JP2006230220A - Microorganism testing kit and microorganism testing method using the kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism testing kit capable of detecting microorganisms with high accuracy even when the amount of collected microorganisms collected by an adhesive sheet is small, and a microorganism testing method using the kit.
A microorganism testing kit comprising: (A) an adhesive sheet for collecting microorganisms provided with an adhesive layer on at least one side of a support; and (B) a medium sheet having at least a medium layer containing a dye for detecting microorganisms. The adhesive layer of the adhesive sheet for collecting microorganisms of (A) above is pressure-bonded to a subject to collect microorganisms, and then the medium layer of the medium sheet of (B) above to the adhesive surface on which the microorganisms of the adhesive layer are collected The microorganisms are cultured by culturing the microorganisms, and then the microorganisms grown through the culture are observed, for example, under a microscope.
[Selection] None

Description

  The present invention relates to a microorganism testing kit and a microorganism testing method using the kit, and more specifically, a high-accuracy test can be performed by culturing and growing microorganisms collected on an adhesive surface of an adhesive sheet. The present invention relates to a method for testing microorganisms.

  Conventionally, in order to estimate the number of bacteria in a test material, for example, an agar stamp method using a commercially available food stamp or the like, it is applied that one bacterium forms one colony, There is a method in which a solid medium is pressed against a test material to cultivate bacteria, and a colony expressed on an agar plate is measured with the naked eye. In addition, as in the membrane filter method using a commercially available membrane filter for microorganisms / microparticle inspection, etc., the test material is washed out with physiological saline, collected by the membrane filter, and absorbed on the liquid medium. There is also a method of forming a colony. Furthermore, Non-Patent Document 1 below reports an example in which a film is coated on a film and a film-coated medium capable of collecting a sample is prepared by simply culturing the film on a test surface.

  However, with the agar stamp method, the size (area) of the agar medium is limited, and since it can usually be used only once per test surface, the collection efficiency varies depending on the moisture content of the agar medium, and reproducibility. In many cases, it is inconvenient in the collection efficiency of bacteria. Furthermore, there is also a problem that the test surface is contaminated by moisture in the agar medium.

  In addition, the membrane filter method requires two steps, a washing-out step and a filtration step with a filter, and thus there are inconveniences such as complicated operations and concern about secondary contamination. That is, in this method, bacteria cannot be collected directly from the test substance, and sterilization work before use, sampling with a cotton swab, adjustment of the washing solution, filter filtration, etc. require a lot of labor, and an increase in cost is inevitable. .

  Further, the film-coated medium disclosed in Non-Patent Document 1 below has a problem that the test surface is contaminated by moisture, as in the agar stamp method. Moreover, since the bending strength of the agar culture medium used is small, utilization as a sheet | seat is restrict | limited and there exist various faults, such as insufficient adhesive force of a culture medium and a film.

On the other hand, the present applicant presses and peels the pressure-sensitive adhesive layer of the pressure-sensitive adhesive sheet, collects (accumulates) microorganisms on the pressure-sensitive adhesive layer, and then contains an aqueous solution containing one or more coloring substances that can stain the microorganisms. A test method was proposed in which microorganisms present in a subject can be detected by contacting the surface of the adhesive layer and observing and counting (image analysis) the stained bacterial cells (Patent Document 1 below). This test method uses a pressure-sensitive adhesive layer of a pressure-sensitive adhesive sheet as a means for collecting microorganisms and a test surface so that microorganisms present on the surface of a solid subject (solid surface) can be easily detected. is there. However, for example, when the surface of the subject is relatively clean, there is a problem that sufficiently high detection accuracy cannot be obtained because the amount of collected microorganisms is small. There is also a problem that it is difficult to distinguish between the collected microorganisms and minute garbage.
JP 2002-142797 A Medical Examination, 1992, Vol. 41, No. 3, p352

  The present invention is regarded as a ship in the above circumstances, and the problem to be solved is a microorganism test that can detect microorganisms with high accuracy even when the amount of microorganisms collected by the adhesive sheet is small. And a method for testing microorganisms using the kit.

In order to solve the above problems, the present invention employs the following configuration.
(1) A kit for microbiological examination comprising the following (A) and (B).
(A): Adhesive sheet for collecting microorganisms provided with an adhesive layer on at least one side of the support (B): Medium sheet having at least a medium layer containing a dye for detecting microorganisms (2) The dye for detecting microorganisms is a fluorescent dye The kit for microbiological examination according to (1) above.
(3) A method for testing microorganisms using the microorganism testing kit according to (1) above,
A step of collecting microorganisms by pressure-bonding the adhesive layer of the adhesive sheet for collecting microorganisms of (A) to a subject;
Stacking the medium layer of the medium sheet of (B) on the adhesive surface that has collected the microorganisms of the adhesive layer, and culturing the microorganisms;
A method for detecting microorganisms, comprising a step of detecting microorganisms grown through the culture step.
(4) The microorganism inspection method according to (3), wherein the microorganism detection operation is an operation of observing the microorganism under a microscope.
(5) The microorganism testing method according to (4), wherein the microorganism detection dye contained in the adhesive layer of the microorganism-collecting adhesive sheet is SYBRGold, and the microscope is a fluorescence microscope.
In the present invention, “microorganism inspection” means “microorganism detection inspection”.

  When the microorganism testing kit of the present invention is used, when the culture medium layer containing the microorganism detection dye of the culture medium sheet is stacked on the adhesive layer (adhesive surface) of the adhesive sheet that has been pressure-bonded and peeled to the subject, it is collected on the adhesive surface. Since the nutrients are supplied to the microorganisms from the medium sheet and the dye for detecting microorganisms is taken in, the collected microorganisms are labeled at the same time as the collected microorganisms are placed in the incubator, for example, by placing a stack of the adhesive sheet and the medium sheet. can do. Therefore, even when the amount of microorganisms (sample) collected by the pressure-sensitive adhesive sheet is small, the microorganism concentration (sample concentration) can be increased and labeled on the pressure-sensitive adhesive sheet. it can. In addition, when a subject with relatively high cleanliness is to be examined, the examination range can be expanded on the same adhesive surface, so that microorganisms can be detected economically and labor-saving. In addition, since the collection of microorganisms depends on the adhesive force of the adhesive, a stable and high collection effect is obtained, and the problem of contamination of the test surface due to moisture, which has been a problem with conventional culture media, is also eliminated. be able to.

Hereinafter, embodiments of the present invention will be described in detail.
[(A) Adhesive sheet for collecting microorganisms]
As shown in FIG. 1, the microorganism-collecting pressure-sensitive adhesive sheet is a pressure-sensitive adhesive sheet 10 in which a pressure-sensitive adhesive layer 2 is provided on at least one surface of a support 1. In general, the adhesive layer 2 is often provided only on one side of the support 1 from the viewpoint of the operability of the adhesive sheet, but may be provided on both sides of the support 1.

  The microorganism-collecting pressure-sensitive adhesive sheet 10 is used for collecting microorganisms present on the surface (test surface) of a specimen. That is, the pressure-sensitive adhesive surface of the pressure-sensitive adhesive layer 2 is pressure-bonded to the surface of the subject (test surface), and microorganisms present on the surface of the subject (test surface) are collected on the pressure-sensitive adhesive surface.

The pressure-sensitive adhesive (polymer compound) used for the pressure-sensitive adhesive layer 2 is not particularly limited as long as it has sufficient adhesiveness to capture microorganisms on the test surface, and various pressure-sensitive adhesives used in known pressure-sensitive adhesive sheets. A high molecular compound (adhesive) exhibiting properties can be used.
Specifically, acrylic pressure-sensitive adhesives, rubber-based pressure-sensitive adhesives, silicone-based pressure-sensitive adhesives, ethylene-vinyl acetate copolymers, polyvinyl ether, polyvinyl esters and other water-insoluble pressure-sensitive adhesives, agar, carrageenan, gum arabic, polyvinyl Pyrrolidone, polyvinyl alcohol (PVA), gelatin, carboxymethyl cellulose, alginic acid, methyl cellulose, polyacrylic acid and / or polyacrylate polymer compounds, hydrophilic polyether urethane polymers, stilbazolated polyvinyl alcohol, xanthan gum, etc. Water-soluble adhesives such as saccharides, polyethylene oxide, acrylamides such as poly (N-isopropylacrylamide) and the like can be mentioned.

  From the viewpoint of fluorescent labeling on the adhesive surface of microorganisms, the adhesive layer is preferably highly transparent and non-fluorescent, and from this point of view, acrylic adhesives and silicone adhesives are preferred. As a suitable specific example of the said acrylic adhesive, (meth) acrylic acid alkyl ester which is a C2-C13 linear or branched alkyl group having an alkyl group, for example, ethyl (meth) acrylate, ( One or more selected from meth) propyl acrylate, butyl (meth) acrylate, hexyl (meth) acrylate, octyl (meth) acrylate, nonyl (meth) acrylate and decyl (meth) acrylate As the main monomer, (meth) acrylic acid, itaconic acid, maleic acid, hydroxyethyl (meth) acrylate, methoxyethyl (meth) acrylate, ethoxyethyl (meth) acrylate, butoxyethyl (meth) acrylate , One or more hydrophilic monomers such as ethylene glycol (meth) acrylate were copolymerized Polymers. In order to improve adhesive properties (particularly adhesive strength), the copolymer is treated with a thermal crosslinking agent such as an isocyanate compound, an organic peroxide, or an epoxy group-containing compound, ultraviolet rays, γ rays, electron beams, etc. Those subjected to crosslinking by irradiation treatment or the like are also preferably used. Moreover, as a suitable specific example of a silicone type adhesive, what a main component consists of dimethylsiloxane is mentioned.

  From the viewpoint of the growth speed of microorganisms during culture, a water-soluble pressure-sensitive adhesive made of an acrylic copolymer having alkoxy acrylate and N-vinyl lactam as main structural units is preferable. Examples of the alkoxyalkyl acrylate in the acrylic copolymer include acrylates having an alkoxy group having 1 to 4 carbon atoms and an alkyl group having 2 to 4 carbon atoms or an alkylene glycol group. Methoxyethyl acrylate, 2-ethoxyethyl acrylate, 2-butoxyethyl acrylate, 3-methoxypropyl acrylate, 3-methoxybutyl acrylate, 3-ethoxypropyl acrylate, 3-ethoxybutyl acrylate, butoxytriethylene glycol, 2- (2- Ethoxyethyl) ethyl acrylate, methoxytriethylene acrylate, methoxydipropylene glycol acrylate and the like. Of these, 2-methoxyethyl acrylate, 2-ethoxyethyl acrylate, and the like are preferable. Further, as the N-vinyl lactam, a 5- to 7-membered ring N-vinyl lactam is preferable, for example, N-vinyl-2-pyrrolidone, N-vinyl-2-piperidone, N-vinyl-2-caprolactam, etc. Among them, N-vinyl-2-pyrrolidone is preferable.

  The blending ratio of the alkoxyalkyl acrylate in the acrylic copolymer is preferably 60 to 80% by weight, more preferably 65 to 75% by weight, based on the entire acrylic copolymer. Further, the blending ratio of N-vinyl lactam is preferably 20 to 40% by weight, more preferably 25 to 35% by weight, based on the entire acrylic copolymer. When the blending ratio of N-vinyl lactam is less than 20% by weight, the strength and water absorption ability of the obtained adhesive layer may be lowered. On the other hand, if it exceeds 40% by weight, the resulting adhesive layer has low flexibility, and the acrylic copolymer may dissolve in moisture and flow may occur. In order to improve the water absorption capacity, the acrylic copolymer may be copolymerized with vinyl carboxylic acid. Examples of vinyl carboxylic acid include acrylic acid, methacrylic acid, crotonic acid, itaconic acid, maleic acid, Examples thereof include fumaric acid, maleic acid half ester, and fumaric acid half ester. Among these, acrylic acid is preferable from the viewpoint of reactivity and versatility. The blending ratio of the vinyl carboxylic acid is preferably 10% by weight or less of the entire acrylic copolymer.

  When using a water-soluble pressure-sensitive adhesive such as the acrylic copolymer, a plasticizer may be added within the range of 5 to 40 parts by weight per 100 parts by weight of the pressure-sensitive adhesive, and a plasticizer is added. Thereby, the softness | flexibility and adhesiveness of an adhesion layer can be improved more. As the plasticizer, a compound having good compatibility with the water-soluble polymer to be used and hydrophilic is preferable, for example, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, glycerin, diglycerin, 2,3-butanediol, 1,2-butanediol, 3-methyl-1,3-butanediol, 3-methyl-1,3,5-pentanetriol, 2-ethyl-1,3-hexanediol, etc. Low molecular weight polyhydric alcohol, polyethylene glycol having an average molecular weight of 1000 or less, polypropylene glycol, polyoxyethylene butyl ether, polyoxyethylene, polyoxypropylene, polyoxypropylene glyceryl ether, polyoxypropylene glycol, polyoxy Examples include lopyrene sorbitol, polyoxyethylene polyoxypropylene pentaerythritol ether, etc., but polyoxypropylene glyceryl ether having an average molecular weight of 200 or more and 800 or less, particularly from the viewpoint of obtaining excellent flexibility, adhesiveness and hydrophilicity, Polyoxypropylene glycol having an average molecular weight of 200 to 800 and polyoxypropylene sorbitol having an average molecular weight of 200 to 800 are preferred. In order to maintain an appropriate water content of the water-soluble pressure-sensitive adhesive, a hydrophilic low-molecular substance may be added in the range of 20 to 300 parts by weight per 100 parts by weight of the water-soluble pressure-sensitive adhesive. Substances include high-boiling liquid substances (for example, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, sugar alcohols such as glycerin and sugar alcohols such as sorbitol) and / or deliquescent inorganic salts (for example, , Lithium nitrate, lithium chloride, etc.) are preferred.

  In order to improve the adhesive properties (especially adhesive strength), the acrylic copolymer is treated with a thermal crosslinking agent such as an isocyanate compound, an organic peroxide, or an epoxy group-containing compound, ultraviolet rays, γ Those subjected to cross-linking by performing irradiation treatment of a wire, electron beam or the like are also preferably used.

  In the present invention, the pressure-sensitive adhesive (polymer compound) used for the pressure-sensitive adhesive layer 2 of the pressure-sensitive adhesive sheet may be one type of pressure-sensitive adhesive or a mixture of two or more types of pressure-sensitive adhesives. Further, the adhesive layer agent can be blended with conventional additives such as an anti-aging agent and a filler, if necessary, together with the adhesive.

  In the present invention, the thickness of the pressure-sensitive adhesive layer 2 of the pressure-sensitive adhesive sheet 10 for collecting microorganisms (thickness due to the solid content after drying) is 5 to 100 μm from the viewpoints of the efficiency of collecting microorganisms, oxygen permeability during culture, and the like. Preferably, it is 10-60 micrometers especially preferably. That is, when the thickness exceeds 100 μm, the pressure-sensitive adhesive layer tends to cohesively break down, the collection efficiency is lowered, and the test surface is easily contaminated. On the other hand, when the thickness is smaller than 5 μm, it is difficult to apply the adhesive with a uniform thickness, which is not preferable.

  As the support 1 in the adhesive sheet 10 for collecting microorganisms of the present invention, known sheets (films) used as a support for an adhesive tape can be applied. In particular, a material made of a flexible material that does not form large irregularities on the surface of the adhesive layer and can be freely crimped to a curved surface or a narrow surface is suitable. For example, polyester, polyethylene, polycarbonate, polyurethane, polychlorinated Examples thereof include solid or porous plastic sheets made of vinyl, ethylene-vinyl acetate copolymer, urethane-acrylic hybrid resin, cloth, nonwoven fabric, paper, polyethylene laminated paper, and the like. Among these, a polyester film and a polycarbonate film are preferable from the viewpoint of flexibility and mechanical strength.

  Further, the thickness of the support 2 is not particularly limited as long as sufficient strength as a support for the pressure-sensitive adhesive sheet is obtained, but is generally about 5 to 5000 μm, preferably 5 to 200 μm. Degree.

  In the pressure-sensitive adhesive sheet 10 for collecting microorganisms of the present invention, the surface of the support 1 opposite to the surface in contact with the pressure-sensitive adhesive layer 2 is increased in color by using a substrate that increases the strength of the base material, optical scattering, or a coloring material. In order to form a background color that improves the visibility of the microorganisms that have been removed, a backing layer may be further provided.

  The adhesive sheet 10 can be manufactured by a conventionally known method. For example, after a solution containing a polymer compound for forming the pressure-sensitive adhesive layer 2 is applied to a release liner and dried, the surface of the pressure-sensitive adhesive layer 2 is bonded with a film, a fabric, or the like, Alternatively, the solution containing the polymer compound is directly applied to a film, fabric, or the like constituting the support 1 and dried, and then a release liner is laminated on the surface of the pressure-sensitive adhesive layer 2 as necessary, and the support is supported. It can be manufactured by obtaining a sheet-shaped raw material including 1 and the pressure-sensitive adhesive layer 2 and punching it in the shape of the outer frame of the pressure-sensitive adhesive sheet.

  In the pressure-sensitive adhesive sheet 10 for collecting microorganisms of the present invention, the surface of the pressure-sensitive adhesive layer 2 is preferably protected with a release liner until it is affixed to a subject, and a release liner is laminated on the pressure-sensitive adhesive layer 2 as necessary. The The material of the release liner is not particularly limited as long as it can be easily peeled from the adhesive layer 2 at the time of use. Specific examples include a solid or porous plastic sheet (film) from which the contact surface with the pressure-sensitive adhesive layer 2 has been peeled, a laminate film of fine paper or glassine paper and a plastic film (for example, a polyolefin film), and the like. It is done. Moreover, it is preferable that the pressure-sensitive adhesive sheet 10 for collecting microorganisms of the present invention is in a sterilized state by, for example, enclosing it in a germ-blocking packaging material in a sterilized state before use. In addition, it is preferable to use the EOG (ethylene oxide gas) method, the electron beam method, and the gamma ray method for this sterilization process. That is, the adhesive sheet for microorganism collection according to the present invention is subjected to sterilization treatment by irradiating with an electron beam or γ-ray after being made into a form capable of maintaining a sterilized state by enclosing the adhesive sheet in a bacteria-blocking packaging material, etc. It is preferable to use 10 for distribution. In this case, the irradiation dose of radiation such as an electron beam or γ-ray is suitably about 25 to 50 kGy in order to achieve sufficient sterilization while preventing denaturation such as coloring.

[(B) Medium sheet]
As shown in FIG. 1, the medium sheet is a sheet 20 having at least a medium layer 12 containing a microorganism detecting dye. Usually, a medium layer 12 containing a microorganism detecting dye is provided on a support 11. Configured. The support 11 supports the medium layer 12 and plays a role of preventing the medium layer 12 from drying.

The type of the medium used for the medium layer 12 is not particularly limited as long as it does not inhibit the growth of the microorganism to be detected, and a known medium for growing (proliferating) microorganisms can be used. For example, standard agar medium, soy bean casein digest agar medium (SCD agar medium), SCDLP agar medium, R2A medium (for water quality test), mannitol salt agar medium (for staphylococci), desoxycholate agar medium, potato Examples include dextrose agar and Sabouraud / glucose agar. In addition, it is possible to detect only specific microorganisms by adding an appropriate selection agent to the medium layer by utilizing the auxotrophy of microorganisms. Examples of the selective agent include a selective medium, an antibacterial agent, and an antibiotic. Specific microorganisms include, for example, Escherichia coli, enterohemorrhagic Escherichia coli, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Listeria, and MRSA, a drug-resistant bacterium that has attracted attention for nosocomial infections. Examples include microorganisms present in water, microorganisms present in pharmaceutical water, yeast, fungi and the like. On the other hand, as antibacterial agents added for detection of resistant bacteria, for example, alcohols, glutaraldehyde, iodophor, popidone iodine, benzalkonium chloride, benzethonium chloride, alkylpolyaminoethylglycine, cyclohexidine gluconate, benzisothiazoline, organic Examples include nitrogen-containing cyclic compounds, organic nitrogen sulfur compounds, organic nitrogen sulfur halogen compounds, pyridine oxide compounds, and antibiotics such as cephem, second generation cephem, and third generation cephem antibiotics. (Cephalosin, cephaloridine, etc.), penicillin antibiotics (methicillin, ampicillin, penicillin G, etc.), tetracycline antibiotics (tetracycline, etc.), peptide antibiotics (vancomycin, etc.), aminoglycoside antibiotics (a) Bekashin, spray script mycin, kanamycin, etc.). The amount of the antibacterial agent and / or antibiotic added to these medium layers is not particularly limited, but is generally about 2 × 10 ⁻⁵ to 2 × 10 ⁻² wt% with respect to the medium (solid content). .

  The dye for detecting microorganisms contained in the medium layer 12 is not particularly limited as long as it does not inhibit the growth of microorganisms, and any known dye used for staining microorganisms can be used without limitation. Examples thereof include enzyme substrates specific to microorganisms to be grown (proliferated), pH indicators, fluorescent dyes that specifically bind to nucleic acids, and the like. A particularly suitable enzyme substrate is the dye “carboxyfluorescein diacetate (CFDA)”. The CFDA is essentially non-fluorescent, but emits fluorescence when it is degraded by an enzyme (esterase) possessed by living microorganisms. Therefore, by using a medium sheet having a medium layer containing the CFDA, for example, when a cultured (growth) microorganism is observed with a fluorescence microscope, only living organisms among the aggregate of microorganisms are observed. It will be dyed and detected. Further, as a fluorescent dye that specifically binds to a nucleic acid, SYBR Green I, SYBR Green II, SYBR Gold, SYTOX Green, DAPI, TOTO-1 Ioide, YOYO-1 Ioide, SYTO 62, SYTO 63, SYTO 64 ( In any case, SYBR Gold is particularly preferable because SYBR Gold is excellent in dyeability of microorganisms and does not inhibit the growth of microorganisms even if it coexists with microorganisms.

The medium layer 12 is preferably provided so that the amount of the medium per unit area is 0.1 to ⁵ mm ³ / mm ^2, and is provided so that the amount of the medium is 0.2 to ² mm ³ / mm ² . Is more preferable. When the amount of the medium per unit area in the medium layer 12 is less than 0.1 mm ³ / mm ² , the nutrient supply to microorganisms tends to decrease, and when it exceeds 5 mm ³ / mm ² , the observability tends to decrease. Become. Moreover, the density | concentration (content) of the said fluorescent dye in the culture medium layer 12 becomes like this. Preferably it is 0.01-100 ppm, More preferably, 0.1-10 ppm is preferable. When the concentration (content) of the fluorescent dye is less than 0.01 ppm, the dyeability of microorganisms tends to deteriorate, and when it exceeds 100 ppm, the background color development tends to increase during observation.

  The support 11 of the medium sheet 20 is not particularly limited as long as appropriate adhesion to the medium layer 12 is obtained and the medium layer 12 is not detached and is useful for preventing the medium layer 12 from drying. However, polyester, polycarbonate, and polypropylene are preferable from the viewpoint of handling properties and clarity during microscopic observation. The thickness of the support 11 is not particularly limited, but is generally about 5 to 200 μm.

  The medium sheet can be obtained, for example, as follows. First, a sheet (film) that protects the surface of the medium layer 12 on the surface of the medium layer 12 is formed, if necessary, after forming a medium layer 12 by applying a solution containing at least a medium and a microorganism detecting dye to the support 11 and drying it. Then, a cloth or the like is attached. Here, the sheet (film) for protecting the surface of the medium layer 12, the fabric, and the like are provided for drying the medium layer of the medium sheet and preventing microbial contamination. The sheet for protecting the surface (film) By providing a fabric, etc., there are advantages of maintaining quality and improving the reliability of test results using the kit of the present invention.

[Microbial inspection method]
The kit for testing microorganisms of the present invention has at least a (A) adhesive sheet for collecting microorganisms provided with an adhesive layer on at least one side of the support and (B) a medium layer containing a dye for detecting microorganisms. In the following, a method for examining microorganisms using a kit including a medium sheet will be described.

  First, the pressure-sensitive adhesive layer 2 of the pressure-sensitive adhesive sheet 10 of this kit is pressed against a subject, and microorganisms adhering to the subject are collected on the pressure-sensitive adhesive layer 2, or an aqueous test substance (that is, a test target) An aqueous solution containing microorganisms (for example, a drinking water, a food suspension, a microorganism-containing liquid collected by a wiping method, etc.) is directly inoculated on the adhesive layer 2 of the adhesive sheet 10.

  Next, the culture medium layer 12 of the culture medium sheet 20 is stacked on the adhesive surface (collecting surface) of the adhesive layer 2, and in this state, the stacked material is placed in an incubator, and the collected microorganisms are cultured. . During this time, the collected microorganisms are fed with nutrients from the medium sheet and grow on the adhesive layer 2, and the microorganisms form aggregates (colony) and at the same time take in the dye for detecting microorganisms from the medium layer 12 and are labeled. . That is, for example, when the microorganism detection dye is a fluorescent dye, the microorganism takes in the fluorescent dye and emits fluorescence.

  In such a culture operation, the stack of the adhesive sheet 10 and the medium sheet 20 is preferably kept in a state of being isolated from these contamination source articles and the environment for the purpose of preventing secondary contamination of human bodies and equipment. For example, it is preferable to immediately put a stack of the adhesive sheet 10 and the medium sheet 20 into a petri dish. In addition, the means for shielding such a stack from the environment is not limited to a petri dish, and may be a film or the like through which microorganisms cannot permeate. The “film through which microorganisms cannot permeate” refers to a film that does not have pores of a size in which microorganisms can move. For example, a plastic film such as polyester, polyurethane, polyethylene, or a pore diameter of 0.2 μm or less. The membrane filter etc. are mentioned. In addition, although culture time changes with microorganisms to be examined, it is generally about 4 to 48 hours.

  Next, the culture medium layer 12 of the culture medium sheet 20 is separated from the adhesive layer 2 of the adhesive sheet 10, and microorganisms are detected by observing the adhesive surface (collecting surface) of the adhesive layer 2 of the adhesive sheet 10 with a microscope. That is, when the test object is contaminated with live microorganisms, the microorganisms are collected on the adhesive surface (collecting surface), proliferated by culture, and labeled. And detection of microorganisms can be performed.

  There are no particular limitations on the microorganisms to be detected in the microorganism testing kit of the present invention. As described above, it is possible to detect only specific bacteria by adding a selective medium or antibiotics. Specific examples include Escherichia coli, enterohemorrhagic Escherichia coli, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Listeria, and drug-resistant bacteria MRSA that are attracting attention for nosocomial infections and natural water. Examples include microorganisms present, microorganisms present in pharmaceutical water, yeast, fungi and the like.

  In addition, in the microorganism inspection using this microorganism inspection kit, the method for observing (detecting) the microorganisms (colony) grown on the pressure-sensitive adhesive sheet is not particularly limited, and visual observation is possible. From this point, it is usually preferable to carry out by microscopic observation such as an optical microscope, a phase contrast microscope, a fluorescence microscope, an electron microscope, and a laser microscope. In addition, a commercially available colony counter or an automatic detection device for various images (for example, an automatic device that combines an autoscan device with a simple microscope, and by simply placing the kit (adhesive sheet) of the present invention on the stage, images are automatically displayed. For example, those that detect uptake and microorganisms). The size of the aggregate (colony) of the grown microorganisms is not particularly limited. The size may be any size as long as it can be properly observed (detected) by the observation (detection) means to be used. In the case of microscopic observation, particularly fluorescence microscopic observation, if the grown microorganisms form at least 4 solid aggregates, they can be determined as grown colonies.

  The subject to which the microorganism testing kit of the present invention is applied is not particularly limited, and various articles can be tested for microorganisms. For example, floors and walls, food and medicine manufacturing devices, meat, fish and vegetable surfaces, homogenized food, cooking utensils, medical utensils, water, and the like can be mentioned.

  The microorganism testing kit of the present invention is other than (A) an adhesive sheet for collecting microorganisms provided with an adhesive layer on at least one side of a support and (B) a medium sheet having at least a medium layer containing a dye for detecting microorganisms. For example, a petri dish or a small incubator used when culturing microorganisms after collection may be included.

Example 1
(Preparation of adhesive sheet)
An acrylic pressure-sensitive adhesive obtained by polymerizing isononyl acrylate / 2-methoxyethyl acrylate / acrylic acid (65/30/5 (weight ratio)) was applied onto a polycarbonate film having a thickness of 100 μm with an applicator. An adhesive layer having a final thickness of 20 μm was formed by drying at 3 ° C. for 3 minutes. This was cut into an appropriate size and γ-ray sterilized to obtain an adhesive sheet.

(Preparation of medium sheet)
A fluorescent dye (SYBR Gold, manufactured by Molecular Probes) was added to SCDA medium (manufactured by Nippon Pharmaceutical Co., Ltd.) so that the final concentration was 1 ppm. This was cast on a 75 μm thick PET film in a clean bench so as to have a thickness of about 0.2 mm to obtain a medium sheet.

(Microbiological examination)
Microorganisms were detected from an aqueous sample using the above adhesive sheet and medium sheet.
E. coli K-121 ml diluted to 6.3 × 10 ⁵ cells / ml with physiological saline is filtered through a filter (Whatman, Nuclepore, pore size 0.2 mm), and an adhesive layer of an adhesive sheet on the filtration surface The (adhesive surface) was lightly pressed with a finger and immediately peeled off. The culture medium layer of the culture medium sheet was stacked on the adhesive surface of the adhesive sheet thus collected and incubated at 25 ° C. for 16 hours. After incubation, it was observed from the medium sheet side with an epi-fluorescence microscope (1000 times, blue excitation). As a result, it was found that an aggregate (colony) of E. coli that emits fluorescence can be detected, and that living microorganisms can be detected using the sheet of the present invention. FIG. 2 is a photograph of an observation image of a microcolony with such an epifluorescence microscope.

Example 2
Using the pressure-sensitive adhesive sheet and the culture medium sheet prepared in Example 1, the microorganisms in the environment were examined. The subjects were the left click part of the mouse, the door knob, the water currant, the handle of the refrigerator door, the water sink, and the palm.

(Microbiological examination)
The pressure-sensitive adhesive layer (pressure-sensitive adhesive surface) of the pressure-sensitive adhesive sheet was lightly pressed with a finger against the test surface of the specimen and immediately peeled off. The medium layer of the medium sheet was stacked on the pressure-sensitive adhesive surface of the collected pressure-sensitive adhesive sheet and incubated at 25 ° C. for 48 hours. After incubation, it was observed from the medium sheet side with an epi-fluorescence microscope (1000 times, blue excitation). As a result, it was found that fluorescent microorganisms (colonies) can be detected, and living microorganisms in the environment can be detected with the sheet of the present invention. Table 1 below shows the detection results. In the table, + indicates that an aggregate of microorganisms could be detected. ++ indicates that an aggregate of microorganisms could be detected and the amount thereof was large. On the other hand, when the same examination was performed on a medium containing no fluorescent dye, no colonies were detected in fluorescence observation.

It is a schematic cross-sectional view of the adhesive sheet for collecting microorganisms and the culture medium sheet of the kit for testing microorganisms of the present invention. 2 is a copy of an observation image photograph of a microcolony obtained by an epi-illumination type fluorescence microscope obtained in Example 1.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Support body 2 Adhesion layer 10 Adhesive sheet for microorganism collection 11 Support body 12 Medium layer 20 Medium sheet

Claims (5)

A kit for microbiological examination comprising the following (A) and (B).
(A): Adhesive sheet for collecting microorganisms provided with an adhesive layer on at least one side of the support (B): Medium sheet having at least a medium layer containing a dye for detecting microorganisms   The microorganism testing kit according to claim 1, wherein the microorganism detecting dye is a fluorescent dye. A microorganism testing method using the microorganism testing kit according to claim 1,
A step of collecting microorganisms by pressing the adhesive layer of the adhesive sheet for collecting microorganisms of (A) to a subject;
Stacking the medium layer of the medium sheet of (B) on the adhesive surface that has collected the microorganisms of the adhesive layer, and culturing the microorganisms;
A method for detecting microorganisms, comprising a step of detecting microorganisms grown through the culturing step.   The microbe inspection method according to claim 3, wherein the microbe detection operation is an operation of microscopically observing the microbe.   The microorganism inspection method according to claim 4, wherein the microorganism detection dye contained in the adhesive layer of the microorganism-collecting adhesive sheet is SYBR Gold, and the microscope is a fluorescence microscope.