Detection method of staphylococcus aureus, culture medium and preparation method of flat plate

Abstract

The invention provides a detection method of staphylococcus aureus, a culture medium and a preparation method of a flat plate. Each liter of the detection culture medium comprises 22 to 60 g of a basic component, 1.0 to 3.0 g of triglyceride, 0.01 to 0.10 g of a fat-soluble dye, 0.5 to 2.0 mL of a nonionic surfactant, 0.05 to 0.20 g of a chromogenic substrate and 0.05 to 0.20 g of a selective inhibitor. The staphylococcus aureus detection culture medium effectively solves a series of problems caused by low contrast of a lysolytic zone and a background in a Bair-Parker chromogenic culture medium, easy leak detection of strains with weak enzyme activity and animal-derived components. When the staphylococcus aureus detection culture medium is used for detecting staphylococcus aureus, the cost is low, the sensitivity is high, the accuracy is high, and the staphylococcus aureus detection culture medium can be widely applied to staphylococcus aureus detection in the fields of food, medical treatment, environment and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism detection, in particular to the technical field of detection of Staphylococcus aureus, and more particularly to a detection method of Staphylococcus aureus, a culture medium and a preparation method of a plate. BACKGROUND

[0002] Staphylococcus aureus, as a common foodborne pathogen, can cause a variety of infections and diseases, and seriously threatens human health. Therefore, it is crucial to detect it accurately and quickly. At present, the detection methods of Staphylococcus aureus include bacterial culture method, molecular biology technology, immunological method, chromogenic medium, electrochemical immunosensor and traditional microbiological method. Among them, the chromogenic medium is a pre-prepared ready-to-use medium product, which combines standard medium, cold water gel and chromogenic indicator, so that the detection process is more convenient and efficient. The chromogenic medium is specially designed for the determination of Staphylococcus aureus in food and beverage. The traditional detection of Staphylococcus aureus by using chromogenic medium mostly relies on Baird-Parker medium. Staphylococcus aureus appears as a circle on the Baird-Parker medium plate, with smooth, convex, wet surface, colony diameter of 2-3 mm, color of gray-black to black, luster, often with light (non-white) edge, and surrounding solubilization circle (because the lecithinase in the medium decomposes the fat in the yolk to form a milky white precipitate ring), which is used to determine the number of Staphylococcus aureus colonies.

[0003] However, the detection of Baird-Parker chromogenic medium has many drawbacks. First, the contrast between the solubilization circle and the background is low, resulting in the need for experience to judge the results, which is prone to subjective errors. For example, in the detection of 50 samples known to contain Staphylococcus aureus, the consistency of the results of different detection personnel using traditional medium is only 60%. Second, weak enzyme activity strains are prone to be missed, and part of the weak enzyme activity strains cannot form obvious solubilization circle, causing detection omission. Research shows that the omission rate of weak enzyme activity strains by Baird-Parker chromogenic medium is as high as 25%. Third, there is a problem of animal-derived ingredients. The addition of yolk and other animal-derived ingredients has batch differences, which affects the stability of the detection results and brings biological safety risks.

[0004] Although, there are also color substrates in Baird-Parker chromogenic medium to improve specificity, but the cost is high, and the problem of solubilization circle visualization has not been solved. Therefore, it is urgent to develop a low-cost and high-sensitivity improved chromogenic medium. SUMMARY

[0005] Based on the above problems, the purpose of the present application is to provide a staphylococcus aureus detection method, a culture medium and a preparation method of the plate. The staphylococcus aureus detection culture medium effectively solves the problems of low contrast between the bacteriolytic circle and the background in the Baird-Parker color developing culture medium, easy detection of weak enzyme activity strains and a series of problems caused by animal-derived components. The staphylococcus aureus detection culture medium is used for staphylococcus aureus detection, which is low in cost, high in sensitivity and accuracy, and can be widely used in staphylococcus aureus detection in the fields of food, medical treatment and environment.

[0006] To achieve the above purpose, the first aspect of the present application provides a staphylococcus aureus detection culture medium, which contains 22-60 g of basic components, 1.0-3.0 g of triglyceride, 0.01-0.10 g of fat-soluble dye, 0.5-2.0 mL of non-ionic surfactant, 0.05-0.20 g of color developing substrate and 0.05-0.20 g of selective inhibitor per liter of culture medium.

[0007] In the staphylococcus aureus detection culture medium adopted by the present application, in addition to the basic components, color developing substrate and selective inhibitor commonly used in conventional culture medium, fat-soluble dye and triglyceride are added. In the detection culture process, the undecomposed fat in the sample to be detected is dyed by the fat-soluble dye, and the bacteriolytic circle area is faded due to the enzymatic hydrolysis of fat by triglyceride, thereby forming an obvious bacteriolytic circle and a high-contrast color developing ring. The visualization degree of the bacteriolytic circle can be significantly improved, the subjective error can be reduced, and the detection accuracy can be greatly improved. Compared with egg yolk, the stability of triglyceride is high, and the detection sensitivity is higher. Further, the introduction of non-ionic surfactant can promote the uniform dispersion of fat-soluble dye, reduce the deviation rate and ensure the consistency of the dyeing effect.

[0008] As a technical solution of the present application, the basic components include 5-15 g of tryptone, 2-10 g of yeast extract, 5-15 g of sodium chloride and 10-20 g of agar.

[0009] As a technical solution of the present application, the fat-soluble dye includes Nile red and / or Sudan red IV.

[0010] As a technical solution of the present application, the non-ionic surfactant includes at least one of Tween 20, Tween 40, Tween 60, Tween 80, Tween 81 and Tween 85.

[0011] As a technical solution of the present application, the color developing substrate includes 5-bromo-4-chloro-3-indole glucoside.

[0012] As a technical solution of the present application, the selective inhibitor includes potassium tellurite and / or sodium azide. The second aspect of the present application provides a preparation method of a Staphylococcus aureus detection culture medium plate, comprising the steps of: dissolving the base component in the Staphylococcus aureus detection culture medium in water, heating sterilization, adding the triglyceride, the fat-soluble dye, the non-ionic surfactant, the chromogenic substrate and the selective inhibitor after cooling, and pouring the mixture into a plate.

[0013] In the preparation method of the Staphylococcus aureus detection culture medium plate of the present application, the base component is dissolved in water, heated and sterilized, and then mixed with the triglyceride, the fat-soluble dye, the non-ionic surfactant, the chromogenic substrate and the selective inhibitor, which can ensure the activity of the fat-soluble dye and the chromogenic substrate.

[0014] As a technical solution of the present application, the triglyceride, the fat-soluble dye, the non-ionic surfactant, the chromogenic substrate and the selective inhibitor are sterilized before mixing.

[0015] As a technical solution of the present application, the heating sterilization is sterilized at 100-140°C for 10-20min, and the cooling reduces the temperature to 50±5°C.

[0016] The third aspect of the present application provides a detection method of Staphylococcus aureus, comprising the steps of: (1) Preparation of sample solution The sample is taken in phosphate buffer or physiological saline to prepare a sample solution; (2) Inoculation and culture The sample solution is coated on the culture medium plate prepared from the Staphylococcus aureus detection culture medium or the Staphylococcus aureus detection culture medium plate prepared by the preparation method of the Staphylococcus aureus detection culture medium plate, and cultured; (3) Colony counting and calculation The number of typical colonies on the culture medium plate is counted, and the number of Staphylococcus aureus colonies in the sample is calculated.

[0017] In the detection method of Staphylococcus aureus of the present application, the fat-soluble dye and the triglyceride in the culture medium can form a clear lysing circle and a high-contrast color ring, which can greatly improve the accuracy of counting the number of typical colonies on the culture medium plate, reduce the counting time, deviation and minimum detection limit. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The staining result of Staphylococcus aureus after inoculation and culture in Example 1 of the present application.

[0019] Figure 2 The staining result of Staphylococcus aureus after inoculation and culture in Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0020] The method for detecting Staphylococcus aureus according to the present application can be used to detect Staphylococcus aureus on products in the fields of food, medicine, environment, etc.

[0021] The method for detecting Staphylococcus aureus according to the present application can comprise the following steps: (1) Preparation of sample solution The sample is placed in phosphate buffer or physiological saline to prepare a sample solution; (2) Inoculation and culture The sample solution is spread on a Staphylococcus aureus detection culture medium plate for culture; (3) Colony counting and calculation The number of typical colonies on the culture medium plate is counted, and the number of Staphylococcus aureus colonies in the sample is calculated.

[0022] In the method for detecting Staphylococcus aureus according to the present application, the preparation of the sample solution can be performed according to the national standard GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination Staphylococcus aureus Examination, and the sample is placed in phosphate buffer or physiological saline to prepare a sample solution.

[0023] If the sample is solid or semi-solid, 25 g of the sample can be taken and placed in a sterile homogenization cup containing 225 mL of phosphate buffer or physiological saline, and homogenized at 8000-10000 r / min for 1-2 min, or placed in a sterile homogenization bag containing 225 mL of diluent, and beaten for 1-2 min with a beating homogenizer to prepare a 1:10 sample homogenate.

[0024] If the sample is liquid, 25 mL of the sample can be taken with a sterile pipette and placed in a sterile conical flask containing 225 mL of phosphate buffer or physiological saline (with an appropriate number of sterile glass beads pre-placed in the flask), and mixed thoroughly to prepare a 1:10 sample homogenate.

[0025] The sample homogenate with a higher concentration can be diluted, for example, 1 mL of the 1:10 sample homogenate is taken with a 1 mL sterile pipette or micropipette, slowly injected along the wall of the tube into a sterile test tube containing 9 mL of phosphate buffer or physiological saline (note that the tip of the pipette or pipette should not touch the surface of the diluent), and the test tube is shaken or a 1 mL sterile pipette is used to repeatedly blow and beat to mix the sample homogenate uniformly to prepare a 1:100 sample homogenate. Further, a 10-fold serially diluted sample homogenate can be prepared. A 1 mL sterile pipette or pipette tip is replaced each time the dilution is increased by one.

[0026] Step (2) Inoculation culture can be selected according to the estimation of sample contamination, 2-3 appropriate dilution sample homogenate (liquid sample can include stock solution), 10-fold incremental dilution is carried out, 1 mL sample homogenate is taken for each dilution, 0.3 mL, 0.3 mL, 0.4 mL inoculation amount is added to three staphylococcus aureus detection medium plates respectively, and then the whole plate is coated with a sterile coating rod, and the edge of the plate is not touched. If there is water droplet on the surface of the staphylococcus aureus detection medium plate before use, it can be placed in a 25-50℃ incubator to dry until the water droplet on the surface of the plate disappears. After coating, the staphylococcus aureus detection medium plate can be placed for 10 min for culture, if the sample homogenate is not easy to absorb, the medium plate can be placed in a 36±1℃ incubator for 1 h, and after the sample homogenate is absorbed, the medium plate is turned over and placed in a 36±1℃ incubator for 24-48 h.

[0027] In step (3), the colony counting and calculation can also refer to the number of typical colonies in GB 4789.10-2016 Food safety national standard Food microbiological examination Staphylococcus aureus test to determine the number of typical colonies and calculate the number of staphylococcus aureus colonies in the sample.

[0028] The staphylococcus aureus detection medium plate used in the staphylococcus aureus detection method of the application can be prepared by pouring the staphylococcus aureus detection medium into the plate. Specifically, the preparation method can include: dissolving the base component in water, heating and sterilizing, adding triglyceride, fat-soluble dye, non-ionic surfactant, color developing substrate and selective inhibitor after cooling, and pouring the plate. The base component is dissolved in water and heated and sterilized, and the triglyceride, fat-soluble dye, non-ionic surfactant, color developing substrate and selective inhibitor are sterilized before mixing. By stepwise sterilization and aseptic addition, the activity of the fat-soluble dye and the color developing substrate can be ensured. The base component can be heated and sterilized at 100-140℃ for 10-20 min, and after the temperature is cooled to 50±5℃, the triglyceride, fat-soluble dye, non-ionic surfactant, color developing substrate and selective inhibitor can be added again.

[0029] In the staphylococcus aureus detection medium of the application, 22-60 g of base component, 1.0-3.0 g of triglyceride, 0.01-0.10 g of fat-soluble dye, 0.5-2.0 mL of non-ionic surfactant, 0.05-0.20 g of color developing substrate and 0.05-0.20 g of selective inhibitor are contained per liter of medium.

[0030] The base component can be, but is not limited to, 22 g, 28 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g. Further, the base component includes 5-15 g of tryptone, 2-10 g of yeast extract, 5-15 g of sodium chloride, and 10-20 g of agar. By way of example, the tryptone can be, but is not limited to, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g. The yeast extract can be, but is not limited to, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g. The sodium chloride can be, but is not limited to, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g. The agar can be, but is not limited to, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g.

[0031] The triglyceride can be, but is not limited to, 1.0 g, 1.2 g, 1.4 g, 1.6 g, 1.8 g, 2.0 g, 2.2 g, 2.4 g, 2.6 g, 2.8 g, 3.0 g. The lipophilic dye can be, but is not limited to, 0.01 g, 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.10 g. The lipophilic dye includes Nile Red and / or Sudan Red IV. If Nile Red alone is used, the Nile Red can be 0.01-0.05 g, and if Sudan Red alone is used, the Sudan Red IV can be 0.03-0.10 g.

[0032] The non-ionic surfactant can be, but is not limited to, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL. The non-ionic surfactant includes at least one of Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, and Tween 85.

[0033] The chromogenic substrate can be, but is not limited to, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.10 g, 0.11 g, 0.12 g, 0.13 g, 0.14 g, 0.15 g, 0.16 g, 0.17 g, 0.18 g, 0.19 g, 0.20 g. The chromogenic substrate includes 5-bromo-4-chloro-3-indolyl glucuronide (X-Glucuronide).

[0034] The selective inhibitor can be, but is not limited to, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.10 g, 0.11 g, 0.12 g, 0.13 g, 0.14 g, 0.15 g, 0.16 g, 0.17 g, 0.18 g, 0.19 g, 0.20 g. The selective inhibitor includes potassium tellurite and / or sodium azide, and the selective inhibitor can inhibit the growth of bacteria.

[0035] For the purpose, technical scheme and beneficial effects of the present application, the present application will be further described below in combination with specific examples. It should be noted that the following implementation of the method is a further explanation of the present application and should not be regarded as a limitation of the present application.

[0036] First part: verification of minimum detection limit of staphylococcus aureus detection medium Example 1 (1) Preparation of staphylococcus aureus detection medium plate Add the base ingredients (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar) to 1000 mL of deionized water, stir uniformly, sterilize at 121°C for 15 min, cool to 50°C, then add 1.5 g of sterile glycerol triglyceride, 0.02 g of nile red, 1 mL of Tween 80, 0.1 g of X-Glucuronide, and 0.1 g of potassium tellurite, mix uniformly, and pour the plate.

[0037] (2) Preparation of TCF color developing sheet Prepare TCF color developing solution: 0.115wt.% calcium chloride solution 20 mL, TrisBase 8 g, NaCl 1 g, toluidine blue 5 g, DNA 6 g, 95% ethanol 300 mL, and deionized water 500 mL, mix uniformly and boil, then place a sterile filter paper sheet with a diameter slightly smaller than the culture dish in the boiled TCF color developing solution after slight cooling, uniformly dye, and dry under sterile conditions, ready for use.

[0038] (3) Inoculation culture Dilute staphylococcus aureus ATCC 6538 bacterial solution with physiological saline to different concentrations, spread 0.2 mL of the diluted bacterial solution on the medium plate, and incubate at 37°C for 20 h.

[0039] (4) Heat treatment and color development Place the plate with colonies on the medium after incubation in a 62°C incubator for 1 h, open the cover of the medium plate, place the TCF color developing sheet on the surface of the medium to avoid air bubbles, cover the cover, incubate at 37°C for 15 min, then remove the TCF color developing sheet and place it in another clean medium plate, and incubate at 37°C for another 30 min.

[0040] (5) Results and analysis From the structure of Figure 1 The results show that the minimum detection limit of the medium plate-TCF chromogenic piece is 10 CFU / mL.

[0041] Table 1 Medium plate-TCF chromogenic piece minimum detection limit results

[0042] In the table, "+" indicates that Staphylococcus aureus is detected, and "-" indicates that it is not detected.

[0043] The results of Table 1 show that the minimum detection limit of the medium plate-TCF chromogenic piece is 10 CFU / mL.

[0044] Example 2 (1) Preparation of Staphylococcus aureus detection medium plate Add the base ingredients (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar) to 1000 mL of deionized water, stir until uniform, and sterilize at 121°C for 15 min. After cooling to 50°C, add 1.5 g of sterile glycerol triglyceride, 0.05 g of Sudan red IV, 1 mL of Tween 80, 0.1 g of X-Glucuronide, and 0.1 g of potassium tellurite, mix well, and pour the plate.

[0045] (2) Preparation of TCF chromogenic piece Prepare the TCF chromogenic solution: 20 mL of 0.115 wt.% calcium chloride solution, 8 g of TrisBase, 1 g of NaCl, 5 g of toluidine blue, 6 g of DNA, 300 mL of 95% ethanol, and 500 mL of deionized water. Mix well and boil. After slight cooling, place a sterile filter paper piece with a diameter slightly smaller than the culture dish in the boiled TCF chromogenic solution, evenly dye, and dry under sterile conditions. Prepare for use.

[0046] (3) Inoculation culture Dilute the Staphylococcus aureus ATCC 6538 bacterial solution with physiological saline to different concentrations, spread 0.2 mL of the diluted bacterial solution on the medium plate, and incubate at 37°C for 20 h.

[0047] (4) Heat treatment and color development Place the plate with colonies on the medium after incubation in a 62°C incubator for 1 h. Open the medium plate cover, place the TCF chromogenic piece on the surface of the medium to avoid air bubbles, cover the cover, incubate at 37°C for 15 min, then remove the TCF chromogenic piece and place it in another clean medium plate, and incubate at 37°C for another 30 min.

[0048] (5) Results and analysis The presence of blue colonies surrounded by a transparent zone of lysis in the TCF staining slide indicates the presence of Staphylococcus aureus.

[0049] Table 2. Limits of detection for culture medium plates-TCF chromogenic slides.

[0050] In the table, "+" indicates that Staphylococcus aureus was detected, and "-" indicates that it was not detected.

[0051] The results in Table 2 indicate that the limit of detection for the culture medium plate-TCF chromogenic plate is 10 CFU / mL.

[0052] Example 3 (1) Preparation of Staphylococcus aureus detection medium plates Add the base ingredients (15g tryptone, 8g yeast extract, 13g sodium chloride, and 12g agar) to every 1000mL of deionized water, stir well, sterilize at 120℃ for 20min, cool to 45℃, and then add 2.5g of aseptically treated triglycerides, 0.05g of Nile Red, 1.5mL of Tween 60, 0.15g of X-Glucuronide, and 0.15g of sodium azide in sequence. Mix well and pour into plates.

[0053] (2) Preparation of TCF colorimetric slides Prepare TCF staining solution: Mix 20 mL of 0.115 wt.% calcium chloride solution, 8 g of TrisBase, 1 g of NaCl, 5 g of toluene blue, 6 g of DNA, 300 mL of 95% ethanol and 500 mL of deionized water, boil thoroughly, and after slightly cooling, place sterile filter paper with a diameter slightly smaller than the petri dish into the boiled TCF staining solution, stain evenly, and then air dry under sterile conditions for later use.

[0054] (3) Inoculation and culture Staphylococcus aureus ATCC 6538 bacterial suspension was diluted with physiological saline to different concentrations, and 0.2 mL of the diluted bacterial suspension was spread onto agar plates and incubated at 37°C for 20 h.

[0055] (4) Heat treatment and color development Place the plates with colonies on the culture medium after incubation in a 62°C incubator for 1 hour. Open the plate lid, place the TCF chromogenic strip on the surface of the medium, avoiding the formation of air bubbles, close the lid, and incubate at 37°C for 15 minutes. Then remove the TCF chromogenic strip and place it in another clean culture medium plate. Incubate at 37°C for another 30 minutes.

[0056] (5) Results and Analysis The presence of blue colonies surrounded by a transparent zone of lysis in the TCF staining slide indicates the presence of Staphylococcus aureus.

[0057] Table 3 Medium plate-TCF chromogenic sheet minimum detection limit results

[0058] " + " in the table indicates that Staphylococcus aureus is detected, and "-" indicates that it is not detected.

[0059] The results of Table 1 show that the minimum detection limit of the medium plate-TCF chromogenic sheet is 10 CFU / mL.

[0060] Comparative Example 1 (1) Preparation of Staphylococcus aureus detection medium plate Add the base ingredients (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar) to 1000 mL of deionized water, stir until uniform, and sterilize at 121°C for 15 min. After cooling to 50°C, add 1.5 g of sterile egg yolk, 0.1 g of X-Glucuronide, and 0.1 g of potassium tellurite, mix well, and pour the plate.

[0061] (2) Preparation of TCF chromogenic sheet Prepare the TCF chromogenic solution: 20 mL of 0.115 wt.% calcium chloride solution, 8 g of Tris Base, 1 g of NaCl, 5 g of toluidine blue, 6 g of DNA, 300 mL of 95% ethanol, and 500 mL of deionized water. Mix well and boil. After slight cooling, place a sterile filter paper sheet with a diameter slightly smaller than the culture dish in the boiled TCF chromogenic solution, evenly dye, and dry under sterile conditions. Prepare for use.

[0062] (3) Inoculation culture Dilute the Staphylococcus aureus ATCC 6538 bacterial solution with physiological saline to different concentrations, spread 0.2 mL of the diluted bacterial solution on the medium plate, and incubate at 37°C for 20 h.

[0063] (4) Heat treatment and color development Place the plate with colonies on the medium after incubation in a 62°C incubator for 1 h. Open the cover of the medium plate, place the TCF chromogenic sheet on the surface of the medium to avoid air bubbles, cover the plate, incubate at 37°C for 15 min, remove the TCF chromogenic sheet, and place it on another clean medium plate. Incubate at 37°C for another 30 min.

[0064] (5) Results and analysis From the structure of Figure 2 , it can be seen that Staphylococcus aureus exists in the TCF chromogenic sheet, and the Staphylococcus aureus appears as a blue colony with a transparent lysed zone around it. The diameter of the lysed zone is 1.6-2.5 mm, which is 15-20% smaller than that of Example 1.

[0065] Table 4 Medium plate-TCF chromogenic sheet minimum detection limit results

[0066] " + " in the table indicates that Staphylococcus aureus is detected, and "-" indicates that it is not detected.

[0067] The results of Table 4 show that the minimum detection limit of the medium plate-TCF chromogenic sheet is 100 CFU / mL, indicating that the minimum detection limit of the medium of Comparative Example 1 is one order of magnitude lower than that of Examples 1 to 3.

[0068] Second part: Detection of Staphylococcus aureus in contaminated food Example 4 (1) Preparation of Staphylococcus aureus detection medium plate Add the base ingredients (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar) to 1000 mL of deionized water, stir until uniform, and sterilize at 121°C for 15 min. After cooling to 50°C, add 1.5 g of glycerol triester, 0.02 g of Nile red, 1 mL of Tween 80, 0.1 g of X-Glucuronide, and 0.1 g of potassium tellurite, which have been sterilized, and mix well. Pour the mixture into a plate.

[0069] (2) Preparation of TCF chromogenic sheet Prepare the TCF chromogenic solution: 30 mL of 0.115 wt.% calcium chloride solution, 5 g of Tris Base, 8 g of NaCl, 2.5 g of toluidine blue, 3 g of DNA, 400 mL of 95% ethanol, and 400 mL of deionized water. Mix well and boil. After slight cooling, place a sterile filter paper sheet with a diameter slightly smaller than the culture dish in the boiled TCF chromogenic solution, and evenly dye it. Dry it under sterile conditions, and store it for use.

[0070] (3) Preparation of sample solution Pick fresh bacterial lawn of Staphylococcus aureus strain ATCC6538 to prepare a bacterial suspension. Dilute it with physiological saline to 7.5 x 10 4 CFU / mL, and mix equal volumes to obtain a contaminated bacterial solution. Use chocolate cake, frozen dumplings, cold dishes, and oil tofu as detection samples. Immediately cut them into about 0.2 cm 3 pieces under sterile conditions after purchase. Contaminate 25 g of sample with 2 mL of contaminated bacterial solution and mix well. Place it at room temperature for 10 min, and store it for use. Take 25 g of contaminated sample and 225 mL of sterile physiological saline, shake well to obtain a detection sample solution, and prepare 200 portions of sample solution.

[0071] (4) Inoculation culture Take 0.2 mL sample solution to coat on the medium plate of step (1), incubate at 37℃ for 24 h, and repeat each sample and medium for 2 times. Take 2 mL of contaminated bacteria solution, add to 248 mL of normal saline, mix well, take 0.2 mL to coat NA, incubate at 37℃ for 24 h, as positive control, set two repeats.

[0072] (4) Heat treatment and color development Put the medium plate with colonies after incubation into 62℃ incubator for 1 h, open the cover of the medium plate, paste the TCF color development sheet on the surface of the medium to avoid bubble generation, cover the cover, incubate at 37℃ for 15 min, then take off the TCF color development sheet and put it on another clean medium plate, incubate at 37℃ for another 30 min.

[0073] (5) Counting Detect 91 samples out of 200 weak positive samples.

[0074] Comparative Example 2 (1) Preparation of Staphylococcus aureus detection medium plate Add basic ingredients (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar) to 1000 mL of deionized water, stir uniformly, sterilize at 121℃ for 15 min, cool to 50℃, then sequentially add 1.5 g of sterile processed egg yolk, 0.1 g of X-Glucuronide, and 0.1 g of potassium tellurite, mix well, and pour the plate.

[0075] (2) Preparation of TCF color development sheet Prepare TCF color development solution: 0.115 wt.% calcium chloride solution 30 mL, TrisBase 5 g, NaCl 8 g, toluidine blue 2.5 g, DNA 3 g, 95% ethanol 400 mL, and deionized water 400 mL, mix well and boil, then place sterile filter paper with a diameter slightly smaller than the culture dish in the boiled TCF color development solution after slight cooling, uniformly dye, and dry under sterile conditions, ready for use.

[0076] (3) Preparation of sample solution Pick fresh bacterial lawn of Staphylococcus aureus strain ATCC6538 to prepare bacterial suspension, dilute to 7.5×10 4 CFU / mL with normal saline, mix equal volumes to serve as contaminated bacteria solution. Use chocolate cake, frozen dumplings, cold dishes, and oil tofu as detection samples, cut into about 0.2 cm 3 pieces under sterile conditions immediately after purchase. Contaminate 25 g of sample with 2 mL of contaminated bacteria solution and mix well, stand at room temperature for 10 min, ready for use. Take 25 g of contaminated sample and 225 ml of sterile normal saline, shake well to make detection sample solution, and prepare 200 sample solutions.

[0077] (4) Inoculation culture Take 0.2 mL sample solution to spread on the culture medium plate of step (1), and culture at 37℃ for 24 h, with 2 repeats for each sample and each culture medium. Take 2 mL contaminated bacteria solution, add to 248 mL normal saline, mix well, take 0.2 mL to spread on NA, and culture at 37℃ for 24 h, as positive control, with 2 repeats.

[0078] (4) Heat treatment and color development Put the culture medium plate with colonies after culture into a 62℃ incubator for 1 h, open the cover of the culture medium plate, put the TCF color development sheet on the surface of the culture medium, avoid air bubbles, cover the cover, culture at 37℃ for 15 min, then take off the TCF color development sheet and put it on another clean culture medium plate, and culture at 37℃ for another 30 min.

[0079] (5) Counting Detect 200 weak positive samples, and 70 samples are detected.

[0080] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited to the scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, it is not limited to the examples listed in the embodiments. Those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application.

Claims

1. A culture medium for detecting Staphylococcus aureus, characterized in that, Each liter of culture medium contains 22–60 g of basic components, 1.0–3.0 g of triglycerides, 0.01–0.10 g of lipid-soluble dyes, 0.5–2.0 mL of nonionic surfactants, 0.05–0.20 g of chromogenic substrates, and 0.05–0.20 g of selective inhibitors.

2. The Staphylococcus aureus detection culture medium according to claim 1, characterized in that, The basic ingredients include 5-15g tryptone, 2-10g yeast extract, 5-15g sodium chloride, and 10-20g agar.

3. The Staphylococcus aureus detection culture medium according to claim 1, characterized in that, The fat-soluble dyes include Nile Red and / or Sudan IV.

4. The Staphylococcus aureus detection culture medium according to claim 1, characterized in that, The nonionic surfactant includes at least one of Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, and Tween 85.

5. The Staphylococcus aureus detection culture medium according to claim 1, characterized in that, The chromogenic substrate includes 5-bromo-4-chloro-3-indoleglucoside.

6. The Staphylococcus aureus detection culture medium according to claim 1, characterized in that, The selective inhibitors include potassium tellurite and / or sodium azide.

7. A method for preparing Staphylococcus aureus detection medium plates, characterized in that, The method includes the following steps: dissolving the basic components of the Staphylococcus aureus detection culture medium according to any one of claims 1 to 6 in water, then heating and sterilizing the solution, and after cooling, adding the triglyceride, the lipid-soluble dye, the nonionic surfactant, the chromogenic substrate and the selective inhibitor, mixing well and then pouring the mixture into a plate.

8. The method for preparing Staphylococcus aureus detection culture medium plates according to claim 7, characterized in that, The triglycerides, the fat-soluble dyes, the nonionic surfactants, the chromogenic substrates, and the selective inhibitors are sterilized before mixing.

9. The method for preparing Staphylococcus aureus detection culture medium plates according to claim 7, characterized in that, The heating sterilization is performed at 100~140℃ for 10~20 minutes, and the cooling reduces the temperature to 50±5℃.

10. A method for detecting Staphylococcus aureus, characterized in that, Including the following steps: (1) Preparation of sample solution Prepare a sample solution by placing the sample in phosphate buffer or physiological saline; (2) Inoculation and culture The sample solution is spread onto a medium plate prepared by the Staphylococcus aureus detection medium according to any one of claims 1 to 6 or a Staphylococcus aureus detection medium plate prepared by the method of preparing Staphylococcus aureus detection medium plate according to any one of claims 7 to 9 and cultured thereon. (3) Colony counting and calculation Count the typical colony count on the culture medium plate and calculate the number of Staphylococcus aureus colonies in the sample.

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