DE2941471A1 - Test agent for detection of dental caries bacteria - esp. Streptococcus mutans contg. a saccharide, e.g. sucrose, and a pH indicator in water or aq. alcohol at pH 6.5.7.0 - Google Patents

Abstract

New testing agent contains (a) 2.5-40 wt.% of a saccharide, (b) 0.0001-0.05wt. % of a pH-indicator with a colour change point in the pH range 5-7, and (c) the remainder a medium consisting of water or an aq. alcohol; the pH of the agent being 6.5-7.0. Pref. saccharides are sucrose, glucose and fructose; sucrose in a concn. of 5-20wt. % is particularly preferred is resazurin in a concn. of 0.001-0.01wt. %. Simple and rapid determination of dental caries activity by detection in the saliva of bacteria which metabolise saccharides to form acids and thus cause a change in ph and a change in the colour of the pH indicator.

Description

Description

The invention relates to a diagnostic test means for determination a dental caries activity. The invention particularly relates to an agent that contains a specific amount of a saccharide and a specific pH indicator and that is capable of simply and unambiguously creating a Determine dental caries activity by observing the color change of the agent will.

The test for dental caries activity is done to predict or determine whether dental caries is developing or whether dental caries is in the future is to be expected. The test is therefore very good from the standpoint of oral hygiene important.

The test for dental caries activity is usually done when by monitoring any factor involved in dental caries. It is known that dental caries is mainly induced by microorganisms that are able to form acids in the mouth. Because of this knowledge are already various methods for testing dental caries activity have been proposed, for example by measuring the number of lactobacilli in saliva and by cultivation of bacteria in saliva in a medium that contains a pH indicator and by Observation of the degree of color change of the medium when the pH value is lowered (this method is known as the Snyder test).

Recently an improvement of the Snyder test has been developed, in which the dental caries activity is determined in such a way that one selectively acids measures that by acid producing bacteria, particularly Streptococcus mutans believed to be primarily responsible for the occurrence of dental caries are involved. It has also been suggested that the dental caries activity using a buffer solution containing diazoresorcinol (resazurin) as an indicator contains, to be determined (see US Pat. No. 3,332,743).

These known methods disadvantageously require a highly trained Technique for testing the bacteria, a specific reagent to determine the specificity versus the agent used in the process, or a specific one Device or a fairly long measurement time (usually 24 to 72 hours). It is therefore to seek an improved method for testing dental caries activity, the easier and within a very short period of time by people without special Training can be done.

Extensive studies of tests for dental caries activity, which are built on the acid producing capacity of bacteria in the mouth show that the determination of dental caries activity easily within a very can be carried out in a short period of time and by people without any special training can, if one uses a special agent that uses a saccharide as a 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 from 5 to 7. With this method a sample of dental plaque is collected and inoculated into the agent. On that becomes visible to the naked eye the change in color of the pH indicator that is in the agent is included, observed.

The object of the invention is to provide a new test means for easy determination of dental caries -Activity suitable is. Furthermore, an improved process for Determination of dental caries activity is available within a very short period of time be asked.

The test agent according to the invention for dental caries activity contains a specific amount of a saccharide and a specific amount of a specific one pH indicator and it has a pH of 6.5 to 7.0.

Because the acids produced by bacteria in the mouth are weak acids and their amount is therefore very small, the pH indicators that should be used should be one Test used for dental caries activity, their color within a pH range changed from 3.0 to 7.0. The inventors made the following experiments Facts Established: (1) The change in the color of the pH indicator is different, if the pH value is experimentally adjusted by adding an acid (e.g. lactic acid, which is the Main acid is among the acids produced by bacteria in the mouth) too the remedy is lowered and when the pH value with acids, exactly by bacteria generated in the mouth is lowered.

(2) A carbon source is essential to the acid generating Capacity to promote bacteria in the mouth.

(3) No additional nutrient is required for the test. It is rather so that - if another nutrient, e.g. yeast extract, is added to the agent is given - the determination of the change in color of the pH indicator due to the discoloration of the other nutrient is inhibited.

(4) Some specific pH indicators, e.g. Resazurin, Lakmoid resp. Resorcinol blue, methyl red and alizarin, which have a color change point in one pH range of 5 to 7 are against the acids produced by bacteria in the Mouths are made very delicate and they can change their color within one change sharply in a very short period of time.

(5) When the pH indicator with a color change point in the pH range from 5 to 7 is first dissolved in an aqueous alkaline solution and the aqueous Solution is adjusted to pH 6.7 to 7.0 with a weak acid and if bacteria in the mouth are subsequently inoculated into it, then the Color change of the indicator sharper.

(6) The test agent can be kept stable by adjusting the pH with specific acids such as acetic acid, lactic acid and tartaric acid.

(7) It is preferred to incorporate an antiseptic agent into the agent.

The above facts were found through the following experiments: 1. Experiment 1: Illactic acid was added to aqueous solutions of various indicators given to lower the pH. The indicator was changing color observed. The results are shown in Table I. Tabel I indicators pH value type concentration 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 tration (% by weight) Resazurin 0.02 blue violet violet reddish reddish carmine carmine red red violet violet red red Resazurin 0.002 violet reddish- reddish- carmine- carmine- dark- red red red violet violet red red red methyl red 0.02 yellow yellow light yellow light yellow orange red red red red alizarin 0.002 red red red orange light yellow yellow yellow yellow yellow lakmoid 0.02 blue blue violet reddish carmine carmine red reddish weak violet red red orange reddish orange lakmoid 0.002 blue violet reddish carmine carmine weak pink weak weak purple red red red pink pink BCG * 1 0.002 blue blue blue-blue-green green yellow- yellow- yellowish- green green green green BPB * 2 0.02 blue blue blue blue blue violet violet reddish-reddish brown Lich brown Footnotes: * 1) Bromocresol Green * 2) Bromophenol Blue 2. Experiment 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 made with lactic acid adjusted to a fixed pH. To the aqueous 0.002% strength by weight obtained in this way Solution of the indicator (2 ml) was a suspension of Streptococcus mutans (OMZ 176) (number of bacterial cells: 2 x 109 to 4 x 109, 0.4 ml) and the mixture was incubated at 57 ° C.

The change in pH and the change in color were made observed over time. The results are shown in Table II.

Table II Indicator incubation time at 37 ° C prior to inoculation 0 min 15 min 30 min 1 h 24 g of the bacteria pH color pH color pH color pH color pH Color pH Color Resazurin 6.8 blue 6.7 blue 5.9 reddish-5.2 pink 4.8 light pink 4.5 colorless violet Lakmoid 6.8 blue 6.8 blue 6.3 bluish 5.2 weak 5.0 weak 4.7 colorless - red pink violet methyl red 6.8 yellow 6.6 yellow 5.6 light - 4.7 pink 4.5 weak 4.3 weak yellow pink pink alizarin 6.8 red 6.7 red 5.9 weak 5.5 yellow 5.0 yellow 4.8 weak pink yellow BCG 6.8 blue 6.8 blue 6.3 blue 6.0 blue 5.7 blue 5.5 bluish green BPB 6.8 blue 6.8 blue 6.3 blue 6.1 blue 5.8 blue 5.1 bluish purple Resazurin * 6.8 blue 6.7 blue 6.4 purple 6.0 reddish - 5.4 red 4.8 weak purple pink footnote: *) no addition of sucrose From the results Table I and Table II it can be seen that - if the pH of the test solution was lowered with ilactic acid - all indicators a sharp change in color showed. It can also be seen that inoculated as Streptococcus mutans the special pH indicators with a color change point in the pH range from 5 to 7, e.g. resazurin, lakmoid, methyl red and alizarin, especially resazurin and Lakmoid, could respond sharply to the acids generated by the bacteria and could change their color clearly in a very short period of time. If no sucrose was added, the color change was delayed. It it is therefore essential to add a saccharide. When the indicator first resolved and then the pH was adjusted with an acid, then the Color change sharper.

The effect of concentration on the pH indicator was also observed tested. As a result, it was found that - when the indicator is in a too small amount was used - the color change was indistinct and that on the other hand - if the amount of the indicator was too large - too long a time for the color change was necessary and that the hue became dark and hence the color change indistinct was. The pH indicator is therefore preferably at a concentration of 0.0001 to 0.05% by weight, more preferably from 0.001 to 0.01% by weight, used.

3. Experiment 3: The same suspension (0.2 ml) of Streptococcus mutans as in Experiment 2 above, was in a 0.002% aqueous solution from Resazurin (1 ml), the 10% by weight of the various saccharides according to Table III contained, inoculated. The mixture was incubated at 370 c and the color change measured over time. The results obtained are shown in Table III compiled.

Table III Saccharides Incubation Time at 370C 0 min 15 min 30 min Sucrose blue red pale pink (transparent) glucose blue red dark pink fructose blue reddish-dark pink violet Strength blue reddish-dark pink violet From the From the above results, it can be seen that when sucrose is used, the color of the indicator changed the fastest and that the rate of color change decreased in the order of glucose, fructose and starch. Using the color change was clearest from sucrose.

The effect of the concentration of the saccharides was also tested. As a result, it was found that if the saccharides are in too small an amount - the amount of acids produced by the bacteria was too little and therefore the color did not change in a short period of time changed. On the other hand, if the amount of saccharides was too great, then that was Color change speed too slow. Accordingly will the saccharides preferably in an amount of 2.5 to 40% by weight, more preferably 5 to 20 wt. -5 'is used.

4. Experiment 4: An aqueous solution containing 10% by weight of sucrose and 0.003% by weight of resazurin (not containing an antiseptic agent) was used with various organic acids adjusted to a pH value of 6.8 to 7.0. Each agent (1 ml) was placed in a small test tube and the test tube was sealed with a silicone stopper and held at 370C or 550C. With the color change of the agent with the lapse of time was observed to the naked eye. The results are summarized in Tables IV and V. The evaluation criteria are as follows: -: no color change +: little change in color +: slightly Change in color ++: clear change in color, significant change in Color Table IV Organic acids Hold time at 370C (days) such as 3 10 20 30 60 90 150 No.

1 Lactic acid - - + - + ++ +++ +++ 2 Oxalic acid - + - + - + ++ +++ +++ 3 Citric acid - # # + ++ +++ +++ 4 Tartaric acid - - # + ++ +++ +++ 5 Acetic acid - - - + - + ++ +++ Table V Ver organic acids hold time at 550C (days) search 3 10 20 30 60 90 150 No.

6 Lactic acid - + + ++ +++ +++ +++ 7 Oxalic acid - + + ++ +++ +++ +++ 8 Citric acid - + + ++ +++ +++ +++ 9 Tartaric acid - + + ++ +++ +++ +++ 10 Acetic acid - - + + ++ +++ +++ From the above results it can be seen that the speed the change in color during holding according to the kind of organic acids that used to adjust the pH of the agent varies. Using of acetic acid, tartaric acid or lactic acid, especially acetic acid, could die Color change can be inhibited over a long period of time. After the above test the agents were subjected to the bacterial test. As a result, in the means, that had been kept at 370C, bacterial growth was observed, which means that the color change is not only due to the hold temperature, but also due to the growth of bacteria is influenced.

5. Experiment 5: Different antiseptics became the same Averages as used in Example 4, added, the pH being adjusted with acetic acid or lactic acid has been set.

The means were held at 37 ° C and the color change was determined accordingly. The results obtained are shown in Table VI.

Table VI Verorganic Antiseptic Hold Time at 370C (days) such acids medium 3 10 20 30 60 90 150 No.

11 Lactic acid sodium benzoate - - - + + ++ +++ 12 Acetic acid (0.5 Weight -5 ') + - - - + + ++ +++ 13 lactic acid methyl-p-hydro- - - - - + + +++ 14 acetic acid xybenzoate (0.05 - - - - + - + +% by weight) 15 lactic acid 1,4-dithio- + + ++ +++ +++ +++ +++ 16 acetic acid threitol (0.02 + + ++ +++ +++ +++ +++ wt. -5 ') 17 lactic acid Potassium tellu- - - - - # + ++ 18 acetic acid rit (0.05 - - - - - # +% by weight) 19 lactic acid Sodium Azide - - - - + + ++ 20 Acetic Acid (0.05 wt%) - - - - - - # From the above It can be seen from the results that the agents were kept stable over long periods of time by using antiseptics, especially potassium tellurite and sodium trinitride, incorporated.

6. Experiment 6: In means, the 10 wt. - Sucrose, 0.003 wt .-% Resazurin and various amounts of potassium tellurite (pH 6.8, adjusted with acetic acid) contained, a fixed amount of a suspension of Streptococcus mutans (GS-5) inoculated and the mixture was incubated at 37 ° C. The change in pH and the color of the agent was observed. The results are shown in Table VII.

Table VII Content of Ka holding time at 370C (min) lium tellurite 0 15 30 60 (wt. -5 ') pH color pH color pH color pH color none (comparison) 6.8 blue 6.0 reddish - 5.3 pink 5.0 weak violet pink 0.1 6.8 blue 6.7 violet 6.6 violet 6.5 reddish purple 0.075 6.8 blue 6.3 purple 5.8 red 5.4 pink 0.05 6.8 blue 6.2 violet 5.6 red 5.3 pink 0.025 6.8 blue 6.1 reddish - 5.4 pink 5.2 weak violet pink 0.01 6.8 blue 6.0 reddish-5.4 pink 5.2 weak purple pink From the above It can be seen from the results that the addition of antiseptic agents is not undesirable Effect on the determination of the dental caries activity results, since this means the color change of the agent in an amount of 0.1 to 0.01 wt. 5 ', in particular 0.075 wt. 5' or less, do not affect.

The agents according to the invention therefore contain 2.5 to 40% by weight of one Saccharide and 0.0001 to 0.05% by weight, 6 of a pH indicator with a color change point in the pH range from 5 to 7, the agent having a pH of 6.5 to 7.0, the has been adjusted with an alkali or an acid.

The agent according to the invention is prepared in such a way that the fixed amount of the saccharide and the pH indicator in water or an aqueous one Alcohol (e.g. ethanol) with a concentration of alcohol that affects the bacteria in the Mouth has no undesirable effect, e.g., about 1 wt. Or less, preferably dissolves in water or a 0.1 to 1 wt. 5% aqueous ethanol solution and then adjusts the pH value. In a preferred embodiment, the pH indicator in an aqueous solution or an aqueous alcohol solution of an alkali compound dissolved and in the obtained mixture the saccharide is dissolved. After that, will the solution adjusted to the desired pH value with an acid.

Saccharides suitable for the invention are, for example, sucrose, clucose and fructose, among which sucrose is most preferred. The saccharides will be used in an amount of 2.5 to 40% by weight, preferably 5 to 20% by weight.

Suitable pH indicators are e.g. Resazurin, Lakmoid or

Resorcinol blue, methyl red and alizarin, of which resazurin and lakmoid, particularly resazurin, are preferred. The pH indicators are in a lot from 0.0001 to 0.05% by weight, preferably 0.001 to 0.01% by weight, more preferred 0.001 to 0.005 wt% is used.

Examples of alkali compounds used to adjust the pH of the agent are alkali metal hydroxides (e.g. sodium hydroxide, Potassium hydroxide and lithium hydroxide), alkaline earth metal hydroxides (e.g. magnesium hydroxide and barium hydroxide), alkali metal carbonates or bicarbonates (e.g. sodium carbonate, Potassium carbonate and sodium bicarbonate), of which sodium hydroxide is the most suitable is. Examples of acids for regulating the pH are lactic acid, tartaric acid, Citric acid, malonic acid, Oxalic acid, acetic acid, formic acid and the like, with lactic acid, tartaric acid and acetic acid being most suitable.

The agent works with these alkali compounds and acids pH range from 6.5 to 7.0, preferably 6.7 to 7.0, adjusted.

In the agent according to the invention can optionally an antiseptic Agents, e.g. sodium azide or a tellurite, can be incorporated to stimulate bacterial growth not adversely affected in the mouth. Suitable examples of tellurites are potassium tellurite, Sodium tellurite, magnesium tellurite and calcium tellurite, of which potassium tellurite is most suitable. The antiseptic agent is used in an amount from 0.01 to 0.1 5 'by weight, preferably 0.025 to 0.075% by weight, based on the weight of the agent, used.

The test for determining the dental caries activity using the agent according to the invention is carried out in such a way that a sample of plaque collects from the patient to be tested, the plaque into the agent incorporated and the color change of the agent after incubation of the agent over observed for a fixed period of time (e.g. 30 min). The change in color varies according to the amount of acid produced by the bacteria. The color changes in the order blue reddish violet R solid pink i colorless in the case of Resazurin, blue> purple. ) red i pink in the case of lakmoid, yellow; light yellow> + dark pink 9 pink in the case of methyl red and red "pale pink" light yellow yellow in the case of Alizarin. Because the test was carried out within a very short period of time (e.g. 30 min) will can, the test is not due to the incubation temperature significantly influenced and it can at a temperature of room temperature (approx 250C) up to 37 ° C.

The agents according to the invention also have a selectivity towards Streptococcus mutans, which results from the following experiment.

To a 0.002% by weight aqueous solution of resazurin (pH 6.7, 1 ml), which contained 10% by weight of sucrose, became a suspension of various Bacteria (0.2 ml) were added and the mixture was incubated at 37 ° C. The change the color of the agent was observed. The results are shown in Table VIII.

Table VIII Bacteria Incubation Time at 370C (min) 5 10 30 Streptococcus reddish-red weak mutans violet-pink Streptococcus violet reddish-reddish sanguis violet violet Streptococcus violet violet reddish mitis violet Streptococcus violet reddish-red salivarius violet Lactobacillus violet violet reddishcasei purple From the above results, it can be seen that when Streptococcus mutans the color of the remedy changed within a very short period of time, but that in the case of other bacteria the color change delayed was. The agent according to the invention thus has a selectivity for Streptococcus mutans.

The preparation of the agent according to the invention is as follows Examples explained.

Example 1 Components wt. -5 'sucrose 10 resazurin 0.002 aqueous 0.1N sodium hydroxide solution 5 distilled water about 80 0.1N lactic acid to one pH adjusted from 6.7 to 7.0 According to the above approach, resazurin is aqueous Dissolved 0.1N sodium hydroxide solution and distilled water is added to this. The sucrose is dissolved in the solution and afterwards the agent is applied to one pH adjusted from 6.7 to 7.0, creating a test agent for the determination the dental caries activity is maintained.

Example 2 Using the same approach as in Example 1, except that, however, 0.002 wt. -5 'Lakmoid is used in place of resazurin similarly becomes a test agent for determining dental caries activity manufactured.

EXAMPLE 3 Using the same approach as in the example 1, except that 0.02% by weight of methyl red is used instead of resazurin, a test agent for determining the activity of dental caries is prepared in a similar manner.

Example 4 Using the same approach as in the example 1, but with the exception that 0.02 wt. Alizarin is used in place of resazurin similarly becomes a test agent for determining dental caries activity manufactured.

Example 5 components by weight -5 'sucrose 10 resazurin 0.003 aqueous 0.1N sodium hydroxide solution 5 antiseptic (sodium azide) 0.05 distilled Water about 80 0.1N lactic acid adjusted to a pH of 6.7 to 7.0.

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

Example 6 Components by weight 5: sucrose 10 Re sazurin 0.003 aqueous 0.1N sodium hydroxide solution 5 antiseptic (potassium tellurite) 0.05 distilled Water a total of 100 0.1N acetic acid adjusted to a pH of 6.8 to 7.0 According to the above approach, resazurin is in the 0.1N aqueous sodium hydroxide solution dissolved and distilled water is added to this. In the solution there is sucrose and potassium tellurite are dissolved and the mixture is adjusted to a pH of 6.8 to 7.0 adjusted, making a test agent for determining the dental caries activity is obtained.

End of description.

Claims (11)

Test means for determining a dental caries activity. Patent claims Test means for determining a dental caries activity, thereby g e k e n n z e i c h n e t that it is 2.5 to 40% by weight, of a saccharide, 0.0001 to 0.05% by weight a pH indicator with a color change point in the pH range from 5 to 7 and the remainder contains a medium from the group of water and an aqueous alcohol solution, wherein the agent has a pH range of 6.5 to 7.0. 2. Test means according to claim 1, characterized in that g e k e n n -z e i c h n e t that the saccharide is a substance selected from the group consisting of sucrose, glucose and fructose is. 3. Test means according to claim 1, characterized in that g e k e n n -z e i c h n e t that the saccharide is 5 to 20 wt. g of sucrose. 4. Test means according to claim 1, characterized in that g e k e n n -z e i c h n e t that the pH indicator is a substance from the group resazurin, lakmoid, methyl red and alizarin is. 5. Test means according to claim 4, characterized in that g e k e n n -z e i c h n e t that the pH indicator is a substance from the group resazurin and lakmoid. 6. Test means according to claim 1, characterized in that g e k e n n -z e i c h ne t that the pH indicator is 0.001 to 0.01 wt. -5 'resazurin. 7. Test means according to claim 1, characterized in that g e k e n n -z ei c h ne t that there is 5 to 20% by weight of sucrose, 0.001 to 0.01% by weight of 5 'resazurin and the remainder Contains water, the agent with sodium hydroxide and an acid from the group Lactic acid, tartaric acid and acetic acid to a pH in the range of 6.7 to 7.0 has been adjusted. 8. Test agent according to claim 7, characterized in that g e k e n n -z e i c h n e t that it was made in such a way that sucrose and resazurin have been dissolved in an aqueous sodium hydroxide solution and that the pH has been adjusted to the range of 6.7 to 7.0 with the acid. 9. Test agent according to one of claims 1, 2, 3, 4, 5, 6, 7 or 8, in that it is an antiseptic agent from the group Sodium azide and a tellurite in an amount of 0.01 to 0.1% by weight c has been incorporated. 10. Test means according to claim 9, characterized in that g e k e n n -z e i c h n e t that the tellurite is potassium tellurite. 11. Test means according to claim 10, characterized in that g e k e n n -z e i c h n e t that the potassium tellurite incorporated in an amount of 0.025 to 0.075 wt has been.