JP6066274B2 - Microorganism culture device and microbe inspection method using microorganism culture device - Google Patents

Description

  The present invention relates to a microorganism culture device for culturing microorganisms. The present invention also relates to a microorganism testing method for testing microorganisms using a microorganism culture device.

  As a method for confirming the presence of microorganisms or measuring the number of microorganisms, there is an agar plate mixing method. In this agar plate pour method, an agar medium formed in a petri dish previously sterilized is used. For this reason, an autoclave for autoclaving the agar medium and an inspection room for performing microbial tests aseptically are required. Moreover, skill is required for the operation of the microorganism inspection from the sampling of the microorganism to the preparation of the sample solution, dispensing, mixing with the medium, culture, and counting. Therefore, a microorganism culture device having a culture layer that does not require highly skilled skills and can be easily tested for microorganisms has been developed.

  For example, Patent Document 1 discloses a microorganism culture device including a culture layer including an adhesive composition based on water and a cold water-soluble powder attached on the adhesive composition and containing a gelling agent. ing. In this inspection method using a microorganism culture device, first, a test solution containing E. coli is supplied to the culture layer, and then the test solution is covered with a cover film fixed to the main body of the microorganism culture device. Thereafter, by using a spreader, the test solution is uniformly spread on the cold water soluble powder of the culture layer.

  Further, in Patent Document 2, a microorganism culture comprising: a culture layer comprising a circular porous matrix layer and a water-soluble polymer compound layer laminated on a square pressure-sensitive adhesive sheet; and a transparent cover film disposed thereon A tool is disclosed. In this microorganism culture device, a test liquid is supplied to the porous matrix layer, and the test liquid is held in the porous matrix layer. And the water | moisture content of the hold | maintained test solution melt | dissolves the water-soluble polymer compound layer which contact | connected the porous matrix layer, As a result, polymer compound aqueous solution arises. Based on this polymer compound aqueous solution and the growth nutrient component, an environment in which microorganisms can grow is realized.

Japanese Patent No. 3383304 Japanese Patent No. 4396078

  By the way, in factories and research laboratories that conduct microorganism testing, the presence or number of microorganisms is examined for a large number of test solutions obtained from various samples. Therefore, it is preferable that the inspection method using the microorganism culture device is simple and the time required for the inspection is short. In addition, microorganism inspection is performed on a work bench using a microorganism culture device, but the inspection is generally performed on a large number of test liquids using a large number of microorganism culture tools on the same work table. The However, the work area on the work table is limited, and therefore, it is preferable to efficiently move the microorganism culture tool on which the test liquid is dropped from the work area to the storage area.

  However, in the inspection method using the microorganism culture device described in Patent Document 1, it is necessary to spread the test solution on the culture layer using the spreader as described above. For this reason, the work becomes complicated, and as a result, the time required for the inspection process may be increased. In addition, the spread of the test solution on the culture layer is likely to vary, and it is considered difficult to measure the number of microorganisms with high accuracy.

  Moreover, in the microorganism culture device described in Patent Document 2, as described above, the culture layer includes a porous matrix layer. In this inspection method using the microorganism culture device, usually, after the porous matrix layer has absorbed the test solution, the subsequent steps, for example, the step of transporting the microorganism culture device are performed. That is, the time for waiting for the porous matrix layer to absorb the test liquid is included in the inspection method, and as a result, the time required for the inspection may be increased. In the microorganism culture device described in Patent Document 2, as described above, the culture layer is laminated on the adhesive sheet, and the culture layer is covered with a transparent cover film. In this case, a step of peeling the cover film from the adhesive sheet is required before supplying the test solution to the culture layer. In addition, when covering the culture layer after supplying the test solution, it is necessary to cover the cover film while taking care to prevent the cover film from adhering to the adhesive sheet and causing wrinkles into the cover film. is there. For this reason, it is considered that the time required for the inspection process becomes long.

  The object of the present invention is to provide a microorganism culture device and a microorganism testing method using the microorganism culture device that can effectively solve such problems.

  The present invention includes a base sheet, a culture layer provided on the top surface of the base sheet and configured to culture microorganisms, and a frame provided on the top surface of the base sheet so as to surround the culture layer. A cover film capable of covering the culture layer and the frame, wherein the culture layer is a dry culture layer containing at least a gelling agent, and the cover film is formed by the cover film being the culture layer. And when covering the said frame, it is a microorganism culture tool comprised so that the area | region of the upper surface of the said base material sheet may be exposed at least partially, and this may form an exposed area | region.

  The microorganism culture device according to the present invention may further include a joining portion that joins a part of the cover film to a part of the base sheet.

  In the microorganism culture device according to the present invention, the base sheet has a contour including a first outer edge and a second outer edge facing the first outer edge when viewed from the normal direction of the base sheet. The joining portion is disposed in the vicinity of the first outer edge of the base sheet, and the exposed region of the base sheet is formed in the vicinity of the second outer edge of the base sheet. Good.

  In the microorganism culture device according to the present invention, when the direction from the first outer edge of the base sheet toward the second outer edge is the first direction, the maximum distance in the first direction of the exposed region is 2 to 20 mm. It may be within the range.

  In the microorganism culture device according to the present invention, when the direction from the first outer edge of the base sheet toward the second outer edge is a first direction, the space between the frame body and the exposed region of the base sheet. The distance in the first direction is preferably in the range of 3 to 17 mm, and the height of the frame is preferably in the range of 300 to 1400 μm.

  In the microorganism culture device according to the present invention, the cover film may be configured such that the lower surface of the cover film has water repellency.

The present invention is a microorganism testing method for testing microorganisms using a microorganism culture tool on a work table, and the microorganism culture tool comprises the microorganism culture tool according to any one of claims 1 to 5. The work table includes a work area and a storage area, and the microorganism testing method includes:
A step of disposing the microorganism culture tool in the working area, a supply step of supplying a test solution onto the culture layer of the microorganism culture tool, and after the supplying step, the culture layer of the microorganism culture tool and A covering step of covering the frame with the cover film of the microorganism culture device, and after the covering step, applying a force to the exposed region of the microorganism culture device, And a transporting process for transporting to the storage area.

  In the microorganism testing method according to the present invention, the work area and the storage area of the work table may be on the same plane.

  According to the microorganism culture device of the present invention, when the cover film covers the culture layer and the frame, the region of the upper surface of the base material sheet is at least partially exposed, whereby an exposed region is formed. It is configured. For this reason, it can touch a base material sheet, without touching a cover film. Accordingly, the microorganism culture device can be moved by applying a force to the exposed region of the base sheet while preventing stress from being transmitted to the culture layer and the test solution via the cover film. For this reason, a microorganism culture tool can be conveyed, without waiting for a culture layer to gelatinize, and, thereby, the time required for a test | inspection can be shortened.

FIG. 1 is a plan view showing a microorganism culture device according to an embodiment of the present invention. FIG. 2 is a side view showing the microbial culture device of FIG. 1 as viewed from the direction of arrow A. FIG. 3 is a perspective view showing the microorganism culture device in a state where the cover film is lifted. FIG. 4 is a longitudinal sectional view showing a case where the microorganism culture device of FIG. 1 is viewed from the BB direction. FIGS. 5A to 5F are views showing an example of a method for producing a microorganism culture device. 6A to 6E are diagrams showing an example of a microorganism testing method using a microorganism culture device. FIG. 7 is a plan view showing microorganism culture devices arranged on a work table. 8 (a) and 8 (b) are diagrams showing another example of a microorganism testing method using a microorganism culture device. FIG. 9 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. FIG. 10 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. FIG. 11 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. FIG. 12 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. FIG. 13 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. FIG. 14 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. 15A is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention, and FIG. 15B is a view of the microorganism culture device shown in FIG. FIG. FIG. 16 is a side view showing a modification of the cover film of the microorganism culture device in the embodiment of the present invention. FIG. 17 is a longitudinal sectional view showing a modified example of the microorganism culture device in the embodiment of the present invention. FIG. 18 is a plan view showing a modification of the microorganism culture device according to the embodiment of the present invention. FIG. 19 is a diagram illustrating how air bubbles are formed around bacteria in the example. FIG. 20 is a diagram illustrating a state in which bleeding is visually recognized around bacteria in the example.

  Hereinafter, embodiments of the present invention will be described with reference to FIGS. 1 to 6. In the drawings attached to the present specification, for the sake of illustration and ease of understanding, the scale, the vertical / horizontal dimension ratio, and the like are appropriately changed and exaggerated from those of the actual product.

First, microorganisms will be described. Microorganisms are divided into aerobic organisms and anaerobic organisms.
Aerobic organisms are organisms with a metabolic mechanism based on oxygen. For aerobic organisms, obligate aerobic organisms that cannot grow without oxygen, and in the presence of oxygen, ATP is produced by aerobic respiration, but energy can be obtained by fermentation even in the absence of oxygen. Includes facultative aerobic organisms that can switch metabolism. Examples of obligate aerobic organisms include mold, Nocardia genus, and Pseudomonas genus. Examples of facultative aerobic organisms include Staphylococcus genus, Escherichia genus, Citrobacter genus, Klebsiella genus, Enterobacter genus, Proteus genus and yeast.
Anaerobic organisms are organisms that can grow only in the absence of oxygen and are called obligate anaerobes. Examples of obligate anaerobes include the genus Clostridium and the genus Bifidobacterium.
The microorganism culture device according to the present embodiment, which will be described below, can be suitably used for testing aerobic bacteria (aerobic organisms). In addition, the microorganism culture device according to the present embodiment can also be used for inspection of anaerobic bacteria (anaerobic organisms).

Microorganism culture tool Next, the structure of the microorganism culture tool 1 according to the present embodiment will be described with reference to FIGS. FIG. 1 is a plan view showing a microorganism cultivating device 1, and FIG. 2 is a side view showing the microorganism culturing device 1 of FIG. FIG. 3 is a perspective view showing the microorganism culture device 1. In FIG. 1, constituent elements of the microorganism culturing tool 1 such as the frame body 20, the joint portion 25, and the culture layer 30 located below the cover film 40 described later are seen through and indicated by dotted lines.

  As shown in FIG. 1, the microorganism culture device 1 has a substantially rectangular outline when viewed in a plan view. As shown in FIGS. 1 and 2, the microorganism culture tool 1 includes a base sheet 10, a culture layer 30 provided on the top surface of the base sheet 10, and an upper surface of the base sheet 10 so as to surround the culture layer 30. And a frame body 20 provided on the frame. Moreover, the microorganism culture tool 1 further includes a cover film 40 that can cover the culture layer 30 and the frame body 20.

  As shown in FIG. 1, both the base sheet 10 and the cover film 40 have a substantially rectangular outline when viewed in a plan view, that is, when viewed from the normal direction of the base sheet 10. ing. Specifically, when the base sheet 10 is viewed from the normal direction of the base sheet 10, the linear first outer edge 11, the linear second outer edge 12 facing the first outer edge 11, and The first outer edge 11 and the second outer edge 12 have a contour that includes a third outer edge 13 and a fourth outer edge 14 that are linear and extend from each other. Similarly, the cover film 40 has a contour including a first outer edge 41, a second outer edge 42, a third outer edge 43, and a fourth outer edge 44 that are each linear.

  As shown in FIG. 2, the microorganism culture device 1 may further include a joining portion 25 that joins a part of the cover film 40 to a part of the base sheet 10. Thereby, the cover film 40 can be displaced with respect to the base material sheet 10 with the joint portion 25 as an axis. Thereby, the operation and handling of the cover film 40 can be facilitated. Moreover, the position of the cover film 40 with respect to the base material sheet 10 can be determined reliably, and thereby, an exposed region 10A described later can be accurately formed. In FIG. 3, the microorganism culture device 1 is shown in which the cover film 40 is lifted with respect to the base sheet 10 while a part of the cover film 40 is fixed to the base sheet 10 by the joint portion 25. ing.

  The joining part 25 is arrange | positioned in the vicinity of the 1st outer edge 11 of the base material sheet 10, for example. Here, “near” means that the joint portion 25 is arranged so that the joint portion 25 is in contact with the first outer edge 11, and the joint portion 25 is not in contact with the first outer edge 11, This is a concept that includes both cases where the distance between the first outer edge 11 is smaller than the distance between the joint portion 25 and the other outer edges 12, 13, and 14.

  The frame 20 is for surely limiting the water absorption range of a test liquid to be described later supplied on the culture layer 30. FIG. 3 shows an example in which there is no gap between the frame body 20 and the culture layer 30 and both are in contact with each other. However, the positional relationship between the frame 20 and the culture layer 30 is not particularly limited as long as the test liquid can be prevented from leaking outside the frame 20.

  FIG. 3 shows an example in which the frame 20 has a circular outline. However, the outline of the frame body 20 is not particularly limited as long as the water absorption range of the test solution supplied onto the culture layer 30 can extend over a predetermined area. For example, the frame 20 may have a contour such as a rectangle, an ellipse, a polygon, and an indefinite shape.

  As shown in FIG. 3, the cover film 40 may have a grid line 40 a. The grid line 40a serves as a standard for calculating the number of microorganisms cultured in the culture layer 30. Such a grid line 40 a can be provided by a print layer 47 described later included in the cover film 40. Such grid lines may be formed on the base sheet 10 instead of the cover film 40.

  Next, with reference to FIG. 1 and FIG. 2, the positional relationship between the base material sheet 10 and the cover film 40 is demonstrated. As shown in FIGS. 1 and 2, the cover film 40 is configured such that when the base sheet 10 covers the culture layer 30 and the frame body 20, the region of the upper surface of the base sheet 10 is at least partially exposed. ing. For example, when the direction from the first outer edge 11 to the second outer edge 12 of the base sheet 10 is the first direction, the dimension of the cover film 40 in the first direction is larger than the dimension of the base sheet 10 in the first direction. It is getting smaller. In the following description, a region exposed without being covered by the cover film 40 in the region of the upper surface of the base sheet 10 is referred to as an exposed region 10A. By forming such an exposed area 10 </ b> A on the base sheet 10, as will be described later, the time required for the inspection using the microorganism culture tool 1 can be shortened.

  As shown in FIGS. 1 and 2, the exposed region 10 </ b> A is formed in the vicinity of the second outer edge 12 of the base sheet 10, for example. Here, the term “near” refers to the case where the exposed region 10A is formed so that the exposed region 10A is in contact with the second outer edge 12 and the exposed region 10A is the second outer edge 12 as in the case of the joint portion 25 described above. A concept that includes both cases in which the distance between the exposed region 10A and the second outer edge 12 is smaller than the distance between the exposed region 10A and the other outer edges 11, 13, and 14 although not in contact with It is. FIG. 1 shows an example in which the exposed region 10 </ b> A is formed along the entire area of the second outer edge 12. Such an exposed region 10 </ b> A is obtained by arranging the cover film 40 so that the second outer edge 42 of the cover film 40 is positioned closer to the frame body 20 than the second outer edge 12 of the base sheet 10.

1 and 2, the maximum distance of the exposed region 10A in the first direction described above is indicated by reference numeral s _1, the minimum distance between the exposed region 10A and the frame body 20 in the first direction code It is shown in s _2. The height of the frame 20 is shown at h _1. Among these, the distance s ₁ is appropriately set so that the microorganism culture device 1 can be appropriately conveyed by applying a force to the exposed region 10A in the microorganism testing method using the microorganism culture device 1. For example, the distance s ₁ is in the range of 2 to 20 mm, preferably 4 to 10 mm. Distance s ₁ is not preferable because the finger is easily brought into contact with the cover film 40 when applying a force to 2mm smaller the exposed area 10A. In addition, if the distance s ₁ is greater than 20 mm, the length of the microorganism culture tool 1 in the first direction becomes long. Therefore, the number of the microorganism culture tools 1 that can be arranged in the work area on the work table and / or the storage area. Is not preferred because of a decrease in Appropriate ranges for the distance s ₂ and the height h ₁ will be described later.

Layer Configuration of Microorganism Culture Tool Next , the layer configuration of the microorganism culture tool 1 will be described with reference to FIG. FIG. 4 is a longitudinal sectional view showing a case where the microorganism culture tool 1 of FIG. 1 is viewed from the BB direction. Hereinafter, each component will be described in order.

(Substrate sheet)
The base sheet 10 is required to have a solvent resistance that is not deteriorated by a later-described medium solution 30 </ b> A applied on the base sheet 10 in order to form the culture layer 30, and a water resistance necessary for culture. In addition, heat resistance that can withstand the drying process when the culture layer 30 is formed is also required. Moreover, when the microorganisms cultured in the culture layer 30 are aerobic bacteria, the base material sheet 10 is required to have oxygen permeability. When the base sheet 10 has oxygen permeability, oxygen can be supplied to the aerobic bacteria.

  The material constituting the base sheet 10 is not particularly limited as long as it has water resistance and heat resistance and, if necessary, oxygen permeability. For example, polyester, polyethylene, polypropylene, Plastic sheets such as polystyrene, polycarbonate, and polyvinyl chloride can be used. These plastic sheets are preferable in terms of excellent transparency. As will be described later, the microorganism culture tool 1 is for observing and counting colonies of microorganisms grown in the culture layer 30. When the transparency of the base material sheet 10 is high, the visibility of the colonies can be increased by the projection light from the lower side (base material sheet 10 side) of the microorganism culture device 1 or the incident light from the side surface, and the colony measurement is possible. Becomes easy. However, it does not necessarily need to be transparent, and the optical characteristics of the base sheet 10 can be appropriately selected depending on the type of microorganism, the presence or absence of colony coloring, and the like. For example, the base sheet 10 may be white or colored by foaming.

  The base sheet 10 may be a single layer or a laminated sheet of two or more layers. As a laminated sheet, what laminated | stacked 2 or more types of the said plastic sheet is mentioned. In addition, a laminated sheet in which a paper base material is laminated on the plastic sheet, a laminated sheet in which a paper base material is coated with a synthetic resin, and the like can be suitably used. Examples of such a laminated sheet include a laminated sheet of a polyethylene film and a paper base material. Moreover, when the above-mentioned oxygen permeability is calculated | required by the base material sheet 10, the synthetic paper which used the synthetic resin as the main raw material can be used conveniently. Examples of such commercially available synthetic paper include “YUPO” manufactured by Yupo Corporation, “Chrisper” manufactured by Toyobo Co., Ltd., and the like.

  In order to improve the adhesion with the culture layer 30, surface treatment may be performed on the upper surface of the substrate sheet 10 in advance. As the surface treatment, for example, a known surface treatment such as a corona discharge treatment, an ozone treatment, a glow discharge treatment, an oxidation treatment using chemicals, or a low temperature plasma treatment using oxygen gas or nitrogen gas can be used.

  Moreover, you may perform the surface treatment of apply | coating an anchor coating agent to the upper surface of the base material sheet 10. FIG. Examples of such anchor coating agents include isocyanate (urethane), polyethyleneimine, polybutadiene, organic titanium, and other anchor coating agents. More preferably, for example, aromatic polyisocyanates such as tolylene diisocyanate, diphenylmethane diisocyanate, polymethylene polyphenylene polyisocyanate, or aliphatic polyisocyanates such as hexamethylene diisocyanate, xylylene diisocyanate, etc. Mainly used are polyether polyurethane resins, polyester polyurethane resins, and polyacrylate polyurethane resins obtained by reacting polyfunctional isocyanates with polyether polyols, polyester polyols, polyacrylate polyols, and other hydroxyl group-containing compounds. Ingredients.

  The substrate sheet 10 is preferably flat without curling or the like. Although the thickness of the base material sheet 10 is not specifically limited, Usually, it exists in the range of 25-1500 micrometers, Preferably it exists in the range of 50-500 micrometers.

(Culture layer)
The culture layer 30 is a dry culture layer containing at least a gelling agent, and is a layer configured to culture microorganisms. Here, the dry culture layer is obtained by drying a medium solution containing a solvent to volatilize the solvent. For example, the culture layer 30 includes a fixing agent, a gelling agent, a nutrient component, and a plasticizer. The culture layer 30 may further contain a color development indicator, a selection agent, and the like. Each component of the culture layer 30 is appropriately selected according to the microorganism to be cultured. Hereinafter, the role of each component and an example of the material constituting each component will be described.

[Sticking agent]
The fixing agent has a role of fixing other components such as a gelling agent, a nutrient component, a plasticizer, a color development indicator, and a selection agent to the base sheet 10. Examples of the fixing agent include polyvinyl pyrrolidone, polyethylene oxide, hydroxypropyl cellulose, polyvinyl alcohol, and the like, and polyvinyl pyrrolidone is particularly preferable. When polyvinyl pyrrolidone is used, it is soluble in an alcohol solvent, so that the viscosity of the medium solution can be easily adjusted. In addition, when polyvinylpyrrolidone is used, an alcohol-based solvent can be used, so that a dispersed medium solution can be prepared without dissolving the gelling agent. Thereby, the remarkable viscosity raise of the culture medium liquid resulting from a gelatinizer can be suppressed. Furthermore, after apply | coating a culture solution on the base material sheet 10 by a predetermined pattern, a solvent can be removed from a culture solution without heating at high temperature. Moreover, since the film property of polyvinylpyrrolidone is high, it is possible to form a film by incorporating other components such as a gelling agent, a nutrient component, a plasticizer, a coloring indicator, and a selective agent. Moreover, the adhesiveness with the base material sheet 10 is excellent.

[Gelling agent]
The gelling agent gels the culture layer 30 to which the test solution 35A is supplied, thereby making the viscosity of the culture layer 30 favorable for culturing microorganisms. In addition, since the culture layer 30 contains the gelling agent, the culture layer 30 easily adheres to the cover film 40, so that evaporation of moisture from the culture layer 30 during culture can be suppressed.

  As the gelling agent, those having water absorbability (or water swellability) and capable of substantially retaining the shape when water is absorbed or swollen by water can be used. Examples of gelling agents include water-soluble polymers such as polyvinyl alcohol (PVA), modified PVA, cellulose derivatives, starch and derivatives thereof, cellulose derivatives and polysaccharides other than starch, acrylic acid and derivatives thereof, polyethers, and proteins. Cross-linked product; starch-based water-absorbing polymer; cellulose-based water-absorbing polymer; acrylic water-absorbing polymer; maleic anhydride-based copolymer; vinyl pyrrolidone-based copolymer; polyether-based water-absorbing polymer; acrylic fiber hydrolyzate; Examples thereof include polymers, guar gum, psyllium seed gum, xanthan gum, hydroxyethyl cellulose, carboxymethyl cellulose, polyacrylamide, locust bean gum, and algin. These may be used alone or in combination of two or more.

  When the microorganism cultured in the culture layer 30 is an aerobic bacterium, a mixture of guar gum and psyllium seed gum may be used as a gelling agent. Hereinafter, the advantage of using a mixture of guar gum and psyllium seed gum as a gelling agent will be described.

  Guar gum exhibits very high viscosity at low concentrations. For this reason, the culture layer 30 which can be gelled by dropping the test solution and can create an environment suitable for the growth of bacteria can be provided. However, if the ratio of guar gum in the gelling agent becomes too high, the flexibility of the culture layer 30 is lowered, and as a result, the culture layer 30 may become brittle. For example, it is conceivable that the culture layer 30 is torn by the gas generated from aerobic bacteria during culture, and the visibility of the colonies is reduced. On the other hand, psyllium seed gum is capable of forming a gel with elasticity, and thus can provide an appropriate elasticity to the culture layer 30. For this reason, it becomes possible to confine the gas generated from the aerobic bacteria in the culture layer 30 during the culture. That is, it is possible to suppress the culture layer 30 from being partially crushed by the gas. The mixing ratio of guar gum and psyllium seed gum in the gelling agent is appropriately set according to the type of aerobic bacteria to be cultured, etc., for example, the ratio of the weight part of guar gum to the weight part of psyllium seed gum is 7 : Within the range of 3-5: 5.

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

[Plasticizer]
The plasticizer is added when the film of the culture layer 30 composed of a fixing agent such as polyvinylpyrrolidone is hard and brittle. Examples of the plasticizer include glycerin, glycerin derivative plasticizer, and polyethylene glycol. By adding such a plasticizer, the film of the culture layer 30 can be given flexibility. Thereby, for example, the vicinity of the outer edge of the base sheet 10 can be prevented from curling. In addition, when the content rate of the plasticizer in the culture layer 30 becomes high too much, it can be considered that the culture characteristics and visibility of the microorganisms deteriorate. Therefore, the content ratio of the plasticizer in the culture layer 30 is determined in consideration of various points such as desired flexibility, culture characteristics, and visibility in the culture layer 30. For example, when glycerin is used as a plasticizer, the weight percentage of glycerin relative to the entire solid content of the culture layer 30 (a component excluding solvents such as alcohol) is preferably in the range of 10 to 15%.

[Coloring indicator]
The coloring indicator is colored by coloring with a reaction with a specific substance produced by metabolism of microorganisms that develop during the culturing process, recognition of pH change, reaction with an enzyme, and the like. By using a color development indicator, the number of colonies can be counted very easily. Examples of the color development indicator include tetrazorim salts such as triphenyltetrazolium chloride (hereinafter referred to as TTC), p-tolyltetrazolium red, tetrazolium violet, petetylyltetrazolium blue, neutral red mixtures, phenol red, bromthymol blue, and thymol. PH indicator such as blue mixture. Further, as a color developing indicator, 5-bromo-4-chloro-3-indolyl-BD-glucuronide cyclohexylammonium hydrate, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5 -Bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl phosphate and the like can also be used.

(Selective agent)
The selective agent is added to the culture layer 30 in order to suppress the growth of microorganisms that are not detection targets. Examples of such a selective agent include antibiotics such as methicillin, ceftamezole, cefixime, ceftazidime, cefsulosin, bacitracin, polymyxin B, rifampicin, novobiocin, colistin, lincomycin, chloramphenicol, tetracycline, streptomycin, azactam, acriflavine, Synthetic antibacterial agents such as sulfa drugs, nalidixic acid and olaquindox, dyes having bacteriostatic or bactericidal activity such as crystal violet, brilliant green, malachite green, methylene blue, Tergitol 7, dodecyl sulfate, lauryl sulfate, sodium cholate, etc. Surfactant, selenite, tellurite, sulfite, sodium nitride, lithium chloride, oxalate, sodium azide, high concentration sodium chloride, etc. Inorganic salts, and the like.

  The solvent used in the medium solution 30A applied on the base sheet 10 in order to form the culture layer 30 containing the above constituent components is not particularly limited as long as it can dissolve or disperse the selected constituent components. Although not intended, it is preferable to use an alcohol solvent. Unlike a high boiling point solvent such as water or toluene, the alcohol solvent does not need to be heated at a high temperature when removing the solvent. For this reason, there is no possibility that each component is thermally decomposed, and it does not require a long time for solvent removal, which is preferable in terms of excellent production efficiency. As the alcohol solvent, an alcohol having 1 to 5 carbon atoms is preferable, and examples thereof include methanol, ethanol, isopropyl alcohol and the like. Among these, methanol is most preferable because it has a low boiling point, can be easily removed by drying, and does not inhibit the growth of microorganisms. In addition, the said solvent can be used individually or in mixture of 2 or more types. The amount of solvent used may be appropriately selected depending on the components of the medium and the type of solvent.

  In FIG. 4, an example in which the culture layer 30 is composed of one layer is shown, but the present invention is not limited to this, and the culture layer 30 may be composed of two or more layers. For example, a first culture layer is formed using a medium solution containing a fixing agent and a gelling agent, and a second culture layer is formed thereon using a medium solution containing a fixing agent, a gelling agent and / or a nutrient component. It may be formed.

(Frame)
As described above, the frame body 20 is provided on the upper surface of the substrate sheet 10 so as to surround the culture layer 30. By providing such a frame 20, when supplying the test solution 35 A onto the culture layer 30, the test solution 35 A can be prevented from spreading beyond the frame 20, and the test solution The area where 35 extends can be made constant. Therefore, since the test solution 35A can be expanded only by covering the cover film 40, the work of expanding the test solution 35A on the culture layer 30 by using a spreader can be omitted, and this requires the inspection process. Time can be shortened. Further, it is possible to prevent the test liquid 35A from leaking onto the base sheet 10.

  The frame body 20 may be formed before the culture layer 30 is formed, or may be formed after the culture layer 30 is formed. When the frame body 20 is formed after the culture layer 30 is formed, the frame body 20 and the culture layer 30 are formed. It is conceivable that a gap is easily generated between the two. In this case, when the microorganism is cultured, the bacteria migrate to the gap, and it may be difficult to accurately measure the number of colonies. Therefore, it is preferable that the frame body 20 is formed before the culture layer 30 is formed. Further, by forming the frame body 20 before the formation of the culture layer 30, it is possible to delimit the range in which the medium solution 30A spreads, that is, the range in which the culture layer 30 is formed, so that expensive microorganism culture material is wasted. Can be reduced.

  As for the width | variety of the frame 20, it is preferable that the most detail is in the range of 0.5-5.0 mm in width, More preferably, it exists in the range of 1.0-3.0 mm. If it is the said range, even if it is a case where the culture layer 30 absorbs water and swells, there is little possibility that the shape of the frame 20 will collapse.

  The height of the frame 20 is set so that the test liquid 35A comes into contact with the cover film 40 when the test quarantine 35A is dropped and covered with the cover film 40. The frame 20 is set to be 100 to 1200 μm higher than the height of the culture layer 30, more preferably 200 to 1000 μm higher, and particularly preferably 300 to 800 μm higher. By making the difference between the height of the frame 20 and the height of the culture layer 30 larger than 100 μm, it is possible to sufficiently suppress the spread of the test solution, thereby causing leakage onto the base sheet 10. Can be prevented. Further, by making the difference between the height of the frame body 20 and the height of the culture layer 30 smaller than 1200 μm, the adhesion between the test solution 35A and the culture layer 30 and the cover film can be ensured. Thus, the test solution 35A can be spread over the entire culture layer 30 while supplying the test solution 35A to the culture layer 30.

The height h _{1 of} the frame body 20, that is, the distance from the base sheet 10 to the upper end of the frame body 20 is 300 to 1400 μm, preferably 500 to 900 μm. By making the height h ₁ of the frame 20 larger than 300 μm, a finger can easily enter between the base sheet 10 and the cover film 40 when the cover film 40 is turned up to drop the test liquid 35A. Therefore, the cover film 40 can be easily turned up. In addition, by reducing the height h ₁ of the frame 20 to less than 1400 μm, the amount of the expensive medium solution 30A applied onto the base sheet 10 can be reduced, so that the cost of the microorganism culture tool 1 is reduced. Can do.

  As a material constituting the frame body 20, a hydrophobic material is preferably used, and for example, a hydrophobic resin is used. As the hydrophobic resin, for example, a UV curable resin, a hot melt resin, a thermally foamed ink, a UV foaming agent, or the like can be preferably used. These may be colored.

  The UV curable resin is not particularly limited. For example, the radical type includes an acrylate type and an unsaturated polyester type, and the cationic type includes an epoxy acrylate type, an oxetane type, a vinyl ether type, and the like. .

  As the hot melt resin, one having a softening point temperature of 120 ° C. or lower, more preferably 100 ° C. or lower can be suitably used. Examples of the hot melt resin include urethane, polyamide, polyolefin, polyester, ethylene vinyl acetate, synthetic rubber such as styrene butadiene rubber and urethane rubber, but are not limited thereto. Absent.

  In addition, as the hydrophobic resin constituting the frame body 20, a thermal foaming ink, a UV foaming agent, or the like can be suitably used. For example, polyurethane resin, polyamide resin, polyvinyl chloride resin, polyacrylate resin, polyester resin as a binder, polyolefin resin such as chlorinated polypropylene, and rubbers such as chlorinated rubber and cyclized rubber Etc. can be used. These inks are more preferable if they have water repellency. In this respect, inks obtained by adding silicones or waxes to the above resins are more preferable.

  It is preferable that the hydrophobic resin used for the frame 20 has hydrophobicity and water repellency, and at the same time the thickness of the coating film can be increased. From this viewpoint, it is effective to add a conventionally known foaming agent or the like to the above resin and use it as a foamed ink. As such UV foaming ink, at least reactive diluent such as acrylate reactive diluent or methacrylate reactive dilution, urethane acrylate photopolymerizable oligomer, polyester acrylate photopolymerizable oligomer, epoxy acrylate photopolymerizable oligomer, acrylic A UV-curable ink consisting of a photopolymerizable oligomer, a photopolymerizable oligomer such as a special photopolymerizable oligomer, and a photopolymerization initiator, and containing a foaming agent such as an inorganic foaming agent, an organic foaming agent, or a liquid foaming agent. An ultraviolet curable foaming ink can be mentioned.

  In addition, you may add an antifoamer, a polymerization inhibitor, a pigment dispersant etc. to the said hydrophobic resin as an additive. Moreover, since the coloring pigment normally used may be contained in the range less than 15 mass% and exceeds 15 mass%, since there exists a possibility of inhibiting foaming, it may be unpreferable.

  In the microorganism culture device 1 according to the present embodiment, any of the above-mentioned hydrophobic resins can be suitably used as the material of the frame 20, but a UV curable resin or a hot melt resin is preferable, and a hot melt resin is more preferable. preferable. The formation of the frame 20 requires a pattern formation having a certain height, but the UV curable resin or hot melt resin can be easily processed by coating with a dispenser or the like in a non-solvent system. This is because the pattern can be easily formed only by curing by light irradiation or cooling. However, if the culture layer 30 contains a component that changes in quality due to UV irradiation, it is necessary to cover the culture layer 30 with a metal or the like during UV irradiation, and the work becomes complicated. It is good to use resin.

  The frame 20 may be formed by bonding a hollow material of a hydrophobic material to the upper surface of the base sheet 10. Or you may form the frame 20 by shape | molding the base material sheet 10 in a frame shape using thermoforming, press molding, drawing, etc.

(Cover film)
The cover film 40 has an effect of preventing contamination by falling bacteria during culture and preventing moisture evaporation of the culture layer 30. The cover film 40 also has an action of expanding the mass of the test solution 35 </ b> A supplied on the culture layer 30 over the entire area of the culture layer 30. For example, as shown in FIG. 4, the cover film 40 includes a base material 46, a printing layer 47 provided on the base material sheet 10 side of the base material 46, and a repellent provided on the base material sheet 10 side of the printing layer 47. And an aqueous layer 48.

  The cover film 40 preferably has transparency and water vapor impermeability as well as transparency. Thereby, after culture | cultivation of microorganisms, a colony can be observed through the cover film 40, or can be counted. For example, a plastic film such as a polyolefin such as polyethylene or polypropylene, a polyester, a polyamide, a polystyrene, a polycarbonate, or a polyvinyl chloride can be used as a material constituting the substrate 46 of the cover film 40.

  Further, the substrate 46 of the cover film 40 may be subjected to a surface treatment such as a corona discharge treatment described in the section of the substrate sheet 10. The cover film 40 preferably has suitable gas permeability, mainly oxygen permeability, or oxygen non-permeability depending on the type of microorganism to be cultured, and may be selected in consideration of this point.

  When the cover film 40 is lifted from the base material sheet 10 in order to supply the test solution 35A onto the culture layer 30, it is preferable that the cover film 40 has an appropriate flexibility. Therefore, the thickness of the cover film 40 is preferably about 10 to 200 μm, more preferably about 20 to 70 μm.

  When the grid line is not printed on the base material sheet 10, the grid line 40 a may be printed on the cover film 40 using an ink that is insoluble in water and does not affect the growth of microorganisms. In addition, the square line 40a is used to obtain an approximate total number by simply counting the number of bacteria in several squares and multiplying by a number proportional to the total number of squares when a large number of colonies are formed. Is formed. The grid line 40 a may be printed on the base material 46. Alternatively, as shown in FIG. 4, a printing layer 47 having a grid line 40a formed using an ink that is insoluble in water and does not affect the growth of microorganisms is prepared. The grid line 40a may be obtained by stacking layers. A suitable size of the square is about 10 mm square.

  Preferably, the cover film 40 is configured such that the lower surface of the cover film 40, that is, the surface facing the culture layer 30 has water repellency. Thereby, the action of the cover film 40 for expanding the mass of the test solution 35A supplied onto the culture layer 30 over the entire area of the culture layer 30 can be enhanced. As a method for imparting water repellency to the lower surface of the cover film 40, for example, as shown in FIG. 4, it is conceivable to form the lower surface of the cover film 40 using a water-repellent layer 48 having water repellency.

  The water repellent layer 48 is preferably configured such that the contact angle with respect to water droplets is 50 to 105 degrees in accordance with JIS R3257. As a result, the test solution 35A on the culture layer 30 can be held inside the frame 20, and the test solution 35A can be diffused. Even if a slight gap is formed between the cover film 40 and the frame body 20, the test liquid 35 </ b> A passes through the gap and the test liquid 35 </ b> A is outside the frame body 20. It is possible to suppress the leakage. Examples of the material constituting the water repellent layer 48 include known resins such as polyamide resins, poly (meth) acrylic resins, polyolefin resins, and polyester resins. These resins may be used alone or in combination of two or more.

  A specific component layer may be formed in a region of the cover film 40 that faces the culture layer 30. As the specific component layer, for example, a specific component solution in which one or more selected from the group consisting of a gelling agent, a nutritional component, a coloring indicator, a selective agent, and a substrate is mixed in a solution in which a fixing agent is dissolved or dispersed. A layer obtained by coating is used. In addition, when the specific component layer provided in the cover film 40 contains the color development indicator, the effort which adds a color development indicator to the culture layer 30 or the test liquid 35A can be saved.

(Joint part)
The joining portion 25 is for joining the cover film 40 to the base sheet 10. The cover film 40 is displaced with respect to the base sheet 10 with the joint portion 25 as an axis, whereby the frame 20 and the culture layer 30 are covered with the cover film 40, and the frame 20 and the culture layer 30. As long as it is possible to realize an open state in which the cover film 40 is not covered with the cover film 40, the specific configuration of the joint portion 25 is not particularly limited. For example, as illustrated in FIGS. 2 and 4, the joint portion 25 may be configured using a member separate from the base sheet 10 and the cover film 40. In this case, for example, a general double-sided tape or a hot melt agent can be used as the bonding portion 25. Or although not illustrated, you may comprise the junction part 25 using the material same as the base material sheet 10 and the cover film 40. FIG. For example, the base sheet 10 and the cover film 40 may be defined on a series of sheets by preparing a single plastic sheet or a laminated sheet and bending the sheet. In this case, the folded region located between the base sheet 10 and the cover film 40 becomes a joint portion 25 that joins the cover film 40 to the base sheet 10.

  Next, the operation and effect of the present embodiment having such a configuration will be described. Here, first, a method for producing the microorganism culture device 1 will be described. Next, a method for inspecting microorganisms using the obtained microorganism culture tool 1 will be described.

The manufacturing method Introduction microbial culture device, a method for producing the microorganism culture device 1 will be described with reference to FIG. 5 (a) ~ (f) .

  First, as shown in FIG. 5A, a base sheet 10 such as synthetic paper is prepared. Next, a circular frame 20 made of hot melt resin or the like is formed on the upper surface of the base sheet 10 using a dispenser equipped with a single-hole nozzle (see FIG. 5B).

  Also, a medium solution 30A for forming the culture layer 30 is prepared by dissolving or dispersing the above-mentioned fixing agent, gelling agent, nutrient component, coloring indicator, selective agent, and the like in an alcohol solvent. Next, as illustrated in FIG. 5C, the medium solution 30 </ b> A is applied to the upper surface of the base sheet 10 and in a region surrounded by the frame body 20 using a dispenser N having a porous nozzle. Thereafter, the applied medium solution 30A is heated to remove the solvent and dried. Thereby, the culture layer 30 is formed on the upper surface of the base sheet 10 as shown in FIG.

  Next, as shown in FIG. 5E, the joint portion 25 made of a double-sided tape, a hot melt agent, or the like is disposed in the vicinity of the first outer edge 11 of the base sheet 10. Then, the cover film 40 whose dimension in the above-mentioned 1st direction is smaller than the base material sheet 10 is prepared, and the 1st outer edge 41 vicinity of the said cover film 40 is joined to the junction part 25 as shown in FIG.5 (f). To do. Thereby, the microorganism culture tool 1 having the exposed region 10A formed along the entire region of the second outer edge 12 is obtained.

  By the way, the culture layer 30 needs to have a certain thickness. For example, the thickness of the culture layer 30 is preferably 50 to 1000 μm, more preferably 200 to 600 μm after drying. As a conventional method for forming the culture layer 30 having such a thickness on the base sheet 10, there is screen printing. However, in screen printing using a mesh plate, the solvent volatilizes during the formation of the culture layer 30 and the viscosity of the medium solution 30A increases, so the mesh portion is likely to be clogged, and the plate of the medium solution 30A. Since the separation is also bad, it is difficult to form the culture layer 30 continuously. Even when screen printing using a metal mask plate is employed, the plate separation of the medium solution 30A is poor as in the case of the mesh plate, and the continuous formation of the culture layer 30 is also difficult. In addition, uneven thickness tends to occur in the moving direction of the squeegee, and it is difficult to form the culture layer 30 having a uniform thickness. On the other hand, if the amount of the solvent is increased in consideration of the increase in the viscosity of the medium solution 30A due to the volatilization of the solvent, the viscosity of the medium solution 30A becomes lower, so that it becomes difficult to form the culture layer 30 only in a predetermined region. On the other hand, according to the present embodiment, since the medium solution 30A with an increased amount of solvent is supplied into the region surrounded by the frame 20, even when the viscosity of the medium solution 30A is low, The culture layer 30 having an appropriate thickness and a uniform thickness can be formed in a predetermined region.

  The culture layer 30 having a sufficient thickness can sufficiently contain an amount of nutrient components necessary for the growth of microorganisms and an amount of a gelling agent that does not cause colony bleeding. For this reason, while growing microorganisms appropriately, a colony with few bleeding can be formed. Further, in the culture layer 30 having a uniform thickness, the content of each component such as a fixing agent, a gelling agent, and a nutrient component is not easily biased, so that the growth environment of microorganisms can be made almost constant. . Thereby, it is possible to suppress the occurrence of a difference in colony formation speed for each region of the culture layer 30. By these things, microbe inspections, such as measurement of the number of colonies, can be performed more correctly. In the culture layer 30 formed using the dispersion medium solution 30A in which at least one of the components (excluding the solvent) of the medium solution 30A is dispersed without dissolving in the solvent, the thickness of the culture layer 30 is Has a great influence on the distribution of each constituent component in the culture layer 30. Therefore, the formation of the culture layer 30 by the above-described method is particularly effective when the dispersion medium solution 30A is used.

Microbial testing method Next, a method for inspecting microbes using a microorganism culture device 1 will be described with reference to FIG. 6 (a) ~ (e) and FIG.

  First, as shown in FIG. 6A, the microorganism culture tool 1 is placed on the work table 5. Here, as shown in FIG. 7, the work table 5 includes a work area 5 a for performing the work of supplying the test liquid onto the culture layer 30 of the microorganism culture device 1, and the test liquid after the test liquid is supplied. A storage area 5b for temporarily storing the microorganism culture device 1. In the step shown in FIG. 6A, the microorganism culture tool 1 is disposed in the work area 5 a of the work table 5.

(Supply process)
Next, as shown in FIG. 6 (b), a supplying step of supplying the test solution 35 </ b> A onto the culture layer 30 of the microorganism culture device 1 is performed. The test liquid 35A is a liquid that is a target for confirming the presence of microorganisms or measuring the number of microorganisms. The test solution 35A is supplied as a lump to a part of the culture layer 30, for example.

In the supplying step, the cover film 40 is lifted from the base sheet 10 as shown in FIG. Here, in the present embodiment, as described above, the cover film 40 is configured such that the dimension in the first direction is smaller than that of the base sheet 10. Preferably, the minimum distance s ₂ between the exposed region 10A in the first direction (that is, the second outer edge 42 of the cover film 40) and the frame 20 and the height h ₁ of the frame 20 are determined by the cover film 40. Is set appropriately in consideration of the ease of the work of lifting the sheet from the substrate sheet 10. For example, the second outer edge 42 of the cover film 40 is displaced below the upper end of the frame body 20 due to the flexibility of the cover film 40, but when the degree of this displacement is small, for example, the second outer edge 42 is the base material. When it is not in contact with the upper surface of the sheet 10, it is considered that the operation of lifting the cover film 40 from the base sheet 10 is facilitated. Accordingly, the distance s ₂ and the height h ₁ are preferably set so that the second outer edge 42 does not contact the upper surface of the base sheet 10 in the covered state. Incidentally, the distance s ₂ is too small, it fingers will touch the culture layer 30 to lift the cover film 40 for dropping the test fluid 35A, or hardly picked second outer edge 42 of the cover film 40 Therefore, it is conceivable that it is difficult to turn up the cover film 40. Considering these points, the distance s ₂ is in the range of ₃ to 17 mm, preferably 4 to 12 mm, and the height h ₁ is in the range of 300 to 1400 mm μm, preferably 500 to 900 μm. Preferably it is. By setting the distance s ₂ and the height h ₁ in this way, the operation of lifting the cover film 40 from the base sheet 10 while reducing the possibility that the finger touches the culture layer 30 when lifting the cover film 40. Ease can be ensured.

(Coating process)
Next, a covering step of covering the culture layer 30 and the frame 20 of the microorganism culture tool 1 with the cover film 40 of the microorganism culture tool 1 is performed. For example, as shown in FIG. 6C, the cover film 40 is displaced toward the base sheet 10 with the joint portion 25 as an axis. At this time, the lower surface of the cover film 40 comes into contact with the test liquid 35A. The test liquid 35 </ b> A spreads on the culture layer 30 by contacting the cover film 40. As a result, as shown in FIG. 6 (d), the test solution 35 A can be spread over the entire culture layer 30. Here, according to the present embodiment, the culture layer 30 is surrounded by the frame body 20 having a larger thickness (height) than the culture layer 30. For this reason, the test liquid 35A can be quickly spread throughout the culture layer 30 while suppressing the test liquid 35A from leaking onto the base sheet 10. In this way, the culture solution 30 can be inoculated with the test solution 35A. In the following description, the microorganism culture tool 1 inoculated with the test solution 35A may be indicated by reference numeral 1A.

(Conveying process)
Next, a carrying step of carrying the microorganism culture tool 1A inoculated with the test solution 35A from the work area 5a to the storage area 5b is performed. For example, as shown in FIG. 6 (e), a force is applied from above to the exposed region 10 </ b> A of the base sheet 10 using the finger 6. Thereafter, by dragging the microorganism culture tool 1A on the work table 5 using the finger 6, the microorganism culture tool 1A is transported to the storage area 5b as shown by arrows in FIG. 6 (e) and FIG. .

  By the way, it takes about several minutes for the culture layer 30 to gel. On the other hand, in factories and research laboratories that conduct microorganism testing, the presence or number of microorganisms is examined for a large number of test liquids 35A obtained from various samples. Further, it is conceivable that the inspection is performed with the n number being increased or the inspection is performed under a plurality of culture conditions. For example, as shown in FIG. 7, in the work table 5, the test liquid 35A is sequentially supplied to the plurality of microorganism culture tools 1, and the microorganism culture tool 1A supplied with the test liquid 35A is sequentially supplied to the storage area 5b. Be transported. In this case, if the microorganism culture tool 1A is transported to the storage area 5b after waiting for the test liquid 35A supplied on the culture layer 30 to gel, the efficiency of the inspection work will deteriorate. Therefore, the microorganism culture tool 1A supplied with the test liquid 35A is transported to the storage area 5b without waiting for the test liquid 35A supplied on the culture layer 30 to gel, and then in the work area 5a. It is preferable to supply the test liquid 35A to the new microorganism culture device 1.

  Here, suppose that the microorganism culture tool in which the base material sheet is covered with the cover film over the whole area is used. In this case, when the microorganism culture instrument supplied with the test liquid 35A is transported, the finger 6 or the like comes into contact with the cover film. However, the lower surface of the cover film is in contact with the test solution 35A or the culture layer 30. For this reason, when the finger 6 or the like comes into contact with the cover film, it is considered that some stress is applied to the cover film 40 or the like due to the impact at that time. For example, it is conceivable that the cover film 40 in a portion in contact with the test solution 35A or the culture layer 30 is lifted. When the cover film 40 is lifted, the test solution 35A leaks outside from the frame body 20, and air is interposed between the culture layer 30 and the cover film 40, so that it is difficult to count colonies. It is done.

  Here, according to the present embodiment, as described above, the microorganism culture tool 1A is transported using the exposed region 10A of the microorganism culture tool 1. For this reason, the finger 6 or the like does not contact the cover film 40 during conveyance. Accordingly, the microorganism culture tool 1A can be moved while preventing stress from being applied to the cover film 40. For this reason, the microorganism culture tool 1A can be conveyed to the storage area 5b without waiting for the culture layer 30 to gel. As a result, the time required for the inspection can be shortened.

  Preferably, the cover film 40 is configured not only to partially expose the upper surface of the base sheet 10, but also so that each outer edge of the cover film 40 is positioned inside each outer edge of the base sheet 10. . That is, the cover film 40 is configured not to protrude from the base sheet 10. Accordingly, the cover film 40 can be prevented from coming into contact with the work table 5, thereby further reliably preventing stress from being applied to the cover film 40 when the microorganism culture tool 1 </ b> A is moved. be able to. Further, the cover film 40 can be prevented from being contaminated due to contact with the work table 5.

  The microorganism culture tool 1A conveyed to the storage area 5b is placed in an incubator after the culture layer 30 is gelled. After culturing the microorganisms for a predetermined time in the incubator, the microorganism culture tool 1A is taken out of the incubator and the state of the generated colonies is observed. In this manner, microorganisms contained in the test liquid 35A can be tested. The time for putting the microorganism culture device 1A into the incubator is appropriately set according to the type of the test solution 35A and the culture layer 30, but is set to, for example, 35 hours and 48 hours.

  Note that various modifications can be made to the above-described embodiment. Hereinafter, an example of modification will be described with reference to the drawings. In the following description and the drawings used in the following description, the same reference numerals as those used for the corresponding parts in the above embodiment are used for the parts that can be configured in the same manner as in the above embodiment. A duplicate description is omitted.

Modified Example of Inspection Method For example, FIG. 6E shows an example in which the microorganism culture tool 1A is conveyed by applying a force from above to the exposed region 10A of the base sheet 10 using the finger 6. . However, the method for conveying the microorganism culture tool 1A using the exposed region 10A is not particularly limited.

  For example, in the present embodiment, as described above, the exposed region 10 </ b> A is formed in the vicinity of the second outer edge 12 of the base sheet 10, and on the other hand, in the vicinity of the first outer edge 11 facing the second outer edge 12. The joint 25 is arranged. Here, the case where the junction part 25 is comprised from the member which fixes a part of cover film 40 with respect to the junction part 25 like the above-mentioned double-sided tape is considered. In this case, a portion of the cover film 40 that is in contact with the joint portion 25 is a portion that is not displaced even when stress is applied, unlike other portions of the cover film 40. Therefore, even if the finger 6 or the like touches the part of the cover film 40 when the microorganism culture tool 1A is conveyed, the impact or stress at that time is not transmitted to the other part of the cover film 40. That is, even when the part of the cover film 40 is touched when the microorganism culture tool 1A is conveyed, stress is applied to the part of the cover film 40 that is in contact with the test solution 35A or the culture layer 30. There is nothing. In this case, as shown in FIGS. 8 (a) and 8 (b), not only the exposed region 10A, but also a part of the base sheet 10 and the cover film 40 that are in contact with the joint portion 25 is used. 1A can be conveyed. For example, as shown in FIGS. 8 (a) and 8 (b), the microorganism culture instrument 1A is supported at two portions of the exposed region 10A and a part of the base sheet 10 and the cover film 40 that are in contact with the joint portion 25. Thus, the microorganism culture tool 1A can be lifted. Therefore, not only when the storage area 5b of the work table 5 is on the same surface as the work area 5a, but also when the storage area 5b is on a different surface from the work area 5a, While preventing stress from being applied to the test solution 35A or the portion in contact with the culture layer 30, the microorganism culture tool 1A can be transported from the work area 5a to the storage area 5b. In addition, it is possible to stack another microorganism culture tool 1A on the microorganism culture tool 1A already arranged in the storage area 5b. As a result, it is possible to increase the number of microorganism culture tools 1A that can be stored in the storage area 5b. That is, the storage area 5b can be used more effectively.

In the modified example of the exposed region and in the present embodiment, the example in which the exposed region 10A is formed along the entire area of the second outer edge 12 is shown. However, the present invention is not limited to this. For example, as shown in FIGS. 9 and 10, the exposed region 10 </ b> A may be formed only on a part of the second outer edge 12. In this case, as shown in FIG. 9, an exposed region 10 </ b> A is formed by drawing a part of the second outer edge 42 of the cover film 40 inward with respect to the linear second outer edge 12 of the base sheet 10. Also good. Alternatively, as shown in FIG. 10, an exposed region 10 </ b> A is formed by a part of the second outer edge 12 of the base sheet 10 projecting outward with respect to the linear second outer edge 42 of the cover film 40. Also good.

  Further, in the present embodiment and the modified examples of FIGS. 9 and 10, the example in which the exposed region 10 </ b> A is formed in the vicinity of the second outer edge 12 is shown. However, the present invention is not limited to this, and as shown in FIGS. 11 and 12, the exposed region 10 </ b> A is in the vicinity of the outer edge other than the outer edge facing the outer edge where the joint portion 25 is disposed, for example, the third outer edge 13. It may be formed in the vicinity. In this case, the exposed region 10A may be formed only in the vicinity of the third outer edge 13 as shown in FIG. 11, or the exposed region 10A is in the vicinity of the third outer edge 13 and the second as shown in FIG. It may be formed both near the outer edge 12. Although not shown, an exposed region 10A may be further formed in the vicinity of the fourth outer edge 14.

  Further, as shown in FIGS. 13 and 14, the cover is formed such that a region including a corner portion between the second outer edge 12 and the third outer edge 13 and / or the fourth outer edge 14 of the base sheet 10 becomes an exposed region 10A. The film 40 may be configured. In this case, among the outer edges of the cover film 40, the second outer edge facing the first outer edge 41 includes an outer portion 42 a extending along the second outer edge 12 of the base sheet 10 and an inner portion extending inside the outer portion 42 a. 42b. The inner portion 42b may extend substantially parallel to the first outer edge 41 as shown in FIG. 13, or may extend so as to be inclined with respect to the first outer edge 41 as shown in FIG. Although the specific method for producing the cover film 40 shown in FIGS. 13 and 14 is not particularly limited, for example, first, the cover film 40 having the same outer shape as the base sheet 10 is prepared, and then, The cover film 40 can be produced by cutting off a corner corresponding to a corner between the second outer edge 12 and the third outer edge 13 and / or the fourth outer edge 14 of the base sheet 10. In addition, the protrusion part 45 which protrudes outside from the inner side part 42b of the 2nd outer edge of the cover film 40 shown in FIG. 13 may exist.

  Further, the exposed region 10A may be formed in the vicinity of the outer edge where the joint portion 25 is disposed. Such a modification will be described with reference to FIGS. FIG. 15A is a plan view showing the microorganism culture tool 1 in the present modification, and FIG. 15B shows a case where the microorganism culture tool 1 of FIG. 15A is viewed from the direction of the arrow A. It is a side view. In this modification, as shown in FIGS. 15A and 15B, the joint portion 25 is disposed in the vicinity of the first outer edge 11 of the base sheet 10 and not in contact with the first outer edge 11. Has been. Further, the cover film 40 is arranged such that the first outer edge 11 is drawn inward with respect to the first outer edge 11 of the base sheet 10. For this reason, the exposed region 10 </ b> A can be formed in the vicinity of the first outer edge 11 of the base sheet 10. In the present modification, the second outer edge 42 of the cover film 40 at a position facing the joint portion 25 does not need to be drawn inward from the second outer edge 12 of the base sheet 10. For example, the second outer edge 42 of the cover film 40 may be located outside the second outer edge 12 of the base sheet 10. For this reason, the operation | work which lifts the cover film 40 from the base material sheet 10 can be performed easily.

Modification Example of Cover Film In the present embodiment and each modification example described above, an example is shown in which the joining portion 25 that joins a part of the cover film 40 to a part of the base sheet 10 is provided. However, it is not restricted to this, You may comprise the microorganism culture tool 1 without providing the junction part 25. FIG. That is, the cover film 40 may not be bonded to the base sheet 10. For example, as shown in FIG. 16, the cover film 40 may be an independent member prepared in the covering process. Also in this modification, the test solution 35A can be spread over the entire culture layer 30 by the cover film 40 coming into contact with the test solution 35A. Moreover, by making the dimension of the cover film 40 smaller than the dimension of the base sheet 10 in at least one direction, the exposed region 10A can be formed on the base sheet 10 as shown in FIG.

  Further, in the above-described embodiment, the example in which the surface of the cover film 40 on the base sheet 10 side is configured by the water-repellent water-repellent layer 48 is shown. However, when the base material 46 itself contained in the cover film 40 has water repellency, the base film 10 side surface of the cover film 40 may be configured by the base material 46 as shown in FIG. In this case, as shown in FIG. 17, the printing layer 47 may be provided on the surface of the base material 46 that is located on the side opposite to the base material sheet 10. For example, the base material having water repellency includes polypropylene resin, polyamide resin, and the like.

  Further, the color of the region including at least the second outer edge 42 in the cover film 40 is different from the color of the region adjacent to the region including the second outer edge 42 when viewed in plan view in the base sheet 10. It may be. For example, as shown in FIG. 18, a color indicated by a reference numeral 40 </ b> C extending along the second outer edge 42 so as to include the second outer edge 42 in the cover film 40 is colored with a color different from that of the base sheet 10. The region 40C may be used. Accordingly, when the cover film 40 is turned up, the boundary between the base sheet 10 and the cover film 40 is easily recognized, and thus the cover film 40 can be more easily grasped.

  In addition, although some modified examples with respect to the above-described embodiment have been described, naturally, a plurality of modified examples can be applied in combination as appropriate.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to this Example.

Example 1
As the base sheet 10, a synthetic paper (manufactured by YUPO Corporation, YUPO) using a synthetic resin as a main raw material was prepared. The thickness of the base sheet 10 is 270 μm, the length of the base sheet 10 (the dimension from the first outer edge 11 to the second outer edge 12) is 90 mm, and the width of the base sheet 10 (from the third outer edge 13 to the second outer edge 13). 4 to the outer edge 14) was 72 mm.

  A circular frame 20 made of an ethylene vinyl acetate-based hot melt resin was provided on the upper surface of the base sheet 10. The width of the frame body 20 (interval between the outer edge and the inner edge of the frame body 20) is 1 mm, the inner diameter of the frame body 20 (diameter of a circle formed by the inner edge) is 50 mm, and the height of the frame body 20 Was 750 μm.

A medium solution 30 </ b> A for forming the culture layer 30 was applied to the upper surface of the substrate sheet 10 and in the region surrounded by the frame body 20. The medium solution 30A was composed of a solid content including a gelling agent, a nutrient component, a fixing agent and a plasticizer, and a solvent. The breakdown of each component was as follows.
Gelling agent: Guar gum: 7: 3 parts by mass of psyllium seed gum Nutritional component: Protein, sugar, inorganic salt Fixing agent: Polyvinylpyrrolidone (PVP)
Plasticizer: Glycerin Solvent: Methanol Also, the weight of the main component of the solid content contained in the medium solution 30A was as follows.
Gelling agent + nutrient component + PVP: 281.2g
Glycerin: 36.0g
The weight percent of glycerin relative to the total solid content of the medium 30A was 11%. Moreover, the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 7: 3.

  The applied medium solution 30A was dried, whereby the culture layer 30 was formed on the base sheet 10. The thickness of the obtained culture layer 30 was 250 μm.

  On the upper surface of the base sheet 10, a joining portion 25 made of a double-sided tape having a length of 6 mm and a width of 72 mm was provided along the first outer edge 11 of the base sheet 10.

  Next, as the cover film 40, a cover film 40 (see FIG. 4) having a substrate 46, a printed layer 47, and a water repellent layer 48 was prepared. The base material 46 was made of transparent oriented polypropylene having a thickness of 40 μm. The spacing between the grid lines 40a in the printed layer 47 was 10 mm. The water repellent layer 48 was made of a resin mainly composed of polyamide. The length of the cover film 40 (the dimension from the first outer edge 41 to the second outer edge 42) was 85 mm, and the width (the dimension from the third outer edge 43 to the fourth outer edge 44) was 72 mm. Next, the portion in the vicinity of the first outer edge 41 of the cover film 40 was bonded to the base sheet 10 via the bonding portion 25. Thus, the microorganism culture tool 1 including the exposed region 10A formed in the vicinity of the first outer edge 11 of the base sheet 10 was produced.

(Example 2)
A microorganism culture device 1 was produced in the same manner as in Example 1 except that the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 6: 4.

(Example 3)
A microorganism culture device 1 was produced in the same manner as in Example 1 except that the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 5: 5.

Example 4
A microorganism culture device 1 was produced in the same manner as in Example 1 except that the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 8: 2.

(Comparative Example 1)
A microorganism culture device 1 was produced in the same manner as in Example 1, except that the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 4: 6.

(Example 5)
A microorganism culture device 1 was produced in the same manner as in Example 2 except that the weight percent of glycerin relative to the entire solid content of the medium 30A was 10%.

(Example 6)
A microorganism culture device 1 was produced in the same manner as in Example 2 except that the weight percent of glycerin relative to the entire solid content of the medium 30A was 15%.

(Comparative Example 2)
A microorganism culture device 1 was produced in the same manner as in Example 2 except that the weight percentage of glycerin relative to the entire solid content of the medium 30A was 8%.

(Comparative Example 3)
A microorganism culture device 1 was produced in the same manner as in Example 2 except that the weight percentage of glycerin relative to the entire solid content of the medium 30A was 20%.

<< Culture performance evaluation >>
Using the microorganism culture device 1 obtained in Examples 1 to 4 and Comparative Example 1, the performance of cultivating the bacteria in the test solution in each microorganism culture device 1 was evaluated. First, as a test solution 35A, a bacterial solution having a concentration of 100 to 150 cfu / ml of Proteus mirabilis, which is an aerobic bacterium, was prepared. Next, 1 ml of the test solution 35A was supplied onto the culture layer 30 of each microorganism culture device 1 obtained in Examples 1 to 4 and Comparative Example 1. Thereafter, the cover film 40 was placed on the frame 20 and the culture layer 30, thereby spreading the test solution 35 </ b> A over the entire culture layer 30. After the culture layer 30 was gelled, the microorganism culture tool 1A inoculated with the test solution 35A was placed in an incubator, and the bacteria were cultured at 35 degrees for 48 hours. Thereafter, the number of cultured bacteria was measured, and thereby the culture performance of each microorganism culture device 1 was evaluated.

  The evaluation of the culture performance was made based on the number of bacteria observed when the bacteria in the same test solution were cultured using the agar plate mixing method, which was carried out for comparison. Specifically, the level 1 is the case where 80% or more of the number of bacteria in the agar plate pour method is observed, the level 2 is the case where the number of bacteria is 70% or more, 70% Level 3 was observed when less than the number of bacteria was observed. The evaluation results are shown in Table 1.

<< Visibility evaluation >>
Using the microorganism culture device 1 obtained in Examples 1 to 6 and Comparative Examples 1 to 3, the bacteria in the test solution 35A were cultured in the same manner as in the above-described culture performance evaluation. Next, colonies that developed color in response to the coloring indicator were visually observed to confirm whether each colony was visible, that is, whether it could be counted. TTC was used as the color development indicator. This TTC was provided on the lower surface of the cover film 40. Based on the confirmation results, the visibility of each microorganism culture device 1 was evaluated at levels 1 to 3. The evaluation results are shown in Tables 1 and 2.

  In addition, the above-mentioned levels 1 to 3 are such that the visibility is hindered by the overlap of the gas 51 generated by the gas-producing bacteria and the colony 50 or the bleeding 52 generated in the culture layer 30. Was determined based on the degree of inhibition. In FIG. 19, the parameter | index about the relationship between the grade of the gas 51 and the levels 1-3 is shown. Among the three images shown in FIG. 19, the image on the left is an example evaluated as level 1 in which there is almost no inhibition of the visibility due to the gas 51, and the center image is the gas 51. This is an example evaluated as level 2 in which the visibility hindrance caused by the gas is slightly generated, and the right image is evaluated as level 3 in which the hindrance in the visibility due to the gas 51 is significantly generated. It is. FIG. 20 shows an index about the relationship between the degree of bleeding 52 and levels 1 to 3. Of the three images shown in FIG. 20, the image on the left is an example evaluated as level 1 in which the visibility impediment due to the blur 52 hardly occurs, and the center image is the blur 52. This is an example evaluated as level 2 in which the visibility hindrance is slightly caused by the image, and the image on the right side is evaluated as level 3 in which the visibility hindrance is significantly caused by the blur 52. It is.

<< Warpage evaluation >>
The degree of warpage (curl) of the substrate sheet 10 was evaluated for the microorganism culture devices 1 obtained in Examples 2, 5, and 6 and Comparative Examples 2 and 3. Specifically, the microorganism culture tool 1 is placed on the work table 5, and at this time, among the outer edges 11, 12, 13, and 14 of the base material sheet 10 of the microorganism culture tool 1, the microorganism culture tool 1 floats most from the work table 5. For the outer edge, the distance from the upper surface of the work table 5 was measured. The results are shown in Table 2. The causes of curling include a difference in thermal contraction rate between the culture layer 30 and the base sheet 10, and contraction of the culture layer 30 due to evaporation of the solvent in the medium solution 30A and the test solution 35A. Conceivable.

  In the above-described embodiment, as one method for transporting the microorganism culture tool 1A to the storage area 5b, the microorganism culture tool on the work table 5 while applying a force to the exposed area 10A of the base sheet 10 from above. It is considered to drag 1A. Accordingly, if the curl at the outer edge of the base sheet 10, particularly the curl near the exposed area 10 </ b> A, is large, a force is applied to the exposed area 10 </ b> A from above, thereby cultivating the microorganism culture device located distal to the exposed area 10 </ b> A It is conceivable that the outer edge of 1A is lifted from the work table 5. Therefore, it is preferable that the amount of curl does not exceed a certain limit, for example 3 mm.

  As shown in Table 1, when the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent is within the range of 7: 3 to 5: 5, both culture performance and visibility Could be level 2 or higher. On the other hand, when the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 8: 2, the visibility decreased to level 3. As the ratio of guar gum increases, the culture layer 30 becomes brittle. As a result, it is considered that the culture layer 30 is partially broken by the gas 51 generated from the bacteria. In addition, when the ratio of the weight part of guar gum to the weight part of psyllium seed gum in the gelling agent was 4: 6, the culture performance was lowered to level 3. It is thought that the increase in the ratio of the psyllium seed gum improved the elastic modulus of the culture layer 30 and made it difficult for the bacteria to grow.

  As shown in Table 2, when the solid content of the medium solution 30A, that is, the ratio of the solid content of glycerin to the total solid content of the culture layer 30 is 10 to 15% by weight, the curl is set to 3 mm or less, Both visibility could be level 2 or higher. On the other hand, when the ratio of the solid content of glycerin to the total solid content of the culture layer 30 was 8% by weight, the curl was 3.4 mm. It is conceivable that the lower the weight percent of glycerin, the lower the flexibility of the culture layer 30 and, as a result, the culture layer 30 easily contracts. In addition, when the ratio of the solid content of glycerin to the total solid content of the culture layer 30 was 20% by weight, the acceptability decreased to level 3. It is conceivable that bleeding 52 is likely to occur in the culture layer 30 by increasing the ratio of glycerin.

DESCRIPTION OF SYMBOLS 1,1A Microorganism culture tool 10 Base sheet 10A Exposed area 20 Frame body 25 Joint part 30 Culture layer 30A Medium solution 35A Test liquid 40 Cover film 40C Colored area 41-44 1st outer edge-4th outer edge 45 Protrusion part 46 group Material 47 Printing layer 48 Water repellent layer 50 Colony 51 Gas 52 Bleeding

Claims (7)

A base sheet;
A culture layer provided on an upper surface of the base sheet and configured to culture microorganisms;
A frame provided on the upper surface of the base sheet so as to surround the culture layer;
A cover film capable of covering the culture layer and the frame;
A joining portion for joining a part of the cover film to a part of the base sheet ,
The culture layer is a dry culture layer containing at least a gelling agent,
The cover film is configured such that when the cover film covers the culture layer and the frame body, an area of the upper surface of the base sheet is at least partially exposed, thereby forming an exposed area ,
The joint portion includes a cover state in which the cover film is displaced with respect to the base sheet with the joint portion as an axis, and the frame body and the culture layer are covered with a cover film, and the frame body. And a microorganism culture device configured to realize an open state in which the culture layer is not covered with a cover film . The base sheet has a contour including a first outer edge and a second outer edge facing the first outer edge when viewed from the normal direction of the base sheet,
The joint is disposed in the vicinity of the first outer edge of the base sheet,
The microorganism culture device according to claim 1 , wherein the exposed region of the base sheet is formed so as to be at least partially in contact with the second outer edge of the base sheet. The maximum distance in the first direction of the exposed region is in the range of 2 to 20 mm, where the direction from the first outer edge of the base sheet toward the second outer edge is the first direction. 2. The microorganism culture device according to 2. When the direction from the first outer edge of the base sheet toward the second outer edge is the first direction, the distance in the first direction between the frame and the exposed region of the base sheet is 3 In the range of ~ 17 mm,
The microorganism culture device according to claim 2 or 3 , wherein a height of the frame body is in a range of 300 to 1400 µm. The microorganism culture device according to any one of claims 1 to 4 , wherein the cover film is configured such that a lower surface of the cover film has water repellency. A microorganism testing method for testing microorganisms using a microorganism culture tool on a workbench,
The microorganism culture device comprises the microorganism culture device according to any one of claims 1 to 5 ,
The workbench includes a work area and a storage area,
The microorganism testing method includes:
Placing the microorganism culture tool in the work area;
A supplying step of supplying a test liquid onto the culture layer of the microorganism culture tool;
After the supplying step, a covering step of covering the culture layer and the frame body of the microorganism culture device with the cover film of the microorganism culture device;
A microorganism testing method comprising: a transporting step of transporting the microorganism culture tool from the work area to the storage area by applying a force to the exposed area of the microorganism culture tool after the covering step. The microorganism testing method according to claim 6 , wherein the work area and the storage area of the work table are on the same plane.