JPH0975062A - Apparatus for culturing - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus for culturing a microorganism, good in keeping quality, simple in culturing operations, capable of readily spreading a test solution to a prescribed area and excellent in simplicity and performances such as the ability to precisely count developed colonies with a good productivity. SOLUTION: This apparatus for culturing is obtained by applying a water absorbing ink using a water absorbing resin to the inner surface on the side of at least the substrate sheet 1 into a prescribed shape by a means such as printing and forming a water holding layer (5a) of a prescribed area in order to keep the spread of a test solution to be inoculated constant in the apparatus for culturing equipped with the waterproof substrate sheet 1 and a moisture- impermeable and transparent covering film 2 covering the substrate sheet 1 and further provided with a culture medium layer 4 for microorganisms on the inner surface of either of inner surfaces of the substrate sheet 1 and covering film 2 or the inner surfaces of both.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention relates to a technique of a culturing apparatus for microorganisms used for bacterial count inspection, and more specifically, it can be stored for a long period of time and is easy to use at the time of use.
The present invention relates to a culturing apparatus for microorganisms having excellent accuracy in measuring the number of bacteria.

[0002]

2. Description of the Related Art As a method for measuring the number of microorganisms such as foods, the agar plate pour method has been conventionally used. In this method, a predetermined amount of agar medium having a temperature of about 45 ° C. after high-pressure steam sterilization and a test liquid prepared separately are dispensed into a petri dish that has been sterilized in advance, and the mixture is sufficiently mixed and left to stand. After confirming that the medium has solidified, the medium is put into an incubator or the like and cultured, and after a predetermined time, the number of colonies that have grown in the petri dish is counted. However, in the case of this method, an autoclave for high-pressure steam sterilization of the agar medium and a laboratory capable of performing a series of microbial tests aseptically are required, and the adjustment of the test liquid from the sampling of microorganisms, Skills are required for operations of microbiological examinations such as dispensing, pour with medium, culture, and counting, and it is difficult for a beginner to perform accurately.

[0003] Therefore, as a result of research on a method for conducting a microbial test easily and without requiring a high degree of skill, a dry culture medium has been developed and used as one of the culture devices. As such a culture device, for example, Petri film plate [trade name of 3M] or B
ACcT [Shimadaku Yakuhin Co., Ltd. product name] etc.

The former Petri film plate is composed of a waterproof flat plate and a cover film that overlaps with the flat plate. An adhesive is applied to the inner surfaces facing each other, and a cold water-soluble substance (gelling agent) is further applied. ) Powder and medium components or powder of cold water soluble substance (gelling agent) is sprayed, and then excess powder is shaken off to form a dry medium, which is further sterilized. It is a product. Then, the outline of the method of use is as follows. First, the cover film film laminated on the flat plate is lifted, and the test is performed on a dry medium composed of a cold water-soluble substance and a medium component formed on the flat plate via an adhesive layer. A predetermined amount of the liquid is inoculated, then the cover film is lowered, and a plastic spreader is pressed onto the cover film to spread the test liquid circularly and uniformly so that the liquid is absorbed. Since the gelling agent, which is a cold water-soluble substance, solidifies in about 1 minute, it is stored in the incubator as it is after being solidified, cultured at a predetermined temperature and time, and the number of generated colonies is counted. .

The latter BACcT has a waterproof flat plate and a transparent cover film covering the waterproof flat plate, and the surface of the waterproof flat plate facing the cover film is cold water soluble in the solution of the adhesive. A mixture of a gelling agent and a microbial culture medium is added, and a coating solution that is kneaded in a mud form is applied and dried to form a coating, and a fibrous water absorbent sheet is laminated on at least a part of the coating solution, and a cover film is also used. On the surface opposite to the waterproof flat plate, a mixture of a cold water-soluble gelling agent and a microbial culture medium, or a cold water-soluble gelling agent is added and kneaded to apply a coating solution to the surface of the sticking agent, and dried. After forming a coating, these are sterilized to obtain a product. The method of using this BACcT is to lift a cover film covering a waterproof flat plate, and to test on a fibrous water-absorbent sheet laminated on a coating layer of a mixture of a cold water-soluble gelling agent and a microorganism culture medium on the waterproof flat plate. Inoculate a predetermined amount of liquid. Then remove the cover film. As a result, the test liquid is diffused throughout the water-absorbent sheet by the capillary phenomenon due to the overlapping of the fibers of the fibrous water-absorbent sheet, the culture medium of the microorganisms thereunder is dissolved, and the cold water-soluble gelling agent absorbs water and gels. . At this time, the fibers of the fibrous water absorbent sheet are wrapped in the gel or adhere to the gel. After that, it is placed in an incubator, cultured at a predetermined temperature and for a certain period of time, and the number of colonies generated is counted.

[0006]

The culturing apparatus as described above is convenient in use because it is not necessary to prepare a Petri dish or a medium each time when culturing a microorganism. . However, in the case of the former, in the production thereof, an adhesive is applied to each of the flat plate and the cover film and dried, and then the cold water-soluble substance (gelling agent) and the powder of the medium component are applied to the flat plate and the cover film. There is a process of spraying cold water soluble substance (gelling agent) powders and removing excess powder, which is not always efficient in terms of productivity and also causes unevenness in adhesion of the sprayed powders. However, there is a problem in that the production amount tends to be unstable, and the adhesion amount tends to be unstable, and the manufacturing cost becomes high. Further, the method of use is to lift the cover film, inoculate a predetermined amount of the test liquid on a dry medium composed of a cold water-soluble substance and a medium component formed on the flat plate through an adhesive layer, and cover the cover film. After lowering, spread the test liquid evenly by pressing it with a plastic spreader and let the cold water-soluble substance absorb water by leaving the spreader on for about 1 minute to form a gel and at the same time to remove the medium components. To dissolve. Therefore, with one spreader, only one sample can be processed in one minute, and if the spreader shifts, the test liquid will spread further on the dry medium.

On the other hand, in the latter case, as described above, by dropping the test liquid onto the fibrous water-absorbent sheet on the waterproof flat plate side, the test liquid spreads throughout the sheet due to the capillary phenomenon between fibers, The gelation of the cold water-soluble gelling agent and the dissolution of the culture medium thereunder are carried out. At this time, the fibers of the fibrous water absorbent sheet are wrapped in the gel or adhered to the gel. Therefore, the spread area of the test liquid can be made substantially constant,
The fungus body has penetrated into the fibers of the fibrous water-absorbent sheet, and the growth proceeds in the sheet. At this time, the substantially used water-soluble fixing agent is dissolved and exists on the surface of the gel, which causes the generated colonies to become blurred and unclear. Therefore, there is a problem that the counting becomes difficult especially when the bacterial cells grow close to each other.

The present invention has been made in view of the above problems, and an object of the present invention is to simplify the manufacturing process, save labor, improve production efficiency, and apply medium components and the like. The amount can be made uniform and stable,
Further, it has a storage property as a culture device, and the operation of culture is simple, and particularly when inoculating a test liquid, it can be surely spread to a desired area without using a device such as a spreader, and generated. It is an object of the present invention to provide a culturing device which is capable of clearly discriminating colonies, which is excellent in productivity, storability, ease of use, and precision of microbial culture.

[0009]

The above-mentioned problems can be solved by the present invention described below. That is, the invention of claim 1 is provided with a waterproof base sheet and a water-impermeable transparent cover film which is placed on the base sheet, and either one of the base sheet and the cover film is provided. The inner surface of the, or, in a microorganism culture device comprising a microorganism culture base layer provided on both inner surfaces, in order to make the spread of the test liquid to be inoculated into a predetermined area, a water-retaining layer made of a water-absorbent ink, at least a cover. The culture device is provided on the inner surface of the base sheet that overlaps with the film.

The invention according to claim 2 is the culture apparatus according to claim 1, wherein the resin used in the water-absorbent ink is electron beam cross-linking type polyacrylamide. The invention according to claim 3 is the culture apparatus according to claim 1, wherein the resin used in the water-absorbent ink is polyethylene oxide. The invention of claim 4 is the culture apparatus according to claim 1, wherein the resin used in the water-absorbent ink is carboxymethyl cellulose. And the invention of claim 5 is that the water-retaining layer of a predetermined area made of the water-absorbent ink,
The culture device according to any one of claims 1 to 4, wherein the culture device is patterned in a concentric circle shape, a spiral shape, a lattice shape, a checkered pattern, or the like. Incidentally, in the above-mentioned present invention,
The culture substratum of a microorganism is water-soluble or amphoteric (both soluble means that it is both water-soluble and simultaneously soluble in an organic solvent, and the same terms will be used hereinafter)
Is a layer composed of a mixed system of the resin of (1) and one or more kinds of nutrient components of microorganisms, and the details thereof will be described again in the following section "Embodiment of the Invention".

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic perspective view illustrating the configuration of an embodiment of the culture device of the present invention. FIG.
FIG. 2 is a schematic cross-sectional view taken along the line AA showing the configuration of the base material sheet portion of the culture device shown in FIG. 1. (A) of FIG.
(B), (C), (D) is a partial schematic sectional view explaining the layer structure of one Example of the culture apparatus of this invention, respectively.
In addition, FIG. 4 is a plan view showing an example of the shape in the case where the water retention layer is patterned and formed in the culture device of the present invention. The present invention is not limited to the above figures.

A detailed description will be given below step by step. FIG.
FIG. 1 is a schematic perspective view for explaining the configuration of an embodiment of a culture device of the present invention, in which a grid printed pattern 3 is printed on a base material sheet 1 and a culture base layer 4 for microorganisms is provided thereon, A water-retaining layer 5a made of a water-absorbent ink for providing a predetermined area for spreading the test liquid to be inoculated thereon is provided as a main sheet 7a of the culture apparatus, and a cover film 2 is joined to the end of the main sheet 7a with an adhesive or the like. It is the structure provided. The inner surface of the cover film 2 (the surface that overlaps the main body sheet) may be provided with a microorganism culture base layer and a water-retaining layer of a predetermined area formed by a water-absorbent ink, in that order. Only one may be provided. When a water-retaining layer of a predetermined area made of water-absorbent ink is also provided on the cover film 2 side, it is preferable that the water-retaining layer 5a on the body sheet 7a side is vertically aligned with the water-retaining layer 5a on the body sheet 7a. . Further, the cover film 2 can be used as a separate body without being joined to the main body sheet 7a. When inoculating the test solution and spreading it uniformly in the area surrounded by the water retention layer 5a, or
It is preferable to bond the cover film 2 in order to prevent the cover film 2 from being displaced or peeled off in the subsequent culture process.

FIG. 2 is a cross-sectional view of the main body sheet 7a of the culturing apparatus shown in FIG. 1 taken along the line AA, in which a grid printed pattern (not shown) is printed on the base sheet 1. The microbial culture base layer 4 is provided thereon, and the water retention layer 5a having a predetermined area made of the water-absorbent ink is further provided thereon. In this case, the water-retaining layer 5a absorbs the water content of the test solution inoculated thereon to form a gel suitable for culturing, and at the same time, the water absorption and the thickness allow the test solution to spread over a predetermined area. It functions to stop in the area surrounded by the water retention layer 5a.

FIGS. 3A to 3D are partial schematic cross-sectional views for explaining the layer structure of an embodiment of the culture apparatus of the present invention, and FIG. 3A shows the base sheet 1 The culture base layer 4 and the water retention layer 5a having a predetermined area made of a water-absorbent ink are sequentially provided only on the upper side, and the cover film 2 has neither a culture base layer nor a water retention layer. Then, FIG. 3B shows that the water-retaining layer 5 of a predetermined area formed of the water-absorbent ink on the base material sheet 1.
Only the a is provided, and the culture substrate 4 is provided on the inner surface of the cover film 2.
Is provided. FIG. 3 (C) shows a structure in which a culture base layer 4 and a water retaining layer 5a of a predetermined area made of a water-absorbent ink are sequentially provided on a substrate sheet 1, and a water retaining layer 5b is also provided on the inner surface of the cover film 2. is there. In FIG. 3D, a water-retaining layer 5c having a predetermined area is formed on the base sheet 1 by a system in which a nutrient component of a microorganism is added to a water-absorbent ink, and the inside of the cover film 2 is metabolized by the microorganism. This is a structure in which a dye layer 6 containing a dye that can be processed is provided. Further, FIG. 4 is a plan view showing an example of a shape in the case where the water retaining layer is formed by patterning in the culturing apparatus of the present invention, which is a concentric circular, spiral, lattice-like, or checkered pattern of the water retaining layer. showed that.

The constituent materials of the culture apparatus of the present invention will be described below. (Base material sheet) The base material sheet 1 needs to have waterproofness, for example, polyester, polypropylene,
Polyethylene, polystyrene, polycarbonate, polyvinyl chloride, polyvinylidene chloride, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, polyacrylate, polyvinyl alcohol, polyvinyl butyral, polyamide, polyurethane, polyimide, triacetyl cellulose, etc. A plastic sheet or a laminate of these can be used. In the case of using the above-mentioned substrate sheet 1 and cover film 2 in combination as a culture device, at least one of them is preferably transparent in order to observe and count grown colonies. Usually, since a transparent film is used for the cover film 2, the base material sheet 1 may be a non-colored transparent material, but it is easy to observe colonies grown by white coloring and easy to count. It is preferable in that In addition to the plastic sheet, water-resistant paper can also be used. For example, paper coated with synthetic resin or extrusion-coated, or laminated with plastic film can be used.

The base sheet 1 is preferably flat without curling, and its thickness is not particularly limited, but is usually in the range of about 50 to 500 μm, and 80 to.
About 200 μm is more preferable. In addition, it is preferable to print the grid printed pattern 3 on the base sheet 1 in advance with an ink that is insoluble in water and does not affect the growth of microorganisms, in order to facilitate the counting of colonies. The printing plate type is not particularly limited and may be any one, but it has a wide selection range of colorants, resins, solvents, etc., and a method of continuously processing in a roll shape when laminating the culture substrate 4 etc. in the post-processing. However, in consideration of high productivity and the like, rotary gravure printing capable of roll printing is preferable. As for the size of the cells, it is usually appropriate that the pitch is about 10 mm both vertically and horizontally.

(Cover Film) The cover film 2 is
It is preferably transparent because it has waterproofness and water impermeability, and at the same time, it is common to count colonies through the cover film 2 after culturing microorganisms.
A plastic film such as polypropylene, polyethylene, polyester, polystyrene, polycarbonate, polyvinyl chloride, or polyvinylidene chloride as mentioned in the above-mentioned base sheet 1, or a laminate thereof can be used. When culturing aerobic microorganisms, the cover film 2 preferably has oxygen permeability, and for example, an oxygen permeable film such as a stretched or unstretched polypropylene film, polyethylene film, or polystyrene film. Can be used. When culturing anaerobic microorganisms, it is preferable that the base material sheet 1 and the cover film 2 have an oxygen barrier property, for example, polyvinylidene chloride film, polyester film, or ethylene- Use a film or sheet having gas barrier properties (mainly oxygen barrier properties), such as a multi-layer laminated film in which a vinyl alcohol copolymer is used as a core layer and water-impermeable resin layers such as polyethylene and polypropylene are laminated on both sides. be able to. Also, during the culture test, the cover film 2 is the main body sheet 7
Since the test liquid is dropped by lifting from a, 7b, 7c, it is preferable that the material and thickness have appropriate flexibility. Therefore, the thickness of the cover film 2 is preferably about 10 to 200 μm, more preferably about 20 to 70 μm.

When the base sheet 1 or the cover film 2 is provided with the culture base layer 4 or the water-retaining layers 5a, 5b, 5c of a predetermined area made of a water-absorbent ink by coating or the like, it is necessary to adhere them appropriately. However, if the adhesiveness is insufficient, it is preferable to perform a pretreatment such as corona discharge treatment or primer coating on the coated surface of the substrate sheet 1 or the cover film 2. In addition, it is preferable that the base material sheet 1 and the cover film 2 have suitable gas permeability, mainly oxygen permeability, depending on the kind of microorganism to be cultured, as described above. .

(Culture base layer) In the culture apparatus of the present invention,
The microorganism culture base layer 4 provided on the base sheet 1 or the cover film 2 is composed of a water-soluble or amphoteric resin and a nutrient component of the microorganism. The water-soluble or amphoteric resin absorbs water in the test solution inoculated during culturing, so that the nutrient component dissolves and supplies the nutrient component necessary for the microorganism, and the applied substrate is It is necessary to have appropriate adhesiveness with the material sheet 1 or the cover film 2. From this point, as the water-soluble or amphoteric resin, for example, polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl cellulose, poly-N-vinylacetamide, polyethylene glycol, polyvinyl alcohol and the like can be preferably used. Further, as the nutrient component of the microorganism, for example, yeast extract, peptone, dipotassium hydrogen phosphate, glucose, lactose, sodium chloride and the like can be used, and are appropriately selected according to the type of the microorganism to be cultured, and the predetermined Can be mixed and used in the ratio.

As a method for providing the culture base layer 4 of the microorganism composed of the above materials on the base sheet 1 or the cover film 2, the above-mentioned water-soluble or amphoteric resin is used.
Nutrient components of microorganisms, and, if necessary, dyes that can be metabolized by microorganisms are dissolved or suspended in water or an organic solvent, mixed at a predetermined mixing ratio and stirred to form a coating solution, and then roll coat, wire The culture substrate 4 can be formed by applying by a known method such as bar coating, micro bar coating, air knife coating, gravure coating, and drying by heating. Microbial culture substrate 4 as described above
Usually, the total amount of nutrients of microorganisms is 4 g / m
^{It is} preferable to adjust the coating amount to about ² (solid content) and then apply it, since a standard medium can be formed by inoculating 1 ml of the test liquid. The advantage of such a coating method is that the coating amount can be stabilized uniformly, and the material loss is small and the productivity is good.

(Water Retaining Layer) In the culturing apparatus of the present invention, the water retaining layers 5a, 5b, 5c of a predetermined area formed by the water-absorbent ink provided on the substrate sheet 1 or the cover film 2 are the test liquid inoculated during the culturing. It is provided to keep the spread area within a certain area surrounded by the water retention layer and to hold the test liquid within that area for a long time. It is formed by a water-absorbent ink made of a water-absorbent resin that can be used. Therefore, the water retention layer 5a,
The water-absorbent resin used for 5b and 5c has good adhesiveness to the substrate sheet 1, the cover film 2, or the culture substrate 4, and at the same time, the water-absorbent resin itself absorbs the water content of the test solution during culturing. Culture substrate 4 that absorbs quickly and contacts with it
It has the function of dissolving the nutrient components of microorganisms and forming a gel medium suitable for culture.

Specific examples of such a water-absorbent resin include polyethylene oxide, polyacrylamide resins such as polydiethylacrylamide and polyisopropylacrylamide (electron beam cross-linking polyacrylamide),
Poly-N-vinylacetamide, polyvinyl alcohol-based resin, polyvinylpyrrolidone-based resin, and cross-linked products thereof, vinyl alcohol / acrylic acid (salt)
Copolymer resins, polyacrylic acid (salt) resins, polyethylene glycol, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carrageenan, guar gum, xanthan gum, etc., may be used alone or in combination. it can.

When the water-retaining layers 5a, 5b, 5c are formed of these resins, the water-soluble resin is used as the water-soluble resin, and the organic solvent-soluble resin is used as the organic solvent-based solution. Can be made into ink. In particular, in the case of using a mixture of a plurality of resins, in the case of a mixed system of a resin soluble only in water and a resin soluble in an organic solvent, first, a solution of the latter organic solvent is prepared. The water-absorbent ink can be composed of a mixed system in which a powder of a resin soluble in only water is dispersed, and in the case of a mixed system of a resin insoluble in water and a resin soluble in an organic solvent, The water absorbing ink can be constituted by a mixed system in which a solution of the latter organic solvent is prepared, and water-insoluble water absorbing resin powder is dispersed therein. When the water-absorbent ink having such a structure is used, since it is an organic solvent system, the wetting effect on the coating substrate is improved and the drying after coating is facilitated, so that the quality and productivity can be improved.

Further, the water-absorbent ink used for the water-retaining layers 5a, 5b and 5c may contain nutrients necessary for the growth of microorganisms. When the water-retaining layer 5c is formed with such a water-absorbent ink, The culture substrate 4 can be omitted. Water-retaining layers 5a, 5b, 5c formed by using such water-absorbent ink
The shape is not particularly limited and is free, but in order to spread the test liquid inoculated in the inside surrounded by the water retention layer in the frame as much as possible, for example, a circular shape is easier to spread than a rectangular shape. Further, in order to sufficiently retain the test liquid, the water retention layers 5a, 5b,
5c may be a circular solid pattern, but the inside of the circle is preferably patterned into concentric circles, spirals, lattices, checkered patterns, or the like. The size of the circle is generally about 5 cm in diameter. The thickness (height) of the water retention layers 5a, 5b, 5c is 5 to 500.
The range of about μm is preferable. When the thickness is 5 μm or less, it is not preferable because when the test liquid is spread out, it easily protrudes from the water retention layers 5a, 5b, 5c, and when it is 500 μm or more, it is not necessary and the productivity is lowered and the manufacturing cost is low. However, it is not preferable because it is disadvantageous.

Further, as described above, the water retaining layers 5a, 5b, 5c are formed so that the entire shape is circular and the inside is patterned into a pattern, so that the entire surface not patterned is formed. Compared with the one formed in a shape, the surface area can be expanded, and water absorption from the side also occurs, so that the water absorption efficiency is improved and a gel medium can be formed by absorbing a predetermined amount of the test liquid in a short time. Like Water retention layer 5a, 5b,
As a method for providing 5c on the substrate sheet 1 or the cover film 2, it can be formed by applying a water-absorbent ink in a pattern by a printing means such as screen printing or gravure printing or a dispenser, and drying by heating. In this case, when the application cannot be completed by printing once, the application may be performed several times. When an electron beam cross-linking type polyacrylamide is used as the water-absorbent resin, it is coated and dried by an ordinary method and then irradiated with an electron beam of about 10 Mrad to cross-link, thereby absorbing water at the time of use and forming a good gel. To form.

(Addition of Dye) In the culture apparatus of the present invention, it is also effective to add a dye capable of being metabolized by microorganisms, in addition to the above-mentioned constituents of the gel medium. Since such a dye is metabolized by a microorganism that grows during the culture process, colonies are colored, which has the effect of making it easy to count the number of colonies. Specific examples of such dyes include triphenyltetrazolium chloride (hereinafter referred to as T
Displayed as TC), p-tolyl tetrazolium red,
Tetrazolium violet etc. can be used.

The method of applying such a dye to the culture apparatus of the present invention is not particularly limited, and for example,
A method in which a water-soluble resin such as poly-N-vinylacetamide is used as a binder to prepare a coating solution in which a necessary amount of a dye is added to the binder, and the coating solution is applied to the inner surface of the cover film 2 and dried to form the dye layer 6. When laminating the culture substrate 4, a required amount of dye is added to the coating solution in advance and the dye is added to the culture substrate 4 for lamination, and the water retaining layer 5 is used.
There is a method in which a dye is added in advance to the water-absorbent ink used for a, 5b, and 5c, and the dye is contained in the water-retaining layer and laminated, and any method may be used. The amount of the dye added is an area used for culturing, and in the present invention, the area of the water retaining layer is a circle having a diameter of 5 cm (approximately 20 cm ² ), and 0.01 to 0.04 mg (5 per square meter in terms of this area). It is preferable to adjust so as to contain uniformly.

(Others) In the culturing apparatus of the present invention, the main body sheets 7a, 7b, 7c and the cover film 2 can also be used by stacking them as separate bodies.
For example, if the positions of the two are displaced during culturing or the like, the gel medium may be contaminated by falling bacteria or may be dried, so it is preferable to join the both at one end. The joining method is not particularly limited, and it suffices to freely select and join a method suitable for the materials of the joining surfaces of both, usually, a heat seal agent, a hot melt material, an adhesive,
It can be joined using double-sided adhesive tape. The culture device prepared as described above is packaged and sterilized to obtain a finished product. For example, a method in which it is put in a bag made of a plastic laminated film, sealed, and sterilized by γ-ray irradiation is reliable and preferable. .

[0029]

EXAMPLES The culturing apparatus of the present invention will be described below with reference to specific examples, in which a culturing test is actually carried out and the performance thereof is compared with a pour method using a standard agar medium. [Example 1] A 100 μm thick biaxially stretched polyethylene phthalate film (hereinafter referred to as PET film) [Lumirror (single-sided corona discharge treatment) manufactured by Toray Industries, Inc.] was used as a substrate sheet, and its corona discharge treatment surface was used. Gravure printing is used to print a grid print pattern with vertical and horizontal pitches of 10 mm each, and a culture base layer coating solution adjusted to the following composition is applied thereon by a micro bar coater (comma coater) to achieve a dry coating amount of 9 g / A culture base layer was provided by coating and drying so as to be m ² .

Composition of Coating Solution for Culture Substrate Water-Soluble Resin Polyvinylpyrrolidone 20% by weight aqueous solution 50 parts by weight Nutrient components of microorganisms yeast extract 10 parts by weight Peptone 5 parts by weight Glucose 2 parts by weight Dye TTC 0.03 parts by weight A plate in which concentric patterns having an outer diameter of 50 mm, a line width of 3 mm, and an interval of 3 mm are arranged at a predetermined pitch from the outside by a screen printing method in order to provide a water retaining layer with a water-absorbent ink on the culture base layer of the base material sheet. Using a 10 wt% toluene solution of polyethylene oxide [Aquacork Sumitomo Seika Co., Ltd.] as a water-absorbent ink, it was printed on the culture base layer of the base material sheet to a dry thickness of 100 μm. So as to obtain a main body sheet by repeating printing at a drying temperature of 80 ° C. As the cover film, a biaxially oriented polypropylene film having a thickness of 50 μm (hereinafter referred to as OPP film) [OP U-1 (single-sided corona discharge treatment) manufactured by Tokyo Cellophane Paper Co., Ltd.] was used together with the main body sheet. , 10 cm in height
Cut to a width of 10 cm. The body sheet is
It was cut so that the water retaining layer would enter the center. The above 10 cm square main body sheet and cover film were combined one by one to prepare 5 sets of culture devices, each of which was irradiated with γ-rays for sterilization to obtain the culture device of Example 1.

[Embodiment 2] In the constitution of the above Embodiment 1, the water-absorbent ink of the water-retaining layer provided on the culture base layer of the substrate sheet was replaced with 10% by weight of electron beam cross-linking type polyacrylamide.
Change to an aqueous solution, and the water retention layer pattern has an outer diameter of 50 m.
m, 4mm square square inside the circle with line width 1mm
The pattern was changed to a grid pattern arranged at intervals of 2 mm horizontally, and the drying temperature was 80 to 1 so that the film thickness during drying was 100 μm.
Except for overprinting at 00 ° C., the same process as in Example 1 was performed, and 5
A set of culture devices was created. In addition, since the water-absorbent resin of the water retaining layer of the above-mentioned culturing device (5 sets) is electron beam cross-linking type polyacrylamide, each of them has 10 Mrad.
Example 2 in which the cross-linking of polyacrylamide and the sterilization are performed together (the sterilization by γ-ray is omitted).
Was used as the culture device.

[Example 3] A substrate sheet having a thickness of 100
Using a PET film of μm [Lumirror (one-sided corona discharge treatment) manufactured by Toray Co., Ltd.], a corrugated pattern with a vertical and horizontal pitch of 10 mm is printed on the corona discharge treated surface by gravure printing, and water absorbency is printed on it. In order to provide a water-retaining layer with ink, by a screen printing method, a pattern in which squares of 2 mm square are arranged in a checkered pattern inside a circle having an outer diameter of 50 mm and a line width of 1 mm is used with a plate provided at a predetermined pitch,
Polyethylene oxide [Aquacork Sumitomo Seika Co., Ltd.
10% by weight toluene solution of [Production] was used as a water-absorbent ink, printed on the grid printed pattern of the base sheet, dried at 80 ° C., and overprinted so that the film thickness when dried was 100 μm. I made a body sheet.

On the other hand, in order to prepare a cover film provided with a culture substrate of microorganisms, an OPP film having a thickness of 50 μm [OPU-1 (single-sided corona discharge treatment) manufactured by Tokyo Cellophane Paper Co., Ltd.] was used and its corona discharge was used. On the treated surface, a coating liquid having the same composition as the coating liquid for culture base layer used in Example 1 was applied with a micro bar coater (comma coater) so that the same coating amount (the coating amount when dried was 9 g / m ² ). A cover film having a culture base layer was prepared by coating and drying the entire surface. Then, the main body sheet and the cover film were cut into a size of 10 cm in length and 10 cm in width.
In this case as well, the main body sheet was cut so that the water retention layer would be located substantially in the center. The above-obtained base sheet and cover film were combined one by one to prepare five sets of culture devices, each of which was irradiated with γ-rays for sterilization to obtain the culture device of Example 3.

[Culturing Test] (Preparation of Bacterial Solution) Escherichia coli was used as a strain, and shaking culture was carried out at 37 ° C. for 24 hours in a liquid medium (NUTRIENT BROTH). ^It was diluted to ² / ml to prepare a bacterial solution used for the culture test.

The above-mentioned bacterial solution was used as a test solution, and Examples 1 and 2 were used.
1 ml of each was inoculated with a sterilized pipette on the water-retaining layer of the water-absorbent ink on the base material sheet of 5 sets for each of the culture devices 3 and 3, and after overlaying the cover film, lightly press from the top of the cover film for inspection. Spread the liquid over the entire inner surface of the water retention layer,
The culture samples of Examples 1, 2 and 3 were prepared by allowing the gel to stand for about 1 minute (5 samples each). on the other hand,
For comparison, 1 ml of the above-mentioned bacterial solution was added by the conventional pour method to 5
20 ml of standard agar medium prepared in each Petri dish was poured into each to prepare a culture sample for comparison (the number of samples was 5).

The culture samples of Examples 1, 2 and 3 prepared as described above and the culture sample for comparison were placed in an incubator adjusted to 37 ° C. and cultured for 48 hours to find the number of colonies generated. Were counted, and each data and average value are shown in Table 1. (Below margin)

[0037]

[Table 1] Bacteria count measurement result (E. coli) [unit: pieces]

As is clear from the results of the culture tests shown in Table 1, the results of the culture tests using the culture devices of Examples 1, 2 and 3 of the present invention were carried out using the standard agar medium prepared by the conventional pour method. The results are substantially in agreement with the results of the culture test of the comparative culture sample, which shows that the culture performance is good, the operation is easy, and the culture device is sufficiently practical. In addition, the colonies that occurred were clearly observed, and counting was easy.

[0039]

As described in detail above, the culturing apparatus of the present invention comprises a waterproof base sheet, and a water-impermeable and transparent cover film which is placed on the base sheet,
The inner surface of either one of the base sheet and the cover film,
Alternatively, in a microorganism cultivating apparatus having a microorganism culture base layer provided on both inner surfaces, a water-retaining layer made of a water-absorbent ink overlaps at least the cover film in order to spread a test solution to be inoculated to a predetermined area. The culture device is provided on the inner surface of the base material sheet, and each layer for forming a gel medium is provided as a dry coating film layer by coating or printing, and the entire device is sterilized in advance. Therefore, the culture device is storable and does not require any special device or equipment such as an autoclave or a spreader even when it is used, and it is possible to culture microorganisms at any time with extremely simple operation. Easily available.

In terms of manufacturing, the culture apparatus of the present invention coats or prints a coating solution for a culture base layer, a water-absorbent ink for a water retaining layer, or a coating solution for a dye layer on a substrate sheet or a cover film. By applying by such means as above, and heating and drying, it is possible to form a culture base layer, water retention layer, or dye layer of microorganisms, which simplifies the manufacturing process, saves labor, improves productivity, and is uniform and stable. A dry coating film is formed, and an effect that a culture device having excellent performance can be provided is exerted.

By using any one of electron beam cross-linking type polyacrylamide, polyethylene oxide and carboxymethyl cellulose as the resin used for the water-absorbent ink of the water-retaining layer, the water-retaining layer absorbs water and retains the test liquid. Is further improved, and a good gel medium can be formed within a predetermined area. In particular, among the above, when electron beam cross-linking type polyacrylamide is used, it is applied to a base material sheet or a cover film film, processed into a culture device in a heat-dried state, and then irradiated with an electron beam. Thus, there is an advantage that the crosslinking of the resin and the sterilization of the entire culture device can be performed simultaneously in one step. Furthermore, by patterning the water-retaining layer of a predetermined area made of water-absorbent ink into concentric circles, spirals, grids, checkerboard patterns, etc., the retention of the test liquid is improved and the spread area is retained. It can be easily stopped within the range surrounded by the layers, and the efficiency of water absorption can be improved, and the gel medium can be formed in a short time. In addition, sterilization and incineration can be easily carried out in terms of disposability after being used in a culture test and the like, which is also excellent in simplicity.

[Brief description of drawings]

FIG. 1 is a schematic perspective view illustrating the configuration of an embodiment of a culture device of the present invention.

FIG. 2 is a schematic cross-sectional view taken along the line AA showing the configuration of the main body sheet portion of the culture device shown in FIG.

3 (A), (B), (C), and (D) of FIG. 3 are partial schematic cross-sectional views for explaining the layer structure of an example of the culture apparatus of the present invention.

FIG. 4 is a plan view showing an example of the shape of a water retention layer formed by patterning in the culture device of the present invention.

[Explanation of symbols]

 1 Base Material Sheet 2 Cover Film 3 Cell Print Pattern 4 Culture Base Layer 5a, 5b, 5c Water Retaining Layer 6 Dye Layer 7a, 7b, 7c Main Sheet

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kiyoshi Oguchi 1-1-1, Ichigaya-Kagacho, Shinjuku-ku, Tokyo Dai Nippon Printing Co., Ltd.

Claims (5)

[Claims] 1. A waterproof base sheet, and a water-impermeable and transparent cover film which covers the base sheet, and the inner surface of either or both of the base sheet and the cover film. In an apparatus for culturing microorganisms in which a culture base layer for microorganisms is provided on the inner surface of the substrate, a water-retaining layer made of a water-absorbent ink for spreading a test solution to be inoculated into a predetermined area is a substrate at least overlapping with a cover film. A culture device, which is provided on the inner surface of a sheet. 2. The culture apparatus according to claim 1, wherein the resin used in the water-absorbent ink is electron beam cross-linking type polyacrylamide. 3. The culture apparatus according to claim 1, wherein the resin used in the water-absorbent ink is polyethylene oxide. 4. The culture apparatus according to claim 1, wherein the resin used in the water-absorbent ink is carboxymethyl cellulose. 5. The water-retaining layer having a predetermined area, which is made of the water-absorbent ink, is patterned in a concentric circle shape, a spiral shape, a lattice shape, a checkered pattern, or the like. Culture device.