JPH054077B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for easily determining caries activity in a healthy person or patient during caries prevention or caries treatment. (Prior Art) Conventionally, microorganisms, especially bacteria such as Streptococcus spp. (hereinafter referred to as S. .
(abbreviated as mutans) and Lactobacillus (lactic acid bacteria)
Various methods have been reported to test the number and activity of bacteria. In Patent No. 539011 (Japanese Patent Publication No. 19817-1981), which was obtained for general bacteria, the applicant disclosed a rapid discrimination method that utilizes the fact that bacteria change the color of specific drugs, but the patented invention can be summarized as follows: As a drug, a certain amount of (a) resazurin, (b) neotetrazolium chloride, or (c) triphenyltetrazolium chloride is added to the culture solution (a nutrient source for microbial growth), and a test paper is immersed in the solution and dried. A certain volume of the bacterial suspension to be tested is dropped onto the test piece, and then incubated at 37°C for a certain period of time. The change in color of the test piece indicates the activity of the bacteria in the bacterial suspension. It is characterized by a method for determining. On the other hand, various methods for determining caries activity have been recently reported by Tsutomu Shimono et al. [Journal of Pediatric Dentistry, 18(3)]
606-611, 1980] The gist of the paper can be summarized as follows. That is, a fixed volume (0.1 ml) of dental plaque collected from the oral cavity and suspended in physiological saline is used as the sample.
Separately prepared sucrose 10%, bromothymol blue (BTB) 0.006%, trisodium nitride ( _Na3N )
Prepare a solution with a concentration of 0.05% and pH 7.2, inoculate it with the sample from above, and leave it at 37°C for 30 minutes. The color tone of the liquid changes to yellow-green, pale yellow-green, and yellow, which can be interpreted as negative, false positive, or positive. When compared with changes in conventional culture-based tests conducted in parallel, a high degree of positive correlation with childhood caries prevalence was observed. Furthermore, Shoichi Nakamura et al. (Journal of the Oral Hygiene Society, 30(4) December 1980) describes the discrimination method and results of oral examinations conducted on kindergarten children as follows. That is, color indicators include resazurin, methylred, alizarinred, lacmalt, bromcresol green, bromophenol blue, etc., and indicators containing these
A culture solution consisting of 0.003% sucrose, 10% sucrose, and 0.05% Na ₃ N was inoculated with a fixed volume of saline suspension of dental plaque and incubated at 37℃ for 30 minutes. As a result of determining caries activity, a high positive correlation was found with the caries-affected tooth rate among kindergarten children. It is stated that among these indicators used in the above drug solution, resazurin has the most vivid color change. As mentioned above, various reports have confirmed that resazurin, which is used as a method for determining caries activity, is an effective indicator. All of these methods are considered to be simple methods, but they are wet methods in which saliva or dental plaque is added to a small amount of coloring solution containing resazurin or a PH indicator, heated to 37°C, and the discoloration is checked 30 minutes later. It is something. However, since this method uses resazurin as a PH indicator, there is a problem in that the coloration is unclear and visual judgment is difficult, so it has not been widely used in clinical practice. Therefore, the most common conventional caries activity test method is to collect 0.1 to 0.2 ml of saliva or dental plaque and mix it with about 3 ml of a medium.
This is a wet method that consists of culturing in an incubator at 37°C for 24 to 72 hours. This method is popular nationwide. However, this method has the limitation that it must be cultured, so an incubator is required, some skill is required for the determination, and the culture results cannot be determined on the spot, so it cannot be used for research. However, there was a problem in that it was difficult to motivate the subjects. (Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide a method for determining the activity of caries-causing microorganisms, which does not have the above-mentioned drawbacks. (Means for Solving the Problems) The present inventors further simplified the method for determining caries activity using resazurin and investigated a method that could be determined in a short time. The present invention has been achieved. That is, the present invention provides a test piece containing a reagent composition for determining the amount of caries-causing microorganisms in saliva (or dental plaque, hereinafter referred to as dental plaque). A certain amount of infiltration is infiltrated, this infiltrated material is sandwiched between a film having an adhesive layer, sealed, and kept at a constant temperature for a certain period of time to culture caries-causing microorganisms, and then the color change of the test piece is measured. The purpose of the present invention is to provide a simple dry caries discrimination method characterized by the following. The present invention will be explained in detail below. "Caries-causing microorganisms in saliva" refers to microorganisms present in saliva that cause caries, especially bacteria, such as S.mutans, Lactobacillus,
Examples include Actinomyces and the like. The reagent composition in the present invention refers to an enzyme reaction caused by caries-causing microorganisms in saliva;
A composition containing a reagent that can detect changes in redox potential, etc., and a culture component that promotes the growth of caries-causing microorganisms. Examples of the reagent include resazurin, triphenyltetrazolium, methyl orange, salts thereof, and mixtures thereof. Among these, resazurin, triphenyltetrazolium, neotetrazolium, 2,6-dichlorophenol indophenol, and methyl orange are preferred. The above-mentioned culture components that promote the growth of caries-causing microorganisms include any culture components that promote the growth of caries-causing microorganisms, but examples include:
Examples include sucrose, glucose, and fructose. Among these, sucrose is preferred. An example of the ratio of reagents and culture components is 1:100 ~
1:500 is listed, among which 1:300 to 1:
400 is preferred. Furthermore, in addition to these components, a sizing agent such as polyvinylpyrrolidone, methylcellulose, hydroxypropylcellulose, carboxymethylcellulose, starch paste, or gelatin may be included for the purpose of spreading the reagent. The proportion of these components is 0.5 to 5% by weight based on the total weight of the reagent composition.
is preferred. A test piece can be prepared by incorporating the above reagent composition into an inorganic or organic porous body. Examples of the porous body include paper, nonwoven fabric, and sheet-like materials made of natural or synthetic materials. Among these, water-absorbing sheet-like materials such as paper and sponge are preferred. The shape of the test piece can be any shape, but circular, rectangular, etc. are preferable. The area of the test piece is preferably 25 to 80 ^mm2 . A test piece containing the above reagent composition can be produced as follows. First, a reagent composition solution containing 5 to 20% by weight of the reagent composition is prepared. The solvent for the above solution includes water, alcohol, isopropyl alcohol,
Mention may be made of acetone and mixtures thereof. Among these, water and alcohol are preferred. After immersing the porous body in the above reagent composition solution for 1 to 3 minutes, the porous body was taken out and heated at 50 to 80°C for 30 to 120 minutes.
Let dry for a minute. A certain amount of saliva to be tested is infiltrated into the above test piece. The fixed amount may be any amount by which a change in color tone of the test piece can be measured, and may be, for example, 10 to 100μ. These amounts are in the dropper.
Equivalent to 0.5 to 2 drops. The saliva to be tested can be infiltrated into the test piece by dropping it with a dropper, draining it from a capillary, or applying saliva entwined with a plastic or glass rod. Among these, the method of dropping with a dropper is preferred. The above-mentioned infiltrated material is sandwiched between films having an adhesive layer, and the air inside is removed by pressing to form a sealed state. The film can be anything that can keep the infiltrate sealed. Here, the sealed state refers to a state in which there is no risk of gas or microorganisms entering. Examples of the film material include polyvinyl chloride, polyvinylidene chloride, polyethylene, polypropylene, and the like. this house,
Polyvinyl chloride is preferred. An example of the thickness of the film is 50 to 300 μm, preferably 125 to 175 μm. The film can be any size that will keep the infiltrate sealed. Generally, it is preferable to have an area 5 to 10 times the area of the test piece. The shape of the film may be any shape capable of keeping the infiltrate sealed. For example, even if two separate films sandwich the infiltrate like a sandwich,
It may be one continuous film in which the film is folded to sandwich the infiltrate. In addition, the supporting film does not necessarily need to be a film, but a glass plate,
You can also use plate-like materials that do not absorb moisture, such as chinaware, metal plates, or waterproof wood. The adhesive layer refers to a layer capable of adhering between films and between films and infiltrated matter. The adhesive layer is made of natural resins such as rosin and copal, synthetic hydrocarbon resins such as polyethylene, lanolin,
Synthetic rubber pressure-sensitive adhesives made by blending and kneading synthetic rubber or natural rubber with a waxy substance such as beeswax can be preferably used. The adhesive layer can be present on all or part of the film, provided that it can keep the infiltrate sealed. For example, the adhesive layer can be provided on the entire surface of two separate films or one continuous film, or around the position on the film where the infiltrate is placed. Of these, using two separated films,
Preferably, an adhesive layer is provided on one entire surface of the two films. Furthermore, it is preferable that the caries-causing microorganism can be adhered to a warmer or the like via an adhesive layer when culturing the caries-causing microorganism. Further, the test piece may be previously present on the film with or without an adhesive layer interposed therebetween. The sealed infiltrate is kept at a constant temperature for a certain period of time to cultivate caries-causing microorganisms. The constant temperature is 25 to 40℃, of which
35-37°C is preferred. Examples of the certain period of time include 10 to 30 minutes, with 10 to 15 minutes being preferred. Further, the culture can be carried out in air or warm water, for example, in a warmer, incubator, human skin, warm bath, etc. After the above culture, the amount of caries-causing microorganisms is determined by measuring the change in color of the test piece. To explain this in detail, there are many S. mutans and lactic acid bacteria, which are said to be the main cause of dental caries, in saliva or dental plaque.In particular, the enzyme of S. mutans condenses sucrose in the culture solution to produce glucan. (dextran) is made. Sugar is also enzymatically broken down to lactose. It is thought that the color tone of resazurin changes based on this active enzymatic reaction, changes in redox potential, etc. For example, the color tone changes from purple to bluish-red to red depending on the test solution. As shown in the examples below, this color change and caries activity are correlated. Therefore, caries activity can be determined based on this. When produced in the same manner as above using triphenyltetrazolium chloride as an indicator, the color tone changes from red to colorless. This color change corresponds to a change in caries activity from negative to positive. When methyl orange is used as an indicator, the color changes from yellow-green to red-yellow. The caries activity in this case corresponds to a change from negative to positive. Among these indicators, resazurin has the most vivid color change, changes color in stages, and is easy to distinguish with the naked eye. Therefore, it is desirable to mainly use resazurin as an indicator. The change in color tone can be measured visually, using an absorbance meter, a color densitometer, or the like. Among these, it is preferable to visually judge using a colorimetric table. In one embodiment of the device for carrying out the method of the present invention, a 10% by weight aqueous solution containing sucrose and resazurin in a ratio of 1:350 is prepared, and a small circular or square piece of paper is soaked in the solution and dried. Place it on the mount,
The small pieces of paper are sandwiched in a sandwich pattern and overlapped with a circular or rectangular covering film coated with an adhesive on the inside, or when the small pieces of paper are not to come into contact with the adhesive, the adhesive is applied around the inside. A circular or rectangular covering film applied only to the area is overlapped, and a supporting film (a film that supports the small piece of paper and the covering film during the test) is placed side by side on the same mount, and Both the film and the covering film are formed by being removably adhesively attached to the mount. In FIG. 1 of the drawings showing a specific example of the discrimination tool for carrying out the method of the present invention, symbol 1 is a small piece of paper containing a reagent composition and having a diameter of 5 to 10 mm or a rectangular piece of paper with a side of 4 mm or more. The material for the small pieces of paper is commonly used filter paper, preferably filter paper with a thickness of about 0.8 mm or less. This is covered with a covering film 2 having an adhesive layer on the inner side 2A of the peripheral portion, and a supporting film 3 is placed side by side with these on a mount 4 so that it can be peeled off. However, if an adhesive layer is provided on the entire inside of the covering film, one side of the small paper piece will always remain fixed to the inside of the covering film, which is often convenient for handling during testing. When formed in this manner, the small piece of paper 1 before use is completely isolated from the outside air, no air flows through it, and moreover, it is held aseptically.
The supporting film 3 may have a circular shape with a diameter of 5 to 10 cm, or a square or rectangle with a similar area, and the film 2 may have a circular shape with a diameter of 3 to 5 cm or a rectangular shape with a similar area. The size of the mount is film 2
and the film 3 are large enough to be juxtaposed. Next, the small piece of paper 1 contains sucrose, resazurin,
Triphenyltetrazolium, neotetrazolium, 2,6-dichlorophenol indophenol or methyl orange or their salts in 1:
It is contained in a ratio of 100 to 1:500. Next, an adhesive layer containing an adhesive is applied to the film 2. Although the adhesive is mainly made of synthetic rubber, it can be easily removed from the supporting film 3 and the mount 4 at room temperature, and it can be used together with small pieces of paper to heat the heating equipment, such as metal, wood, plastic, etc. Any material that can be attached to the heating equipment for use is sufficient. That is, the small piece of paper soaked with saliva or dental plaque must be incubated at 32-37°C for 10-15 minutes while blocking air supply as much as possible and preventing evaporation of water in saliva. Since the device for carrying out the method of the present invention is a simple small device, it may be used as a substitute for a heating device by attaching the film together with the surface of the human skin to heat it. Therefore, it is desirable to select an adhesive to be included in the adhesive layer on the film that does not harm the skin as a whole. Note that when the supporting film 3 is not attached to a heating device or a human skin surface, but is used in a manner that it is housed in a warming device, the supporting film 3 does not need to have an adhesive layer. In order to use the identification tool for implementing the method of the present invention, collect a small amount of saliva (usually one drop) with a dropper, etc. Separately, while peeling half of the film 2 from the mount of the tool, pour the saliva from the dropper onto a small piece of paper 1 inside. After the droplets have soaked and spread sufficiently uniformly, cover the small piece of paper 1 with the film 2 and apply the supporting film 3.
Apply adhesive on top. FIG. 2 shows a case where the film 2 has an adhesive layer around its inner surface. Peel off the supporting film 3 with the small piece of paper 1 and the covering film 2 still supported from the mount 4, and apply it to the surface of the warmer or an appropriate exposed part of the human body as a substitute for it.15 Observe the change in color of small piece of paper 1 after a minute. The caries activity is determined as negative, weakly positive, or positive according to this color change, ie, blue, purple-red, or red. A feature of the above-mentioned device is that it can use human body temperature as a substitute for a warmer to maintain the culture temperature at around 37°C. When the covering film 2 has an adhesive layer on the entire inner surface, when half of the adhesive layer is peeled off, the small piece of paper 1 inside is removed.
Since its front surface is adhered to the film 2, its back surface is exposed, and therefore saliva is added to this back surface. After saliva infiltration, peel off the small piece of paper 1 together with the film 2 from the mount 4,
This is adhered onto the supporting film 3. This state is shown in FIG. The supporting film 3 and the supporting film 3 are peeled off from the mount 4 and attached to the surface of the warmer or the surface of the exposed part of the human body as a substitute thereof. If the supporting film 3 is to be stored in the warmer without being pasted, the support film 3 may be stored in the warmer together with the mount 4 without peeling it off. (Effects of the Invention) Based on the above, the identification method of the present invention can be used to easily and quickly determine caries activity not only in caries sufferers but also as a preventive or therapeutic measure for caries in children. Summer. Furthermore, since it is easy to manufacture tools for carrying out the method of the present invention, it has become possible to discriminate between negative and positive caries activity at a low cost. The method of the present invention has made it possible to test with a small amount of saliva. Further, according to the method of the present invention, saliva collection does not require an operator and can be easily performed by an amateur. Furthermore, the method of the present invention does not involve operations such as licking or applying the small piece of paper containing the reagent composition into the oral cavity, and the reagent composition does not enter the body orally, or is applied to the small piece of paper containing the reagent composition. It also has the advantage that it does not have any physiological effects on the human body because it does not involve touching with fingers. Furthermore, the method of the present invention has the advantage that it is extremely clean because the small piece of paper remains sandwiched between the films after inoculation with saliva and can be disposed of in this state after the determination. (Example) Next, an example will be shown. Reference example: A piece of filter paper with a diameter of 8 mm is immersed in an aqueous solution of 0.025% by weight of resazurin and 10% by weight of sucrose for 5 minutes at 70℃.
By drying for 90 minutes, a small piece of paper 1 (disk) for identification was produced. This has a diameter of 4 mm with an adhesive layer containing an adhesive (synthetic rubber type) on the entire inner surface.
Cover with 2cm transparent plastic covering film (150μ polyvinyl chloride film), 4.5cm
A 4.5 cm x 4.5 cm rectangular transparent plastic support film (200μ polyvinyl chloride plate) 3 (with an adhesive layer on the back side) is juxtaposed and pasted on a 5 cm x 10 cm rectangular mount 4, and this is then attached to a transparent plastic A single-use product was prepared by heat-sealing the product in a packaging sachet or small plastic bag. Example: For the saliva sample, collect saliva from each subject into a mini-cup, then use a plastic dropper to collect a drop (approx.
0.05 ml) was removed and exposed by peeling off the film 2 of the tool manufactured in Reference Example, or sufficiently permeated into the blue disk (small piece of paper) 1 taken out, and then these were pasted onto the supporting film 3 and sealed. These were peeled off from the mount 4 and pasted on the subject's arm, and 15 minutes later, the change in color tone of the disc 1 was examined. The results of comparing this with the previous testing methods of using ST media and using MSBB are shown in the first section.
Shown in the table.

[Table] According to the results of this example, the results of "ST Media" and "MSBB" (which have been proven to show a positive correlation with the caries prevalence status in adults) and the judgment based on the color tone change of this tool clearly shows a positive correlation. From this, the caries discrimination method of the present invention is easy to implement and can be carried out in a short time using a dry method instead of a wet method.
It is found to be effective for easily determining caries activity.

[Brief explanation of drawings]

FIG. 1 is a plan view of a discriminating tool for carrying out the method of the present invention, and FIGS. 2 and 3 are side views of the same tool in use. Symbols, 1... Dry small piece of paper for testing, 2... Covering film, 2A... Inner peripheral portion having adhesive layer of film 2, 3... Supporting film, 4... Mounting paper.

Claims (1)

[Scope of Claims] 1. A test piece containing a reagent composition for determining the amount of caries-causing microorganisms in saliva is infiltrated with a certain amount of saliva to be tested, and this infiltrated material is coated with an adhesive layer. A dry method characterized in that the test piece is sandwiched between films and pressed to exclude the air inside to create a sealed state, kept at a constant temperature for a certain period of time to culture caries-causing microorganisms, and then measures the color change of the test piece. How to identify caries. 2. The method according to claim 1, wherein the reagent composition contains resazurin triphenyltetrazolium or methyl orange or their salts and sucrose. 3. The method according to claim 1, wherein the test piece is a small piece of paper. 4. The method according to claim 1, wherein the constant temperature is around 37°C and the holding time is about 15 minutes. 5 The constant temperature is room temperature (around 20℃) and the holding time is 20 to 30 minutes.
2. The method according to claim 1, wherein the method takes about a minute.