Dental caries pathogenic bacterium culture medium and preparation method thereof

Abstract

The invention discloses a decayed tooth pathogenic bacterium culture medium and a preparation method thereof, and relates to the field of microorganism culture and detection, and the key point of the technical scheme is that the culture medium comprises a culture solution and a hydrogen ion sensing membrane; the hydrogen ion sensing membrane is arranged in the culture solution; the formula of the hydrogen ion sensing membrane comprises a hydrogen ion probe, a cross-linkable ionic polymer, a cross-linking agent and a nano inorganic filler, the hydrogen ion probe is a mixture of at least one of bromcresol purple, bromcresol green, phenol red and methyl red and resorufin amyl ether; the hydrogen ion probe, the cross-linkable ionic polymer, the nano inorganic filler and the cross-linking agent are mixed and then cured to form the hydrogen ion sensing membrane. The aim of conveniently detecting the pathogenic bacteria is fulfilled.

Description

Technical Field

[0001] This invention relates to a microbial culture and detection technology, and more specifically, to a method for preparing a culture medium for caries-causing bacteria. Background Technology

[0002] Dental caries has a high incidence and wide distribution, making it a major common oral disease and one of the most prevalent diseases in humans. It can lead to secondary pulpitis and periapical periodontitis, and even inflammation of the alveolar bone and jawbone. If left untreated, the lesions continue to progress, forming cavities and eventually causing complete destruction and loss of the tooth crown. Bacteria are a necessary condition for the development of dental caries. The main bacteria causing caries include Streptococcus mutans, Streptococcus salivarius, and Lactobacillus, hereinafter referred to as cariogenic bacteria. These bacteria share the characteristic of producing acid, breaking down sugars in the oral cavity and forming acidic metabolic products. These acidic metabolic products demineralize the tooth surface, thereby damaging the tooth and causing caries. Culture and detection of the amount of cariogenic acid-producing bacteria is an important method for determining susceptibility to cariogenic acid-producing bacteria. Caries culture media are mainly used to detect and assess the risk of dental caries. By detecting the number and activity of bacteria in the oral cavity, it helps determine an individual's risk of developing dental caries.

[0003] Most existing culture media use tryptone as the main ingredient. These media have a relatively simple composition and cannot meet all the requirements for the growth of caries-causing bacteria. Therefore, the culture speed is slow, requiring more than 48 hours, and a colorimetric card is needed to obtain results after culture. Therefore, there is an urgent need for a culture medium that can rapidly culture and screen for caries-causing bacteria. Summary of the Invention

[0004] Firstly, the purpose of this invention is to provide a culture medium for caries-causing bacteria. By setting a hydrogen ion sensing membrane in the culture medium, the level of caries-causing bacteria can be determined directly by detecting changes in pH within the culture medium. The hydrogen ion probe is specifically designed, which has higher detection sensitivity compared to traditional pH reagents.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a caries-causing bacteria culture medium, characterized in that: the culture medium comprises a culture solution and a hydrogen ion sensing membrane; the hydrogen ion sensing membrane is disposed in the culture solution; the formulation of the hydrogen ion sensing membrane comprises a hydrogen ion probe, a crosslinkable ionomer, a crosslinking agent, and a nano-inorganic filler; the hydrogen ion probe is a mixture of at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with halogenated ether; the hydrogen ion probe, the crosslinkable ionomer, the nano-inorganic filler, and the crosslinking agent are mixed and then cured to form the hydrogen ion sensing membrane.

[0006] Furthermore, the crosslinkable ionic polymer is one of quaternary ammonium silane and methacrylate; the crosslinking agent is one of dicumyl peroxide and benzoyl peroxide; and the nano-inorganic filler is nano-silica or nano-titanium dioxide.

[0007] Furthermore, the mass fractions of each component in the formulation of the hydrogen ion sensing membrane are as follows: 92-103 parts of crosslinkable ionomer, 35-55 parts of nano-inorganic filler, 0.1-1 parts of crosslinking agent, and 0.1-1 parts of hydrogen ion probe; wherein the mass fraction of the halogenated ether in the hydrogen ion probe is 0.1-0.6 parts.

[0008] Furthermore, the formulation and mass fractions of the hydrogen ion sensing membrane are as follows: 95 parts quaternary ammonium silane, 45 parts nano silica, 0.4 parts dicumyl peroxide, 0.5 parts bromocresol purple, and 0.5 parts halogenated ether.

[0009] Furthermore, the composition of each component in the culture medium is as follows: peptone 0.5%~3%, sucrose 10%~50%, glucose 0.05%~0.5%, dipotassium hydrogen phosphate 0.1%~1%, buffer 0.05%~0.5%, antibacterial agent 5%~50%, and the remainder is purified water.

[0010] Furthermore, the selective antibacterial agent is at least one selected from phenylethyl alcohol, sodium azide, actinomycete ketone, and potassium tellurate.

[0011] Furthermore, the buffer is one of phosphate buffer or MES buffer.

[0012] Furthermore, the culture medium also includes a culture flask; the culture flask body is a colorless and transparent cylindrical container; the culture flask is pre-filled with culture medium and a hydrogen ion sensing membrane; the hydrogen ion sensing membrane is disposed at the bottom of the culture flask.

[0013] In a second aspect, the present invention provides a method for preparing the culture medium for caries-causing bacteria as described in the first aspect, comprising the following steps: A. Preparation of culture medium: peptone, dipotassium hydrogen phosphate, and glucose are added sequentially to purified water and stirred at 60°C until completely dissolved; sucrose is added and stirred continuously until a transparent solution is obtained; buffer solution is added and stirred evenly; a selective antibacterial agent is added; the solution is brought to a final volume and then sterilized to obtain a culture medium; B. Preparation of hydrogen ion sensing membrane: a hydrogen ion probe, a crosslinkable ionomer, nano-inorganic filler, and a crosslinking agent are mixed and injected into a culture flask to solidify in the culture flask and form a hydrogen ion sensing membrane; the hydrogen ion probe is at least one of bromocresol purple, bromocresol green, phenol red, and methyl red mixed with halogenated ether; C. After sterilizing the culture flask with the solidified hydrogen ion sensing membrane, the culture medium is injected.

[0014] Furthermore, the mass fractions of each component in the formulation of the hydrogen ion sensing membrane are as follows: 92-103 parts of crosslinkable ionomer, 35-55 parts of nano-inorganic filler, 0.1-1 parts of crosslinking agent, and 0.1-1 parts of hydrogen ion probe; wherein the mass fraction of the halogenated ether in the hydrogen ion probe is 0.1-0.6 parts; the preparation method of the hydrogen ion sensing membrane is as follows: A. The crosslinkable ionomer and nano-inorganic filler are stirred and mixed until the nano-inorganic filler is completely wetted to obtain the first product; B. At least one of bromocresol purple, bromocresol green, phenol red, and methyl red is dissolved with halogenated ether in anhydrous ethanol to obtain the second product; C. The crosslinking agent is mixed with hydroxyl silicone oil until it reaches a fluid state to obtain the third product; D. The first product, the second product, and the third product are mixed and stirred for 1-5 hours, and degassed to obtain the fourth product; E. The fourth product is injected into a culture flask and cured by heating.

[0015] In summary, the present invention has the following beneficial effect: 1. By setting up a hydrogen ion sensing membrane in the culture medium, the content of caries-causing bacteria can be directly determined by detecting changes in pH within the culture medium; the hydrogen ion probe is designed specifically for this purpose, and its detection sensitivity is higher than that of traditional pH reagents.

[0016] 2. By optimizing the culture medium composition, the metabolism and acid production of cariogenic bacteria (such as Streptococcus mutans) are significantly accelerated, shortening the culture time to less than 24 hours; the selective antibacterial agent effectively inhibits interference from non-target bacteria, improving detection specificity; the rapid culture medium culture and the high-sensitivity hydrogen ion probe work together to achieve the purpose of rapid detection of cariogenic bacteria. Detailed Implementation

[0017] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the embodiments.

[0018] Example 1: This example provides a culture medium for detecting caries-causing bacteria and its preparation method. The culture medium preparation method includes the following steps: Step 1: Culture medium preparation. Peptone, dipotassium hydrogen phosphate, and glucose are added to purified water in sequence and stirred at 60°C until completely dissolved. Sucrose is added and stirred continuously until a clear solution is obtained. Buffer solution is added and stirred evenly. Selective antibacterial agent is added. After adjusting the volume, sterilize to obtain the culture medium.

[0019] By adding peptone, dipotassium hydrogen phosphate, and glucose in stages, the system ensures that each component is fully dissolved and does not decompose at 60°C, gradually constructing the complex nutrient environment required for the proliferation of cariogenic bacteria. High-concentration sucrose is then added to simulate the high-sugar conditions in the oral cavity, promoting bacterial fermentation and acid production. Simultaneously, a buffer stabilizes the initial pH, preventing premature acidification that could interfere with the sensitivity of the sensor membrane. A selective antibacterial agent is used to specifically inhibit the growth of non-target bacteria. After adjusting the volume, sterilization ensures the culture medium is sterile, laying the foundation for subsequent pure culture of the target bacteria.

[0020] Step 2: Preparation of the hydrogen ion sensing membrane. A mixture of a hydrogen ion probe, a crosslinkable ionomer, nano-inorganic filler, and a crosslinking agent is injected into a culture flask and solidified inside to form a hydrogen ion sensing membrane. The hydrogen ion probe is a mixture of at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with halogenated ether. The culture flask is a colorless, transparent cylindrical container. A colorimetric card is placed at the bottom of the culture flask. The height of the mixed solution is one-tenth to one-fifth of the height of the culture flask.

[0021] Crosslinkable ionomers, when mixed with a crosslinking agent and heated, undergo a crosslinking reaction to form a substrate loaded with hydrogen ion probes. The addition of nano-inorganic materials enhances the substrate's strength. The crosslinked ionomers possess ion-exchange properties, creating conditions for the diffusion of hydrogen ions in the culture medium within the substrate and their reaction with the hydrogen ion probes. After the mixed solution is injected into the culture flask and solidifies, the sensing membrane adheres tightly to the flask wall or bottom, ensuring that bacterial metabolic acid production directly contacts the membrane surface. Color changes reflect the degree of pH decrease in real time, eliminating the need for external equipment. The uniform distribution of the hydrogen ion probes and the substrate's permeability design enable the membrane to respond rapidly to minute changes in hydrogen ion levels. The color gradient shows a linear correlation with bacterial load / acid production activity, improving the accuracy of quantitative detection. In this embodiment, a mixture of at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with halogenated ether is commonly used as a composite hydrogen ion probe. The molecular resonance system formed by the cation and anion (R-, R+) groups of the composite hydrogen ion probe maintains equilibrium with the (R-OH, RH) groups formed by the combination of these groups with water molecules. When the number of protons [H+] in the system continuously increases, the resonance system will protonate, forming a photoluminescent body. Using the molecular resonance hydrogen ion probe as a chemical sensor, its detection sensitivity is more than 1000 times that of pH reagents, as evaluated by the response slope.

[0022] Step 3: After sterilizing the culture bottle with the solidified hydrogen ion sensing membrane, inject the culture medium.

[0023] After the culture bottle with the cured membrane is sterilized separately, it is then filled with pre-sterilized culture medium. This double sterilization ensures aseptic operation throughout the entire process, preventing contamination from other microorganisms that could interfere with the test results. Once injected, the culture medium comes into direct contact with the sensing membrane. The cariogenic bacteria metabolize and produce acid, rapidly lowering the pH. The membrane color change is synchronized with the bacterial activity, achieving integrated "culture-detection" and shortening the time required for traditional step-by-step culture and colorimetric card operations. The pre-prepared culture medium is ready to use; users only need to inoculate the sample and observe the color development, simplifying the operation and making it suitable for chairside rapid screening or home self-testing scenarios.

[0024] Optionally, the culture flask includes a body, a cap, and a rubber stopper; the body is a cylindrical structure with a closed bottom and an open top, and the body is made of a transparent material; optionally, it is made of glass, plexiglass, plastic, or other materials; the interior of the flask provides a sterile environment. The cap is used to cover the mouth of the flask; optionally, the cap is made of aluminum alloy or plastic. The rubber stopper is made of rubber or plastic and is used to plug the mouth of the flask to prevent leakage. Optionally, those skilled in the art should know that the same technical effect can be achieved by replacing the culture flask with a culture tube. The colorimetric card is directly attached to the bottom of the flask, and is in the same light source environment as the sensing membrane, avoiding misjudgment caused by differences in light from external colorimetric cards. The colorimetric card indicates the pH gradient (e.g., 4.0–7.0) and the corresponding color, allowing users to directly compare the membrane color with the colorimetric card to quantify the degree of acid production.

[0025] The composition of each component in the culture medium is as follows: peptone 0.5%~3%, sucrose 10%~50%, glucose 0.05%~0.5%, dipotassium hydrogen phosphate 0.1%~1%, buffer 0.05%~0.5%, antibacterial agent 5%~50%, and the remainder is purified water.

[0026] Peptone provides nitrogen, amino acids, and growth factors, supporting the proliferation of cariogenic bacteria. Sucrose, a high-concentration carbon source, promotes bacterial fermentation and acid production, mimicking the cariogenic environment of the oral cavity. Glucose, a rapidly metabolized carbon source, assists in initial bacterial colonization. Dipotassium hydrogen phosphate provides nutrients and promotes microbial growth. A buffer stabilizes the initial pH, ensuring the sensitivity of the sensor membrane. A selective antibacterial agent inhibits the growth of non-target bacteria. Purified water, a solvent, dissolves all components.

[0027] The mass fractions of each component in the formulation of the hydrogen ion sensing membrane are as follows: 92-103 parts of crosslinkable ionomer, 35-55 parts of nano-inorganic filler, 0.1-1 parts of crosslinking agent, and 0.1-1 parts of hydrogen ion probe; wherein the mass fraction of the hydrogen ion probe is 0.1-0.6 parts of halogenated pentylenetetrazol.

[0028] The hydrogen ion probe is a reaction product of at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with fluorescein; the crosslinkable ionic polymer is one of quaternary ammonium silane and methacrylate; the crosslinking agent is one of dicumyl peroxide and benzoyl peroxide; and the nano-inorganic filler is nano-silica or nano-titanium dioxide.

[0029] Quaternary ammonium silanes are a class of hybrid compounds whose molecular structure contains both silane coupling agent groups and organic quaternary ammonium salt cationic groups. They combine the stability and crosslinkability of organosilicon materials with the strong polarity, hydrophilicity, and ion exchange capacity of quaternary ammonium salt compounds. As the main polymer of sensing membranes, they form the three-dimensional network structure framework supporting dyes and fillers.

[0030] Methacrylates, as another film-forming material option, form a free radical curing system with crosslinking agents.

[0031] Dicumyl peroxide, as a thermosetting initiator, decomposes during the heating and curing process to generate free radicals, which initiate the polymerization and crosslinking reaction of methacrylate monomers or other unsaturated groups, transforming the liquid resin into a solid polymer network.

[0032] Benzoyl peroxide, as an alternative to DCP, can be used in medium and low temperature curing systems.

[0033] Nano-silica, dispersed in a polymer matrix, can significantly improve the mechanical strength, hardness, and wear resistance of the sensing membrane, preventing it from being damaged or scratched during cultivation, transportation, or cleaning.

[0034] Nano-titanium dioxide, similar to nano-silicon dioxide, serves as an enhancer and filler. Being white, it can act as a background brightener, making the sensor film's color more vibrant and bright, thus improving color contrast and reading accuracy.

[0035] Bromocresol purple is yellow in acidic conditions (pH < 5.2), transitions to purple in neutral to alkaline conditions (pH 5.2–6.8), and turns deep purple at pH > 6.8. The color change range (pH 5.2–6.8) covers the typical pH after acid production by cariogenic bacteria (e.g., the pH can drop to 4.5–5.5 after acid production by Streptococcus mutans), making it suitable for detecting the early acid-producing activity of cariogenic bacteria.

[0036] Bromocresol green is yellow in acidic environments (pH < 3.8), transitions to blue-green in neutral environments (pH 3.8–5.4), and turns blue in alkaline environments (pH > 5.4). Its wide color change range (pH 3.8–5.4) makes it suitable for testing in strongly acidic environments and can help determine high caries risk (e.g., when lactobacilli produce large amounts of acid).

[0037] Phenol red is yellow in acidic conditions (pH < 6.8), red in neutral conditions (pH 6.8–8.4), and turns purplish-red in alkaline conditions (pH > 8.4). It is routinely used for pH monitoring in cell culture media and can be combined with other indicators to extend the acid detection range. It is suitable for acid-producing environments with mixed bacterial strains.

[0038] Methyl red appears red in acidic conditions (pH < 4.4), transitions to orange in neutral conditions (pH 4.4–6.2), and is yellow in alkaline conditions (pH > 6.2). It is sensitive to low pH (4.0–4.5) and is often used to verify the presence of highly acid-producing bacteria such as Lactobacillus.

[0039] Halogen ether, as a fluorescein, exhibits significantly reduced fluorescence intensity in acidic conditions (pH < 4.0) and emits green fluorescence in neutral to alkaline conditions (pH > 6.0). Fluorescence changes can be used to quantitatively detect minute fluctuations in pH.

[0040] The selective antibacterial agent is at least one of phenylethanol, sodium azide, actinomycete ketone, and potassium tellurate. Phenylene disrupts the lipid structure of bacterial cell membranes, increases membrane permeability, and leads to leakage of intracellular substances. It preferentially inhibits the membrane stability of Gram-negative bacteria (such as Escherichia coli and Pseudomonas), and has less effect on Gram-positive cariogenic bacteria (such as Streptococcus mutans).

[0041] Sodium azide inhibits cytochrome oxidase, blocks the electron transport chain, and interferes with energy metabolism. It has a strong inhibitory effect on aerobic bacteria (such as Bacillus and some fungi), but a weaker effect on facultative anaerobic cariogenic bacteria (such as Lactobacillus).

[0042] Actinomycin binds to eukaryotic ribosomes (80S type), inhibiting protein synthesis and blocking fungal growth. It has no effect on prokaryotic (bacterial) ribosomes (70S type), therefore it does not affect the proliferation of cariogenic bacteria.

[0043] Potassium tellurate is reduced to non-toxic elemental tellurate (a black precipitate) by certain bacteria (such as Staphylococcus and Lactobacillus), while other bacteria are inhibited due to the lack of reductase. It inhibits Gram-negative bacteria and some Gram-positive bacteria (such as bacteria other than Streptococcus), but is non-toxic to cariogenic bacteria that can metabolize tellurate (such as some Lactobacillus).

[0044] The buffer is either phosphate buffer or MES buffer. Phosphate buffer, composed of dihydrogen phosphate (KH₂PO₄) and dihydrogen phosphate (K₂HPO₄), buffers pH changes through proton exchange (H⁺ / OH⁻). With a pH of 5.8–8.0 (optimal pH 6.8–7.4), it is suitable for initial pH stability under neutral conditions. It has a high buffering capacity, suitable for suppressing pH fluctuations during culture medium storage or sterilization, but its buffering capacity for sustained acid production by highly acid-producing bacteria is limited. Lactobacillus, a highly acid-producing bacterium in caries pathogens, can be used as a buffer to achieve rapid results. MES buffer is a Good's buffer that maintains pH stability through reversible protonation of the morpholine ring and sulfonic acid groups. With a pH of 5.5–6.7, it approximates the typical acidic environment (pH 4.5–5.5) after caries-causing bacteria produce acid. It is chemically inert and does not complex with metal ions, thus avoiding interference with bacterial metabolism. In the early stage of acid production, it slows down the rate of pH decrease, making the color gradient easier to observe (such as the gradual change from pH 6.5 to 5.0). It is used as a buffer in the acid production stage, prolongs the color response time, and improves the detection sensitivity.

[0045] The method for using the caries-causing bacteria culture medium of the present invention includes the following steps: Dental plaque samples are collected using sterile swabs, and a bacterial suspension is prepared with physiological saline. The suspension is then quantitatively inoculated into a culture bottle, mixing the bacterial suspension with a culture medium containing a selective antibacterial agent, a buffer, and a carbon and nitrogen source. The culture bottle is placed in a constant temperature environment of 35-37°C and incubated statically for 12-24 hours. The color change of the built-in hydrogen ion sensing membrane is observed in real time through the colorless, transparent bottle. The hydrogen ion probe is cross-linked and solidified into a film, which can exhibit a gradient color change (e.g., yellow → purple) with the H⁺ released by acid-producing bacteria metabolism. The activity and risk level of caries-causing bacteria are assessed by directly comparing the pH color chart pre-placed at the bottom of the bottle with the corresponding pH value (e.g., pH ≤ 5.0 indicates high risk), without the need for external equipment or step-by-step operations.

[0046] This invention uses selective antibacterial agents to inhibit non-target bacteria, and combines MES buffer to stabilize the initial pH and extend the colorimetric response window, enabling simultaneous detection of rapid proliferation and dynamic acid production of cariogenic bacteria. With the transparent cylindrical bottle and embedded colorimetric card design, the detection sensitivity is increased by 50%, the time is shortened to within 24 hours, and the operation threshold is low, making it suitable for accurate screening in clinical, community and home settings.

[0047] Example 2: This example provides a formulation and preparation method for a hydrogen ion sensing membrane.

[0048] 95 parts of quaternary ammonium silane, 45 parts of nano silica, 0.4 parts of dicumyl peroxide, 0.5 parts of bromocresol purple, and 0.5 parts of halogenated ether.

[0049] The preparation method includes the following steps: A. The crosslinkable ionomer and the nano-inorganic filler are stirred and mixed until the nano-inorganic filler is completely wetted to obtain the first product; B dissolves at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with halogenated ether in anhydrous ethanol to obtain a second product; C. The crosslinking agent is mixed with hydroxyl silicone oil until it reaches a fluid state to obtain the third product; D. Mix and stir the first product, the second product and the third product for 1 to 5 hours, and then degas to obtain the fourth product; E injects the fourth product into a culture flask and heats it to solidify.

[0050] In this embodiment, a quaternary ammonium salt silane and nano-silica system is used to achieve high strength, low shrinkage and fast response of the sensing membrane, resulting in optimal overall performance.

[0051] Example 3: This example provides a formulation for a hydrogen ion sensing membrane, which is prepared using the method provided in Example 2.

[0052] 100 parts methacrylate, 50 parts nano titanium dioxide, 0.5 parts benzoyl peroxide, 0.2 parts bromocresol purple, and 0.2 parts halogenated ether.

[0053] In this embodiment, a system of methacrylate and nano-titanium dioxide is used, which provides excellent optical contrast and makes color interpretation clearer and more intuitive.

[0054] Example 4: This example provides a formulation for a hydrogen ion sensing membrane, which is prepared using the method provided in Example 2.

[0055] 100 parts methacrylate, 40 parts nano silica, 0.8 parts benzoyl peroxide, 0.1 parts bromocresol green, and 0.1 parts halogenated ether.

[0056] In this embodiment, a system of methacrylate and high-content crosslinking agent is used, which optimizes curing efficiency and production cost, making it suitable for large-scale preparation.

[0057] Example 5: Based on Examples 1 and 2, this example provides a culture medium for caries-causing bacteria. The culture medium comprises a nutrient solution and a hydrogen ion sensing membrane.

[0058] The mass ratio of each component in the culture medium is as follows: peptone 0.5%, sucrose 10%, glucose 0.5%, dipotassium hydrogen phosphate 1%, buffer 0.05%, selective antibacterial agent 5%, and the remainder is purified water; the selective antibacterial agent is phenylethanol; the buffer is phosphate buffer. In the testing of multiple samples, the culture medium provided in this embodiment was used, and the testing time was less than 18 hours. The following criteria were used to determine the level of pathogenic bacteria in the samples: High risk: yellow after 6-8 hours of incubation; Medium risk: purple-yellow transition after 10-12 hours of incubation; Low risk: still purple after 18 hours of incubation.

[0059] The pathogenic bacteria culture medium provided in this embodiment is suitable for samples with high cariogenic bacterial load, and risk assessment can be completed in as little as 6 hours, meeting the needs of rapid clinical screening.

[0060] Example 6: Based on Examples 1 and 2, this example provides a culture medium for caries-causing bacteria. The culture medium comprises a nutrient solution and a hydrogen ion sensing membrane.

[0061] The mass ratio of each component in the culture medium is as follows: peptone 3%, sucrose 50%, glucose 0.05%, dipotassium hydrogen phosphate 0.1%, buffer 0.5%, selective antibacterial agent 5%, and the remainder is purified water; the selective antibacterial agent is sodium azide; the buffer is MES buffer.

[0062] During the testing of multiple samples using the culture medium provided in this embodiment, the testing time was consistently less than 12 hours. It is speculated that the high concentration of sucrose provided an extreme environment, stimulating rapid acid production by bacteria. The following criteria were used to determine the level of pathogenic bacteria in the samples: High risk: yellow after 4-6 hours of incubation; Medium risk: blue-green after 8-12 hours of incubation; Low risk: still blue after 12 hours of incubation.

[0063] Example 7: Based on Examples 1 and 2, this example provides a culture medium for caries-causing bacteria. The culture medium comprises a nutrient solution and a hydrogen ion sensing membrane.

[0064] The mass ratio of each component in the culture medium is as follows: peptone 1%, sucrose 20%, glucose 0.3%, dipotassium hydrogen phosphate 0.5%, buffer 0.2%, selective antibacterial agent 5%, and the remainder is purified water; the selective antibacterial agent is sodium azide and phenylethanol, with a mass ratio of 1:1; the buffer is phosphate buffer. In the testing of multiple samples, the culture medium provided in this embodiment was used, and the testing time was less than 16 hours. The following criteria were used to determine the level of pathogenic bacteria in the samples: High risk: yellow after 8-10 hours of incubation; Medium risk: yellowish-purple after 12-14 hours of incubation; Low risk: still purple after 16 hours of incubation.

[0065] Example 8: Based on Examples 1 and 2, this example provides a culture medium for caries-causing bacteria. The culture medium comprises a nutrient solution and a hydrogen ion sensing membrane.

[0066] The mass ratio of each component in the culture medium is as follows: peptone 1%, sucrose 20%, glucose 0.3%, dipotassium hydrogen phosphate 0.5%, buffer 0.2%, selective antibacterial agent 5%, and the remainder is purified water; the selective antibacterial agent is sodium azide; the buffer is MES buffer. In the testing of multiple samples, the culture medium provided in this embodiment was used, and the testing time was less than 16 hours. The following criteria were used to determine the level of pathogenic bacteria in the samples: High risk: yellow after 7-10 hours of incubation; Medium risk: yellowish-purple after 10-14 hours of incubation; Low risk: still purple after 16 hours of incubation.

[0067] Nano-silica significantly improves the mechanical strength of the membrane through physical filling and interfacial bonding, preventing membrane rupture or deformation during immersion or agitation in the culture medium. Nanoparticles reduce the membrane's hydrophilic swelling; after 16 hours of immersion, the thickness swelling rate is less than 5%, maintaining pore structure stability and preventing color drift. Nano-silica is uniformly dispersed in the substrate, forming uniform nanoscale pores, promoting rapid H⁺ diffusion and shortening the colorimetric response time. The adsorption of some bromocresol violet isomers on the nanoparticle surface prevents their aggregation, resulting in a clearer colorimetric gradient and improved semi-quantitative accuracy. The nanoporous structure delays the neutralization of H⁺ by the MES buffer, extending the colorimetric dynamic window and facilitating the observation of gradual acid production processes.

[0068] In the above embodiments, by optimizing the culture medium composition, the metabolic acid production of cariogenic bacteria (such as Streptococcus mutans) is significantly accelerated, shortening the culture time to less than 24 hours; the selective antibacterial agent effectively inhibits interference from non-target bacteria, improving detection specificity; the rapid culture medium culture and the high-sensitivity hydrogen ion probe work together to achieve the purpose of rapid detection of cariogenic bacteria.

[0069] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A culture medium for caries-causing bacteria, characterized in that: The culture medium comprises a culture solution and a hydrogen ion sensing membrane; the hydrogen ion sensing membrane is disposed within the culture solution. The formulation of the hydrogen ion sensing membrane includes a hydrogen ion probe, a crosslinkable ion polymer, a crosslinking agent, and nano-inorganic fillers; the hydrogen ion probe is a mixture of at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with halogenated ether; the hydrogen ion probe, crosslinkable ion polymer, nano-inorganic fillers, and crosslinking agent are mixed and cured to form the hydrogen ion sensing membrane.

2. The caries-causing bacteria culture medium according to claim 1, characterized in that: The crosslinkable ionic polymer is one of quaternary ammonium silane and methacrylate; the crosslinking agent is one of dicumyl peroxide and benzoyl peroxide; and the nano-inorganic filler is nano-silica or nano-titanium dioxide.

3. The caries-causing bacteria culture medium according to claim 2, characterized in that: The mass fractions of each component in the formulation of the hydrogen ion sensing membrane are as follows: 92-103 parts of crosslinkable ionomer, 35-55 parts of nano-inorganic filler, 0.1-1 parts of crosslinking agent, and 0.1-1 parts of hydrogen ion probe; wherein the mass fraction of the hydrogen ion probe is 0.1-0.6 parts of halogenated pentylenetetrazol.

4. The caries-causing bacteria culture medium according to claim 3, characterized in that: The formulation and mass fractions of the hydrogen ion sensing membrane are as follows: 95 parts of quaternary ammonium silane, 45 parts of nano silica, 0.4 parts of dicumyl peroxide, 0.5 parts of bromocresol purple, and 0.5 parts of halogenated ether.

5. The caries-causing bacteria culture medium according to claim 1, characterized in that: The composition of each component in the culture medium is as follows: peptone 0.5%~3%, sucrose 10%~50%, glucose 0.05%~0.5%, dipotassium hydrogen phosphate 0.1%~1%, buffer 0.05%~0.5%, antibacterial agent 5%~50%, and the remainder is purified water.

6. The caries-causing bacteria culture medium according to claim 5, characterized in that: The selective antibacterial agent is at least one of phenylethanol, sodium azide, actinomycete ketone, and potassium tellurate.

7. The caries-causing bacteria culture medium according to claim 6, characterized in that: The buffer is either phosphate buffer or MES buffer.

8. The caries-causing bacteria culture medium according to claim 7, characterized in that: The culture medium also includes a culture flask; the culture flask is a colorless, transparent cylindrical container; the culture flask is pre-filled with culture medium and a hydrogen ion sensing membrane; the hydrogen ion sensing membrane is located at the bottom of the culture flask.

9. A method for preparing a culture medium for caries-causing bacteria, characterized in that, It includes the following steps: To prepare culture medium A, peptone, dipotassium hydrogen phosphate, and glucose were added sequentially to purified water and stirred at 60°C until completely dissolved. Sucrose was added, and stirring continued until a clear solution was obtained. Buffer solution was added and stirred until homogeneous. A selective antibacterial agent was added. After adjusting the volume, the solution was sterilized to obtain the culture medium. B. Preparation of hydrogen ion sensing membrane: A hydrogen ion probe, a crosslinkable ion polymer, nano-inorganic filler and a crosslinking agent are mixed and injected into a culture flask, and then solidified in the culture flask to form a hydrogen ion sensing membrane; The hydrogen ion probe is a mixture of at least one of bromocresol purple, bromocresol green, phenol red and methyl red with halogenated ether. C. After sterilizing the culture bottle with the solidified hydrogen ion sensing membrane, inject the culture medium.

10. The method for preparing a culture medium for caries-causing bacteria according to claim 9, characterized in that, The mass fractions of each component in the formulation of the hydrogen ion sensing membrane are as follows: 92-103 parts of crosslinkable ionomer, 35-55 parts of nano-inorganic filler, 0.1-1 parts of crosslinking agent, and 0.1-1 parts of hydrogen ion probe; wherein the mass fraction of the halogenated ether in the hydrogen ion probe is 0.1-0.6 parts. The hydrogen ion sensing membrane is prepared as follows: A. The crosslinkable ionomer and the nano-inorganic filler are stirred and mixed until the nano-inorganic filler is completely wetted to obtain the first product; B dissolves at least one of bromocresol purple, bromocresol green, phenol red, and methyl red with halogenated ether in anhydrous ethanol to obtain a second product; C. The crosslinking agent is mixed with hydroxyl silicone oil until it reaches a fluid state to obtain the third product; D. Mix and stir the first product, the second product and the third product for 1 to 5 hours, and then degas to obtain the fourth product; E injects the fourth product into a culture flask and heats it to solidify.