DE2941471C2 - - Google Patents

Description

Tests for the determination of dental caries activity are carried out to predict or determine if a Dental caries is developed or expected in the future. The Tests are therefore very important from the point of view of oral hygiene. They are usually carried out by some Monitor factor that participates in dental caries. It is Dental caries is known mainly by microorganisms caused in the mouth To form acids.

There are already various methods for the determination of dental caries Activity has been developed, for example, as Snyder test known measurement of the number of lactobacilli in saliva and culturing of bacteria in saliva a medium containing a pH indicator, and the Observation of the degree of color change of the medium at lowering of the pH. One suitable for this test Culture medium and the procedure used are described in Difco Mannual (1974), pp. 190-192. More recently, there has been an improvement in the Snyder test developed in which the dental caries activity in the way it is determined that one selectively measures acids that pass through acid-producing bacteria, especially Streptococcus mutans, which are thought to be mainly Occurrence of dental caries are involved.

According to the US-PS 37 46 624, the presence of the cariogenic Bacterium Streptococcus mutans is determined by a Dental plaque sample of the patient in a special medium Cultivated for 48 hours and then the color of the sample determined becomes. It has also been tried to prevent the dental caries  Activity using a buffer solution, the diazoresorcinol (Resazurin) as an indicator to determine. The used for this, from US-PS 33 32 743 known test agent contains a phosphate buffer in addition to the indicator and as substrate sodium chloride, tryptose and dextrose or Lactose. An indicator, like phenolphthalein, is also in the toothpaste known from US-PS 11 12 180, indicating to the user whether his mouth is sour or sour alkaline state.

The known methods for the determination of dental caries activity unfavorably require a sophisticated technique to test the bacteria, a specific reagent the specificity to that used in the process To get funds, or a specific device or a fairly long measuring time of usually 24 to 72 hours. There is therefore a need for an improved method To test the dental caries activity, the easier and within a very short period of time by persons can be done without special training.

The object of the invention is to provide a new test agent which is used for the simple determination of dental caries Activity is suitable and this provision within a very short period of time.

Extensive testing of dental caries activity tests on the acid-producing capacity of bacteria in the mouth, have revealed that the provision the tooth circle activity easily within a very short time span and by persons without special education can be done if you have a special one Agent uses a saccharide as carbon source to promote the acid-producing capacity of the bacteria  in the mouth and a pH indicator with a color change point in the pH range of 5 to 7 contains. In this method a sample of plaque is collected and placed in the middle hineinokuliert. This is followed by the color change with the naked eye of the pH indicator contained in the agent is, watched.

The invention is defined in claim 1 Test agent for the determination of dental caries activity. Advantageous embodiments are in the dependent claims specified.

Because the acids produced by bacteria in the mouth are weak acids and their amount is therefore very low, should the pH indicators used in a test for dental caries activity be used, their color within a pH range from 3.0 to 7.0. The inventors became experimental the following facts have been established:

• (1) The change in the color of the pH indicator is different if the pH is determined experimentally by adding one Acid (for example, lactic acid, which is the main acid among the acids which are produced by bacteria in the mouth) to the Medium is lowered and when the pH with acids, the accurately produced by bacteria in the mouth, humiliated becomes.
• (2) A carbon source is essential to the acid-producing To promote capacity of bacteria from the mouth.  
• (3) No other nutrient is required for the test. It is rather that if another nutrient, z. B. Yeast extract to which agent is added - the determination the color change of the pH indicator because of the discoloration of the other nutrient is inhibited.
• (4) Some specific pH indicators, e.g. B. resazurin, Lakmoid or resorcinol blue, methyl red and alizarin, the a color change point at a pH range of 5 to 7 are against the acids that are caused by bacteria in the Mouth are made very sensitive and they can do theirs Change color sharply within a very short period of time.
• (5) If the pH indicator with a color change point in the pH range of 5 to 7 first in an aqueous alkaline Solution is dissolved and the aqueous solution adjusted a pH of 6.7 to 7.0 with a weak acid and then bacteria from the mouth in these are inoculated, then the color change of the Indicator sharper.
• (6) The test agent can be kept stable by: the pH with specific acids, such as acetic acid, Lactic acid and tartaric acid.
• (7) It is preferable to use an antiseptic agent in the To incorporate means.

The above facts were confirmed by the following experiments determined:

1. Experiment 1

To aqueous solutions of various indicators was added lactic acid to lower the pH. The color change of the indicator was observed. The results are summarized in Table I.

2nd attempt 2

Various indicators were each dissolved in an aqueous 0.1N sodium hydroxide solution and to the mixture was added 10% by weight of sucrose. The solution was adjusted to a fixed pH with lactic acid. To the resulting aqueous 0.002 wt% solution of the indicator (2 ml) was added a suspension of Streptococcus mutans (number of bacterial cells: 2 x 10⁹ to 4 x 10⁹, 0.4 ml) and the mixture was incubated at 37 ° C incubated. The change in pH and the change in color were observed over time. The results are summarized in Table II.

The results of Tables I and II show the following: If the pH of the test solution was lowered with lactic acid, All indicators showed a sharp change in color. At the Inoculations of Streptococcus mutans could be specific pH indicators with a color change point in the pH range from 5 to 7 resazurin, lakmoid, methyl red and alizarin, especially Resazurin and Lakmoid, keen on the the bacteria produce acids and their color appeal clearly change within a very short period of time. Without Addition of sucrose, the color change was slower. It is therefore essential to add a saccharide. If the Indicator first dissolved and then the pH with a Acid was adjusted, the color change appeared sharper.

The effect of the concentration of the pH indicator was also tested. As a result, it was found that if the amount of indicator is too small, the color change is unclear was and on the other hand with too much amount of Indicator too long a period for the color change it was necessary that the hue became dark and therefore that the color change was unclear. The pH indicator is therefore in a concentration of 0.0001 to 0.05 wt .-%, preferably from 0.001 to 0.01% by weight.

3rd attempt 3

The same suspension (0.2 ml) of Streptococcus mutans As in the above experiment 2 was in a 0.002% aqueous solution  Resazurin (1 ml) containing 10% by weight of the various Contained saccharides according to Table III, inoculated. The mixture was incubated at 37 ° C and the color change in Measured over time. The results are summarized in Table III.

Table III

The results show that when used of sucrose the color of the indicator on the fastest changed and that the speed of the Color change in the order of glucose, fructose and strength decreased. When using sucrose was the color change most clearly.

There was also the effect of saccharide concentration tested and found that too low an amount Saccharides the amount of those produced by the bacteria Acids were too low and therefore the color was not within a short period of time changed; that on the other hand if the amount of saccharides is too high, the speed the color change was too low. Accordingly, the Saccharides in an amount of 2.5 to 40 wt .-%, preferably from 5 to 20% by weight.  

4th attempt 4

An aqueous solution containing 10% by weight of sucrose and 0.003 wt .-% resazurin (without content of an antiseptic Means) was made up with various organic acids adjusted to a pH of 6.8 to 7.0. Every means (1 ml) was placed in a small test tube and the Test tube closed with a silicone stopper and kept at 37 ° C or 55 ° C. With the naked eye became observed the color change of the agent over time. The results are summarized in Tables IV and V. The evaluation criteria are as follows:

-: no color change
±: slight change in color
+: a little change in color
++: clear change of color
+++: significant change in color

Table IV

Table V

The results show that the speed the color change while holding according to the type of organic acids used to adjust the pH of the agent used varies. at Use of acetic acid, tartaric acid or lactic acid, in particular of acetic acid, the color change could over be inhibited for a long period of time. After the above Test the media were subjected to the bacteria test. When Result was in the remedies, which were kept at 37 ° C were detected, bacterial growth, which means that the color change not only by the holding temperature, but also influenced by the growth of bacteria becomes.

5th experiment 5

Different antiseptic agents became the same Means as used in Experiment 4 added, the pH was adjusted with acetic acid or lactic acid. The agents were kept at 37 ° C and the color change was determined accordingly. The results are compiled in Table VI.  

Table VI

The results show that the funds can be kept stable for long periods by antiseptic agents, in particular potassium tellurite and Sodium trinitride, incorporated.

6th attempt 6

In medium, the 10 wt% sucrose, 0.003 wt% resazurin and various amounts of potassium tellurite (pH 6.8, with Acetic acid adjusted) became a fixed amount inoculated into a suspension of Streptococcus mutans and the mixture incubated at 37 ° C. The change the pH and color of the agent was observed. The results are summarized in Table VII.  

Table VII

The results show that the addition of antiseptic Means no adverse effect on the determination of Dental caries activity results as these remedies in an amount from 0.1 to 0.01 wt .-%, in particular from 0.075 wt .-% or less, do not affect the color change of the test agent.

The test agent according to the invention therefore contains 2.5 to 40% by weight of the specific saccharide and 0.0001 to 0.05% by weight the special pH indicator with a color change point in the pH range of 5 to 7, wherein the agent has a pH of 6.5 to 7.0, which with an alkali or an acid has been adjusted.

The test agent according to the invention is prepared in the manner that is the fixed amount of the saccharide and the pH indicator in water or an aqueous alcohol (eg ethanol) with a concentration of alcohol on the bacteria  in the mouth exerts no undesirable effect, eg. B. about 1 wt .-% or less, preferably in water or one 0.1 to 1 wt.% Aqueous ethanol solution dissolves and then adjust the pH. In a preferred embodiment First, the pH indicator and then the Saccharide in an aqueous solution or an aqueous-alcoholic Solution of an alkali compound dissolved. After that The solution is acidified to the desired pH set.

Among the saccharides used according to the invention, sucrose, Glucose and fructose, sucrose is preferred. The Saccharides are used in an amount of 2.5 to 40% by weight, preferably from 5 to 20% by weight.

The pH indicators are Resazurin, Lakmoid or Resorcinblau, Methyl red and alizarin, of which resazurin and lakmoid, especially resazurin, are preferred. The pH indicators are in an amount of 0.0001 to 0.05 wt .-%, preferably from 0.001 to 0.01% by weight, more preferably from 0.001 to 0.005 wt .-%, used.

Examples of alkalis used to adjust the pH of the agent can be used are alkali metal hydroxides (For example, sodium hydroxide, potassium hydroxide and Lithium hydroxide), alkaline earth metal hydroxides (eg, magnesium hydroxide and barium hydroxide), alkali metal carbonates or bicarbonates (eg sodium carbonate, potassium carbonate and sodium bicarbonate), among those most suitable are sodium hydroxide is. Examples of acids for adjusting the pH Values are lactic acid, tartaric acid, citric acid, malonic acid,  Oxalic acid, acetic acid and formic acid, Lactic acid, tartaric acid and acetic acid are the most suitable are.

The test agent is used with these alkali compounds and acids to a pH range of 6.5 to 7.0, preferably from 6.7 to 7.0, discontinued.

If necessary, an antiseptic Means, e.g. As sodium azide or tellurite, incorporated That will not be the growth of bacteria from the mouth adversely affected. Suitable examples of tellurites are potassium tellurite, sodium tellurite, magnesium tellurite and Calcium tellurite, of which potassium tellurite is the most suitable is. The antiseptic agent is used in an amount of 0.01 to 0.1 wt .-%, preferably from 0.025 to 0.075 wt .-%, based on the weight of the agent used.

The test for determining dental caries activity is described in The method performed is to take a sample of the plaque of the collects patients to be tested, incorporated into the test agent and the color change of the test agent after incubation during a specified period of time of z. B. 30 minutes observed. The change of color varies accordingly the amount of acid produced by the bacteria becomes. The color changes in the order blue → reddish purple → dark pink → colorless in the case of resazurin, blue → purple → red → pink in the case of Lakmoid, yellow → pale yellow → dark pink → pink in the case of methyl red and red → pale pink → pale yellow → yellow in the case of Alizarin. Because the test is within one a very short period of time (eg 30 min)  can not, he will not by the incubation temperature significantly affected and can in a temperature range from room temperature (about 25 ° C) to 37 ° C.

The test agent also has a selectivity towards Streptococcus mutans, which results from the following Trial results.

To a 0.002 wt .-% aqueous solution of resazurin (pH 6.7, 1 ml) containing 10% by weight of sucrose became Given suspension of different bacteria (0.2 ml) and the mixture incubated at 37 ° C. The change the color of the agent was observed. The results are summarized in Table VIII.

Table VIII

The results show that inoculation of Streptococcus mutans the color of the remedy changed within a very short period of time, but in the case of other bacteria, the color change  took longer. Thus, the test agent proves to be selective against Streptococcus mutans.

The preparation of the test agent is in the following examples explained.

Example 1

components Wt .-% sucrose 10 resazurin 0,002 aqueous 0.1 N sodium hydroxide solution 5 distilled water about 80 0.1 N-lactic acid adjusted to a pH of 6.7 to 7.0

According to the above approach, resazurin is dissolved in aqueous 0.1N Dissolved sodium hydroxide solution and distilled to this Given water. The sucrose is dissolved in the solution and then the pH is adjusted to 6.7 to 7.0.

example 2

Using the same approach as in Example 1, but with 0.002 wt% lakmoid instead of resazurin, Similarly, a test agent for determination the dental caries activity produced.  

example 3

Using the same approach as in Example 1, except that 0.02% by weight of methyl red instead of resazurin is used in the same way a test agent for the determination of dental caries activity.

example 4

Using the same approach as in Example 1, however, with 0.02 wt% alizarin instead of resazurin, In the same way, a test agent for determining the Dental caries activity produced.

example 5

components Wt .-% sucrose 10 resazurin 0,003 aqueous 0.1 N sodium hydroxide solution 5 antiseptic agent (sodium azide) 0.05 distilled water about 80 0.1 N-lactic acid adjusted to a pH of 6.7 to 7.0.

According to the above approach, a test agent for determination the dental caries activity in the same way as in the Example 1, prepared.  

example 6

components Wt .-% sucrose 10 resazurin 0,003 aqueous 0.1 N sodium hydroxide solution 5 antiseptic agent (potassium tellurite) 0.05 distilled water a total of 100 0.1 N acetic acid adjusted to a pH of 6.8 to 7.0

According to the above approach, resazurin is dissolved in the aqueous 0.1N Dissolved sodium hydroxide solution and distilled to this Given water. In the solution is sucrose and potassium tellurite dissolved and the mixture is brought to a pH Value adjusted from 6.8 to 7.0, creating a test agent to determine the dental caries activity.

Claims (9)

1. A test agent for the determination of dental caries activity of a dental plaque sample comprising a carbohydrate and a pH color indicator, characterized in that the agent from 2.5 to 40 wt .-% of a saccharide from the group sucrose, glucose and fructose, 0, 0001 to 0.05 wt .-% of a pH indicator from the group Resazurin, Lakmoid, Methyl red and alizarin and the remainder of a medium from the group consisting of water and an aqueous alkolische solution, wherein the means with alkali or acid to a pH Value has been adjusted in the range of 6.5 to 7.0. 2. test agent according to claim 1, characterized that the saccharide 5 to 20 wt .-% sucrose is. 3. test agent according to claim 1, characterized that the pH indicator is a substance the group Resazurin and Lakmoid is. 4. test agent according to claim 1, characterized the pH indicator is 0.001 to 0.01 Wt .-% Resazurin is.   5. test agent according to claim 1, characterized it is 5 to 20% by weight of sucrose, 0.001 to 0.01 wt .-% resazurin and the balance contains water, wherein the agent with sodium hydroxide and an acid from the Group of lactic acid, tartaric acid and acetic acid to the pH Value has been adjusted in the range of 6.7 to 7.0. 6. test agent according to claim 5, characterized that it has been made in the way is that the sucrose and resazurin in an aqueous Sodium hydroxide solution have been resolved and that the pH with the acid to the range of 6.7 to 7.0 has been adjusted. 7. test agent according to one of claims 1 to 6, characterized characterized in that an antiseptic Agents from the group sodium azide and a tellurite incorporated in an amount of 0.01 to 0.1 wt .-% is. 8. test agent according to claim 7, characterized that tellurite is potassium tellurite. 9. test agent according to claim 8, characterized that the potassium tellurite in an amount from 0.025 to 0.075 wt .-% has been incorporated.