WO2022039054A1 - Culturing device for microorganisms, culturing device for microorganisms and including culture medium components, and method for counting microorganisms using same - Google Patents

Culturing device for microorganisms, culturing device for microorganisms and including culture medium components, and method for counting microorganisms using same Download PDF

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Publication number
WO2022039054A1
WO2022039054A1 PCT/JP2021/029299 JP2021029299W WO2022039054A1 WO 2022039054 A1 WO2022039054 A1 WO 2022039054A1 JP 2021029299 W JP2021029299 W JP 2021029299W WO 2022039054 A1 WO2022039054 A1 WO 2022039054A1
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Prior art keywords
culture
medium
microorganisms
convex portion
medium component
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PCT/JP2021/029299
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French (fr)
Japanese (ja)
Inventor
哉 寺村
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高崎 真一
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Publication of WO2022039054A1 publication Critical patent/WO2022039054A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the present invention relates to a culture device for microorganisms, a culture device for microorganisms containing a medium component, and a method for measuring the number of microorganisms using the same.
  • Non-Patent Document 1 As a method for measuring the number of microorganisms, a pour culture method, an agar plate smearing method, and the like are known (Non-Patent Document 1).
  • the agar medium used for culturing microorganisms in these methods is a solidified medium in which nutritional components and selective components are heated and dissolved together with agar, and must be prepared and sterilized in advance prior to culturing and measurement. be.
  • the pour culture method it is necessary to complete these medium preparations in advance before the test of the sample and keep the medium warm at about 50 ° C.
  • Patent Document 1 discloses a sheet-like culture apparatus provided with an adhesive layer, a cold water-soluble gelling agent powder layer containing a nutritional component, and a cover sheet that can be peeled off on the upper surface of a waterproof substrate. ..
  • Patent Document 2 discloses a simple medium provided with a water-soluble gelling agent and a fibrous water-absorbent sheet having a mesh on the upper surface of a waterproof base material.
  • Patent Document 3 discloses a sheet-like culture device in which a water-absorbent polymer layer and a porous matrix layer are sequentially laminated on the upper surface of a waterproof base material.
  • Patent Document 4 discloses a sheet-like culture device in which a frame on which a sample spreads is provided on a base sheet, and a medium solution containing an adhesive component and a gelling agent is formed in the frame.
  • the frame is made of a hydrophobic resin whose contact angle is set to a specific value, and the sample liquid spreads only within the frame.
  • Patent Document 5 describes a microorganism having a space surrounded by the upper surface of the convex portion of the upper member and the bottom surface and the side surface of the concave portion of the lower member as a medium region in a state where the upper member and the lower member having the concave portion are fitted. Culture equipment is disclosed.
  • a sample is tested between the waterproof substrate and the top film, and the sample liquid is spread over a predetermined area by pressing the sample from the top of the top film with a device called a spreader.
  • This operation requires a flat surface, and if not done carefully, the sample may not spread evenly but may flow out to the surroundings, which is not only difficult to operate, but also the area of the medium formed by the spreader. Since they are not always the same, there is a problem that the concentration of the formed medium fluctuates and the culture conditions are not stable.
  • the microorganisms in the sample cannot be cultured by a simple operation, and the number thereof cannot be easily measured.
  • Patent Document 2 Since a porous matrix such as a non-woven fabric is used in the culture equipment described in Patent Documents 2 and 3, it is difficult to control the dry state of the solvent during its production due to the convenience of using the porous matrix.
  • Patent Document 3 the procedure for laminating the porous matrix layer and the culture medium layer is complicated (Patent Document 3), and there are some difficulties in manufacturing. Further, the diffused reflection generated on the surface of the medium due to the unevenness of the surface of the porous matrix and the opacity of the porous matrix itself make it difficult to see the colonies after culturing. It is difficult to measure the number easily.
  • the culture equipment described in Patent Document 4 does not use a porous matrix, but it requires a flat place and caution during operation so that the sample does not spill from the frame that serves as the bank when the liquid sample spreads. It is done.
  • the frame is formed of a material set to a specific contact angle, but depending on the type of the sample, for example, when a food or drink containing oil, protein, etc. is used as the sample, the water repellency of the frame changes. It is assumed that there is a problem in versatility as a culture equipment, and since a member considering the contact angle is required, it is necessary to configure the member from a plurality of members, which can be said to be complicated to manufacture.
  • Patent Document 5 the upper member having a convex portion and the lower member having a concave portion are fitted to each other, and the upper surface of the convex portion of the upper member and the concave portion of the lower member are combined.
  • the volume of the space surrounded by the bottom surface and these side surfaces to include the spatial volume integral of the medium region, the sample containing water and the medium component are brought into contact with each other while being pressed in this space, and the medium is contacted.
  • a culture medium for microorganisms capable of forming a culture medium.
  • the incubator of Patent Document 5 is a space called a culture medium region surrounded by the upper surface of the convex portion of the upper member and the bottom surface and the side surface of the concave portion of the lower member in a state where the convex portion of the upper member and the concave portion of the lower member are fitted. Is provided as an indispensable configuration.
  • the present inventor examines the culture equipment of Patent Document 5, when an appropriate amount of the medium component is not put in the space of this medium region, at least one of the upper surface and the bottom surface and the medium component are used.
  • the present inventor has noticed that an air layer is likely to be generated between them.
  • the present inventor has found that when the air layer is generated, the bacteria existing on the culture medium gel spread on the gel surface and overlap with the surrounding colonies, making measurement difficult.
  • Using the culture equipment of Patent Document 5 to operate so as not to generate an air layer is not a simple operation, and it is easy to measure the number of microorganisms in a sample by generating an air layer. Is difficult.
  • the sample liquid is applied to the medium component, the upper member is closed to diffuse the sample liquid, and at the same time, the upper member and the lower member come into contact with each other via water. Capillary action occurred. Due to this capillarity, the applied sample liquid moves from the medium region of the cylindrical part composed of the upper member and the lower member to the outside at an earlier stage than the gelling agent in the medium component captures and solidifies the water content of the sample liquid. The present inventor has found that it leaks. Furthermore, the present inventor has found that there is a problem that a medium having an intended concentration cannot be formed due to leakage of the sample solution to the outside of the medium area.
  • test solution which may contain microorganisms
  • spontaneously leaks to the outside of the culture medium region which makes it impossible to cultivate the microorganisms in the sample by a simple operation, and the number of microorganisms can be easily measured.
  • the present inventor has also found that there is a problem in that the microbiological test is easily carried out.
  • the present inventor makes the upper area of the convex portion of the upper member smaller than the bottom area of the concave portion of the lower member, so that the sample liquid is around the portion to be the medium region. I tried to set up an escape route for the water. However, even in such a culture device, it takes time for the gelling agent in the medium component to capture and solidify the water content of the sample solution, so that the capillary phenomenon caused by the contact between the upper member and the lower member via the water is caused. The present inventor has found that it cannot be prevented and that water leaks to the outside.
  • the present inventor has noticed that the concentration of the medium component becomes non-uniform every time the culture is used because water accumulates in the outer peripheral portion of the medium region in the culture equipment of Patent Document 5.
  • the present inventor has found that the culture equipment of Patent Document 5 has an adverse effect that the microorganisms in the sample cannot be cultured by a simple operation and the number thereof cannot be easily measured. rice field.
  • the present inventor has made a configuration in which the top surface of the convex portion and the bottom of the concave portion are in contact with each other in a microbial culture equipment in a state where the convex portion is fitted into the concave portion. It was found that the microorganisms in the sample can be cultivated by a simple operation and the number of microorganisms can be easily measured. Further, the present inventor provides a groove in the outer peripheral region of the concave and / or convex portions, regardless of the space volume formed by the concave portions and the convex portions and the contact angle at the time of fitting the equipment, and also the instrument and the capillary phenomenon.
  • a first member having a recess capable of accommodating a medium component It has a second member having a convex portion that can be fitted into the concave portion, and has.
  • An incubator for microorganisms having a structure in which the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state.
  • the hinge portion has a structure in which the top surface of the convex portion can be fitted while maintaining a parallel state with respect to the bottom surface.
  • the term "specimen” is not particularly limited in the present specification, it is usually a sample liquid, and specifically, it is an aqueous solution such as drinking water, soft drink, industrial water, pharmaceutical water, dialysate, and urine. It is a liquid sample or the like. Further, a solid sample made into an emulsion or a suspension by a diluted solution or the like is also included.
  • the term "microorganism” as used herein usually refers to bacteria and fungi, and generally refers to general bacteria, bacilli, coliforms, coliforms, staphylococci, Vibrio bacteria, enterococci, yeasts, molds and the like. ..
  • the microorganisms in the sample can be cultured by a simple operation, and the number of microorganisms in the sample can be easily measured.
  • the effects described here are not necessarily limited, and may be any of the effects described in the present specification.
  • FIG. 1A is an orthogonal projection view of the culture equipment in a state where the concave portion of the first member and the convex portion of the second member are not fitted, and is a view on the side where the bottom surface and the top surface can be seen.
  • FIG. 1B represents an example of a cross-sectional view taken along the line I-I'of FIG. 1A.
  • FIG. 1C is a cross-sectional view showing a state in which the convex portion of the second member is to be fitted into the concave portion of the first member. It is a figure which shows one aspect of the culture equipment of this invention.
  • FIG. 1A is an orthogonal projection view of the culture equipment in a state where the concave portion of the first member and the convex portion of the second member are not fitted, and is a view on the side where the bottom surface and the top surface can be seen.
  • FIG. 1B represents an example of a cross-sectional view taken along the line I-I'of FIG. 1A.
  • FIG. 2A is an orthogonal projection view of the incubator in a state where the concave portion of the first member and the convex portion of the second member are fitted, and is a view seen from the outside of the second member.
  • FIG. 2B shows an example of a cross-sectional view taken along the line I-I'of FIG. 2A.
  • FIG. 2C shows an example of a cross-sectional view in which the culture medium component is contained in the concave portion of the incubator and the convex portion is fitted. It is a figure which shows one aspect of the 1st member of the culture equipment of this invention.
  • 3A is a view seen from the bottom surface side of the first member, and FIGS.
  • FIG. 3B and 3C each represent an example of a cross-sectional view taken along the line I-I'of FIG. 3A. It is a figure which shows one aspect of the 2nd member of the culture equipment of this invention.
  • FIG. 4A is a top view when viewed from the opposite side of the top surface of the second member, and FIG. 4B shows an example of a cross-sectional view taken along the line I-I'of FIG. 4A. It is a figure which shows one aspect of the culture equipment of this invention.
  • 5A to 5C are examples of cross-sectional views in which the first member and the second member are connected by various hinge portions, and the concave portion of the first member and the convex portion of the second member are not fitted. show.
  • FIGS. 5A to 5C The cross-sectional shapes of the hinge portions of FIGS. 5A to 5C are C-shaped, triangular-shaped, and square-shaped at the time of fitting, respectively.
  • FIG. 5D shows an example of a state in which the first member and the second member are not integrated but separated separately. It is a figure which shows one aspect of the culture equipment of this invention.
  • FIG. 6 is a diagram showing an example of a culture device having a hinge portion whose cross-sectional shape becomes quadrangular at the time of fitting, and in a state where the concave portion of the first member and the convex portion of the second member are fitted.
  • the front view, the back view, the left view (cross-sectional view), the right view (cross-sectional view), the plan view, and the bottom view of the culture equipment are shown. It is a figure which shows one aspect of the culture equipment of this invention.
  • 7A and 7B are views when the incubator at the time of fitting is viewed from the opposite side of the top surface of the second member, respectively.
  • 7A and 7B show an example of a mode in which a plurality of stacking convex portions are formed in the outer peripheral region of the groove portion.
  • 8A and 8B are views when the circular second member is viewed from the opposite side of the top surface, and are views showing an example of a cross section on the side surface side thereof, respectively.
  • FIG. 8C is a diagram showing a cross section on the side surface side when the stacking convex portions are formed on the first member at equal intervals in the outer peripheral region.
  • FIG. 8D is a diagram showing a side surface (cross-sectional view) of an example of the culture equipment at the time of fitting.
  • FIG. 8E is a view showing an example of the culture equipment at the time of fitting when viewed from the opposite side of the top surface of the second member. It is a figure which shows one aspect of the culture equipment containing a culture medium component of this invention.
  • FIG. 9 shows a cross-sectional view when a plurality of culture equipment containing a medium component at the time of insertion are stacked.
  • the upper and lower culture equipment is fixed by the groove portion that is convex on the outside and the convex portion for stacking on the outside, which indicates that the stacking collapse of the culture equipment containing a plurality of medium components is prevented. It is a figure showing the photograph of the detected colony using the culture equipment containing the culture medium component of Example 1. FIG. It is a figure showing the photograph of the detected colony using the culture equipment containing the culture medium component of Example 1. FIG.
  • the present invention has a first member having a concave portion capable of accommodating a medium component and a second member having a convex portion that can be fitted into the concave portion, and the top surface of the convex portion in the fitted state. It is possible to provide a culturing equipment for microorganisms or a culturing equipment for microorganisms containing a culture medium component, which is provided with a structure in which the surface is in contact with the bottom surface of the recess. By using the culture equipment of the present invention, microorganisms in a sample can be cultured by a simple operation and the number thereof can be easily measured.
  • the incubator of the present invention does not have to adopt a complicated structure, it can be easily manufactured.
  • the culturing equipment for microorganisms of the present invention has high operability, can be used safely, can be easily manufactured, and can easily measure the number of microorganisms in a sample.
  • the incubator 1 of the present invention has a first member 10 having a concave portion 11 capable of accommodating a medium component, and a second member 20 having a convex portion 21 that can be fitted into the concave portion 11 (see FIGS. 1 and 2 and the like). ..
  • the convex portion 21 of the second member 20 can be fitted into the concave portion 11 of the first member 10, and the top surface 22 of the convex portion 21 is said to be in the fitted state. It is preferable to have a configuration in which the bottom surface 12 of the recess 11 is in contact with the bottom surface 12 (FIGS.
  • the longitudinal direction of the incubator is the X direction
  • the lateral direction is the Y direction
  • the height direction is the Z axis direction.
  • the line I-I' is a line passing through the center of the recess along the longitudinal direction.
  • the incubator 1 of the present invention is a microorganism in a state in which the bottom surface 12 and the top surface 22 are inside by closing the concave portion 11 of the first member 10 so that the convex portion 21 of the second member 20 is fitted.
  • Incubator 1 for use can be obtained (see FIGS. 1 and 2 and the like).
  • the culture equipment 1 for microorganisms in the fitted state can be opened by using the end portions and the like of these members, and the concave portion 11 and the convex portion 21 in the fitted state can be removed at the time of opening.
  • the medium component may be applied to the bottom surface 12 and / or the top surface 22, and the sample solution may be applied to the medium component.
  • the groove 30 in at least one outer peripheral region of the concave portion 11 of the first member 10 or the convex portion 21 of the second member 20 (see FIGS. 1 and 2 and the like).
  • the outer peripheral region means a region from the outer circumference of the concave portion to the end of the first member or a region from the outer circumference of the convex portion to the end of the second member.
  • the incubator 1 of the present invention has a first member 10 and a second member 20, and these members may be integrally molded, or may be composed of a separately separated first member 10 and a second member 20. It may have been done.
  • FIG. 3A is a view when the first member 10 is viewed from the bottom surface 12 (Z-axis direction) of the recess 11.
  • the surface of the outer peripheral region on the bottom surface side is preferably a flat surface from the viewpoint of making good contact between the facing surfaces.
  • examples other than the first member of FIG. 1B are shown in FIGS. 3B, 3C, 8C, etc. Not limited.
  • the height (Z-axis direction) of the member in the outer peripheral region of the recess is not particularly limited and can be appropriately changed.
  • the height of the recess is not particularly limited and can be appropriately changed.
  • the width between the recess 11 and the groove portion 30 described later is not particularly limited and can be appropriately changed, and the height of the member of the width portion (Z-axis direction) is the member. It can be changed as appropriate according to the height of.
  • FIG. 4A is a view when the second member 20 is viewed from the opposite side (Z-axis direction) of the top surface 22 of the convex portion 21.
  • the surface of the outer peripheral region on the top surface side is preferably a flat surface from the viewpoint of making good contact between the facing surfaces.
  • examples other than the second member of FIG. 1B are shown in FIGS. 4B, 8B and the like, but the cross section of the second member is not particularly limited thereto.
  • the height (Z-axis direction) of the member in the outer peripheral region of the convex portion is not particularly limited and can be appropriately changed.
  • the height of the convex portion (Z-axis direction) is not particularly limited and can be appropriately changed, and the height of the convex portion can be appropriately changed so as to be easily fitted into the concave portion.
  • the bottom surface 12 of the concave portion 11 and the top surface 22 of the convex portion 21 may be flat or curved, respectively, but a flat surface is preferable from the viewpoint of operability. Since it is flat, the sample liquid can be easily spread evenly in the recess of the first member.
  • the surface shapes of the bottom surface and the top surface are not particularly limited, and examples thereof include a circular shape, an elliptical shape, and a polygonal shape. Of these, the circular shape is preferable from the viewpoint of operability and culture measurement.
  • the polygon includes, but is not limited to, an octagon, a hexagon, a quadrangle, a triangle, and the like.
  • the bottom surface and the top surface are both the same plane and have the same surface shape, and more preferably, the area of the bottom surface and the area of the top surface are substantially the same. Further, it is preferable that the concave portion having a circular bottom surface has a bottom area of 20 to 30 cm 2 because it is suitable for application of 1 mL sample solution.
  • the second member 20 is configured to have a concave portion 11 of the first member 10 and a convex portion 21 having a three-dimensional shape that can be fitted to each other (FIG. 2C).
  • the convex portion and the concave portion at this time may be any three-dimensional shape as long as they can be fitted, and the three-dimensional shape may be, for example, a polygonal columnar shape (for example, a hexagonal column), an elliptical columnar shape, a columnar shape, or a truncated cone shape (for example). (Conical table, etc.), etc., but are not limited to these. Of these, a columnar or truncated cone shape is preferable from the viewpoint of ease of manufacturing and ease of fitting.
  • the height (Z-axis direction) of the recessed portion is not particularly limited, but is preferably 0.5 to 5 mm, more preferably 1 to 3 mm, and the maximum length of the bottom surface is not particularly limited, but is preferable. It is 30 to 80 mm, more preferably 40 to 70 mm, still more preferably 50 to 60 mm, and the maximum length is preferably a diameter.
  • the second member 20 has a convex portion 21 having a cylindrical shape that can be completely fitted into the concave portion 11 of the first member 10 (FIG. 2C).
  • the cylindrical shape of the convex portion of the second member can be fitted into the cylindrical shape (hollow) of the concave portion of the first member slightly larger than that.
  • the recess of the first member has a bottom area of 20 to 30 cm 2 because it is suitable for application of 1 mL sample solution.
  • the incubator 1 of the present invention is preferably configured so that the top surface 22 of the convex portion 21 is in contact with the bottom surface 12 of the concave portion 11 in the fitted state. .. It is preferable that the contact state does not have a space of a certain distance or more between the top surface and the bottom surface, and more specifically, the distance between the top surface and the bottom surface is preferably 0. It is less than 0.01 mm, more preferably 0.005 mm or less, still more preferably 0.001 mm or less, and it is even more preferable that it is in a completely adherent state.
  • the culture equipment of the present invention closes the culture equipment after accommodating the culture medium components in the recesses, so that the first member and the second member and the culture medium components are in a more contact state. Due to this contact state, the space of the medium component can be newly formed independently. Then, by adopting the incubator of the present invention, the medium components are in close contact with each other between the top surface of the convex portion of the second member and the bottom surface of the concave portion of the first member (in the Z-axis direction). It is possible to provide a culture device containing a medium component.
  • the sample solution added to the medium component can be easily spread from the outside of the culture equipment to the medium component in the concave portion with a finger or the like, and can be used for culturing.
  • the medium can be prepared easily and easily. Furthermore, the contact state of the medium component with each member prevents the developed colonies from spreading significantly during use of the culture, and contributes to creating an optimum situation for measuring the number of colonies.
  • a handle for opening and closing the first member and the second member may be provided at any of the ends of the outer peripheral region of the incubator of the present invention, or the handle may be provided due to the difference in height of the members. Further, the handle portion may be provided at one end of the culture equipment in the longitudinal direction, and it is preferable that the handle portion is provided at the end opposite to the hinge portion. Further, it is preferable that the center of the recess arranged in the first member is arranged at 3/10 to 6 of the length of the first member (X-axis direction or Y-axis direction).
  • the culture equipment of the present invention it is preferable to provide a single or a plurality of groove portions 30 in at least one outer peripheral region of the concave portion 11 of the first member or the convex portion 21 of the second member. It is preferable that the groove portion is formed on the facing surface side, and it is more preferable that the groove portion is formed so as to open in the facing surface direction. Further, it is preferable that the recessed portion of the groove portion is formed so as to form a protrusion in the Z-axis direction, whereby when used in combination with the stacking convex portion described later, a plurality of culture equipments are stacked. Each member can be positioned (FIGS. 1, FIG. 2, FIG. 8, FIG.
  • the number of grooves is not particularly limited, but it is preferable that the number of grooves is one or two or more in each of the outer peripheral region of the concave portion and the outer peripheral region of the convex portion, and more preferably one. One may be provided in at least one outer peripheral region of the concave portion or the convex portion.
  • the groove portion is configured to prevent the sample liquid added to the medium component from leaking out of the recess due to the capillary phenomenon.
  • the groove is not provided as in the conventional culture member configuration, the water content of the sample liquid added to the medium component wets the surface of the first member and the surface of the second member in contact with each other, so that the space between the two is reached. Moisture present in the culture medium immediately exceeds the outside of the recess and reaches the end of the member.
  • the groove portion in the outer peripheral region of the concave portion or the convex portion, the phenomenon that the sample liquid between the surfaces of both members moves toward the end portion due to the capillary phenomenon can be suppressed in front of the groove portion. can.
  • the groove can prevent the sample liquid from leaking out of the recess (more preferably, the medium at the time of use). It is also possible to prevent the medium concentration caused by the sample liquid added to the medium component leaking out of the concave portion from becoming non-uniform, thereby forming a medium having an intended concentration.
  • the water content of the sample liquid may be suppressed in front of the groove portion, and the groove portion may function as an isolation groove portion for isolating the outflow of water to the outer peripheral region.
  • the groove portion is provided in the outer peripheral region of the concave portion of the first member so as to surround all or a part of the outer peripheral portion of the concave portion. Further, it is preferable that the groove portion is provided in the outer peripheral region of the convex portion of the second member so as to surround all or a part of the outer peripheral portion of the convex portion. Further, grooves may be appropriately provided in both the outer peripheral region of the first member and the outer peripheral region of the second member. Further, it is preferable that the groove portion completely surrounds the outer periphery of the concave portion or the convex portion by at least 50% or more, more preferably 80% or more, further preferably 90% or more, and even more preferably completely. Further, the groove portion may be formed continuously or intermittently along the outer periphery of the concave portion of the first member or the convex portion of the second member when viewed from the facing surface side (Z-axis direction). ..
  • the position where the groove portion is provided is not particularly limited as long as it is outside the outer periphery (outer peripheral end: 0 mm) of the concave portion or the convex portion (in the direction of the end portion of the member).
  • the position where the groove is provided is preferably 0.5 mm or more, more preferably 1 mm or more away from the outer circumference (0 mm) of the concave or convex portion, and the upper limit thereof is appropriately set depending on the size of the member. However, for example, it is 10 mm or less, 5 mm or less, or 3 mm or less.
  • the groove depth (Z-axis direction) of the groove portion may be sufficient as long as it can block the capillary phenomenon, and can be appropriately changed depending on the height of the concave portion or the convex portion (Z-axis direction) in terms of container design. ..
  • the groove depth of the groove portion is preferably, for example, 1 to 5 mm, and more preferably 1 to 3 mm.
  • the width of the groove of the groove portion (X-axis direction or Y-axis direction) is not particularly limited and can be set arbitrarily, for example, preferably about 0.5 to 3 mm, more preferably about 2 to 3 mm. be.
  • the cross-sectional shape (Z-axis direction) of the groove portion is not particularly limited, but a shape that opens toward the facing surface is preferable.
  • Examples of the cross-sectional shape of the groove include a V-shape, a U-shape, an upward C-shape, a semicircular shape, a quadrangular shape, a trapezoidal shape, and the like, and a U-shape or an upward C-shape is preferable.
  • the shape of the bottom surface of the groove is not particularly limited, but a curved surface is preferable from the viewpoint of ease of molding and strength.
  • 1 mL of the sample liquid is inoculated into the medium component contained in the concave portion of the first member, and the sample liquid is spread over the medium component by the convex portion of the second member fitted in the concave portion. If so, the gelling agent in the contained medium component absorbs the water content of the sample solution and solidifies. Further, when the groove is not provided in the outer peripheral region of the concave portion of the first member, the capillary phenomenon with the first member in contact with the second member occurs at an earlier stage than the gelling agent absorbs the water in the sample liquid. Water easily escapes from the recess toward the surrounding outer peripheral area.
  • the test solution spreads over the entire concave part of the first member, and the gelling agent in the medium component absorbs water, and each medium component as designed. It is possible to reconstitute the medium of concentration.
  • the groove portion in the outer peripheral region of the concave portion of the first member is separated from the outer peripheral region of the concave portion installed in the first member by 1 mm or more, but is too far from the vicinity of the outer peripheral portion of the concave portion in order to prevent the capillary phenomenon. It is preferable not to do so. Further, it is also applicable to further put a medium component between the bottom surface and the top surface of the culture equipment in a state where the concave portion of the first member and the convex portion of the second member are completely in close contact with each other and have no space. At 1 mL sample solution, the vertical volume increase of the medium component itself due to the swelling of water is almost negligible.
  • the first member 10 and the second member 20 are continuously provided by the hinge portion 50.
  • the length of the hinge portion in the lateral direction (Y-axis direction) may be formed shorter than the length of the end portion of the member.
  • the hinge portion may be arranged on the line I-I'passing through the center of the recess.
  • the cross-sectional shape of the hinge portion in the closed state of the incubator is not particularly limited, and is, for example, a polygonal shape (for example, a quadrangular shape, a pentagonal shape, a hexagonal shape, etc.), a semicircular shape, a semi-elliptical shape, an I-shaped shape, or the like. Can be mentioned.
  • this side is used as a hinge part, and when this side is bent and fitted with this side as an axis, the cross-sectional shape is I-shaped.
  • a shaped hinge portion may be formed.
  • the hinge portion 50 has a structure in which the top surface 22 of the convex portion 21 of the second member 20 can be fitted while maintaining substantially parallel to the bottom surface 12 of the concave portion 11 of the first member 10. be.
  • 5A, 5B, and 5C are culture equipment 1a, 1b, and 1c having a hinge portion 50, respectively, and represent a cross-sectional view taken along the line I-I'with the first member and the second member open. ..
  • the cross-sectional shapes of the hinge portions are C-shaped, triangular, and square, respectively.
  • the cross-sectional shape of the hinge portion is a quadrangular shape from the viewpoint that it can be fitted while maintaining substantially parallel with a simpler operation (for example, FIGS. 5C and 6).
  • the structure may be such that the first member and the second member are configured as separate members without providing the hinge portion.
  • the stacking convex portion 60 is formed in the outer peripheral region of either the first member 10 or the second member 20. It is preferable that the stacking convex portion is formed so as to form a protrusion on the outer surface (Z-axis direction) of the first member and / or the second member.
  • the arrangement of the stacking convex portions (in the XY axis direction) is not particularly limited, and may be, for example, singular or plurally arranged in the peripheral end region of the concave portion or the convex portion of the member (for example, FIG. 7A), or the end region of the member. It may be arranged singularly or plurally (for example, FIG.
  • the culture equipment arranged below can support the culture equipment arranged above, and a plurality of culture equipments can be stacked (as a result). For example, FIG. 9).
  • the number of stacking protrusions is not particularly limited, and may be singular or plural, preferably 1 or 2 or more, more preferably about 3 to 10, more preferably 3 to 6, still more preferably 3 or 4. be.
  • the shape of the stacking convex portion includes, for example, a three-dimensional shape such as a square shape, a rectangular shape, a conical shape, and a conical shape, but is not particularly limited thereto.
  • a support portion for positioning the stacking convex portion on the surface facing the surface on which the stacking convex portion is formed.
  • the support portion for example, a groove portion formed in a protrusion shape, a recess shape formed between the recess and the groove portion, and an outer peripheral region are formed on the surface of the culture equipment in the outer direction (Z-axis direction). Examples thereof include support portions that can be positioned such as recessed shapes and protrusions, but the present invention is not limited thereto.
  • the first member and / or the second member in the present invention is preferably formed of a synthetic resin material, and more preferably formed of a light-transmitting synthetic resin material.
  • the material of the first member and / or the second member is not particularly limited, and for example, synthesis of polystyrene-based, polyacrylic-based, polyvinyl-based, polyethylene-based, polyester-based, polylactic acid-based polymers and the like.
  • a resin material can be adopted, and one or more of them can be used.
  • the first member or the second member is transparent, and it is more preferable that both the first member and the second member are transparent.
  • the first member and / or the second member preferably have light transmission.
  • the colony of the microorganism to be measured can be easily observed and measured from the outside without decomposing the culture equipment.
  • transparent means that the opposite side of the member can be visually seen through, and more specifically, the visible light transmittance is preferably 70% or more, but the present invention is not limited to this. ..
  • the first member and the second member may be separately separated or integrated.
  • the first member 10 and the second member 20 are integrally molded.
  • a part of the first member and a part of the second member may be connected by sharing one side or the like.
  • a hinge portion described later may be provided between the end portion of the first member and the end portion of the second member, and these members may be continuously provided.
  • the molded incubator is used by overlapping and bending the first member and the second member so as to fit into the convex portion of the second member and the concave portion of the first member. (For example, FIGS. 1 and 2), and from the viewpoint of operability such as coating of medium components and opening / closing of members during use of culture, it is preferable that only one member is required. I can say.
  • the medium component that can be accommodated in the recess of the culture equipment of the present invention will be described.
  • the medium component used in the present invention is uniformly applied to the portion that comes into contact with each other when the second member is placed on the first member, that is, the bottom surface of the concave portion of the first member and / or the top surface of the convex portion of the second member. It is preferably coated.
  • the medium component is preferably in a dry state, and a dry medium component that can be reconstituted into a culture medium by containing the water content of the sample solution is more preferable.
  • the application site of this medium component is usually such that the sample liquid is uniformly spread in the concave portion of the first member by using the top surface of the convex portion of the second member, and the spread sample liquid is smooth. It should be uniformly applied or coated on the bottom surface of the concave portion of the first member and / or the top surface of the convex portion of the second member in order to achieve both diffusion and uniform reconstruction on the medium during use of the culture. Is preferable.
  • the medium component in the culture medium of the present invention preferably contains a gelling agent and a nutritional component.
  • the medium component is for preparing a medium for culturing microorganisms. The above preparation is usually carried out by adding the water content in the sample solution containing the microorganism to be measured to the medium component as it is as the solvent of the gel constituting the medium and allowing it to permeate.
  • the gelling agent is preferable because the non-heated gelling agent can gel the liquid by applying the sample liquid to the medium component without heating.
  • a heating gelling agent such as agar or carrageenan
  • heating is required when the sample solution is solidified together with the medium components, so a heating step is required when using the culture, or the microorganisms in the sample are killed by heating. Measurements tend to vary due to factors such as.
  • the non-heated gelling agent can form a gel only by adding water without undergoing dissolution by heating and without cooling, so that the operation of medium formation is simple and that the target microorganism can be formed. It has the advantage of not hindering growth.
  • a component that can be gelled at the time of standing is more preferable, and a thickening polysaccharide is further suitable as the gelling component at the time of standing, but the present invention is not limited thereto.
  • the thickening polysaccharide can play a role of a gelling agent constituting the medium, and when the sample solution is spread and applied uniformly to the medium components at room temperature (about 10 to 30 ° C), these are applied.
  • a medium containing a sample capable of solidifying and then maintaining a gel state in a stationary state can be formed.
  • the medium formed by the gelling agent in the medium components has almost no fluidity and can retain water firmly, so that the number of microorganisms present is quantitatively more accurate depending on the number of colonies that have grown. Can be measured.
  • the thickening polysaccharide is not particularly limited, and examples thereof include guar gum, xanthan gum, locust bean gum, gum arabic, tamarind gum, curdlan, tara gum, pullulan, and the like, and one or two selected from the group consisting of these. More than seeds are more preferred.
  • Other gelling agents may be further used in combination with the thickening polysaccharide as long as the effects of the present invention are not impaired.
  • guar gum one or more selected from guar gum, xanthan gum, and mixtures thereof are preferable.
  • the mixture is more preferable because it is easy to adjust the gelling ability at the time of using the culture by adjusting these ratios.
  • Other gelling agents may be used in combination with these as long as the effects of the present invention are not impaired.
  • guar gum and / or xanthan gum have a relatively slow rate of water absorption and solidification, it is not necessary to perform an operation for immediately mixing the medium component and the gelling agent. Therefore, even if the sample solution is added to the medium component containing guar gum and / or xanthan gum and diffused uniformly throughout the medium component, a thinly spread gel medium can be formed.
  • the medium used for culture has almost no fluidity and can firmly retain water, so that the number of microorganisms present can be quantitatively and accurately measured by the number of colonies that have grown.
  • the gel formed of guar gum, xanthan gum, or a mixture thereof is transparent, the colonies of microorganisms and their numbers in the culture medium after culturing can be accurately and easily measured from the outside without decomposing the culture equipment. Can be detected.
  • the concentration of the gelling agent (more preferably guar gum and / or xanthan gum) used in the present invention is not particularly limited, but from the viewpoint of solidification ability with respect to 1 mL of water, when 1 mL of water is added.
  • the total concentration (concentration per 1 mL) is preferably 0.01 to 0.2 g / mL, more preferably 0.01 to 0.1 g / mL.
  • the nutritional component contained in the medium component is for developing the target microorganism.
  • the nutritional component is not particularly limited, and preferably includes peptone, animal meat extract, yeast extract, fish meat extract and the like.
  • There are two types of media for measuring the number of microorganisms an agar medium containing agar and a liquid medium not containing agar.
  • a component of a liquid medium containing no agar or a component equivalent thereto is used. It is preferable to include it in the medium component.
  • the medium component used in the present invention contains an adhesive component to the first member and / or the second member.
  • the adhesive component By including the adhesive component, all of the medium components can be applied to at least one side of the bottom surface of the concave portion or the top surface of the convex portion of the incubator of the present invention.
  • the medium component plays a role of stably applying the medium component to the first member and / or the second member, and further, the medium component is brought into a state of being in closer contact with the top surface and the bottom surface. Can be done. Then, by putting the convex portion into the concave portion, the medium component is accommodated in the concave portion, and the first member and the second member are adhered to each other via the medium component containing the adhesive component. As a result, it is also possible to obtain a culture medium-containing culture medium containing a medium component in a state in which the convex portion of the second member is fitted into the concave portion of the first member. In addition, when the culture is used, an air layer is less likely to be generated between the medium and the top surface and the bottom surface, so that the operability is simpler and the overlap with the surrounding colonies during the culture can be reduced. , Can be easily measured.
  • the adhesive component is not particularly limited, and examples thereof include polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxymethyl cellulose, and methyl cellulose, from which one or more can be selected.
  • the concentration of the adhesive component at the time of use is not particularly limited, but from the viewpoint of viscosity and adhesive ability when the medium component is applied as an emulsion or suspension, the concentration at the time of use with 1 mL of water added (per 1 mL).
  • the total amount of adhesive components is preferably 0.1 to 10 mg / mL, more preferably 0.5 to 5 mg / mL.
  • the medium component used in the present invention preferably further contains a color-developing reagent.
  • a color-developing reagent This is to make the colonies of microorganisms produced by the culture colored so that they can be more easily detected and / or measured.
  • the color-developing reagent include redox indicators such as 2,3,5-triphenyltetrazolium chloride (TTC) and tetrazolium violet, and one or more of them can be selected. can.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • tetrazolium violet tetrazolium violet
  • the color-developing reagent can be preferably used when it is desired to measure all kinds of microorganisms present in a sample.
  • the concentration (concentration per 1 mL) when 1 mL of water is added is preferably 1 mg to 100 mg / L, more preferably 10 to 50 mg / L.
  • the color-developing reagent a compound which is a substrate for an enzyme possessed only by a specific microbial species (hereinafter referred to as an enzyme substrate) and which can release a chromogen compound by decomposition may be used.
  • the chromogen compound may be either a colored compound or a fluorescent compound under visible light.
  • the functional group that can be liberated as a colored compound under visible light include a 5-bromo-4-chloro-3-indoxyl group, and the liberated 5-bromo-4-chloro-3-indole is oxidatively condensed.
  • the functional group that can be liberated as the fluorescently colored compound include a 4-methylumbelliferyl group, and the liberated 4-methylumbelliferone fluoresces under ultraviolet irradiation.
  • the target microorganism is coliform bacteria, 5-bromo-4-chloro-3-indoxyl- ⁇ -D-galactopyranoside (X-GAL), etc., and if it is E. coli, , 5-bromo-4-chloro-3-indoxyl- ⁇ -D-glucuronic acid (X-GLUC), etc., and in the case of Escherichia coli, 5-bromo-4-chloro-3 indoxyl-phosphate ( X-phos), etc., 5-bromo-4-chloro-3-indoxyl- ⁇ -D-glucopyranoside, etc.
  • the concentration (concentration per 1 mL) when 1 mL of water is added is preferably 0.01 to 1.0 g / L, more preferably 0.2 to 0.5 / L.
  • the medium component used in the present invention may further optionally contain a selective substance, an antibacterial substance, an inorganic salt, a saccharide, a thickener, a pH adjuster and the like, as long as the effect of the present invention is not impaired. From these arbitrary components, one kind or two or more kinds can be appropriately selected.
  • the selective substance include antibiotics such as polymyxin B and vancomycin, and surfactants such as bile salts such as sodium lauryl sulfate (SDS), Tween80 and sodium cholic acid.
  • the antibacterial substance include polylysine, protamine sulfate, glycine, sorbic acid and the like.
  • inorganic salts include inorganic acid metal salts such as sodium chloride and sodium thiosulfate, and organic acid metal salts such as sodium pyruvate, ammonium iron citrate and sodium citrate.
  • saccharides include glucose, lactose, sucrose, xylose, cellobiose, maltose and the like.
  • pH adjuster include sodium carbonate, sodium hydrogencarbonate, tartaric acid and the like.
  • the medium composition applied to the incubator of the present invention preferably has a pH at the time of use of 6.0 to 8.0, more preferably 6.5 to 7.5, from the viewpoint of growth of the target microorganism. Is prepared to be.
  • the culture equipment of the present invention can be produced by any method, but an example thereof will be described, but the present invention is not limited thereto.
  • a synthetic resin flat plate such as an acrylic plate having an appropriate size can be used as the first member and the second member.
  • the concave portion of the first member and / or the convex portion of the second member can be produced by using a synthetic resin processing and molding method. It can be manufactured by injection molding or the like.
  • the medium component used in the present invention is prepared by dissolving or suspending it in a non-aqueous solvent, uniformly applying it to the entire surface of the concave portion of the first member and / or the convex portion of the second member, and then quickly forcibly drying or the like.
  • the non-aqueous solvent is preferably a volatile solvent that can rapidly volatilize under normal temperature and pressure, and is preferably one or more lower alcohols selected from, for example, ethanol, methanol, propanol, butanol and the like (preferably). The number of carbon atoms 1 to 4) is preferably mentioned.
  • the medium component can be applied without gelling the gelling agent at the time of production, so that the culture medium containing the medium component can be easily and efficiently produced.
  • Example of culture equipment according to the embodiment of the present invention Examples of embodiments relating to the culture equipment for microorganisms of the present invention will be described below, but the present invention is not limited thereto. Further, it may be appropriately applied to a culture medium for microorganisms containing a medium component.
  • a culture medium for microorganisms containing a medium component e.g., a culture medium for microorganisms containing a medium component.
  • ⁇ 1 in the description of the culture equipment of the first embodiment and the second embodiment of the present invention, ⁇ 1.
  • the description also applies to the present embodiment, and the description can be appropriately adopted. Further, the configurations of the first embodiment and the second embodiment may be appropriately combined.
  • the first embodiment is a culture device having a rectangular cross-sectional shape of a hinge portion, and has a first member having a concave portion capable of accommodating a medium component and a second member having a convex portion that can be fitted into the concave portion. , And it is preferable to have a configuration in which the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state.
  • the hinge portion may be connected to the first member and the second member in series, or may be arranged on a line I-I'passing through the center of the recess.
  • the groove portion is preferably all around the concave portion or the convex portion, and the number of the groove portions is preferably one. It is preferable that the cross-sectional shape (line I-I') of the groove portion has a curved surface (for example, a U-shape) on the bottom surface.
  • the shapes of the first member and the second member when viewed from the Z-axis direction are preferably triangular or rectangular, and more preferably rectangular.
  • the convex portion of the second member may be a hollow columnar shape when viewed from the outside on the opposite side of the top surface. It is preferable that the outer peripheral region of the first member and the second member is in a contact state, and it is more preferable that the surfaces of the outer peripheral region on the top surface side and the bottom surface side are flat. A single or a plurality of stacking protrusions may be appropriately formed in the outer peripheral region of either the first member or the second member. It is preferable to arrange the plurality of stacking convex portions at equal intervals, and it is more preferable to arrange them in the peripheral end region of the groove portion.
  • the first member and the second member may have a plurality of convex portions and concave portions, respectively. That is, a plurality of contact states of the concave portion and the convex portion may be formed at the time of fitting, and it is suitable for processing a plurality of samples in parallel at one time. At this time, it is more preferable to provide a part or all of the groove portion in the outer peripheral region of the concave portion of the second member.
  • the first member and the second member in the fitted state may be regarded as one fraction, and the culture equipment may have a plurality of the fractions.
  • the present invention has a first member having a plurality of recesses in which a medium component can be useful, and a second member having a plurality of protrusions that can be fitted into the recesses. It is possible to provide a culture equipment for microorganisms having a configuration in which the top surface of the recess is in contact with the bottom surface of the recess in the fitted state.
  • the second member has a plurality of the convex portions so that the concave portions of the first member can be fitted in a one-to-one correspondence.
  • the outer peripheral region of each of the plurality of concave portions is provided with one or more grooves, and / or the outer peripheral region of each of the plurality of convex portions is provided with one or more grooves.
  • Microbial culture equipment containing medium components according to the present invention As another aspect of the present invention, it is possible to provide a culture medium-containing culture medium-containing culture medium having a structure in which the convex portion is fitted while the medium component is housed in the concave portion of the culture medium.
  • the above-mentioned ⁇ 1.
  • the description of each configuration of the first member, the second member, the medium component, etc., which overlaps with the example of the culture equipment according to the embodiment of the present invention, will be omitted as appropriate, but the above ⁇ 1. > ⁇ 2. > The description also applies to the present embodiment, and the description can be appropriately adopted.
  • the culture equipment of the present invention and the culture equipment containing a medium component described above can be suitably used for a method of culturing microorganisms in a sample and measuring the number of the microorganisms.
  • the present invention has a first member having a concave portion capable of accommodating a medium component and a second member having a convex portion that can be fitted into the concave portion, and the top surface of the convex portion in the fitted state.
  • the measuring method is a step of adding a sample solution to a recess of a culture device.
  • the fitting step by covering the first member with the second member, the convex portion of the second member is fitted into the concave portion of the first member, and by fitting, the sample liquid added to the concave portion of the first member is applied. It can be spread evenly over the entire medium component.
  • the water content of the sample solution that has been uniformly spread is absorbed by the gelling agent in the medium components and rapidly gels, so that a medium that contains the sample and is solidified when used in culture is easily formed.
  • the culture conditions of the microorganism are not particularly limited, but are appropriately selected depending on the type of the target microorganism. For example, in the case of culturing a bacterium, 24 to 48 hours at 35 ⁇ 2 ° C. is preferable. Growth colonies of the target microorganisms appear in the medium after culturing, and these are measured. The number of colonies of microorganisms can be measured by visually confirming the number of colonies from the outside without disassembling the culture equipment, or by analyzing what is imaged by a camera or the like with image analysis software. According to the measuring method of the present invention, the number of colonies can be accurately measured.
  • the sample to which the measurement method of the present invention can be applied is not particularly limited, and preferably includes liquid samples such as drinking water, soft drinks, industrial water, pharmaceutical water, dialysate, and urine. Further, a solid sample made into an emulsion by a diluted solution or the like is also included. Further, a culture solution in which these samples are previously cultured in trypto-soy bouillon or the like may be used. Further, the measuring method of the present invention can be preferably applied to the measuring method of the present invention even for a sample obtained by diluting the above sample with a diluted solution such as a phosphate buffer solution.
  • a diluted solution such as a phosphate buffer solution.
  • Example 1 As the incubator of Example 1, a polystyrene sheet was produced in which the incubator shown in FIG. 6 was provided with three stacking convex portions at equal intervals at the outer peripheral end of the concave portion (see FIGS. 10 and 11). Specifically, as the polystyrene sheet culture equipment of Example 1, a transparent 0.2 mm thick polystyrene rectangular sheet is molded, and has a diameter of 56.43 mm (see D (mm) in FIG. 1) and a height. A lower member having a 2.00 mm hollow cylindrical recess (bottom area 25 cm 2 ) and an upper member having a cylindrical convex portion (height 2.00 mm) of the same volume that completely fits the recess.
  • the lower member and the upper member are designed to have a rectangular shape having substantially the same area.
  • the incubator of Example 1 is designed so that the convex portion is fitted into the concave portion and the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state, and the height of the fitted portion of the convex portion is increased.
  • the mm is designed to be the same mm as the mm of the depth of the fitting portion of the recess, and the distance between the top surface and the bottom surface is 0.001 mm or less in the fitted state.
  • the upper member and the lower member are connected to each other by a hinge portion, and the hinge portion at the end portion is configured such that the cross section of the end portion has a quadrangular cross section by the line I-I'in the fitted state.
  • the opposite side of this hinge portion can be a handle portion for opening and closing the incubator.
  • the center of the recess is placed on the position of half the length of the member in the lateral direction, and this center is placed so as to be slightly closer to the hinge portion side, and the distance between the end of the hinge portion and the end of the handle portion is 3/10 to 3/10. It is arranged in 5.
  • the incubator of Example 1 is provided with a groove portion for isolation in the lower member so as to completely surround the outer peripheral region of the recess, and the groove portion is arranged in a range of 1 mm or more and 3 mm or less from the outer circumference (0 mm) of the recess.
  • the groove depth is designed to be 1 to 3 mm
  • the groove width is designed to be 2 to 3 mm.
  • the shape of the bottom surface of the groove is a curved surface, and the cross-sectional shape is U-shaped.
  • the polystyrene sheet culture equipment of Comparative Example 1 reproduces the aspect of Patent Document 5 (Japanese Unexamined Patent Publication No. 2019-180369), and is a medium in a state where the convex portion of the upper member and the concave portion of the lower member are fitted. No groove is provided in the lower member so that a space called a region is formed.
  • Example 1 two types of the polystyrene sheet culture equipment of Example 1 and the polystyrene sheet culture equipment of Comparative Example 1 were prepared.
  • test strain used Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739, and after culturing in trypto-soy agar medium for 24 hours, it was equivalent to McFarland turbidity # 1 (about 3.0 ⁇ 10). 8 CFU / mL) was suspended in sterile physiological saline using a sterile cotton swab to prepare a bacterial stock solution. Using each bacterial stock solution, 10-fold serial dilution with sterile physiological saline was repeated up to 10-8 to prepare a bacterial diluted solution of several tens of CFU / mL.
  • FIG. 10 shows the growth colony status when the Bacillus subtilis ATCC 6633 strain is used
  • FIG. 11 shows the growth colony status when the Escherichia coli ATCC 8739 strain is used.
  • FIGS. 10 and 11 use the polystyrene sheet culture equipment of Example 1.
  • the sample solution when the sample solution is inoculated into the concave portion of the lower member and covered so that the convex portion of the upper member is completely fitted, the sample immediately spreads uniformly over the entire bottom surface of the concave portion. Moisture was rapidly absorbed and a clear medium was formed. At this time, if the concave portion and the convex portion are completely fitted, the sample volume for 1 mL is not available, so that the inoculated sample solution is expected to overflow to the outside. It was confirmed that the gel that had swollen to the extent that the convex part was slightly lifted solidified rapidly, and that no water in the sample solution overflowed, and that a uniform and transparent medium was formed.
  • the capillary phenomenon is blocked by the groove of the culture medium of the present invention, a small amount of water exuded from the recess does not fall into the groove and stops between the outer peripheral edge of the recess and the inner peripheral edge of the groove. After that, the gelling agent in the medium component was attracted into the recess by absorbing the water in between. In this way, the water content of the sample solution added to the medium components does not fall into the groove, the medium at the time of use for culture has an accurate medium concentration, and all the microbial colonies when observed after culture are formed in the recesses. rice field.
  • the culture equipment produced as a comparative example which does not have a groove for isolation that completely surrounds the concave portion, when the concave portion and the convex portion are completely fitted, an air layer is generated in the concave portion, and the upper and lower members come into contact with each other. It was found that water leaked to the periphery of the recess due to the capillary phenomenon. Moreover, it was found that this phenomenon leaks to the surroundings at an earlier stage than gelation even if the uneven parts of the upper and lower members are fitted very carefully. As a result, the applied medium was not uniformly reconstituted, and it was difficult to make a clean culture example. Further, it is not preferable that the bacterial solution flows out and leaks to the outside. In the culture equipment of the comparative example, there were many cases in which the bacteria existing on the medium gel during culturing spread on the gel surface of the air layer and overlapped with the surrounding colonies, making measurement difficult.
  • the culture equipment of the present invention when used, regardless of the space volume formed by the uneven portion when the 1 mL sample is applied and the contact angle of the equipment, and even if it is not due to the capillary phenomenon caused by an instrument such as a spreader or a non-woven fabric, etc.
  • the liquid sample could be spread evenly over the entire recess without generating an air layer in the recess.
  • the gel of the gelling agent in the medium component was rapidly solidified, and the medium at the time of use containing the sample solution could be easily constructed without overflowing the sample solution. In this way, it was possible to culture by a simple operation.
  • the incubator of the present invention does not have a complicated structure, it could be easily and efficiently produced.
  • the present inventor has a dish-shaped member having a concave portion having a circular bottom area of 20 to 30 cm 2 , and a lid member having a convex portion having the same volume as the concave portion and completely fitting with the concave portion.
  • a culture device having a groove for isolation that completely surrounds one of the convex portions is provided, and the medium component is applied to at least one side of the concave portion or the convex portion of the culture device.
  • the liquid sample does not leak to the outside evenly regardless of the space volume formed by the uneven part and the contact angle of the equipment, and without using equipment or capillary phenomenon.
  • guar gum, xanthan gum or a mixture thereof is suitable as a gelling agent for forming a medium from the viewpoint of ease of operation and high visibility from the outside.
  • the culture equipment of the present invention can be molded by a general resin molding method such as a vacuum forming method or an injection molding method.
  • the molded equipment structure itself can improve the defects existing in the conventional culture equipment and the microorganism measurement method using the same. Therefore, as another aspect, the present invention is also an invention capable of providing an extremely simple and inexpensive method for producing a culture device for microorganisms.
  • the present invention can provide a novel microbial culture equipment, a microbial culture equipment containing a medium component, and a method for measuring the number of microorganisms using the same. Further, since the present invention is not a complicated structure, it is possible to provide a culture equipment for microorganisms that can be easily produced. As described above, the culturing equipment for microorganisms of the present invention has high operability, can be used safely, can be easily manufactured, and can easily measure the number of microorganisms in a sample.
  • microorganisms in a sample can be cultured by a simple operation and the number thereof can be easily measured.
  • the incubator of the present invention is industrially useful because it does not have a complicated structure and is easy to manufacture.

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Abstract

Provided is a culturing device that has high operability and can be easily produced, and that is capable of easily counting microorganisms in a specimen.　The present invention provides a culturing device that is for microorganisms, that has a first member having a recessed portion capable of accommodating culture medium components and a second member having a protruding portion capable of fitting into the recessed portion, and that is configured such that the top surface of the protruding portion, when being fitted, comes in contact with the bottom surface of the recessed portion. The present invention provides a culturing device that is for microorganisms, that contains culture medium components, and that is configured such that the protruding portion is fitted into the recessed portion in a state where the culture medium components are contained in the recessed portion of the culturing device. The present invention provides a method for culturing microorganisms in a specimen, and counting the microorganisms, using the culturing device that is for microorganisms and that includes culture medium components.

Description

微生物用培養器材、培地成分入り微生物用培養器材、及びこれを用いた微生物数計測法Microbial culture equipment, microbial culture equipment containing medium components, and microbial count measurement method using the same
　本発明は、微生物用培養器材、培地成分入り微生物用培養器材、及びこれを用いた微生物数計測法に関する。 The present invention relates to a culture device for microorganisms, a culture device for microorganisms containing a medium component, and a method for measuring the number of microorganisms using the same.
　微生物数を計測する方法としては、混釈培養法や寒天平板塗抹法等が知られている（非特許文献１）。
　これらの方法において微生物を培養するのに用いる寒天培地は、栄養成分や選択成分を寒天と共に加熱溶解した培地を固化させたものであり、培養・計測に先立って予め調製、滅菌しておく必要がある。特に混釈培養法においては、これらの培地調製を検体の供試前に予め完了し、培地を約５０℃に保温した状態にしておく必要がある。この混釈培養法においては、比較的高温に保温された培地を検体と直接混合することから、検体中の微生物の損傷状態によっては微生物の発育に影響を与え、十分に検出できない可能性がある。一方、寒天平板塗抹法においては、熱による検体中の微生物への影響はないが、検体を平板培地に供試するに際して、培地に検体を完全に吸収させながら塗布させるため、予め検体の供試前に培地表面を乾燥させる必要があり、また塗抹操作自体が煩雑であり、時間を要するという問題もある。 As a method for measuring the number of microorganisms, a pour culture method, an agar plate smearing method, and the like are known (Non-Patent Document 1).
The agar medium used for culturing microorganisms in these methods is a solidified medium in which nutritional components and selective components are heated and dissolved together with agar, and must be prepared and sterilized in advance prior to culturing and measurement. be. In particular, in the pour culture method, it is necessary to complete these medium preparations in advance before the test of the sample and keep the medium warm at about 50 ° C. In this pour culture method, since a medium kept at a relatively high temperature is directly mixed with the sample, it may affect the growth of the microorganism depending on the damaged state of the microorganism in the sample and may not be sufficiently detected. .. On the other hand, in the agar plate smearing method, heat does not affect the microorganisms in the sample, but when the sample is tested on the plate medium, the sample is applied in advance while being completely absorbed by the medium. There is also a problem that the surface of the medium needs to be dried before, and the smearing operation itself is complicated and takes time.
　近年、微生物の検出・計測をより簡便かつ効率的に行うため、予め培地の調製が不要な乾燥培地成分入りの乾燥簡易培養器材が種々開発されている。かかる乾燥簡易培養器材では、使用時に液体検体を乾燥培地成分に添加すると、その検体中の水分により培地を形成させてそのまま培養に供することができる。
　例えば、特許文献１には、防水性基体の上面部に、接着剤層、栄養成分を含む冷水可溶性ゲル化剤粉末層、及び剥がすことができるカバーシートを備えるシート状培養装置が開示されている。特許文献２には、防水性基材の上面に水溶性ゲル化剤とメッシュを有する繊維質吸水性シートとを具備する簡易培地が開示されている。特許文献３には、防水性基材の上面に、吸水性ポリマー層、多孔質マトリックス層を順次積層した、シート状培養器材が開示されている。特許文献４には、基材シート上に検体が広がる枠を設け、その枠内に接着成分、ゲル化剤を含む培地液をパターン形成した、シート状培養器材が開示されている。かかる枠は、接触角を特定の値に設定した疎水性樹脂からなり、試料液は枠内にだけ広がる。特許文献５には、上部材と凹部を有する下部材とが嵌合した状態において、上部材の凸部の上面と下部材の凹部の底面及び側面とで囲まれる空間を培地領域として有する微生物の培養器材が開示されている。 In recent years, in order to detect and measure microorganisms more easily and efficiently, various dry simple culture equipment containing a dry medium component that does not require preparation of a medium in advance has been developed. In such a dry simple culture equipment, when a liquid sample is added to a dry medium component at the time of use, a medium can be formed by the water content in the sample and used as it is for culture.
For example, Patent Document 1 discloses a sheet-like culture apparatus provided with an adhesive layer, a cold water-soluble gelling agent powder layer containing a nutritional component, and a cover sheet that can be peeled off on the upper surface of a waterproof substrate. .. Patent Document 2 discloses a simple medium provided with a water-soluble gelling agent and a fibrous water-absorbent sheet having a mesh on the upper surface of a waterproof base material. Patent Document 3 discloses a sheet-like culture device in which a water-absorbent polymer layer and a porous matrix layer are sequentially laminated on the upper surface of a waterproof base material. Patent Document 4 discloses a sheet-like culture device in which a frame on which a sample spreads is provided on a base sheet, and a medium solution containing an adhesive component and a gelling agent is formed in the frame. The frame is made of a hydrophobic resin whose contact angle is set to a specific value, and the sample liquid spreads only within the frame. Patent Document 5 describes a microorganism having a space surrounded by the upper surface of the convex portion of the upper member and the bottom surface and the side surface of the concave portion of the lower member as a medium region in a state where the upper member and the lower member having the concave portion are fitted. Culture equipment is disclosed.
特公平２－４９７０５号公報Special Fair 2-49705 Gazette 特開２０００－３２５０７２号公報Japanese Unexamined Patent Publication No. 2000-325072 ＷＯ９７／２４４３２パンフレットWO97 / 24432 Pamphlet 特開２０１５－２０４８４５号公報JP-A-2015-204845 特開２０１９－１８０３６９号公報Japanese Unexamined Patent Publication No. 2019-180369
　特許文献１記載の培養器材では、防水性基体と上面フィルムの間に検体を供試し、上面フィルムの上からスプレッダーという器具で押さえつけて試料液を所定の面積に広げる操作を行うものである。この操作は平らな面を必要とし、かつ慎重に行わないと、試料が均一に広がるどころか、周囲へ流出する恐れがあり、操作性に難があるだけでなく、スプレッダーにより形成される培地面積が常に同一ではないため、形成された培地の濃度が変動し、培養条件が安定的でない難点がある。特許文献１記載の培養器材では、検体中の微生物を、簡便な操作で培養することができず、その数を容易に計測することができない。 In the culture equipment described in Patent Document 1, a sample is tested between the waterproof substrate and the top film, and the sample liquid is spread over a predetermined area by pressing the sample from the top of the top film with a device called a spreader. This operation requires a flat surface, and if not done carefully, the sample may not spread evenly but may flow out to the surroundings, which is not only difficult to operate, but also the area of the medium formed by the spreader. Since they are not always the same, there is a problem that the concentration of the formed medium fluctuates and the culture conditions are not stable. In the culture equipment described in Patent Document 1, the microorganisms in the sample cannot be cultured by a simple operation, and the number thereof cannot be easily measured.
　特許文献２及び３記載の培養器材には、不織布をはじめとする多孔質マトリックスが用いられているため、多孔質マトリックスを用いる都合上、その製造の際に、溶媒の乾燥状態を制御する困難さがあったり（特許文献２）、多孔質マトリックス層と培地層とを積層する手順が繁雑であったり（特許文献３）、製造する場合に難点が存在する。また、多孔質マトリックスの表面の凹凸により培地表面に生じる乱反射や、多孔質マトリックス自体の不透明さによって、培養後のコロニーが見にくくなり、特許文献２及び３記載の培養器材では、検体中の微生物の数を容易に計測することが難しい。
　特許文献４記載の培養器材は多孔質マトリックスを用いていないが、液体検体が広がる際の土手となる枠から検体がこぼれないように、操作の際に平らな場所を要したり慎重性が求められたりする。また、該枠は、特定の接触角に設定した材料で形成されるが、検体の種類によっては、例えば油分やタンパク質等を含む飲食品等を検体とする場合には、枠の撥水性が変わることが想定され、培養器材としての汎用性に問題があるだけでなく、接触角を考慮した部材が必要であるため、複数の部材から構成させる必要があり製造が複雑であるともいえる。 Since a porous matrix such as a non-woven fabric is used in the culture equipment described in Patent Documents 2 and 3, it is difficult to control the dry state of the solvent during its production due to the convenience of using the porous matrix. (Patent Document 2), the procedure for laminating the porous matrix layer and the culture medium layer is complicated (Patent Document 3), and there are some difficulties in manufacturing. Further, the diffused reflection generated on the surface of the medium due to the unevenness of the surface of the porous matrix and the opacity of the porous matrix itself make it difficult to see the colonies after culturing. It is difficult to measure the number easily.
The culture equipment described in Patent Document 4 does not use a porous matrix, but it requires a flat place and caution during operation so that the sample does not spill from the frame that serves as the bank when the liquid sample spreads. It is done. Further, the frame is formed of a material set to a specific contact angle, but depending on the type of the sample, for example, when a food or drink containing oil, protein, etc. is used as the sample, the water repellency of the frame changes. It is assumed that there is a problem in versatility as a culture equipment, and since a member considering the contact angle is required, it is necessary to configure the member from a plurality of members, which can be said to be complicated to manufacture.
　これらの特許文献１～４に記載の先行発明を鑑みて特許文献５には、凸部を有する上部材と凹部を有する下部材を勘合させ、上部材の凸部の上面と下部材の凹部の底面及びこれらの側面とで囲まれる空間の体積を、培地領域の空間体積分まで含ませた設計にすることで、この空間内で水分を含む検体と培地成分とを押圧しながら接触させて培地を形成できる、微生物用の培養器材が開示されている。 In view of the prior inventions described in Patent Documents 1 to 4, in Patent Document 5, the upper member having a convex portion and the lower member having a concave portion are fitted to each other, and the upper surface of the convex portion of the upper member and the concave portion of the lower member are combined. By designing the volume of the space surrounded by the bottom surface and these side surfaces to include the spatial volume integral of the medium region, the sample containing water and the medium component are brought into contact with each other while being pressed in this space, and the medium is contacted. Disclosed is a culture medium for microorganisms capable of forming a culture medium.
　特許文献５の培養器材は、上部材の凸部と下部材の凹部が嵌合した状態において、上部材の凸部の上面と下部材の凹部の底面及び側面とで囲まれた培地領域という空間を、必須の構成として設けている。ところが、特許文献５の培養器材を本発明者が検討すると、この培地領域の空間に適切な量の培地成分を入れない場合には、上面及び底面の少なくともいずれか一方の面と培地成分との間に空気層が発生しやすくなることに、本発明者は気がついた。当該空気層が発生した場合、培養使用時に培地ゲル上に存在する菌がゲル表面上で広がり、周囲のコロニーと重なり、計測が困難になることを本発明者は見出した。特許文献５の培養器材を用いて、空気層が発生しないように操作することは簡便な操作とはいえず、また、空気層が発生することで検体中の微生物の数を容易に計測することが難しい。 The incubator of Patent Document 5 is a space called a culture medium region surrounded by the upper surface of the convex portion of the upper member and the bottom surface and the side surface of the concave portion of the lower member in a state where the convex portion of the upper member and the concave portion of the lower member are fitted. Is provided as an indispensable configuration. However, when the present inventor examines the culture equipment of Patent Document 5, when an appropriate amount of the medium component is not put in the space of this medium region, at least one of the upper surface and the bottom surface and the medium component are used. The present inventor has noticed that an air layer is likely to be generated between them. The present inventor has found that when the air layer is generated, the bacteria existing on the culture medium gel spread on the gel surface and overlap with the surrounding colonies, making measurement difficult. Using the culture equipment of Patent Document 5 to operate so as not to generate an air layer is not a simple operation, and it is easy to measure the number of microorganisms in a sample by generating an air layer. Is difficult.
　さらに、かかる特許文献５の培養器材を本発明者が再現すると検体液を培地成分に適用し上部材を閉めて検体液を拡散させたと同時に、上部材と下部材が水分を介して接触することによる毛細管現象が生じた。この毛細管現象により、培地成分中のゲル化剤が検体液の水分を捕捉し固化させるよりも早い段階で、適用した検体液が上部材と下部材により構成される円筒部分の培地領域から外部に漏れ出してしまうことを本発明者は見出した。さらに、検体液が培地領域の外部に漏れ出すことで、意図した濃度の培地を形成させることができない問題があることを本発明者は見出した。加えて微生物が存在するかもしれない被検液が培地領域の外部に自発的に漏れ出すことで、検体中の微生物を、簡便な操作で培養することができず、その数を容易に計測することができない。微生物検査を容易に実施するという点で問題があることも本発明者は見出した。 Further, when the present inventor reproduces the culture equipment of Patent Document 5, the sample liquid is applied to the medium component, the upper member is closed to diffuse the sample liquid, and at the same time, the upper member and the lower member come into contact with each other via water. Capillary action occurred. Due to this capillarity, the applied sample liquid moves from the medium region of the cylindrical part composed of the upper member and the lower member to the outside at an earlier stage than the gelling agent in the medium component captures and solidifies the water content of the sample liquid. The present inventor has found that it leaks. Furthermore, the present inventor has found that there is a problem that a medium having an intended concentration cannot be formed due to leakage of the sample solution to the outside of the medium area. In addition, the test solution, which may contain microorganisms, spontaneously leaks to the outside of the culture medium region, which makes it impossible to cultivate the microorganisms in the sample by a simple operation, and the number of microorganisms can be easily measured. Can't. The present inventor has also found that there is a problem in that the microbiological test is easily carried out.
　さらに、本発明者は、特許文献５の培養器材の再現において、上部材の凸部の上面積を下部材の凹部の底面積より小さくすることで、培地領域とされる部分の周囲に検体液の水分の逃げ道を設けてみた。しかしながら、このような培養器材でも、培地成分中のゲル化剤が検体液の水分を捕捉し固化するのに時間を要するため、上部材と下部材が水分を介して接触することによる毛細管現象を防ぐことはできず、水分が外部へ漏れ出すことを本発明者は見出した。さらには特許文献５の培養器材にある培地領域の外周部分に水分が溜まるため、培地成分の濃度が培養使用時ごとに不均一になることを本発明者は気づいた。
　このように、特許文献５の培養器材では、検体中の微生物を、簡便な操作で培養することができず、その数を容易に計測することができないという弊害が生じることを本発明者は見出した。 Further, in the reproduction of the culture equipment of Patent Document 5, the present inventor makes the upper area of the convex portion of the upper member smaller than the bottom area of the concave portion of the lower member, so that the sample liquid is around the portion to be the medium region. I tried to set up an escape route for the water. However, even in such a culture device, it takes time for the gelling agent in the medium component to capture and solidify the water content of the sample solution, so that the capillary phenomenon caused by the contact between the upper member and the lower member via the water is caused. The present inventor has found that it cannot be prevented and that water leaks to the outside. Furthermore, the present inventor has noticed that the concentration of the medium component becomes non-uniform every time the culture is used because water accumulates in the outer peripheral portion of the medium region in the culture equipment of Patent Document 5.
As described above, the present inventor has found that the culture equipment of Patent Document 5 has an adverse effect that the microorganisms in the sample cannot be cultured by a simple operation and the number thereof cannot be easily measured. rice field.
　このような状況を鑑みて、本発明は、検体中の微生物を簡便な操作で培養することができ、検体中の微生物数を容易に計測することができる微生物の培養器材を提供することを目的とする。 In view of such a situation, it is an object of the present invention to provide a microorganism culture device capable of culturing microorganisms in a sample by a simple operation and easily measuring the number of microorganisms in the sample. And.
　本発明者は、上記課題を解決するために鋭意研究の末、微生物用培養器材において、凸部が凹部に嵌入がなされた状態で凸部の天面と凹部の底部とが接触状態となる構成を備えることで、検体中の微生物を簡便な操作で培養することができ、微生物数を容易に計測することができることを見出した。さらに、本発明者は、凹部及び／又は凸部の外周領域に溝部を備えることで、凹部及び凸部により形成される空間体積や器材の嵌入時の接触角に関係なく、また器具や毛細管現象等を気にしなくとも、検体液が凹部（より具体的には使用時の培地）の外に漏れ出すことを抑制でき、培地中に均一に拡散し一定面積及び一定濃度の培地形成が可能となり、より安定的で簡便な微生物の培養及び計測を実現できることをさらに見出した。 In order to solve the above-mentioned problems, the present inventor has made a configuration in which the top surface of the convex portion and the bottom of the concave portion are in contact with each other in a microbial culture equipment in a state where the convex portion is fitted into the concave portion. It was found that the microorganisms in the sample can be cultivated by a simple operation and the number of microorganisms can be easily measured. Further, the present inventor provides a groove in the outer peripheral region of the concave and / or convex portions, regardless of the space volume formed by the concave portions and the convex portions and the contact angle at the time of fitting the equipment, and also the instrument and the capillary phenomenon. It is possible to prevent the sample solution from leaking out of the recess (more specifically, the medium at the time of use) without worrying about such factors, and it is possible to uniformly diffuse into the medium and form a medium having a certain area and a certain concentration. , Further found that more stable and convenient culture and measurement of microorganisms can be realized.
　すなわち、本発明は以下の通りである。
［１]
　培地成分を収容可能な凹部を有する第１部材と、
　前記凹部に嵌入し得る凸部を有する第２部材と、を有し、
　前記嵌入がなされた状態で前記凸部の天面が前記凹部の底面に接触状態となる構成を備える、微生物用培養器材。
［２]
　前記凹部又は前記凸部の少なくとも一方の外周領域に溝部を備える、前記［１］記載の微生物用培養器材。
［３]
　前記溝部は、前記培地に添加された検体液が毛細管現象により前記凹部の外へ漏れ出することを抑制する、前記［２］記載の微生物用培養器材。
［４]
　前記第１部材及び／又は前記第２部材は光透過性の合成樹脂材で形成される、前記［１］から[３]のいずれか一つに記載の微生物用培養器材。
［５]
　前記第１部材と前記第２部材は一体成形されている、前記［１］から[４]のいずれか一つに記載の微生物用培養器材。
［６]
　前記第１部材と前記第２部材とはヒンジ部により連設されている、前記［１］から[５]のいずれか一つに記載の微生物用培養器材。
［７]
　前記ヒンジ部は、前記凸部の前記天面を前記底面に対して平行状態を維持しつつ嵌入し得る構造を備える、前記［６］に記載の微生物用培養器材。
［８]
　前記第１部材又は前記第２部材のいずれかの外周領域に、スタッキング用凸部が形成される、前記［１］から[７]のいずれか一つに記載の微生物用培養器材。
［９]
　前記［１］から[８]のいずれか一つに記載の培養器材の前記凹部に培地成分が収容された状態で前記凸部が嵌入された構成を備える、培地成分入り微生物用培養器材。
［１０]
　前記培地成分は、ゲル化剤と栄養成分とを少なくとも含む、前記［９］に記載の培地成分入り微生物用培養器材。
［１１]
　前記ゲル化剤は、グアーガム、キサンタンガム、及びそれらの混合物から選択される１種以上である、前記［１０］に記載の培養器材。
［１２]
　前記培地成分には、前記第１部材及び前記第２部材に対する接着性成分が含まれている、前記［９］から[１１]のいずれか一つに記載の培養器材。
［１３]
　前記［９］から[１２]のいずれか一つに記載の培地成分入り微生物用培養器材を用いて、検体中の微生物を培養し、微生物数を計測する方法。
［１４]
　前記培養器材の凹部に検体を添加する工程、
　添加後に、前記第１部材の凹部に前記第２部材の凸部を嵌入する工程、
　嵌入後に、前記検体に含まれる微生物を培養する工程、及び
　培養後に、前記微生物のコロニー数を計測する工程、
を含む、前記［１３］に記載の方法。 That is, the present invention is as follows.
[1]
A first member having a recess capable of accommodating a medium component,
It has a second member having a convex portion that can be fitted into the concave portion, and has.
An incubator for microorganisms having a structure in which the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state.
[2]
The microorganism culture equipment according to the above [1], wherein the concave portion or the groove portion is provided in at least one outer peripheral region of the convex portion.
[3]
The microbial culture device according to the above [2], wherein the groove portion suppresses the sample liquid added to the medium from leaking out of the recess due to a capillary phenomenon.
[4]
The microorganism culture equipment according to any one of [1] to [3], wherein the first member and / or the second member is made of a light-transmitting synthetic resin material.
[5]
The microorganism culture equipment according to any one of [1] to [4], wherein the first member and the second member are integrally molded.
[6]
The microorganism culture equipment according to any one of [1] to [5], wherein the first member and the second member are connected to each other by a hinge portion.
[7]
The microorganism culture equipment according to the above [6], wherein the hinge portion has a structure in which the top surface of the convex portion can be fitted while maintaining a parallel state with respect to the bottom surface.
[8]
The microorganism culture equipment according to any one of [1] to [7], wherein a stacking convex portion is formed in the outer peripheral region of either the first member or the second member.
[9]
A culture medium-containing culture medium-containing culture medium having a structure in which the convex portion is fitted in a state in which the medium component is contained in the concave portion of the culture medium according to any one of [1] to [8].
[10]
The culture medium component containing a medium component according to the above [9], wherein the medium component contains at least a gelling agent and a nutritional component.
[11]
The incubator according to the above [10], wherein the gelling agent is one or more selected from guar gum, xanthan gum, and a mixture thereof.
[12]
The culture medium according to any one of [9] to [11], wherein the medium component contains an adhesive component to the first member and the second member.
[13]
A method for culturing microorganisms in a sample and measuring the number of microorganisms by using the culture medium-containing culture equipment containing a medium component according to any one of [9] to [12].
[14]
The step of adding a sample to the recess of the culture equipment,
A step of fitting the convex portion of the second member into the concave portion of the first member after the addition.
A step of culturing the microorganism contained in the sample after embedding, and a step of measuring the number of colonies of the microorganism after culturing.
The method according to the above [13].
　なお、本明細書において「検体」とは、特に限定されないが、通常は、検体液であり、具体的には飲料水、清涼飲料水、工業用水、製薬用水、透析水、尿等の水性の液体検体等である。また、固形の検体を希釈液等により乳剤又は懸濁液としたものも含まれる。
　また、本明細書において「微生物」とは、通常は、細菌類、真菌類を指し、一般細菌、枯草菌、大腸菌、大腸菌群、ブドウ球菌、ビブリオ属細菌、腸球菌、酵母、カビ等をいう。 Although the term "specimen" is not particularly limited in the present specification, it is usually a sample liquid, and specifically, it is an aqueous solution such as drinking water, soft drink, industrial water, pharmaceutical water, dialysate, and urine. It is a liquid sample or the like. Further, a solid sample made into an emulsion or a suspension by a diluted solution or the like is also included.
In addition, the term "microorganism" as used herein usually refers to bacteria and fungi, and generally refers to general bacteria, bacilli, coliforms, coliforms, staphylococci, Vibrio bacteria, enterococci, yeasts, molds and the like. ..
　本発明によれば、検体中の微生物を、簡便な操作で培養することができ、検体中の微生物数を容易に計測することができる。なお、ここに記載された効果は、必ずしも限定されるものではなく、本明細書中に記載されたいずれかの効果であってもよい。 According to the present invention, the microorganisms in the sample can be cultured by a simple operation, and the number of microorganisms in the sample can be easily measured. The effects described here are not necessarily limited, and may be any of the effects described in the present specification.
本発明の培養器材の一態様を表す図である。図１Ａは第１部材の凹部と第２部材の凸部が嵌入していない状態の培養器材の正投影図であり、底面及び天面がみえる側の図である。図１Ｂは、図１Ａの線Ｉ－Ｉ’での断面図の一例を表す。図１Ｃは、第２部材の凸部を第１部材の凹部に嵌入させようとしている状態を表す断面図である。It is a figure which shows one aspect of the culture equipment of this invention. FIG. 1A is an orthogonal projection view of the culture equipment in a state where the concave portion of the first member and the convex portion of the second member are not fitted, and is a view on the side where the bottom surface and the top surface can be seen. FIG. 1B represents an example of a cross-sectional view taken along the line I-I'of FIG. 1A. FIG. 1C is a cross-sectional view showing a state in which the convex portion of the second member is to be fitted into the concave portion of the first member. 本発明の培養器材の一態様を表す図である。図２Ａは、第１部材の凹部と第２部材の凸部とが嵌入した状態の培養器材の正投影図であり、第２部材の外側からみたときの図である。図２Ｂは、図２Ａの線Ｉ－Ｉ’での断面図の一例を示す。図２Ｃは、培養器材の凹部に培地成分が収容され、凸部が嵌入された状態での断面図の一例を示す。It is a figure which shows one aspect of the culture equipment of this invention. FIG. 2A is an orthogonal projection view of the incubator in a state where the concave portion of the first member and the convex portion of the second member are fitted, and is a view seen from the outside of the second member. FIG. 2B shows an example of a cross-sectional view taken along the line I-I'of FIG. 2A. FIG. 2C shows an example of a cross-sectional view in which the culture medium component is contained in the concave portion of the incubator and the convex portion is fitted. 本発明の培養器材の第１部材の一態様を表す図である。図３Ａは、第１部材の底面側からみたときの図であり、図３Ｂ及び図３Ｃは、それぞれ、図３Ａの線Ｉ－Ｉ’での断面図の一例を表す。It is a figure which shows one aspect of the 1st member of the culture equipment of this invention. 3A is a view seen from the bottom surface side of the first member, and FIGS. 3B and 3C each represent an example of a cross-sectional view taken along the line I-I'of FIG. 3A. 本発明の培養器材の第２部材の一態様を表す図である。図４Ａは、第２部材の天面の反対側からみたときの上面図であり、図４Ｂは、図４Ａの線Ｉ－Ｉ’での断面図の一例を表す。It is a figure which shows one aspect of the 2nd member of the culture equipment of this invention. FIG. 4A is a top view when viewed from the opposite side of the top surface of the second member, and FIG. 4B shows an example of a cross-sectional view taken along the line I-I'of FIG. 4A. 本発明の培養器材の一態様を表す図である。図５Ａ～図５Ｃは、第１部材と第２部材とが種々のヒンジ部により連結され、第１部材の凹部と第２部材の凸部とが嵌入していない状態の断面図の一例をそれぞれ表す。図５Ａ～Ｃのヒンジ部の断面形状は、それぞれ、嵌入時に、Ｃ形状、三角形状、四角形状である。図５Ｄは、第１部材と第２部材とが、一体とならず、別々に分離している状態の一例を示す。It is a figure which shows one aspect of the culture equipment of this invention. 5A to 5C are examples of cross-sectional views in which the first member and the second member are connected by various hinge portions, and the concave portion of the first member and the convex portion of the second member are not fitted. show. The cross-sectional shapes of the hinge portions of FIGS. 5A to 5C are C-shaped, triangular-shaped, and square-shaped at the time of fitting, respectively. FIG. 5D shows an example of a state in which the first member and the second member are not integrated but separated separately. 本発明の培養器材の一態様を表す図である。図６は、嵌入時に断面形状が四角形状になるヒンジ部を有する培養器材であり、第１部材の凹部と第２部材の凸部とが嵌入した状態の培養器材の一例を表す図であり、当該培養器材の正面、背面、左図（断面図）、右図（断面図）、平面図、底面図をそれぞれ表す。It is a figure which shows one aspect of the culture equipment of this invention. FIG. 6 is a diagram showing an example of a culture device having a hinge portion whose cross-sectional shape becomes quadrangular at the time of fitting, and in a state where the concave portion of the first member and the convex portion of the second member are fitted. The front view, the back view, the left view (cross-sectional view), the right view (cross-sectional view), the plan view, and the bottom view of the culture equipment are shown. 本発明の培養器材の一態様を表す図である。図７Ａ及び図７Ｂは、それぞれ、嵌入時の培養器材を第２部材の天面の反対側からみたときの図である。図７Ａ及び図７Ｂには、溝部の外周領域にスタッキング用凸部が複数形成される態様の一例を表す。It is a figure which shows one aspect of the culture equipment of this invention. 7A and 7B are views when the incubator at the time of fitting is viewed from the opposite side of the top surface of the second member, respectively. 7A and 7B show an example of a mode in which a plurality of stacking convex portions are formed in the outer peripheral region of the groove portion. 本発明の培養器材の一態様を表す図である。図８Ａ及び図８Ｂは、円状の第２部材を天面の反対側からみたときの図、及びこの側面側の断面の一例をそれぞれ表す図である。図８Ｃは、スタッキング用凸部が外周領域に均等間隔で第１部材に形成されたときの側面側の断面を表す図である。図８Ｄは、嵌入時の培養器材の一例の側面（断面図）を表す図である。図８Ｅは嵌入時の培養器材の一例を第２部材の天面の反対側からみたときの図である。It is a figure which shows one aspect of the culture equipment of this invention. 8A and 8B are views when the circular second member is viewed from the opposite side of the top surface, and are views showing an example of a cross section on the side surface side thereof, respectively. FIG. 8C is a diagram showing a cross section on the side surface side when the stacking convex portions are formed on the first member at equal intervals in the outer peripheral region. FIG. 8D is a diagram showing a side surface (cross-sectional view) of an example of the culture equipment at the time of fitting. FIG. 8E is a view showing an example of the culture equipment at the time of fitting when viewed from the opposite side of the top surface of the second member. 本発明の培地成分入り培養器材の一態様を表す図である。図９は、嵌入時の培地成分入り培養器材を、複数積み重ねたときの断面図を表す。外側に凸状になっている溝部と外側にあるスタッキング用凸部とで上下の培養器材が固定され、複数の培地成分入り培養器材の積み重ね崩れが防止されていることを表す。It is a figure which shows one aspect of the culture equipment containing a culture medium component of this invention. FIG. 9 shows a cross-sectional view when a plurality of culture equipment containing a medium component at the time of insertion are stacked. The upper and lower culture equipment is fixed by the groove portion that is convex on the outside and the convex portion for stacking on the outside, which indicates that the stacking collapse of the culture equipment containing a plurality of medium components is prevented. 実施例１の培地成分入り培養器材を用いて、検出されたコロニーの写真を表す図である。It is a figure showing the photograph of the detected colony using the culture equipment containing the culture medium component of Example 1. FIG. 実施例１の培地成分入り培養器材を用いて、検出されたコロニーの写真を表す図である。It is a figure showing the photograph of the detected colony using the culture equipment containing the culture medium component of Example 1. FIG.
　以下、本発明を実施するための好適な実施形態について説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本技術の範囲が狭く解釈されることはない。なお、各数値範囲の上限値と下限値は、所望により、任意に組み合わせることができる。 Hereinafter, suitable embodiments for carrying out the present invention will be described. It should be noted that the embodiments described below show an example of a typical embodiment of the present invention, and the scope of the present technology is not narrowly interpreted by this. The upper limit value and the lower limit value of each numerical value range can be arbitrarily combined as desired.
＜１．本発明に係る微生物用培養器材＞
　本発明は、培地成分を収容可能な凹部を有する第１部材と、前記凹部に嵌入し得る凸部を有する第２部材と、を有し、前記嵌入がなされた状態で前記凸部の天面が前記凹部の底面に接触状態となる構成を備える、微生物用培養器材又は培地成分入り微生物用培養器材を提供することができる。
　本発明の培養器材を用いることで、検体中の微生物を、簡便な操作で培養し、その数を容易に計測することができる。また、本発明の培養器材は、複雑な構成を採用しなくともよいため、製造も簡単にできる。
　また、本発明の微生物用培養器材は、操作性が高く、安全に使用でき、かつ簡単に製造することができ、さらに、検体中の微生物数を容易に計測することができる。 <1. Microbial culture equipment according to the present invention>
The present invention has a first member having a concave portion capable of accommodating a medium component and a second member having a convex portion that can be fitted into the concave portion, and the top surface of the convex portion in the fitted state. It is possible to provide a culturing equipment for microorganisms or a culturing equipment for microorganisms containing a culture medium component, which is provided with a structure in which the surface is in contact with the bottom surface of the recess.
By using the culture equipment of the present invention, microorganisms in a sample can be cultured by a simple operation and the number thereof can be easily measured. Further, since the incubator of the present invention does not have to adopt a complicated structure, it can be easily manufactured.
In addition, the culturing equipment for microorganisms of the present invention has high operability, can be used safely, can be easily manufactured, and can easily measure the number of microorganisms in a sample.
　以下、本発明の培養器材を、図面を参照してより詳細に説明するが、本発明はこれに限定されない。
　本発明の培養器材１は、培地成分を収容可能な凹部１１を有する第１部材１０、前記凹部１１に嵌入し得る凸部２１を有する第２部材２０を有する（図１及び図２等参照）。
　本発明の培養器材１は、第１部材１０の凹部１１に、第２部材２０の凸部２１を嵌入することができ、当該嵌入がなされた状態で、当該凸部２１の天面２２が当該凹部１１の底面１２に接触状態となるような構成を備えることが好適である（図１Ａ～Ｃ、図２Ａ～Ｂ）。
　なお、本明細書において、培養器材の長手方向をＸ方向、短手方向をＹ方向、高さ方向をＺ軸方向とする。また、線Ｉ－Ｉ’は、長手方向に沿って凹部の中心を通過する線である。 Hereinafter, the incubator of the present invention will be described in more detail with reference to the drawings, but the present invention is not limited thereto.
The incubator 1 of the present invention has a first member 10 having a concave portion 11 capable of accommodating a medium component, and a second member 20 having a convex portion 21 that can be fitted into the concave portion 11 (see FIGS. 1 and 2 and the like). ..
In the incubator 1 of the present invention, the convex portion 21 of the second member 20 can be fitted into the concave portion 11 of the first member 10, and the top surface 22 of the convex portion 21 is said to be in the fitted state. It is preferable to have a configuration in which the bottom surface 12 of the recess 11 is in contact with the bottom surface 12 (FIGS. 1A to 1C and FIGS. 2A to 2B).
In the present specification, the longitudinal direction of the incubator is the X direction, the lateral direction is the Y direction, and the height direction is the Z axis direction. Further, the line I-I'is a line passing through the center of the recess along the longitudinal direction.
　本発明の培養器材１は、第１部材１０の凹部１１に第２部材２０の凸部２１が嵌入するように閉じることで、底面１２と天面２２とを内側にして、嵌入した状態の微生物用の培養器材１を得ることができる（図１及び図２等参照）。この嵌入した状態の微生物用の培養器材１は、これら部材の端部等を用いて開くことができ、開くときに嵌入した状態の凹部１１と凸部２１とを外すことができる。培養器材の開閉時に、培地成分を底面１２及び／又は天面２２に塗着してもよく、検体液を培地成分に適用してもよい。 The incubator 1 of the present invention is a microorganism in a state in which the bottom surface 12 and the top surface 22 are inside by closing the concave portion 11 of the first member 10 so that the convex portion 21 of the second member 20 is fitted. Incubator 1 for use can be obtained (see FIGS. 1 and 2 and the like). The culture equipment 1 for microorganisms in the fitted state can be opened by using the end portions and the like of these members, and the concave portion 11 and the convex portion 21 in the fitted state can be removed at the time of opening. When opening and closing the culture equipment, the medium component may be applied to the bottom surface 12 and / or the top surface 22, and the sample solution may be applied to the medium component.
　さらに、第１部材１０の凹部１１又は第２部材２０の凸部２１の少なくとも一方の外周領域に溝部３０を備えることが好適である（図１及び図２等参照）。当該外周領域とは、凹部の外周から第１部材の端までの領域又は凸部の外周から第２部材の端までの領域をいう。 Further, it is preferable to provide the groove 30 in at least one outer peripheral region of the concave portion 11 of the first member 10 or the convex portion 21 of the second member 20 (see FIGS. 1 and 2 and the like). The outer peripheral region means a region from the outer circumference of the concave portion to the end of the first member or a region from the outer circumference of the convex portion to the end of the second member.
　本発明の培養器材１は、第１部材１０及び第２部材２０を有しており、これら部材が一体成形されていてもよいし、別々に分離した第１部材１０及び第２部材２０から構成されていてもよい。 The incubator 1 of the present invention has a first member 10 and a second member 20, and these members may be integrally molded, or may be composed of a separately separated first member 10 and a second member 20. It may have been done.
＜第１部材及び第２部材＞
　第１部材１０は、培地成分を収容可能な凹部１１を有することが好適であり、当該培地成分については後述する。
　図３Ａは、第１部材１０を、凹部１１の底面１２（Ｚ軸方向）からみたときの図である。底面側にある外周領域の面は、対向面同士を良好に接触させる等の観点から、平面であることが好適である。
　さらに、第１部材１０の線Ｉ－Ｉ’での断面について、図１Ｂの第１部材以外の例を、図３Ｂ、図３Ｃ、図８Ｃ等に示すが、第１部材の断面はこれらに特に限定されない。例えば、凹部の外周領域の部材の高さ（Ｚ軸方向）は、特に限定されず、適宜変更が可能である。また、凹部の高さ（Ｚ軸方向）は、特に限定されず、適宜変更可能である。また、凹部１１と後述する溝部３０との間の幅（例えばＸ軸方向）は、特に限定されず、適宜変更が可能であり、当該幅部分の部材の高さ（Ｚ軸方向）は、部材の高さに応じて、適宜変更が可能である。 <First member and second member>
It is preferable that the first member 10 has a recess 11 capable of accommodating the medium component, and the medium component will be described later.
FIG. 3A is a view when the first member 10 is viewed from the bottom surface 12 (Z-axis direction) of the recess 11. The surface of the outer peripheral region on the bottom surface side is preferably a flat surface from the viewpoint of making good contact between the facing surfaces.
Further, regarding the cross section of the first member 10 along the line I-I', examples other than the first member of FIG. 1B are shown in FIGS. 3B, 3C, 8C, etc. Not limited. For example, the height (Z-axis direction) of the member in the outer peripheral region of the recess is not particularly limited and can be appropriately changed. Further, the height of the recess (Z-axis direction) is not particularly limited and can be appropriately changed. Further, the width between the recess 11 and the groove portion 30 described later (for example, in the X-axis direction) is not particularly limited and can be appropriately changed, and the height of the member of the width portion (Z-axis direction) is the member. It can be changed as appropriate according to the height of.
　第２部材２０は、凹部１１に嵌入し得る凸部２１を有することが好適である。
　図４Ａは、第２部材２０を、凸部２１の天面２２の反対側（Ｚ軸方向）からみたときの図である。天面側にある外周領域の面は、対向面同士を良好に接触させる等の観点から、平面であることが好適である。
　さらに、第２部材２０の線Ｉ－Ｉ’での断面について、図１Ｂの第２部材以外の例を、図４Ｂ、図８Ｂ等に示すが、第２部材の断面はこれに特に限定されない。例えば、凸部の外周領域の部材の高さ（Ｚ軸方向）は、特に限定されず、適宜変更が可能である。また、凸部の高さ（Ｚ軸方向）は、特に限定されず、適宜変更可能であり、凹部に嵌入しやすいように、凸部の高さを適宜変更可能である。 It is preferable that the second member 20 has a convex portion 21 that can be fitted into the concave portion 11.
FIG. 4A is a view when the second member 20 is viewed from the opposite side (Z-axis direction) of the top surface 22 of the convex portion 21. The surface of the outer peripheral region on the top surface side is preferably a flat surface from the viewpoint of making good contact between the facing surfaces.
Further, with respect to the cross section of the second member 20 along the line I-I', examples other than the second member of FIG. 1B are shown in FIGS. 4B, 8B and the like, but the cross section of the second member is not particularly limited thereto. For example, the height (Z-axis direction) of the member in the outer peripheral region of the convex portion is not particularly limited and can be appropriately changed. Further, the height of the convex portion (Z-axis direction) is not particularly limited and can be appropriately changed, and the height of the convex portion can be appropriately changed so as to be easily fitted into the concave portion.
　また、凹部１１の底面１２及び凸部２１の天面２２は、それぞれ、平面でも曲面でもよいが、操作性の観点から、平面が好適である。平面であることにより、検体液を均一に第１部材の凹部に簡便に広げることができる。
　底面の及び天面の面形状は、特に限定されず、例えば、円状、楕円状、多角形状等が挙げられるが、このうち円状が、操作性及び培養計測の観点から好適である。なお、多角形には、八角形、六角形、四角形、三角形等が挙げられるがこれらに限定されない。
　底面及び天面は、両方が同じ平面であって同じ面形状であることが好適であり、さらに好適には、底面の面積及び天面の面積が実質的に同じである。また、円状の底面を有する凹部は、２０～３０ｃｍ２の底面積を有することが、１ｍＬ検体液の適用に適しているので、好適である。 The bottom surface 12 of the concave portion 11 and the top surface 22 of the convex portion 21 may be flat or curved, respectively, but a flat surface is preferable from the viewpoint of operability. Since it is flat, the sample liquid can be easily spread evenly in the recess of the first member.
The surface shapes of the bottom surface and the top surface are not particularly limited, and examples thereof include a circular shape, an elliptical shape, and a polygonal shape. Of these, the circular shape is preferable from the viewpoint of operability and culture measurement. The polygon includes, but is not limited to, an octagon, a hexagon, a quadrangle, a triangle, and the like.
It is preferable that the bottom surface and the top surface are both the same plane and have the same surface shape, and more preferably, the area of the bottom surface and the area of the top surface are substantially the same. Further, it is preferable that the concave portion having a circular bottom surface has a bottom area of 20 to 30 cm 2 because it is suitable for application of 1 mL sample solution.
　好ましい態様では、第２部材２０は、第１部材１０の凹部１１と互いに嵌入しうる立体形状である凸部２１を有するように構成されている（図２Ｃ）。このときの凸部及び凹部は、嵌入しうる形状であれば任意の立体形状でもよく、当該立体形状として、例えば、多角柱状（例えば六角柱等）、楕円柱状、円柱状、錐台状（例えば円錐台等）等が挙げられるが、これらに限定されない。このうち、円柱状又は円錐台状が、製造容易の観点及び嵌入のしやすさの観点から好適である。
　凹部の嵌入部分の高さ（Ｚ軸方向）は、特に限定されないが、好ましくは０．５～５ｍｍ、より好ましくは１～３ｍｍであり、底面の最大長さは、特に限定されないが、好ましくは３０～８０ｍｍ、より好ましくは４０～７０ｍｍ、さらに好ましくは、５０～６０ｍｍであり、当該最大長さは、直径であることが好適である。 In a preferred embodiment, the second member 20 is configured to have a concave portion 11 of the first member 10 and a convex portion 21 having a three-dimensional shape that can be fitted to each other (FIG. 2C). The convex portion and the concave portion at this time may be any three-dimensional shape as long as they can be fitted, and the three-dimensional shape may be, for example, a polygonal columnar shape (for example, a hexagonal column), an elliptical columnar shape, a columnar shape, or a truncated cone shape (for example). (Conical table, etc.), etc., but are not limited to these. Of these, a columnar or truncated cone shape is preferable from the viewpoint of ease of manufacturing and ease of fitting.
The height (Z-axis direction) of the recessed portion is not particularly limited, but is preferably 0.5 to 5 mm, more preferably 1 to 3 mm, and the maximum length of the bottom surface is not particularly limited, but is preferable. It is 30 to 80 mm, more preferably 40 to 70 mm, still more preferably 50 to 60 mm, and the maximum length is preferably a diameter.
　また、第２部材２０は、第１部材１０の凹部１１と互いに完全に嵌入しうる円柱形状である凸部２１を有することがさらに好適である（図２Ｃ）。これにより、第２部材の凸部の円柱形状が、それよりやや大きい第１部材の凹部の円柱形状（中空）に嵌入し得る。この第１部材の凹部は、２０～３０ｃｍ２の底面積を有することが、１ｍＬ検体液の適用に適しているので、好適である。 Further, it is more preferable that the second member 20 has a convex portion 21 having a cylindrical shape that can be completely fitted into the concave portion 11 of the first member 10 (FIG. 2C). As a result, the cylindrical shape of the convex portion of the second member can be fitted into the cylindrical shape (hollow) of the concave portion of the first member slightly larger than that. It is preferable that the recess of the first member has a bottom area of 20 to 30 cm 2 because it is suitable for application of 1 mL sample solution.
　本発明の培養器材１は、上述のように、嵌入がなされた状態において、凸部２１の天面２２が、凹部１１の底面１２に接触状態となるように構成されていることが好適である。当該接触状態とは、天面と底面との間に一定距離以上の空間を持たないことが好適であり、より具体的には、当該天面と当該底面との間の距離は、好ましくは０．０１ｍｍ未満、より好ましくは０．００５ｍｍ以下、さらに好ましくは０．００１ｍｍ以下であり、完全に密着状態であることがよりさらに好適である。 As described above, the incubator 1 of the present invention is preferably configured so that the top surface 22 of the convex portion 21 is in contact with the bottom surface 12 of the concave portion 11 in the fitted state. .. It is preferable that the contact state does not have a space of a certain distance or more between the top surface and the bottom surface, and more specifically, the distance between the top surface and the bottom surface is preferably 0. It is less than 0.01 mm, more preferably 0.005 mm or less, still more preferably 0.001 mm or less, and it is even more preferable that it is in a completely adherent state.
　本発明の培養器材は、上述した構成を採用することにより、培地成分を凹部に収容した後に培養器材を閉じることで、第１部材及び第２部材と培地成分とがより接触状態になる。この接触状態により、培地成分の空間が独立して新たに形成されうる。そして、本発明の培養器材を採用することにより、第２部材の凸部の天面と第１部材の凹部の底面との間（Ｚ軸方向）で、培地成分が両者によって密着した状態となる培地成分入り培養器材を提供することができる。
　培地成分が各部材と接触状態になることで、培地成分に添加した検体液を、培養器材の外部から指等にて、凹部の培地成分に均一に広げる操作が容易にでき、培養に使用できる培地を簡便に容易に調製することができる。さらに、培地成分が各部材と接触状態になることで、培養使用時に、発育したコロニーが大きく広がることを防止し、コロニー数を計測するのに最適な状況を生み出すことに寄与する。 By adopting the above-mentioned configuration, the culture equipment of the present invention closes the culture equipment after accommodating the culture medium components in the recesses, so that the first member and the second member and the culture medium components are in a more contact state. Due to this contact state, the space of the medium component can be newly formed independently. Then, by adopting the incubator of the present invention, the medium components are in close contact with each other between the top surface of the convex portion of the second member and the bottom surface of the concave portion of the first member (in the Z-axis direction). It is possible to provide a culture device containing a medium component.
When the medium component is in contact with each member, the sample solution added to the medium component can be easily spread from the outside of the culture equipment to the medium component in the concave portion with a finger or the like, and can be used for culturing. The medium can be prepared easily and easily. Furthermore, the contact state of the medium component with each member prevents the developed colonies from spreading significantly during use of the culture, and contributes to creating an optimum situation for measuring the number of colonies.
　なお、本発明の培養器材における外周領域の端部のいずれかに、第１部材及び第２部材の開閉用の取手部を設けてもよく、部材の高さの差で取手部としてもよい。また、培養器材の長手方向のいずれか一方の端部に取手部を設けてもよく、取手部はヒンジ部とは反対側の端部に設けることが好適である。また、第１部材内に配置する凹部の中心は、第１部材の長さ（Ｘ軸方向又はＹ軸方向）の１０分の３～６に配置されていることが好適である。 A handle for opening and closing the first member and the second member may be provided at any of the ends of the outer peripheral region of the incubator of the present invention, or the handle may be provided due to the difference in height of the members. Further, the handle portion may be provided at one end of the culture equipment in the longitudinal direction, and it is preferable that the handle portion is provided at the end opposite to the hinge portion. Further, it is preferable that the center of the recess arranged in the first member is arranged at 3/10 to 6 of the length of the first member (X-axis direction or Y-axis direction).
＜溝部＞
　さらに、本発明の培養器材の好ましい態様として、第１部材の凹部１１又は第２部材の凸部２１の少なくとも一方の外周領域に、単数又は複数の溝部３０を備えることが好適である。当該溝部は、対向する面側に形成されることが好適であり、対向する面方向に開口するように形成されることがより好適である。また、溝部の窪み部分は、Ｚ軸方向に突起状になるように形成されることが好適であり、これにより、後述するスタッキング用凸部と併用することで、複数の培養器材を積み重ねる際の各部材の位置決めをすることができる（図１、図２、図８、図９等）。
　溝部の数は、特に限定されないが、前記凹部の外周領域及び前記凸部の外周領域のそれぞれに、１つ又は２つ以上であることが好適であり、より好ましくは１つであるが、前記凹部又は前記凸部の少なくとも一方の外周領域に１つ備えてもよい。 <Groove>
Further, as a preferred embodiment of the culture equipment of the present invention, it is preferable to provide a single or a plurality of groove portions 30 in at least one outer peripheral region of the concave portion 11 of the first member or the convex portion 21 of the second member. It is preferable that the groove portion is formed on the facing surface side, and it is more preferable that the groove portion is formed so as to open in the facing surface direction. Further, it is preferable that the recessed portion of the groove portion is formed so as to form a protrusion in the Z-axis direction, whereby when used in combination with the stacking convex portion described later, a plurality of culture equipments are stacked. Each member can be positioned (FIGS. 1, FIG. 2, FIG. 8, FIG. 9, etc.).
The number of grooves is not particularly limited, but it is preferable that the number of grooves is one or two or more in each of the outer peripheral region of the concave portion and the outer peripheral region of the convex portion, and more preferably one. One may be provided in at least one outer peripheral region of the concave portion or the convex portion.
　溝部は、培地成分に添加された検体液が毛細管現象により凹部の外へ漏れ出すことを抑制することができるように構成されている。従来の培養部材の構成のように溝部を設けない場合、培地成分に添加された検体液の水分が、接触状態の第１部材の表面と第２部材の表面とを濡らすことで、両者の間に存在する水分が直ちに凹部の外を超え部材端にまで到達する。本発明では、溝部を凹部又は凸部の外周領域に形成することで、毛細管現象によって、両部材の表面間にある検体液が端部方向に移動する現象を、溝部の手前で抑止することができる。このとき、この溝部によって、凹部（より好適には使用時の培地）の外に、検体液が漏れ出すことを抑制することができる。
　培地成分に添加した検体液が凹部の外に漏れ出すことにより生じる培地濃度が不均一になるのを防ぐこともでき、これにより意図した濃度の培地を形成させることができる。このように溝部の付加的機能として、検体液の水分を溝部の手前で抑制し、外周領域へ水分の流出を隔離するための隔離用の溝部として機能させてもよい。 The groove portion is configured to prevent the sample liquid added to the medium component from leaking out of the recess due to the capillary phenomenon. When the groove is not provided as in the conventional culture member configuration, the water content of the sample liquid added to the medium component wets the surface of the first member and the surface of the second member in contact with each other, so that the space between the two is reached. Moisture present in the culture medium immediately exceeds the outside of the recess and reaches the end of the member. In the present invention, by forming the groove portion in the outer peripheral region of the concave portion or the convex portion, the phenomenon that the sample liquid between the surfaces of both members moves toward the end portion due to the capillary phenomenon can be suppressed in front of the groove portion. can. At this time, the groove can prevent the sample liquid from leaking out of the recess (more preferably, the medium at the time of use).
It is also possible to prevent the medium concentration caused by the sample liquid added to the medium component leaking out of the concave portion from becoming non-uniform, thereby forming a medium having an intended concentration. As described above, as an additional function of the groove portion, the water content of the sample liquid may be suppressed in front of the groove portion, and the groove portion may function as an isolation groove portion for isolating the outflow of water to the outer peripheral region.
　溝部は、第１部材の凹部の外周領域に、当該凹部の外周の全部又は一部を囲うように備えることが好適である。また、溝部は、第２部材の凸部の外周領域に、当該凸部の外周の全部又は一部を囲うように備えることが好適である。また、第１部材の外周領域及び第２部材の外周領域の両方に、それぞれ溝部を適宜設けてもよい。
　また、溝部は、凹部又は凸部の外周を少なくとも５０％以上、より好適には８０％以上、さらに好適には９０％以上、よりさらに好適には完全に囲うことが好適である。また、溝部は、対向面側（Ｚ軸方向）からみたときに、第１部材の凹部又は第２部材の凸部の外周に沿って、連続的に又は断続的に、形成されていてもよい。 It is preferable that the groove portion is provided in the outer peripheral region of the concave portion of the first member so as to surround all or a part of the outer peripheral portion of the concave portion. Further, it is preferable that the groove portion is provided in the outer peripheral region of the convex portion of the second member so as to surround all or a part of the outer peripheral portion of the convex portion. Further, grooves may be appropriately provided in both the outer peripheral region of the first member and the outer peripheral region of the second member.
Further, it is preferable that the groove portion completely surrounds the outer periphery of the concave portion or the convex portion by at least 50% or more, more preferably 80% or more, further preferably 90% or more, and even more preferably completely. Further, the groove portion may be formed continuously or intermittently along the outer periphery of the concave portion of the first member or the convex portion of the second member when viewed from the facing surface side (Z-axis direction). ..
　溝部を備える位置は、凹部又は凸部の外周（外周端：０ｍｍ）より外側（部材の端部方向）であれば、特に限定されない。溝部を備える位置は、凹部又は凸部の外周（０ｍｍ）より、好ましくは０．５ｍｍ以上、より好ましくは１ｍｍ以上離れていることが好適であり、その上限値は、部材の大きさによって適宜設定することができるが、例えば１０ｍｍ以下、５ｍｍ以下、又は３ｍｍ以下等である。 The position where the groove portion is provided is not particularly limited as long as it is outside the outer periphery (outer peripheral end: 0 mm) of the concave portion or the convex portion (in the direction of the end portion of the member). The position where the groove is provided is preferably 0.5 mm or more, more preferably 1 mm or more away from the outer circumference (0 mm) of the concave or convex portion, and the upper limit thereof is appropriately set depending on the size of the member. However, for example, it is 10 mm or less, 5 mm or less, or 3 mm or less.
　溝部の溝の深さ（Ｚ軸方向）は、毛細管現象を遮断できる程度の深さがあれば良く、容器設計上、凹部又は凸部の高さ（Ｚ軸方向）によって適宜変更することができる。溝部の溝の深さは、例えば１～５ｍｍが好適であり、１～３ｍｍがより好適である。また、溝部の溝の幅（Ｘ軸方向又はＹ軸方向）は、特に限定されず、任意に設定することができ、例えば、好ましくは０．５～３ｍｍ程度、より好ましくは２～３ｍｍ程度である。
　また、溝部の断面形状（Ｚ軸方向）は、特に限定されないが、対向する面に向かって開口する形状が好適である。溝部の断面形状として、例えば、Ｖ字状、Ｕ字状、上向きＣ字状、半円状、四角形状、台形状等が挙げられるが、Ｕ字状又は上向きＣ字状が好適である。また、溝部の底面の形状は特に限定されないが、成形の容易性と強度の点から曲面であることが好ましい。 The groove depth (Z-axis direction) of the groove portion may be sufficient as long as it can block the capillary phenomenon, and can be appropriately changed depending on the height of the concave portion or the convex portion (Z-axis direction) in terms of container design. .. The groove depth of the groove portion is preferably, for example, 1 to 5 mm, and more preferably 1 to 3 mm. Further, the width of the groove of the groove portion (X-axis direction or Y-axis direction) is not particularly limited and can be set arbitrarily, for example, preferably about 0.5 to 3 mm, more preferably about 2 to 3 mm. be.
Further, the cross-sectional shape (Z-axis direction) of the groove portion is not particularly limited, but a shape that opens toward the facing surface is preferable. Examples of the cross-sectional shape of the groove include a V-shape, a U-shape, an upward C-shape, a semicircular shape, a quadrangular shape, a trapezoidal shape, and the like, and a U-shape or an upward C-shape is preferable. The shape of the bottom surface of the groove is not particularly limited, but a curved surface is preferable from the viewpoint of ease of molding and strength.
　本発明の培養器材に係る態様として、１ｍＬの検体液を第１部材の凹部に収容されている培地成分に接種し、凹部に嵌入した第２部材の凸部によって検体液が培地成分に押し広げられた場合、収容されている培地成分中のゲル化剤が検体液の水分を吸収し、固化される。さらに、第１部材の凹部の外周領域に溝部を設けなかった場合、ゲル化剤が検体液中の水分を吸収するよりも早い段階で、第２部材と接触した第１部材との毛細管現象により凹部から水が周囲の外周領域方向に逃げていきやすい。このため、溝部を備えなかった場合には、水が周囲に逃げることで培地成分を膨潤させるための必要な水分がない状態で膨潤し培地が再構成され、再構成された培地が塗布面から剥離するため良好な培養結果が得られにくいだけでなく、設計通りの濃度の培地が得られにくい。一方、凹部又は凸部の外周領域に、凹部又は凸部の外周に沿って溝部を少なくとも１つ備えることで、毛細管現象が断ち切られ、凹部の外への水の流出を防ぐことが可能となる。１ｍＬの検体液接種後に第２部材の凸部を嵌め込むだけで、第１部材の凹部全体に被検液が広がると共に培地成分中のゲル化剤が水を吸収し、設計通りの各培地成分濃度の培地を再構成させることが可能となる。 As an embodiment of the culture equipment of the present invention, 1 mL of the sample liquid is inoculated into the medium component contained in the concave portion of the first member, and the sample liquid is spread over the medium component by the convex portion of the second member fitted in the concave portion. If so, the gelling agent in the contained medium component absorbs the water content of the sample solution and solidifies. Further, when the groove is not provided in the outer peripheral region of the concave portion of the first member, the capillary phenomenon with the first member in contact with the second member occurs at an earlier stage than the gelling agent absorbs the water in the sample liquid. Water easily escapes from the recess toward the surrounding outer peripheral area. For this reason, when the groove is not provided, water escapes to the surroundings and swells in a state where there is no necessary water for swelling the medium components, and the reconstituted medium is reconstituted from the coated surface. Not only is it difficult to obtain good culture results due to exfoliation, but it is also difficult to obtain a medium with the designed concentration. On the other hand, by providing at least one groove along the outer periphery of the concave or convex portion in the outer peripheral region of the concave or convex portion, the capillary phenomenon can be cut off and water can be prevented from flowing out of the concave portion. .. By simply fitting the convex part of the second member after inoculating 1 mL of the sample solution, the test solution spreads over the entire concave part of the first member, and the gelling agent in the medium component absorbs water, and each medium component as designed. It is possible to reconstitute the medium of concentration.
　従って、第１部材の凹部の外周領域に溝部は、第１部材に設置した凹部の外周から１ｍｍ以上離れていることが好適であるが、毛細管現象を堰き止めるために凹部の外周付近から離れすぎないようにすることが好ましい。また、第１部材の凹部と第２部材の凸部が完全に密着した状態で勘合し、空間を持たない状態の培養器材に、底面及び天面の間に培地成分をさらに入れても、適用時１ｍＬ検体液では、水の膨潤による培地成分自体の上下方向への体積増加はほとんど無視できるほど小さい。 Therefore, it is preferable that the groove portion in the outer peripheral region of the concave portion of the first member is separated from the outer peripheral region of the concave portion installed in the first member by 1 mm or more, but is too far from the vicinity of the outer peripheral portion of the concave portion in order to prevent the capillary phenomenon. It is preferable not to do so. Further, it is also applicable to further put a medium component between the bottom surface and the top surface of the culture equipment in a state where the concave portion of the first member and the convex portion of the second member are completely in close contact with each other and have no space. At 1 mL sample solution, the vertical volume increase of the medium component itself due to the swelling of water is almost negligible.
＜ヒンジ部＞
　さらに、好ましい態様として、第１部材１０及び第２部材２０はヒンジ部５０により連設されていることである。当該ヒンジ部の短手方向（Ｙ軸方向）の長さは、部材の端部の長さよりも短く形成されていてもよい。当該ヒンジ部は、凹部の中心を通過する線Ｉ－Ｉ’上に配置してもよい。
　培養器材が閉じた状態でのヒンジ部の断面形状は、特に限定されず、例えば、多角形状（例えば、四角形状、五角形状、六角形状等）、半円状、半楕円状、I字状等が挙げられる。例えば、第１部材の端部と第２部材の端部とが一辺を共有するような構成で、この一辺をヒンジ部とし、この一辺を軸として折り曲げて嵌入したときに、断面形状がＩ字状のヒンジ部が形成されてもよい。 <Hinge part>
Further, as a preferred embodiment, the first member 10 and the second member 20 are continuously provided by the hinge portion 50. The length of the hinge portion in the lateral direction (Y-axis direction) may be formed shorter than the length of the end portion of the member. The hinge portion may be arranged on the line I-I'passing through the center of the recess.
The cross-sectional shape of the hinge portion in the closed state of the incubator is not particularly limited, and is, for example, a polygonal shape (for example, a quadrangular shape, a pentagonal shape, a hexagonal shape, etc.), a semicircular shape, a semi-elliptical shape, an I-shaped shape, or the like. Can be mentioned. For example, in a configuration in which the end of the first member and the end of the second member share one side, this side is used as a hinge part, and when this side is bent and fitted with this side as an axis, the cross-sectional shape is I-shaped. A shaped hinge portion may be formed.
　ヒンジ部５０は、第２部材２０の凸部２１の天面２２を、第１部材１０の凹部１１の底面１２に対して略平行を維持しつつ嵌入し得る構造を備えることが、より好適である。このように嵌入させることで、培養使用時に、検体液を培地成分に適用した後に凹部に凸部を嵌入させたときに、検体液を培地成分への均一適用させること、毛細管現象の発生の抑制、空気層の発生抑制等ができる。
　このような構造を備えるヒンジ部として、例えば、図５Ａ～図５Ｃに示すような構造が挙げられるが、これらに限定されない。図５Ａ、図５Ｂ、図５Ｃは、それぞれ、ヒンジ部５０を有する培養器材１ａ、１ｂ、１ｃであり、第１部材及び第２部材を開いた状態でのＩ－Ｉ’線の断面図を表す。培養器材１ａ、１ｂ、１ｃは閉じた状態で、ヒンジ部の断面形状は、それぞれ、Ｃ字形状、三角形状、四角形状となる。
　このうち、ヒンジ部の断面形状が四角形状であることが、より簡便な操作で略平行を維持しつつ嵌入できる観点から、より好適である（例えば図５Ｃ及び図６）。 It is more preferable that the hinge portion 50 has a structure in which the top surface 22 of the convex portion 21 of the second member 20 can be fitted while maintaining substantially parallel to the bottom surface 12 of the concave portion 11 of the first member 10. be. By inserting in this way, when the sample solution is applied to the medium component during use in culture and then the convex portion is fitted into the concave portion, the sample solution is uniformly applied to the medium component and the occurrence of capillarity is suppressed. , It is possible to suppress the generation of the air layer.
Examples of the hinge portion having such a structure include, but are not limited to, the structures shown in FIGS. 5A to 5C. 5A, 5B, and 5C are culture equipment 1a, 1b, and 1c having a hinge portion 50, respectively, and represent a cross-sectional view taken along the line I-I'with the first member and the second member open. .. With the culture equipment 1a, 1b, and 1c closed, the cross-sectional shapes of the hinge portions are C-shaped, triangular, and square, respectively.
Of these, it is more preferable that the cross-sectional shape of the hinge portion is a quadrangular shape from the viewpoint that it can be fitted while maintaining substantially parallel with a simpler operation (for example, FIGS. 5C and 6).
　なお、第２部材２０の凸部２１の天面２２を、第１部材１０の凹部１１の底面１２に対して略平行を維持しつつ嵌入し得る構造として、例えば、図５Ｄに表すように、ヒンジ部を設けずに、第１部材と第２部材とが別々の部材として構成されている構造であってもよい。 As a structure capable of fitting the top surface 22 of the convex portion 21 of the second member 20 while maintaining substantially parallel to the bottom surface 12 of the concave portion 11 of the first member 10, for example, as shown in FIG. 5D. The structure may be such that the first member and the second member are configured as separate members without providing the hinge portion.
＜スタッキング用凸部＞
　好ましい態様として、第１部材１０又は第２部材２０のいずれかの外周領域に、スタッキング用凸部６０が形成されることである。
　スタッキング用凸部は、第１部材及び／又は第２部材の外側（Ｚ軸方向）の面に突起状になるように形成されることが好適である。
　スタッキング用凸部の配置（ＸＹ軸方向）は、特に限定されず、例えば、部材の凹部又は凸部の周端領域に単数又は複数配置されてもよいし（例えば図７Ａ）、部材の端領域に単数又は複数配置されてもよし（例えば図７Ｂ）、部材の溝部の周端領域に単数又は複数配置してもよい（例えば図８）。
　このように、培養器材ごとにスタッキング用凸部を設けることで、下に配置した培養器材が上に配置した培養器材を、支持することができ、これにより複数の培養器材を積み重ねることができる（例えば図９）。 <Convex for stacking>
As a preferred embodiment, the stacking convex portion 60 is formed in the outer peripheral region of either the first member 10 or the second member 20.
It is preferable that the stacking convex portion is formed so as to form a protrusion on the outer surface (Z-axis direction) of the first member and / or the second member.
The arrangement of the stacking convex portions (in the XY axis direction) is not particularly limited, and may be, for example, singular or plurally arranged in the peripheral end region of the concave portion or the convex portion of the member (for example, FIG. 7A), or the end region of the member. It may be arranged singularly or plurally (for example, FIG. 7B), and may be arranged singularly or plurally in the peripheral end region of the groove portion of the member (for example, FIG. 8).
In this way, by providing the stacking convex portion for each culture equipment, the culture equipment arranged below can support the culture equipment arranged above, and a plurality of culture equipments can be stacked (as a result). For example, FIG. 9).
　スタッキング用凸部の数は、特に限定されず、単数又は複数であり、好ましくは１又は２以上、さらに好ましくは３～１０程度であり、より好ましくは３～６、さらに好ましくは３又は４である。スタッキング用凸部の形状は、例えば、正方形状、長方形状、円錐形状、円錐形状等の立体形状が挙げられるが、これらに特に限定されない。 The number of stacking protrusions is not particularly limited, and may be singular or plural, preferably 1 or 2 or more, more preferably about 3 to 10, more preferably 3 to 6, still more preferably 3 or 4. be. The shape of the stacking convex portion includes, for example, a three-dimensional shape such as a square shape, a rectangular shape, a conical shape, and a conical shape, but is not particularly limited thereto.
　なお、スタッキング用凸部が形成される面に対向する面上に、スタッキング用凸部を位置決するための支持部を形成することが好適である。当該支持部として、例えば、培養器材の外側方向（Ｚ軸方向）の面上に、突起状になるように形成された溝部、凹部と溝部との間に形成された窪み状、外周領域に形成された窪み状や突起状等の位置決め可能な支持部等が挙げられるが、これらに限定されない。 It is preferable to form a support portion for positioning the stacking convex portion on the surface facing the surface on which the stacking convex portion is formed. As the support portion, for example, a groove portion formed in a protrusion shape, a recess shape formed between the recess and the groove portion, and an outer peripheral region are formed on the surface of the culture equipment in the outer direction (Z-axis direction). Examples thereof include support portions that can be positioned such as recessed shapes and protrusions, but the present invention is not limited thereto.
＜部材材料＞
　本発明における第１部材及び／又は第２部材は、合成樹脂材で形成されることが好適であり、光透過性の合成樹脂材で形成されることがより好適である。
　本発明の培養器材において、第１部材及び／又は第２部材の材料は特に限定されず、例えば、ポリスチレン系、ポリアクリル系、ポリビニル系、ポリエチレン系、ポリエステル系、ポリ乳酸系のポリマー等の合成樹脂材を採用でき、これらから１種又は２種以上を用いることができる。 <Material material>
The first member and / or the second member in the present invention is preferably formed of a synthetic resin material, and more preferably formed of a light-transmitting synthetic resin material.
In the culture equipment of the present invention, the material of the first member and / or the second member is not particularly limited, and for example, synthesis of polystyrene-based, polyacrylic-based, polyvinyl-based, polyethylene-based, polyester-based, polylactic acid-based polymers and the like. A resin material can be adopted, and one or more of them can be used.
　本発明において、第１部材又は第２部材のいずれか一方は透明であることが好ましく、第１部材と第２部材の両方が透明であることがより好ましい。第１部材及び／又は第２部材は、光透過性を有することが好ましい。これにより、計測対象の微生物のコロニーを、培養器材を分解することなく、外部から容易に観察・計測することができる。
　なお、ここで透明（光透過性）とは、目視により部材の反対側を透視できる程度でよく、より具体的には可視光透過率が７０％以上であることが好ましいが、これに限定されない。 In the present invention, it is preferable that either the first member or the second member is transparent, and it is more preferable that both the first member and the second member are transparent. The first member and / or the second member preferably have light transmission. As a result, the colony of the microorganism to be measured can be easily observed and measured from the outside without decomposing the culture equipment.
Here, the term "transparent" means that the opposite side of the member can be visually seen through, and more specifically, the visible light transmittance is preferably 70% or more, but the present invention is not limited to this. ..
　また、本発明の培養器材において、第１部材と第２部材は、別個に分離していてもよいし、一体となっていてもよい。
　好ましい態様として、第１部材１０及び第２部材２０が一体成形されているものである。
　例えば、第１部材の一部と第２部材の一部とが、一辺を共有する等して、連結していてもよい。例えば、後述するヒンジ部を第１部材の端部と第２部材の端部との間に設けて、これら部材を連設してもよい。
　このような一体形成の態様の場合、成形した培養器材を第２部材の凸部と第１部材の凹部とに嵌入するように第１部材と第２部材とを重ね合わせて折り曲げることにより使用することが可能となり（例えば図１及び２）、製造する上で、培地成分塗着や培養使用時の部材の開閉等の操作性等の観点からも、部材が１つで済むことは好ましい態様といえる。 Further, in the incubator of the present invention, the first member and the second member may be separately separated or integrated.
As a preferred embodiment, the first member 10 and the second member 20 are integrally molded.
For example, a part of the first member and a part of the second member may be connected by sharing one side or the like. For example, a hinge portion described later may be provided between the end portion of the first member and the end portion of the second member, and these members may be continuously provided.
In the case of such an integral formation mode, the molded incubator is used by overlapping and bending the first member and the second member so as to fit into the convex portion of the second member and the concave portion of the first member. (For example, FIGS. 1 and 2), and from the viewpoint of operability such as coating of medium components and opening / closing of members during use of culture, it is preferable that only one member is required. I can say.
＜培地成分＞
　本発明の培養器材の凹部に収容可能な培地成分について説明する。
　本発明に用いられる培地成分は、第２部材を第１部材に被せたときに互いに接触する部分、即ち第１部材の凹部分の底面及び／又は第２部材の凸部分の天面に均一に塗着されていることが好ましい。当該培地成分は、乾燥状態のものが好適であり、検体液の水分を含むことで、培養用の培地に再構成できるような乾燥培地成分がより好適である。
　この培地成分の塗着部位は、通常、検体液は、第２部材の凸部の天面を利用することにより均一に第１部材の凹部に広げられることと、広げられた検体液のスムーズな拡散と培養使用時の培地への均一な再構成を両立させる点で、第１部材の凹部分の底面及び／又は第２部材の凸部分の天面に均一に塗布又は塗着されていることが好適である。 <Medium component>
The medium component that can be accommodated in the recess of the culture equipment of the present invention will be described.
The medium component used in the present invention is uniformly applied to the portion that comes into contact with each other when the second member is placed on the first member, that is, the bottom surface of the concave portion of the first member and / or the top surface of the convex portion of the second member. It is preferably coated. The medium component is preferably in a dry state, and a dry medium component that can be reconstituted into a culture medium by containing the water content of the sample solution is more preferable.
The application site of this medium component is usually such that the sample liquid is uniformly spread in the concave portion of the first member by using the top surface of the convex portion of the second member, and the spread sample liquid is smooth. It should be uniformly applied or coated on the bottom surface of the concave portion of the first member and / or the top surface of the convex portion of the second member in order to achieve both diffusion and uniform reconstruction on the medium during use of the culture. Is preferable.
　本発明の培養器材における培地成分は、ゲル化剤及び栄養成分を含有することが好ましい。
　本発明において、培地成分は、微生物を培養するための培地を調製するためのものである。前記調製は、通常、計測対象の微生物を含む検体液中の水分をそのまま培地を構成するゲルの溶媒として、培地成分に添加し浸透させることにより行われる。 The medium component in the culture medium of the present invention preferably contains a gelling agent and a nutritional component.
In the present invention, the medium component is for preparing a medium for culturing microorganisms. The above preparation is usually carried out by adding the water content in the sample solution containing the microorganism to be measured to the medium component as it is as the solvent of the gel constituting the medium and allowing it to permeate.
　前記ゲル化剤は、非加熱ゲル化剤が、加熱することなく検体液を培地成分に適用することで液体をゲル化できるので好ましい。寒天やカラギーナンといった加熱ゲル化剤を用いる場合、検体液を培地成分と共に固化させる際に加熱が必要であるため、培養使用時に加熱工程が必要となったり、検体中の微生物を加熱することによる死滅等にて計測にばらつきが生じやすい。 The gelling agent is preferable because the non-heated gelling agent can gel the liquid by applying the sample liquid to the medium component without heating. When using a heating gelling agent such as agar or carrageenan, heating is required when the sample solution is solidified together with the medium components, so a heating step is required when using the culture, or the microorganisms in the sample are killed by heating. Measurements tend to vary due to factors such as.
　一方、非加熱ゲル化剤は、加熱による溶解を経ずに、また冷却によらず、水分の添加のみによりゲルを形成させることができるため、培地形成の操作が簡便であり、また対象微生物の生育を妨げないという利点がある。
　さらに、静置時にゲル化できる成分がより好適であり、当該静置時のゲル化成分として、増粘多糖類がさらに好適であるが、これらに限定されない。当該増粘多糖類は、培地を構成するゲル化剤の役割を担うことができ、常温（１０～３０℃程度）で検体液を培地成分に均一になるように押し広げて適用したときにこれらを固化でき、その後、静置状態でゲル状態を維持可能な検体を含む培地が形成できることから、好ましい。
　これにより、培地成分中のゲル化剤により形成される培養使用時の培地は、流動性がほとんどなくまた水分を強固に保持できるため、微生物の存在数をその発育したコロニー数により定量的により正確に計測することができる。 On the other hand, the non-heated gelling agent can form a gel only by adding water without undergoing dissolution by heating and without cooling, so that the operation of medium formation is simple and that the target microorganism can be formed. It has the advantage of not hindering growth.
Further, a component that can be gelled at the time of standing is more preferable, and a thickening polysaccharide is further suitable as the gelling component at the time of standing, but the present invention is not limited thereto. The thickening polysaccharide can play a role of a gelling agent constituting the medium, and when the sample solution is spread and applied uniformly to the medium components at room temperature (about 10 to 30 ° C), these are applied. It is preferable because a medium containing a sample capable of solidifying and then maintaining a gel state in a stationary state can be formed.
As a result, the medium formed by the gelling agent in the medium components has almost no fluidity and can retain water firmly, so that the number of microorganisms present is quantitatively more accurate depending on the number of colonies that have grown. Can be measured.
　増粘多糖類として、特に限定されないが、例えば、グアーガム、キサンタンガム、ローカストビーンガム、アラビアガム、タマリンドガム、カードラン、タラガム、プルラン等が挙げられ、これらからなる群から選択される１種又は２種以上がより好ましい。当該増粘多糖類には、本発明の効果を妨げない限りにおいて、さらに他のゲル化剤を併用してもよい。 The thickening polysaccharide is not particularly limited, and examples thereof include guar gum, xanthan gum, locust bean gum, gum arabic, tamarind gum, curdlan, tara gum, pullulan, and the like, and one or two selected from the group consisting of these. More than seeds are more preferred. Other gelling agents may be further used in combination with the thickening polysaccharide as long as the effects of the present invention are not impaired.
　増粘多糖類のうち、グアーガム、キサンタンガム、及びそれらの混合物から選択される１種又は２種以上が好ましい。混合物の方が、これらの割合を調整して培養使用時のゲル化能を調整しやすいのでより好適である。これらに、本発明の効果を妨げない限りにおいて、さらに他のゲル化剤を併用してもよい。
　ここで、グアーガム及び／又はキサンタンガムは、吸水、固化の速度が比較的緩やかであるため、培地成分及びゲル化剤を早急に混合させるような操作を行わなくともよい。このため、検体液をグアーガム及び／又はキサンタンガムを含む培地成分に添加し、培地成分の全体に均一になるように拡散させる操作をしても、薄く広がったゲル培地を形成することができるため、本発明の培養器材により良好に適する。これにより、培養使用時の培地は、流動性がほとんどなくまた水分を強固に保持できるため、微生物の存在数をその発育したコロニー数により定量的に正確に計測することができる。
　また、グアーガム、キサンタンガム、又はそれらの混合物にて形成されるゲルが、透明であることにより、培養後の培地中の微生物のコロニー及びその数を、培養器材を分解することなく外部から正確かつ容易に検出することができる。 Among the thickening polysaccharides, one or more selected from guar gum, xanthan gum, and mixtures thereof are preferable. The mixture is more preferable because it is easy to adjust the gelling ability at the time of using the culture by adjusting these ratios. Other gelling agents may be used in combination with these as long as the effects of the present invention are not impaired.
Here, since guar gum and / or xanthan gum have a relatively slow rate of water absorption and solidification, it is not necessary to perform an operation for immediately mixing the medium component and the gelling agent. Therefore, even if the sample solution is added to the medium component containing guar gum and / or xanthan gum and diffused uniformly throughout the medium component, a thinly spread gel medium can be formed. It is better suited to the culture medium of the present invention. As a result, the medium used for culture has almost no fluidity and can firmly retain water, so that the number of microorganisms present can be quantitatively and accurately measured by the number of colonies that have grown.
In addition, since the gel formed of guar gum, xanthan gum, or a mixture thereof is transparent, the colonies of microorganisms and their numbers in the culture medium after culturing can be accurately and easily measured from the outside without decomposing the culture equipment. Can be detected.
　本発明に用いられるゲル化剤（より好適にはグアーガム及び／又はキサンタンガム）の使用時の濃度は、特に限定されないが、１ｍLの水に対する固化能の観点から、１ｍLの水を添加した使用時の濃度（１ｍＬ当たりの濃度）として合計量で０．０１～０．２ｇ／ｍＬが好ましく、０．０１～０．１ｇ／ｍＬがより好ましい。 The concentration of the gelling agent (more preferably guar gum and / or xanthan gum) used in the present invention is not particularly limited, but from the viewpoint of solidification ability with respect to 1 mL of water, when 1 mL of water is added. The total concentration (concentration per 1 mL) is preferably 0.01 to 0.2 g / mL, more preferably 0.01 to 0.1 g / mL.
　培地成分に含まれる栄養成分は、対象微生物を発育させるためものである。栄養成分としては、特に限定されないが、ペプトン、獣肉エキス、酵母エキス、魚肉エキス等が好ましく挙げられる。
　微生物数を計測する培地には、寒天を含む寒天培地と寒天を含まない液体培地の２種の培地形態が存在するが、寒天を含まない液体培地の成分かそれと同等の成分を、本発明における培地成分に含有させることが好ましい。 The nutritional component contained in the medium component is for developing the target microorganism. The nutritional component is not particularly limited, and preferably includes peptone, animal meat extract, yeast extract, fish meat extract and the like.
There are two types of media for measuring the number of microorganisms, an agar medium containing agar and a liquid medium not containing agar. In the present invention, a component of a liquid medium containing no agar or a component equivalent thereto is used. It is preferable to include it in the medium component.
　本発明に用いられる培地成分には、第１部材及び／又は第２部材に対する接着性成分が含まれることが好適である。接着性成分を含ませることで、培地成分の全ては、本発明の培養器材の凹部の底面又は凸部の天面の少なくとも片側に塗着することができる。 It is preferable that the medium component used in the present invention contains an adhesive component to the first member and / or the second member. By including the adhesive component, all of the medium components can be applied to at least one side of the bottom surface of the concave portion or the top surface of the convex portion of the incubator of the present invention.
　接着性成分を用いることで、培地成分を、第１部材及び／又は第２部材に、安定的に塗着させる役割を果たし、さらに天面と底面とに培地成分をより密着した状態にすることができる。そして、凸部を凹部に嵌入させた状態にすることで培地成分が、凹部に収容されると共に第１部材と第２部材とが接着性成分を含む培地成分を介して接着される。これにより、第１部材の凹部に第２部材の凸部が嵌入した状態を維持した状態の培地成分入り微生物用培養器材を得ることもできる。また、培養使用時に、培地と天面と底面との間に空気層がより発生しづらくなるため、操作性がより簡便であり、培養時の、周囲のコロニーとの重なりを低減することができ、容易に計測することができる。 By using the adhesive component, the medium component plays a role of stably applying the medium component to the first member and / or the second member, and further, the medium component is brought into a state of being in closer contact with the top surface and the bottom surface. Can be done. Then, by putting the convex portion into the concave portion, the medium component is accommodated in the concave portion, and the first member and the second member are adhered to each other via the medium component containing the adhesive component. As a result, it is also possible to obtain a culture medium-containing culture medium containing a medium component in a state in which the convex portion of the second member is fitted into the concave portion of the first member. In addition, when the culture is used, an air layer is less likely to be generated between the medium and the top surface and the bottom surface, so that the operability is simpler and the overlap with the surrounding colonies during the culture can be reduced. , Can be easily measured.
　接着性成分として、特に限定されないが、例えば、ポリビニルピロリドン、ヒドロキシプロピルセルロース、ヒドロキシメチルセルロース、及びメチルセルロース等が挙げられ、これらから１種又は２種以上を選択することができる。
　接着性成分の使用時の濃度は、特に限定されないが、培地成分を乳剤又は懸濁液として塗布する際の粘度と接着能の観点から、１ｍLの水を添加した使用時の濃度（１ｍＬ当たりの濃度）として接着性成分合計量で０．１～１０ｍｇ／ｍＬが好ましく、０．５～５ｍｇ／ｍＬがより好ましい。 The adhesive component is not particularly limited, and examples thereof include polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxymethyl cellulose, and methyl cellulose, from which one or more can be selected.
The concentration of the adhesive component at the time of use is not particularly limited, but from the viewpoint of viscosity and adhesive ability when the medium component is applied as an emulsion or suspension, the concentration at the time of use with 1 mL of water added (per 1 mL). The total amount of adhesive components is preferably 0.1 to 10 mg / mL, more preferably 0.5 to 5 mg / mL.
　本発明に用いられる培地成分は、さらに呈色試薬を含有することが好ましい。これは、培養によって生じた微生物のコロニーを有色のものとして、より検出及び／又は計測しやすくするためである。
　呈色試薬としては、例えば、２,３,５－トリフェニルテトラゾリウムクロライド（ＴＴＣ）やテトラゾリウムバイオレット等をはじめとする酸化還元指示薬等が挙げられ、これらから１種又は２種以上を選択することができる。呈色試薬は、検体中に存在する全ての種類の微生物を計測したい場合に好ましく用いることができる。ＴＴＣを用いる場合は、１ｍＬの水を添加した使用時の濃度（１ｍＬ当たりの濃度）として１ｍｇ～１００ｍｇ／Ｌが好ましく、１０～５０ｍｇ／Ｌがより好ましい。 The medium component used in the present invention preferably further contains a color-developing reagent. This is to make the colonies of microorganisms produced by the culture colored so that they can be more easily detected and / or measured.
Examples of the color-developing reagent include redox indicators such as 2,3,5-triphenyltetrazolium chloride (TTC) and tetrazolium violet, and one or more of them can be selected. can. The color-developing reagent can be preferably used when it is desired to measure all kinds of microorganisms present in a sample. When TTC is used, the concentration (concentration per 1 mL) when 1 mL of water is added is preferably 1 mg to 100 mg / L, more preferably 10 to 50 mg / L.
　また、呈色試薬としては、特定の微生物種のみが保有する酵素に対する基質（以下、酵素基質という）であって、分解されることにより色原体化合物を遊離し得る化合物を用いてもよい。これは、当該特定の微生物を計測したい場合に好ましく用いることができる。
　ここで、色原体化合物とは、可視光下で有色のもの及び蛍光発色するもののいずれでもよい。可視光下で有色の化合物として遊離され得る官能基として、例えば５－ブロモ－４－クロロ－３－インドキシル基等が挙げられ、遊離した５－ブロモ－４－クロロ－３－インドールは酸化縮合して２量体の５，５’－ジブロモ－４，４’－ジクロロ－インディゴとなり、青色を呈する。蛍光発色する化合物として遊離され得る官能基としては、４－メチルウンベリフェリル基等が挙げられ、遊離した４－メチルウンベリフェロンは紫外線照射下で蛍光を発する。 Further, as the color-developing reagent, a compound which is a substrate for an enzyme possessed only by a specific microbial species (hereinafter referred to as an enzyme substrate) and which can release a chromogen compound by decomposition may be used. This can be preferably used when it is desired to measure the specific microorganism.
Here, the chromogen compound may be either a colored compound or a fluorescent compound under visible light. Examples of the functional group that can be liberated as a colored compound under visible light include a 5-bromo-4-chloro-3-indoxyl group, and the liberated 5-bromo-4-chloro-3-indole is oxidatively condensed. Then, it becomes a dimer of 5,5'-dibromo-4,4'-dichloro-indigo, which has a blue color. Examples of the functional group that can be liberated as the fluorescently colored compound include a 4-methylumbelliferyl group, and the liberated 4-methylumbelliferone fluoresces under ultraviolet irradiation.
　酵素基質の例を挙げると、対象微生物が大腸菌群の場合は、５－ブロモ－４－クロロ－３－インドキシル－β－Ｄ－ガラクトピラノシド（Ｘ－ＧＡＬ）等を、大腸菌の場合は、５－ブロモ－４－クロロ－３－インドキシル－β－Ｄ－グルクロン酸（Ｘ－ＧＬＵＣ）等を、黄色ブドウ球菌の場合は、５－ブロモ－４－クロロ－３インドキシル－リン酸（Ｘ－ｐｈｏｓ）等を、腸球菌等の場合は、５－ブロモ－４－クロロ－３－インドキシル－β－Ｄ－グルコピラノシド等を、真菌の場合は、Ｘ－ｐｈｏｓ、５－ブロモ－４－クロロ－３－インドキシル－酢酸や５－ブロモ－４－クロロ－３－インドキシル－酪酸等を、それぞれ好ましく用いることができる。さらに、全ての微生物種を検出したい場合には、これら全てを組み合わせて使用してもよい。
　酵素基質を用いる場合には、１ｍＬの水を添加した使用時の濃度（１ｍＬ当たりの濃度）として０．０１～１．０ｇ／Ｌが好ましく、０．２～０．５／Ｌがより好ましい。 To give an example of an enzyme substrate, if the target microorganism is coliform bacteria, 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-GAL), etc., and if it is E. coli, , 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid (X-GLUC), etc., and in the case of Escherichia coli, 5-bromo-4-chloro-3 indoxyl-phosphate ( X-phos), etc., 5-bromo-4-chloro-3-indoxyl-β-D-glucopyranoside, etc. in the case of enterobacteria, X-phos, 5-bromo-4-in the case of fungi, etc. Chloro-3-indoxyl-acetic acid, 5-bromo-4-chloro-3-indoxyl-butyric acid and the like can be preferably used. Furthermore, if it is desired to detect all microbial species, all of these may be used in combination.
When an enzyme substrate is used, the concentration (concentration per 1 mL) when 1 mL of water is added is preferably 0.01 to 1.0 g / L, more preferably 0.2 to 0.5 / L.
　本発明に用いられる培地成分は、本発明の効果を妨げない限りにおいて、さらに、選択物質、抗菌性物質、無機塩類、糖類、増粘剤、ｐＨ調整剤等を任意に含有してもよい。これら任意の成分から、適宜、１種又は２種以上選択することができる。
　選択物質としては、例えば、ポリミキシンＢやバンコマイシンなどの抗生物質や、ラウリル硫酸ナトリウム（ＳＤＳ）、Ｔｗｅｅｎ８０、コール酸ナトリウム等の胆汁酸塩等の界面活性剤等が挙げられる。
　抗菌性物質としては、例えば、ポリリジン、プロタミン硫酸塩、グリシン、ソルビン酸等が挙げられる。
　無機塩類としては、例えば、塩化ナトリウム、チオ硫酸ナトリウム等の無機酸金属塩、ピルビン酸ナトリウム、クエン酸鉄アンモニウム、クエン酸ナトリウム等の有機酸金属塩等が挙げられる。
　糖類としては、例えば、グルコース、ラクトース、スクロース、キシロース、セロビオース、マルトース等が挙げられる。
　ｐＨ調整剤としては、例えば、炭酸ナトリウム、炭酸水素ナトリウム、酒石酸等が挙げられる。なお、本発明の培養器材に塗布される培地組成物は、対象微生物の生育の観点から、使用時のｐＨが好ましくは６．０～８．０に、より好ましくは６．５～７．５になるように調製される。 The medium component used in the present invention may further optionally contain a selective substance, an antibacterial substance, an inorganic salt, a saccharide, a thickener, a pH adjuster and the like, as long as the effect of the present invention is not impaired. From these arbitrary components, one kind or two or more kinds can be appropriately selected.
Examples of the selective substance include antibiotics such as polymyxin B and vancomycin, and surfactants such as bile salts such as sodium lauryl sulfate (SDS), Tween80 and sodium cholic acid.
Examples of the antibacterial substance include polylysine, protamine sulfate, glycine, sorbic acid and the like.
Examples of the inorganic salts include inorganic acid metal salts such as sodium chloride and sodium thiosulfate, and organic acid metal salts such as sodium pyruvate, ammonium iron citrate and sodium citrate.
Examples of saccharides include glucose, lactose, sucrose, xylose, cellobiose, maltose and the like.
Examples of the pH adjuster include sodium carbonate, sodium hydrogencarbonate, tartaric acid and the like. The medium composition applied to the incubator of the present invention preferably has a pH at the time of use of 6.0 to 8.0, more preferably 6.5 to 7.5, from the viewpoint of growth of the target microorganism. Is prepared to be.
　本発明の培養器材は、任意の方法で製造することができるが、一例を説明するが、これに限定されない。
　適当な大きさのアクリル板等の合成樹脂平板を用いて、第１部材及び第２部材とすることができる。第１部材の凹部及び／又は第２部材の凸部は、合成樹脂の加工成形方法を利用して作製することができ、例えば、アクリル板の接着やくり抜き、又は金型等を用いた押圧や射出による成形などにより、作製することができる。
　本発明に用いられる培地成分は、非水系溶媒に溶解又は懸濁させたものを、第１部材の凹部及び／又は第２部材の凸部の面全体に均一に塗布した後、素早く強制乾燥等乾燥することにより、培養器材の第１部材の凹部及び／又は第２部材の凸部に塗着させることができる。
　ここで、非水系溶媒は、常温常圧下で速やかに揮発し得る揮発性溶媒がよく、例えば、エタノール、メタノール、プロパノール、及びブタノール等から選択される１種又は２種以上の低級アルコール（好適には炭素数１～４）を好ましく挙げられる。これらの非水系溶媒を用いれば、製造時にゲル化剤をゲル化させることなく培地成分を塗着させることができるので、容易かつ効率良く培地成分入り培養器材を製造することができる。 The culture equipment of the present invention can be produced by any method, but an example thereof will be described, but the present invention is not limited thereto.
A synthetic resin flat plate such as an acrylic plate having an appropriate size can be used as the first member and the second member. The concave portion of the first member and / or the convex portion of the second member can be produced by using a synthetic resin processing and molding method. It can be manufactured by injection molding or the like.
The medium component used in the present invention is prepared by dissolving or suspending it in a non-aqueous solvent, uniformly applying it to the entire surface of the concave portion of the first member and / or the convex portion of the second member, and then quickly forcibly drying or the like. By drying, it can be applied to the concave portion of the first member and / or the convex portion of the second member of the culture medium.
Here, the non-aqueous solvent is preferably a volatile solvent that can rapidly volatilize under normal temperature and pressure, and is preferably one or more lower alcohols selected from, for example, ethanol, methanol, propanol, butanol and the like (preferably). The number of carbon atoms 1 to 4) is preferably mentioned. By using these non-aqueous solvents, the medium component can be applied without gelling the gelling agent at the time of production, so that the culture medium containing the medium component can be easily and efficiently produced.
＜２．本発明の実施形態に係る培養器材の例＞
　本発明の微生物用培養器材に関する実施形態の例を以下に説明するが、これらに限定されない。また、適宜、培地成分入り微生物用培養器材に適用してもよい。
　以下、本発明の第一実施形態及び第二実施形態の培養器材の説明において、＜１．本発明に係る微生物用培養器材＞の構成と重複する、第１部材、第２部材、溝部、ヒンジ部、スタッキング用凸部、培地成分などの各構成などの説明については適宜省略するが、当該＜１．＞の説明が、本実施形態にも当てはまり、当該説明を適宜採用することができる。また、第一実施形態及び第二実施形態の構成を適宜、組み合わせてもよい。 <2. Example of culture equipment according to the embodiment of the present invention>
Examples of embodiments relating to the culture equipment for microorganisms of the present invention will be described below, but the present invention is not limited thereto. Further, it may be appropriately applied to a culture medium for microorganisms containing a medium component.
Hereinafter, in the description of the culture equipment of the first embodiment and the second embodiment of the present invention, <1. The description of each configuration of the first member, the second member, the groove portion, the hinge portion, the convex portion for stacking, the medium component, etc., which overlaps with the configuration of the culture device for microorganisms according to the present invention, will be omitted as appropriate. <1. > The description also applies to the present embodiment, and the description can be appropriately adopted. Further, the configurations of the first embodiment and the second embodiment may be appropriately combined.
　＜本第一実施形態＞
　本発明の第一実施形態の培養器材として、ヒンジ部の断面形状が四角形状を有する培養器材の１例を、図６に表すが、これに限定されない。
　本第一実施形態は、ヒンジ部の断面形状が四角形状を有する培養器材であり、培地成分を収容可能な凹部を有する第１部材と、当該凹部に嵌入し得る凸部を有する第２部材と、を有し、前記嵌入がなされた状態で当該凸部の天面が前記凹部の底面に接触状態となる構成を備えることが好適である。当該ヒンジ部は、第１部材と第２部材に連設されてもよく、凹部の中心を通過する線Ｉ－Ｉ’上に配置されてもよい。 <First Embodiment>
As the culture equipment of the first embodiment of the present invention, an example of the culture equipment having a rectangular cross-sectional shape of the hinge portion is shown in FIG. 6, but is not limited thereto.
The first embodiment is a culture device having a rectangular cross-sectional shape of a hinge portion, and has a first member having a concave portion capable of accommodating a medium component and a second member having a convex portion that can be fitted into the concave portion. , And it is preferable to have a configuration in which the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state. The hinge portion may be connected to the first member and the second member in series, or may be arranged on a line I-I'passing through the center of the recess.
　さらに、本第一実施形態の凹部又は凸部の少なくとも一方の外周領域に溝部を単数又は複数備えることがより好適である。当該溝部は、前記凹部又は前記凸部を全周することが好適であり、溝部の数は１つが好適である。当該溝部の断面形状（線Ｉ－Ｉ’）は、底面が曲面（例えばＵ字状等）であることが好適である。
　Ｚ軸方向（図６の平面方向又は底面方向）からみたときの第１部材及び第２部材の形状は、三角形状、長方形状が好適であり、長方形状がより好適である。第２部材の凸部は、天面の反対側の外部からみたときに中空の円柱状であってもよい。第１部材と第２部材との外周領域は、接触状態となる構成であることが好適であり、天面側及び底面側にある外周領域の面は平面であることがより好適である。
　前記第１部材又は前記第２部材のいずれかの外周領域に、スタッキング用凸部を、単数又は複数、適宜形成してもよい。複数のスタッキング用凸部は、等間隔で配置することが好適であり、溝部の周端領域に配置することがより好適である。 Further, it is more preferable to provide one or more grooves in at least one outer peripheral region of the concave portion or the convex portion of the first embodiment. The groove portion is preferably all around the concave portion or the convex portion, and the number of the groove portions is preferably one. It is preferable that the cross-sectional shape (line I-I') of the groove portion has a curved surface (for example, a U-shape) on the bottom surface.
The shapes of the first member and the second member when viewed from the Z-axis direction (planar direction or bottom surface direction in FIG. 6) are preferably triangular or rectangular, and more preferably rectangular. The convex portion of the second member may be a hollow columnar shape when viewed from the outside on the opposite side of the top surface. It is preferable that the outer peripheral region of the first member and the second member is in a contact state, and it is more preferable that the surfaces of the outer peripheral region on the top surface side and the bottom surface side are flat.
A single or a plurality of stacking protrusions may be appropriately formed in the outer peripheral region of either the first member or the second member. It is preferable to arrange the plurality of stacking convex portions at equal intervals, and it is more preferable to arrange them in the peripheral end region of the groove portion.
＜本第二実施形態＞
　また、本発明の第二実施形態の培養器材において、第１部材及び第２部材は、それぞれ、複数の凸部及び凹部を有していてもよい。すなわち、嵌入時に凹部及び凸部の接触状態が複数形成される態様であってもよく、一度に複数の検体を並行して処理するのに適する。このとき、第２部材の凹部の外周領域に、溝部を一部又は全部に備えることがより好適である。
　また、上記＜１．＞で説明した、嵌入がなされた状態の第１部材及び第２部材を１画分として、当該画分を複数有する、培養器材であってもよい。
　本発明の第二実施形態として、培地成分を有用可能な凹部を複数有する第１部材と、前記凹部に嵌入し得る凸部を複数有する第２部材と、を、有し、
　前記嵌入がなされた状態で前記凹部の天面が前記凹部の底面に接触状態となる構成を、複数備える微生物用培養器材を提供することができる。
　より好ましい態様として、前記第１部材の凹部ごとに１対１で対応し嵌入可能なように前記凸部を、前記第２部材に複数有することが好適である。
　より好ましい態様として、前記複数の凹部ごとの外周領域に溝部を単数又は複数備える、及び／又は、前記複数の凸部ごとの外周領域に溝部を単数又は複数備える。 <The second embodiment>
Further, in the culture equipment of the second embodiment of the present invention, the first member and the second member may have a plurality of convex portions and concave portions, respectively. That is, a plurality of contact states of the concave portion and the convex portion may be formed at the time of fitting, and it is suitable for processing a plurality of samples in parallel at one time. At this time, it is more preferable to provide a part or all of the groove portion in the outer peripheral region of the concave portion of the second member.
In addition, the above <1. >, The first member and the second member in the fitted state may be regarded as one fraction, and the culture equipment may have a plurality of the fractions.
As a second embodiment of the present invention, it has a first member having a plurality of recesses in which a medium component can be useful, and a second member having a plurality of protrusions that can be fitted into the recesses.
It is possible to provide a culture equipment for microorganisms having a configuration in which the top surface of the recess is in contact with the bottom surface of the recess in the fitted state.
As a more preferable embodiment, it is preferable that the second member has a plurality of the convex portions so that the concave portions of the first member can be fitted in a one-to-one correspondence.
As a more preferable embodiment, the outer peripheral region of each of the plurality of concave portions is provided with one or more grooves, and / or the outer peripheral region of each of the plurality of convex portions is provided with one or more grooves.
＜３．本発明に係る培地成分入り微生物用培養器材＞
　本発明の別の側面として、上記培養器材の前記凹部に培地成分が収容された状態で前記凸部が嵌入された構成を備える、培地成分入り微生物用培養器材を提供することができる。
　本実施形態の説明において、上述した＜１．本発明に係る微生物用培養器材＞＜２．本発明の実施形態に係る培養器材の例＞と重複する、第１部材、第２部材、培地成分などの各構成などの説明については適宜省略するが、当該＜１．＞＜２．＞の説明が、本実施形態にも当てはまり、当該説明を適宜採用することができる。 <3. Microbial culture equipment containing medium components according to the present invention>
As another aspect of the present invention, it is possible to provide a culture medium-containing culture medium-containing culture medium having a structure in which the convex portion is fitted while the medium component is housed in the concave portion of the culture medium.
In the description of this embodiment, the above-mentioned <1. Microbial culture equipment according to the present invention><2. The description of each configuration of the first member, the second member, the medium component, etc., which overlaps with the example of the culture equipment according to the embodiment of the present invention, will be omitted as appropriate, but the above <1. ＞ ＜ 2. > The description also applies to the present embodiment, and the description can be appropriately adopted.
＜４．本発明に係る微生物数の計測方法＞
　本発明の別の側面として、上記説明した本発明の培養器材及び培地成分入り培養器材は、検体中の微生物を培養し、該微生物数を計測する方法に好適に用いることができる。
　本発明は、培地成分を収容可能な凹部を有する第１部材と、前記凹部に嵌入し得る凸部を有する第２部材と、を有し、前記嵌入がなされた状態で前記凸部の天面が前記凹部の底面に接触状態となる構成を備える、培地成分入り微生物用培養器材を用いて、検体中の微生物を培養し、微生物数を計測する方法を提供することができる。
　本実施形態の説明において、上述した＜１．本発明に係る微生物用培養器材＞＜２．本発明の実施形態に係る培養器材の例＞＜３．本発明に係る培地成分入り微生物用培養器材＞と重複する、第１部材、第２部材、培地成分などの各構成などの説明については適宜省略するが、当該＜１．＞＜２．＞＜３．＞の説明が、本実施形態にも当てはまり、当該説明を適宜採用することができる。 <4. Method for measuring the number of microorganisms according to the present invention>
As another aspect of the present invention, the culture equipment of the present invention and the culture equipment containing a medium component described above can be suitably used for a method of culturing microorganisms in a sample and measuring the number of the microorganisms.
The present invention has a first member having a concave portion capable of accommodating a medium component and a second member having a convex portion that can be fitted into the concave portion, and the top surface of the convex portion in the fitted state. It is possible to provide a method for culturing a microorganism in a sample and measuring the number of microorganisms by using a culture medium-containing culture medium-containing culture medium having a structure in which the invention is in contact with the bottom surface of the recess.
In the description of this embodiment, the above-mentioned <1. Microbial culture equipment according to the present invention><2. Example of culture equipment according to the embodiment of the present invention><3. The description of each configuration of the first member, the second member, the medium component, etc., which overlaps with the culture medium-containing culture medium-containing culture medium component according to the present invention, will be omitted as appropriate, but the above <1. ＞ ＜ 2. ＞ ＜ 3. > The description also applies to the present embodiment, and the description can be appropriately adopted.
　該計測方法は、具体的には、培養器材の凹部に検体液を添加する工程、
　添加後に、前記第１部材の凹部に前記第２部材の凸部を嵌入する工程、
　嵌入後に、前記検体に含まれる微生物を培養する工程、及び
　培養後に、前記微生物のコロニー数を計測する工程、を含むことが好ましい。
　前記嵌入工程において、第２部材を第１部材に被せることで、第１部材の凹部に第２部材の凸部が嵌入し、嵌入することで、第１部材の凹部に添加された検体液を培地成分全体に均一に押し広げることができる。また、均一に押し広げられた検体液の水分が、培地成分中のゲル化剤により吸収され、速やかにゲル化することで、検体を含み固化した状態の培養使用時の培地が容易に形成される。 Specifically, the measuring method is a step of adding a sample solution to a recess of a culture device.
A step of fitting the convex portion of the second member into the concave portion of the first member after the addition.
It is preferable to include a step of culturing the microorganism contained in the sample after embedding, and a step of measuring the number of colonies of the microorganism after culturing.
In the fitting step, by covering the first member with the second member, the convex portion of the second member is fitted into the concave portion of the first member, and by fitting, the sample liquid added to the concave portion of the first member is applied. It can be spread evenly over the entire medium component. In addition, the water content of the sample solution that has been uniformly spread is absorbed by the gelling agent in the medium components and rapidly gels, so that a medium that contains the sample and is solidified when used in culture is easily formed. To.
　微生物の培養条件は、特に限定されないが、対象微生物の種類により適正に選ばれるが、例えば、細菌を培養する場合等では、３５±２℃で２４～４８時間が好ましい。
　培養後の培地中には、対象微生物の生育コロニーが出現するので、これを計測する。微生物のコロニー数の計測は、培養器材を分解することなく外部から目視によって確認したり、カメラ等で撮像したものを画像解析ソフトで解析したりすることによって、計測することができる。本発明の計測方法によれば、正確にコロニー数を計測することができる。 The culture conditions of the microorganism are not particularly limited, but are appropriately selected depending on the type of the target microorganism. For example, in the case of culturing a bacterium, 24 to 48 hours at 35 ± 2 ° C. is preferable.
Growth colonies of the target microorganisms appear in the medium after culturing, and these are measured. The number of colonies of microorganisms can be measured by visually confirming the number of colonies from the outside without disassembling the culture equipment, or by analyzing what is imaged by a camera or the like with image analysis software. According to the measuring method of the present invention, the number of colonies can be accurately measured.
　本発明の計測方法を適用しうる検体としては、特に限定されないが、飲料水、清涼飲料水、工業用水、製薬用水、透析水、尿等の液体検体等が好ましく挙げられる。また、固形の検体を希釈液等により乳剤としたものも含まれる。さらに、これらの検体を予めトリプトソイブイヨン等で培養した培養液であってもよい。
　また、本発明の計測方法は上記検体をリン酸食塩緩衝液等の希釈液で希釈した検体にも、本発明の計測方法に好ましく供することができる。 The sample to which the measurement method of the present invention can be applied is not particularly limited, and preferably includes liquid samples such as drinking water, soft drinks, industrial water, pharmaceutical water, dialysate, and urine. Further, a solid sample made into an emulsion by a diluted solution or the like is also included. Further, a culture solution in which these samples are previously cultured in trypto-soy bouillon or the like may be used.
Further, the measuring method of the present invention can be preferably applied to the measuring method of the present invention even for a sample obtained by diluting the above sample with a diluted solution such as a phosphate buffer solution.
　次に実施例を挙げて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
　（１）培養器材の作製
＜実施例１＞
　実施例１の培養器材として、図６に示す培養器材に、スタッキング用凸部を凹部の外周端部に等間隔に３箇所設けたポリスチレンシート製を作製した（図１０及び図１１参照）。具体的には、実施例１のポリスチレンシート製培養器材として、透明な０．２ｍｍ厚ポリスチレン製の長方形状のシートを成形し、直径５６．４３ｍｍ（図１のＤ（ｍｍ）参照）、高さ２．００ｍｍの中空円柱状の凹部（底面積２５ｃｍ２）を有する下部材と、この凹部と完全に勘合する同体積の円柱状の凸部（高さ２．００ｍｍ）を中央に有する上部材とをそれぞれ設計し作製した。下部材及び上部材は略同じ面積の長方形状に設計されている。実施例１の培養器材は、凸部が凹部に嵌入し、嵌入がなされた状態で凸部の天面が凹部の底面に接触状態となる構成に設計され、凸部の嵌入部分の高さのｍｍが凹部の嵌入部分の深さのｍｍと同じｍｍに設計されており、嵌入がなされた状態で天面と底面との距離０．００１ｍｍ以下になるような構成に設計されている。 (1) Preparation of culture equipment <Example 1>
As the incubator of Example 1, a polystyrene sheet was produced in which the incubator shown in FIG. 6 was provided with three stacking convex portions at equal intervals at the outer peripheral end of the concave portion (see FIGS. 10 and 11). Specifically, as the polystyrene sheet culture equipment of Example 1, a transparent 0.2 mm thick polystyrene rectangular sheet is molded, and has a diameter of 56.43 mm (see D (mm) in FIG. 1) and a height. A lower member having a 2.00 mm hollow cylindrical recess (bottom area 25 cm 2 ) and an upper member having a cylindrical convex portion (height 2.00 mm) of the same volume that completely fits the recess. Was designed and manufactured respectively. The lower member and the upper member are designed to have a rectangular shape having substantially the same area. The incubator of Example 1 is designed so that the convex portion is fitted into the concave portion and the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state, and the height of the fitted portion of the convex portion is increased. The mm is designed to be the same mm as the mm of the depth of the fitting portion of the recess, and the distance between the top surface and the bottom surface is 0.001 mm or less in the fitted state.
　実施例１の培養器材は、上部材と下部材はヒンジ部で連設され、嵌入した状態で末端部分のヒンジ部は線Ｉ－Ｉ’による断面が四角形状になるように構成されている。このヒンジ部の反対側は、培養器材の開閉用の取手部とすることができる。短手方向の部材長さの半分の位置上に凹部の中心を配置し、この中心はヒンジ部側方向にやや近いように配置され、ヒンジ部末端及び取手部末端の距離の１０分の３～５に配置されている。
　実施例１の培養器材は、下部材に、凹部の外周領域を完全に囲うような隔離用の溝部を設けており、溝部は、凹部の外周（０ｍｍ）から１ｍｍ以上３ｍｍ以下の範囲に配置され、溝部の深さは１～３ｍｍ、溝部の幅は２～３ｍｍで設計されている。溝部の底面の形状は曲面であり、断面形状はＵ字状となっている。 In the incubator of the first embodiment, the upper member and the lower member are connected to each other by a hinge portion, and the hinge portion at the end portion is configured such that the cross section of the end portion has a quadrangular cross section by the line I-I'in the fitted state. The opposite side of this hinge portion can be a handle portion for opening and closing the incubator. The center of the recess is placed on the position of half the length of the member in the lateral direction, and this center is placed so as to be slightly closer to the hinge portion side, and the distance between the end of the hinge portion and the end of the handle portion is 3/10 to 3/10. It is arranged in 5.
The incubator of Example 1 is provided with a groove portion for isolation in the lower member so as to completely surround the outer peripheral region of the recess, and the groove portion is arranged in a range of 1 mm or more and 3 mm or less from the outer circumference (0 mm) of the recess. The groove depth is designed to be 1 to 3 mm, and the groove width is designed to be 2 to 3 mm. The shape of the bottom surface of the groove is a curved surface, and the cross-sectional shape is U-shaped.
＜比較例１＞
　一方、比較例１のポリスチレンシート製培養器材は、特許文献５（特開２０１９－１８０３６９号公報）の態様を再現したものであり、上部材の凸部と下部材の凹部が嵌入した状態において培地領域という空間が形成されるように、かつ下部材に溝部を設けていない。 <Comparative Example 1>
On the other hand, the polystyrene sheet culture equipment of Comparative Example 1 reproduces the aspect of Patent Document 5 (Japanese Unexamined Patent Publication No. 2019-180369), and is a medium in a state where the convex portion of the upper member and the concave portion of the lower member are fitted. No groove is provided in the lower member so that a space called a region is formed.
　上述のように、実施例１のポリスチレンシート製培養器材及び比較例１のポリスチレンシート製培養器材の２種を用意した。
　１００ｍＬ調製用量（３ｇ）のトリプトソイブイヨン培地粉末（ベクトン・ディッキンソン製）、０．００２５ｇのＴＴＣ、それぞれ１.５ｇのキサンタンガム及びグアーガム
、及び０．１ｇのヒドロキシプロピルセルロースを、１００ｍＬのエタノールに懸濁し、混合溶液を得た。該混合溶液１０００μＬを、上記作製した２種のポリスチレンシート製培養器材の下部材の凹部の底面に添加し、底面上に均一に広げた後、これをヒートブロック上に置き７０℃で５分間乾燥させ、実施例及び比較例の培地成分入り培養器材をそれぞれ作製した。 As described above, two types of the polystyrene sheet culture equipment of Example 1 and the polystyrene sheet culture equipment of Comparative Example 1 were prepared.
A 100 mL preparation dose (3 g) of trypto soy bouillon medium powder (Becton Dickinson), 0.0025 g of TTC, 1.5 g of xanthan gum and guar gum, respectively, and 0.1 g of hydroxypropyl cellulose were suspended in 100 mL of ethanol. , A mixed solution was obtained. 1000 μL of the mixed solution is added to the bottom surface of the concave portion of the lower member of the two types of polystyrene sheet culture equipment prepared above, spread uniformly on the bottom surface, and then placed on a heat block and dried at 70 ° C. for 5 minutes. Then, the culture equipment containing the medium components of Examples and Comparative Examples was prepared.
（２）菌株の供試
　供試菌株はＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＡＴＣＣ　６６３３及びＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＡＴＣＣ　８７３９を使用し、トリプトソイ寒天培地で２４時間前培養した後、マクファーランド比濁＃１相当（約３．０×１０８ＣＦＵ／ｍＬ）になるように滅菌綿棒を用いて滅菌生理食塩水に懸濁し、菌原液とした。各菌原液を用いて、滅菌生理食塩水にて１０倍段階希釈を１０－８まで繰り返し、数１０ＣＦＵ／ｍＬの菌希釈液を調製した。この菌希釈液１ｍＬを上記「（１）培養器材の作製」で作製した実施例及び比較例の培養器材の下部材の凹部にそれぞれ接種し、すぐに上部材の凸部を嵌入して、菌希釈液を凹部に均一に広げ、培地成分に菌希釈液を浸透させて、培地を形成させた。検体を含んだ培地入り培養器材（実施例、比較例）を、３５℃で２４時間、インキュベータ内にて培養した後、実施例及び比較例の培養器材における菌株の発育の状況（有無、コロニー数）を確認した。
　なお、使用したＡＴＣＣ菌株は、ＡＴＣＣ（American Type Culture Collection）に保存され、ここから入手可能である。 (2) Test of strain The test strain used Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739, and after culturing in trypto-soy agar medium for 24 hours, it was equivalent to McFarland turbidity # 1 (about 3.0 × 10). 8 CFU / mL) was suspended in sterile physiological saline using a sterile cotton swab to prepare a bacterial stock solution. Using each bacterial stock solution, 10-fold serial dilution with sterile physiological saline was repeated up to 10-8 to prepare a bacterial diluted solution of several tens of CFU / mL. 1 mL of this diluted solution of bacteria was inoculated into the concave portions of the lower members of the culture equipment of the examples and comparative examples prepared in the above "(1) Preparation of culture equipment", and immediately the convex portions of the upper members were inserted to inoculate the bacteria. The diluted solution was spread evenly in the recesses, and the bacterial diluted solution was infiltrated into the medium components to form a medium. After culturing the culture medium-containing culture equipment (Example, Comparative Example) containing the sample in the incubator at 35 ° C. for 24 hours, the growth status (presence / absence, number of colonies) of the strains in the culture equipment of the Examples and Comparative Examples. )It was confirmed.
The ATCC strain used is stored in the ATCC (American Type Culture Collection) and can be obtained from here.
　図１０にＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＡＴＣＣ　６６３３菌株を用いた場合の発育コロニー状況を、図１１にＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＡＴＣＣ　８７３９菌株を用いた場合の発育コロニー状況を示す。このとき、図１０及び１１は、実施例１のポリスチレンシート製培養器材を用いている。 FIG. 10 shows the growth colony status when the Bacillus subtilis ATCC 6633 strain is used, and FIG. 11 shows the growth colony status when the Escherichia coli ATCC 8739 strain is used. At this time, FIGS. 10 and 11 use the polystyrene sheet culture equipment of Example 1.
　実施例として作製した本発明の培養器材では、試料液を下部材の凹部に接種し、上部材の凸部が完全に嵌入するように被せると、直ちに検体が凹部の底面全体に均一に広がり、速やかに水分が吸収され、透明な培地が形成された。このとき、凹部と凸部が完全に勘合されると１ｍL分の検体体積がないため、接種した試料液は外部に溢れ出ることが予想されるが、本発明の培養器材では、驚くべきことに若干凸部が持ち上がる程度で膨潤したゲルが速やかに固化し、一切の試料液の水分が溢れ出ることを認めず、均一で透明な培地が構成されることを認めた。また、ゲル化させた培地の培養後の様子は図１０及び１１に示すように、明瞭な発色コロニーを形成することを認め、コロニーが離水等により拡散することを認めなかった。
　本発明の培養器材に備えた溝の役割についてより詳細に観察した。検体液を添加したとき、本発明の培養器材に収容された培地成分中のゲル化剤が水にて膨潤し固化するには若干の時間差があるため、培地成分に添加した検体液の水分が凹部から若干染み出すこともあった。しかし、本発明の培養器材の溝により毛細管現象が遮断されるため、凹部から染み出した少量の水は、溝に落ち込むことなく、凹部の外周縁部と溝の内周縁部との間で止まり、その後培地成分中のゲル化剤がこの間にある水分を吸収することにより、凹部内に吸い寄せられた。このように、培地成分に添加された検体液の水分は溝に落ち込むことなく、培養使用時の培地は正確な培地濃度となると共に、培養後に観察するときの微生物コロニーは全て凹部内に形成された。 In the culture medium of the present invention produced as an example, when the sample solution is inoculated into the concave portion of the lower member and covered so that the convex portion of the upper member is completely fitted, the sample immediately spreads uniformly over the entire bottom surface of the concave portion. Moisture was rapidly absorbed and a clear medium was formed. At this time, if the concave portion and the convex portion are completely fitted, the sample volume for 1 mL is not available, so that the inoculated sample solution is expected to overflow to the outside. It was confirmed that the gel that had swollen to the extent that the convex part was slightly lifted solidified rapidly, and that no water in the sample solution overflowed, and that a uniform and transparent medium was formed. In addition, as shown in FIGS. 10 and 11, it was observed that the gelled medium formed clear colored colonies, and that the colonies did not diffuse due to water separation or the like.
The role of the groove provided in the incubator of the present invention was observed in more detail. When the sample solution is added, there is a slight time lag for the gelling agent in the medium component contained in the culture medium of the present invention to swell and solidify with water, so that the water content of the sample solution added to the medium component is increased. It sometimes exuded from the recess. However, since the capillary phenomenon is blocked by the groove of the culture medium of the present invention, a small amount of water exuded from the recess does not fall into the groove and stops between the outer peripheral edge of the recess and the inner peripheral edge of the groove. After that, the gelling agent in the medium component was attracted into the recess by absorbing the water in between. In this way, the water content of the sample solution added to the medium components does not fall into the groove, the medium at the time of use for culture has an accurate medium concentration, and all the microbial colonies when observed after culture are formed in the recesses. rice field.
　一方、比較例として作製した、凹部を完全に囲うような隔離用の溝を設けない培養器材では、凹部と凸部が完全に勘合されると凹部に空気層が発生し、さらに上下部材が接触する際の毛細管現象により、凹部の周囲へ水分が漏れ出してしまうことを認めた。しかも、本現象は上下部材の凹凸部を相当慎重に勘合させてもゲル化よりも早い段階で周囲へ漏れ出してしまうことを認めた。これにより、塗布された培地が均一に再構成されず、きれいな培養例を作ることが困難であった。また、菌液が外部へ流出し漏れることは好ましくはない。
　比較例の培養器材ではさらに培養時に培地ゲル上に存在する菌が空気層のゲル表面上で広がり、周囲のコロニーと重なり、計測が困難になる例が多数見られた。 On the other hand, in the culture equipment produced as a comparative example, which does not have a groove for isolation that completely surrounds the concave portion, when the concave portion and the convex portion are completely fitted, an air layer is generated in the concave portion, and the upper and lower members come into contact with each other. It was found that water leaked to the periphery of the recess due to the capillary phenomenon. Moreover, it was found that this phenomenon leaks to the surroundings at an earlier stage than gelation even if the uneven parts of the upper and lower members are fitted very carefully. As a result, the applied medium was not uniformly reconstituted, and it was difficult to make a clean culture example. Further, it is not preferable that the bacterial solution flows out and leaks to the outside.
In the culture equipment of the comparative example, there were many cases in which the bacteria existing on the medium gel during culturing spread on the gel surface of the air layer and overlapped with the surrounding colonies, making measurement difficult.
　すなわち、本発明の培養器材を用いると、１ｍＬ検体の適用時に凹凸部により形成される空間体積や器材の接触角に関係なく、さらにスプレッダー等の器具や不織布等による毛細管現象によらずとも、上部材と下部材を完全に勘合させるだけで、凹部に空気層を発生させずに均一に凹部全体に液体試料を広げることができた。また、培地成分中のゲル化剤のゲルは速やかに固化し、試料液が溢れることなく試料液を含む使用時の培地を簡便に構築することができた。このように、簡便な操作で培養することができた。培養後は図１０及び図１１に示すように凹部のみに形成された透明なゲルの中に明瞭な赤色のコロニーが目視により確認でき、容易にそのコロニー数を計測することができた。また、本発明の培養器材は複雑な構成ではないため、容易かつ効率的に作製することができた。 That is, when the culture equipment of the present invention is used, regardless of the space volume formed by the uneven portion when the 1 mL sample is applied and the contact angle of the equipment, and even if it is not due to the capillary phenomenon caused by an instrument such as a spreader or a non-woven fabric, etc. By simply fitting the member and the lower member completely, the liquid sample could be spread evenly over the entire recess without generating an air layer in the recess. In addition, the gel of the gelling agent in the medium component was rapidly solidified, and the medium at the time of use containing the sample solution could be easily constructed without overflowing the sample solution. In this way, it was possible to culture by a simple operation. After culturing, clear red colonies could be visually confirmed in the transparent gel formed only in the recesses as shown in FIGS. 10 and 11, and the number of colonies could be easily measured. Moreover, since the incubator of the present invention does not have a complicated structure, it could be easily and efficiently produced.
　そして、本発明者は、２０～３０ｃｍ２の円状の底面積の凹部を有する皿状部材と、その凹部と同体積で凹部と完全に勘合する凸部を有する蓋部材を有し、凹部又は凸部のいずれか片方を完全に囲うような隔離用の溝を有する培養器材を設け、この培養器材の凹部あるいは凸部の少なくとも片側に、培地成分を塗布することで、１ｍＬ検体液の適用時にフタを閉めるという簡単な操作のみで、凹凸部により形成される空間体積や器材の接触角に関係なく、また器具や毛細管現象等を利用せずとも、液体検体が外部に漏れ出すことなく均一に拡散し一定面積の培地形成が可能となり、より簡便な微生物の培養及び計測を実現できることに至った。そして、培地を形成するゲル化剤として、グアーガム、キサンタンガム又はそれらの混合物が、操作の簡便性や外部からの視認性の高さの観点から好適であることを見出した。 The present inventor has a dish-shaped member having a concave portion having a circular bottom area of 20 to 30 cm 2 , and a lid member having a convex portion having the same volume as the concave portion and completely fitting with the concave portion. When a 1 mL sample solution is applied, a culture device having a groove for isolation that completely surrounds one of the convex portions is provided, and the medium component is applied to at least one side of the concave portion or the convex portion of the culture device. With just a simple operation of closing the lid, the liquid sample does not leak to the outside evenly regardless of the space volume formed by the uneven part and the contact angle of the equipment, and without using equipment or capillary phenomenon. It diffused and made it possible to form a medium with a certain area, which led to the realization of simpler culture and measurement of microorganisms. Then, they have found that guar gum, xanthan gum or a mixture thereof is suitable as a gelling agent for forming a medium from the viewpoint of ease of operation and high visibility from the outside.
　さらに、本発明の培養器材は、真空成形法や射出成形法などの一般的な樹脂成形法により成形することができる。当該成形された器材構造自体が、従来技術の培養器材及びこれを用いた微生物計測方法に存在していた欠点をも改善できる。このため、本発明は、別の側面として、極めて簡単で安価な微生物用培養器材の製造方法を提供できる発明でもある。 Further, the culture equipment of the present invention can be molded by a general resin molding method such as a vacuum forming method or an injection molding method. The molded equipment structure itself can improve the defects existing in the conventional culture equipment and the microorganism measurement method using the same. Therefore, as another aspect, the present invention is also an invention capable of providing an extremely simple and inexpensive method for producing a culture device for microorganisms.
　よって、本発明は、新規な微生物用培養器材、培地成分入り微生物培養用器材、及びこれを用いた微生物数計測法を提供することができる。また、本発明は、複雑な構成ではないため、製造も簡単にできる微生物用培養器材を提供することができる。
　このように、本発明の微生物用培養器材は、操作性が高く、安全に使用でき、かつ簡単に製造することができ、さらに、検体中の微生物数を容易に計測することができる。 Therefore, the present invention can provide a novel microbial culture equipment, a microbial culture equipment containing a medium component, and a method for measuring the number of microorganisms using the same. Further, since the present invention is not a complicated structure, it is possible to provide a culture equipment for microorganisms that can be easily produced.
As described above, the culturing equipment for microorganisms of the present invention has high operability, can be used safely, can be easily manufactured, and can easily measure the number of microorganisms in a sample.
　本発明によれば、検体中の微生物を、簡便な操作で培養し、その数を容易に計測することができる。また、本発明の培養器材は、複雑な構成ではないため、製造も簡単であるため、産業上有用である。 According to the present invention, microorganisms in a sample can be cultured by a simple operation and the number thereof can be easily measured. In addition, the incubator of the present invention is industrially useful because it does not have a complicated structure and is easy to manufacture.
１：微生物用培養器材、１０：第１部材、１１：凹部、１２：底面、２０：第２部材、２１：凸部、２２天面３０：溝部、４０：培地成分、５０：ヒンジ部、６０：スタッキング用凸部、１００：培地成分入り微生物用培養器材 1: Culture equipment for microorganisms, 10: 1st member, 11: concave part, 12: bottom surface, 20: 2nd member, 21: convex part, 22 top surface 30: groove part, 40: medium component, 50: hinge part, 60 : Convex for stacking, 100: Culture equipment for microorganisms containing medium components

Claims (14)

  1. 　培地成分を収容可能な凹部を有する第１部材と、
    　前記凹部に嵌入し得る凸部を有する第２部材と、を有し、
    　前記嵌入がなされた状態で前記凸部の天面が前記凹部の底面に接触状態となる構成を備える、微生物用培養器材。     A first member having a recess capable of accommodating a medium component,
    It has a second member having a convex portion that can be fitted into the concave portion, and has.
    An incubator for microorganisms having a structure in which the top surface of the convex portion is in contact with the bottom surface of the concave portion in the fitted state.
  2. 　前記凹部又は前記凸部の少なくとも一方の外周領域に溝部を備える、請求項１に記載の微生物用培養器材。 The microbial incubator according to claim 1, wherein a groove is provided in the concave portion or at least one outer peripheral region of the convex portion.
  3. 　前記溝部は、前記培地成分に添加された検体液が毛細管現象により前記凹部の外へ漏れ出することを抑制する、請求項２に記載の微生物用培養器材。 The microbial culture device according to claim 2, wherein the groove portion suppresses the sample liquid added to the medium component from leaking out of the recess due to a capillary phenomenon.
  4. 　前記第１部材及び／又は前記第２部材は光透過性の合成樹脂材で形成される、請求項１から３のいずれか一項に記載の微生物用培養器材。 The microbial incubator according to any one of claims 1 to 3, wherein the first member and / or the second member is made of a light-transmitting synthetic resin material.
  5. 　前記第１部材と前記第２部材は一体成形されている、請求項１から４のいずれか一項に記載の微生物用培養器材。 The microorganism culture equipment according to any one of claims 1 to 4, wherein the first member and the second member are integrally molded.
  6. 　前記第１部材と前記第２部材はヒンジ部により連設されている、請求項１から５のいずれか一項に記載の微生物用培養器材。 The microorganism culture equipment according to any one of claims 1 to 5, wherein the first member and the second member are connected by a hinge portion.
  7. 　前記ヒンジ部は、前記凸部の前記天面を前記底面に対して平行状態を維持しつつ嵌入し得る構造を備える、請求項６に記載の微生物用培養器材。 The microorganism culture device according to claim 6, wherein the hinge portion has a structure capable of fitting the top surface of the convex portion while maintaining a parallel state with respect to the bottom surface.
  8. 　前記第１部材又は前記第２部材のいずれかの外周領域に、スタッキング用凸部が形成される、請求項１から７のいずれか一項に記載の微生物用培養器材。 The microorganism culture equipment according to any one of claims 1 to 7, wherein a stacking convex portion is formed in the outer peripheral region of either the first member or the second member.
  9. 　請求項１から８のいずれか一項に記載の微生物用培養器材の前記凹部に培地成分が収容された状態で前記凸部が嵌入された構成を備える、培地成分入り微生物用培養器材。 A microbial culture device containing a medium component, comprising a configuration in which the convex portion is fitted in a state in which the medium component is housed in the concave portion of the microbial culture device according to any one of claims 1 to 8.
  10. 　前記培地成分は、ゲル化剤と栄養成分とを少なくとも含む、請求項９に記載の培地成分入り微生物用培養器材。 The culture medium component for microorganisms according to claim 9, wherein the medium component contains at least a gelling agent and a nutritional component.
  11. 　前記ゲル化剤は、グアーガム、キサンタンガム、及びそれらの混合物から選択される１種以上である、請求項１０に記載の培地成分入り微生物用培養器材。 The culturing device for microorganisms containing a medium component according to claim 10, wherein the gelling agent is one or more selected from guar gum, xanthan gum, and a mixture thereof.
  12. 　前記培地成分には、前記第１部材及び前記第２部材に対する接着性成分が含まれている、請求項９から１１のいずれか一項に記載の培地成分入り微生物用培養器材。 The culture medium component-containing culture medium component according to any one of claims 9 to 11, wherein the medium component contains an adhesive component to the first member and the second member.
  13. 　請求項９から１２のいずれか一項に記載の培地成分入り微生物用培養器材を用いて、検体中の微生物を培養し、微生物数を計測する方法。 A method for culturing microorganisms in a sample and measuring the number of microorganisms by using the culture medium-containing culture equipment for microorganisms according to any one of claims 9 to 12.
  14. 　前記培地成分入り微生物用培養器材の凹部に検体を添加する工程、
    　添加後に、前記第１部材の凹部に前記第２部材の凸部を嵌入する工程、
    　嵌入後に、前記検体に含まれる微生物を培養する工程、及び
    　培養後に、前記微生物のコロニー数を計測する工程、
    を含む、請求項１３に記載の方法。     The step of adding a sample to the recess of the culture medium-containing culture medium containing a medium component,
    A step of fitting the convex portion of the second member into the concave portion of the first member after the addition.
    A step of culturing the microorganism contained in the sample after embedding, and a step of measuring the number of colonies of the microorganism after culturing.
    13. The method of claim 13.
PCT/JP2021/029299 2020-08-21 2021-08-06 Culturing device for microorganisms, culturing device for microorganisms and including culture medium components, and method for counting microorganisms using same WO2022039054A1 (en)

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