JP2001275651A - Sheet-like microorganism incubator and culture medium using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sheet-like microorganism incubator which improves the dispersibility and visibility of microorganisms in a sheet-like microorganism culture medium comprising a fibrous sheet and a gelling agent and with which a microorganism test can thereby simply be performed, and to provide a culture medium used therefor. SOLUTION: This sheet-like microorganism incubator in which a transparent film and a resin film are laminated in a state nipping a fibrous sheet having a smaller size than those of the films is characterized in that the transparent film and the fibrous sheet are adhered to each other with an adhesive coated on the transparent film and in that a gelling agent adhered to the resin sheet is intervened between the fibrous sheet and the resin sheet.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sheet-shaped microorganism incubator for culturing microorganisms and a medium using the same. More specifically, the present invention relates to a sheet-shaped microorganism incubator for testing microorganisms in foods and the environment, and a medium using the same.

[0002]

2. Description of the Related Art Conventionally, microbial culture tests have been carried out, for example, in the case of measuring the number of viable bacteria in a food test, after dissolving and sterilizing a powdered agar medium, make sure that the agar medium does not solidify. Keep at about 45 ° C, aliquot aliquots of the agar medium into a sterile Petri dish or the like in which a fixed amount of test sample solution (food suspension, etc.) has been previously placed, pour, agar Or a test sample solution is applied to a previously prepared solid agar medium, cultured at a constant temperature, and the number of colonies of the resulting microorganisms is counted. In such a conventional microorganism culturing method, it is extremely time-consuming and time-consuming, for example, it is necessary to prepare a medium in advance and maintain the agar medium at a temperature at which the adjusted medium does not solidify. In order to carry out a microorganism culture test more simply and quickly, it is preferable that such a laborious and time-consuming preparation of a medium can be omitted. Glass or plastic Petri dishes are used as sterile Petri dishes.
In the case of food testing, it is necessary to perform a large number of microbial culture tests, and when glass Petri dishes are used for culture,
The petri dishes must be washed, sterilized, etc. for reuse. This is very labor intensive, and in recent years, sterile plastic petri dishes that can be disposed of have been used. However, in this case, since a large amount of plastic waste is generated, there has been a problem in disposal and the like. In addition, when measuring the number of general viable bacteria in a microbial test in the environment, usually, a predetermined area of the test object is wiped off with a gauze or a cotton swab, and the gauze or the cotton swab is washed in sterile water or sterile physiological saline, and a cotton swab is used. The cells adhered to the suspension are suspended in sterile water or sterile physiological saline, and the suspension is applied to a previously prepared solid agar medium, or poured into an agar medium in the same manner as described above. After culturing, the resulting colonies are counted. In this case, too, it took a lot of trouble and time as in the case of the microorganism culture test. From such a situation, a simple medium in which the trouble of adjusting the medium is omitted has been studied.

[0003] 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 as a simple medium. this is,
It is a microorganism culturing device that combines a gelling agent and a fibrous sheet.
Since the added sample solution is also absorbed by the gelling agent, it is difficult to uniformly disperse the sample solution on the surface of the culture medium. Furthermore, colonies are not always formed on the upper surface of the fibrous sheet. In some cases, microorganisms grow on the lower surface or inside of the sheet, and in such a case, it is difficult to visually confirm the presence of the microorganisms, and there is a disadvantage that the quantitativeness is impaired.
For this reason, when using this type of microbial culture medium device, in practice, in order to facilitate observation of grown microorganisms, a fibrous water-absorbing sheet thinned to be transparent or translucent is used. Had to be used. In addition, when this microorganism culture medium device is used, since the fibrous sheet and the layer of the cold-water-soluble gelling agent are not bonded, it is difficult to use the device for directly wiping the test object.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to suitably use the present invention for testing microorganisms in ordinary foods and the environment.
Microorganism testing of curved or non-smooth surfaces can also be performed by directly stamping or wiping the test object, and the microbial dispersibility and visual properties of the sheet-shaped microbial medium combining a fibrous sheet and a gelling agent An object of the present invention is to provide a sheet-shaped microorganism incubator capable of easily performing a microorganism test by performing an improvement and a medium using the same.

[0005]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, have found that the following objects are achieved to achieve the intended purpose. The present invention has been achieved. (1) A sheet-shaped microbial incubator in which a transparent film and a resin sheet are laminated so as to sandwich a fibrous sheet of a smaller size, wherein the transparent film and the fibrous sheet are formed by an adhesive applied to the transparent film. Glued,
A sheet-shaped microorganism incubator, wherein a gelling agent attached to the resin sheet is interposed between the fibrous sheet and the resin sheet. (2) The transparent film is made of a thermoplastic resin having a thickness of 20 to 200 μm, and the fibrous sheet has a basis weight of 10 to 100 g / m.
^2. The sheet-shaped microorganism incubator according to the above (1), comprising the nonwoven fabric of (2). (3) The sheet-shaped microorganism incubator according to the above (1) or (2), wherein the resin sheet and the transparent film are bonded at an end of the resin sheet and the transparent film. (4) A sheet-shaped microbial medium comprising the sheet-shaped microbial incubator according to any one of the above (1) to (3), containing a nutrient component for microbial growth. (5) The sheet-shaped microorganism culture medium according to the above (4), wherein a nutrient component for microbial growth is mixed with a gelling agent. (6) The sheet-shaped microbial medium according to the above (4) or (5), wherein a nutrient component for microbial growth is contained in the fibrous sheet.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The sheet-shaped microorganism incubator of the present invention has a structure in which a fibrous sheet smaller in size than the transparent film and the resin sheet is inserted between the transparent film and the resin sheet. Further, it is preferable to use a nonwoven fabric as the fibrous sheet.
It is preferably 00 g / m ² . In addition, the sheet-shaped microbial incubator of the present invention can be made into a sheet-shaped microbial medium by including nutrients for microbial growth in a gelling agent and / or a fibrous sheet, and by adding water containing nutrients. Is also a sheet-shaped microorganism medium. In addition, the sheet medium of the present invention becomes an environment where microorganisms can grow by adding water or water containing nutrient components.

[0007] In the sheet-shaped microorganism incubator of the present invention,
An adhesive is attached to the inner surface of the transparent film laminate,
It is adhered to the fibrous sheet via this adhesive. At this time, the pressure-sensitive adhesive may be uniformly applied on the transparent film, or the pressure-sensitive adhesive may be applied to a portion or a part thereof to be bonded to the fibrous sheet. Further, a gelling agent is attached to the inner surface of the laminate of the resin sheet, and the laminate is laminated with the fibrous sheet.

[0008] In the sheet-shaped microorganism incubator of the present invention, one end of the transparent film and the resin sheet may be adhered with an adhesive tape or the like. By bonding with a pressure-sensitive adhesive tape or the like, it becomes possible to use the transparent film and the resin sheet without peeling them, or to completely peel them apart and use them. As a method of bonding with an adhesive tape, a method of bonding one end of a transparent film and a resin sheet with a double-sided tape. A method in which an overlapping portion of a transparent film and a resin sheet is shifted, and an adhesive tape is adhered to a surface where the transparent film or the resin sheet is not in contact, or a back surface of the transparent film and a surface of the resin sheet coated with a gelling agent. For example, a method in which the adhesive tape is bent without shifting the overlapping portion between the transparent film and the resin sheet and bonded so as to cover the transparent film and the resin sheet can be exemplified. In addition, the sheet-shaped microbial medium adhered by bending the adhesive tape to cover the transparent film and the resin sheet, the transparent film and the resin sheet are adhered by the adhesive tape even when the resin sheet and the transparent film are completely opened. Therefore, it is particularly preferably used.

When opening the sheet-shaped microbial medium, there is no problem if the transparent film and the resin sheet are completely peeled off.
When re-adhesion is necessary, it is preferable that a part remains without peeling. However, when direct stamping or wiping is performed, workability may be degraded due to the fact that both are attached, so it is necessary to ensure that they can be completely peeled off.

[0010] The transparent film used in the present invention is not particularly limited as long as it has transparency enough to allow the grown microorganisms (colonies) to be observed on the transparent film. Examples of the transparent film include polyolefin, polyester, nylon, poly (chlorinated) vinyl and the like. A transparent film produced from a thermoplastic resin of the formula (1) can be preferably used, and the thickness thereof is preferably in the range of 20 to 200 μm. Furthermore, a polyolefin film or a polyester film is more preferable because workability is improved because the film is flexible, and a polypropylene film having a thickness of 40 to 80 μm can be particularly preferably used. Also,
The adhesive used in the present invention may be any adhesive that does not inhibit the growth of microorganisms and does not become cloudy when water is added. Further, a transparent adhesive film to which an adhesive has been applied in advance may be used.

The resin sheet used in the present invention includes:
For example, a resin sheet manufactured from a resin such as a thermoplastic resin or a thermosetting resin can be used. As the adhesive for adhering the gelling agent, any adhesive can be used as long as it does not inhibit the growth of microorganisms.

Fibers can be used as a material for forming the fibrous sheet used in the present invention. For example, nylon fibers, polyacrylonitrile fibers, polyester fibers (especially those subjected to hydrophilization treatment), and polyolefin fibers (especially hydrophilization treatment) Processed fibers), synthetic fibers such as polyurethane fibers, semi-synthetic fibers such as rayon, natural fibers such as wool (animal hair), silk, cotton fibers, cellulose fibers and pulp fibers, and inorganic fibers such as glass fibers. Among the above fibers, nylon fibers, cotton fibers, cellulose fibers, and rayon are more preferable and can be used. The fibrous sheet is
It is preferable to use a knitted fabric, a nonwoven fabric, or the like formed from the above fibers, but it is more preferable to use a nonwoven fabric whose basis weight can be easily adjusted. The fibrous sheet is preferably subjected to a hydrophilic or hydrophilizing treatment, and it is more preferable to attach a water-soluble substance to the surface of the fibrous sheet, since diffusion of added water becomes faster. The basis weight of the fibrous sheet is not particularly limited, but is preferably in the range of 10 to 100 g / m ² from the viewpoint of flexibility and water retention.

The water-soluble substance to be attached to the surface of the fibrous sheet can be used without any problem as long as it does not hinder the growth of microorganisms.
Salts such as salts for adjusting pH or osmotic pressure are preferred. As nutrients, various peptones, yeast extracts, meat extracts, casamino acids, amino acids, amino acid mixtures, sugars such as glucose and maltose, organic acids, organic acid salts and the like can be used. Examples of the salts include hydrochlorides such as sodium chloride, phosphates such as dipotassium phosphate, and carbonates such as sodium hydrogen carbonate. In addition, these single components or the mixed components adhere to the surface of the fibrous sheet. At this time, these water-soluble substances may be applied in a powder state, but are preferably attached as a solution or a suspension. . As a method of adhering, a method of impregnating a fibrous sheet or a method of applying to the surface of a fibrous sheet is used. The solvent used as the solution or suspension may be any solvent that can dissolve or partially dissolve the water-soluble substance and further volatilizes or evaporates, but water, ethanol, methanol or a mixed solvent thereof is preferable.

The gelling agent used in the present invention may be any as long as it can gel by absorbing cold water and does not inhibit the growth of microorganisms, and may be partially dissolved. For example, guar gum, xanthan gum, canagilan, cross-linked polyvinyl alcohol, cross-linked polyacrylic acid and the like are preferably used.

The nutrient component contained in the sheet-shaped microorganism incubator of the present invention can be used without any particular problem as long as it is suitable for the microorganism to be cultured. For example, agar is removed from a general-purpose liquid medium or agar medium. Medium components and the like can be used. Further, a substance that suppresses the growth of microorganisms other than the object of detection may be added, an indicator for facilitating the appearance of the resulting colony, a dye, etc. may be added, and a color-forming or fluorescent enzyme may be added to detect a specific microorganism. Substrates may be added.

Nutrient components for the growth of microorganisms include, for example,
For general viable bacteria testing, yeast extract / peptone / glucose mixture, meat extract / peptone mixture, peptone / soybean peptone / glucose mixture, etc., and those obtained by adding dipotassium phosphate and / or sodium chloride thereto, etc.・ Sodium desoxycholate, peptone, iron ammonium citrate, sodium chloride, dipotassium phosphate, lactose, neutral red mixture, peptone, lactose, dipotassium phosphate, eosin Y
・ Methylene blue mixture is a mixture of meat extract, peptone, sodium chloride, mannitol, phenol red and egg yolk for staphylococci, peptone, meat extract, yeast extract, sodium pyruvate, glycine, lithium chloride, and yolk liquid of tellurite The mixture etc. are used for Vibrio, such as yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, sodium cholate, citric acid second
Iron, sodium chloride, beef bile, bromthymol blue,
Thymol blue mixture, etc., for enterococci, bovine brain extract, heart extract, peptone, glucose, dipotassium phosphate, sodium nitride, bromthymol blue, 2,3,5-triphenyltetrazolium chloride mixture, etc.
For fungi, a mixture of peptone / glucose, a mixture of potato extract / glucose and the like are used.

The sheet-shaped microorganism incubator of the present invention and the method for producing the sheet-shaped medium will be described below.

(1) An adhesive is applied on a transparent film made of a thermoplastic resin such as polypropylene, and a fibrous sheet smaller than the transparent film is adhered almost at the center of the transparent film. At this time, the pressure-sensitive adhesive applied surface may be smaller than the area of the fibrous sheet. In addition, the nutritional component may be contained in the fibrous sheet in advance, or the nutritional component may be contained after the fibrous sheet is adhered to the transparent film. (2) The gelling agent is adhered to the resin sheet coated with the adhesive, or the gelling agent dissolved or suspended in a solvent is applied to the resin sheet and dried. At this time, the application range of the gelling agent is preferably wider than that of the fibrous sheet. (3) A transparent film to which a fibrous sheet is adhered and a resin sheet coated with a gelling agent are combined so that the fibrous sheet and the gelling agent are in contact with each other, and sterilized by ethylene oxide gas sterilization or the like to obtain a medium. When one end of the transparent film and the resin sheet are adhered to each other with an adhesive tape or the like, the transparent film and the resin sheet can be used without being separated, or can be completely separated and separated.

An example of using the sheet-shaped microorganism culture device and the sheet-shaped medium of the present invention will be described below.

Sterile physiological saline or sterile water is added to the food or the like, and the homogenized suspension or the food production environment is wiped off with a gauze or the like, and the suspension or the like suspended in the sterile physiological saline or sterile water is appropriately diluted. . The diluted solution obtained is added to the fibrous sheet on the transparent film of the sheet medium, and after the diluent spreads on the fibrous sheet, the microorganism is grown by covering with a resin sheet coated with a gelling agent and culturing. Let it. Alternatively, sterile saline or sterile water is added to the fibrous sheet, and the fibrous sheet is directly stamped on the test object.
Alternatively, the surface to be inspected is directly wiped, and as it is, or after adding sterile physiological saline or sterile water, a microorganism sheet is grown by covering with a resin sheet coated with a gelling agent and culturing. When inspecting a wet surface, it is not necessary to wet the fibrous sheet in advance.Wipe or stamp the fibrous sheet as it is, or add sterile physiological saline or sterile water, and then cover with a resin sheet coated with a gelling agent and culture. To grow the microorganisms. The grown microorganisms (colonies) are observed from the transparent film side.

Example 1 A 100 μm-thick polypropylene film was used as a transparent film, and an acrylic-based slightly-adhesive type adhesive (manufactured by Lintec Co., Ltd.) was applied to one surface of the polypropylene film.
The transparent film was cut into a size of 0 mm × 95 mm and coated with an acrylic-based slightly adhesive type pressure-sensitive adhesive. In addition, a rayon needle punched nonwoven fabric having a basis weight of 50 g / m ² is used as the fibrous sheet, and a 20 g / L peptone solution is applied and dried on one surface of the sheet as a nutrient for growing microorganisms so that the diameter becomes 50 mm. To produce a fibrous sheet to which nutrients for growing microorganisms were attached. A 120 μm-thick polypropylene synthetic paper was used as the resin sheet, and a rubber-based adhesive (manufactured by Lintec Co., Ltd.) was applied to one surface of the resin sheet. 7 parts of peptone, 3 parts of meat extract, 0.1 part of sodium carbonate,
Using a mixed powder consisting of 0.5 part of glucose and 0.01 part of 2,3,5-triphenyltetrazolium chloride, these were dropped on the applied rubber-based adhesive, adhered and fixed, and 80 mm × 90 mm in size. By cutting to size, a resin sheet to which the gelling agent was attached was manufactured. One side of the fibrous sheet, on which the peptone solution is not applied, is adhered to a substantially central portion of the transparent film, and the surface of the resin sheet, on which the gelling agent is adhered, faces the fibrous sheet, and is 9 mm wide. The back sealing tape (manufactured by Kyowa Co., Ltd.) is folded in two, and a polypropylene film and 4.5 mm each of the outer surface of one end of the polypropylene synthetic paper are adhered.
Finally, sterilization was performed with ethylene oxide gas to prepare a sheet-like medium for general viable bacteria. Put 10g of various foods such as meat, cut vegetables and delicatessen in a sterilized bag, 100m
L of sterile physiological saline was added thereto, homogenized with a stomacher, and sterile physiological saline was further added to prepare a 10-fold dilution series. Open the polypropylene film of the sheet-shaped medium to the portion where the back sealing tape is adhered, and apply the 10-fold dilution line to a rayon needle-punched nonwoven fabric for 1 m.
After adding L, the polypropylene film was covered again and sealed. In addition, the 10-fold dilution line was placed in a sterilized Petri dish for 1 m.
L, and a sterilized standard agar medium at about 45 ° C. was added and the mixture was pulverized. Incubate each at the same time for 48 hours at 35 ° C.
When the number of growing bacteria was measured and compared, good results were obtained as shown in FIG.

[0022]

EFFECTS OF THE INVENTION By using the medium of the present invention, microorganism testing in foods and the environment can be easily performed.
The inspection can be performed by stamping or wiping the inspection object, and the grown microorganisms can be observed from the transparent film side. Microorganism inspection for various inspection objects and applications can be easily performed. The medium of the present invention is thinner than a medium using a normal petri dish, requires less space for culture, and can greatly reduce waste. Also, when the sample liquid is added to the fibrous sheet, since the fibrous sheet does not contact the gelling agent,
The sample liquid easily diffuses into the fibrous sheet, and the dispersion of microorganisms does not become uneven. Further, after the sample liquid is diffused into the fibrous sheet, the resin sheet coated with the gelling agent is covered on the fibrous sheet, so that the gelling agent swells to the bottom of the fibrous sheet while absorbing the sample liquid. . For this reason,
Microorganisms mainly grow at the bottom of the fibrous sheet, and the fibrous sheet hardly hinders observation of the grown microorganisms, and can be easily observed from the transparent film side.

[Brief description of the drawings]

Fig. 1 Comparison with standard agar medium

Claims (6)

[Claims] 1. A sheet-shaped microorganism incubator in which a transparent film and a resin sheet are laminated so as to sandwich a fibrous sheet of a smaller size, wherein the transparent film and the fibrous sheet are coated on the transparent film. A sheet-shaped microorganism incubator which is adhered by an agent and has a gelling agent attached to the resin sheet interposed between the fibrous sheet and the resin sheet. 2. A transparent film comprising a thermoplastic resin having a thickness of 20 to 200 μm, and a fibrous sheet having a basis weight of 10 to 10 μm.
^2. A non-woven fabric of 0 g / m ^2.
The sheet-shaped microorganism incubator according to the above. 3. The sheet-shaped microorganism incubator according to claim 1, wherein the resin sheet and the transparent film are adhered at ends of the resin sheet and the transparent film. 4. A sheet-shaped microorganism culture medium comprising the sheet-shaped microorganism incubator according to any one of claims 1 to 3 containing a nutrient component for growing microorganisms. 5. The sheet-shaped microorganism medium according to claim 4, wherein a nutrient component for growing the microorganism is mixed with a gelling agent. 6. The sheet-shaped microorganism medium according to claim 4, wherein a nutrient for growing the microorganism is contained in the fibrous sheet.