JP7507591B2 - Microbial culture sheet - Google Patents

Description

The present invention relates to a microbial culture sheet.

A microbial culture sheet, which has a dry culture medium provided on a base sheet, is known as a means for confirming the presence of microorganisms in microbial testing.

Various types of microbial culture sheets that can realize simple microbial testing have been developed. For example, Patent Document 1 proposes a microbial culture sheet that has a base sheet, a culture layer containing a predetermined component formed on the base sheet, and a cover sheet that covers the culture layer.

International Publication No. 2011/007802

However, there is a need for a microbial culture sheet that can culture various types of microorganisms more efficiently and makes it easier to detect the development of microbial colonies.

The present invention was made in consideration of the above-mentioned circumstances, and aims to provide a microbial culture sheet suitable for culturing various types of aerobic microorganisms.

The present inventors have discovered that the above problems can be solved by adjusting the oxygen permeability of the cover sheet within a predetermined range in a microbial culture sheet having a base sheet, a culture layer formed on the base sheet, and a cover sheet covering the culture layer, and have completed the present invention. More specifically, the present invention provides the following.

(1) a base sheet;
A culture layer formed on the base sheet;
A cover sheet that covers the culture layer,
the cover sheet has an oxygen transmission rate of 10,000 cc/ ^m2 ·day·atm or more and 50,000 cc/ ^m2 ·day·atm or less at 23°C and 90% RH, as measured in accordance with JIS K 7126 B method;
Used for culturing aerobic microorganisms,
Microbial culture sheet.

(2) The microorganism culture sheet according to (1), wherein the cover sheet contains polymethylpentene.

(3) The microbial culture sheet according to (1) or (2), wherein the thickness of the cover sheet is 100 μm or less.

(4) The microbial culture sheet according to any one of (1) to (3), wherein the aerobic microorganism is a fungus.

(5) A microbial culture sheet according to any one of (1) to (4), in which a frame layer made of a hydrophobic resin is formed on the outer periphery of the culture layer.

The present invention provides a microbial culture sheet suitable for culturing various types of aerobic microorganisms.

FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is a diagram showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is an external perspective view showing an example of a microorganism culture sheet according to the present invention. FIG. 1 is an external perspective view showing an example of a microorganism culture sheet according to the present invention. FIG. 13 is a diagram showing the state of colony development in the microorganism culture sheet according to the embodiment. FIG. 13 is a diagram showing the state of colony development in the microorganism culture sheet according to the embodiment.

Specific embodiments of the present invention are described in detail below, but the present invention is not limited to the following embodiments and can be modified as appropriate within the scope of the invention.

<Microbial culture sheet>
The microorganism culture sheet of the present invention comprises a base sheet, a culture layer formed on the base sheet, and a cover sheet covering the culture layer, and the oxygen permeability of the cover sheet at 23°C and 90% RH in accordance with JIS K 7126 B method is 10,000 cc/ ^m2 -day-atm or more and 50,000 cc/ ^m2 -day-atm or less, and is used for culturing aerobic microorganisms.

(Example of the composition of the microbial culture sheet)
Hereinafter, examples of the configuration of the microorganism culture sheet of the present invention will be described with reference to the drawings as appropriate.

An example of a representative embodiment of the microorganism culture sheet of the present invention is shown in Figures 1(a) and (b), where Figure 1(a) is a plan view and Figure 1(b) is a cross-sectional view taken along line AA' of Figure 1(a).
FIG. 1 shows an embodiment in which a culture layer (30) is formed approximately in the center of a rectangular base sheet (10), and a rectangular cover sheet (40) is provided so as to cover the culture layer (30).

As shown in the cross-sectional view of FIG. 2(a), the base sheet (10) may be a multi-layer sheet consisting of two layers, a base sheet (11) and a base sheet (13), or may be a multi-layer sheet in which a layer other than plastic is laminated. Also, as shown in FIG. 2(b), the culture layer (30) may be composed of two or more layers.

The shape of the culture layer (30) is not particularly limited, and may be circular as shown in FIG. 1, or may be square, rectangular, other polygonal, or irregular.

As shown in the development view of Figure 3 (a), the microorganism culture sheet of the present invention may have a culture layer (30) and a specific component layer (30') containing a binder component, a nutrient component and other components formed on both the base sheet (10) and the cover sheet (40).
For example, when a gelling agent is contained in both the culture layer (30) and the specific component layer (30'), the culture layer (30) on the base sheet and the specific component layer (30') provided on the cover sheet (40) are arranged facing each other. Therefore, for example, when a test liquid is inoculated into the culture layer (30) on the base sheet (10) and then the culture layer (30) is covered with the cover sheet (40), the test liquid is absorbed into the culture layer (30) and the specific component layer (30'), and microorganisms can be observed in both the culture layer (30) and the specific component layer (30').
In addition, depending on the type of indicator, if it is contained in the culture layer (30), the coloring may become noticeable during the sterilization treatment. In such a case, by containing the indicator in the specific component layer (30'), the coloring during the sterilization treatment can be prevented.
FIG. 3(b) is a cross-sectional view of a microorganism culture sheet provided with a specific component layer (30').

The microbial culture sheet of the present invention may have a culture layer (30) formed approximately in the center of a base sheet (10) and a circular frame layer (20) formed on the outer periphery of the culture layer, as shown in the plan view of FIG. 4(a) and the cross-sectional view of line A-A' of FIG. 4(b).
The frame layer (20) makes it easier to limit the range in which the test liquid inoculated in the culture layer (30) spreads, and prevents leakage of the test liquid from the microorganism culture sheet. Furthermore, the frame layer (20) makes it easier to spread the test liquid inoculated in the culture layer (30) evenly within a predetermined range. The shape of the frame layer (20) is not particularly limited, and may be circular as shown in FIG. 4, or may be rectangular, elliptical, polygonal, or irregular.

In the embodiment shown in FIG. 4, as shown in FIG. 5, a specific component layer (30') may be further formed on the cover sheet (40) so as to face the culture layer (30) on the base sheet (10).

As shown in FIG. 6, an adhesive layer (50) may be formed on the cover sheet (40) so as to face the frame layer (20). By bringing the frame layer (20) and the adhesive layer (50) into close contact with each other, drying and contamination of the culture layer (30) can be more effectively prevented.

The base sheet (10) and the culture layer (30) may each be a single layer or a multilayer.
For example, as shown in the cross-sectional view of FIG. 7(a), when a frame layer (20) made of a hydrophobic resin is formed on the surface of a base sheet (10), the base sheet (10) may be a multilayer sheet consisting of two layers, a base sheet (11) and a base sheet (13), or may be a multilayer sheet in which a layer other than plastic is laminated.
As shown in the cross-sectional view of Figure 7 (b), when a frame layer (20) made of a hydrophobic resin is formed on the surface of the base sheet (10), the culture layer (30) may be composed of two or more layers.

A plurality of culture layers (30) may be present on the base sheet (10).
For example, Figures 8(a) and (b) show an embodiment in which a plurality of culture layers (30), a frame layer (20) made of a convex hydrophobic resin surrounding the outer periphery of the culture layers (30), and a cover sheet (40) covering the culture layers (30) are fixed at the end of a base sheet (10). Figure 8(a) is a plan view, and Figure 8(b) is a cross-sectional view taken along the line A-A' of Figure 8(a).
In FIG. 8, two corners (C1, C2) are formed so that the cover sheet (40) can cover the culture layer (30) and be in close contact with the top of the frame layer (20).
In this embodiment, a specific component layer (30') may be formed on the cover sheet (40) so as to face the culture layer (30) formed on the base sheet (10).

When the base sheet and the cover sheet described later are made of the same material, as shown in the development view of FIG. 9(a) and the cross-sectional view of FIG. 9(b), the base sheet (10) and the cover sheet (40) may be connected together, and the culture layer (30) and, optionally, the frame layer (20) may be formed on the base sheet (10), and the cover sheet (40) may be folded to form a microbial culture sheet. FIG. 9(c) shows a cross-sectional view of the folded state. According to this method, even when a specific component layer (30') or an adhesive layer (50) is formed on the cover sheet, the base sheet (10) and the cover sheet (40) are connected together, so that the specific component layer (30') can be accurately and easily formed at a position facing the culture layer (30) on the base sheet (10).

Also, as shown in the cross-sectional views of Figures 10 and 11, the cover sheet (40) and the base sheet (10) may be made of different materials, and the cover sheet (40) and the base sheet (10) on which the culture layer (30) is formed may be attached together with a double-sided adhesive tape (60) or the like.

Figures 12 and 13 show external perspective views of a microbial culture sheet in which a lattice print is formed on a cover sheet (40) and the cover sheet (40) and a base sheet (10) on which a culture layer (30) is formed are bonded at the ends. In Figure 13, a frame layer (20) is formed around the culture layer (30).

The microorganism culture sheet of the present invention may have any configuration, such as a gripping portion, as long as it does not impede the object of the present invention.

(Base sheet)
The base sheet in the microorganism culture sheet of the present invention can be any shape, size, material, etc. used in the culture of microorganisms. The base sheet preferably has solvent resistance, printability, water resistance, heat resistance that can withstand the drying treatment when forming the culture layer, etc.

The substrate sheet may be a single layer or a laminated sheet of two or more layers.

The base sheet may be made of the same material as the cover sheet described below, or it may be made of a different material.

When the base sheet is a single layer, plastic sheets (e.g., sheets of polymethylpentene, polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyvinyl chloride, etc.), paper base materials, etc. can be suitably used.

Since plastic sheets are usually transparent, microbial colonies can be easily observed by light transmitted through the base sheet, improving visibility of the colony development state. However, this does not exclude the base sheet being opaque (ones that are white due to foaming, colored ones, etc.). The transparency of the base sheet can be appropriately selected depending on the ease of counting the cultured bacteria, etc.

Examples of paper substrates include synthetic paper made primarily from synthetic resin. Examples of such synthetic paper include "Yupo" manufactured by Yupo Corporation and "Crisper" manufactured by Toyobo.

When the base sheet is a laminated sheet, a laminate of two or more of the above-mentioned plastic sheets or paper base materials can be preferably used. For example, a laminated sheet in which a paper base material is laminated to the above-mentioned plastic sheet, or a laminated sheet in which a paper base material is coated with a synthetic resin can be preferably used. More specifically, for example, a laminated sheet of a polyethylene film and a paper base material can be exemplified.

To improve adhesion to the culture layer, the substrate sheet may be surface-treated on the surface on which the culture layer is to be provided. Examples of such surface treatments include application of an anchor coating agent, corona discharge treatment, ozone treatment, low-temperature plasma treatment (plasma treatment using oxygen gas or nitrogen gas, etc.), glow discharge treatment, and oxidation treatment (oxidation treatment using chemicals, etc.).

Examples of anchor coating agents include isocyanate-based (urethane-based), polyethyleneimine-based, polybutadiene-based, and organic titanium-based agents. Examples include those mainly composed of polyether polyurethane-based resins, polyester polyurethane-based resins, and polyacrylate polyurethane-based resins obtained by reaction with aromatic polyisocyanates (tolylene diisocyanate, diphenylmethane diisocyanate, polymethylene polyphenylene polyisocyanate, etc.), polyfunctional isocyanates (aliphatic polyisocyanates such as hexamethylene diisocyanate and xylylene diisocyanate), and hydroxyl group-containing compounds (polyether polyols, polyester polyols, polyacrylate polyols, etc.).

It is preferable that the base sheet is flat and free from curling, and its thickness is not particularly limited, but is usually 25 μm or more and 1500 μm or less, preferably 50 μm or more and 500 μm or less.

The base sheet may be printed with a grid pattern or the like.

(Culture layer)
The shape, composition, etc. of the culture layer are not particularly limited and can be appropriately set depending on the type of microorganism to be cultured, etc.

The culture layer is usually obtained by applying a medium liquid (such as an aqueous medium solution) to a base sheet by any method, followed by drying, etc. After dropping the test liquid onto the culture layer, the test liquid can be allowed to naturally spread over a certain area by quickly covering the cover sheet, realizing good operability.

The method of applying the medium liquid to the base sheet is not particularly limited, and examples include application by printing (screen printing, gravure printing, letterpress printing, transfer printing, flexographic printing, etc.), application using a dispenser or inkjet, coating using a bar, bar coat, knife coat, die coat, etc., and spraying using a spray method, etc.

The method for drying the medium liquid is not particularly limited, and as long as it is possible to remove the solvent, drying can be performed in accordance with conventionally known methods such as adjusting temperature and pressure.

The culture layer may be a single layer or may be a multi-layer consisting of two or more layers.

The shape of the culture layer is not particularly limited, but is preferably a circle. The diameter of the circle is preferably 20 mm or more and 80 mm or less, more preferably 30 mm or more and 70 mm or less. The amount of test liquid dropped onto the microbial culture sheet is generally 1 ml, and if the amount is within the above range, the test liquid can be sufficiently absorbed.

The thickness of the culture layer (height from the surface of the base sheet) is 50 μm to 1000 μm when dry, more preferably 200 μm to 600 μm. The coating amount is 5 g/m ² to 400 g/m ² when dry, more preferably 100 g/m ² to 300 g/m ^2. Adjusting the thickness to the above range makes it easy to obtain a culture layer optimal for the growth of bacteria.

The medium solvent is not particularly limited, but it is preferable to use a solvent that can be removed by drying. Examples include water and alcohol (ethanol, etc.).

The composition of the medium liquid constituting the culture layer is not particularly limited, but may, for example, contain polyvinylpyrrolidone and at least one selected from the group consisting of a gelling agent, a nutrient component, a color indicator, a selection agent, and a substrate.

[Polyvinylpyrrolidone]
Polyvinylpyrrolidone is a component that enables adjustment of the viscosity of the medium liquid and also functions as a binder.

[Gelling agent]
The gelling agent thickens or gels when inoculated with the test liquid, providing a viscosity suitable for the growth of bacteria. In addition, after thickening, the gelling agent can adhere closely to the cover sheet, preventing the culture layer from drying out during the culture.

As the gelling agent, a polymeric polysaccharide can be suitably used, and examples thereof include carrageenan, guar gum, xanthan gum, hydroxyethyl cellulose, carboxymethyl cellulose, locust bean gum, algin, etc. These polymeric polysaccharides thicken or gel with water when the test liquid is dropped, which promotes good bacterial growth, and also provides excellent visibility of the colonies due to their high transparency. Furthermore, the thickening allows the liquid to adhere closely to the cover sheet, making it possible to suppress drying during cultivation.

[Nutritional Information]
The nutritional components are components that enable the growth of microorganisms, and can be used in combination with known components (peptones, sugars, extracts, inorganic salts, etc.) The type, amount, combination, etc. of the nutritional components to be used can be appropriately set depending on the type of microorganism, etc.

As nutritional components, for example, for general live bacteria testing, a mixture of yeast extract, peptone, and glucose, a mixture of meat extract and peptone, a mixture of peptone, soy peptone, and glucose, or mixtures of these with added dipotassium phosphate and/or sodium chloride may be used.
For testing for E. coli and coliform bacteria, sodium desoxycholate, peptone, ferric ammonium citrate, sodium chloride, dipotassium phosphate, lactose, peptone, lactose, dipotassium phosphate, etc. are used.
For Staphylococcus aureus, a mixture of meat extract, peptone, sodium chloride, mannitol and egg yolk, or a mixture of peptone, meat extract, yeast extract, sodium pyruvate, glycine, lithium chloride and tellurite egg yolk liquid, etc., can be used.
For Vibrio, yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, sodium cholate, ferric citrate, sodium chloride, ox bile, etc. are used.
For enterococci, beef brain extract, heart extract, peptone, glucose, dipotassium phosphate, sodium nitride, etc. are used.
For fungi, a peptone/glucose mixture, a yeast extract/glucose mixture, a potato extract/glucose mixture, etc. are used.

[Color indicator, selective agent, substrate, and other components]
In the microorganism culture sheet of the present invention, the medium liquid may contain other components such as a color indicator, a selection agent, and a substrate.

Color indicators color colonies by reacting with specific substances produced by the metabolism of microorganisms growing during the culture process, by recognizing pH changes, by reacting with enzymes, etc., making it extremely easy to count the number of colonies.
Specific examples of such color indicators include tetrazolium salts (triphenyltetrazolium chloride (hereinafter referred to as TTC), p-tolyltetrazolium red, tetrazolium violet, petetryltetrazolium blue, etc.), pH indicators (neutral red mixture, phenol red, bromthymol blue, thymol blue mixture, etc.), color-developing enzyme substrates (5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl phosphate, etc.), and the like.

The medium may contain a selective agent that suppresses the growth of microorganisms other than those to be detected. Examples of such selective agents include antibiotics, synthetic antibacterial agents, dyes, surfactants, inorganic salts, etc.
Examples of antibiotics include methicillin, ceftamethazole, cefixime, ceftazidime, cefsulodin, bacitracin, polymyxin B, rifampicin, novobiocin, colistin, lincomycin, gentamicin, chloramphenicol, tetracycline, streptomycin, and the like.
Examples of synthetic antibacterial agents include sulfa drugs, nalidixic acid, and olaquindox.
Examples of dyes include crystal violet, brilliant green, malachite green, methylene blue, and the like, which have bacteriostatic or bactericidal properties.
Examples of the surfactant include Tergitol 7, dodecyl sulfate, and lauryl sulfate.
Examples of inorganic salts include selenite, tellurite, sulfite, sodium nitride, lithium chloride, oxalate, and high-concentration sodium chloride.
In addition to the above, taurocholate, glycine, bile powder, bile salts, deoxycholate, etc. can also be used.

Other ingredients that can be added to the culture medium include plasticizers (glycerin, polyethylene glycol, etc.).

(Cover sheet)
The cover sheet covers the culture layer and has an oxygen permeability of 10,000 cc/ ^m2 ·day·atm or more and 50,000 cc/ ^m2 ·day·atm or less, preferably 15,000 cc/ ^m2 ·day·atm or more and 40,000 cc/ ^m2 ·day·atm or less.

In this invention, "oxygen permeability" refers to the oxygen permeability at 23°C and 90% RH according to JIS K 7126 B method.

The microbial culture sheet of the present invention is suitable for culturing aerobic microorganisms because the cover sheet satisfies the above requirements. Furthermore, the cover sheet can prevent contamination by falling bacteria during the culture, and can also prevent water evaporation from the culture layer.

The composition of the cover sheet is not particularly limited as long as it satisfies the above requirements, but it is usually made of a resin. The cover sheet preferably contains polymethylpentene, and more preferably consists of polymethylpentene.

The cover sheet as a whole may contain any combination of resins as long as it has an oxygen transmission rate of 10,000 cc/m ² ·day·atm or more and 50,000 cc/m ² ·day·atm or less.

The cover sheet is preferably transparent so that colonies can be observed and counted through the cover sheet after culturing the microorganisms.

The cover sheet may or may not be fixed to the base sheet via a joint. An example of the joint is an adhesive layer such as a double-sided tape. The cover sheet and base sheet may be bonded together by heat sealing or lamination.

The cover sheet may be subjected to a surface treatment such as corona discharge treatment as described in the section on the base sheet.

The thickness of the cover sheet is preferably 100 μm or less, and more preferably 20 μm or more and 50 μm or less, from the viewpoint of easily adjusting the oxygen permeability to the above range.

The shape of the cover sheet is not particularly limited, but it is preferable that the size of the cover sheet is large enough to cover the entire culture layer in order to prevent the infiltration of bacteria into the culture layer.

The cover sheet may have a grid pattern or the like printed on it.

(Frame layer)
It is preferable that a frame layer made of a hydrophobic resin is formed on the outer periphery of the culture layer. When the frame layer is formed, the test liquid is rapidly diffused to the frame layer when the culture layer is inoculated with the test liquid and covered with a cover sheet, so that the cover sheet can be covered without waiting for the culture layer to absorb the test liquid, and the inoculation operation can be performed in a short time. In addition, the frame layer can more reliably prevent leakage of the test liquid, and improve the work efficiency during inoculation.

The width of the frame layer (the distance between the surface of the frame layer facing the culture layer and the surface on the opposite side) may be uniform or non-uniform. For example, the narrowest part of the frame is preferably 0.5 mm to 5.0 mm in width, and more preferably 1.0 mm to 3.0 mm in width. Within the above range, even if the culture layer absorbs water and swells, there is little risk of the shape of the frame layer being distorted, and the test liquid can be spread within the area within the frame layer.

The height of the frame layer (height from the surface of the base sheet) is not particularly limited, but is preferably higher than the height of the culture layer (height from the surface of the base sheet) from the viewpoint of preventing leakage of the test liquid, etc. When the height of the frame layer is higher than the height of the culture layer, the difference between the height of the frame layer and the height of the culture layer is, for example, preferably 100 μm or more and 1200 μm or less, more preferably 200 μm or more and 1000 μm or less, and particularly preferably 300 μm or more and 800 μm or less.
Within the above range, the test liquid can be absorbed into the culture layer and spread over the entire surface, leakage of the test liquid can be more reliably prevented, and adhesion to the cover sheet can be ensured.

If the height of the frame layer is excessively lower than the height of the culture layer, the spread of the diffused test liquid cannot be suppressed, and leakage may occur.
If the height of the frame layer is excessively higher than that of the culture layer, an excessive space is generated between the culture layer and the cover sheet when the test liquid is inoculated, making it difficult for the surface of the culture layer to adhere to the cover sheet, which may result in the culture layer drying out easily during culture.

If a frame layer is formed on the culture layer, there is a risk that the culture layer will collapse when it absorbs the test liquid, swells, and becomes gel-like. Therefore, from the viewpoint of improving operability and stability of the operation, it is preferable that the frame layer is formed directly on the base sheet.

The shape of the frame layer is not particularly limited as long as it is disposed around the outer periphery of the culture layer. For example, the following shapes can be mentioned.
A frame layer is formed by applying a hydrophobic resin by any method such as printing or coating so as to surround the portion of the surface of the base sheet where the culture layer is to be formed.
A frame layer formed by adhering a hollowed-out piece of hydrophobic material to the outer periphery of the culture layer.
A frame layer formed by embossing a base sheet.
A frame layer is formed by carving a portion of a base sheet (such as a thick hydrophobic base material) where a culture layer is to be formed into a concave shape, and forming the frame layer as the outer periphery of the concave portion.

When the frame layer is formed by embossing, the following two methods are possible.
(1) On the surface of a base sheet, a convex shape is formed as a frame layer at a position corresponding to the outer periphery of a culture layer. The culture layer is formed within the area surrounded by the convex shape.
(2) A recess is formed on the surface of the base sheet at a position where a culture layer is to be formed. The outer periphery of the recess corresponds to a frame layer.

The frame layer may be a single one, or may be a double frame layer with an additional frame layer formed on the outside of one frame layer.

The hydrophobic resin that forms the frame layer is not particularly limited, but UV-curable resins, hot-melt resins, thermal foaming inks, UV foaming agents, etc. can be suitably used. These resins may or may not be colored.

The UV-curable resin may be either radical or cationic. Examples of the radical type include acrylate-based resins and unsaturated polyester-based resins. Examples of the cationic type include epoxy acrylate-based resins, oxetane-based resins, and vinyl ether-based resins.

As hot melt resins, those with a softening point temperature of preferably 120°C or less, more preferably 100°C or less can be used. If the softening point temperature exceeds 120°C, the base sheet may curl. Examples of hot melt resins include synthetic rubbers such as urethane resins, polyamide resins, polyolefin resins, polyester resins, ethylene vinyl acetate resins, styrene butadiene rubber resins, and urethane rubber resins.

As the thermally foamable ink, for example, inks using any binder (polyurethane resin, polyamide resin, polyvinyl chloride resin, polyacrylate resin, polyester resin, polyolefin resin such as chlorinated polypropylene, rubbers such as chlorinated rubber and cyclized rubber, etc.) can be used. These inks are more preferable if they are water-repellent. From the viewpoint of higher water repellency, inks in which silicones or waxes are added to the above resins are even more preferable.

Examples of UV foaming agents include resins that can be used in thermal foaming inks to which conventionally known foaming agents have been added. Examples of preferred UV foaming inks include ultraviolet curing inks that contain at least a reactive diluent (acrylate reactive diluent, methacrylate reactive diluent, etc.), a photopolymerizable oligomer (urethane acrylate photopolymerizable oligomer, polyester acrylate photopolymerizable oligomer, epoxy acrylate photopolymerizable oligomer, acrylic photopolymerizable oligomer, special photopolymerizable oligomer, etc.), and a photopolymerization initiator, and a foaming agent (inorganic foaming agent, organic foaming agent, liquid foaming agent, etc.) is blended therein.
When a UV foaming ink is used, the frame layer can be formed by curing the resin through UV irradiation.

Additives such as defoamers, polymerization inhibitors, and pigment dispersants may be added to the hydrophobic resin. The types and amounts of additives can be set according to the intended purpose.

(Culture subject)
The microorganism culture sheet of the present invention can be used for culturing various kinds of aerobic microorganisms, including, but not limited to, fungi, Pseudomonas genus, Bacillus genus, and the like.
Preferred culture subjects of the microorganism culture sheet of the present invention include fungi such as Penicillium citrinum, Wallemia sebi, Aspergillus niger, Cladosporium cladosporioides, Fusarium solani, Candida albicans, etc. Preferred culture subjects of the microorganism culture sheet of the present invention are Penicillium citrinum.

The present invention makes it possible to culture a wider variety of aerobic microorganisms than with the microbial culture sheets that have been used conventionally for culturing aerobic microorganisms.

(Microbial culture kit)
The present invention also includes a microorganism culture kit comprising the above-mentioned microorganism culture sheet in combination with any test liquid.

The test liquid is not particularly limited, and may be a liquid sample in which the presence or absence of the aerobic microorganisms to be detected is unknown.

(Method for producing a microorganism culture sheet)
The method for producing the microorganism culture sheet of the present invention is not particularly limited. For example, the microorganism culture sheet can be obtained by applying and drying a medium liquid on a base sheet to form a culture layer, and covering the medium layer with a cover sheet.

When the microbial culture sheet has a frame layer, the frame layer and the culture layer can be prepared in any order.

The microorganism culture sheet of the present invention may be sterilized by gamma ray irradiation, ethylene oxide gas sterilization, or the like to produce the microorganism culture sheet of the present invention.

The present invention will now be described in detail with reference to examples, but these examples are not intended to limit the present invention in any way.

<Preparation of microbial culture sheet>
A microbial culture sheet was prepared in the following manner.

(Preparation of base sheet)
A synthetic paper (product name "Yupo", manufactured by Yupo Corporation) whose main raw material was synthetic resin was prepared as the base sheet (10). The dimensions of the base sheet (10) were set to 72 mm wide x 90 mm deep x 270 μm thick.

A joint made of double-sided tape measuring 6 mm in length and 72 mm in width was provided on the upper surface of the base sheet (10) along one side of the base sheet (10). This joint joins the base sheet to the cover sheet described below.

(Preparation of frame layer)
A circular frame layer (20) made of ethylene vinyl acetate-based hot melt resin was provided on the upper surface of the base sheet (10). The width of the frame layer (20) (the distance between the outer edge and the inner edge of the frame layer (20)) was 1 mm, the inner diameter of the frame layer (20) (the diameter of the circle formed by the inner edge) was 50 mm, and the height of the frame layer (20) (height from the surface of the base sheet (10)) was set to 750 μm.

(Preparation of culture layer)
A medium liquid for forming a culture layer (30) was applied to the upper surface of the base sheet (10) and within the area surrounded by the frame layer (20) using a dispenser. The applied medium liquid was dried to form the culture layer (30) on the base sheet (10). The thickness of the obtained culture layer (30) (height from the surface of the base sheet (10)) was 250 μm.

The medium solution contains solid components including a gelling agent, nutrients, a color indicator, a selection agent, a fixing agent, and a plasticizer, and a solvent (methanol) for adjusting viscosity. The composition of the medium solution is shown in Table 1.
[Solid components of culture medium]
Gelling agent: mixture of guar gum and psyllium seed gum (guar gum:psyllium seed gum (mass ratio) = 3:2)
Nutrients: mixture of peptone, sugar, extracts, and inorganic salts Color indicator: 5-bromo-4-chloro-3-indoxyl phosphate Selective agent: chloramphenicol Fixative: polyvinylpyrrolidone (PVP)
Plasticizer: Polyethylene glycol

(Creating the cover sheet)
As the cover sheet (40), a sheet was prepared made of a material having the thickness and oxygen permeability shown in Table 2. The dimensions of the cover sheet (40) were set to 72 mm width x 95 mm depth.

Details of the materials for each cover sheet are as follows:
Polypropylene: biaxially oriented polypropylene, product name "OP U-1", manufactured by Tocello Co., Ltd. Polymethylpentene: product name "Opulent TPX Film", manufactured by Mitsui Chemicals, Inc. Polystyrene: biaxially oriented polystyrene sheet, product name "OPS Film", manufactured by Asahi Kasei Chemicals Corporation Polyethylene: low-density polyethylene, manufactured by Dai Nippon Printing Co., Ltd.

The oxygen permeability of each cover sheet was determined in accordance with JIS K 7126 B method.

One side of each cover sheet 40 was attached to the joint of the base sheet 10 having the frame layer 20 and the culture layer 30, and a microbial culture sheet according to the embodiment or comparative example was obtained, which has a common configuration except for the material of the cover sheet.

<Microbial culture test>
Test solutions for culture tests were prepared from various strains by the following method, and culture tests were carried out using the microbial culture sheets obtained above.

(Preparation of test solution for culture test)
A spore suspension of each strain shown in Table 3 was diluted with sterile saline (containing 0.05% Tween 80) to give a bacterial count of 10 to 10 ² /ml to prepare a test solution for the culture test.

All of the bacteria shown in Table 3 are aerobic microorganisms (fungi).

(Culture test method)
The obtained test liquid for culture test was inoculated onto the surface of the culture layer (30) of each microbial culture sheet in an amount of 1 ml using a sterilized pipette. A cover sheet (40) was placed on top of the test liquid, and the test liquid was spread over the entire culture layer.
Next, each microbial culture sheet was placed in an incubator and cultured at 25° C. for 48 hours, 66 hours, or 72 hours, and the occurrence of colonies at each time point was visually confirmed. In this example, colonies were confirmed as blue dots.

The state of colony occurrence on each microbial culture sheet is shown in Figures 14 and 15. The state of colony occurrence was evaluated based on the following criteria.
[Evaluation criteria]
◯: The color is developed enough to allow the number of bacteria to be counted, and visibility is high.
△: The color is developed enough to count the number of bacteria, but visibility is low.
×: No color developed to the extent that the number of bacteria could be counted.

The microbial culture sheet of the present invention, which is provided with a cover sheet made of polymethylpentene, was able to effectively detect all aerobic bacteria. It was also found that the microbial culture sheet of the present invention was able to effectively detect various fungi.

On the other hand, some aerobic bacteria could not be detected sufficiently with the microbial culture sheet according to the comparative example. In particular, Penicillium citrinum could only be detected with the microbial culture sheet according to the present invention.

From the above, it has been found that the microbial culture sheet of the present invention is suitable for culturing various types of aerobic microorganisms.

REFERENCE SIGNS LIST 10 Base sheet 20 Frame layer 30 Culture layer 30' Specific component layer 40 Cover sheet 50 Adhesive layer 60 Double-sided adhesive tape

Claims (5)

A base sheet;
A culture layer formed on the base sheet;
A cover sheet that covers the culture layer,
the cover sheet comprises polymethylpentene;
the cover sheet has an oxygen transmission rate of 15,000 cc/ ^m2 ·day·atm or more and 40,000 cc/ ^m2 ·day·atm or less at 23°C and 90% RH, as measured in accordance with JIS K 7126 B method;
Used for culturing aerobic microorganisms,
Microbial culture sheet. The microorganism culture sheet according to claim 1 , wherein the cover sheet is made of polymethylpentene. The microorganism culture sheet according to claim 1 or 2, wherein the thickness of the cover sheet is 100 μm or less. The microbial culture sheet according to any one of claims 1 to 3, wherein the aerobic microorganism is a fungus. The microbial culture sheet according to any one of claims 1 to 4, in which a frame layer made of a hydrophobic resin is formed on the outer periphery of the culture layer.