JP2011250742A - Microbiological culture sheet, method of manufacturing the same, and injector of medium liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microbiological culture sheet which can spread suspected liquid on a culture sheet to a predetermined area without requiring complicated operations, nor generating the loss of operation time, and capable of performing quick inoculation operation, is equipped with a culture layer required with even thickness enough for growing microorganisms, can perform an accurate microbiological test, and reduces wastes of expensive microbiological culture materials.SOLUTION: The microbiological culture sheet includes a dried culture layer in a predetermined area on a base material sheet. The culture layer has a predetermined area virtually divided in a plurality of division areas. After individually or continuously injected to form the medium layer in the respective division areas, the medium liquid is dried.

Description

  The present invention relates to a microorganism culture sheet that can perform a microorganism test quickly and accurately, a method for producing the microorganism culture sheet, and a medium liquid injection device for producing the microorganism culture sheet.

  As a method for confirming the presence of microorganisms or measuring the number of microorganisms, there is an agar plate mixing method. In this method, since an agar medium formed in a petri dish previously sterilized is used as a medium, an autoclave for autoclaving the agar medium under high-pressure steam sterilization and a laboratory capable of performing microbial inspection aseptically are required. Moreover, skill is required for the operation of the microorganism test from the sampling of the microorganism to the preparation of the sample solution, dispensing, mixing with the medium, culture, and counting. Therefore, development of a microorganism culture sheet having a dried culture layer that can be easily tested for microorganisms without requiring a high degree of skill has been promoted.

  So far, as a sheet-shaped microorganism incubator reported so far, for example, a water-based adhesive composition layer formed on the upper surface of a support and a gelling agent attached to the adhesive composition layer There is a culture substrate device comprising a cold water-soluble powder containing a cover sheet and a cover sheet covering the cold water-soluble powder (see Patent Document 1). In addition, there is a microorganism incubator in which a circular water-soluble polymer compound layer and a porous matrix layer are laminated on a square pressure-sensitive adhesive sheet, and a square transparent film is disposed on the top (see Patent Document 2).

Japanese Patent No. 3383304 International Publication No. 01/044437

  However, in the culture medium apparatus described in Patent Document 1, in order to spread the test solution over a certain area on the culture layer (adhesive composition layer to which the cold water-soluble powder containing the gelling agent is attached) It is necessary to use a spreader that requires skill, and the work becomes complicated when performing a large amount of inspection. Moreover, since the microorganism culture material is expensive, it is preferable that the amount used can be reduced. However, in the culture substrate apparatus described in Patent Document 1, a culture layer is formed over the entire upper surface of the base film as a support. This wastes expensive microbial culture material. In the microorganism incubator described in Patent Document 2, the culture layer is composed of a porous matrix in order to suppress the spread of the test solution. For this reason, it takes time to wait for the porous matrix to absorb the test solution, which causes a loss of time for the operator. In addition, the microorganism incubator described in Patent Document 2 is prepared by cutting a microorganism culture medium produced by laminating a water-soluble polymer compound layer and a porous matrix layer on an adhesive sheet and pasting it on a mount. The manufacturing method is complicated.

  The present invention has been made in view of the above-described problems, and can expand a test solution to a certain area on a culture layer without requiring complicated work and without causing loss of work time. An object of the present invention is to provide a microorganism culture sheet that can be quickly inoculated. It is another object of the present invention to provide a microorganism culture sheet that is necessary and sufficient for the growth of microorganisms and has a culture layer having a uniform thickness and can perform an accurate microorganism test. It is another object of the present invention to provide a microorganism culture sheet in which waste of expensive microorganism culture material is reduced. And it aims at providing the method which can manufacture the said microorganisms culture sheet reliably and continuously. It is another object of the present invention to provide a medium solution injection device for easily producing the microorganism culture sheet.

  The present inventors directly apply a medium solution containing components necessary for the growth of microorganisms to a predetermined region on the base sheet and dry it to form a culture layer. It does not require a complicated operation such as using a spreader as in the described culture medium apparatus, and waits until the porous matrix absorbs the test liquid as in the microorganism culture device described in Patent Document 2. Without incurring a loss of work time, the test solution can be spread over a certain range on the culture layer by inoculating the test solution on the culture layer and then immediately covering with a cover sheet (2 ) It has been found that waste of expensive microbial culture material can be eliminated.

  By the way, in order to grow microorganisms appropriately, a certain amount of thickness is required for the culture layer. Screen printing can be considered as a method of forming a thick culture layer in a predetermined range on the base sheet. However, as a result of studies by the inventors, it was difficult to continuously produce a culture layer having a uniform thickness by screen printing. As a result of further research, the culture layer formation region on the base sheet determined in advance is virtually divided into a plurality of divided regions, and a medium solution is individually injected into each divided region, or continuously. It was found that the above-mentioned problems can be solved by injecting into the solution, and the present invention was completed.

  Specifically, the present invention provides the following.

  (1) A microorganism culture sheet comprising a dried culture layer in a predetermined region on a base sheet, wherein the culture layer virtually divides the predetermined region into a plurality of divided regions, and each divided region A microorganism culture sheet obtained by injecting a medium solution for forming the culture layer individually or continuously and then drying.

  (2) The microorganism culture sheet according to (1), wherein a medium solution is injected into each of the divided regions from above the predetermined region through a porous nozzle.

  (3) The culture medium sheet according to (1) or (2), wherein the medium solution contains an alcohol solvent.

  (4) The microorganism culture sheet according to any one of (1) to (3), wherein the medium solution is a dispersion system in which at least one of the components of the culture layer is dispersed in a solvent.

  (5) The microorganism culture sheet according to any one of (1) to (4), wherein a concave portion is formed in advance in the predetermined region, and the concave portion is the predetermined region.

  (6) A method for producing a microorganism culture sheet comprising a dried culture layer in a predetermined region on a substrate sheet, wherein the predetermined region is virtually divided into a plurality of divided regions, A method for producing a microorganism culture sheet comprising a step of individually or continuously injecting a culture solution for forming the culture layer and then drying to form a culture layer.

  (7) The method for producing a microorganism culture sheet according to (6), further comprising a step of forming a frame layer made of a hydrophobic resin on the outer periphery of the culture layer.

  (8) A method for producing a microorganism culture sheet comprising a dried culture layer in a predetermined region on a base sheet, wherein the predetermined layer is formed by forming a frame layer made of a hydrophobic resin on the base sheet. Forming a recess surrounded by the frame layer in the region, and virtually dividing the recess into a plurality of divided regions, and separately forming a culture solution for forming the culture layer in each divided region, Or after continuously injecting, and drying to form a culture layer.

  (9) The method for producing a microorganism culture sheet according to any one of (6) to (8), wherein a medium solution is injected into the respective divided regions from above the predetermined region through a porous nozzle.

  (10) A medium solution injecting apparatus for injecting a medium solution for forming a culture layer into a predetermined region on a base sheet, wherein the predetermined region is virtually divided into a plurality of divided regions, A medium solution injecting apparatus comprising a nozzle for individually or continuously injecting the medium solution into divided areas.

  (11) The culture medium injecting device according to (10), wherein the nozzle is a porous nozzle.

(12) The medium solution injecting device according to (11), wherein each of the porous nozzles has a pore diameter of 0.1 to 3.0 mm / piece and a hole arrangement density of 5 to 20 pieces / cm ² .

According to the microorganism culture sheet of the present invention, the test solution can be spread over a certain area on the culture layer without requiring complicated work and without loss of work time. Work can be performed. And, according to the microorganism culture sheet of the present invention, it is necessary and sufficient for the growth of microorganisms, and since it has a culture layer with a uniform thickness, colonies are difficult to spread, and differences in colony formation speed are unlikely to occur, It is possible to accurately perform microbial tests such as counting the number of colonies. Furthermore, according to the microorganism culture sheet of the present invention, since the culture layer is limited to a predetermined region on the base sheet, waste of expensive microorganism culture material is reduced.
Moreover, according to the method for producing a microorganism culture sheet of the present invention, the microorganism culture sheet can be produced reliably and continuously.
Furthermore, according to the medium solution injection device of the present invention, the above microorganism culture sheet can be easily produced.

It is the partial perspective top view (A) and sectional drawing (B) which show the microorganism culture sheet which concerns on 1st embodiment of this invention. It is sectional drawing which shows the microorganisms culture sheet which concerns on 2nd embodiment of this invention. It is a figure which shows the discharge hole of the porous nozzle with which the culture medium injection apparatus which concerns on 1st embodiment of this invention is provided. It is sectional drawing which shows the manufacturing method of the microorganism culture sheet which concerns on 1st embodiment of this invention sequentially. It is a top view which shows sequentially the manufacturing method of the microorganism culture sheet which concerns on 1st embodiment of this invention. It is a figure which shows a culture test result.

  Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and may be implemented with appropriate modifications within the scope of the object of the present invention. can do.

[Microbe culture sheet]
The microorganism culture sheet of the present invention includes a dried culture layer in a predetermined region on the base sheet. Then, the culture layer virtually divides the predetermined region into a plurality of divided regions, and after individually or continuously injecting a medium solution for forming the culture layer into each divided region It is formed by drying.

  A microorganism culture sheet according to the first embodiment of the present invention is shown in FIGS. 1A is a partially transparent plan view, and FIG. 1B is a cross-sectional view taken along the arrow XX shown in FIG. In addition, each figure (FIGS. 1-5) shown below is the figure typically shown, and the magnitude | size and shape of each part are exaggerated suitably for easy understanding.

  In the microorganism culture sheet according to the first embodiment of the present invention, the culture layer 30 is provided on the rectangular base sheet 10. A circular frame layer 20 made of a hydrophobic resin is formed on the outer periphery of the culture layer 30, and the culture layer 30 is formed in a recessed region surrounded by the frame layer 20. Further, there is no gap between the frame layer 20 and the culture layer 30, and both layers are in contact with each other. A rectangular transparent cover sheet 40 is provided so as to cover the frame layer 20 and the culture layer 30.

  According to the microorganism culture sheet according to the first embodiment of the present invention, since the circular frame layer 20 is formed on the outer periphery of the culture layer 30, it is ensured within a predetermined range without leaking the test liquid. Can be expanded. Moreover, since the frame layer 20 is formed of a hydrophobic resin, the frame layer 20 is not broken by the test solution even when the test solution is inoculated on the culture layer 30. Furthermore, since the frame layer 20 and the culture layer 30 are in contact with each other, the test solution is not stored in the gap, and the test solution can be spread uniformly on the culture layer 30.

  FIG. 2 shows a cross-sectional view of the microorganism culture sheet according to the second embodiment of the present invention. In the microorganism culture sheet according to the second embodiment of the present invention, a grid print layer 50 is provided so as to cover the frame layer 20 and the culture layer 30, and the transparent cover sheet 40 is provided on the grid print layer 50. Are stacked.

  According to the microorganism culture sheet according to the second embodiment of the present invention, since the square print layer 50 is provided, when many colonies are formed, the number of bacteria in several squares is measured and distributed. Just to get an approximate total number.

  Hereinafter, the substrate sheet, the culture layer, the frame layer, the cover sheet, and the square print layer will be described in order.

<Base material sheet>
The substrate sheet is required to have water resistance as a microorganism culture sheet. Moreover, the heat resistance which can endure the drying process at the time of forming a culture layer is calculated | required. The base sheet is not particularly limited as long as it has water resistance and heat resistance. For example, a plastic sheet such as polyester, polyethylene, polypropylene, polystyrene, polycarbonate, and polyvinyl chloride is preferably used. be able to. These plastic sheets are preferable in terms of excellent transparency. The microorganism culture sheet is for observing and counting the colonies of microorganisms that have grown in the culture layer. If the substrate sheet is highly transparent, the projection light from the back side (substrate sheet side) of the microorganism culture sheet In addition, the visibility of the colonies can be enhanced by the incident light from the side surface and the colonies can be easily measured. However, it does not necessarily need to be transparent, and those that are white due to foaming or those that are colored can be used, and may be appropriately selected depending on the type of microorganism, the presence or absence of coloring of colonies, and the like.

  The base sheet may be a single layer or a laminated sheet of two or more layers. As a laminated sheet, what laminated | stacked 2 or more types of the said plastic sheet is mentioned. In addition, a laminated sheet obtained by laminating a paper base material on the plastic sheet, a laminated sheet obtained by coating a paper base material with a synthetic resin, and the like can be suitably used. Examples of such a laminated sheet include a laminated sheet of a polyethylene film and a paper base material. In addition, synthetic paper using a synthetic resin as a main raw material can also be used. Examples of such commercially available synthetic paper include “YUPO” manufactured by Yupo Corporation, “Chrisper” manufactured by Toyobo Co., Ltd., and the like.

  In order to improve the adhesiveness with the culture layer, the base sheet may have been subjected to surface treatment on the surface on which the culture layer is formed in advance. The surface treatment is not particularly limited. For example, a known surface such as corona discharge treatment, ozone treatment, glow discharge treatment, oxidation treatment using chemicals, low temperature plasma treatment using oxygen gas or nitrogen gas, etc. Processing. Further, the surface treatment may be performed by applying an anchor coating agent.

  The substrate sheet may have a recessed area formed by embossing. By setting this recessed region as a predetermined region for forming a culture layer, a culture layer having a predetermined shape can be reliably formed on the base sheet, and expensive microorganism culture material is not wasted. Moreover, if a culture layer having a predetermined shape can be formed, the test solution can be expanded to a certain area by a simple operation. In addition, this recessed part area | region may be formed by shaving the base material sheet | seat of a thick hydrophobic material into a concave shape.

The thickness of a base material sheet is not specifically limited, Usually, 25-1500 micrometers, Preferably it is 50-500 micrometers. Moreover, in this embodiment, although the shape of a base material sheet is a square, it is not limited to this, Circular, an ellipse, a polygon, etc. may be sufficient. The magnitude | size of a base material sheet is also not specifically limited, Usually, it is 30-100 cm < ² >.

  The base sheet may be printed in advance with an ink that is insoluble in water and does not affect the growth of microorganisms. This is because, according to the base sheet on which the checkerboard pattern is printed, when a large number of colonies are formed, the approximate total number can be obtained simply by measuring and apportioning the number of bacteria in a few checkered points. . The printing plate type is not particularly limited, but gravure printing is preferable in terms of a wide selection range of colorants, resins, solvents and the like. The size of the mesh is about 1 cm square.

<Culture layer>
In the microorganism culture sheet of the present invention, the culture layer is limited to a predetermined region on the base sheet in order to reduce waste of expensive microorganism culture material and to enable quick inoculation work. Is formed. By forming a culture layer of a predetermined shape on the base material sheet, it is possible to immediately inoculate the culture layer with a cover sheet without requiring a complicated operation such as the use of a spreader or the like. As a result, the test solution can be spread over a certain area on the culture layer.

  In addition, the culture layer is virtually divided into a plurality of divided areas on the base sheet in order to make the thickness necessary and sufficient for the growth of microorganisms and uniform, and each divided area On the other hand, the medium solution for forming the culture layer is injected individually or continuously and then dried. In order to accurately perform a microorganism test such as measurement of the number of colonies, it is necessary to appropriately grow microorganisms in a certain environment to form colonies. By giving the culture layer a sufficient thickness, the culture layer can contain a sufficient amount of nutrients necessary for the growth of microorganisms and an amount of gelling agent that does not cause colony bleeding. . In addition, by making the thickness of the culture layer uniform, the content of each component such as nutrients and gelling agent in the culture layer becomes uniform, and the growth environment of microorganisms becomes constant, Differences in colony formation rate are less likely to occur. According to the microorganism culture sheet of the present invention, since it is necessary and sufficient for the growth of microorganisms and has a culture layer having a uniform thickness, colonies are difficult to spread and the difference in colony formation speed is less likely to occur. Microbiological tests such as measurement can be performed accurately.

  The constituents of the medium solution for forming the culture layer are not particularly limited, and can be appropriately selected according to the type of microorganism to be cultured, the purpose of use, and the like. Preferably, a medium solution containing a binder and other components such as a gelling agent, a nutrient component, a color development indicator, and a selection agent is used.

  Here, the binder has a role of fixing other components such as a gelling agent, a nutritional component, a coloring indicator, and a selective agent to the base material. For example, polyvinylpyrrolidone, polyethylene oxide, hydroxypropylcellulose, Examples thereof include polyvinyl alcohol, and polyvinyl pyrrolidone is particularly preferable. Since polyvinylpyrrolidone is soluble in an alcohol solvent, the viscosity of the medium solution can be easily adjusted. Moreover, according to polyvinylpyrrolidone, an alcohol-based solvent can be used, so that a dispersed medium solution can be prepared without dissolving the gelling agent. As a result, it is possible to suppress a significant increase in the viscosity of the medium solution by the gelling agent. Furthermore, after the pattern formation, the solvent can be removed without heating at a high temperature. And since polyvinylpyrrolidone has a high film property, it can be formed into a film by incorporating other components such as a gelling agent, a nutritional component, a coloring indicator, a selective agent, etc. Excellent.

  Examples of the gelling agent include carrageenan, guar gum, chitansan gum, locust bean gum, algin, carboxymethylcellulose, hydroxyethylcellulose and the like. These can be used individually or in mixture of 2 or more types. According to these gelling agents, the culture layer is thickened or gelled by dropping the test solution, and an environment suitable for the growth of microorganisms is obtained. And if a culture layer becomes thickened or gelatinized, since adhesiveness with a cover sheet will improve, evaporation of the water | moisture content at the time of culture | cultivation can also be suppressed.

  The nutrient component can be appropriately selected according to the type of microorganism. For example, for the inspection of general viable bacteria, yeast extract / pepton / glucose mixture, meat extract / pepton mixture, peptone / soybean peptone / glucose mixture, etc., and mixtures obtained by adding dipotassium phosphate and / or sodium chloride to these Can be mentioned. Examples of the test for Escherichia coli and coliform group include sodium desoxycholate / peptone / ammonium iron citrate / sodium chloride / dipotassium phosphate / lactose mixture, and peptone / lactose / dipotassium phosphate mixture. Examples of staphylococci include meat extract, peptone, sodium chloride, mannitol, egg yolk mixture, peptone, meat extract, yeast extract, sodium pyruvate, glycine, lithium chloride, tellurite egg yolk mixture, and the like. For vibrio use, yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, sodium cholate, ferric citrate, sodium chloride, bovine bile mixture and the like can be mentioned. Examples of enterococci include bovine brain extract, heart extract, peptone, glucose, dipotassium phosphate and sodium nitride. Examples of fungi include peptone / glucose mixture, yeast extract / glucose mixture, potato extract / glucose mixture, and the like. From these, one or more types can be selected and mixed for use according to the type of microorganism to be grown.

  The color indicator is colored by coloring with the reaction with specific substances produced by metabolism of microorganisms that grow during the culture process, recognizing pH changes, reaction with enzymes, etc., so counting the number of colonies is extremely easy There is an effect to. Examples of the color development indicator include tetrazorim salts such as triphenyltetrazolium chloride (hereinafter referred to as TTC), p-tolyltetrazolium red, tetrazolium violet, petetylyltetrazolium blue, neutral red mixtures, phenol red, bromthymol blue, and thymol. PH indicator such as blue mixture.

  In addition, the culture layer may contain a selective agent that suppresses the growth of microorganisms other than those for detection purposes. As such a selective agent, methicillin, ceftamezole, cefixime, ceftazidime, cefsulosin, bacitracin, polymyxin B, rifampicin, novobiocin, colistin, lincomycin, chloramphenicol, tetracycline, streptomycin and other antibiotics, sulfa drugs, nalidixic acid, Synthetic antibacterial agents such as olaquindox, dyes having bacteriostatic or bactericidal activity such as crystal violet, brilliant green, malachite green, methylene blue, surfactants such as Tergitol 7, dodecyl sulfate, lauryl sulfate, selenite, Examples thereof include inorganic salts such as tellurite, sulfite, sodium nitride, lithium chloride, oxalate, and high-concentration sodium chloride.

  The solvent used in the culture medium containing the above-described constituents is not particularly limited as long as it can dissolve or disperse the selected constituents, but an alcohol solvent is preferably used. Alcohol-based solvents do not need to be heated at a high temperature when removing the solvent after pattern formation, as in high-boiling solvents such as water and toluene, so that the components contained in the culture layer are not likely to be thermally decomposed. Since it does not take a long time to remove the solvent, it is preferable in terms of excellent production efficiency. As the alcohol solvent, an alcohol having 1 to 5 carbon atoms is preferable, and examples thereof include methanol, ethanol, isopropyl alcohol and the like. Among these, methanol is most preferable because it has a low boiling point, can be easily removed by drying, and does not inhibit the growth of microorganisms. In addition, the said solvent can be used individually or in mixture of 2 or more types. The amount of solvent used may be appropriately selected depending on the components of the medium and the type of solvent.

<Frame layer>
In the microorganism culture sheet of the present invention, a frame layer made of a hydrophobic resin may be formed. If a frame layer is formed, when the test solution is inoculated on the culture layer, the test solution spreads quickly to the frame layer, so the cover sheet is covered without waiting for the test solution to absorb water. The inoculation operation can be performed in a short time. It is also possible to prevent leakage of the test liquid. The frame layer may be formed on the culture layer, or may be directly formed on the base sheet. However, when the frame layer is formed on the culture layer, the culture layer is absorbed by water absorption of the test solution. Since it may collapse when swollen into a gel, it is preferably formed directly on the substrate sheet.

  Moreover, it is more preferable that the frame layer is formed directly on the base material sheet and surrounds the outer periphery of the culture layer. In this case, the frame layer may be formed before the formation of the culture layer, or may be formed after the formation of the culture layer, but if formed after the formation of the culture layer, there is a gap between the frame layer and the culture layer. It is easy to occur, and when microorganisms are cultured, the bacteria migrate in the gaps, and it may be difficult to accurately measure the number of colonies. Therefore, it is preferable to form them before the formation of the culture layer. Before forming the culture layer, by forming a frame layer on the substrate sheet in advance, a recess surrounded by the frame layer is formed in a predetermined region on the substrate sheet, and a medium solution is injected into the recess region. When the culture layer is formed in this manner, a culture layer having a predetermined shape can be reliably formed, so that expensive microorganism culture material is not wasted.

A hydrophobic resin is used as the material of the frame layer. As the hydrophobic resin, for example, UV curable resin, hot melt resin, thermally foamed ink, UV foaming agent and the like can be suitably used. The UV curable resin is not particularly limited. For example, the radical type includes an acrylate type and an unsaturated polyester type, and the cationic type includes an epoxy acrylate type, an oxetane type, and a vinyl ether type. it can. The irradiation conditions for curing the UV curable resin are an integrated light amount of 50 to 2000 mJ / cm ² , more preferably 100 to 1000 mJ / cm ² .

  As the hot melt resin, one having a softening point temperature of 120 ° C. or lower, more preferably 100 ° C. or lower can be suitably used. If it exceeds 120 ° C, the substrate sheet may be curved. Examples of the hot melt resin include, but are not limited to, urethane, polyamide, polyolefin, polyester, ethylene vinyl acetate, styrene butadiene rubber, urethane rubber, and other synthetic rubber is not.

  In addition, a thermal foaming ink, a UV foaming agent, or the like can be suitably used. For example, polyurethane resins, polyamide resins, polyvinyl chloride resins, polyacrylate resins, polyester resins, polyolefin resins such as chlorinated polypropylene, and rubbers such as chlorinated rubber and cyclized rubber are used as binders. Ink can also be used. Since these inks are more preferable if they are water-repellent, inks obtained by adding silicones or waxes to the above resins are more preferable. The hydrophobic resin used in the frame layer is hydrophobic and water-repellent, and at the same time it is necessary to increase the thickness of the coating film. It is effective to do. Such UV foamed ink includes at least a reactive diluent such as an acrylate reactive diluent or a methacrylate reactive dilution, a urethane acrylate photopolymerizable oligomer, a polyester acrylate photopolymerizable oligomer, an epoxy acrylate photopolymerizable oligomer, an acrylic. Ultraviolet curable ink composed of photopolymerizable oligomers such as photopolymerizable oligomers and special photopolymerizable oligomers, and a photopolymerization initiator, contains foaming agents such as inorganic foaming agents, organic foaming agents, and liquid foaming agents. And ultraviolet curable foamed ink. In addition, you may add an antifoamer, a polymerization inhibitor, a pigment dispersant etc. to the said hydrophobic resin as an additive. Moreover, you may contain the coloring pigment used normally in less than 15 mass%. In addition, foaming may be inhibited when it is 15% by mass or more.

  In order to form the frame layer, after patterning the hydrophobic resin, UV irradiation or cooling is performed using a metal halide lamp, a mercury lamp, or the like. The pattern formation is not particularly limited as long as the hydrophobic resin can be printed, applied, and coated in a predetermined shape on a substrate sheet or the like, and is not limited to screen printing, gravure printing, letterpress printing. And printing such as transfer printing and flexographic printing, application by a dispenser, coating using a bar or the like, bar coating, knife coating, die coating or the like. Among these, application by a dispenser is preferable in that it is not necessary to prepare a plate, and various shapes can be easily drawn by creating a program.

  In the microorganism culture sheet of the present invention, any of the above hydrophobic resins can be suitably used as the frame layer material, but UV curable resins or hot melt resins are preferable, and hot melt resins are more preferable. Forming a frame layer requires pattern formation with a certain height, but UV curable resins and hot melt resins can be easily processed by application with a dispenser in a non-solvent system. This is because the pattern can be easily formed only by curing by light irradiation or cooling. However, when the culture layer contains a component that is altered by UV irradiation, it is necessary to cover the culture layer with metal or the like during UV irradiation, and the work becomes complicated. .

  In the microorganism culture sheet of the present invention, a frame layer may be formed by bonding a hollow material of a hydrophobic material to the outer periphery of the culture layer.

  The width of the frame layer is not limited to a uniform width, but the most detailed width is preferably 0.5 to 5.0 mm, more preferably 1.0 to 3.0 mm. . If it is the said range, even if it is a case where a culture layer absorbs water and swells, there is no possibility that the shape of a frame layer may collapse, and a test solution can be spread over a predetermined region. Moreover, although the height of a frame layer is dependent also on the thickness of a culture layer, Preferably it is 600-1200 micrometers based on a base material sheet. If there is no difference in height from the culture layer, when the test solution is dropped, it may protrude from the frame layer and leak. If the difference in height is large, a space is likely to be formed between the culture layer and the cover sheet after inoculation with the test solution, and the culture layer may be dried during the culture.

  In the microorganism culture sheet according to the first embodiment of the present invention shown in FIG. 1 (A), the shape of the frame layer 20 is circular, but is not limited thereto, and is rectangular, elliptical, polygonal, etc. It may be.

<Cover sheet>
The cover sheet has the effect of preventing contamination by falling bacteria during culture and preventing moisture evaporation of the culture layer. The cover sheet is waterproof and water vapor impermeable, and is preferably transparent so that colonies can be observed and counted through the cover sheet after culturing microorganisms, for example, polyethylene, polypropylene, etc. Plastic films such as polyolefin, polyester, polyamide, polystyrene, polycarbonate, and polyvinyl chloride can be used.

  In the microorganism culture sheet of the present invention, a cover sheet coated with a binder solution containing an unstable substance whose physical properties are likely to change when contained in the culture layer may be used on the surface facing the substrate sheet. . Examples of the material whose physical properties are likely to change when contained in the culture layer include TTC whose physical properties change by a reduction reaction. As the binder, water or alcohol-soluble resins such as polyethyleneimine, polyethylene oxide, and acrylic acid polymers can be preferably used. However, if the function of the contained substance can be exhibited, the hydrophobic binder can be used. A solution, a dispersion, or the like can also be used.

  Since the cover sheet is lifted when the test solution is inoculated, it is preferable that the cover sheet has an appropriate flexibility. The thickness of the cover sheet is preferably about 10 to 200 μm, and more preferably about 20 to 70 μm. In view of workability of the cover sheet, a polyester film or a polyolefin film is particularly preferable.

  The cover sheet may have any shape, but it is necessary to have a size larger than that of the culture layer so as to cover the culture layer in order to prevent invasion of various bacteria.

  In the microorganism culture sheet according to the first embodiment of the present invention shown in FIG. 1 (A), the cover sheet is bonded to the end of one side of the base sheet, but is fixed to the microorganism culture sheet. It is not limited to a case, You may coat | cover the culture layer which inoculated the test liquid with the cover sheet prepared separately. A mode in which the cover sheet is fixed to the base material sheet in advance is preferable in terms of easy operation.

  In addition, when the grid pattern is not printed on the base material sheet, the grid pattern may be printed on the cover sheet in advance. This is because, according to the cover sheet on which the checkerboard pattern is printed, when a large number of colonies are formed, an approximate total number can be obtained simply by measuring and apportioning the number of bacteria in a few checkers. The ink used is insoluble in water and does not affect the growth of microorganisms. As with the base sheet, the printing plate is preferably gravure printing because it has a wide selection range of colorants, resins, solvents, and the like. The size of the mesh is about 1 cm square.

<Rule print layer>
In the microorganism culture sheet of the present invention, a square print layer may be formed. Examples of the grid print layer include a resin film or a paper substrate on which a grid pattern is printed with an ink that is insoluble in water and does not affect the growth of microorganisms. As in the case of the base sheet or cover sheet, the printing plate type is preferably gravure printing because it has a wide selection range of colorants, resins, solvents, and the like. The size of the mesh is about 1 cm square. In addition, the formation position of a square print layer is not specifically limited, For example, the aspect laminated | stacked on the lower layer of a base material sheet is mentioned.

[Medium fluid injection device]
Next, the medium solution injection device of the present invention will be described. In addition, description about the structure which is common in the microorganism culture sheet | seat of this invention mentioned above is abbreviate | omitted. The medium solution injecting device of the present invention is a medium solution injecting device that injects a medium solution for forming a culture layer into a predetermined region on a base sheet, and the predetermined region is virtually divided into a plurality of divided regions. And a nozzle for individually or continuously injecting the medium solution into each divided region. According to the medium solution injection device of the present invention, a culture layer having a uniform thickness can be formed continuously and with good reproducibility in a predetermined region on a substrate sheet. The nozzle provided in the medium liquid injection device of the present invention may be either a single-hole nozzle having one discharge hole or a multi-hole nozzle having a plurality of discharge holes, but is preferably a multi-hole nozzle. According to the multi-hole nozzle having a plurality of discharge holes, it is possible to inject a medium solution into a plurality of divided regions at the same time by one discharge, so that an efficient culture layer can be formed. Note that the porous nozzle has a number of ejection holes equal to the number of divided areas when a predetermined area on the base sheet is virtually divided, and is a porous nozzle capable of injecting a medium solution into the predetermined area at a time. The nozzle has a smaller number of discharge holes than the number of divided areas when virtual fractionation is performed, and a porous nozzle that injects a medium solution into a part of the predetermined area can be mentioned. A porous nozzle having the same number of ejection holes as the number of divided regions is most preferable in that a more efficient culture layer can be formed.

  As a method of individually injecting the medium solution into the divided areas, for example, (1) using a single hole nozzle, injecting a predetermined amount of medium liquid into one divided area, An injection method in which the single-hole nozzle is moved after stopping the discharge, the discharge hole is arranged on the next divided area, and a predetermined amount of medium solution is injected into the divided area; (2) virtual fractionation Using a porous nozzle having a smaller number of discharge holes than the number of divided areas, and disposing each discharge hole of the porous nozzle on each divided area, and simultaneously supplying a predetermined amount of medium solution to each divided area. After the injection, the discharge of the medium solution from each discharge hole is stopped, and then the porous nozzle is moved to dispose each discharge hole on each next divided region, and a predetermined amount of the medium solution is simultaneously provided in each divided region. Injection method that repeats injection, (3) divided region when virtual fractionation There is an injection method in which a multi-hole nozzle having the same number of discharge holes is used, each discharge hole of the multi-hole nozzle is disposed on each divided region, and a predetermined amount of medium solution is simultaneously injected into each divided region. .

  As a method for continuously injecting the medium liquid into the divided areas, for example, (4) using a single-hole nozzle and moving the medium liquid on each divided area while continuously discharging the medium liquid from the discharge holes. Thus, a method of injecting a predetermined amount of medium solution into each divided region can be mentioned. Specifically, in the case where the medium is applied in a circular shape on the base sheet, the medium is continuously discharged from the discharge hole of the single-hole nozzle, and concentrically from the central portion of the circle. The method of drawing circles at the same interval or drawing a spiral from the center of the circle. In addition, (5) using a multi-hole nozzle having a smaller number of discharge holes than the number of divided areas at the time of virtual fractionation, the medium liquid is continuously discharged from the discharge holes and moved on each divided area. Thus, a method of injecting a predetermined amount of medium solution into each divided region can be mentioned. In addition, when continuously injecting the culture medium into the divided areas, the discharge rate per unit time of the medium liquid discharged from the nozzle discharge holes and the moving speed of the nozzles between the divided areas are constant. It is desirable to be.

  When the nozzle provided in the culture medium injecting apparatus of the present invention is a perforated nozzle, the discharge holes of the perforated nozzle may be arranged at equal intervals or may be partially densely arranged. In FIG. 3, the preferable aspect of the discharge hole of a perforated nozzle is shown. In the present embodiment, the arrangement density of the discharge holes K of the multi-hole nozzle is partially increased at a location corresponding to the vicinity of the outer periphery of the culture layer 30. According to such an aspect, when the frame layer 20 is formed on the outer periphery of the culture layer 30, the medium solution can be spread without a gap between the frame layer 20 and the culture layer 30.

The hole diameter of each discharge hole of the porous nozzle is preferably 0.1 to 3.0 mm / piece, more preferably 0.1 to 2.0 mm / piece, and 0.5 to 2.0 mm / piece. Most preferably. If it is the said range, it will be hard to produce the sag and clogging of a culture solution. In particular, in the case of a dispersed medium solution, if the pores are too large, liquid sag and unevenness after application are likely to occur, and if the pores are too small, clogging is likely to occur depending on the particle size of dispersed nutrients and the like. The individual hole diameters do not have to be the same size as long as they are within the above range. The arrangement density of the discharge holes of the porous nozzle is preferably 5 to 20 / cm ^2, more preferably from 5 to 13 pieces / cm ^2, to be 8 to 13 pieces / cm ² Most preferred. When the arrangement density is less than 5 pieces / cm ² , the medium solution does not spread sufficiently, and it may be difficult to form a culture layer having a uniform thickness or a culture layer having a thickness. When the arrangement density exceeds 20 pieces / cm ² , the gap between the holes becomes narrow, so that the medium solutions may come into contact with each other at the time of dispensing and cannot be dispensed satisfactorily. In addition, as a perforated nozzle, what is generally marketed, such as the multi-nozzle MN series made by Musashi Engineering, can be used, for example.

[Method for producing microorganism culture sheet]
Next, the manufacturing method of the microorganism culture sheet of this invention is demonstrated in detail. As an example, a manufacturing method in the case of using the above-described medium solution injection device of the present invention will be described with reference to the drawings.

  First, the above-mentioned binder, gelling agent, nutrient component, color indicator, selective agent, and the like are dissolved or dispersed in an alcohol solvent to prepare a culture layer forming medium solution 30A. Next, a circular frame layer 20 made of hot melt resin is formed on a base sheet 10 such as synthetic paper using a dispenser having a single-hole nozzle (FIGS. 4A and 4B). , FIG. 5 (A), FIG. 5 (B)). Then, the concave region surrounded by the frame layer 20 on the base sheet 10 is virtually fractionated into a plurality of divided regions P (FIG. 5C), and the culture layer forming medium is divided into each divided region P. The liquid 30A is individually dispensed and applied using a dispenser including a multi-hole nozzle N having the same number of discharge holes as the number of divided regions (FIGS. 4C, 4D, and 5D). ). Thereafter, the applied medium solution 30A is dried at 60 ° C. for 15 minutes to form the culture layer 30 (FIG. 5E). Then, a microorganism culture sheet can be produced by laminating a cover film 40 such as a biaxially stretched polypropylene film on the base material sheet 10 on which the culture layer 30 is formed, and adhering one end.

In the microorganism culture sheet, the culture layer needs to have a certain thickness. For example, after drying, the thickness is preferably 50 to 1000 μm, and more preferably 200 to 600 μm. Moreover, as an application quantity of a culture solution, it is preferable that it is 5-400 g / m < ² > after drying, and it is more preferable that it is 100-300 g / m < ² >. As a conventional method for forming a culture layer having such a thickness in a predetermined region on a substrate sheet, there is screen printing. However, in screen printing using a mesh plate, the solvent volatilizes during the formation of the culture layer, and the viscosity of the medium solution increases. Therefore, the mesh part is likely to be clogged, and the medium solution is not easily separated. For this reason, it was difficult to continuously form the culture layer. Even with screen printing using a metal mask plate, the plate separation of the medium solution is poor as with the mesh plate, and continuous formation of the culture layer is still difficult, even if the culture layer is printed in a predetermined area, Unevenness of thickness tends to occur in the moving direction of the squeegee, and it has been difficult to form a culture layer having a uniform thickness. Therefore, when the amount of the solvent is increased in consideration of the increase in the viscosity of the medium due to the volatilization of the solvent, the viscosity of the medium is low, so that the medium expands between the plate and the substrate when applied, It was difficult to form a culture layer only in the region. On the other hand, according to the method for producing a microorganism culture sheet of the present invention, it is possible to form a culture layer having an appropriate thickness and a uniform thickness necessary for the growth of microorganisms, which was difficult by screen printing. Become.

  When the culture layer is thick enough, the culture layer contains enough nutrients for the growth of microorganisms and an amount of gelling agent that prevents colony bleeding, so that the microorganisms grow properly. As a result, a colony without bleeding is formed. If the thickness of the culture layer is uniform, the content of each component such as the binder, gelling agent and nutrients in the culture layer will not be biased, so the growth environment of microorganisms will be constant and colonies will be formed. There is no difference in speed. Therefore, according to the microorganism culture sheet of the present invention having a culture layer that is uniform and has a sufficient thickness, colonies are difficult to bleed, and differences in colony formation speed are unlikely to occur. Microbial testing can be performed accurately. In addition, in a culture layer formed from a dispersed medium solution in which at least one of the components (excluding the solvent) of the medium solution is not dissolved in the solvent, the uniformity of the thickness of the culture layer is The influence on the distribution of each component in the layer is large. Therefore, the formation of the culture layer by the above-described method is particularly effective when a dispersed medium solution is used.

  In the method for producing a microorganism culture sheet according to the first embodiment of the present invention shown in FIGS. 4 and 5, after forming the frame layer 20, the culture layer 30 is formed in the recessed region surrounded by the frame layer 20. However, after forming the culture layer 30, a frame layer may be formed so as to surround the outer periphery of the culture layer 30. By forming the frame layer 20 on the base material sheet 10, a recess surrounded by the frame layer 20 is formed in a predetermined region on the base material sheet 10, and then culture is performed by injecting a medium solution into the recess. The mode of forming the layer 30 is preferable in that a culture layer having a predetermined shape can be reliably formed, so that expensive microorganism culture material is not wasted.

  Moreover, in the manufacturing method of the microorganism culture sheet which concerns on 1st embodiment of this invention shown in FIG.4 and 5, using the culture medium liquid injection | pouring apparatus provided with the one porous nozzle N, predetermined | prescribed on the base material sheet 10 Although the medium solution 30A was individually injected into each divided region obtained by virtually fractionating the region, the method is not limited to this method. The medium solution may be injected into the divided regions individually or continuously. Since the method for injecting the medium solution individually or continuously into the divided regions has been described above, the description thereof is omitted here. In addition, from the viewpoint of uniformity of the thickness of the formed culture layer, production efficiency, and the like, an injection method in which a medium solution is simultaneously discharged into each divided region using a single porous nozzle is preferable.

  The microorganism culture sheet of the present invention is made into a product by performing sterilization treatment such as γ-ray irradiation and ethylene oxide gas sterilization. According to the microorganism culture sheet of the present invention, various microorganisms can be cultured according to the type of nutrient components contained in the culture layer, the base sheet used, the air permeability of the cover sheet, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention does not receive a restriction | limiting at all by these description.

[Production Example 1] Culture medium for forming culture layer (A)
Dissolve 20 g of polyvinyl pyrrolidone (trade name “polyvinyl pyrrolidone K-90”, manufactured by Nippon Shokubai Co., Ltd.) in 130 g of methanol, and disperse 60 g of guar gum powder (trade name “NEOVISCO G”, 400 mesh type, manufactured by Sanki Co., Ltd.). did. As a nutrient component, tryptone (trade name “BACT TRYPTONE”, Becton, Dickinson and Company) 3.26 g, meat extract (trade name “Lab-Lemco”, Oxoid) 0.82 g, yeast extract ( Trade name “YEAST EXTRACT”, Becton, Dickinson and Company 0.03 g, glucose (trade name “D-(+)-GLUCOSE”, Wako Pure Chemical Industries, Ltd.) 0.16 g, sodium chloride (Wako Pure Chemical) 0.41 g (manufactured by Kogyo Co., Ltd.) and 0.37 g of disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) were added and mixed to prepare a culture layer forming medium solution (A).

[Production Example 2] Culture medium for forming culture layer (B)
Dissolve 20 g of polyvinylpyrrolidone (trade name “polyvinylpyrrolidone K-90”, manufactured by Nippon Shokubai Co., Ltd.) in 230 g of methanol, and disperse 60 g of guar gum powder (trade name “NEOVISCO G”, 400 mesh type, manufactured by Sanki Co., Ltd.). did. As a nutrient component, tryptone (trade name “BACT TRYPTONE”, Becton, Dickinson and Company) 3.26 g, meat extract (trade name “Lab-Lemco”, Oxoid) 0.82 g, yeast extract ( Trade name “YEAST EXTRACT”, Becton, Dickinson and Company 0.03 g, glucose (trade name “D-(+)-GLUCOSE”, Wako Pure Chemical Industries, Ltd.) 0.16 g, sodium chloride (Wako Pure Chemical) 0.41 g (manufactured by Kogyo Co., Ltd.) and 0.37 g of disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) were added and mixed to prepare a culture layer forming medium solution (B).

[Production Example 3] Bacterial liquid for culture test Preliminary culture was carried out for 18 hours under a temperature condition of 36 ± 1 ° C. using a liquid medium (TRYPTONE SOYA BROTH). The bacterial suspension of coli (ATCC 25922), was diluted in sterile saline and adjusted to the number of bacteria per 1ml is 10 ^2. To this was added TTC (triphenyltetrazolium chloride) adjusted to a concentration of 0.05 g / L and mixed to prepare a bacterial solution for culture test.

<Comparative Example 1>
On the base material sheet (polyethylene terephthalate film, trade name “Teijin Tetron Film”, manufactured by Teijin DuPont Films Ltd.), the culture medium forming medium solution (A) prepared in Production Example 1 is applied in a circular shape having a diameter of 50 mm. Therefore, using a plate (mesh plate, diameter: 49 mm, thickness: 0.5 mm, Tetoron 70 mesh, manufactured by Murakami Co., Ltd.) on which a circular pattern was formed, the medium solution (A) was screen printed. It was impossible.

<Comparative example 2>
On the base material sheet (polyethylene terephthalate film, trade name “Teijin Tetron Film”, manufactured by Teijin DuPont Films Ltd.), the culture medium forming medium solution (A) prepared in Production Example 1 is applied in a circular shape having a diameter of 50 mm. For this purpose, the medium solution (A) was screen-printed using a plate on which a circular pattern was formed (metal mask plate, diameter: 49 mm, thickness: 0.5 mm, manufactured by Murakami Co., Ltd.). Then, the applied medium solution was dried at 60 ° C. for 15 minutes to form a culture layer. Thereafter, a cover sheet (biaxially stretched polypropylene film, trade name “OP U-1”, film thickness: 40 μm, manufactured by Tosero Co., Ltd.) is laminated on the base material sheet on which the culture layer is formed, and the end of one side is formed. After being adhered, the microorganism culture sheet of Comparative Example 2 was obtained by sterilization by γ-ray irradiation.

  When the formation of the culture layer by screen printing was attempted, when the mesh plate was used (Comparative Example 1), the mesh plate was clogged, and the separation of the plate was bad and coating was difficult. . When a metal mask was used (Comparative Example 2), clogging of the plate was eliminated and printing was possible, but the plate separation was poor and continuous productivity was low. Moreover, when the culture layer of the comparative example 2 was observed visually, it was confirmed that there is unevenness in thickness. A possible cause of this phenomenon is an increase in the viscosity of the medium solution due to the volatilization of methanol. Then, the amount of methanol was increased, the viscosity of the culture solution for culture layer formation was lowered, and formation of a culture layer was tried.

<Comparative Example 3>
On the base material sheet (polyethylene terephthalate film, trade name “Teijin Tetron Film”, manufactured by Teijin DuPont Films Ltd.), the culture layer forming medium solution (B) prepared in Production Example 2 is applied in a circular shape having a diameter of 50 mm. Therefore, using a plate with a circular pattern (metal mask plate, diameter: 49 mm, thickness: 0.9 mm, manufactured by Murakami Co., Ltd.), an attempt was made to screen-print the medium solution (B). Since the viscosity was lowered by increasing the amount of methanol, the medium solution spread between the plate and the substrate before leaving the plate from the coated medium solution, and it was difficult to apply only to a predetermined area.

<Example 1>
A circular frame layer (width: 1 mm) made of a thermoplastic resin (trade name “MP973”, manufactured by Konishi Co., Ltd.) on a base sheet (polyethylene terephthalate film, trade name “Teijin Tetron Film”, manufactured by Teijin DuPont Films) , Height: 750 μm, diameter: 52 mm) was formed using a dispenser (product name “SHOTmini200α”, manufactured by Musashi Engineering Co., Ltd.). Next, a porous nozzle (pore diameter: about 0.9 mm / piece, pore arrangement density: about 9 pieces / cm ² ) is formed in the concave portion (concave shape region) surrounded by the frame layer formed on the base sheet. Using a dispenser (product name “SHOTmini200α”, manufactured by Musashi Engineering Co., Ltd.), the culture layer forming medium solution (B) prepared in Production Example 2 was discharged once from the porous nozzle and injected. The injected medium solution was dried at 60 ° C. for 15 minutes to form a culture layer (coating amount after drying: 160 g / m ² ). Thereafter, a cover sheet (biaxially stretched polypropylene film, trade name “OP U-1”, film thickness: 40 μm, manufactured by Tosero Co., Ltd.) is laminated on the base material sheet on which the culture layer is formed, and the end of one side is formed. After bonding, the microorganism culture sheet of Example 1 was obtained by sterilization by γ-ray irradiation.

<Example 2>
On the base material sheet (polyethylene terephthalate film, trade name “Teijin Tetron Film”, manufactured by Teijin DuPont Films Ltd.), the culture layer forming medium solution (B) prepared in Production Example 2 is applied in a circular shape having a diameter of 50 mm. Therefore, using a dispenser (product name “SHOTmini200α”, manufactured by Musashi Engineering Co., Ltd.) equipped with a porous nozzle (pore diameter: about 0.9 mm / piece, hole arrangement density: about 9 pieces / cm ² ) The medium solution (B) was discharged once from the nozzle. Then, the applied medium solution was dried at 60 ° C. for 15 minutes to form a culture layer (amount applied after drying: 160 g / m ² ). Next, a circular frame layer (width: 1 mm, height) made of ultraviolet curable ink (trade name “Reicure GA 4100-2”, manufactured by Jujo Chemical Co., Ltd.) is provided on the outer periphery of the culture layer on the substrate sheet. : 750 μm, diameter: 52 mm) was formed using a dispenser (product name “SHOTmini200α”, manufactured by Musashi Engineering Co., Ltd.). Then, the frame layer was cured by irradiating with an ultraviolet ray having an accumulated light amount of 250 mJ / cm ² using a mercury lamp. Thereafter, a cover sheet (biaxially stretched polypropylene film, trade name “OP U-1”, film thickness: 40 μm, manufactured by Tosero Co., Ltd.) is laminated on the base material sheet on which the culture layer is formed, and the end of one side is formed. After bonding, the microorganism culture sheet of Example 2 was obtained by sterilization by γ-ray irradiation.

[Evaluation] Culture Test The microorganism culture sheets obtained in Comparative Example 2 and Examples 1 and 2 were used for the test. On the culture layer of the microorganism culture sheet, 1 ml of the culture test bacterial solution prepared in Production Example 3 was inoculated using a sterilized pipette. Next, a cover sheet was placed on the culture layer inoculated with the culture test bacterial solution, and the culture test bacterial solution was spread over the entire culture layer in the frame. And after leaving still for about 1 minute and forming a gel, it put into the incubator, it culture | cultivated at 37 degreeC for 48 hours, and the state of the generated colony was observed. The results are shown in FIGS.

  In the microorganism culture sheet of Comparative Example 2 in which the culture medium was applied by screen printing to form a culture layer, and then a frame layer was formed on the outer periphery of the culture layer, a culture layer having a uniform thickness could not be obtained (150 to 260 μm). ), Colony bleeding was observed at a location where the culture layer was thin and the amount of the gelling agent was small (FIG. 6A).

  By forming a frame layer in a predetermined region on the substrate sheet, a recess surrounded by the frame layer is formed, and then a culture solution is injected by injecting a medium solution using a dispenser having a porous nozzle in the recess. In the formed microorganism culture sheet of Example 1, the thickness of the culture layer became uniform (250 μm), and the colonies grew in the entire culture layer (FIG. 6B).

  In the microorganism culture sheet of Example 2 in which a culture layer is formed by applying a medium solution using a dispenser equipped with a porous nozzle, and then a frame layer is formed on the outer periphery of the culture layer, the boundary line between the culture layer and the frame layer Although some colony bleeding was observed in the vicinity, a culture layer having a substantially uniform thickness (250 μm) was formed, and the colonies grew on almost the entire culture layer (FIG. 6C).

DESCRIPTION OF SYMBOLS 10 Base material sheet 20 Frame layer 30 Culture layer 30A Medium solution 40 Cover sheet 50 Grid print layer K Discharge hole N Porous nozzle P Divided area

Claims (12)

A microorganism culture sheet having a dry culture layer in a predetermined region on the base sheet,
The culture layer virtually divides the predetermined region into a plurality of divided regions, and is dried after individually or continuously injecting a medium solution for forming the culture layer into each divided region. A microorganism culture sheet.   The microorganism culture sheet according to claim 1, wherein a medium solution is dispensed into each of the divided regions from above the predetermined region via a porous nozzle.   The microorganism culture sheet according to claim 1 or 2, wherein the medium solution contains an alcohol solvent.   The microorganism culture sheet according to any one of claims 1 to 3, wherein the medium solution is a dispersion system in which at least one of the components of the culture layer is dispersed in a solvent.   The microorganism culture sheet according to any one of claims 1 to 4, wherein a concave portion is formed in advance in the predetermined region, and the concave portion is the predetermined region. A method for producing a microorganism culture sheet comprising a dried culture layer in a predetermined region on a substrate sheet,
The predetermined region is virtually divided into a plurality of divided regions, and a medium solution for forming the culture layer is individually or continuously injected into each divided region, and then dried to dry the culture layer. A method for producing a microorganism culture sheet comprising the step of forming.   The method for producing a microorganism culture sheet according to claim 6, further comprising a step of forming a frame layer made of a hydrophobic resin on an outer periphery of the culture layer. A method for producing a microorganism culture sheet comprising a dried culture layer in a predetermined region on a substrate sheet,
Forming a recess surrounded by the frame layer in the predetermined region by forming a frame layer made of a hydrophobic resin on the base sheet;
The concave portion is virtually divided into a plurality of divided regions, and a culture solution for forming the culture layer is individually or continuously injected into each divided region, and then dried to form a culture layer. And a method for producing a microorganism culture sheet.   The method for producing a microorganism culture sheet according to any one of claims 6 to 8, wherein a medium solution is injected into each of the divided regions from above the predetermined region through a porous nozzle. A medium solution injection device for injecting a medium solution for forming a culture layer into a predetermined region on a base sheet,
A medium solution injection device comprising a nozzle for virtually dividing the predetermined region into a plurality of divided regions and individually or continuously injecting the medium solution into each divided region.   The medium injection device according to claim 10, wherein the nozzle is a porous nozzle. The medium solution injecting device according to claim 11, wherein each hole diameter of the porous nozzle is 0.1 to 3.0 mm / piece, and a hole arrangement density is 5 to 20 pieces / cm ² .