JP2011072279A - Microorganism culture sheet - Google Patents

Abstract

The present invention provides a microorganism culture sheet that is used for bacterial count inspection and the like and has excellent operability and sealing properties.
A base sheet, a culture layer formed on the base sheet, and a cover sheet that covers the culture layer, the base sheet and the cover sheet being openable and closable via a hinge portion. Are microbial culture sheets connected to each other,
The base material sheet and / or the cover sheet is provided with a lock mechanism for maintaining the closed state of the cover sheet between the base material sheet and the cover sheet. Can provide.
[Selection] Figure 1

Description

  The present invention relates to a compact microorganism-cultivated sheet that is used for bacterial count inspection and the like and is easy to handle.

As a method for confirming the presence of microorganisms or measuring the number of microorganisms, there is an agar plate pour method, and an agar medium formed in a petri dish previously sterilized is used. This method requires an autoclave for high-pressure steam sterilization of the agar medium and a laboratory that can perform microbiological tests aseptically. From microbial sampling to sample liquid preparation, dispensing, and mixing with the medium. Skillfulness is required for the operation of the microbiological examination from the culture to the counting. Therefore, a dry culture medium has been developed as a microorganism culture sheet that allows simple microorganism testing without requiring a high degree of skill.
For example, a cold water-soluble powder containing a water-based adhesive composition layer formed on the upper surface of a support, a gelling agent attached to the adhesive composition layer, and the cold water-soluble powder are coated. There is a culture substrate device comprising a cover sheet (Patent Document 1). When the cover sheet is peeled off and the aqueous test sample is placed on a cold water soluble powder layer, the gelling agent quickly hydrates to form a reconstitution medium that maintains microbial growth, and covers the cover sheet for a specified time. When incubated, colonies can be counted through a transparent cover sheet.

There is also a sheet-like culture container comprising a base material having one or more water-absorbing portions, and a portion other than the water-absorbing portions being water-repellent, and a film that seals the water-absorbing portions (Patent Document 2). ). It consists of a sheet-like base material coated with a medium and a water-absorbing resin, and a transparent film with a water-absorbing resin formed on the side of the base material. Since it can be cultured by sticking, it has the advantage of being lighter and less bulky than conventional Petri dishes and petri dishes. When the container is immersed in a liquid medium so that all the water absorption parts are peeled off and pulled up, the liquid medium is absorbed only in the water absorption part, and the liquid medium does not adhere to the water repellent parts other than the water absorption part. An independent culture medium can be obtained easily and reliably, and culture of microorganisms can be observed in this state if the water-absorbing part is sealed with a film after inoculating a desired microorganism into the medium.
In addition, in the microorganism culture sheet of a microorganism having a waterproof base sheet and a cover sheet placed on the base sheet, and at least a layer of the medium mixture is provided on the base sheet, the medium mixture There is also a microorganism culture sheet characterized in that an outer frame layer made of hydrophobic ink is provided on the layer to make the spread of the test solution to be inoculated constant (Patent Document 3). In the examples, a transparent biaxially stretched polyester film having a thickness of 100 μm is used as a base sheet, and a coating solution for the medium mixture layer is applied thereon with a comma coater so that the coating amount when dried is 30 g / m ^2. On the medium mixture layer, a donut-shaped pattern having an inner diameter of 50 mm and an outer diameter of 70 mm is screen-printed with a hydrophobic ink RAM (Seiko Advance Co., Ltd.) to form an outer frame layer.

  Furthermore, there is a microorganism culture device in which a circular porous matrix layer and a water-soluble polymer compound layer are laminated on a square pressure-sensitive adhesive sheet, and a square transparent film is disposed on the top (Patent Document 4). When the sample solution is added onto the porous matrix layer, the sample solution is retained in the porous matrix layer, and the retained water dissolves the water-soluble polymer compound layer in contact with the porous matrix layer, resulting in water solubility. It becomes an environment where microorganisms can grow by the polymer compound aqueous solution and the growth nutrient component. In this microorganism incubator, since the microorganisms do not penetrate into the water-soluble polymer compound layer and are further pushed up to the surface of the porous matrix layer, colonies are formed on or near the surface of the porous matrix layer, so that a transparent film When the cover sheet made of is adhered to the adhesive sheet, it can be used as a sheet-shaped microorganism incubator, which is lightweight, low in volume and easy to handle. In this case, the periphery of the area where microorganisms grow is sealed by the cover sheet and the adhesive sheet.

Japanese Patent Publication No. 3383304 JP-A-7-289235 JP-A-8-280377 International Publication No. WO01 / 044437

It is necessary to seal the water-absorbing part with a cover sheet after inoculating the culture medium with a desired microorganism.
When the base sheet is composed of a flexible sheet or film, the sealing between the cover sheet and the base sheet is caused by deformation such as the float or slack of the cover sheet or the curl of the base sheet provided with the culture medium. And has the potential to affect the growth of colonies to be observed due to changes in the inflow of air into the medium.

  Although the sealing property may be improved by using an adhesive sheet as in the above-mentioned Patent Document 4, an operation of peeling the cover sheet and the adhesive sheet occurs at the start of culture of the bacteria, resulting in a problem that the workability is deteriorated. . In addition, it is necessary to adhere the cover sheet after dropping the bacterial solution and then covering the cover sheet. Moreover, wrinkles of the cover sheet due to the thickness of the medium are likely to occur, and once the cover sheet is adhered in the state of wrinkles, it is necessary to remove it once and re-stretch it so that there are no wrinkles.

  In addition, in other conventional examples, there are cases where the medium region containing water in the microbial growth region is substantially in close contact with the cover sheet due to wetness. There is a possibility that it is not sufficient as the adhesion strength to counteract.

  Therefore, before the start of culture, the operator can easily release the cover sheet and the base sheet, and after dropping the bacterial solution, a feeling of completion of sealing between the cover sheet and the base sheet is obtained. It is an object of the present invention to be able to be locked so that the sealing property can be maintained during the cultivation of.

In view of the above-mentioned problems, the following invention has been completed as a result of detailed investigations in order to obtain a sealing property necessary for culture with good operability and ease.
That is, the present invention comprises a base sheet, a culture layer formed on the base sheet, and a cover sheet that covers the culture layer, and the base sheet and the cover sheet can be opened and closed via a hinge portion. The base plate and / or the cover sheet is provided with a lock mechanism for maintaining the closed state between the base sheet and the cover sheet. It is providing the microorganism culture sheet characterized by the above-mentioned.

Since the pressure-sensitive adhesive sheet is not used, the operator can easily open the cover sheet and the base sheet before starting the culture, and the workability is good. After dropping the fungus solution, a feeling of completion of sealing between the cover sheet and the base sheet is obtained, and then locking is possible so that the sealing property can be maintained during the culture of the fungus.
Furthermore, the present invention has a structure in which the lock mechanism is formed by cutting the base sheet and / or the cover sheet, so that the cover sheet is only opened through the hinge portion before the start of culture, compared to the case where an adhesive sheet is used. It is a work and has good workability, and it is possible to provide a microorganism culture sheet without a further increase in cost compared to using a base sheet and / or a cover sheet to form a locking mechanism by cutting and using an adhesive sheet.
The base sheet is formed such that the notch provided with the locking mechanism in either the base sheet or the cover sheet can be locked to the edge of the other base sheet or cover sheet not provided with the notch. Alternatively, the lock mechanism is configured by only cutting one of the cover sheets, and the microorganism culture sheet can be provided without any further increase in cost compared to the use of the adhesive sheet.
Since the shape of the cut is a cut of a figure that is convex with respect to the hinge portion direction, it is easy to lock, and it is difficult to come off after locking, and a microorganism culture sheet with high locking performance can be provided.

It is a perspective view of the microorganisms culture sheet in the state where the cover sheet was opened. It is a front view of the microorganism culture sheet in a state where the cover sheet is opened. It is sectional drawing of the AA line of the microorganism culture sheet | seat in FIG. It is a rear view of the microorganism culture sheet | seat locked by the example of a locking mechanism in the state in which the cover sheet and the base material sheet were closed. It is a front view of the microorganism culture sheet in a state where the cover sheet is closed and the notch is not yet locked to the edge of the base sheet.

(1) Structure of microorganism culture sheet An example of a preferred embodiment of the microorganism culture sheet of the present invention is shown in FIG. FIG. 1 is a perspective view with the cover sheet open, and FIG. 2 is a front view of the microorganism culture sheet with the cover sheet open. 3 is a cross-sectional view taken along the line AA of the microorganism culture sheet in FIG.

In FIG. 1, a culture layer (30) is formed in the approximate center of a square base sheet (10), and a square cover sheet (40) is provided so as to cover the culture layer (30). An aspect is shown. Further, on the cover sheet (40), when the base sheet and the cover sheet are closed, a notch (50) serving as a locking mechanism is provided at a position facing the hinge portion across the region where the culture layer is formed. ) Is provided. In this embodiment, the culture layer (30) is patterned on the substrate sheet (10), and the substrate sheet (10) may be a multilayer sheet composed of two layers, and further, other layers than plastic. The multilayer sheet which laminates may be sufficient. Moreover, the culture layer (30) may be composed of two or more layers.

The shape of the culture layer (30) prepared by pattern formation is not limited to the circle shown in FIG. 1, but may be a square, a rectangle, other polygons, or an indeterminate shape. This also applies to the shape of the base sheet (10).

The microorganism culture sheet of the present invention is not limited to the culture layer (30) provided only on the base sheet (10), and is provided on both the base sheet (10) and the cover sheet (40). The culture layer (30) and a specific component layer (not shown, the same applies hereinafter) containing a binder component, a medium component, a nutrient component, and other components are formed so as to face each other and face each other. May be.
For example, when a gelling agent is contained in both the culture layer (30) and the specific component layer, the culture layer (30) on the base sheet and the cover sheet (40) are provided. The specific component layer is disposed so as to face the culture layer (30) on the substrate sheet (10), and then the culture layer (30) is covered with the cover sheet (40) after inoculating the test solution. Then, the test solution is absorbed by the culture layer (30) and the specific component layer, and microorganisms can be observed in both the culture layer (30) and the specific component layer. In addition, the culture medium substrate constituting the culture layer (30) is a cover sheet without forming an adhesive sheet or the like because the culture layer (30) exhibits adhesiveness and adheres to the cover sheet (40) by gelation. (40) can be fixed to the base sheet (10) side. Thereby, drying of a culture layer (30) can be prevented. Further, when the specific component layer contains a coloring indicator, the indicator is colored by microorganisms in the specific component layer on the cover sheet (40), thereby facilitating detection of microorganisms from the cover sheet (40) side. it can.
Moreover, as shown in FIG. 3 which represents the front view of FIG. 2 and sectional drawing of the AA line in FIG. 2, the microorganism culture sheet of this invention is a culture layer (30) in the approximate center on a base material sheet (10). And a circular frame layer (20) may be formed on the outer periphery of the culture layer. The water absorption range of the test solution inoculated into the culture layer (30) can be limited by the frame layer (20). In addition, the shape of the frame layer (20) is not limited to a circle, and may be any of a square, an ellipse, a polygon, an indeterminate shape, etc., but the test solution inoculated into the culture layer (30) The frame layer is preferably formed so that the water absorption range can ensure a predetermined area. In FIG. 1, FIG. 2, and FIG. 5, only the region where the culture layer (30) is arranged is shown.
A plurality of culture layers (30) may exist on the base sheet (10).
The diameter of the equivalent circle of the culture layer (30) is 20 to 80 mm, more preferably 30 to 70 mm. The test solution dripped onto the microorganism culture sheet is generally 1 ml, and the test solution can be sufficiently absorbed within the above range.

(2) Base sheet The base sheet used in the present invention needs to have solvent resistance and printability. Moreover, as a microorganism culture sheet, water resistance is also required, and when it is accompanied by a drying process by heat when forming a culture layer, it is also required to have heat resistance. The base sheet may be a single layer or a laminated sheet having two or more layers.

When the base sheet is a single layer, for example, a plastic sheet such as polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyvinyl chloride or the like can be suitably used. Since these plastic sheets are excellent in transparency, microbial colonies can be observed even by transmitted light from the base material sheet, visibility can be improved, and the selection range of measurement methods can be expanded. . Specifically, the microorganism culture sheet of the present invention is for observing and counting colonies grown in the culture layer. If the base sheet or cover sheet is transparent, projection from the back of the microorganism culture sheet is possible. It can be observed with light, and can also be observed with incident light from the side. Therefore, it is possible to observe and count from both the cover sheet side and the base sheet side. However, those that are white due to foaming or those that are colored in any way can also be used. Depending on the culture, it is not limited to transparent, and there are cases where colonies grown by coloring can be easily counted, and can be appropriately selected.
The base sheet may be a laminate of two or more of the plastic sheets.
In addition, what is called synthetic paper formed into a sheet like paper by using synthetic resin as a main raw material can also be used as the plastic sheet.
The base material sheet used in the present invention may have been surface-treated in advance on the side on which the culture layer is patterned in order to improve the adhesion with the culture layer. Examples of such surface treatment include corona discharge treatment, ozone treatment, low temperature plasma treatment using oxygen gas or nitrogen gas, glow discharge treatment, oxidation treatment using chemicals, and other pretreatments. . Moreover, in order to improve the adhesiveness with the culture layer, an anchor coat agent can be applied in advance to the surface of the base material sheet and surface treatment can be performed.
The base sheet is preferably flat without curling, and the thickness is not particularly limited, but is usually 25 to 1500 μm, more preferably 50 to 500 μm.

(3) Culture layer Although the culture layer provided in a base material sheet in this invention is not specifically limited, A preferable example is shown below.

  In the present invention, the culture layer provided on the substrate sheet forms a pattern of a medium solution containing at least one selected from the group consisting of a gelling agent, a nutrient component, a coloring indicator, a selection agent, and a substrate in a polyvinylpyrrolidone solution. It is preferable to be prepared. By selecting a polyvinylpyrrolidone solution, it is possible to uniformly disperse at least one selected from the group consisting of a gelling agent, a nutritional component, a coloring indicator, a selective agent and a substrate by adjusting the concentration, and easily This is because the viscosity can be adjusted and pattern formation can be performed using the obtained medium solution. Moreover, when the solvent is removed after pattern formation, polyvinylpyrrolidone has high film properties, so it can be formed by incorporating gelling agents, nutrients, and other components, and has good adhesion to the substrate. A culture layer can be formed without using an agent. The polyvinyl pyrrolidone film may be hard and brittle, and a plasticizer may be added. Plasticizers that can be added include ether ester derivative plasticizers, glycerin and glycerin derivative plasticizers, propylene glycol and glycol derivative plasticizers, glycol ether derivative plasticizers, polyhydroxycarboxylic acid derivative plasticizers, and phthalic acid. Although a derivative plasticizer etc. can be used, it is not limited to these.

(I) Polyvinylpyrrolidone Solution In the present invention, the solvent used for the polyvinylpyrrolidone solution is not particularly limited as long as it can dissolve polyvinylpyrrolidone. On the other hand, alcohol can be suitably used as a solvent.
As alcohol, C1-C5 alcohol is suitable, for example, 1 type, or 2 or more types of mixed liquids, such as methanol, ethanol, isopropyl alcohol, butanol, can also be used. Of these, methanol, ethanol, and isopropyl alcohol can be preferably used in the present invention. Conventionally, high-boiling solvents such as water and toluene have been used to dissolve the medium components. However, it is necessary to heat at a high temperature to remove the solvent, which may cause thermal decomposition of the components contained in the culture layer. was there. In addition, it is necessary to use excessive heat energy for solvent removal, which contributes to an increase in cost, and also causes a decrease in production efficiency because the solvent removal time becomes longer. In the present invention, polyvinyl pyrrolidone is used, and the above alcohol is used as a solvent thereof, so that thermal denaturation of components contained in the culture layer can be avoided and production efficiency can be improved. It should be noted that a solvent obtained by mixing a secondary solvent such as chloroform with alcohol can also be used within a range not impairing the above-mentioned purpose.
Although the ratio of the solvent with respect to polyvinylpyrrolidone can be suitably selected according to the solvent to be used, it is generally 100 to 10,000 parts by mass, more preferably 300 to 2000 parts by mass, and particularly preferably 600 to 100 parts by mass with respect to 100 parts by mass of polyvinylpyrrolidone. 1100 parts by mass. When the amount exceeds 10,000 parts by mass, the dispersibility of the gelling agent and the like is remarkably reduced. On the other hand, when the amount is less than 100 parts by mass, the viscosity of the mixed solution increases and the suitability for pattern formation decreases.

(Ii) Gelling agent In the present invention, a gelling agent can be added to the polyvinylpyrrolidone solution. When the culture layer contains a gelling agent, it thickens or gels when the test solution is inoculated into the culture layer, so that a good viscosity can be secured for the growth of bacteria, Since it can adhere | attach, drying of the culture layer at the time of culture | cultivation can be suppressed.

As such a gelling agent, a high molecular polysaccharide can be preferably used, and carrageenan, guar gum, chitansan gum, hydroxyethyl cellulose, carboxymethyl cellulose, locust bean gum, algin and the like can be exemplified. Of these, guar gum can be preferably used in the present invention. Guar gum is produced from the endosperm part of annual seed legumes and guar seeds. Guar gum is particularly suitable because it exhibits an extremely high viscosity at a low concentration and can create an environment suitable for the growth of bacteria with a small amount.
In the present invention, the gelling agent constituting the medium solution need not be dissolved in the polyvinylpyrrolidone solution as long as pattern formation is possible. For this reason, in this invention, a gelatinizer can be added with a powder. When added as a powder, the gelling agent has an average particle size of 5 to 500 μm, more preferably 10 to 200 μm. If it exceeds 500 μm, the dispersibility may be lowered, and the pattern formation suitability may be impaired. However, these may be dissolved in a solvent and mixed with the polyvinylpyrrolidone solution to form a medium solution.
The compounding amount of the gelling agent is 100 to 600 parts by mass, more preferably 250 to 400 parts by mass with respect to 100 parts by mass of polyvinylpyrrolidone. If it is less than 100 parts by mass, the viscosity of the culture layer may be insufficient. On the other hand, if it exceeds 600 parts by mass, the culture layer may become hard and the growth of bacteria may be reduced.

(Iii) Nutritional component In addition to the polyvinylpyrrolidone solution and the gelling agent, a nutrient component can be blended in the culture layer of the present invention.
Examples of nutritional components include, for example, yeast extract / peptone / glucose mixture, meat extract / pepton mixture, peptone / soybean peptone / glucose mixture, and dipotassium phosphate and / or sodium chloride. As a test mixture for Escherichia coli and coliform bacteria, sodium desoxycholate, peptone, ammonium iron citrate, sodium chloride, dipotassium phosphate, lactose, peptone, lactose, dipotassium phosphate, etc. for staphylococci For example, meat extract, peptone, sodium chloride, mannitol, egg yolk mixture, peptone, meat extract, yeast extract, sodium pyruvate, glycine, lithium chloride, telluric acid egg yolk mixture, etc. Peptone, sucrose, sodium thiosulfate, sodium citrate Sodium cholate, ferric citrate, sodium chloride, bovine bile, etc. for enterococci, bovine brain extract, heart extract, peptone, glucose, dipotassium phosphate, sodium nitride, etc. for fungi Peptone / glucose mixture, yeast extract / glucose mixture, potato extract / glucose mixture and the like are used. One or more can be selected from these according to the microorganism to be grown and mixed for use. In addition, it is good also as a culture medium which can grow microorganisms by adding the water containing the nutrient component of microorganisms to the test liquid containing microorganisms.
These components may be added in powder form, or they may be dissolved in a solvent and mixed with the polyvinyl pyrrolidone solution to form a medium solution.

(Iv) Other components such as a color development indicator, a selection agent, and a substrate In addition to the polyvinyl pyrrolidone solution and the gelling agent, the culture layer of the present invention contains other components such as a color development indicator, a selection agent, and a substrate, A culture layer can be formed.

The coloring indicator is metabolized by microorganisms that grow during the culturing process, and the colonies are colored, so that it has the effect of facilitating counting of the number of colonies. Specific examples of such a color development indicator include tetrazorim salts such as triphenyltetrazolium chloride (hereinafter referred to as TTC), p-tolyltetrazolium red, tetrazolium violet, and petetylyltetrazolium blue, neutral red mixtures, phenol red, and bromthymol blue. PH indicators such as thymol blue mixtures. As the microorganism grows, the color develops or changes color so that the growth of the microorganism becomes easy to see, and the specific microorganism can be detected by adding the color developed or fluorescent enzyme substrate of the enzyme possessed by the particular microorganism.
Furthermore, a selective agent that suppresses the growth of microorganisms other than the detection purpose may be added, and a color developing or fluorescent enzyme substrate may be added to detect a specific microorganism. As such a selective agent, antibiotics such as antibiotics and synthetic antibacterial agents, dyes, surfactants, inorganic salts and the like are used. Examples of antibiotics include methicillin, ceftamethazole, cefixime, ceftazidime, cefsulosin, bacitracin, polymyxin B, rifampicin, novobiocin, colistin, lincomycin, chloramphenicol, tetracycline, streptomycin, etc. Examples thereof include nalidixic acid and olaquindox. Examples of the pigment include bacteriostatic or bactericidal crystal violet, brilliant green, malachite green, and methylene blue. Examples of the surfactant include Tergitol 7, dodecyl sulfate, lauryl sulfate and the like. Examples of inorganic salts include selenite, tellurite, sulfite, sodium nitride, lithium chloride, oxalate, and high-concentration sodium chloride. Besides these, taurocholate, glycine, bile powder, bile salt, deoxycholate, and the like can be used.

  These components may be added in powder form, or they may be dissolved in a solvent and mixed with the polyvinyl pyrrolidone solution to form a medium solution.

Depending on the type of coloring indicator, coloring may be conspicuous at the time of sterilization, and the medium may not be contained in the culture layer but may be developed using a coloring solution containing a coloring indicator in the test solution.
In the culture layer of the present invention, if the polyvinylpyrrolidone solution (i) and the gelling agent (ii) are contained, the nutrient component (iii) and / or other components (iv) such as a coloring indicator, a selective agent, and a substrate are further contained. You may contain.
Moreover, in this invention, it is not limited to forming a culture layer by one layer, It can comprise by the multilayer of two or more layers. For example, a first culture layer is formed of a medium solution containing a gelling agent (ii) in a polyvinylpyrrolidone solution (i), and then the gelling agent (ii) and a nutrient component (iii) are added to the polyvinylpyrrolidone solution (i). The second culture layer may be formed with a medium solution containing) to form a culture layer used in the present invention. Further, the gelling agent may be blended only in the first culture layer, and the first culture layer is formed with a medium solution containing the gelling agent (ii) in the polyvinylpyrrolidone solution (i), and the polyvinylpyrrolidone is formed thereon. The second culture layer may be formed from a medium solution containing the nutrient component (iii) in the solution (i), and may be used as the culture layer used in the present invention.
Further, depending on the combination of the coloring indicator and the selective agent, there is a case where the reaction occurs by mixing, and the mixing reaction can be prevented by containing these in another layer. Moreover, the visibility of colonies can be improved by forming the color development indicator in the uppermost layer. Therefore, for example, when the components A and B that form a mixed reaction are contained in the nutrient component, the A component is removed from the polyvinylpyrrolidone solution (i) + gelling agent (ii) + nutrient component (iii). A culture layer is formed by laminating two layers of one culture layer and a polyvinyl pyrrolidone solution (i) + gelling agent (ii) + coloring indicator (iv) + second culture layer from which the B component is removed from the nutrient component (iii) The first culture layer obtained by removing the A component from the polyvinylpyrrolidone solution (i) + gelling agent (ii) + nutrient component (iii), polyvinylpyrrolidone solution (i) + gelling agent (ii) + The second culture layer from which the B component was removed from the nutritional component (iii), and the third culture layer consisting of the polyvinylpyrrolidone solution (i) + gelling agent (ii) + coloring indicator (iv) were sequentially laminated. It may be a culture layer. In addition, the mixing reaction is not limited to the case where it is contained in a nutritional component, and may occur due to a relationship with a selective agent or a color indicator. In any case, mixing reaction can be avoided by separating components A and B into separate layers.
In the present invention, when the culture layer is composed of multiple layers, the polyvinyl pyrrolidone solution is composed of a gelling agent, a nutrient component, a coloring indicator, a selective agent, and a substrate in any of the culture layers constituting the multilayer structure. Any medium solution containing at least one selected from the group may be patterned, and the composition of the other layers is not limited to the above, and the pattern forming method is not limited.

(V) Pattern formation The culture layer used by this invention is prepared by pattern-forming the said culture solution on a base material sheet. In the present invention, the “pattern formation” is not particularly limited as long as the medium solution can be printed, applied, applied, and sprayed on the shape of the base sheet. Therefore, screen printing, gravure printing, letterpress printing, transfer printing, flexographic printing, other printing, application by dispenser or inkjet, bar coating, knife coating, die coating, etc. coating using a bar, spray method Spraying and other methods can be used.
In addition, it is sufficient that at least the culture layer can be patterned on the base material sheet. When the culture layer is a multilayer of two or more layers, it is not necessary that all of them are formed by the same pattern formation method. Therefore, the first culture layer may be patterned by application with a dispenser, and the second culture layer may be patterned by screen printing.

In this invention, the thickness of the culture layer formed on a base material sheet is 50-1000 micrometers at the time of drying, More preferably, it is 200-600 micrometers. Further, the coating amount is 5 to 400 g / m ² when dried, more preferably 100 to 300 g / m ² . Within the above range, the culture layer can have a viscosity that is optimal for the growth of bacteria.

(4) Cover sheet A cover sheet has the effect | action which prevents the contamination by the falling bacteria etc. during culture | cultivation, and prevents the water | moisture content evaporation of a culture layer. The cover sheet is waterproof and water vapor impermeable, and at the same time is preferably transparent so that colonies can be observed and counted through the cover sheet after culturing microorganisms, for example, as mentioned in the base sheet. Plastic sheets such as polyolefin such as polyethylene and polypropylene, polyester, polyamide, polystyrene, polycarbonate, and polyvinyl chloride can be used. In view of workability such as opening and closing of the cover sheet, a polyester film, a polyolefin film or an easily peelable polyolefin film is particularly preferable. The cover sheet may be subjected to a surface treatment such as a corona discharge treatment described in the section of the base material sheet. The cover sheet preferably has suitable gas permeability, mainly oxygen permeability, or oxygen non-permeability depending on the type of microorganism to be cultured, and can be selected in consideration of this point.
When the cover sheet is lifted from the base sheet in order to inoculate the test liquid, it is preferable that moderate flexibility is secured. Therefore, the thickness of the cover sheet is preferably about 10 to 200 μm, and more preferably about 20 to 70 μm. Further, when the lock mechanism cover sheet is applied, the cover sheet needs to have appropriate strength and stiffness. In addition, although a cover sheet may be what kind of shape, in order to prevent miscellaneous bacteria from entering, it is necessary to make it a size larger than a culture layer so that a culture layer can be coat | covered.
In the present invention, one type selected from the group consisting of a gelling agent, a nutrient component, a color indicator, a selective agent, and a substrate in a binder solution at a position facing the culture layer formed on the base sheet on the cover sheet. You may have the specific component layer (not shown) formed by apply | coating the specific component solution containing the above.
The specific component liquid may contain a gelling agent. The specific component layer is not limited to a single layer, and may be a multilayer of two or more layers.
In addition, when the grid print pattern is not printed on the base material sheet, the grid print pattern may be previously printed on the cover sheet with an ink that is insoluble in water and does not affect the growth of microorganisms. As for the printing plate type, gravure printing or the like is preferable because the selection range of a colorant, a resin, a solvent, and the like is wide, like the base sheet. The size of the mesh is about 1 cm square.

(5) Frame layer In the present invention, a frame layer made of a hydrophobic resin may be formed on or on the outer periphery of the culture layer. Thereby, the leakage of the test liquid can be prevented. By providing the frame layer in a double bank shape on the outer periphery of the culture layer (30) as shown in FIG. 3, leakage of the test solution can be further prevented. The frame layer may be formed on the culture layer or on the base material sheet, but is preferably formed so as to surround the outer periphery of the culture layer on the base material sheet.
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 when a culture layer absorbs water, the shape of a culture layer can be maintained and it can function as a division part between the inner side and the outer side of a frame layer. In general, the height of the frame layer depends on the thickness of the culture layer, but is preferably 600 to 1000 μm based on the base sheet. If it is less than 600 μm, it will be easy to protrude from the frame layer when the test solution is spread. On the other hand, when it exceeds 1000 μm, a space is likely to be generated in the upper part of the culture layer after water absorption, and it becomes difficult to ensure adhesion with the cover sheet.
Further, a hollow material made of a hydrophobic material can be adhered to a base sheet on the outer periphery of the culture layer to form a frame layer.
There is also a method of using an embossed product as a base sheet. For example, in the case of an embossed product in which a convex shape is formed in the shape of the frame layer, a culture layer may be formed inside the convex shape, while a portion where the culture layer is formed is formed by a concave portion, and the convex portion is formed by this. The remainder formed in the shape can be used as a frame layer.
Furthermore, a thick hydrophobic substrate is used as the substrate sheet, and the portion where the culture layer is formed is cut into a concave shape, and the remainder is formed into a frame layer, or a convex shape is formed in the shape of the frame layer. If the culture layer is formed inside the shape, the convex shape can be used as a frame layer. However, it is not limited to these.
As the hydrophobic resin to be used, UV curable ink, hot melt, thermally foamed ink, UV foaming agent and the like can be suitably used. In particular, UV curable ink and hot melt can form a frame layer having a desired height, which is particularly preferable.
(6) Hinge part The microorganism culture sheet of the present invention covers the culture layer with the cover sheet after inoculating the test solution into the culture layer, and incubates the culture layer. A part of the base sheet is continuously opened and closed. The part having the connecting function is referred to as a hinge part (60). Therefore, the hinge part is formed at the end of the microorganism culture sheet.
As a means for imparting a specific connecting function, a cover sheet and a base sheet prepared separately are connected by means of heat sealing, laminate bonding, adhesive tape, or the like.
The frame layer (20) and the culture layer (30) are formed on the same sheet, that is, in the region to be the base sheet (10) with the cover sheet and the base sheet being continuously provided, and the base sheet (10 ) And the region to be the cover sheet, and the microorganism culture sheet may be formed using the substantially bent portion as a hinge portion.
(7) Locking mechanism The locking mechanism of the present invention covers the cover sheet after dropping the bacterial solution on the culture layer of the microorganism culture sheet in which the base sheet and the cover sheet are connected to each other through a hinge part so as to be freely opened and closed. A structure for maintaining a state in which the cover sheet and the base sheet are in close contact with each other, that is, a structure provided on the base sheet and / or the cover sheet is referred to as a lock mechanism.
It is effective to provide a specific locking mechanism at a position facing the hinge portion across the region where the culture layer is formed when the base sheet and the cover sheet are closed. However, it is not limited to this.
As a specific locking mechanism, a plastic occlusion member can be provided between the cover sheet and the base sheet. The plastic occlusion member can be exemplified by what is known as “Lami Zip (Nippon Production Co., Ltd .: registered trademark)” as a packaging bag application.
In this case, it is necessary to complete the occlusion by dropping the bacterial solution and then covering the culture layer with a cover sheet and then pressing the occlusal member.
In addition, it is also possible to engage each other depending on the shape of the edge of the cover sheet and the base sheet.
As another locking mechanism, the locking mechanism can cut the base sheet and / or the cover sheet, insert the other sheet part of the part facing the notch, and lock it. The shape of the cut is not particularly limited, but it is not limited to a linear shape, and may be a hole shape or a combination of a linear shape and a hole.
As an example of the locking mechanism, a cut (50) is formed in FIGS. 1, 2, 4, and 5. FIG. This incision (50) is formed at a position that can be locked to the edge of the base sheet, and the shape of the incision is a notch of a figure that is convex with respect to the hinge portion direction. FIG. 5 is a front view of the microorganism culture sheet in a state where the cover sheet is closed and the notch is not yet locked to the edge of the base sheet. The microorganism culture sheet is supplied in the state shown in FIG. 5 when not in use. The user flips the cover sheet (40) from this state, and when inoculating the culture layer with the bacterial solution as the test solution on the surface of the culture layer (30) that appears, it thickens or gels, so it is good for the growth of bacteria A high viscosity can be ensured, and can be brought into close contact with the cover sheet after thickening. After the child, the user turns the tip of the convex cut around the back surface of the base sheet (10) to lock the cover sheet to the base sheet.
FIG. 4 is a rear view of the microorganism culture sheet that is locked in a state in which the cover sheet and the base sheet are closed according to this example, and the incision goes around the back surface of the base sheet (10) at the edge of the base sheet. It is locked. In this case, the cover sheet is slightly longer than the edge of the base sheet, and the position where the incision (50) becomes the starting point when the base sheet and the cover sheet are closed from the edge of the base sheet. It is necessary that the region (position not on the base sheet) be the starting point. Furthermore, the top part of the convex figure needs to be an area on the base sheet when the base sheet and the cover sheet are closed. In view of this condition, that is, by providing a cut of a figure whose cut shape is convex with respect to the hinge part direction, the cover sheet and the base sheet become a secure locking mechanism in a close contact state and locked. It is obvious to the user whether or not
By forming the cover sheet slightly longer than the edge of the base sheet, and having a long formed region, it is easy to lift only the cover sheet, and there is also an effect that the cover sheet can be easily opened. .

  Regarding the convex shape, it is only necessary to show the same function and effect, and when it is a straight line or a curved line, it may be formed by a combination of these or a plurality of convex figures.

The incision may be not only linear but also a hole or a combination thereof.
According to this lock mechanism, the operator can freely open the cover sheet and the base sheet, and after dripping the bacterial solution, a feeling of completion of sealing between the cover sheet and the base sheet, that is, locking The cover mechanism is strong, the lock mechanism is strong, and unless the lock is released artificially, the cover sheet is slightly lifted or loosened, and the base sheet on which the medium is provided may be deformed. Even if there is, the sealing performance can be maintained.
In addition to the locking mechanism shown in FIG. 1, the roles of the base sheet and cover sheet locking mechanisms may be interchanged. That is, the base sheet is formed longer than the cover sheet, and the cut is formed on the base sheet side (not shown).
However, this configuration has the following disadvantages when the convex cut formed in the base sheet is locked to the edge of the cover sheet. When locking, it is necessary for the user to deform the sheet in the vicinity of the convex cut, and considering the operation of turning up the cover sheet when dripping the bacterial solution, the area formed longer than the base sheet is In addition, it is preferable to provide a cut in a cover sheet that often selects a softer material than the base sheet.

EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

Example 1
UV curable ink (manufactured by Jujo Chemical Co., Ltd., trade name “Reicure GA 4100-2”) is placed on a polyethylene terephthalate film (manufactured by Teijin DuPont, trade name “Teijin Tetron Film”) by a dispenser with a width of 1.0 mm and a height. A circular frame layer having a diameter of 750 μm and a diameter of 52 mm was formed, and the frame layer was cured by irradiation with a mercury lamp with an integrated light amount of 250 mJ / cm ² .
Next, 20 g of polyvinylpyrrolidone K-90 was dissolved in 210 ml of methanol, and 60 g of guar gum powder (trade name “NEOVISCO G”, 400 mesh type, manufactured by Sankisha Co., Ltd.) was dispersed therein, and further, tryptone (Becton, Dickinson) as a nutritional component. and Company, trade name “BACT TRYPTONE” 5.16 g, yeast extract (Becton, Dickinson and Company, trade name “YEST EXTRAACT”) 2.58 g, glucose (Wako Pure Chemicals, trade name “D”) -(+)-GLUCOSE ") 1.03 g was mixed to prepare a coating solution.

The coating solution containing the nutritional component and the gelling agent was applied by a dispenser into the frame of the base material on which the frame layer was formed. Subsequently, the applied layer was dried at 50 ° C. for 15 minutes to form a culture layer. The layer thickness upon drying was 250 μm.
Next, as the cover sheet, an OPP film having a thickness of 40 μm (manufactured by Tosero Co., Ltd., model number OP U-1 (single-sided corona discharge treatment) was used. The cover sheet was cut in advance so as to be engaged with the edge of the base sheet. 1 so as to form a convex shape with respect to the hinge direction, as shown in Fig. 1. Laminate on the PET base material sheet on which the frame layer and the culture layer are formed. The end part was bonded to form a hinge part as shown in Fig. 1, and sterilized with γ-rays to obtain a microorganism culture sheet of the present invention, the height of the obtained frame layer was 750 µm (difference from the culture layer height + 500 µm), The width was 1 mm.
In Example 1, a notch was provided and locking as shown in FIG. 4 was performed, so that operability was good and adhesion between the cover sheet and the base sheet was also good.

  INDUSTRIAL APPLICABILITY The present invention is used for bacterial count inspection and the like, and is compact and easy to handle, and can be effectively used for a sheet-like microorganism culture sheet.

DESCRIPTION OF SYMBOLS 10 Base material sheet 20 Frame material layer 30 Culture layer 40 Cover sheet 50 Cutting 60 Hinge part

Claims (4)

A base sheet, a culture layer formed on the base sheet, and a cover sheet covering the culture layer, and the base sheet and the cover sheet are connected to each other through a hinge part so as to be opened and closed. A microorganism culture sheet,
A microorganism culture sheet, wherein the base sheet and / or the cover sheet is provided with a lock mechanism for maintaining a closed state between the base sheet and the cover sheet. 2. The microorganism culture sheet according to claim 1, wherein the lock mechanism is formed by cutting a base sheet and / or a cover sheet. The incision in which the lock mechanism is provided in either the base sheet or the cover sheet is formed so as to be able to be locked to the edge of the other base sheet or cover film in which the incision is not provided. Item 3. The microorganism culture sheet according to Item 2. The microorganism culture sheet according to claim 3, wherein the cut shape is a cut shape of a convex shape with respect to the hinge portion direction.

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