Medium for selectively detecting and monitoring streptococcus sobrinus as well as preparation method and application of medium

Abstract

The invention discloses a selective detection and monitoring culture medium for streptococcus sobrinus as well as a preparation method and application of the selective detection and monitoring culture medium, and belongs to the technical field of microorganisms. The culture medium comprises a carbon source, a nitrogen source, 4-5g / L of monopotassium phosphate, an additive and 15g / L of agar, the carbon source comprises 1-5g / L of glucose and 40-50g / L of sucrose; the nitrogen source comprises 12-18g / L of multivalent peptone and 3-7g / L of casein tryptone; the additive is prepared from 0.9-1.2 mg / L of crystal violet, 0.05-0.08 g / L of trypan blue, 2-10 mg / L of potassium tellurite, 40-60 mg / L of carumonan and 70-100 mg / L of sulfadimidine. The selective detection and monitoring culture medium for streptococcus sobrinus is high in growth selectivity, good in gram-positive bacterium growth inhibition effect, capable of well inhibiting fungus growth, sensitive in streptococcus sobrinus detection, high in specificity, simple in preparation procedure and suitable for industrial production. The practical operation feasibility is high, and the application of the culture medium for selectively detecting and monitoring the distant streptococcus is also included.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a selective detection and monitoring culture medium for Streptococcus sobrinus and a preparation method and application thereof. Background Art

[0002] Dental plaque is a bacterial group that adheres to the tooth surface, between teeth or on the surface of dentures. It is a bacterial biofilm that cannot be washed off or rinsed off with water. The cariogenic effect of dental plaque is closely related to the metabolic activity of cariogenic bacteria in dental plaque. Common cariogenic bacteria include Streptococcus, Lactobacillus and Actinomyces. Streptococcus sobrinus ) is a Gram-positive facultative anaerobic bacterium and part of the oral flora. Streptococcus sobrinus is acidogenic and acid-resistant, and can ferment carbohydrates to produce acid to demineralize teeth. It can maintain growth, reproduction and metabolic activities in an acidic environment. It has strong adhesion to the tooth surface and is prone to forming plaque. It can also produce glycosyltransferases and synthesize extracellular polysaccharides such as glucan and fructan using sucrose as a substrate, which can accelerate the formation of plaque. The intracellular polysaccharides synthesized by Streptococcus sobrinus can be degraded when the bacteria lack carbohydrates, providing energy for the bacteria and enhancing their caries-causing ability. It can adapt to the ever-changing oral environment and survive in it, so it is closely related to the occurrence of dental caries.

[0003] However, due to its strict nutritional requirements and slow growth, it is usually difficult to detect when cultured on general culture medium plates. The existing technology for detecting Streptococcus sobrinus requires first constructing a bacterial genomic library, designing and synthesizing specific primers and probes for specific genes, and then extracting DNA for amplification detection. The entire procedure is cumbersome and costly. The pretreatment of the sample requires professional instruments and equipment, which is not suitable for large-scale screening. It has the disadvantages of insufficient sensitivity and susceptibility to nonspecific binding caused by impurities in the sample, which affects the accuracy of detection. At present, there is still a lack of convenient and reliable methods to selectively isolate and culture Streptococcus sobrinus in human dental plaque, which has hindered the study of Streptococcus sobrinus in caries. In order to further strengthen the laboratory's understanding of this pathogen, it is urgent to develop a selective culture medium that can effectively meet the growth, metabolism, and reproduction of Streptococcus sobrinus for isolating Streptococcus sobrinus from human dental plaque. This will help to more clearly elucidate the cause of the pathogenesis of dental caries caused by Streptococcus sobrinus, better understand the incidence of dental caries in preschool children, and thus promptly discover new therapeutic drugs. Summary of the Invention

[0004] The present invention aims to provide a selective detection and monitoring culture medium for improving the isolation rate of Streptococcus sobrinus in dental plaque, as well as a preparation method and application thereof. The culture medium is prepared by optimizing the carbon source, nitrogen source, antibiotics and other nutrients in the selective detection and monitoring culture medium for Streptococcus sobrinus, thereby preparing a culture medium suitable for quickly screening and isolating Streptococcus sobrinus from dental plaque.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a selective detection and monitoring culture medium for Streptococcus sobrinus, wherein the selective detection and monitoring culture medium for Streptococcus sobrinus comprises: a carbon source, a nitrogen source, 4-5 g / L of potassium dihydrogen phosphate, an additive, and 15 g / L of agar; The carbon source composition is: glucose 1-5g / L, sucrose 40-50g / L; The nitrogen source is composed of: 12-18 g / L of polyvalent peptone and 3-7 g / L of trypticase peptone; The additive comprises: 0.9-1.2 mg / L of crystal violet, 0.05-0.08 g / L of trypan blue, 2-10 mg / L of potassium tellurite, 40-60 mg / L of calumonan, and 70-100 mg / L of sulfamethazine.

[0006] A method for preparing a culture medium for selective detection and monitoring of Streptococcus sobrinus comprises the following steps: 1-5 g glucose, 40-50 g sucrose, 12-18 g polyvalent peptone, 3-7 g trypticase peptone, 4-5 g potassium dihydrogen phosphate, 0.9-1.2 mg crystal violet, 0.05-0.08 g trypan blue, and 15 g agar are weighed and dissolved in deionized water, the volume is adjusted to 1000 mL, and the pH is adjusted to 6.4-6.8 with sodium carbonate and citric acid. The mixture is sterilized at 121° C. and 0.1 MPa pressure for 15 minutes, cooled to 50° C., 2-10 mg potassium tellurite, 40-60 mg calumonam, and 70-100 mg sulfamethazine are added and mixed, and the mixture is aseptically filled into a 90 mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

[0007] The selective detection and monitoring culture medium for Streptococcus sobrinus is applied to the separation of Streptococcus sobrinus from dental caries plaque in human oral cavity, that is, the microorganism is Streptococcus sobrinus ( Streptococcus sobrinus ), ATCC 33478.

[0008] Preferably, the culture temperature is 37°C.

[0009] Preferably, the culture time is 72 h.

[0010] A method for using the above-mentioned Streptococcus sobrinus selective detection monitoring culture medium is: (1) Sample the carious area of ​​the child using sterile dental floss, immediately place the sample in 15 mL of pH 7.2 phosphate buffer, and disperse it by ultrasonication in an ice bath for 30 seconds to obtain a uniform suspension; (2) The suspension was diluted 10-fold with 0.9% sterile saline to 1 / 10000 of the initial concentration. 0.1 mL of the diluted solution was spread on a monitoring culture medium plate and cultured in a 37°C constant temperature incubator under anaerobic conditions for 72 h. The anaerobic conditions were an environment of 95% nitrogen and 5% carbon dioxide.

[0011] The Streptococcus sobrinus selective detection and monitoring culture medium provided by the invention is used for enrichment culture and separation screening of Streptococcus sobrinus for non-disease diagnosis purposes.

[0012] The beneficial effects of the present invention are: (1) Since Streptococcus sobrinus has strict nutritional requirements and grows slowly, it is usually difficult to be detected when cultured on general culture medium plates. The culture medium of the present invention optimizes the nutritional components of the culture medium, better meets the nutritional requirements of Streptococcus sobrinus, is conducive to the growth of Streptococcus sobrinus, improves detection efficiency, and has a targeted screening effect.

[0013] (2) Potassium tellurite can inhibit the growth of most Gram-positive and Gram-negative bacteria at a specific concentration, and has a good screening effect on other bacteria isolated from the oral cavity, but has no inhibitory effect on Streptococcus sobrinus, thereby improving the selectivity of the culture medium; at the same time, by utilizing the differences in the sensitivity of different microorganisms in dental plaque to different drugs, other additives that have a weak inhibitory effect on Streptococcus sobrinus but a strong inhibitory effect on other miscellaneous bacteria or competing bacteria are added to the culture medium, thereby improving the detection sensitivity.

[0014] (3) Adjusting the pH of the culture medium to a weak acid not only reduces the growth and reproduction of acid-intolerant bacteria to a certain extent, but also provides a relatively suitable growth environment for Streptococcus sobrinus, thereby increasing the growth rate of Streptococcus sobrinus.

[0015] (4) After inoculation with the bacterial solution, the culture medium is placed in an anaerobic environment (95% nitrogen, 5% carbon dioxide) for cultivation. This can reduce the growth and reproduction of some aerobic bacteria without affecting the growth of Streptococcus sobrinus, and can effectively improve the success rate of isolating Streptococcus sobrinus from dental plaque. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 1 is a comparative chart of the recovery rates of Streptococcus sobrinus on the monitoring culture medium in Examples 1-4; Figure 2 This is a comparison chart of the average colony diameters of Streptococcus sobrinus on the monitoring culture medium in Examples 1-4; Figure 3 1 is a comparative chart of the growth inhibition ability of the monitoring culture medium in Examples 1-4 on non-sobrinus Streptococcus; Figure 4This is a comparison chart of the effects of using the monitoring culture medium to screen Streptococcus sobrinus from dental plaque in Examples 1-4. DETAILED DESCRIPTION

[0017] Four examples are described below to more clearly illustrate the technical solutions and advantages of the present invention. By adjusting the nutrient composition of the culture medium of the present invention and using a counting recovery method, the selective separation effect of the culture medium was evaluated based on its growth-promoting ability and its inhibitory ability against non-sobacterial Streptococci.

[0018] The dental plaque samples in the following examples were obtained from the oral cavity of untreated caries in children aged 7 to 10 years old.

[0019] The standard strains used in the following examples were all purchased from institutions with strain management qualifications, and the instruments and reagents used were all commercially available conventional products.

[0020] Table 1 Reference strains used in experiments

[0021] A selective detection and monitoring culture medium for Streptococcus sobrinus and a preparation method thereof are as follows: 1. Culture medium formulation Each liter of culture medium contains: carbon source, nitrogen source, 4-5g of potassium dihydrogen phosphate, additives, and 15g of agar.

[0022] The carbon source composition is: 1-5 g / L glucose and 40-50 g / L sucrose; glucose and sucrose provide the carbon source required for the growth of Streptococcus sobrinus.

[0023] The nitrogen source composition is: polyvalent peptone 12-18 g / L, trypsin peptone 3-7 g / L; polyvalent peptone and trypsin peptone provide nitrogen source, vitamins and amino acids for the growth of Streptococcus sobrinus.

[0024] The additive composition is: crystal violet 0.9-1.2 mg / L, trypan blue 0.05-0.08 g / L, potassium tellurite 2-10 mg / L, calumonan 40-60 mg / L, sulfamethazine 70-100 mg / L. Under the nutritional formula of the present invention, the nutritional needs of Streptococcus sobrinus are better met, which is conducive to the growth of Streptococcus sobrinus and provides a basis for later selective detection. Crystal violet has a killing effect on Gram-positive bacteria such as Staphylococcus, Corynebacterium diphtheriae, Pseudomonas aeruginosa and Epidermophyton. Potassium tellurite can inhibit the growth of most Gram-positive and Gram-negative bacteria at a specific concentration. Calumonan shows greater antibacterial activity against a variety of aerobic Gram-negative bacteria. Sulfamethazine has an inhibitory effect on Staphylococcus aureus, Shigella, Escherichia coli and other Enterobacteriaceae. The present invention uses a plurality of components to form an additive to synergistically inhibit the growth of other microorganisms.

[0025] 2. Preparation of culture medium Preparation of the culture medium of the present invention: 1-5g glucose, 40-50g sucrose, 12-18g polyvalent peptone, 3-7g tryptic peptone, 4-5g potassium dihydrogen phosphate, 0.9-1.2mg crystal violet, 0.05-0.08g trypan blue, and 15g agar are weighed and dissolved in deionized water, the volume is adjusted to 1000ml, and the pH is adjusted to 6.4-6.8 with sodium carbonate and citric acid. The mixture is sterilized at 121°C and 0.1MPa pressure for 15 minutes, cooled to 50°C, and 2-10mg potassium tellurite, 40-60mg calumonam, and 70-100mg sulfamethazine are added and mixed. The mixture is aseptically filled into a 90mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

[0026] 3. Inoculation and cultivation (1) Standard strain recovery test: The activated Streptococcus sobrinus strain was diluted step by step with 0.9% sterile saline to no more than 100 cfu·mL -1 , spread on TSA and the selective detection and monitoring medium for Streptococcus sobrinus, spread 3 plates on each of the two culture media, culture at 37°C for 72 h, observe the growth of the bacteria, count the colonies, and calculate the recovery rate.

[0027] Recovery rate = (number of colonies in selective detection monitoring medium / number of colonies in TSA) × 100% (2) Selective detection of Streptococcus sobrinus to monitor the inhibitory ability of the culture medium: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Lactobacillus salivarius, and Actinomyces dentistry, which are common microorganisms in dental plaque, were used as test strains to detect the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 3 CFU were inoculated on the monitoring medium and cultured in a 37 °C constant temperature incubator for 72 h to observe whether colonies appeared.

[0028] (3) Practical application method of monitoring culture medium for selective detection of Streptococcus sobrinus: Use sterile dental floss to sample the caries area of ​​children, immediately put the sample into 15 mL of pH 7.2 phosphate buffer, disperse it by ultrasonic wave in an ice bath for 30 seconds to obtain a uniform suspension, dilute the suspension 10 times with 0.9% sterile saline to 1 / 10000 of the initial concentration, take 0.1 mL of the dilution and spread it on the monitoring culture medium plate, and culture it in a constant temperature incubator at 37°C under anaerobic conditions (95% nitrogen, 5% carbon dioxide) for 72 hours.

[0029] Specific embodiments are as follows.

[0030] Example 1

[0031] This embodiment provides a culture medium for selective detection and monitoring of Streptococcus sobrinus.

[0032] 1. The culture medium formula is as follows Each liter of culture medium contains: 3.5g glucose, 45g sucrose, 15g polyvalent peptone, 4.5g tryptic peptone, 4.5g potassium dihydrogen phosphate, 15g agar; additives are 1mg crystal violet, 0.07g trypan blue, 7.6mg potassium tellurite, 48mg calumonam, and 75mg sulfamethazine.

[0033] 2. Preparation of culture medium Weigh 3.5 g of glucose, 45 g of sucrose, 15 g of polyvalent peptone, 4.5 g of trypticase peptone, 4.5 g of potassium dihydrogen phosphate, 15 g of agar, 1 mg of crystal violet, and 0.07 g of trypan blue, dissolve them in deionized water, dilute to 1000 mL, adjust the pH to 6.5 with sodium carbonate and citric acid, sterilize at 121 ° C and 0.1 MPa pressure for 15 minutes, cool to 50 ° C, add 10 mg of potassium tellurite, 48 mg of calumonam, and 75 mg of sulfamethazine, and aseptically fill into a 90 mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

[0034] 3. Inoculation and cultivation (1) Dilute the activated Streptococcus sobrinus strain in 0.9% sterile saline to no more than 100 cfu·mL -1 , spread on TSA and the selective detection and monitoring medium for Streptococcus sobrinus, spread 3 plates on each of the two culture media, culture at 37°C for 72 h, observe the growth of the bacteria, count the colonies, and calculate the recovery rate.

[0035] Recovery rate = (number of colonies on selective medium / number of colonies on TSA) × 100% (2) Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus salivarius, and Actinomyces dentistry, which are common microorganisms in dental plaque, were used as test strains to test the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 3 CFU were inoculated on selective isolation medium and cultured in a 37 °C constant temperature incubator for 72 h to observe whether colonies appeared.

[0036] (3) Use sterile dental floss to sample the caries area of ​​the child, immediately place the sample in 15 mL of pH 7.2 phosphate buffer, disperse it by ultrasonication in an ice bath for 30 seconds to obtain a uniform suspension, dilute the suspension 10-fold with 0.9% sterile saline to 1 / 10000 of the initial concentration, take 0.1 mL of the dilution and spread it on a selective detection monitoring culture medium plate, and place it in an anaerobic condition (95% nitrogen, 5% carbon dioxide) in a constant temperature incubator at 37°C for 72 hours.

[0037] Example 2

[0038] This embodiment provides a culture medium for selective detection and monitoring of Streptococcus sobrinus.

[0039] 1. The culture medium formula is as follows Each liter of culture medium contains: 2g glucose, 42.5g sucrose, 17.5g polyvalent peptone, 6g trypticase peptone, 4.7g potassium dihydrogen phosphate, 15g agar; additives are 0.9mg crystal violet, 0.06g trypan blue, 5mg potassium tellurite, 55mg calumonam, and 90mg sulfamethazine.

[0040] 2. Preparation of culture medium Weigh 2 g of glucose, 42.5 g of sucrose, 17.5 g of polyvalent peptone, 6 g of trypticase peptone, 4.7 g of potassium dihydrogen phosphate, 15 g of agar, 0.9 mg of crystal violet, and 0.06 g of trypan blue, dissolve them in deionized water, dilute to 1000 mL, and adjust the pH to 6.8 with sodium carbonate and citric acid. Sterilize at 121° C. and 0.1 MPa pressure for 15 minutes, cool to 50° C., add 5 mg of potassium tellurite, 55 mg of calumonam, and 90 mg of sulfamethazine, and aseptically fill into a 90 mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

[0041] 3. Inoculation and cultivation (1) Dilute the activated Streptococcus sobrinus strain in 0.9% sterile saline to no more than 100 cfu·mL -1 , spread on TSA and the selective detection and monitoring medium for Streptococcus sobrinus, spread 3 plates on each of the two culture media, culture at 37°C for 72 h, observe the growth of the bacteria, count the colonies, and calculate the recovery rate.

[0042] Recovery rate = (selective detection monitoring colony number / TSA colony number) × 100% (2) Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus salivarius, and Actinomyces dentistry, which are common microorganisms in dental plaque, were used as test strains to test the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 3CFU were inoculated on selective detection monitoring medium and cultured in a 37 ℃ constant temperature incubator for 72 h to observe whether colonies appeared.

[0043] (3) Use sterile dental floss to sample the caries area of ​​the child, immediately place the sample in 15 mL of pH 7.2 phosphate buffer, disperse it by ultrasonication in an ice bath for 30 seconds to obtain a uniform suspension, dilute the suspension 10-fold with 0.9% sterile saline to 1 / 10000 of the initial concentration, take 0.1 mL of the dilution and spread it on a selective detection monitoring culture medium plate, and place it in an anaerobic condition (95% nitrogen, 5% carbon dioxide) in a constant temperature incubator at 37°C for 72 hours.

[0044] Example 3

[0045] This embodiment provides a culture medium for selective detection and monitoring of Streptococcus sobrinus.

[0046] 1. The culture medium formula is as follows Each liter of culture medium contains: 4.5g of glucose, 48g of sucrose, 13g of polyvalent peptone, 3.5g of trypticase peptone, 4.2g of potassium dihydrogen phosphate, and 15g of agar; additives are 1.2mg of crystal violet, 0.05g of trypan blue, 9.5mg of potassium tellurite, 43mg of carumonam, and 96mg of sulfamethazine.

[0047] 2. Preparation of culture medium Weigh 4.5 g of glucose, 48 g of sucrose, 13 g of polyvalent peptone, 3.5 g of trypticase peptone, 4.2 g of potassium dihydrogen phosphate, 15 g of agar, 1.2 mg of crystal violet, and 0.05 g of trypan blue, dissolve them in deionized water, dilute to 1000 mL, adjust the pH to 6.8 with sodium carbonate and citric acid, sterilize at 121 ° C and 0.1 MPa pressure for 15 minutes, cool to 50 ° C, add 9.5 mg of potassium tellurite, 43 mg of calumonam, and 96 mg of sulfamethazine, and aseptically fill into a 90 mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

[0048] 3. Inoculation and cultivation (1) Dilute the activated Streptococcus sobrinus strain in 0.9% sterile saline to no more than 100 cfu·mL -1 , spread on TSA and the selective detection and monitoring medium for Streptococcus sobrinus, spread 3 plates on each of the two culture media, culture at 37°C for 72 h, observe the growth of the bacteria, count the colonies, and calculate the recovery rate.

[0049] Recovery rate = (selective detection monitoring colony number / TSA colony number) × 100% (2) Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus salivarius, and Actinomyces dentistry, which are common microorganisms in dental plaque, were used as test strains to test the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 3 CFU were inoculated on selective detection monitoring medium and cultured in a 37 ℃ constant temperature incubator for 72 h to observe whether colonies appeared.

[0050] (3) Use sterile dental floss to sample the caries area of ​​the child, immediately place the sample in 15 mL of pH 7.2 phosphate buffer, disperse it by ultrasonication in an ice bath for 30 seconds to obtain a uniform suspension, dilute the suspension 10-fold with 0.9% sterile saline to 1 / 10000 of the initial concentration, take 0.1 mL of the dilution and spread it on a selective detection monitoring culture medium plate, and place it in an anaerobic condition (95% nitrogen, 5% carbon dioxide) in a constant temperature incubator at 37°C for 72 hours.

[0051] Example 4

[0052] This embodiment provides a culture medium for selective detection and monitoring of Streptococcus sobrinus.

[0053] 1. The culture medium formula is as follows Each liter of culture medium contains: 1.5g glucose, 41g sucrose, 17g polyvalent peptone, 6.5g tryptic peptone, 4.8g potassium dihydrogen phosphate, 15g agar; additives are 1mg crystal violet, 0.075g trypan blue, 4mg potassium tellurite, 50mg carumonam, and 80mg sulfamethazine.

[0054] 2. Preparation of culture medium Weigh 1.5 g of glucose, 41 g of sucrose, 17 g of polyvalent peptone, 6.5 g of trypticase peptone, 4.8 g of potassium dihydrogen phosphate, 15 g of agar, 1 mg of crystal violet, and 0.075 g of trypan blue, dissolve them in deionized water, dilute to 1000 mL, and adjust the pH to 6.4 with sodium carbonate and citric acid. Sterilize at 121° C. and 0.1 MPa pressure for 15 minutes, cool to 50° C., add 4 mg of potassium tellurite, 50 mg of calumonam, and 80 mg of sulfamethazine, and aseptically fill into a 90 mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

[0055] 3. Inoculation and cultivation (1) Dilute the activated Streptococcus sobrinus strain in 0.9% sterile saline to no more than 100 cfu·mL -1 , spread on TSA and the selective detection and monitoring medium for Streptococcus sobrinus, spread 3 plates on each of the two culture media, culture at 37°C for 72 h, observe the growth of the bacteria, count the colonies, and calculate the recovery rate.

[0056] Recovery rate = (number of colonies on selective medium / number of colonies on TSA) × 100% (2) Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus salivarius, and Actinomyces dentistry, which are common microorganisms in dental plaque, were used as test strains to test the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 3 CFU were inoculated on selective detection monitoring medium and cultured in a 37 ℃ constant temperature incubator for 72 h to observe whether colonies appeared.

[0057] (3) Use sterile dental floss to sample the caries area of ​​the child, immediately place the sample in 15 mL of pH 7.2 phosphate buffer, disperse it by ultrasonication in an ice bath for 30 seconds to obtain a uniform suspension, dilute the suspension 10-fold with 0.9% sterile saline to 1 / 10000 of the initial concentration, take 0.1 mL of the dilution and spread it on a selective detection monitoring culture medium plate, and place it in an anaerobic condition (95% nitrogen, 5% carbon dioxide) in a constant temperature incubator at 37°C for 72 hours.

[0058] like Figure 1 As shown, the average recovery rates of the selective culture media of Examples 1-4 were 91%, 82%, 70%, and 67%, respectively. The above results show that the recovery rates of the monitoring culture media of Examples 1-4 for Streptococcus sobrinus were all higher than 50%, but compared with Examples 2-4, the recovery rate of the culture media in Example 1 was significantly better. Figure 2 It can be seen that the average colony diameter of Streptococcus sobrinus in Example 1 on the monitoring culture medium is larger than that in Examples 2-4. Figure 3 As can be seen from the results, the test strain Streptococcus mutans ( Streptococcus mutans ), Streptococcus mitis ( streptococcus midis ), Streptococcus sanguis ( Streptococcus sanguinis ), Lactobacillus salivarius ( Lactobacillus salivarius )、Caries Actinomycetes( Actinomyces Odontolyticus ) did not grow on the monitoring culture medium, indicating that the monitoring culture medium of Examples 1-4 can meet the inhibitory capacity requirements. Figure 4 As shown, Streptococcus sobrinus can effectively inhibit other bacterial groups on the monitoring culture media of Examples 1-4, among which the inhibitory ability of Example 1 is the strongest, and its nutrients are more conducive to the growth of Streptococcus sobrinus. The colony morphology of Streptococcus sobrinus on this culture medium is more characteristic, and a larger number of Streptococcus sobrinus grows.

[0059] In summary, the present invention optimizes the nutrient composition of the culture medium to better meet the growth requirements of Streptococcus sobrinus, and at the same time adds other additives to the culture medium that have a weak inhibitory effect on Streptococcus sobrinus but a strong inhibitory effect on other bacteria or competing bacteria, thereby obtaining a selective detection and monitoring culture medium for Streptococcus sobrinus. Figures 1-4 The content shows that the monitoring culture medium of Example 1 is more conducive to the growth and detection of Streptococcus sobrinus, and Example 1 is the best embodiment of the present invention.

[0060] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A selective detection and monitoring culture medium for Streptococcus sobrinus, characterized in that: The selective detection and monitoring culture medium for Streptococcus sobrinus consists of: a carbon source, a nitrogen source, 4-5 g / L potassium dihydrogen phosphate, additives, and 15 g / L agar; The carbon source composition is: glucose 1-5g / L, sucrose 40-50g / L; The nitrogen source is composed of: 12-18 g / L of polyvalent peptone and 3-7 g / L of trypticase peptone; The additive comprises: 0.9-1.2 mg / L of crystal violet, 0.05-0.08 g / L of trypan blue, 2-10 mg / L of potassium tellurite, 40-60 mg / L of calumonan, and 70-100 mg / L of sulfamethazine.

2. A method for preparing a selective detection and monitoring culture medium for Streptococcus sobrinus according to claim 1, characterized in that: The following steps are involved: 1-5 g glucose, 40-50 g sucrose, 12-18 g polyvalent peptone, 3-7 g tryptic peptone, 4-5 g potassium dihydrogen phosphate, 0.9-1.2 mg crystal violet, 0.05-0.08 g trypan blue, and 15 g agar were weighed and dissolved in deionized water, the volume was adjusted to 1000 mL, and the pH was adjusted to 6.4-6.8 with sodium carbonate and citric acid. The mixture was sterilized at 121° C. and 0.1 MPa pressure for 15 minutes, cooled to 50° C., 2-10 mg potassium tellurite, 40-60 mg calumonam, and 70-100 mg sulfamethazine were added and mixed, and the mixture was aseptically filled into a 90 mm sterile culture dish to prepare the Streptococcus sobrinus selective detection and monitoring medium.

3. The use of the Streptococcus sobrinus selective detection monitoring culture medium according to claim 1, characterized in that, The Streptococcus sobrinus selective detection and monitoring culture medium is used for separating Streptococcus sobrinus from dental caries plaque in human oral cavity.

4. The use of the Streptococcus sobrinus selective detection monitoring culture medium according to claim 3, characterized in that, The culture temperature for culturing Streptococcus sobrinus in the monitoring culture medium is 37°C.

5. The use of the Streptococcus sobrinus selective detection monitoring culture medium according to claim 3, characterized in that, The monitoring medium is used to culture Streptococcus sobrinus for 72 hours.

6. A method for using the Streptococcus sobrinus selective detection and monitoring culture medium according to claim 1, characterized in that: The following steps are involved: (1) Sample the carious area of ​​the child using sterile dental floss, immediately place the sample in 15 mL of pH 7.2 phosphate buffer, and disperse it by ultrasonication in an ice bath for 30 seconds to obtain a uniform suspension; (2) The suspension was diluted 10-fold with 0.9% sterile saline to 1 / 10000 of the initial concentration. 0.1 mL of the diluted solution was spread on a monitoring culture medium plate and cultured in a 37°C constant temperature incubator under anaerobic conditions for 72 h. The anaerobic conditions were an environment of 95% nitrogen and 5% carbon dioxide.

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