JP2002142797A - Method for examining microorganism on surface of solid and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a method for examining microorganisms with which existence of and/or the number of microbial cells of microorganisms on the surface of a solid can be simply monitored in real time and a kit for examining microorganisms used for the method. SOLUTION: This method for examining microorganisms existing in a specimen comprises sticking an adhesive layer of an adhesive sheet for examining microorganism, having the adhesive layer consisting essentially of a water- insoluble polymer compound, to the surface of a specimen through the force of pressure, releasing the sheet, accumulating microorganisms and bringing an aqueous solution containing one or more kinds of coloring substances capable of dyeing the microorganisms to detect the microorganisms. This kit for examining microorganisms comprises the adhesive sheet for examining microorganisms and one or more kinds of the coloring substances.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel microorganism test method. More specifically, the present invention relates to a microorganism test method for collecting microorganisms using an adhesive layer and detecting the microorganisms by staining the collected microorganisms.

[0002]

2. Description of the Related Art Conventionally, in order to observe and count microorganisms such as bacteria that cannot be observed with the naked eye on a test surface, a culture method, that is, a solid plate medium formed with agar or the like is tested. By pressing against the surface, the microorganisms on the test surface are transferred to the plate medium, and the microorganisms are cultured as they are on the plate medium under an optimal environment, and the colonies appearing are measured while visually or by a stereoscopic microscope. The method used is generally used. For example, there is an agar stamp method using a food stamp (manufactured by Nissui Pharmaceutical Co., Ltd.).

In the membrane filter method using a membrane filter or the like capable of trapping microorganisms, microorganisms are washed out by accumulating the test surface while wiping it sufficiently with a physiological saline solution, a phosphate buffer solution or the like, and washing out the microorganisms. After collecting the microorganisms on the membrane filter by filtering the accumulated liquid through a membrane filter, the microorganisms are brought into sufficient contact with the liquid medium to form colonies on the filter, and the number of colonies is counted. . The membrane filter method is also used as a method for detecting microorganisms without culturing by contacting the microorganisms collected on the filter with an appropriate staining solution and counting the number of colored cells with a microscope or the like. can do.

However, in the agar stamp method and the like,
Usually, since it can be used only once for one test surface, the collection efficiency changes depending on the water content of the agar medium, and there are cases where inconvenience is caused in the collection efficiency of microorganisms such as poor reproducibility. In addition, as a common problem of the culture method, contamination between microorganisms occurs, and pure culture cannot be performed due to interaction between microorganisms on a medium, which may cause inconvenience in subsequent determination. In addition, of course, the culture method has a restriction that it is limited to only living bacteria, and there is a problem that detection is lost. Furthermore, since the culturing method requires a culturing time of 1 to 2 days or more, there is a serious limitation that real-time microorganism monitoring cannot be performed.

[0005] In the membrane filter method, if the subject is a liquid such as an aqueous solution, it can be filtered as it is. However, in the case of a non-liquid subject, sampling with a cotton swab, preparation of a washing solution, and the like greatly increase the accumulation of microorganisms. There was a disadvantage that it took a lot of effort. Further, there is another problem that the collected matter other than the microorganisms swells due to the washing and filtering operations, which hinders subsequent observation and measurement.

[0006] Recently, a technique for detecting ATP (adenosine triphosphate) in microbial cells has also been developed. However, this technique is also limited to microorganisms dispersed in water, which also complicates the method of collecting bacteria. Was an issue.

[0007]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a novel method for testing microorganisms which has solved the above-mentioned drawbacks of the conventional method, and in particular, to measure the presence of microorganisms on a solid surface and / or the number of the microorganisms in real time. It is an object of the present invention to provide a microorganism test method which can be easily monitored by using the above method. Another object of the present invention is to provide a kit for testing a microorganism for use in the method.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, a microbial test pressure-sensitive adhesive sheet having a pressure-sensitive adhesive layer containing a water-insoluble polymer compound as a main component has been tested. After the microorganisms are accumulated by pressure bonding and peeling on the surface of the solid, the aqueous solution containing one or more color-forming substances capable of staining the microorganisms is brought into contact with the surface of the adhesive layer, and the stained cells are observed. -By counting, microorganisms on the solid surface were successfully detected quickly and easily, and the present invention was completed.

That is, the present invention provides a microorganism testing pressure-sensitive adhesive sheet having a pressure-sensitive adhesive layer containing a water-insoluble polymer compound as a main component. A method for testing a microorganism present in the subject, comprising detecting the microorganism by contacting an aqueous solution containing one or more color-forming substances capable of staining the microorganism with the surface of the adhesive layer.

In the method of the present invention, microorganisms captured and accumulated on the surface of an adhesive layer of an adhesive sheet (hereinafter also referred to as an adhesive surface) are colored with a color-forming substance while being retained on the adhesive surface without culturing. For this purpose, microorganisms such as bacteria, fungi, and viruses can be detected and / or counted in real time by visually judging with the naked eye or observing the coloring state and the coloring amount using a microscope or an optical device.

[0011] The present invention also provides a kit for testing microorganisms, which is suitable for easily and quickly performing the method for testing microorganisms of the present invention. Accordingly, another aspect of the present invention is a microbial test pressure-sensitive adhesive sheet having a pressure-sensitive adhesive layer containing a water-insoluble polymer compound as a main component, and a microbial test including an aqueous solution containing one or more color-forming substances capable of dyeing microorganisms. Kit.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The pressure-sensitive adhesive sheet for a microorganism test used in the present invention has a structure in which a pressure-sensitive adhesive layer mainly composed of a water-insoluble polymer compound is laminated on a substrate. The adhesive layer is a layer having a smooth surface structure, which has sufficient adhesiveness to capture microorganisms on the test surface and is characterized in that the adhesive does not dissolve even when immersed in an aqueous solution for staining microorganisms. is there.

[0013] The pressure-sensitive adhesive sheet has a surface opposite to the surface in contact with the pressure-sensitive adhesive layer of the substrate, which increases the strength of the substrate, prevents optical scattering, and improves the visibility of microorganisms that have been colored using a coloring substance. A backing layer may be further provided to form a background color to be formed.

The base material of the pressure-sensitive adhesive sheet for microbial test is preferably a water-insoluble, flexible material that does not form large irregularities on the surface of the pressure-sensitive adhesive layer and that can be freely pressed on curved or narrow surfaces. Without limitation, polyester, polyethylene,
Examples thereof include polyurethane, vinyl chloride, cloth, nonwoven fabric, paper, and polyethylene laminated paper. Among them, smooth polyester, polyethylene, vinyl chloride, and polyurethane are preferable as the base material. The thickness of the substrate is not particularly limited as long as it has sufficient strength as a support.
It is preferably about μm.

The pressure-sensitive adhesive of the pressure-sensitive adhesive layer has a tackiness capable of capturing microorganisms on the test surface, and is not particularly limited as long as it is not dissolved in an aqueous solution. For example, natural rubber, synthetic rubber, silicone-based pressure-sensitive adhesive, or Polymer compounds such as polyacrylic acid-based polymer compounds, polyacrylate-based polymer compounds, ethylene acetic acid copolymers, polyvinyl ethers, polyvinyl esters, and the like can be mentioned, and mixtures and copolymers of two or more compounds can be used. May be. The production of these polymer compounds can be performed by known methods. For example,
Polyacrylic acid-based polymer compound, liquid polymerization, emulsion polymerization,
It may be produced by any method such as suspension polymerization, bulk polymerization, and photopolymerization. If necessary, conventional additives such as a tackifier, an antioxidant, a crosslinking agent, and a filler can be added to these polymer compounds.

When the captured microorganisms are measured with a microscope or the like, the smoothness (roughness difference) of the surface is preferably 20 μm or less in order to focus on the microorganisms accumulated on the surface of the adhesive layer. When the smoothness is 20 μm or less, the range of the focal point of the microscope is widened, and the counting becomes easy. The smoothness can be determined by observing the cross section of the pressure-sensitive adhesive sheet for microbial test using a surface roughness meter or an electron microscope, and measuring the height difference from the top of the convex portion to the lowest point of the concave portion on the surface of the pressure-sensitive adhesive layer.

The pressure-sensitive adhesive sheet for a microorganism test used in the present invention is produced by a method known per se. For example, it is manufactured by applying a solution containing a polymer compound to be used for the adhesive layer on a base material and drying the solution at room temperature to 200 ° C.
In addition, a method such as a calendering method, a casting method, or an extrusion molding method can be used. The sheet thus obtained can be cut into an arbitrary shape and used.

In the present invention, the polymer sheet used for the adhesive layer can be cross-linked simultaneously with sterilization by irradiating the adhesive sheet for microbial test with radiation such as an electron beam or γ-ray. In addition, sterilization with a gas such as ethylene oxide can be performed, and a sterile state can be maintained by enclosing the sterilized state in a microorganism-blocking packaging material.

The microorganism test method of the present invention will be described. Microorganisms to be tested include prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and mold, lower algae, viruses, and cultured cells of animals and plants.

The pressure-sensitive adhesive sheet for testing microorganisms is pressed against a test surface such as a floor or a wall, and the microorganisms adhering to the test surface are efficiently transferred and accumulated. When the test surface which is considered to have relatively few microorganisms is pressure-bonded, the pressure-sensitive adhesive sheet may be pressure-bonded a plurality of times on the same surface. Since the method of the present invention does not require culturing unlike the agar stamp method, there is no need to worry about colony contamination, and there is no need to worry about changes in the bacterial flora during culturing. be able to. Therefore, by increasing the number of times of pressure bonding, many microorganisms can be collected as in the case of filtering and concentrating microorganisms dispersed in water in the membrane filter method.

Next, the pressure-sensitive adhesive sheet on which the microorganisms are accumulated is cut into a predetermined size as necessary, and the surface on which the microorganisms are accumulated is immersed in an aqueous solution containing a coloring substance to stain the microorganisms. If it is necessary to remove excess color-forming substances, the surface on which the microorganisms are accumulated is rinsed and washed with sterile water or the like. If it is necessary to dry the surface on which the microorganisms are accumulated after staining the microorganisms, the surface can be dried by air drying, natural drying, drying under reduced pressure, or the like. By observing this under a microscope, particularly when the color-forming substance is a fluorescent dye, by observing under a microscope under excitation light, a detection function is provided and microorganisms can be directly counted. According to the present invention, since no culturing operation is required, the microorganism can be detected within a few minutes.

The color-forming substance used in the present invention is not particularly limited as long as it is capable of forming a color by acting on cell components contained in a microorganism to be tested. Fluorescent staining solution. More specifically, in the case of microbes in general, a fluorescent nucleobase analog, a fluorescent stain for staining nucleic acids, a staining solution for staining proteins, an environment used for structural analysis of proteins, etc. In the case of an aerobic bacterium, a staining solution used for analysis of cell membrane or membrane potential, a staining solution used for labeling fluorescent antibody, etc. For microorganisms, stains for mitochondria, stains for the Golgi apparatus, stains for the endoplasmic reticulum, stains that react with intracellular esterases, and their modifying compounds, etc. are targeted at higher animal cells. In this case, a staining solution used for observing bone tissue, a staining solution that is a nerve cell tracer, and the like can be mentioned, and these can be observed with a fluorescence microscope.

By selecting the types of these chromogenic substances, the total number of bacteria to be detected for all microorganisms, the assay for staining and counting only microorganisms having respiratory activity, and the staining and counting for only microorganisms having esterase activity are counted. This method can be applied to a wide range of fields, such as an assay that performs a double staining method using a combination of a plurality of chromogenic substances, or an assay that stains and counts microorganisms of a specific genus or species.

The detection or counting of microorganisms is performed by visual judgment with the naked eye, or by forming an optical image using a microscope such as an optical microscope, a fluorescent microscope, a laser microscope or other appropriate optical equipment, and analyzing this image by image analysis. Can be performed. As another optical device, a laser scanning cytometer that scans microorganisms at high speed with laser light and graphically displays signals obtained from individual microorganisms is exemplified. Since the present invention does not require a culturing operation, the microorganisms on the adhesive surface of the adhesive sheet can be substantially detected within several minutes to several tens of minutes.

The present invention also provides a microorganism test kit for use in the above-described microorganism test method. The microorganism test kit of the present invention is a microorganism test pressure-sensitive adhesive sheet having a pressure-sensitive adhesive layer containing a water-insoluble polymer compound as a main component,
It includes an aqueous solution containing one or more chromogenic substances capable of staining microorganisms. As the microorganism test sheet and the color-forming substance, those similar to those exemplified in the method of the present invention can be used.

As an example of an application of the present invention, an adhesive surface is attached to a test surface to transfer microorganisms present on the test surface,
Since the microorganisms can be stained without pre-culture and the microorganisms can be observed as a single cell, it can be used for environmental investigation to quickly measure the cleanliness of a subject. Furthermore, since the recovery is performed at a single cell level, the pressure-sensitive adhesive sheet can be pressed against the test surface a plurality of times to accumulate and concentrate microorganisms, which is practical. As an application field, it can be applied to microbial testing of the environment in medical and food fields.

[0027]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the scope of the present invention.

Example 1 1) Preparation of pressure-sensitive adhesive sheet for microbial test 70 parts by weight of 2-ethylhexyl acrylate 25 parts by weight of ethoxyethyl acrylate 5 parts by weight of acrylic acid 150 parts by weight of ethyl acetate 0.3 parts by weight of azoisobutyronitrile Was charged into a polymerization reaction vessel, and the mixture was stirred while replacing the inside of the reaction vessel with nitrogen. After that, the internal bath temperature
The polymerization was maintained at 65 ° C. for about 10 hours. Next, the inner bath temperature was raised to 70 ° C., and stirring was continued for about 2 hours. The glass transition temperature of the obtained copolymer is 212K,
The gel fraction was 31.5%. Next, the pressure-sensitive adhesive was dried on a release paper having a surface treated with silicone to a thickness of 20 μm.
And dried at 130 ° C. for 5 minutes. A 75 μm-thick polyester film that had been subjected to a single-sided corona treatment was attached to the pressure-sensitive adhesive surface of the release paper containing the pressure-sensitive adhesive, followed by pressure bonding, and cut so that the effective pressure-sensitive adhesive surface was about 10 cm ² .

2) Measurement A solution obtained by diluting a staphylococcal culture solution with a sterile phosphate buffer 4
μL was dropped on a surface of plastic or the like, and the naturally dried surface was used as a test object. The pressure-sensitive adhesive sheet from which the release paper was peeled off was repeatedly pressed and peeled off the test object three times to accumulate staphylococci. Next, the adhesive surface on which the bacteria were accumulated was cleaned with sterile deionized water for 1 hour.
SYBR Green II (SYBR Green II RNA Gel)
Stain, manufactured by Molecular Probes Inc.) was dropped and air-dried, and a fluorescence microscope with an excitation wavelength of 490 nm (magnification: 1,
The number of bacteria stained green was measured using 2,000 times).
The results are shown in Table 1 together with Comparative Example 1. Compared with the wiping-membrane filter method, the same or more bacteria were detected.

Comparative Example 1 As a comparative example of the present invention, a wiping-membrane filter method was performed. A test substance prepared in the same manner from the same culture solution as in 2) of Example 1 was wiped off by a wiping method, that is, wiped with a sterilized hydrous cotton swab (trade name: Wiping cloth check, manufactured by Eiken Instruments) and suspended in sterile physiological saline. . Next, add SYBR Green II so that the dilution is 10,000 times, stain for 15 minutes, filter the suspension with a polycarbonate filter with a pore size of 0.2 μm, capture the stained bacteria on the filter, and air dry. Thereafter, the number of bacteria was measured in the same manner as in the examples. The results are shown in Table 1 together with Example 1.

[0031]

[Table 1]

Example 2 The number of microorganisms in a natural environment was measured without using any thing around the body as a test object without artificially adding bacteria. Except for the conditions of the test substance, the bacteria on the test substance surface were accumulated, stained and measured in the same manner as in 2) of Example 1. Table 2 shows the results. Compared with the wiping-membrane filter method, the same or more bacteria were detected.

Comparative Example 2 In the same manner as in Comparative Example 1, the same test substance as in Example 2 was wiped off the surface of the test substance, stained, filtered, and measured. Table 2 shows the results. Although it does not appear in the number of bacteria, the wiping-membrane filter method also collects dirt and the like swelled with water in addition to the bacteria.

[0034]

[Table 2]

Example 3 The viable cell count according to the present invention was measured using staphylococci. Staphylococci on the surface of the test article produced in the same manner as in 2) of Example 1 were accumulated on an adhesive sheet. Next, 6-carboxyfluor prepared at 150 μg / mL was applied to the adhesive surface on which the bacteria were accumulated.
Escein diacetate (manufactured by Sigma, hereinafter abbreviated as 6CFDA) was added dropwise, and after natural drying, the number of bacteria emitting green fluorescence was measured using a fluorescence microscope as in 2) of Example 1.
The results are shown in Table 3 together with the results obtained by staining with the same SYBR Green II as in Example 1 and measuring the number of bacteria. Bacteria with esterase activity can be detected in more than 10 minutes, and SYBR Green II
Approximately 60 to 95% of the total number of bacteria determined in the above.

[0036]

[Table 3]

[0037]

According to the present invention, microorganisms on a solid surface can be efficiently collected without performing culturing or filtration of a washing solution, and the collected microorganisms can be easily detected and / or detected in real time. Can be counted.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shuji Senda 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation (72) Inventor Shinyasu Yamaguchi 4-9-1, Kamisumi, Ibaraki-shi, Osaka No. (72) Inventor Masao Nasu 3-15-9 Miyakojimahondori, Miyakojima-ku, Osaka-shi, Osaka F-term (reference) 4B029 AA03 AA07 AA08 AA09 BB02 CC07 EA16 FA01 FA11 GA08 GB09 HA06 HA10 4B063 QA01 QA18 QQ06 QQ16 QR66 QR69 QR84 QS10 QS39 QX02 4C085 JJ12 KA23 KB37 KB99 LL20

Claims (7)

[Claims] 1. A pressure-sensitive adhesive sheet for a microorganism test having a pressure-sensitive adhesive layer containing a water-insoluble polymer compound as a main component. A method for testing microorganisms present in the subject, comprising detecting the microorganisms by contacting the obtained aqueous solution containing one or more coloring substances with the surface of the adhesive layer. 2. The method according to claim 1, wherein the aqueous solution containing the coloring substance is not filtered. 3. The method according to claim 1, wherein the color-forming substance is a fluorescent material. 4. The method according to claim 1, wherein the surface of the pressure-sensitive adhesive layer of the pressure-sensitive adhesive sheet for microorganism test has a smoothness of 20 μm or less. 5. A microbial test kit comprising a pressure-sensitive adhesive sheet for microbial test having a pressure-sensitive adhesive layer containing a water-insoluble polymer compound as a main component, and an aqueous solution containing one or more color-forming substances capable of dyeing microorganisms. 6. The kit according to claim 5, wherein the chromogenic substance is a fluorescent material. 7. The kit according to claim 5, wherein the surface of the pressure-sensitive adhesive layer of the pressure-sensitive adhesive sheet for testing microorganisms has a smoothness of 20 μm or less.