JP2001245693A - Sheet-like medium for general viable cell detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sheet-like medium having resolved such problems involved in conventional media for detecting microorganisms that both labor and time are required for culture operation, a plenty of plastic wastes are produced, and a various test uses cannot be dealt with. SOLUTION: This sheet-like medium has such a structure that a medium laminate composed of a porous matrix layer and two- or three-layer water-soluble macromolecular compound layer is bonded to an adhesive sheet, which is then laminated with transparent film; wherein peptone, meat extract, (yeast extract), glucose, pH adjustor and color developer are included in the water-soluble macromolecular compound layer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a medium for culturing microorganisms. More specifically, the present invention relates to a culture medium for measuring the number of general viable bacteria for judging the degree of contamination by microorganisms in food and the environment.

[0002]

2. Description of the Related Art A conventional method for examining general viable bacteria will be described taking measurement of the number of general viable bacteria in a food test as an example. First, a powdered agar medium is dissolved and sterilized, and then kept at about 45 ° C. An aliquot of the agar medium is dispensed in advance into a sterilized petri dish or the like containing 1 ml of a test sample such as a food suspension, and the mixture is solidified by agitation. Count the number of colonies. As described above, the conventional general viable cell inspection method is a very laborious and time-consuming method, for example, after preparing and sterilizing a medium in advance, it is necessary to keep the temperature at which the agar medium does not solidify. In order to more easily and quickly carry out a general viable cell test, it is preferable to omit such a laborious and time-consuming preparation of a medium. In addition, a large amount of plastic waste is usually generated after culturing because a large amount of plastic petri dishes are used. In the environmental microbiological test, usually, a predetermined area of the test object is wiped with a cotton swab or gauze, the cotton swab or gauze is washed in sterile water or sterile physiological saline, and the cells adhered to the cotton swab are sterilized in sterile water or sterile physiological saline. Suspended in Then, the suspension is applied to an agar medium prepared in advance, or is mixed with an agar medium and cultured in the same manner as described above, and the resulting colonies are counted.

[0003] On the other hand, a simple medium for detecting general viable bacteria which does not require the preparation of a medium is produced and marketed. If the simple culture medium is classified for convenience, a stamp type (Japanese Unexamined Patent Publication No. 4-117)
299), a filter type, a film type (Japanese Patent Publication No. 2-49705, JP-A-3-15379), and a test paper type. The stamp type is a medium dispensed in a state in which the agar medium is raised in a plastic container, and is used for directly contacting the surface of the agar medium with an object to be inspected. It is easy to use to easily check for microbial contamination in the environment, but due to the small area of the culture medium, it has poor quantitative properties, and it is difficult to test objects with curved or uneven surfaces. Some disadvantages are not available. In addition, since a plastic container is used, a large amount of plastic waste is produced after culturing as in the conventional method. The filter type is suitable for testing liquid samples, but it is difficult to test samples other than liquid. Test paper types have the disadvantage of poor quantitative properties. The film type has a quantitative property in the inspection of food and is easy to use, but when inspecting the environment or the like, the inspection cannot be performed by directly contacting the inspection target like the stamp type.

[0004] Several types of simple media have been produced so as to be easy to use for each purpose, but one type of simple media that can be used for many purposes such as food inspection and direct inspection of inspection objects. There is no present. Japanese Patent Application Laid-Open No. 6-181741 proposes a microorganism culturing apparatus in which a cold water-soluble gelling agent and a fibrous water-absorbing sheet are laminated, but in the case of a combination of a gelling agent and a fibrous sheet, the sample is uniformly dispersed on the medium surface. However, the microorganisms do not always form colonies on the surface of the fibrous sheet, and the microorganisms that grow on the lower surface or inside the fibrous sheet are difficult to confirm, so that there is a disadvantage that the quantitativeness is impaired.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a sheet or film-shaped incubator or medium comprising a porous matrix layer and a water-soluble polymer compound layer proposed by the present inventors in WO97 / 24432. It is an object of the present invention to solve the above-mentioned problems related to a conventional microorganism medium for detecting general viable bacteria in food and the environment.

[0006]

The sheet-form medium of the present invention is a medium in which microorganisms can grow by adding water or physiological saline, and has the following constitution. (1) A laminate comprising a porous matrix layer and two or three water-soluble polymer compound layers, and optionally including a mount in contact with the water-soluble polymer compound layer farthest from the porous matrix layer. The porous matrix layer is adhered to the center of the pressure-sensitive adhesive sheet larger than the laminate so that the porous matrix layer faces upward, and a transparent film larger than the laminate is in contact with the porous matrix layer, and the laminate and the center are bonded together. A portion of the transparent film that is covered so as to fit together and that protrudes from the laminate is a sheet-like medium adhered to a portion of the adhesive sheet to which the laminate is not adhered, and is a water-soluble medium that contacts the porous matrix layer. Nutritional component consisting of peptone, meat extract and optional yeast extract, potassium phosphate, sodium phosphate, sodium carbonate , A water-soluble polymer layer containing at least one salt selected from the group consisting of potassium carbonate and calcium carbonate, and a color former, wherein the water-soluble polymer layer next to the layer in contact with the porous matrix layer is peptone. , A nutrient component comprising meat extract, glucose and yeast extract as an optional component, and at least one salt selected from the group consisting of potassium phosphate, sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate. A sheet-shaped culture medium for detecting general viable bacteria.

(2) There are three water-soluble polymer compound layers, and the water-soluble polymer compound layer furthest from the porous matrix layer is a layer composed of only the water-soluble polymer compound, or peptone, meat extract, glucose And a nutrient component comprising a yeast extract as an optional component, and at least one salt selected from the group consisting of potassium phosphate, sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate. The sheet-like medium for detecting general viable bacteria according to the above (1).

(3) The water-soluble polymer compound layer has a peptone weight of 4 to 15 g / m ² , based on the total weight of all layers in the medium.
1-8 g / m ² of meat extract, of 0.5 to 3 g / m ² glucose, potassium phosphate 0.1 to 0.8 g / m ^2, sodium phosphate, sodium carbonate, consisting of potassium carbonate and calcium carbonate One or more salts selected from the group,
Characterized in that it contains a 0.02~0.15g / m ² of 2,3,5-triphenyltetrazolium chloride or tetrazolium violet and yeast extract 1-8 g / m ² as an optional component, the ( The sheet medium for detecting general viable bacteria according to the item 1) or 2).

(4) The water-soluble polymer compound contained in the water-soluble polymer compound layer has a saponification degree of 75 to 95% and a molecular weight of 25.
The sheet-shaped medium for detecting general viable bacteria according to any one of the above items (1) to (3), which is 2,000 to 250,000 polyvinyl alcohol.

(5) The above (1) to (4), wherein the water-soluble polymer compound layer in contact with the porous matrix layer contains a water-soluble polymer compound having a basis weight of 1 to 20 g / m ^2.
Item 4. The sheet-form medium for detecting general viable bacteria according to any one of Items 1 to 4.

(6) The porous matrix layer has a basis weight of 40 to
100 g / m ² , air permeability 70 to 240 L / (m ² · se
The sheet-shaped medium for detecting general viable bacteria according to any one of the above (1) to (5), which is the nylon meltblown nonwoven fabric of c).

(7) The porous matrix layer is characterized in that one or more components selected from the group consisting of peptone, potassium phosphate, sodium phosphate, sodium carbonate and potassium carbonate are applied to the surface thereof. The sheet-shaped medium for detecting general viable bacteria according to any one of the above (1) to (6).

(8) The total application amount of one or more components selected from the group consisting of peptone, potassium phosphate, sodium phosphate, sodium carbonate and potassium carbonate to the surface of the porous matrix layer is from 0.3 to 0.3. The sheet-form medium for detecting general viable bacteria according to the above (7), which is 3 g / m ² .

(9) The pressure-sensitive adhesive sheet is a polyester film, a white polyester film, a polyolefin-based synthetic paper or a polyolefin laminated paper coated with an acrylic pressure-sensitive adhesive or a rubber-based pressure-sensitive adhesive, and the transparent film is a polyolefin film or a release-treated polyolefin film. (1) to (8),
Item 4. The sheet-form medium for detecting general viable bacteria according to any one of Items 1 to 4.

(10) The thickness of the pressure-sensitive adhesive sheet is 0.07-
0.5 mm, and the thickness of the transparent film is 20 to 80 μm
m. The sheet medium for detecting general viable bacteria according to any one of the above items (1) to (9), wherein m is m.

(11) The above-mentioned (1) to (10), wherein a part of an adhesive portion between the adhesive sheet and the transparent film is adhered so as to have an adhesive strength stronger than the adhesive strength of the adhesive sheet. Item) The sheet-shaped medium for detecting general viable bacteria according to any one of the above items.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The sheet-shaped medium of the present invention takes a form in which a porous matrix layer is laminated on two or three water-soluble polymer compound layers, and a film of polyester or the like is used as a mount. When the number of the water-soluble polymer compound layers is three, the layer farthest from the porous matrix layer may be a layer composed of only the water-soluble polymer compound. This is because even if the nutrients are contained in the layer, they are rarely diffused to the porous matrix layer and used by microorganisms. The total thickness of the water-soluble polymer compound layer up to the porous matrix layer may be adjusted, and the nutrients and the like may be dispersed and added to the layer farthest from the porous matrix layer.

The method for producing the sheet-shaped medium of the present invention will be described for the case where the number of the water-soluble polymer compound layers is three. Using a film of polyester or the like as a mount, an aqueous solution of a polymer compound is applied thereon and dried to form a first water-soluble polymer compound layer. On this layer, a nutritional component consisting of peptone, meat extract, glucose and yeast extract as an optional component, and at least one selected from the group consisting of potassium phosphate, sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate And drying. When a nutrient component or the like is also contained in the first water-soluble polymer compound layer, the formation conditions of the second water-soluble polymer compound layer are preferably followed.

On the second water-soluble polymer compound layer,
Nutrient component consisting of peptone, meat extract and yeast extract as an optional component, potassium phosphate, sodium phosphate, one or more salts selected from the group consisting of sodium carbonate, potassium carbonate and calcium carbonate, and an aqueous solution containing a color former An aqueous solution of a water-soluble polymer compound is applied, and a porous matrix is overlaid thereon and dried. At this time, the water-soluble polymer compound layer in contact with the porous matrix layer has a water-soluble polymer compound content of 1 to 20 per unit area.
g / m ² . The water-soluble polymer compound layer in contact with the porous matrix layer can be laminated with the porous matrix layer in an undried or semi-dried state in order to adhere to the porous matrix layer. If the amount is too large, the drying conditions must be strict.

However, in order to achieve adhesion between the porous matrix layer and the water-soluble polymer compound layer in contact therewith, after drying the water-soluble polymer compound layer in contact with the porous matrix layer, appropriate water is removed by some means. It is also possible to adopt a method in which the held porous matrix layer is laminated and dried, and in this case, the content of the water-soluble polymer compound in the water-soluble polymer compound layer in contact with the porous matrix layer is not necessarily a basis weight. It is not necessary to be within the range of ２０20 g / m ² , but may be beyond this range. In addition,
When an aqueous solution containing one or more components selected from the group consisting of peptone, potassium phosphate, sodium phosphate, sodium carbonate, and potassium carbonate is used for retaining moisture in the porous matrix layer, these aqueous solutions may be renewed after drying. Although the step of applying the active substance can be omitted, the porous matrix layer holding only moisture is laminated and dried, and thereafter, it is composed of peptone, potassium phosphate, sodium phosphate, sodium carbonate and potassium carbonate An aqueous solution, an ethanol solution, a methanol solution, or a mixed solvent thereof of one or more components selected from the group may be applied and dried.

A water-soluble substance is applied to the surface of the porous matrix layer by any one of the above-mentioned methods and dried, and then the used mount is peeled off or cut into an appropriate size without being peeled off. And The obtained culture medium laminate is adhered to the center of the adhesive sheet larger than the culture medium laminate so that the porous matrix layer faces upward, and a transparent film larger than the culture medium laminate is brought into contact with the porous matrix layer and the culture medium Cover the laminate so that its center is aligned, and attach the part of this transparent film that protrudes from the culture laminate to the part of the adhesive sheet where the culture laminate is not adhered, and finally sterilize such as ethylene oxide gas sterilization. To give a sheet medium. Note that the center here does not need to be strict, and in some cases, it may be intentionally shifted according to the purpose as described later. Further, it is also effective to configure the transparent film so as to extend beyond the edge of the adhesive sheet in at least a part of the sheet-shaped medium, in order to facilitate opening and closing of the transparent film.

Further, it is preferable that the adhesive force at a part of the adhesive portion between the adhesive sheet and the transparent film is made stronger than the remaining part. By doing so, the transparent film can be opened while a part thereof is adhered, and when it is desired to peel it off, it can be completely peeled off. As a method of making a part with a strong adhesive force, the adhesive sheet and the transparent film are adhered by another adhesive tape having a stronger adhesive force than the adhesive sheet, the adhesive sheet and the transparent film are partially adhered with a double-sided tape, There is a method of applying a pressure-sensitive adhesive having a higher adhesive force than a pressure-sensitive adhesive sheet to a part of a sheet, a method of applying a pressure-sensitive adhesive having a higher pressure than a pressure-sensitive adhesive sheet to a part of a transparent sheet, or the like. When bonding with another adhesive tape, the adhesive sheet and the transparent sheet are staggered and overlapped, and the adhesive-coated side of the adhesive sheet and the surface of the transparent film that are not in contact with the laminate, or the back surface of the adhesive sheet and the transparent film medium Another adhesive sheet is adhered to the surface in contact with the laminate. Alternatively, there is a method in which the pressure-sensitive adhesive sheet and the transparent sheet are overlapped with little displacement, another pressure-sensitive adhesive tape is folded, and the back surface of the pressure-sensitive adhesive sheet and the surface of the transparent film which are not in contact with the medium laminate are bonded. When another adhesive tape is folded and adhered to the back side of the adhesive sheet and the surface of the transparent film that is not in contact with the culture medium laminate, when the transparent film is opened, the back surface of the adhesive sheet and the transparent film are completely adhered to each other. Can be opened and convenient. It is sufficient that the portion having a strong adhesive strength has a stronger adhesive force than the remaining portion, but it is preferable that the adhesive force is twice or more.

It is preferable that the layers between the water-soluble polymer compounds and between the water-soluble polymer layer and the backing when the backing used in the production is not peeled off are in close contact with each other. Preferably, the porous matrix layer is at least partially adhered. Further, it is preferable that there is no gap in the adhesive portion between the pressure-sensitive adhesive sheet and the transparent film.

As a material for forming the water-soluble polymer compound layer of the present invention, when a sample solution is added, it is dissolved in water and exhibits a high viscosity of 10 cps (viscosity measured at 40 ° C.), Those that do not inhibit the growth of microorganisms are preferably used. For example, polyvinyl alcohol, cellulose derivatives, polyacrylic acid derivatives, starch derivatives, proteins, protein derivatives, polysaccharides and the like can be used. Among them, polyvinyl alcohol is more preferable, and the saponification degree is 7
Polyvinyl alcohol having a molecular weight of 25,000 to 250,000 and a molecular weight of 5 to 95% is particularly preferred.

Next, the contents of the components constituting the medium of the present invention will be described. The numerical values shown here indicate the total basis weight of the medium unless otherwise specified.
It is necessary to add a coloring agent for confirming the growth of the microorganism to the medium. As the color former, 2,3,5-triphenyltetrazolium chloride or tetrazolium violet is preferable, but a reagent that develops a color by oxidation-reduction such as another tetrazolium salt, an enzyme commonly possessed by microorganisms such as phosphatase, esterase, A chromogenic enzyme substrate such as lipase or a chromogenic enzyme substrate system may be used. The added amount of the color former is preferably 0.02 to 0.15 g / m ² . If it is less than 0.02 g / m ² , the time required for confirming the growth of microorganisms becomes long, which is not practical. 0.15g /
There is no problem with chromogenic enzyme substrates such as phosphatase, esterase and lipase even if m ² or more, but the increased effect is low. If the tetrazolium salt is 0.15 g / m ² or more, the growth may be inhibited depending on the microorganism.

The nutritional component is 4-15 g / m ^{2 of} peptone,
Meat extract 1-8 g / m ² , yeast extract 1-8 g / m ²
Preferably, m ² and glucose are in the range of 0.5 to 3 g / m ² . Below this concentration, the growth of microorganisms is slow and not practical. Although it may be added above this concentration, even if added above this concentration, the added effect does not appear. Peptones include casein peptone, soy peptone, meat peptone, and egg peptone. The meat extract includes a beef brain extract, a beef heart extract, a powdered meat extract, and a fish meat extract in addition to a normal pasty meat extract. The yeast extract is preferably added, but need not be added. When a tetrazolim salt is used as a color former, if this is added to the same layer as glucose, this layer will be colored, so these must be added to a different layer. One or more salts selected from the group consisting of potassium phosphate, sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate added for pH adjustment are 0.1 to 0.8 g / total when calculated as anhydrous salts. m ² is preferred. Potassium phosphate is dipotassium phosphate or a mixture of dipotassium phosphate and monopotassium phosphate, sodium phosphate is disodium phosphate or a mixture of disodium phosphate and monosodium phosphate, sodium carbonate is sodium carbonate, Sodium bicarbonate or a mixture of sodium carbonate and sodium bicarbonate, and potassium carbonate refers to potassium carbonate, potassium bicarbonate or a mixture of potassium carbonate and potassium bicarbonate, and these salts can be used in combination.

Materials for forming the porous matrix layer used in the present invention include nylon fibers, polyacrylonitrile fibers, polyvinyl alcohol (PVA) fibers, ethylene-vinyl acetate copolymer fibers, and polyester fibers (hydrophilized). Polyester fibers are more preferable), polyolefin fibers (polyolefin fibers subjected to hydrophilic treatment are more preferable), synthetic fibers such as polyurethane fibers, semi-synthetic fibers such as rayon fibers, wool (animal hair), silk, cotton fibers, and cellulose fibers. And natural fibers such as pulp fibers, inorganic fibers such as glass fibers, and the like. Fabrics such as woven fabrics and nonwoven fabrics made using these fibers, and porous films made from the constituent materials of the above fibers And sponge can be used, and porous ceramics can also be used.

Of these, nylon fibers are preferred.
It is a cotton fiber, a cellulose fiber, and a rayon fiber, and it is preferable to use a fabric such as a knitted or woven fabric or a nonwoven fabric, which can easily adjust the basis weight and the air permeability. A nonwoven fabric produced by a production method or a microfiber nonwoven fabric produced from split fibers can be particularly preferably used. Accordingly, a nylon nonwoven fabric produced by a melt blow method is particularly preferred. And the porous matrix layer has a basis weight of 40 to 100 g / m.
^2. It is preferable that the air permeability is 70 to 240 L / (m ² · sec).

The porous matrix layer used in the present invention may be water-repellent, but by using a hydrophilic or hydrophilized porous matrix layer, the water absorption rate is higher than that of a water-repellent porous matrix layer. This increases the efficiency of the test operation. Further, it is preferable to apply a water-soluble substance to the surface of the porous matrix layer since the dispersion of microorganisms can be further improved. As the water-soluble substance, any substance may be used as long as it does not hinder the growth of microorganisms, but nutrients and salts added to the water-soluble polymer compound layer are preferable. A color former may be applied,
When a color former is applied, the storability may decrease. Specifically, it is one or more components selected from the group consisting of the aforementioned peptone, potassium phosphate, sodium phosphate, sodium carbonate and potassium carbonate, and the amount of addition is 0.3 to 3 g / m ^{2 in} basis weight. It is preferred that
These may be applied in powder form, but are preferably applied as a solution or suspension, and the solvent used as the solution or suspension is one that dissolves or partially dissolves a water-soluble substance and volatilizes or evaporates. However, water, ethanol, methanol, or a mixed solvent thereof is preferable.

The material of the pressure-sensitive adhesive sheet used in the present invention may be any material as long as it does not inhibit the growth of microorganisms, but a certain amount of rigidity is required to make the transparent film easy to peel off. In order to wipe off a curved surface or the like, it is preferable that the surface is flexible. 0.07-0.
A 5 mm polyester film, white polyester film, polyolefin-based synthetic paper or polyolefin laminated paper satisfies this condition. The adhesive used for the adhesive sheet may be any adhesive as long as it does not inhibit the growth of microorganisms, but considering workability, as long as the adhesive sheet and the transparent film can be re-adhered,
The lower the adhesive strength, the better. Rubber-based and acrylic-based pressure-sensitive adhesives are preferable, and re-peelable and slightly-adhesive-type acrylic pressure-sensitive adhesives are particularly preferable.

The transparent film may be of any type as long as it does not inhibit the growth of microorganisms. However, since the thick medium laminate is interposed between the adhesive sheet and the transparent film, flexibility is required. A polyolefin film, a polyester film, a nylon film, or the like can be used, and a film subjected to a release agent treatment to facilitate peeling may be used. The transparent film functions as a lid, and a polyolefin film is preferable in consideration of workability such as opening and closing, and a polypropylene film having a thickness of 20 to 80 μm is particularly preferable.

The sheet-form medium of the present invention is a medium in which microorganisms can grow by adding a sample solution onto a porous matrix layer. When the sample liquid is added to the porous matrix layer, the sample liquid is once held in the porous matrix layer, and the water-soluble polymer compound in contact with the porous matrix layer by water in the sample liquid held in the porous matrix layer The layer dissolves, the nutrients dissolve out of the high molecular compound layer, and an environment in which microorganisms can grow is established, and the growth and division of microorganisms is started. At this time, the microorganisms present in the sample solution do not enter the inside of the water-soluble polymer compound layer because the water-soluble polymer compound layer gradually dissolves to form a high-viscosity solution. A colony of microorganisms is formed near the surface and is colored by a coloring agent.

Examples of the use of the sheet medium of the present invention are shown below. Suspensions obtained by adding sterile saline or sterile water to food and homogenizing, or a suspension obtained by wiping the food production environment with gauze or a cotton swab and suspending in sterile saline or sterile water. Dilute the suspension or the like appropriately. Open the transparent film of the sheet-shaped culture medium for detecting general viable bacteria to the portion where the adhesive strength is increased, add the diluent to the culture medium laminate, cover the transparent film again, and grow the microorganism by culturing at 35 to 37 ° C. . Open the transparent film of the sheet culture medium to the part where the adhesive strength is increased, add sterile physiological saline or sterilized water to the culture medium laminate, place the filter on which the sample solution has been filtered on the culture medium laminate, and remove the transparent film again. The microorganism is grown by culturing at 35-37 ° C. Open the transparent film of the sheet-shaped medium to the part where the adhesive strength is increased, or completely peel off the transparent film, add sterile physiological saline or sterilized water to the medium laminate, and leave as it is to collect falling bacteria. As it is, or after adding sterile physiological saline or sterile water, the microorganism is grown by covering again with a transparent film and culturing at 35 to 37 ° C. Open the transparent film of the sheet medium to the part where the adhesive strength is increased, or completely peel off the transparent film, add sterile physiological saline or sterile water to the medium laminate, and stamp the porous matrix layer side directly on the test object. Alternatively, the surface to be inspected is directly wiped off, and as it is or after adding sterile physiological saline or sterile water, a transparent film is again covered and cultured at 35 to 37 ° C. to grow the microorganism.

As described above, when inspecting the wet surface, it is not necessary to wet the medium laminate in advance, and it is necessary to wipe off or stamp the medium laminate as it is, or after adding sterile physiological saline or sterile water, and then remove the transparent film. Put on,
The microorganism is grown by culturing at 35-37 ° C. In addition to normal food and environmental microbiological testing,
It is also characterized in that inspection can be performed by directly contacting the inspection target and directly wiping the inspection target surface. When performing inspection by direct contact or direct wiping, inspection can be performed even on an inspection object having a curved surface or some unevenness. Since the colonies of the microorganisms are colored by the coloring agent, the determination is easy.

[0035]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1 To 250 ml of water was added 30 g of polyvinyl alcohol having a degree of saponification of 89% and a molecular weight of 83000, followed by dissolution by heating. The resulting aqueous solution was applied on a polyester film having a thickness of 20 μm and a size of 0.5 m × 1 m. After drying for 1 minute, the first water-soluble polymer compound layer was formed. 15 g of the polyvinyl alcohol, 2.3 g of peptone, 1.0 g of meat extract, 0.6 g of yeast extract, 0.0 g of sodium carbonate
75 g and glucose 0.4 g were dissolved in 250 ml of water, and the obtained aqueous solution was applied on the first layer and heated to 110 ° C.
For 7 minutes to form a second water-soluble polymer compound layer. On this layer, the polyvinyl alcohol 5
g, peptone 0.8 g, meat extract 0.3 g, yeast extract 0.2 g, sodium carbonate 0.025 g and 2,2 chloride
20 mg of 3,5-triphenyltetrazolim in 100 parts of water
and then apply an aqueous solution obtained by dissolving the solution in
A nylon meltblown nonwoven fabric having a g / m ² and air permeability of 110 L / (m ² · sec) was laminated and dried at 100 ° C. for 30 seconds. 15 g of peptone and 40 of disodium phosphate
g was dissolved in 1 L of water, applied to a nonwoven fabric using a 60-mesh square pyramid gravure roll, and dried at 100 ° C. for 20 seconds. The obtained culture medium laminate was cut into a circular shape having a diameter of 50 mm, and was adhered to the central part of a 100 μm thick white polyester pressure-sensitive adhesive sheet manufactured by Lintec Co., Ltd., which was cut into a size of 80 mm × 90 mm. 0.
A polypropylene film having a size of 06 mm and a size of 80 mm x 90 mm is adhered to the adhesive sheet so that the 90 mm direction is shifted by 5 mm, and a 9 mm wide back sealing tape manufactured by Kyowa Co., Ltd. is adhered so as to cover the shifted part, and ethylene oxide gas sterilization is performed Then, a sheet-like medium for detecting general viable bacteria was prepared.

Various foods 1 such as meat, cut vegetables, and side dishes
0 g was placed in a sterilized bag, 100 ml of sterile physiological saline was added, and the mixture was homogenized with a stomacher. Make a 10-fold dilution line with sterile physiological saline, open the polypropylene film of the sheet medium for detection of general viable bacteria to the portion where the back sealing tape is adhered, add 1 ml of the diluted solution, and cover the polypropylene film again. And 4 at 35 ° C
The cells were cultured for 8 hours. At the same time, 1 ml was added to a sterilized Petri dish, a standard agar medium kept at about 45 ° C. after sterilization was added, and the mixture was mixed and cultured at 35 ° C. for 48 hours, and the number of viable cells was measured and compared. As shown in FIG. 1, good correlation with a correlation coefficient of 0.999 was exhibited.

Example 2 An area of about 100 cm ² was wiped off with a sterile cotton swab soaked in sterile physiological saline and suspended in 9 ml of sterile physiological saline. Add 1 ml of the suspension to a sterilized Petri dish, add SCD agar medium that has been kept at about 45 ° C. after sterilization, and pour.
The cells were cultured for 8 hours. 0.3 in the adjacent place about 100cm ²
Wipe off with a nonwoven fabric part of the sheet medium for detecting general viable bacteria prepared in Example 1 to which ~ 0.4 ml of sterile physiological saline was added, add 0.6 to 0.7 ml of sterile physiological saline, and add
For 48 hours, and the number of growing bacteria was compared. A part of the bacterial solution was previously applied and air-dried, and then wiped off. Since the same location is not inspected, the variation is recognized, but the correlation is recognized as shown in FIG. Compared to the case of swabbing with a cotton swab and mixing with an SCD agar medium, the number of growing bacteria was found to be larger in the sheet-like medium for detecting general viable bacteria.

Example 3 A nonwoven fabric of a sheet-like medium for detecting general viable bacteria prepared in Example 1 in which a commercially available stamp medium and 0.3 to 0.4 ml of sterile physiological saline were added to adjacent places of about 60 cm ² each. The parts were brought into contact with each other, and 0.6 to 0.7 ml of sterile physiological saline was added to the sheet-like medium for detecting general viable bacteria, followed by culturing at 35 ° C for 48 hours, and the number of viable bacteria was compared. A part of the bacterial solution was previously applied and air-dried, and then wiped off. As shown in FIG. 3, the sheet-shaped medium for detecting general viable bacteria tended to have a larger number of viable bacteria than the commercially available stamp medium, but a correlation was observed.

[0039]

According to the sheet-shaped medium of the present invention, microbial contamination inspection of foods and the environment can be easily performed. In addition to ordinary food and environmental inspections, it is also possible to directly inspect or wipe off inspection targets on curved or slightly uneven surfaces. In addition, because it is thin, it does not take up much space for cultivation, and the amount of waste is significantly reduced as compared with the usual coliform group test.

[Brief description of the drawings]

Fig. 1 Comparison with standard agar medium

[Figure 2] Comparison between direct wiping using a sheet-like medium for detection of general viable bacteria and SCD agar medium

Fig. 3 Comparison with stamp medium

Continued on the front page F term (reference) 4B029 AA03 AA08 BB01 CC02 CC07 CC11 GA08 GB05 GB06 GB09 4B063 QA01 QQ05 QQ16 QR66 QR69 QR84 QS07 QS36 QX01 4B065 AA01X AA57X BA23 BB02 BB03 BB23 BB29 BC46 BC46 CA46 CA46

Claims (11)

[Claims] 1. A laminate comprising a porous matrix layer and two or three water-soluble polymer compound layers, which may include a mount in contact with the water-soluble polymer compound layer furthest away from the porous matrix layer. An article is adhered to the center of the adhesive sheet larger than the laminate so that the porous matrix layer is on the top, and a transparent film larger than the laminate is in contact with the porous matrix layer and is centered on the laminate. The transparent medium is covered so that the parts thereof are overlapped, and the portion of the transparent film protruding from the laminate is a sheet-shaped medium adhered to the part of the adhesive sheet to which the laminate is not adhered, and the porous matrix layer Nutrient components consisting of peptone, meat extract and optional yeast extract, potassium phosphate, sodium phosphate, sodium carbonate A water-soluble polymer layer containing at least one salt selected from the group consisting of lithium, potassium carbonate and calcium carbonate, and a color former, wherein the water-soluble polymer layer next to the layer in contact with the porous matrix layer is Nutritional components consisting of peptone, meat extract, glucose and yeast extract as an optional component, and potassium phosphate,
A sheet-like medium for detecting general viable bacteria, comprising at least one salt selected from the group consisting of sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate. 2. A water-soluble polymer compound layer comprising three layers, wherein the water-soluble polymer compound layer furthest from the porous matrix layer comprises only a water-soluble polymer compound, or peptone, meat extract, glucose and Nutrient component consisting of yeast extract as an optional component, and a layer containing one or more salts selected from the group consisting of potassium phosphate, sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate, The sheet-shaped medium for detecting general viable bacteria according to claim 1. 3. The water-soluble polymer compound layer is 4-15 g / m ² of peptone, 1-8, based on the total weight of all layers in the medium.
g / m ² of meat extract, of 0.5~3g / m ² glucose,
0.1 to 0.8 g / m ² , one or more salts selected from the group consisting of potassium phosphate, sodium phosphate, sodium carbonate, potassium carbonate and calcium carbonate, 0.02
3. The composition according to claim 1, which comprises 0.15 g / m ² of 2,3,5-triphenyltetrazolium chloride or tetrazolium violet, and 1 to 8 g / m ² of yeast extract as an optional component. 2. The sheet-shaped medium for detecting general viable bacteria according to item 1. 4. The water-soluble polymer compound contained in the water-soluble polymer compound layer has a saponification degree of 75 to 95% and a molecular weight of 25,000.
The sheet-shaped culture medium for detecting general viable bacteria according to any one of claims 1 to 3, wherein the medium is polyvinyl alcohol of up to 250,000. 5. The water-soluble polymer compound layer in contact with the porous matrix layer contains a water-soluble polymer compound having a basis weight of 1 to 20 g / m ^2.
Item 4. The sheet-shaped medium for detecting general viable bacteria according to item 4. 6. The porous matrix layer has a basis weight of 40 to 100.
The sheet-shaped medium for detecting general viable bacteria according to any one of claims 1 to 5, wherein the medium is a nylon melt-blown nonwoven fabric having a g / m ² and an air permeability of 70 to 240 L / (m ² · sec). . 7. The porous matrix layer, wherein the surface thereof is coated with at least one component selected from the group consisting of peptone, potassium phosphate, sodium phosphate, sodium carbonate and potassium carbonate. The sheet-shaped culture medium for detecting general viable bacteria according to any one of claims 1 to 6. 8. The total coating weight of one or more components selected from the group consisting of peptone, potassium phosphate, sodium phosphate, sodium carbonate and potassium carbonate on the surface of the porous matrix layer is 0.3 to 3 g. / M ² , wherein the sheet-form medium for detecting general viable bacteria according to claim 7, wherein: 9. The adhesive sheet is a polyester film, a white polyester film, a polyolefin synthetic paper or a polyolefin laminate paper coated with an acrylic adhesive or a rubber adhesive, and the transparent film is a polyolefin film or a release-treated polyolefin film. 9. The method according to claim 1, wherein
Item 4. The sheet-shaped medium for detecting general viable bacteria according to item 4. 10. The pressure-sensitive adhesive sheet has a thickness of 0.07 to 0.5 m.
m, and the thickness of the transparent film is 20 to 80 μm, wherein the sheet-shaped medium for detecting general viable bacteria according to any one of claims 1 to 9, wherein 11. The adhesive sheet according to claim 1, wherein a part of an adhesive portion between the adhesive sheet and the transparent film is adhered so as to have an adhesive strength stronger than the adhesive strength of the adhesive sheet.
The sheet-shaped medium for detecting general viable bacteria according to any one of the above.