and rapid partitioning of culture medium containing single microorganisms into multiple wells and performing culture. It is economical because it does not require the use of special devices and allows for efficient culture of single microorganisms within the wells without the need for diluting the culture medium in each well.

[0023] The method disclosed herein for culturing a single microorganism can independently culture a single microorganism in each well because the culture medium is divided into multiple wells and completely separated in a single step. Attached Figure Description

[0024] Figure 1This is a perspective view of a perforated plate according to an embodiment of the present disclosure.

[0025] Figure 2 This is a perspective view of a perforated plate according to another embodiment of the present disclosure.

[0026] Figure 3 The front and back sides of the perforated plate of the perforated plate of this disclosure are shown.

[0027] Figure 4 The front of the lower container of the perforated plate of this disclosure is shown.

[0028] Figure 5 The process of connecting the lower container and the perforated plate of the perforated plate of this disclosure is shown.

[0029] Figure 6 The appearance of the elastomer (green) is shown when the lower container of the perforated plate of this disclosure is connected to the perforated plate, where a is the height of the through hole and h is the height of the through hole. o It is the height of the lower container, and h e It is the thickness of the elastomer.

[0030] Figure 7 A receiving groove on the partition wall is shown in the lower container of the perforated plate of this disclosure. Detailed Implementation

[0031] In the following, exemplary embodiments of the invention will be described in detail with reference to the accompanying drawings to facilitate practice by those skilled in the art. The invention may be embodied in various different forms and is not limited to the exemplary embodiments described herein.

[0032] Conventionally, to culture a single microorganism in a liquid culture medium, a single species of microorganism is cultured by continuously diluting the medium in a multi-well plate. However, this is laborious and inefficient when performed manually. Therefore, this disclosure provides a multi-well plate for culturing single microorganisms, which can culture a single microorganism in the liquid phase without the need for diluting the medium in each well and without preventing cross-contamination between microorganisms growing in each well.

[0033] The multi-well plate disclosed herein for culturing single microorganisms may include: a lower container 100 including a bottom 110, a plurality of sidewalls 120, and a plurality of first connecting portions 130 disposed on the sidewalls; a perforated plate 200 including a plurality of through holes 210 and a plurality of second connecting portions 230 formed in the edges and independently connected to the first connecting portions; and a cover 300 removable from the top of the perforated plate.

[0034] A multi-well plate for culturing individual microorganisms can be formed with multiple pores for culturing microorganisms because the lower container 100 and the perforated plate 200 are attached together by the connection of the first connecting portion 130 and the second connecting portion 230, so that each through hole 210 is firmly attached to the bottom 110 of the lower container 100, and thus individual microorganisms can be cultured independently in each hole without cross-contamination.

[0035] Specifically, the lower container 100 may include four sidewalls 120 and one to three first connecting portions 130 in each sidewall, and the number of second connecting portions 230 of the perforated plate 200 may be the same as the number of first connecting portions 130.

[0036] Furthermore, the bottom 110 of the lower container 100 may have a thickness of 1.0 mm to 4.0 mm, specifically 1.0 mm to 3.0 mm, and more specifically 1.5 mm to 2.5 mm. In this document, when the thickness of the bottom 110 is less than the range described above, there is a risk of deformation when fastening the lower container 100 and the perforated plate 200.

[0037] Specifically, the perforated plate 200 may have 100 to 50,000 through-holes 210, but is not limited to this, as long as a large number of microorganisms can be isolated. Furthermore, the through-holes 210 may have a diameter of 0.5 mm to 5.0 mm, more specifically 0.5 mm to 4.0 mm, or 1.0 mm to 3.0 mm. The size of the through-holes 210 is determined with consideration of the recovery of microorganisms from the holes where microorganisms grow, and when the diameter of the through-holes 210 is smaller than the range described above, it is not easy to recover the microorganisms cultured inside the holes, and when the diameter is larger than the range described above, it is difficult to perform pure culture of many types of microorganisms at once.

[0038] According to one implementation example, the cross-sectional shape of the through hole 210 may be quadrilateral, hexagonal or circular, but is not limited thereto.

[0039] A fine hole can be formed on the top of the through hole 210, and the fine hole can reduce gas leakage inside the through hole 210 and gas inflow from the outside.

[0040] The lid 300 performs a contamination prevention function because it is positioned on top of the perforated plate 200 and is sterilized. Furthermore, the lid 300 prevents contamination from the external environment (such as the user's hands, gases, and contaminants) and prevents liquid evaporation during incubation.

[0041] According to an example of an embodiment, the lid 300 may be formed of a transparent material. Due to the transparent material, light can be used to check whether cells are growing inside the porous plate through turbidity, and the cell proliferation rate can be checked based on the turbidity of the solution.

[0042] According to another example of an embodiment, the lid 300 may be formed of a gas-permeable breathable membrane. The breathable membrane can perform the function of gas exchange into the pores and contribute to microbial culture. In addition, when removing the microorganisms cultured inside the pores, the microbial culture medium can be removed by piercing the breathable membrane with a syringe needle. The breathable membrane can be fixed to the upper surface of the perforated plate 200 and used.

[0043] Meanwhile, in order to spread and inject the culture medium well into the lower container 100 and the plurality of through holes 210, the inner surface of the lower container, the surface of the perforated plate 200, and the through holes 210 may be subjected to hydrophilic surface treatment by plasma treatment or chemical treatment, and thus, the microorganisms and the culture medium can be naturally and evenly injected into the through holes 210 from the lower container.

[0044] According to an example of an embodiment, the lower container 100 includes: one or more partition walls 140 that divide across the container; and one or more receiving grooves 150 that are placed on the partition walls 140; and the perforated plate 200 may include: one or more dividing regions 240 that divide the through holes 210 across the plate; and one or more protruding portions 250 that are placed in the lower part of the dividing regions 240 and connected to the receiving grooves 150.

[0045] The n (0 < n < 10, n = 1, 2, 3, 4, 5, ◎) receiving grooves 150 placed on the partition walls 140 may be respectively formed at the 1 / (n + 1), 2 / (n + 1), 3 / (n + 1),..., n / (n + 1) points of the length of the partition walls 140, and the protruding portions 250 may be formed at the positions corresponding to the plurality of receiving grooves 150 in the lower part of the dividing regions 240 of the perforated plate 200. Specifically, as Figure 7 shown, the red and blue receiving grooves 150 placed on the partition walls 140 may be formed at the 1 / 2 position of the length of the partition walls marked in yellow. Through the connection of the receiving grooves 150 and the protruding portions, the perforated plate 200 is more stably fitted to the lower container 100, and the movement of substances between the holes of the receiving grooves 150 can be blocked.

[0046] Specifically, the lower container 100 may include one or two partition walls 140 and one to five receiving recesses 150 disposed on the partition walls 140, and more specifically, the lower container 100 includes two partition walls 140 and two receiving recesses 150 disposed in one of the partition walls 140. In this document, the perforated plate 200 may include one or two dividing regions 240, and the protruding portion 250 is the region corresponding to the receiving recess 150 disposed in the perforated plate 140 of the lower container 100, and may be placed in the lower portion of the dividing region 240.

[0047] Furthermore, the materials used to form the porous plate are 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; resin materials including at least one of hydrophilic surface-treated resins can be used; or transparent inorganic materials such as glass or quartz can be used, and these materials can be materials that allow identification of the pores where microbial growth occurs.

[0048] According to one embodiment, the lower container 100 may further include an elastomer 160 formed on the surfaces of the bottom 110, sidewalls 120, and partition walls 140. Specifically, the elastomer 160 may be formed on a surface of the lower container 100 where the bottom 110, sidewalls 120, and partition walls 140 contact each other, and thus, in the connection between the perforated plate 200 and the lower container 100, when each through-hole 210 contacts the bottom 110 of the lower container 100, a plurality of holes are formed by the locking action of the elastomer 160. Liquid movement is prevented between the formed holes, thereby preventing contamination of microorganisms cultured inside the holes.

[0049] According to one embodiment example, the elastomer may include a polypropylene copolymer. The polypropylene copolymer may be a copolymer with an α-olefin monomer, and the olefin monomer may include at least one selected from the group consisting of ethylene, 1-butene, 1-pentene, 1-hexene, and combinations thereof.

[0050] The polypropylene copolymer may include homopolymers and copolymers. Specifically, the polypropylene copolymer may include at least one selected from the group consisting of free block polypropylene, atactic polypropylene, homopolymer polypropylene, highly crystalline block polypropylene, highly crystalline homopolymer polypropylene, metocene polypropylene, metallocene polypropylene, and combinations thereof.

[0051] The polypropylene copolymer may have a weight-average molecular weight (Mw) of 200,000 g / mol to 500,000 g / mol, with a specific lower limit of 250,000 g / mol or 300,000 g / mol and an upper limit of 500,000 g / mol, 450,000 g / mol or 400,000 g / mol.

[0052] Specifically, the polypropylene copolymer can have an isotactic index (II) of 97.5% to 99.5%. Furthermore, the polypropylene copolymer can have a content of 0.80 g / cm³. 3 Up to 0.95 g / cm 3 Specifically, 0.85 g / cm 3 Up to 0.92 g / cm 3 And more specifically 0.89 g / cm 3 Up to 0.92 g / cm 3 The density.

[0053] The polypropylene copolymer can have a melt flow index of 0.1 g / 10 min to 1.0 g / 10 min, specifically 0.5 g / 10 min to 1.0 g / 10 min, measured at 230 °C and a load of 2.16 kg.

[0054] According to one embodiment example, the elastomer may have a hardness (A) of 10 to 40, specifically 10 to 30 or 10 to 25.

[0055] According to one embodiment example, the elastomer may have a thickness of 1.0 mm to 2.5 mm, specifically 1.5 mm to 2.5 mm. In this document, when the thickness is less than the range described above, the locking effect via the elastomer may be weak, and when the thickness is greater, the flatness may be reduced.

[0056] According to one implementation example, the height (a) of the through hole 210 of the perforated plate can satisfy the following relationship 1: [Relation 1] h o -h e <a<h o in, a is the height of the through hole 210, h is... o It is the height of the lower container, 100, and h e It is the thickness of the elastomer 160.

[0057] Specifically, the height (h) of the internal storage space of the lower container 100 o -he When the height (a) of the through hole 210 is lower than that of the lower container 100, the perforated plate 200 is installed in the lower container 100, which can apply pressure to the elastomer 160, and thus the thickness of the elastomer 160 on the bottom 110 of the lower container 100 is reduced, so that the through hole 210 can be completely fitted to the bottom 110 of the lower container 100.

[0058] Furthermore, this disclosure provides a method for culturing a single microorganism, which can culture a single microorganism in a liquid phase without the need for diluting the culture medium in each well of a multi-well plate, and does not allow cross-contamination between microorganisms growing in each well.

[0059] Specifically, the method for culturing a single microorganism may include: injecting a culture medium comprising the single microorganism into a lower container 100, the lower container 100 including a bottom 110, a plurality of sidewalls 120 and a plurality of first connecting portions 130 disposed on the sidewalls 120; connecting a perforated plate 200 to the lower container 100 to seal the culture medium in each of the through holes 210, the perforated plate 200 including the plurality of through holes 210 and a plurality of second connecting portions 230 formed in an edge 220 and independently connected to the first connecting portions 130; and covering the top of the perforated plate 200 with a cap 300 and performing the culture.

[0060] The method disclosed herein for culturing a single microorganism improves user convenience by rapidly and easily injecting the microorganism and culture medium into the lower container 100 of the multiwell plate.

[0061] According to one embodiment example, the lower container 100 includes: one or more partition walls 140 dividing the container across; and one or more receiving recesses 150 disposed on the partition walls 140; and the perforated plate may include: one or more dividing regions 240 dividing through holes 210 across the plate; and one or more protrusions 250 disposed in the lower portion of the dividing regions 240 and connected to the receiving recesses 150.

[0062] According to one embodiment example, the lower container 100 may further include an elastomer 160 formed on the surfaces of the bottom 110, sidewalls 120, and partition walls 140.

[0063] Specifically, before use, the perforated plate 200, which includes multiple through holes 210, is separated from the lower container 100 and suspended on the engagement protrusion of the connecting part. During use, when microorganisms and culture medium are first injected into the lower container 100 in a horizontal workbench and then the perforated plate 200 is attached to the lower container 100, the elastomer 160 between the lower container 100 and the perforated plate 200 is pressed and attached, and then pressed by the tightly fitting engagement protrusion of the connecting device to completely separate each through hole 210, thereby forming a porous structure.

[0064] According to one implementation example, the number (N) of individual microorganisms injected into the lower container 100 can satisfy the following relationship 2: [Relation 2] N≥0.1a (unit: CFU / (a)) V))(100 <a<50000) Where a is the number of holes and V is the individual capacity of each hole.

[0065] Specifically, in relation 2, the lower limit of N can 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 can be 1.5a, 1.4a, 1.3a, 1.2a, 1.1a or 1.0a.

[0066] When the required concentration of microorganisms is injected into the lower container 100, after the perforated plate 200 is installed, 1 CFU or more of microorganisms can be injected into each well.

[0067] In this paper, when the number of microorganisms is greater than the number of pores, other microorganisms grow simultaneously in many pores, and therefore the number of microorganisms can be less than the number of pores, and specifically, it is effective for culturing a single microorganism when the number of bacteria contained is 10% of the number of pores.

[0068] Thus, the method of this disclosure for culturing a single microorganism can form several independent environments in which microorganisms can be cultured by a single connection between the perforated plate 200 and the lower container 100, thereby achieving convenient microbial culture.

[0069] In the following text, reference will be made to Figures 1 to 7 This disclosure is described in detail.

[0070] refer to Figure 7According to one embodiment of this disclosure, a multi-well plate for culturing a single microorganism is an assembly of a lower container 100, a perforated plate 200, and a cap 300 assembled in sequence, and can be used to culture a single microorganism. Specifically, the lower container 100 may include a bottom 110, a plurality of sidewalls 120, and a plurality of first connecting portions 130 disposed on the sidewalls 120, and the perforated plate 200 may include a plurality of through holes 210 and a plurality of second connecting portions 230 formed in the edges 220.

[0071] When the perforated plate 200 is connected from the top of the lower container 100, a plurality of first connecting portions 130 placed on the side wall 120 of the lower container 100 and a plurality of second connecting portions 230 formed in the edge 220 of the perforated plate 200 are independently connected to each other, so that the perforated plate 200 can be installed on the lower container 100.

[0072] Specifically, a first connecting portion 130 with a protruding shape and a second connecting portion 230 with a receiving groove shape are connected to each other, such that the perforated plate 200 is mounted on the lower container 100 and connected.

[0073] The perforated plate 200 is formed by a plurality of through holes 210. The cross-sectional shape of the through holes 210 can be any shape that can be separated from the surrounding area and can be used to cultivate microorganisms. That is, as shown in the figures, the holes have a uniform quadrilateral shape, but are not limited to this, and can be, for example, pentagonal, hexagonal, circular, etc., and the shape and size can be varied taking into account the size and type of microorganisms to be cultivated.

[0074] Microorganisms and culture media can be injected into the lower container 100 through an inlet located on one side or near a corner. Alternatively, microorganisms and culture media can be injected directly into the center of the lower container. In this document, the user may use liquid handling equipment such as pipettes and syringes to inject microorganisms and culture media into this inlet.

[0075] The inlet can be installed at an angle, and the angled inlet facilitates the injection or discharge of culture medium. Additionally, one or more inlets can be located on one surface or near the edge of the lower container.

[0076] The lower container 100 may include ribs formed on the inner surface of the sidewall at a specific height. More specifically, the ribs may be formed along the inner transverse axis of the lower container. After the culture medium and microorganisms are injected, the user may apply physical force, such as shaking the lower container from side to side, to allow the microorganisms to spread evenly inside the lower container. Furthermore, the ribs installed inside the lower container at a specific height can prevent the culture medium from overflowing to the outside. Additionally, the amount of culture medium injected can be checked by marking the ribs or dashed lines on the inside of the sidewall of the lower container at a specific height.

[0077] refer to Figures 2 to 4 According to another embodiment of this disclosure, an example of a multi-well plate for culturing a single microorganism includes: one or more partition walls 140 dividing the container across; and one or more receiving grooves 150 disposed on the partition walls 140; and the perforated plate may include: one or more dividing regions 240 dividing through holes 210 across the plate; and one or more protrusions 250 disposed in the lower portion of the dividing regions 240 and connected to the receiving grooves 150.

[0078] refer to Figure 5 When the perforated plate 200 is installed on the lower container 100, the protrusion 250 placed on the lower portion of the dividing area 240 of the perforated plate 200 can be accommodated and connected to the receiving groove 150 of the partition wall 140 of the lower container 100. Therefore, the perforated plate 200 can be more stably fitted and connected to the lower container 100, and the movement of material between the holes of the receiving groove 150 can be prevented.

[0079] Furthermore, the lower container 100 can be formed of an elastomer 160 that adheres to and is attached to the surfaces of the bottom 110, side walls 120, and partition walls 140. Specifically, when the perforated plate 200 is mounted on the lower container 100, the through holes 210 of the perforated plate 200 adhere to the elastomer 160 formed on the bottom of the lower container 100, such that the space between the holes of the through holes 210 can be completely sealed, and the movement of the culture medium between the holes can be prevented.

[0080] refer to Figure 6 The height (a) of the through hole 210 of the perforated plate 200 can satisfy the following relationship 1: [Relation 1] h o -h e <a<h o in, a is the height of the through hole, h o It is the height of the lower container, and h e It is the thickness of the elastomer.

[0081] When the height of the content storage space (h) o -h e When the height (a) of the through hole is below the height of the through hole, the perforated plate 200 is installed in the lower container 100, which can apply pressure to the elastomer 160, and thus the thickness of the elastomer 160 on the bottom 110 of the lower container is reduced, so that the through hole 210 can be completely fitted to the bottom 110 of the lower container.

[0082] In the foregoing, although the invention has been described by way of specified matters and specific exemplary embodiments, these are provided only to aid in the overall understanding of the invention, and the invention is not limited to the exemplary embodiments. Various modifications and changes can be made by those skilled in the art based on the description.

[0083] Therefore, the spirit of the present invention should not be limited to the exemplary embodiments described above, and the appended claims and modifications equivalent to or equivalent to the claims are intended to fall within the scope and spirit of the present invention.

[0084] 100: Lower container 110: Bottom 120: Side wall 130: First connecting part 140: Partition wall 150: Receiving groove 160: Elastomer 200: Perforated plate 210: Through hole 220: Edge 230: Second connecting part 240: Divide the area 250: Highlighted section 300: Cover.

Claims

1. A multi-well plate for culturing single microorganisms, comprising: A lower container, the lower container including a bottom, multiple sidewalls, and multiple first connecting portions placed on the sidewalls; A perforated plate, the perforated plate including a plurality of through holes and a plurality of second connecting portions, the plurality of second connecting portions being formed in the edge and independently connected to the first connecting portion; as well as A cover, which is detachably attached to the top of the perforated plate.

2. The multi-well plate for culturing single microorganisms according to claim 1, in, The lower container includes one or more partition walls that divide the container across it; and one or more receiving recesses placed on the partition walls. The perforated plate includes one or more dividing regions that divide the through holes across the plate; and one or more protrusions that are positioned in the lower portion of the dividing regions and connected to the receiving groove.

3. The multi-well plate for culturing single microorganisms according to claim 2, wherein, The lower container also includes an elastomer formed on the surfaces of the bottom, the sidewalls, and the partition walls.

4. The multi-well plate for culturing single microorganisms according to claim 3, wherein, The elastomer includes polypropylene.

5. The multi-well plate for culturing single microorganisms according to claim 3, wherein, The elastomer has a hardness of 10 to 40.

6. The multi-well plate for culturing single microorganisms according to claim 3, wherein, The elastomer has a thickness of 1.0 mm to 2.5 mm.

7. The multi-well plate for culturing single microorganisms according to claim 1, wherein, The height (a) of the through hole satisfies the following relationship 1: [Relation 1] h o -h e <a<h o in, a is the height of the through hole, h o h is the height of the lower container. e It is the thickness of the elastomer.

8. The multi-well plate for culturing single microorganisms according to claim 1, wherein, The through hole has a diameter of 1.0 mm to 3.0 mm.

9. The multi-well plate for culturing single microorganisms according to claim 1, wherein, The cover includes a gas-permeable membrane.

10. The multi-well plate for culturing single microorganisms according to claim 1, wherein, The cross-sectional shape of the through hole is quadrilateral, hexagonal, or circular.

11. A method for culturing a single microorganism, the method comprising: A culture medium comprising a single microorganism is injected into a lower container, the lower container comprising a bottom, multiple sidewalls, and a first connecting portion placed on the sidewalls; A perforated plate is attached to the lower container to seal the culture medium within each of the through-holes. The perforated plate includes a plurality of through-holes and a plurality of second connecting portions formed in the edges and independently connected to the first connecting portion. as well as Cover the top of the perforated plate with a cap and perform incubation.

12. The method for culturing a single microorganism according to claim 11, wherein, The lower container includes one or more partition walls that divide the container across it; and one or more receiving recesses placed on the partition walls. The perforated plate includes one or more dividing regions that divide the through holes across the plate; and one or more protrusions that are positioned in the lower portion of the dividing regions and connected to the receiving groove.

13. The method for culturing a single microorganism according to claim 11, wherein, The lower container also includes an elastomer formed on the surfaces of the bottom, the sidewalls, and the partition walls.

14. The method for culturing a single microorganism according to claim 11, wherein, The number (N) of individual microorganisms injected into the lower container satisfies the following relationship 2: [Relationship 2] N≥0.1a (unit: CFU / (a)) V))(100 <50000)​ Where a is the number of holes, and V is the individual capacity of each hole.

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