JPH03272696A - Simple discrimination of dental caries - Google Patents

Abstract

PURPOSE:To discriminate dental caries activity simply and in a short time by wetting spittle specimen with a test piece for discriminating a microorganism of causing dental caries in spittle, sandwiching the test piece in between films having an adhesive layer, sealing, culturing the microorganism and then measuring change in color tone of the test piece. CONSTITUTION:A test piece comprising a small paper piece 1 prepared by immersing a filter paper piece in a reagent composition which comprises an aqueous solution of resazurin and sucrose and discriminates the amount of microorganism of causing dental caries in spittle and drying at 70 deg.C for 90 minutes is wetted with a fixed amount of spittle specimen, the wetted material is covered with a covering transparent plastic film 2, placed side by side with a supporting film 3, stuck to rectangular board 4, put in a packing sash made of a transparent plastic, made into a sealed state, maintained at room temperature for a given time, the dental caries causing microorganism is cultured and change in color tone of the test piece is measured to discriminate dental caries activity in a dry method.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for easily determining the caries activity of a healthy person or patient during caries prevention or caries treatment.

<Prior Art> Conventionally, microorganisms, particularly bacteria, such as Streptococcus mutans (hereinafter referred to as S) in saliva or dental plaque in the oral cavity have been used as a means of determining the susceptibility or progress of caries from the standpoint of clinical dentistry or oral hygiene.

mutans) and Lactobacillus (lactic acid bacteria)
Various methods have been reported to test the number and activity of bacteria. The applicant has patent No. 53, which was obtained for general bacteria.
No. 9011 (Japanese Patent Publication No. 43-19817) disclosed a rapid identification method that utilizes the fact that bacteria change the color of a specific drug.
(b) Add a certain amount of resazurin, (b) neotetrazolium chloride, or (c) triphenyltetrazolium chloride to the culture solution (a nutrient source for microbial growth), and soak a test paper in the iron solution: test the dry one. A certain volume of the bacterial suspension to be tested is dropped onto the test piece, and then heated to 37°C.
This method is characterized by a method in which the activity of the bacteria in the bacterial suspension is determined by culturing the test piece for a certain period of time and observing a change in the color tone of the test piece.

On the other hand, various methods for determining hyperactivity have recently been reported. Tsutomu Shimono et al.: Journal of Pediatric Dentistry, 18(3), 606-6.
11. The gist of 1980E can be summarized as follows.

That is, a fixed volume (0.1 m) of dental plaque collected from the oral cavity and suspended in physiological saline is used as a sample. Separately prepared sucrose 10%, bromothymol blue (B, T, B)
0.006%, trisodium nitride (NaJ) 0.0
A solution was prepared with a concentration of 5% and a pH of 7.2, inoculated with the above specimen, and left at 37° C. for 30 minutes. The color of the iron solution changes from yellow-green to pale yellow-green to 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 was observed with children's caries prevalence. Furthermore, Tadashi Nakamura
Others [Journal of the Oral Health Society, 30 (4) December 1980]
describes the discrimination method and results obtained when performing an oral examination on kindergarten children as follows. That is, the color indicators include resazurin, methyl red, alizarin red, rough malt, bromcresol green, fromphenol blue, etc., and the tooth was placed in a culture solution containing 0.003% of the indicator containing these, 10% of sucrose, and 0.5% of Na5NO. A fixed volume of saline suspension of plaque was inoculated at 37°C.
After incubating for 30 minutes, the caries activity was determined by the color change in the culture solution, and a high positive correlation was found with the rate of caries-affected teeth in 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, it has been recognized in various reports that resazurin, which is used as a method for determining irritability, is an effective indicator. Although these methods are considered to be simple methods, 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 and heated to 37°C.
This test detects discoloration after 0 minutes. However, this method;
However, since resazurin is used as a pf (tM indicator), there is a problem that the coloration is unclear and visual judgment is difficult, so it is not widely used in clinical practice. The conventional caries activity test method is saliva 0,1
~0.2- Alternatively, it is a wet method consisting of collecting dental plaque, mixing it with about 3 ml of culture medium, and culturing it in an incubator at 37°C for 24 to 72 hours. This method is popular nationwide. However, this method: Second, it requires an incubator because it cannot survive without culturing, requires some skill to judge, and cannot judge the culture results on the spot, so it is not suitable for research purposes. Even if it were possible, there was a problem 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 that does not have the above-mentioned drawbacks.

(Means for Solving the Problem) The present inventors have further simplified the method of determining hyperactivity using resazurin, and have developed a method that can be determined in a short time.
), we have arrived at the present invention, which is a dry method that does not rely on a wet method.

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). Infiltrating a certain amount of the infiltrated material, sandwiching the infiltrated material between a film having an adhesive layer and sealing it, and maintaining it at a constant temperature for a certain period of time to culture caries-causing microorganisms, and then measuring the color tone change of the test piece. 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, particularly bacteria, that are present in saliva and cause caries development, such as Somutans, Lactobacillus, Actinomyces, and the like.

The reagent composition in the present invention is a composition containing a reagent that can detect enzyme reactions, changes in redox potential, etc. caused by caries-causing microorganisms in saliva, and culture components that promote the growth of caries-causing microorganisms. refer to things

Examples of the reagent include resazurin, triphenyltetrazolium, neotetrazolium, 2,6-dichlorophenolindophenol, methyl orange, salts thereof, and mixtures thereof. Among these, resazurin, triphenyltetrazolium, neotetrathulium, 2,6-dichlorophenolindophenol, and methyl orange are preferred.

The above-mentioned culture components that promote the growth of caries-causing microorganisms include culture components that promote the growth of caries-causing microorganisms, and examples thereof include sucrose, glucose, and fructose. Among these, sucrose is preferred.

Examples of the ratio of reagents and culture components are 1:100 to 1:5.
00 is mentioned, and among these, 1:300 to 1:400 is preferable.

Furthermore, in addition to these ingredients, polyvinylpyrrolidone,
A sizing agent such as methylcellulose, hydroxypropylcellulose, carboxymethylcellulose, starch paste, or gelatin may be included for the purpose of spreading the reagent. The ratio of these components is 0, based on the total weight of the reagent composition.
．． 5 to 5% by weight 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 material 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, including circular,
A rectangular shape etc. is preferable.

The area of the test piece is preferably 25 to 80 mm.

A test piece containing the above reagent composition can be prepared and manufactured as follows.

First, reagent grade TC containing 5 to 20% by weight of the reagent composition.
Prepare a W solution. Examples of the solvent for the above solution include water, alcohol, isopropyl alcohol, 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 is taken out and dried at 50 to 80 tons for 30 to 120 minutes.

A certain amount of saliva to be tested is infiltrated into the above test piece.

Quantitative determination may be any amount by which a change in color tone of a test piece can be measured, and may include, for example, 10 to 100 μm. This amount corresponds to 0.5 to 2 drops from a dropper.

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 sealed.

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. Among these, polyvinyl chloride is preferred.

An example of the thickness of the film is 50 to 300 μm, and preferably 125 to 175 μm.

The size of the film can be any size that will keep the infiltrate sealed. - Inside the shell, 5 to 50% of the area of the specimen
Preferably, the area is 10 times larger.

The shape of the film can be any shape capable of keeping the infiltrate sealed. For example, it may be two separate films that sandwich the infiltrate therein, or one continuous film that sandwiches the infiltrate by folding the film.

Further, the support film does not necessarily need to be a film, and a plate-like material that does not absorb moisture, such as a glass plate, a chinaware plate, a metal plate, or waterproofed wood, can be used instead.

The adhesive layer refers to a layer capable of adhering between films and between films and infiltrated matter.

The adhesive layer is made by blending and kneading synthetic rubber or natural rubber with natural resins such as rosin and copal, synthetic hydrocarbon resins such as polyethylene, lanolin, beeswax, and other waxy substances. A synthetic rubber adhesive using rubber can be suitably used.

The adhesive layer may 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 on the periphery of the position on the film where the infiltrate is placed. Among these, it is preferable to use two separated films and provide an adhesive layer on the entire one side 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.

The sealed infiltrate is kept at a constant temperature for a certain period of time to cultivate caries-causing microorganisms.

Examples of constant temperature include 25 to 40°C, of which 35°C
~37°C is preferred.

Examples of the certain period of time include 10 to 30 minutes, preferably 10 to 15 minutes).

The culture can be carried out in the air or in warm water, for example, in a warmer, an incubator, on human skin, in a warm bath, or the like.

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 in saliva or dental plaque that are said to be the main cause of dental caries.In particular, the S8 mutans enzyme condenses sucrose in the culture solution to produce glucan ( dextran). 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 depending on the test solution: purple, pink, and red. As shown in Examples below, this color tone change correlates with sensitivity activity. Therefore, it is possible to determine the sensitization activity 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, by an absorbance meter, by changing the color density, 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. Placed on a mount, the small paper pieces are sandwiched and overlapped with a circular or rectangular covering film coated with an adhesive on the inside, or when the small paper pieces are prevented from coming into contact with the adhesive. A circular or rectangular covering film with adhesive applied only to the inner periphery is superimposed, and a supporting film (a film that supports the small paper strip and the covering film during the test) is placed on the same mount. They are arranged side by side, and both the supporting film and the covering film are releasably 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 indicates a diameter 5 containing the reagent composition.
It is a small piece of paper in the form of a circle with a diameter of 10 to 10 mm or a square with a side of 4III111 or more, and the material of the small paper piece is a commonly used filter paper, preferably a filter paper with a thickness of 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 the covering film 2 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 paper strip will always remain fixed to the inside of the covering film, which is often convenient for handling during testing. When formed in this way, the small piece of paper 1 before use is completely isolated from the outside air, no air flows through it, and moreover, it is kept sterile.

The supporting film 3 may have a circular shape with a diameter of 5 to 10 cm or a rectangular shape with an area of this size, and the film 2 may have a circular shape with a diameter of 3 to 5 am or a rectangular shape with an area of this size. The thickness of the mount is sufficient to allow films 2 and 3 to be juxtaposed.

Next, the small paper strip 1 contains sucrose and resazurin, triphenyltetrazolium, neotetrazolium, 2,6-dichlorophenolindophenol or methyl orange or their salts in a ratio of 1:100 to 1:500. ing.

The film 2 is then coated with an adhesive layer having an adhesive. The adhesive is mainly made of synthetic rubber, etc., but it can be easily attached and detached from the supporting film 3 and the mount 4 at room temperature, and it can be used together with the small pieces of paper to heat equipment, such as metal, wood, etc.
Any material that can be attached to heating equipment for culturing, such as plastic, is sufficient. That is, a small piece of paper soaked with saliva or dental plaque should be incubated at 32-37° C. for 10-15 minutes while blocking air supply as much as possible and preventing evaporation of water in the saliva. Equipment for carrying out the method of the invention:
Since it is a simple and small device, it can be used as a substitute for a heating device by attaching the film along with the surface of the human skin to heat it. Therefore, it is desirable to select an adhesive that is harmful to human skin as the adhesive contained in the adhesive layer on the film. Note that when the supporting film 3 is not attached to a heating device or the human skin surface and is used in a manner that it is stored in a warming device, the supporting film 3 does not need to have an adhesive layer.

In order to use the identification tool for carrying out the method of the present invention, collect a small amount of saliva (usually one drop) with a dropper etc. Separately, half peel off the film 2 from the mount of the tool, and place the dropper on the small piece of paper 1 inside the tool. After the saliva has been thoroughly and uniformly soaked and spread, the small piece of paper 1 is covered with the film 2 and adhesively pasted onto the supporting film 3. FIG. 2 shows a case where the film 2 has an adhesive layer around its inner surface. Peel off the supporting film 3 that still supports the small paper strip 1 and the covering film 2 from the mount 4, and apply it to the surface of the warmer or a suitable exposed part of the human body as a substitute for it. Observe the change in color of the paper strip after 15 minutes. Depending on this color change, that is, blue, purplish red, or red, the sensitivity to ciliation is determined as negative, weakly positive, or positive. A feature of the use of the above-mentioned device is that human body temperature can be used as a substitute for a warmer to maintain the culture temperature at around 37°C.

If the covering film 2 has an adhesive layer on the entire inner surface, when this is partially peeled off, the small paper piece 1 inside will have its front side glued to the film 2, so its back side will be exposed. It depends on the addition of saliva. After saliva infiltration, the small pieces of paper 1 are peeled off from the mount 4 together with the film 2, and these are adhered onto the support film 3. This state is shown in FIG. The support film 3 and the support film 3 are peeled off from the mount 4 and pasted on the surface of the warmer or the surface of the exposed part of the human body as a substitute thereof. When storing in the warmer without pasting, remove the supporting film 3 from the mount 4.
It may be stored in the warmer together with the mount 4 without removing it.

(Effects of the Invention) Based on the above, it is possible to easily and quickly identify caries-affected individuals, as well as sensitive individuals who use this method as a preventive or therapeutic measure against caries in children, using the determination method of the present invention. Became.

Furthermore, since it is easy to manufacture a tool 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 allows testing 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 a small piece of paper containing the reagent composition or applying it to the oral cavity, and the reagent composition does not enter the body orally. It also has the advantage that there is no physiological effect on the human body because there is no need to touch the pieces of paper with fingers.

Also: 2. In the method of the present invention, after inoculating with saliva,
The small piece of paper remains sandwiched between the films and is discarded in this state after the determination, so it also has the advantage of being extremely clean.

(Example) Next, examples will be shown.

Reference Example A piece of filter paper with a diameter of 8 mm was immersed in an aqueous solution of 0.025% by weight of resazurin and 10% by weight of sucrose for 5 minutes at 70°C.
By drying for 0 minutes, a small paper piece 1 (disc) for identification was produced. This was covered with a transparent plastic covering film (150 μl polyvinyl chloride film) 2 having a diameter of 4 cm and having an adhesive layer containing an adhesive (synthetic rubber type) on the entire inner surface, and the size was 4.50 m x 4.5 cm.
Rectangular transparent plastic support film (200 μl
Polyvinyl chloride board〉3 (with adhesive layer on the back side)
Paste it on a rectangular mount 4 of 5cm x 10cm in parallel with
This was placed in a transparent plastic packaging sachet or small plastic bag and heat-sealed to form a single-use product.

Example Saliva samples: Collect saliva from each subject into a mini cup and add a drop (approximately 0.05 m1'
) was removed and exposed by peeling off the film 2 of the tool manufactured in the reference example, or sufficiently permeated into the blue disk (small piece of paper) 1 taken out, and then these were attached to the supporting film 3 and sealed. These were peeled off from the mount 4, and affixed to the subject's arm, and 15 minutes later, the change in color tone of the disc 1 was examined.

Table 1 shows the results of comparing this with the conventional inspection methods of using ST media and using σMSBB.

Table 1 Note 1 An individual's dental caries status was determined by a dentist's examination A:
It was graded into three levels: poor, B: average, and C: good.

Note 2: Caries was determined as follows based on the degree of discoloration of this identification tool. White to red <=-=>: Strong, slightly pink to pale pink to red (10 or -10): Weak, purple to blue (-):
NoneNote 3 Commercially available caries activity test set, trade name: rST Media @ (Showa Yakuko).Using saliva as a sample, caries-related bacteria and acid-producing bacteria contained in saliva were detected at pH Check for decrease.

Note 4M5BB (Mitis salivarius bacitracin medium,
Product name: ``Mu Count■'' Showa Yakuko>. Identification medium for Streptococcus mutans, a caries-causing bacterium.

According to the results of this example, “rsT Media” and “MSBBJ
The results clearly show a positive correlation between the results (which have been proven to show a positive correlation with the state of caries development in humans) and the judgment based on the color tone change of this tool.

From this, it can be seen that the caries differentiation method of the present invention is easy to carry out and is effective for easily determining caries activity in a short period of time using a dry method instead of a wet method.

[Brief explanation of drawings]

FIG. 1 is a plan view of a discrimination tool for carrying out the method of the present invention, and FIG. 21! Figures 1 and 3 show side views of the tool in use. Symbols 1: Dry paper strip for testing, 2: Covering film, 2A: Inner periphery with adhesive layer of film 2, 3: Supporting film, 4: Mounting paper.

Claims (5)

[Claims] (1) A certain amount of saliva to be tested is infiltrated into a test piece containing a reagent composition for determining the amount of caries-causing microorganisms in saliva, and this infiltrated material is sandwiched between films having an adhesive layer. 1. A dry caries determination method, comprising: culturing caries-causing microorganisms by keeping the test piece in a sealed state at a constant temperature for a certain period of time, and then measuring a change in color of the test piece. (2) Claim No. 1, characterized in that the reagent composition consists of resazurin, triphenyltetrazolium, neotetrazolium, 2,6-dichlorophenolindophenol, methyl orange or their salts, and sucrose ( The method described in section 1). (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 3
Claim No. 1 (
The method described in section 1).