Method for evaluating bacterium killing effect of antibacterial ceramic

Abstract

The invention relates to a method for evaluating the bacterium killing effect of antibacterial ceramic. The method comprises the following test steps: preparing a sample and pre-treating; selecting equipment and reagents, and preparing a culture medium; preserving and activating strains and preparing a bacterial suspension; oD value-viable bacteria content logarithm value lgCB standard curve establishment and inoculation bacteria liquid CB calibration; establishing an lgCB-relative fluorescence intensity logarithm value lgIB standard curve; inoculating a sample, culturing, eluting and recovering; determining the IB of the recovery liquid and calculating the content CB and TB of viable bacteria of the recovery liquid; calculating an antibacterial rate R or an antibacterial activity value A; evaluating a result; the method is characterized in that an ATP fluorophotometer is used for evaluating the bacterium killing effect of the antibacterial ceramic represented by R or A. The method comprises the following steps: determining a control sample and an antibacterial sample after eluting, recovering and contacting for 0 hour and culturing for 24 hours, determining a recovered liquid IB, and representing and calculating R or A by using lgIB; and performing result evaluation. According to the ATP fluorescence rapid detection method for evaluating the bacterium killing effect of the antibacterial ceramic researched and developed by the invention, the product quality improvement and the market order specification are supported by an advanced detection technology.

Description

Technical Field

[0001] The invention relates to a method for testing the antibacterial performance of antibacterial ceramics, in particular to a method for evaluating the antibacterial performance of antibacterial ceramics characterized by an antibacterial rate R or an antibacterial activity value A by using an ATP fluorescence photometer. Background Art

[0002] China has a long history of ceramic production, with annual ceramic production accounting for about 90% of the world's total production and annual exports exceeding 4 billion pieces; among them, daily-use ceramics, building ceramics, and sanitary ceramics account for 65%, 60%, and 40% of the world's total production respectively, but the total output value of ceramics is only 10% of the world's total output value, and the homogeneous competition caused by the low technological content of products is becoming increasingly fierce. In recent years, the use of new antibacterial technology to transform traditional ceramic production processes has become the main direction of transformation and development of domestic advantageous ceramic companies. The application and popularization of antibacterial technology in the ceramic industry is unstoppable, and the antibacterial function has become a distinctive "selling point" of ceramic products; but the lag in existing detection technology has seriously restricted the development of the industry.

[0003] At present, the domestic and foreign antibacterial material performance testing technology system is mainly aimed at bacteria and fungi. The principles of antibacterial performance testing methods in various countries are mostly based on the plate counting method for separating and culturing bacteria. The first is the film culture and immersion quantitative test method proposed by the Japanese Industrial Standard; the second is the antibacterial ring method originated from the American textile industry; the third is the internationally used oscillating flask method; the fourth is the drip method originated from American textile companies; and the fifth is the cover glass method developed by Japanese companies for photocatalytic antibacterial products. So far, my country has successively formulated JC / T 897-2002 (revised in 2014) "Antibacterial Performance of Antibacterial Ceramic Products" and GB / T 28116-2011 "Antibacterial Bone Porcelain" based on the reference of relevant foreign methods. However, the existing antibacterial performance testing methods have the following common problems in technical content and practical application: First, there are too few experimental strains involved, which makes it difficult to meet the company's new product development and quality control needs that keep pace with the times; second, the experimental process is cumbersome, and related operations are affected by the professional experience of the experimenter, resulting in large test errors and lack of comparability; third, the test cycle is between 48 hours and 72 hours, with high time and economic costs; there is an urgent need to study scientifically advanced, reproducible test results, and simple and easy-to-use ceramic antibacterial performance testing technology. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting the antibacterial performance of antibacterial ceramics characterized by antibacterial rate R or antibacterial activity value A using an ATP fluorescence photometer, which can solve the problem of accurate quantitative testing of the antibacterial performance of antibacterial ceramics and even other antibacterial materials and products.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for detecting the antibacterial performance of antibacterial ceramics, comprising: (1) sample preparation and pretreatment; (2) equipment selection and reagent and culture medium preparation; (3) bacterial strain preservation, activation and bacterial suspension preparation; (4) OD value-live bacteria content logarithm lgC B Standard curve establishment and inoculation of bacterial solution C B Calibration; (5) Logarithm of viable bacterial content lgC B -Relative fluorescence intensity log value lgI B Standard curve establishment; (6) Sample inoculation, culture and elution recovery; (7) Relative fluorescence intensity value I of the recovered solution B Determination; (8) Recover viable bacteria C B and T B Calculation and calculation of antibacterial rate R and antibacterial activity value A; (9) result evaluation; characterized in that an ATP fluorescence photometer is used to accurately and quantitatively test the antibacterial performance of antibacterial ceramics characterized by antibacterial rate R or antibacterial activity value A. B -lgI B Standard curve method, specifically:

[0007] The relative fluorescence intensity value I B Measuring:

[0008] The number, size and water absorption requirements of the control samples and antibacterial samples were clarified, and the samples were sterilized and water-absorbed. After the test standard strain was subcultured and activated, fresh bacterial cultures that were transferred twice were used to prepare bacterial suspensions, and the live bacteria were counted using the MTT colorimetric method. The OD value-live bacteria content logarithm lgC was established. B Standard curve, derive the linear equation of the curve Y = a0X + b0 and the correlation coefficient R0 2 ; and the inoculated bacterial solution was tested for live bacterial content C B Calibration: 5.0×10 6 CFU / mL~9.0×10 6 CFU / mL, at the same time, select C B 4.2×10 3 CFU / mL, 4.2×10 4 CFU / mL, 4.2×10 5 CFU / mL dilution was used as the standard series bacterial suspension; its relative fluorescence intensity value I was determined B , draw lgC B -lgI B Standard curve, and derive the linear equation of the curve Y = a B X+b B And the correlation coefficient R B 2Then, 0.3 mL of bacterial solution was added to the glaze of each group of control samples and antibacterial samples; 4.7 mL of eluent was immediately used to elute and recover the 6 groups of 0-hour contact samples, and the relative fluorescence intensity value I of the recovered solution was measured by ATP fluorescence photometer. BC0ij ,I BT0ij , according to the curve equation Y = a B X+b B Calculate the viable bacterial content C BOij and T BOij At the same time, the six groups of 24h contact samples were sealed in sterile plates and cultured at 37℃±1℃ and 90%±2%RH for 24h±2h; the residual bacteria on the glaze surface were recovered in the same way as the 0h contact samples and the relative fluorescence intensity value I of the recovered solution was determined. BCtij ,I BTtij , calculate the corresponding viable bacterial content C Btij and T Btij ;

[0009] In the calculation of antibacterial rate R and antibacterial activity value A:

[0010] According to the standard curve of the test strain lgC B -lgI B The linear equation Y = a B X+b B The relative fluorescence intensity of each control sample and antibacterial sample recovery solution after 0h contact and 24h incubation was measured. I BCtij and I BTtij As basic data; under the effective conditions of the test, calculate the bacterial growth value F after 24h of culture ij , G ij And the antibacterial rate R ij and antimicrobial activity value A ij ; R for each group of samples ij and A ij Take the arithmetic mean to get the corresponding R i and A i ; The antibacterial rate R and antibacterial activity value A of each batch of antibacterial ceramic samples are the three groups of samples R i and A i The arithmetic mean of the relevant data and the measurement uncertainty requirements shall be clearly stated;

[0011] In the results evaluation:

[0012] Refer to the general practices of the health industry and the relevant antibacterial product standards to determine the antibacterial performance classification criteria; when the antibacterial rate R of a group / piece of antibacterial ceramic samples i / R ij Or antimicrobial activity value A i / Aij When the antibacterial performance of the other two groups / four samples is at least one level lower than that of the other two groups / four samples, extract one group / sample again and repeat the experiment; calculate the ATP bioluminescence lgC B ─lgI B If the antibacterial performance level of the two groups / pieces of antibacterial ceramic samples is the same, then discard them; take the antibacterial rate R of the remaining two groups / four pieces of samples. i / R ij Or antimicrobial activity value A i / A ij The arithmetic mean value is taken as the evaluation result of the antibacterial performance of the antibacterial ceramic samples of this batch / group.

[0013] Compared with the prior art, the present invention using the above technical solution has the following beneficial effects:

[0014] (1) Advancedness: Using modern precision instruments—ATP fluorescence photometer and microplate reader as test equipment, the test of viable bacteria content and antibacterial performance of ceramics has been modernized; it can effectively reduce the influence of human factors in the experimental process and avoid the large errors commonly found in traditional plate culture methods; it can ensure the quantification of test results and greatly improve the accuracy of test data; the detection technology is advanced to a certain extent;

[0015] (2) Scientificity: Based on the testing principle of ATP fluorescence analysis, a logarithmic value of viable bacterial content lgC suitable for multiple bacterial species was established. B ─Relative fluorescence intensity logarithm lgI B Standard curve; a mathematical model of the ATP bioluminescence real-time quantitative analysis method for antibacterial ceramic antibacterial performance detection was constructed; at the same time, taking into account the cognitive habits of consumers in different countries, the antibacterial rate R and antibacterial activity value A were used as relevant performance evaluation indicators, which improved the scientificity and versatility of the detection method;

[0016] (3) Innovation: Compared with the current traditional methods with complicated operations, large errors and long cycles, this method introduces an ATP fluorescence photometer with a high level of automation and intelligence in the test process, which greatly simplifies the experimental steps, realizes the precision and quantification of the test results of the antibacterial performance of ceramics, and has good reproducibility and comparability; at the same time, it greatly shortens the test cycle and reduces the test cost; it can effectively fill the gap in the current related test technology field;

[0017] (4) Prospective: Established a B lgI BThe standard curve quantitative analysis method based on linear relationship innovates and enriches the determination method of live bacteria content in bacterial suspension and the pretreatment method of ceramic samples, clarifies the technical contents such as reference samples, standard solution concentration, determination steps, calculation formula, uncertainty, etc.; it is the first to directly measure the relative fluorescence intensity value I of live bacteria in the recovery liquid by the instrument B It is a form of result evaluation to calculate and determine the antibacterial rate R or antibacterial activity value A, and to examine the measurement uncertainty through the coefficient of variation C·V within and between groups, which is technically forward-looking.

[0018] (5) Operability: ATP fluorescence photometer and microplate reader are inexpensive, easy to operate, and widely used. The established MTT colorimetric analysis method for viable bacterial content and ATP fluorescence test method for ceramic antibacterial performance are simple and easy to use. The relevant technical instructions are clear and specific, easy to understand and master. They are highly operable during implementation and suitable for microbiological laboratory personnel of different professional levels, which is conducive to promoting the transfer and transformation of research results and their promotion and application.

[0019] (6) Universality: Since ATP is widely present in living cells, it can provide a broad-spectrum detection technology support for the amplification of experimental bacteria; the introduction of relevant instruments can greatly simplify the experimental steps and reduce the testing cost; it is conducive to expanding the application in the fields of inspection, learning, research, and production, and can support the universalization of ceramic antibacterial performance detection technology. At the same time, it can provide reference for the research on antibacterial performance detection technology in the fields of plastics, glass and other products;

[0020] Further, the preferred embodiment of the present invention is:

[0021] The sample preparation and pretreatment are carried out according to the following steps:

[0022] (1) Control samples: except that no antibacterial ingredients are added, the control samples are identical in type, raw materials and process to the antibacterial ceramic samples to be tested; the antibacterial test surface is glazed and the other side is unglazed; the thickness of bone china and fine porcelain in daily-use ceramic samples is not more than 6 mm, and the water absorption rate is ≤0.5%; the thickness of ordinary porcelain is not more than 8 mm, and the water absorption rate is ≤1.0%; the thickness of stoneware and fine pottery is not more than 10 mm, and the water absorption rate of stoneware is ≤5.0%; the water absorption rate of fine pottery is ≤17.0%; the thickness of sanitary ceramic samples is 6mm~10mm, porcelain water absorption ≤0.5%, 8.0%≤ceramic water absorption<15.5%; building ceramic sample thickness is not more than 10mm, porcelain water absorption ≤0.5%, stoneware water absorption 0.5%~3%, stoneware water absorption 6%~10%, ceramic water absorption ≥10%, sample size is (50±2)mm×(50±2)mm, 6 groups of samples are used for each bacterial species test; 3 groups are used for 0h contact test and 24h culture test, each group has 5 samples;

[0023] (2) Antibacterial samples: ceramic samples with antibacterial agent added to the glaze layer or antibacterial layer applied on the surface, and the other side is unglazed; the sample thickness, water absorption rate, specifications, dimensions, quantity, etc. are the same as those of the control samples. For each group of antibacterial samples, a group of control samples is selected as a reference and effectively marked;

[0024] (3) Sample pretreatment: Before the experiment, the control samples and antibacterial samples of each group were soaked in sterile water for 24 h until the body and glaze layer fully absorbed the water; then soaked in 70% ethanol solution for 5 min, and the samples were fully washed with sterile water on a clean bench to remove the ethanol; then, the excess water on the surface of the sample was absorbed with sterile dry gauze, and the sample was placed in a sterile plate with the glaze surface facing up for later use;

[0025] The equipment selection and reagent and culture medium preparation are carried out according to the following steps:

[0026] (1) General requirements: Analytical reagents and Grade III water, distilled water or deionized water that conforms to GB / T 6682-2008 must be used for the test; the laboratory must have Grade II biosafety qualification and the personnel must have working experience in a formal microbiology laboratory;

[0027] (2) Instruments and equipment: Class II biosafety cabinet or clean bench with cleanliness level not less than 100; ATP bioluminescence rapid detection system for total bacteria including ATP fluorescence photometer, supporting reagent kit, special test tubes, etc. The wavelength range of ATP fluorescence photometer is 300nm to 650nm, and the detection range is 10 1 CFU / mL~10 7 CFU / mL; microplate reader with wavelength range of 400nm~760nm and reading range of 0.0Abs~4.0Abs; constant temperature and humidity incubator at 37℃±1℃, 90%±2%RH; constant temperature water bath at 46℃±1℃; pressure steam sterilizer at 121℃±2℃, 103kPa±5kPa; low temperature refrigerator at -20℃~-70℃; refrigerator at 2℃~8℃; electronic balance with sensitivity of 0.001g; ultrasonic cleaner with frequency of 30kHz~50kHz; vortex oscillator with speed of 500r / min~3000r / min; pH meter with accuracy of ±0.1 at 25℃; electric furnace;

[0028] (3) Materials and instruments: 1mL and 10mL sterile graduated pipettes; 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, and 10mL single-channel variable-range pipettes and sterile pipette tips, with a measurement error of less than 1%; a syringe filter with a pore size of no more than 0.45μm; a sterile conical flask with a capacity of 250mL and 500mL; a sterile culture dish with a diameter of 90mm; a 96-well flat-bottom culture plate; a standard turbidimetric tube for McMaster bacteria and matching test tubes; a sterile test tube; an inoculation loop with a diameter of no more than 4mm; an L-rod; sterile tweezers and sterile gauze; medical tape; a 75% alcohol cotton ball; and a ruler with a graduation value of 1mm. Thermometer; stopwatch with an accuracy of 0.01s; A6 white copy paper; 0.7mm core black gel pen;

[0029] (4) Reagents: 70% ethanol solution; 85% physiological saline, sterilized at 121°C for 15 min, stored at 5°C-10°C for 30 d; 5 mg / mL MTT (methyl thiazolyl tetrazolium) solution, pH 7.4, sterilized by filtration through a 0.45 μm filter membrane, stored at 4°C-6°C in the dark for 15 d;

[0030] (5) Culture medium / liquid: Commercially available culture medium / liquid can be used: the prepared culture medium / liquid should be sterilized by high pressure sterilization at 121°C for 15 min after aliquoting, and then stored at 2°C to 8°C for 30 days;

[0031] Nutrient agar NA: Dissolve 5 g beef extract, 10 g peptone, 5 g sodium chloride, and 15 g agar in 1000 mL water by heating, and adjust the pH to 7.0-7.2;

[0032] Nutrient broth NB: Dissolve 3g beef extract, 10g peptone, and 5g sodium chloride in 1000mL water by heating, and adjust the pH to 7.0-7.2;

[0033] Brain heart infusion broth BHI for Salmonella preservation: Dissolve 10 g peptone, 5 g sodium chloride, 2.5 g disodium hydrogen phosphate, and 2 g glucose in 500 mL of beef heart infusion by heating, and adjust the pH to 7.4 ± 0.2;

[0034] Culture medium for Escherichia coli and Shigella / Staphylococcus aureus and Salmonella: Mix 1:500 / 1:100 nutrient broth NB with 85% sterile saline solution and adjust the pH to 7.0-7.2;

[0035] Plate count agar PCA: Dissolve 5 g tryptone, 2.5 g yeast extract, 1 g glucose, and 15 g agar in 1000 mL water by heating, and adjust the pH to 7.0 ± 0.2;

[0036] (6) ATP fluorescence reaction reagent, or commercially available reagent: except for phosphate buffer solution, the prepared ATP fluorescence reaction reagent should be stored at -20°C to -70°C and used within 6 months;

[0037] Dilution buffer solution: 0.005 mol / L sodium dihydrogen phosphate solution containing 0.037% sucrose, adjust the pH to 7.2±0.2; sterilize at 121℃ for 15min, store at 2℃~8℃ for 30d;

[0038] ATP fluorescent reagent buffer solution: Dissolve 1117 mg of tris(hydroxymethyl)aminomethane, 183 mg of disodium ethylenediaminetetraacetate, 808 mg of magnesium acetate, 6.7 mg of dithiothreitol, 25000 mg of β-cyclodextrin and 925 mg of glucose in 250 mL of water by heating, and adjust the pH to 7.5±0.2; use within 8 hours;

[0039] ATP lysis buffer: Dissolve 4.6 international units / ml of adenosine triphosphate diphosphatase EC:3.6.1.5 and 46 international units / ml of adenosine phosphate deaminase EC:3.5.4.6 or EC:3.5.4.17, 37 mg of sucrose, and 20 mg of bovine serum albumin in 10 mL of 0.05 mol / L 2-morpholineethanesulfonic acid buffer solution, adjust the pH to 6.0±0.5, and use within 8 hours. 1 mL of lysis buffer can reduce the ATP concentration in the nutrient broth to 10 within 15 minutes. -13 mol / L or less;

[0040] ATP extract: Heat and dissolve 45 mg of tris(hydroxymethyl)aminomethane in 9.8 ml of water, mix with 0.2 ml of 10% benzalkonium chloride solution, and adjust the pH to 12.0 ± 0.5;

[0041] ATP fluorescence reagent: Dissolve 16 mg of luciferase EC: 1.13.12.7, 12.6 mg of D-luciferin, and 56 mg of bovine serum albumin in 30 mL of ATP fluorescence reagent buffer solution, mix well, and let stand at room temperature for 15 min. Use within 3 hours.

[0042] The bacterial strain preservation, activation and bacterial suspension preparation are carried out according to the following steps:

[0043] (1) Test bacteria: Escherichia coli ATCC 8739; Staphylococcus aureus ATCC 6538; Salmonella ATCC 14028; Shigella CMCC 51105, or other bacteria provided by the corresponding national culture collection management center and traceable;

[0044] (2) Preservation of bacterial strains: Open the freeze-dried bacterial strain tube aseptically, add an appropriate amount of nutrient broth with a capillary pipette, and use BHI culture medium for Salmonella, pipette several times to melt and disperse the bacterial strains, then drop a small amount of the test bacterial strain suspension into a test tube containing 5 mL to 10 mL of nutrient broth and culture at 37°C ± 1°C for 24 h to 48 h; store the strains for slant preservation at 5°C ± 1°C for no more than 1 month, and the number of inoculations shall not exceed 14 generations;

[0045] (3) Activation of bacterial strains: transfer the slant-preserved bacteria to nutrient agar slant medium and culture at 37°C±1°C for 18 to 24 hours; transfer once a day for no more than 15 days. Use fresh bacterial cultures of the 3rd to 14th generations transferred within 24 hours.

[0046] (4) Preparation of bacterial suspension: Use an inoculation loop to scrape a small amount of fresh bacteria from the culture medium for bacterial activation and add the culture medium of the test bacteria to make a bacterial suspension; shake the test tube at 1000 r / min for 1 min or shake the test tube 80 times with an amplitude of 30 cm to ensure that the bacteria are evenly suspended; transfer an appropriate amount of bacterial suspension to a sterile test tube that matches the McFarland standard turbidimetric tube, and use a 0.7 mm core black neutral pen to draw three parallel lines of 10 cm in length on A6 white copy paper; visually compare the test tube with the nominal concentration of 6.0 × 10 8 The turbidity difference of the CFU / mL standard turbidimetric tube was measured, and the culture medium was added dropwise until the turbidity of the two tubes was the same;

[0047] The OD value-logarithm of viable bacteria content lgC B Standard curve establishment and inoculation of bacterial solution C B To calibrate, follow the steps below:

[0048] (1) Determination of bacterial solution OD value: The total amount of bacteria was 6.0×10 8 The bacterial suspension with a dilution ratio of 1:5, 1:10, 1:15, and 1:20 was serially diluted, and the culture solution was used as the blank control sample solution to zero the microplate reader. Then, 100 μL of the bacterial solution with different dilutions was injected into a 96-well flat-bottom culture plate, and 3 replicates were made for each dilution. 20 μL of MTT solution was added to each well, and cultured at 37°C±1°C and 90%±2%RH for 30 min. The maximum absorption peak was scanned in the wavelength range of 560nm to 610nm to determine the maximum absorption wavelength. The OD value of the live bacteria of the mixed solution in each well was determined. The OD value of the live bacteria of the bacterial suspension with different dilutions was the arithmetic mean of the OD values ​​of the 3 replicates. At the same time, according to the method specified in the national standard GB 4789.2-2016, the above bacterial suspension was appropriately diluted, cultured at 37°C±1°C for 24h±2h, and the colony count of the plate was performed to calibrate its live bacteria content C B ;

[0049] (2) OD value-logarithm of viable bacterial content lgC B Standard curve establishment and inoculation of bacterial solution C B Calibration: The OD value of live bacteria in bacterial suspensions of different dilutions is used as the ordinate, and the logarithm of the live bacteria content is used as the lgC B As the horizontal axis, plot OD value-lgC B Standard curve; the linear equation of the curve Y=a0X+b0 and the correlation coefficient R0 are derived by the least squares fitting method 2 , where Escherichia coli or Shigella and Staphylococcus aureus or Salmonella were selected with dilutions of 1:10, 1:15, 1:20 and 1:5, 1:10, 1:15 respectively as standard series solutions to draw curves. 2 ≥0.98, confidence level ≥0.95, the measured live bacterial content of bacterial suspension C B Then, the culture fluid of the test strain was adjusted to obtain C B The range is 5.0×10 6 CFU / mL~9.0×10 6 CFU / mL of inoculum;

[0050] The logarithmic value of the viable bacteria content lgC B -Relative fluorescence intensity log value lgI B To establish the standard curve, follow the steps below:

[0051] (1) Determination of standard series bacterial suspension and its I B Determination: Use the test bacterial culture solution to mix the known live bacterial content C B The bacterial suspension was diluted to a standard series: 4.2×10 3 CFU / mL, 4.2×10 4 CFU / mL, 4.2×10 5 CFU / mL and mix thoroughly, then, according to the relative fluorescence intensity value I B Determination method: according to the viable bacterial content C B The relative fluorescence intensity values ​​of the above standard series bacterial suspension and 0.3 mL of inoculated bacterial solution diluted with 4.7 mL of eluent within 1 min were measured and recorded in order from low to high, and the latter was recorded as I B0 ;

[0052] (2) Logarithm of viable bacterial content lgC B -Relative fluorescence intensity log value lgI B Standard curve establishment: The relative fluorescence intensity logarithm of the standard series of bacterial suspensions is used as the lgI B As the horizontal axis, the corresponding logarithm of viable bacterial content lgC BDraw a graph for the ordinate; calibrate the mathematical relationship between the two curves, and use the least squares fitting method to derive the linear equation of the curve Y = a B X+b B And the correlation coefficient R B 2 ; When R B 2 When ≥0.98 and confidence level ≥0.95, the determination is valid;

[0053] The sample inoculation, cultivation and elution recovery are carried out according to the following steps:

[0054] (1) Inoculation culture: Place a multi-layer sterile gauze of appropriate size at the bottom of a sterile dish, and pour 10 mL of sterile physiological saline on the gauze to make it just saturated with water; place 6 groups of 24-hour contact samples on the gauze, and 6 groups of 0-hour contact samples in a dish without gauze, then use a sterile pipette to drop 0.3 mL of bacterial solution on the glaze surface of each group of control samples and antibacterial samples to be tested, and mix with 1gC B -lgI B The bacterial solution for curve calibration is taken from the same test strain stock solution test tube, stored at 2℃±0.2℃, used within 2h, and evenly spread with an L-stick attached with the inoculated bacterial solution but without dripping, so that it covers the entire glaze surface of the sample, cover the dish lid, and seal the plate containing 6 groups of 24h contact samples with medical tape; incubate at 37℃±1℃, 90%±2%RH for 24h±2h;

[0055] (2) Elution and recovery: 4.7 mL of the test strain culture fluid was used as the eluent, and 0.2% sterile surfactant could be added. After the 6 groups of 0-hour contact samples were inoculated with bacteria, they were immediately fully eluted using the solid or liquid swab method. The 6 groups of 24-hour cultured samples were eluted using the same elution method as the 0-hour contact samples. The solid swab method was to use a sterile cotton swab with a saturated eluent on the head to evenly rub each sample surface horizontally and vertically 10 times, and then rotate the cotton swab. Cut off the part of the cotton swab that the experimenter's hand touched, place the cotton swab in a sterile test tube containing the remaining eluent, mix well and use it as the recovery liquid. If the recovery liquid is less than 5 mL, add eluent to 5 mL. The liquid swab method was to use a sterile pipette to draw 4.7 mL of eluent, repeatedly rinse each sample surface in a plate at least 4 times, fully elute and transfer the washing liquid into a sterile test tube, mix well and use it as the recovery liquid. If the recovery liquid is less than 5 mL, add eluent to 5 mL.

[0056] The relative fluorescence intensity value I of the recovery solution B The determination is carried out according to the following steps:

[0057] (1) Calibration of the relative fluorescence intensity background value of the instrument and reagent set: Use a sterile pipette to add 0.1 mL of the test bacterial culture solution, 0.8 mL of sodium hydrogen phosphate buffer solution containing 0.037% sucrose, and 0.1 mL of ATP lysis solution into the same sterile test tube in sequence and mix thoroughly. Then, transfer 0.1 mL of the mixed solution to three sterile test tubes dedicated to the instrument and let it stand for 5 min to 30 min as blank test parallel samples; add 0.1 mL of ATP extraction reagent to each of the three parallel samples, mix well, and then add 0.1 mL of ATP fluorescence reagent; mix well again, and immediately measure the relative fluorescence intensity value I using an ATP fluorescence photometer. B And record to ensure that the operation time of each link is consistent to avoid cross contamination; the measurement time of each parallel sample shall not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values ​​of three blank test parallel samples shall be used as the background value of the instrument and reagent group, or the background value shall be calibrated according to the instrument instructions;

[0058] (2) Relative fluorescence intensity value of the recovered liquid I B Determination: If the background level meets the requirements of the instrument, use a sterile pipette to add 0.1mL of recovery solution, 0.8mL of sodium dihydrogen phosphate buffer solution containing 0.037% sucrose, and 0.1mL of ATP lysis solution into the same sterile test tube in sequence. After fully mixing, transfer 0.1mL of the mixed solution to three sterile test tubes dedicated to the instrument, and let it stand for 5min to 30min as parallel samples for ATP fluorescence test; add 0.1mL of ATP extraction reagent to each of the three parallel samples, mix well, and then drop 0.1mL of ATP fluorescence reagent; mix again, and immediately use an ATP fluorescence photometer to measure the relative fluorescence intensity value I B The measurement time of each parallel sample should not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values ​​of the three ATP fluorescence test parallel samples should be used as the I value of the recovered solution of the sample to be tested. B The relative fluorescence intensity values ​​of three parallel samples of the recovery solution can be directly measured by using the instrument matching kit according to the relevant instructions. B ;

[0059] The viable bacteria content of the recovered liquid is C B and T B The calculation of antibacterial rate R and antibacterial activity value A is carried out according to the following steps:

[0060] (1) Live bacterial content of recovered liquid C B and T B Calculation

[0061] According to the standard curve of the test strain lgC B -lgI B The linear equation Y = a BX+b B Calculate the viable bacterial content C of the recovery solution of the control sample and the antibacterial sample after 0h contact and 24h culture B and T B When using the instrument kit, calculate the viable bacterial content C of 1mL of recovery solution based on the actual injection volume. B and T B , the relevant calculations are shown in formulas (1) to (12):

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Where:

[0075] C B0 and C Bt —The average number of viable bacteria recovered from the control samples of the three groups after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0076] C B0i and C Bti —The average number of viable bacteria recovered from the control samples after 0-hour contact and 24-hour incubation in each group, in colony-forming units per milliliter (CFU / mL); sample groups i = 1, 2, 3;

[0077] C B0ij and C Btij —The number of viable bacteria recovered from each control sample after 0-hour contact and 24-hour incubation, in colony-forming units per milliliter (CFU / mL); each group of samples is numbered j=1, 2, 3, 4, 5;

[0078] I BC0ij and I BCtij —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU;

[0079] a B —Standard curve lgC B -lgI B Slope; b B —Standard curve lgC B -lgI B intercept on the ordinate;

[0080] T B0 and T Bt —The average number of viable bacteria recovered from the three groups of antimicrobial samples after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0081] T B0i and T Bti —The average number of viable bacteria recovered from the antimicrobial samples after 0-hour contact and 24-hour incubation in each group, expressed in colony-forming units per milliliter (CFU / mL);

[0082] T B0ij and T Btij —The number of viable bacteria recovered from each antimicrobial sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0083] and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU;

[0084] (2) Test validity conditions

[0085] If the relative fluorescence intensity of each 0h contact control sample recovery solution and 0.3mL inoculated bacterial solution diluted with 4.7mL eluent within 1min is close, that is The logarithm of the relative fluorescence intensity of the recovered solution of each control sample after 24h of incubation C Btij ≥0.1×C B0ij ; When the relative fluorescence intensity of the recovered solution of the three groups of control samples was measured at 0h When the intra-group and inter-group variation coefficient C·V≤10%, the determination is valid. The calculation formula involved can be found in the relevant measurement uncertainty requirements.

[0086] (3) Bacterial growth value F ij , G ij calculate

[0087] After 24 hours of culture, the bacterial growth value F of each control sample and antibacterial sample was ij , G ij Calculate according to formula (13) and (14) respectively:

[0088]

[0089]

[0090] Where:

[0091] F ij and G ij — Bacterial growth value of each control sample and antibacterial sample after 24 hours of culture;

[0092] C B0ij and C Btij —The number of viable bacteria recovered from each control sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0093] and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU;

[0094] T B0ij and T Btij —The number of viable bacteria recovered from each antimicrobial sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0095] and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU;

[0096] (4) Calculation of antibacterial rate R

[0097] Under the effective conditions of the test, the antibacterial rate R of each antibacterial ceramic sample ij , the antibacterial rate R of each group and batch of samples i and R are calculated according to formulas (15), (16), and (17) respectively:

[0098]

[0099]

[0100]

[0101] Where:

[0102] R ij—Antibacterial rate of each antibacterial ceramic sample, %;

[0103] R i —Antibacterial rate of each group of antibacterial ceramic samples, %;

[0104] R—antibacterial rate of each batch of antibacterial ceramic samples, %;

[0105] T Btij and C Btij —The number of viable bacteria recovered from each antimicrobial sample and control sample after 24 h of culture, in colony forming units per milliliter (CFU / mL);

[0106] and —Relative fluorescence intensity of the recovered solution of each antibacterial sample and control sample after 24h of incubation, RLU;

[0107] (5) Calculation of antibacterial activity value A

[0108] Under the effective test conditions, the antibacterial activity value A of each antibacterial ceramic sample ij , antimicrobial activity value A of each group and batch of samples i and A are calculated according to formulas (18), (19), and (20) respectively:

[0109]

[0110]

[0111]

[0112] Where:

[0113] A ij —The antibacterial activity value of each antibacterial ceramic sample;

[0114] A i —The antibacterial activity value of each group of antibacterial ceramic samples;

[0115] A—antibacterial activity value of each batch of antibacterial ceramic samples;

[0116] F ij and G ij — Bacterial growth value of each control sample and antibacterial sample after 24 hours of culture;

[0117] and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU;

[0118] and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU;

[0119] (6) Data revision requirements: Use the plate count method to calibrate the viable bacterial content of the bacterial suspension C B When C B When the concentration is less than 100 CFU / mL, the integer is rounded off. B When it is not less than 100 CFU / mL, the first two digits of the third digit shall be rounded off, and the digits behind shall be replaced by 0; it can also be expressed in the form of 10 exponentials, and two significant digits shall be used after rounding off; the relative fluorescence intensity of the recovery liquid of the control sample and the antibacterial sample after 0h contact and 24h culture shall be integer, and the antibacterial rate R ij , R i , R calculation results are retained to three significant figures; bacterial growth value F ij , G ij and antimicrobial activity value A ij , A i 、A The calculation result shall be rounded to two significant figures;

[0120] (7) Measurement uncertainty: The reproducibility of applying the ATP bioluminescence analysis method to the antibacterial performance test of ceramics is determined by calculating the intra-group and inter-group coefficient of variation C·V = σ÷μ×100% of the relative fluorescence intensity of the recovery liquid measured by the control sample and the antibacterial sample after 0h contact and 24h incubation. The calculation results of μ, σ and C·V are retained to two decimal places. It is stipulated that the intra-group and inter-group coefficient of variation C·V shall not be greater than 10%;

[0121] The arithmetic mean of the relative fluorescence intensity of the recovered fluid of each group of 5 control samples, 5 antibacterial samples, and 5 control samples and 5 antibacterial samples after 24h of incubation was calculated. and Calculate according to formula (21) and (22) respectively; ATP test parallel sample number k = 1, 2, 3; and Relative fluorescence intensity of each ATP test parallel sample after 0h contact and 24h incubation for each group of control samples and antibacterial samples:

[0122]

[0123]

[0124] The arithmetic mean of the relative fluorescence intensity of the recovered fluids of 15 control samples, 15 antibacterial samples, and 15 control samples and 15 antibacterial samples after 24h of incubation in 3 groups. and Calculate according to formula (23) and (24) respectively:

[0125]

[0126]

[0127] The standard deviation of the relative fluorescence intensity of the recovered solution of each group of 5 control samples, 5 antibacterial samples, and 5 control samples and 5 antibacterial samples after 24h of incubation was and Calculate according to formula (25) and (26) respectively:

[0128]

[0129]

[0130] Standard deviation of relative fluorescence intensity of recovered fluids of 15 control samples, 15 antibacterial samples after 0h contact and 15 control samples, 15 antibacterial samples after 24h incubation in 3 groups and Calculate according to formula (27) and (28) respectively:

[0131]

[0132]

[0133] The evaluation of the antibacterial performance of the antibacterial ceramic sample is carried out according to the following steps:

[0134] (1) With reference to the general practices of the health industry and the requirements of relevant antimicrobial product standards, the following grading criteria are adopted:

[0135] If the antibacterial rate R is used as the relevant performance evaluation index: ij / R i / R<90%, or Staphylococcus aureus and Salmonella R ij / R i / R<80%, the sample has no antibacterial effect; when 90%≤R ij / R i / R<99%, or Staphylococcus aureus and Salmonella 80%≤R ij / R i / R<90%, the sample has antibacterial effect; when 99%≤R ij / R i / R<99.9%, or Staphylococcus aureus and Salmonella 90%≤R ij / R i / R<99%, the sample has a strong antibacterial effect; when R ij / R i / R≥99.9%, or Staphylococcus aureus and Salmonella R ij / R i / R≥99%, the sample has a strong antibacterial effect;

[0136] If the antibacterial activity value A is used as the relevant performance evaluation index: ij / A i / A<1.0, Staphylococcus aureus and Salmonella A ij / A i / A<0.5, the sample has no antibacterial effect; when 1.0≤A ij / A i / A<2.0, Staphylococcus aureus and Salmonella 0.5≤A ij / A i / A<1.0, the sample has antibacterial effect; when 2.0≤A ij / A i / A<3.0, Staphylococcus aureus and Salmonella 1.0≤A ij / A i / A<2.0, the sample has a strong antibacterial effect; when A ij / A i / A≥3.0, Staphylococcus aureus and Salmonella A ij / A i / A≥2.0, the sample has strong antibacterial effect;

[0137] (2) When the antibacterial rate R of a group / piece of antibacterial ceramic samples i / R ij Or antimicrobial activity value A i / A ij When the antibacterial performance of the other two groups / four samples is at least one level lower than that of the other two groups / four samples, extract one group / sample again and repeat the experiment; calculate the ATP bioluminescence lgC B ─lgI B The antibacterial rate or antibacterial activity value measured by the standard curve method, if the antibacterial performance level of the two groups of antibacterial ceramic samples is the same, then discard it; take the other two groups / four remaining samples of antibacterial rate R i / R ij Or antimicrobial activity value A i / A ij The arithmetic mean value is taken as the evaluation result of the antibacterial performance of the antibacterial ceramic samples of this batch / group; DETAILED DESCRIPTION

[0138] The present invention is described in detail below in conjunction with preferred embodiments so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0139] This embodiment is described by taking the antibacterial performance test of an antibacterial sanitary ceramic sample whose glaze layer is processed by adding a nano-silver antibacterial agent as an example.

[0140] The specific detection method is carried out according to the following steps:

[0141] (1) Sample preparation and pretreatment

[0142] 1.1 Control samples: Except for the addition of no antibacterial ingredients, the control sample category, raw materials and process are exactly the same as the antibacterial ceramic samples to be tested; the antibacterial test surface is glazed and the other side is unglazed. The thickness of bone china and fine porcelain in daily-use ceramic samples is not more than 6mm, and the water absorption rate is ≤0.5%; the thickness of ordinary porcelain is not more than 8mm, and the water absorption rate is ≤1.0%; the thickness of stoneware and fine pottery is not more than 10mm, and the water absorption rate of stoneware is ≤5.0%; the water absorption rate of fine pottery is ≤17.0%, and the thickness of sanitary ceramic samples is The sample thickness is not more than 10mm, the water absorption rate of porcelain is ≤0.5%, the water absorption rate of pottery is 8.0%≤<15.5%; the sample thickness of building ceramics is not more than 10mm, the water absorption rate of porcelain is ≤0.5%, the water absorption rate of stoneware is 0.5%~3%, the water absorption rate of stoneware is 6%~10%, the water absorption rate of pottery is ≥10%, the sample size is (50±2)mm×(50±2)mm, and 6 groups of samples are used for each bacterial species test; 3 groups are used for 0h contact and 24h culture tests, with 5 samples in each group.

[0143] 1.2 Antibacterial samples: Ceramic samples with antibacterial agent added to the glaze layer or antibacterial layer applied on the surface, and the other side is unglazed; the sample thickness, water absorption rate, specifications, dimensions, quantity, etc. are the same as those of the control samples. Each group of antibacterial samples selects a group of control samples as a reference and effectively marks them.

[0144] 1.3 Sample pretreatment: Before the experiment, soak the control samples and antibacterial samples in sterile water for 24 hours until the body and glaze layer fully absorb water; then soak them in 70% ethanol solution for 5 minutes, and wash the samples with sterile water on an ultra-clean workbench to remove ethanol; then, use sterilized dry gauze to absorb excess water on the surface of the sample, and place it in a sterile flat dish with the glaze surface facing up for later use.

[0145] (2) Equipment selection and reagent and culture medium preparation

[0146] 2.1 General requirements: Analytical reagents and Grade III water, distilled water or deionized water in accordance with GB / T 6682-2008 shall be used for the test. The laboratory shall have Grade II biosafety qualification and the personnel shall have working experience in a formal microbiology laboratory.

[0147] 2.2 Instruments and equipment: Class II biosafety cabinet or clean bench with cleanliness level not less than 100; ATP bioluminescence rapid detection system for total bacteria including ATP fluorescence photometer, supporting reagent kit, special test tubes, etc. The wavelength range of ATP fluorescence photometer is 300nm~650nm, and the detection range is 10 1 CFU / mL~10 7 CFU / mL; microplate reader with wavelength range of 400nm~760nm and reading range of 0.0Abs~4.0Abs; constant temperature and humidity incubator of 37℃±1℃, 90%±2%RH; constant temperature water bath of 46℃±1℃; pressure steam sterilizer of 121℃±2℃, 103kPa±5kPa; low temperature refrigerator of -20℃~-70℃; refrigerator of 2℃~8℃; electronic balance with sensitivity of 0.001g; ultrasonic cleaner with frequency of 30kHz~50kHz; vortex oscillator with rotation speed of 500r / min~3000r / min; pH meter with accuracy of ±0.1 at 25℃; electric furnace.

[0148] 2.3 Materials and instruments: 1mL, 10mL sterile graduated pipettes; 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL single-channel variable range pipettes and sterile pipette tips, with a measurement error of less than 1%; syringe filters with a pore size of no more than 0.45μm; sterile conical flasks with a capacity of 250mL and 500mL; sterile culture dishes with a diameter of 90mm; 96-well flat-bottom culture plates; McLaughlin standard turbidimetric tubes and matching test tubes; sterile test tubes; inoculation loops with a diameter of no more than 4mm; L-bars; sterile tweezers and sterile gauze; medical tape; 75% alcohol cotton balls; ruler with a graduation value of 1mm; Thermometer; stopwatch with an accuracy of 0.01s; A6 white copy paper; 0.7mm core black gel pen.

[0149] 2.4 Reagents: 70% ethanol solution; 85% physiological saline, sterilized at 121℃ for 15min, stored at 5℃~10℃ for 30d; 5mg / mL MTT (methyl thiazolyl tetrazolium) solution, pH 7.4, sterilized by 0.45μm filter membrane, stored at 4℃~6℃ in the dark for 15d.

[0150] 2.5 Culture medium / liquid, commercially available culture medium / liquid can be used: the prepared culture medium / liquid is sterilized at 121℃ for 15min after aliquoting, and stored at 2℃~8℃ for 30d;

[0151] Nutrient agar NA: Dissolve 5 g beef extract, 10 g peptone, 5 g sodium chloride, and 15 g agar in 1000 mL water by heating, and adjust the pH to 7.0-7.2;

[0152] Nutrient broth NB: Dissolve 3g beef extract, 10g peptone, and 5g sodium chloride in 1000mL water by heating, and adjust the pH to 7.0-7.2;

[0153] Brain heart infusion broth BHI for Salmonella preservation: Dissolve 10 g peptone, 5 g sodium chloride, 2.5 g disodium hydrogen phosphate, and 2 g glucose in 500 mL of beef heart infusion by heating, and adjust the pH to 7.4 ± 0.2;

[0154] Culture medium of Escherichia coli and Shigella / Staphylococcus aureus and Salmonella: Mix 1:500 / 1:100 nutrient broth (NB) with 85% sterile saline solution and adjust the pH to 7.0-7.2;

[0155] Plate count agar PCA: Heat and dissolve 5 g of tryptone, 2.5 g of yeast extract, 1 g of glucose, and 15 g of agar in 1000 mL of water and adjust the pH to 7.0 ± 0.2.

[0156] 2.6 ATP fluorescence reaction reagent, or commercially available reagent: except for phosphate buffer solution, the prepared ATP fluorescence reaction reagent should be stored at -20℃~-70℃ and used within 6 months;

[0157] Dilution buffer solution: 0.005 mol / L sodium dihydrogen phosphate solution containing 0.037% sucrose, adjust the pH to 7.2±0.2; sterilize at 121℃ for 15min, store at 2℃~8℃ for 30d;

[0158] ATP fluorescent reagent buffer solution: Dissolve 1117 mg of tris(hydroxymethyl)aminomethane, 183 mg of disodium ethylenediaminetetraacetate, 808 mg of magnesium acetate, 6.7 mg of dithiothreitol, 25000 mg of β-cyclodextrin and 925 mg of glucose in 250 mL of water by heating, and adjust the pH to 7.5±0.2; use within 8 hours;

[0159] ATP lysis buffer: Dissolve 4.6 international units / ml of adenosine triphosphate diphosphatase EC:3.6.1.5 and 46 international units / ml of adenosine phosphate deaminase EC:3.5.4.6 or EC:3.5.4.17, 37 mg of sucrose, and 20 mg of bovine serum albumin in 10 mL of 0.05 mol / L 2-morpholineethanesulfonic acid buffer solution, adjust the pH to 6.0±0.5, and use within 8 hours. 1 mL of lysis buffer can reduce the ATP concentration in the nutrient broth to 10 within 15 minutes. -13 mol / L or less;

[0160] ATP extract: Heat and dissolve 45 mg of tris(hydroxymethyl)aminomethane in 9.8 ml of water, mix with 0.2 ml of 10% benzalkonium chloride solution, and adjust the pH to 12.0 ± 0.5;

[0161] ATP fluorescence reagent: Dissolve 16 mg of luciferase EC: 1.13.12.7, 12.6 mg of D-luciferin, and 56 mg of bovine serum albumin in 30 mL of ATP fluorescence reagent buffer solution, mix well, and let stand at room temperature for 15 minutes. Use within 3 hours.

[0162] (3) Strain preservation, activation and bacterial suspension preparation

[0163] 3.1 Test bacteria: Escherichia coli ATCC 8739; Staphylococcus aureus ATCC 6538; Salmonella ATCC 14028; Shigella CMCC 51105, or other bacteria provided by the corresponding national culture collection management center and traceable.

[0164] 3.2 Preservation of bacterial strains: Open the freeze-dried bacterial strain tube aseptically, add an appropriate amount of nutrient broth with a capillary pipette, and use BHI culture medium for Salmonella, pipette and pump several times to melt and disperse the bacterial strains, then drop a small amount of the test bacterial strain suspension into a test tube containing 5mL~10mL nutrient broth and culture at 37℃±1℃ for 24h~48h; for use as slant preserved bacteria, store at 5℃±1℃ for no more than 1 month, and the number of inoculations shall not exceed 14 generations.

[0165] 3.3 Activation of bacterial strains: transfer the slant-preserved bacteria to nutrient agar slant culture medium and culture at 37℃±1℃ for 18h~24h; transfer once a day and the continuous transfer shall not exceed 15 days. The test shall use fresh bacterial cultures of the 3rd to 14th generations transferred within 24h.

[0166] 3.4 Preparation of bacterial suspension: scrape a small amount of fresh bacteria from the culture medium for bacterial activation with an inoculation loop, add the culture medium of the test bacteria to make a bacterial suspension; shake the test tube at 1000r / min for 1min or shake the test tube at an amplitude of 30cm for 80 times to ensure that the bacteria are evenly suspended; transfer an appropriate amount of bacterial suspension to a sterile test tube that matches the McFarland standard turbidimetric tube, and use a 0.7mm core black neutral pen to draw three parallel lines of 10cm in length on A6 white copy paper; visually compare the test tube with the nominal concentration of 6.0×10 8 The turbidity difference of the CFU / mL standard turbidimetric tube was measured, and the culture medium was added dropwise until the turbidity of the two tubes was the same.

[0167] (4) OD value-logarithm of viable bacterial content lgC B Standard curve establishment and inoculation of bacterial solution C B Calibration

[0168] 4.1 Determination of OD value of bacterial solution: The total amount of bacteria was 6.0×10 8The bacterial suspension with a dilution ratio of 1:5, 1:10, 1:15, and 1:20 was serially diluted, and the culture solution was used as the blank control sample solution to zero the microplate reader. Then, 100 μL of the bacterial solution with different dilutions was injected into a 96-well flat-bottom culture plate, and 3 replicates were made for each dilution. 20 μL of MTT solution was added to each well, and cultured at 37°C±1°C and 90%±2%RH for 30 min. The maximum absorption peak was scanned in the wavelength range of 560nm to 610nm to determine the maximum absorption wavelength. The OD value of the live bacteria of the mixed solution in each well was determined. The OD value of the live bacteria of the bacterial suspension with different dilutions was the arithmetic mean of the OD values ​​of the 3 replicates. At the same time, according to the method specified in the national standard GB 4789.2-2016, the above bacterial suspension was appropriately diluted, cultured at 37°C±1°C for 24h±2h, and the colony count of the plate was performed to calibrate its live bacteria content C BB .

[0169] 4.2OD value-logarithm of viable bacteria content lgC B Standard curve establishment and inoculation of bacterial solution C B Calibration: The OD value of live bacteria in bacterial suspensions of different dilutions is used as the ordinate, and the logarithm of the live bacteria content is used as the lgC B As the horizontal axis, plot OD value-lgC B Standard curve; the linear equation of the curve Y=a0X+b0 and the correlation coefficient R0 are derived by the least squares fitting method 2 , where Escherichia coli or Shigella and Staphylococcus aureus or Salmonella were selected with dilutions of 1:10, 1:15, 1:20 and 1:5, 1:10, 1:15 respectively as standard series solutions to draw curves. 2 ≥0.98, confidence level ≥0.95, the measured live bacterial content of bacterial suspension C B Then, the culture fluid of the test strain was adjusted to obtain C B The range is 5.0×10 6 CFU / mL~9.0×10 6 CFU / mL of inoculum.

[0170] (5) Logarithm of viable bacterial content lgC B -Relative fluorescence intensity log value lgI B Standard curve establishment

[0171] 5.1 Determination of standard series bacterial suspension and its application B Determination: Use the test bacterial culture solution to mix the known live bacterial content C B The bacterial suspension was diluted to a standard series: 4.2×10 3 CFU / mL, 4.2×10 4 CFU / mL, 4.2×105 CFU / mL and mix thoroughly, then, according to the relative fluorescence intensity value I B Determination method: according to the viable bacterial content C B The relative fluorescence intensity values ​​of the above standard series bacterial suspension and 0.3 mL of inoculated bacterial solution diluted with 4.7 mL of eluent within 1 min were measured and recorded in order from low to high, and the latter was recorded as I B0 .

[0172] 5.2 Logarithm of viable bacteria content lgC B -Relative fluorescence intensity log value lgI B Standard curve establishment: The relative fluorescence intensity logarithm of the standard series of bacterial suspensions is used as the lgI B As the horizontal axis, the corresponding logarithm of viable bacterial content lgC B Draw a graph for the ordinate; calibrate the mathematical relationship between the two curves, and use the least squares fitting method to derive the linear equation of the curve Y = a B X+b B And the correlation coefficient R B 2 ; When R B 2 When the value is ≥0.98 and the confidence level is ≥0.95, the determination is valid.

[0173] (6) Sample inoculation, culture and elution recovery

[0174] 6.1 Inoculation culture: Place a multi-layer sterile gauze of appropriate size at the bottom of a sterile dish, and pour 10 mL of sterile saline on the gauze to make it just saturated with water; place 6 groups of 24-hour contact samples on the gauze, and 6 groups of 0-hour contact samples in a dish without gauze, then use a sterile pipette to drop 0.3 mL of bacterial solution on the glaze surface of each group of control samples and antibacterial samples to be tested, and mix with 1gC B -lgI B The bacterial solution for curve calibration is taken from the same test tube of test bacteria stock solution, stored at 2℃±0.2℃, used within 2h, and evenly spread with an L-stick attached with the inoculated bacterial solution but without dripping, so that it covers the entire glaze surface of the sample, cover the dish with a lid, and seal the plate containing 6 groups of 24h contact samples with medical tape; culture at 37℃±1℃, 90%±2%RH for 24h±2h.

[0175] 6.2 Elution and recovery: 4.7 mL of the test strain culture fluid was used as the eluent, and 0.2% sterile surfactant could be added. After the 6 groups of 0-hour contact samples were inoculated with bacteria, solid or liquid swab method was immediately used for full elution. The 6 groups of 24-hour cultured samples were used to recover bacteria in the same elution method as the 0-hour contact samples. The solid swab method was to use a sterile cotton swab with a saturated eluent on the head to rub each sample surface horizontally and vertically for 10 times, and then rotate the cotton swab. Cut off the part of the cotton swab that the experimenter's hand touched, put the cotton swab in a sterile test tube containing the remaining eluent, mix it and use it as the recovery liquid. If the recovery liquid is less than 5 mL, add eluent to 5 mL. The liquid swab method was to use a sterile pipette to absorb 4.7 mL of eluent, repeatedly rinse each sample surface in the plate at least 4 times, fully elute and transfer the washing liquid into a sterile test tube, mix it and use it as the recovery liquid. If the recovery liquid is less than 5 mL, add eluent to 5 mL.

[0176] (7) Relative fluorescence intensity value I of the recovered liquid B Determination

[0177] 7.1 Calibration of the relative fluorescence intensity background value of the instrument and reagent set: Use a sterile pipette to add 0.1 mL of the test bacterial culture solution, 0.8 mL of sodium hydrogen phosphate buffer solution containing 0.037% sucrose, and 0.1 mL of ATP lysis solution into the same sterile test tube in sequence and mix thoroughly. Then transfer 0.1 mL of the mixed solution to three sterile test tubes dedicated to the instrument and let it stand for 5 min to 30 min as blank test parallel samples; add 0.1 mL of ATP extraction reagent to each of the three parallel samples, mix well, and then drop 0.1 mL of ATP fluorescence reagent; mix well again, and immediately measure the relative fluorescence intensity value I using an ATP fluorescence photometer. B And record to ensure that the operation time of each link is consistent to avoid cross contamination; the measurement time of each parallel sample shall not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values ​​of three blank test parallel samples shall be used as the background value of the instrument and reagent group, or the background value shall be calibrated according to the instrument instructions.

[0178] 7.2 Relative fluorescence intensity value I of the recovery solution B Determination: If the background level meets the requirements of the instrument, use a sterile pipette to add 0.1mL of recovery solution, 0.8mL of sodium dihydrogen phosphate buffer solution containing 0.037% sucrose, and 0.1mL of ATP lysis solution into the same sterile test tube in sequence. After fully mixing, transfer 0.1mL of the mixed solution to three sterile test tubes dedicated to the instrument, and let it stand for 5min to 30min as parallel samples for ATP fluorescence test; add 0.1mL of ATP extraction reagent to each of the three parallel samples, mix well, and then drop 0.1mL of ATP fluorescence reagent; mix again, and immediately use an ATP fluorescence photometer to measure the relative fluorescence intensity value I BThe measurement time of each parallel sample should not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values ​​of the three ATP fluorescence test parallel samples should be used as the I value of the recovered solution of the sample to be tested. B The relative fluorescence intensity values ​​of three parallel samples of the recovery solution can be directly measured by using the instrument matching kit according to the relevant instructions. B .

[0179] (8) Live bacteria content of recovered liquid C B and T B Estimation and calculation of antibacterial rate R and antibacterial activity value A

[0180] 8.1 Live bacteria content of recovered liquid C B and T B Calculation

[0181] According to the standard curve of the test strain lgC B -lgI B The linear equation Y = a B X+b B Calculate the viable bacterial content C of the recovery solution of the control sample and the antibacterial sample after 0h contact and 24h culture B and T B When using the instrument kit, calculate the viable bacterial content C of 1mL of recovery solution based on the actual injection volume. B and T B , the relevant calculations are shown in formulas (1) to (12):

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] Where:

[0195] C B0 and C Bt —The average number of viable bacteria recovered from the control samples of the three groups after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0196] C B0i and C Bti —The average number of viable bacteria recovered from the control samples after 0-hour contact and 24-hour incubation in each group, in colony-forming units per milliliter (CFU / mL); sample groups i = 1, 2, 3;

[0197] C B0ij and C Btij —The number of viable bacteria recovered from each control sample after 0-hour contact and 24-hour incubation, in colony-forming units per milliliter (CFU / mL); each group of samples is numbered j=1, 2, 3, 4, 5;

[0198] and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU;

[0199] a B —Standard curve lgC B -lgI B Slope; b B —Standard curve lgC B -lgI B intercept on the ordinate;

[0200] T B0 and T Bt —The average number of viable bacteria recovered from the three groups of antimicrobial samples after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0201] T B0i and T Bti —The average number of viable bacteria recovered from the antimicrobial samples after 0-hour contact and 24-hour incubation in each group, expressed in colony-forming units per milliliter (CFU / mL);

[0202] T B0ij and T Btij —The number of viable bacteria recovered from each antimicrobial sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0203] and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU.

[0204] 8.2 Test validity conditions

[0205] If the relative fluorescence intensity of each 0h contact control sample recovery solution and 0.3mL inoculated bacterial solution diluted with 4.7mL eluent within 1min is close, that is The logarithm of the relative fluorescence intensity of the recovered solution of each control sample after 24h of incubation C Btij ≥0.1×C B0ij ; When the relative fluorescence intensity of the recovered solution of the three groups of control samples was measured at 0h When the intra-group and inter-group coefficient of variation C·V≤10%, the determination is valid. For the calculation formula involved, see the relevant measurement uncertainty requirements.

[0206] 8.3 Bacterial growth value F ij , G ij calculate

[0207] After 24 hours of culture, the bacterial growth value F of each control sample and antibacterial sample was ij , G ij Calculate according to formula (13) and (14) respectively:

[0208]

[0209]

[0210] Where:

[0211] F ij and G ij — Bacterial growth value of each control sample and antibacterial sample after 24 hours of culture;

[0212] C B0ij and C Btij —The number of viable bacteria recovered from each control sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0213] and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU;

[0214] T B0ij and T Btij —The number of viable bacteria recovered from each antimicrobial sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter (CFU / mL);

[0215] and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU.

[0216] 8.4 Calculation of antibacterial rate R

[0217] Under the effective test conditions, the antibacterial rate R of each antibacterial ceramic sample ij , the antibacterial rate R of each group and batch of samples i and R are calculated according to formulas (15), (16), and (17) respectively:

[0218]

[0219]

[0220]

[0221] Where:

[0222] R ij —Antibacterial rate of each antibacterial ceramic sample, %;

[0223] R i —Antibacterial rate of each group of antibacterial ceramic samples, %;

[0224] R—antibacterial rate of each batch of antibacterial ceramic samples, %;

[0225] T Btij and C Btij —The number of viable bacteria recovered from each antimicrobial sample and control sample after 24 h of culture, in colony forming units per milliliter (CFU / mL);

[0226] and —Relative fluorescence intensity of the recovered solution of each antibacterial sample and control sample after 24 hours of incubation, RLU.

[0227] 8.5 Calculation of antimicrobial activity value A

[0228] Under the effective test conditions, the antibacterial activity value A of each antibacterial ceramic sample ij , antimicrobial activity value A of each group and batch of samples i and A are calculated according to formulas (18), (19), and (20) respectively:

[0229]

[0230]

[0231]

[0232] Where:

[0233] A ij —The antibacterial activity value of each antibacterial ceramic sample;

[0234] A i —The antibacterial activity value of each group of antibacterial ceramic samples;

[0235] A—antibacterial activity value of each batch of antibacterial ceramic samples;

[0236] F ij and G ij — Bacterial growth value of each control sample and antibacterial sample after 24 hours of culture;

[0237] and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU;

[0238] and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU.

[0239] 8.6 Data revision requirements: Use plate count method to calibrate the viable bacterial content of bacterial suspension C B When C B When the concentration is less than 100 CFU / mL, the integer is rounded off. B When it is not less than 100 CFU / mL, the first two digits of the third digit shall be rounded off, and the digits behind shall be replaced by 0; it can also be expressed in the form of 10 exponentials, and two significant digits shall be used after rounding off; the relative fluorescence intensity of the recovery liquid of the control sample and the antibacterial sample after 0h contact and 24h culture shall be integer, and the antibacterial rate R ij , R i , R calculation results are retained to three significant figures; bacterial growth value F ij , G ij and antimicrobial activity value A ij , A i 、A The calculation result shall be rounded to two significant figures.

[0240] 8.7 Measurement uncertainty: Mainly by calculating the intra-group and inter-group coefficient of variation C·V=σ÷μ×100% of the relative fluorescence intensity of the control sample and the antibacterial sample after 0h contact and 24h incubation, the calculation results of μ, σ and C·V are retained to two decimal places, to determine the reproducibility of the ATP bioluminescence analysis method applied to the antibacterial performance test of ceramics; it is stipulated that the intra-group and inter-group coefficient of variation C·V shall not be greater than 10%;

[0241] The arithmetic mean of the relative fluorescence intensity of the recovered fluid of each group of 5 control samples, 5 antibacterial samples, and 5 control samples and 5 antibacterial samples after 24h of incubation was calculated. and Calculate according to formula (21) and (22) respectively; ATP test parallel sample number k = 1, 2, 3; and Relative fluorescence intensity of each ATP test parallel sample after 0h contact and 24h incubation for each group of control samples and antibacterial samples:

[0242]

[0243]

[0244] The arithmetic mean of the relative fluorescence intensity of the recovered fluids of 15 control samples, 15 antibacterial samples, and 15 control samples and 15 antibacterial samples after 24h of incubation in 3 groups. and Calculate according to formula (23) and (24) respectively:

[0245]

[0246]

[0247] The standard deviation of the relative fluorescence intensity of the recovered solution of each group of 5 control samples, 5 antibacterial samples at 0h contact and 5 control samples and 5 antibacterial samples after 24h incubation and Calculate according to formula (25) and (26) respectively:

[0248]

[0249]

[0250] Standard deviation of relative fluorescence intensity of recovered fluids of 15 control samples, 15 antibacterial samples after 0h contact and 15 control samples, 15 antibacterial samples after 24h incubation in 3 groups and Calculate according to formula (27) and (28) respectively:

[0251]

[0252]

[0253] (9) Result evaluation

[0254] 9.1 With reference to the general practices of the health industry and the requirements of relevant antimicrobial product standards, the following grading criteria are adopted:

[0255] If the antibacterial rate R is used as the relevant performance evaluation index: ij / R i / R<90%, or Staphylococcus aureus and Salmonella R ij / R i / R<80%, the sample has no antibacterial effect; when 90%≤R ij / R i / R<99%, or Staphylococcus aureus and Salmonella 80%≤R ij / R i / R<90%, the sample has antibacterial effect; when 99%≤R ij / R i / R<99.9%, or Staphylococcus aureus and Salmonella 90%≤R ij / R i / R<99%, the sample has a strong antibacterial effect; when R ij / R i / R≥99.9%, or Staphylococcus aureus and Salmonella R ij / R i / R≥99%, the sample has a strong antibacterial effect;

[0256] If the antibacterial activity value A is used as the relevant performance evaluation index: ij / A i / A<1.0, Staphylococcus aureus and Salmonella A ij / A i / A<0.5, the sample has no antibacterial effect; when 1.0≤A ij / A i / A<2.0, Staphylococcus aureus and Salmonella 0.5≤A ij / A i / A<1.0, the sample has antibacterial effect; when 2.0≤A ij / A i / A<3.0, Staphylococcus aureus and Salmonella 1.0≤A ij / A i / A<2.0, the sample has a strong antibacterial effect; when A ij / A i / A≥3.0, Staphylococcus aureus and Salmonella A ij / A i / A≥2.0, the sample has a strong antibacterial effect.

[0257] 9.2 When the antibacterial rate R of a group / piece of antibacterial ceramic samples i / Rij Or antimicrobial activity value A i / A ij When the antibacterial performance of the other two groups / four samples is at least one level lower than that of the other two groups / four samples, extract one group / sample again and repeat the experiment; calculate the ATP bioluminescence lgC B ─lgI B The antibacterial rate or antibacterial activity value measured by the standard curve method, if the antibacterial performance level of the two groups of antibacterial ceramic samples is the same, then discard it; take the other two groups / four remaining samples of antibacterial rate R i / R ij Or antimicrobial activity value A i / A ij The arithmetic mean value is taken as the evaluation result of the antibacterial performance of the antibacterial ceramic samples in this batch / group.

[0258] Laboratory biosafety qualifications, instruments and equipment, culture media / fluids, chemical reagents, and standard strains used in this example:

[0259] (1) Laboratory biosafety qualification

[0260] The relevant experiments are completed by microbial testing professionals with associate senior technical titles in a Class II biosafety laboratory with institutional registration number CNAS BL0059.

[0261] (2) Instruments and Equipment

[0262] 2.1 Class II biological safety cabinet: Thermo Scientific 1300 series Class II B2 biological safety cabinet, workbench surface area 0.55m 2 , exhaust volume 1130m 3 / h, filtration efficiency 99.99%, 0.3μm.

[0263] 2.2 Clean bench: American brand Thermo Scientific TM Heraguard TM , Model ECO clean bench, internal width × depth × height = 920mm × 585mm × 645mm; air flow rate is 0.15m / s~0.25m / s and 0.36m / s~0.45m / s.

[0264] 2.3 Rapid detection system for total bacterial count ATP bioluminescence: portable system SUREATP fluorescence detector from Hygiena, USA, including matching test kits, special plastic test tubes, etc.; the detection limit of ATP content is 4×10 -18 mol / ml, total microbial detection limit 1.0 CFU / ml, RLU reading range 0-9999, detection time 10s, sampling rate 1000 times / s, measurement error ±5%.

[0265] 2.4 Microplate reader: American brand Thermo Scientific, model Multiskan FC, fully automatic microplate reader, wavelength range 340nm±1nm~850nm±1nm, reading range 0.0Abs±0.001Abs~6.0Abs±0.001Abs; the measurement accuracy corresponding to 405nm is ±1% for 0.0Abs~3.0Abs or ±2% for 3.0Abs~4.0Abs.

[0266] 2.5 Constant temperature and humidity incubator: Guansen Biotechnology Co., Ltd. model WS-380H, volume 380L, temperature control range 0℃±0.8℃~50℃±0.8℃, humidity control range 30%±2%~95%±2%RH.

[0267] 2.6 Constant temperature water bath: Shanghai Jiwei Testing Instrument Equipment Co., Ltd. model DK-S420 electric constant temperature water bath, volume 15L, temperature control range 5℃±0.5℃~99.9℃±0.5℃, timing range 1min~999min.

[0268] 2.7 Pressure steam sterilizer: Japan Sanyo company's high pressure sterilizer model MLS-3780, volume 75L, sterilization temperature 105℃±2℃~135℃±2℃, maximum pressure 0.235MPa, timing range 1min~250min.

[0269] 2.8 Low-temperature refrigerator: Ultra-low-temperature freezer storage box model DW-HL398 of Zhongke Meiling Low-temperature Technology Co., Ltd., with an effective volume of 398L and a storage temperature of -10℃ to -86℃.

[0270] 2.9 Refrigerator: Medical refrigerator model YC-968L manufactured by Zhongke Meiling Cryogenic Technology Co., Ltd., with an effective volume of 968L and a storage temperature of 2℃±0.1℃~8℃±0.1℃.

[0271] 2.10 Electronic balance: Japanese Shimadzu model AUX220 electronic balance, range 220g, accuracy ±0.1mg.

[0272] 2.11 Ultrasonic cleaner: Model YQ-120C ultrasonic cleaner produced by Shanghai Yijing Ultrasonic Instrument Co., Ltd., with a capacity of 3.2L, ultrasonic frequency of 40KHz / 28KHz / 25KHz, and a timing range of 1min to 30min / 99min.

[0273] 2.12 Vortex oscillator: American brand Coleparmer Votex-Genie2 vortex oscillator, speed range 500r / min±3r / min~3000r / min±3r / min, timing range 1s~60s.

[0274] 2.13 pH meter: Shanghai Precision Instruments Co., Ltd. model MP512-03 precision pH meter, range -2.000pH±0.002pH~19.999pH±0.002pH, temperature range -10℃±0.4℃~110℃±0.4℃.

[0275] 2.14 Electric furnace: 1KW closed temperature-controlled electric furnace model DLD-1KW produced by Changzhou Deke Instrument Co., Ltd.

[0276] (3) Culture medium / fluid

[0277] Culture medium / fluid produced by Beijing Luqiao Technology Co., Ltd.

[0278] (4) Chemical reagents

[0279] Tetramethyl azobium salt, adenosine triphosphate disodium standard and a series of biochemical reagents required for preparing ATP fluorescent reagent buffer solution, ATP lysate, ATP extract and ATP fluorescent reagent were purchased from the American Amresco brand represented by Shanghai Jinpan Biotechnology Co., Ltd.

[0280] (5) Standard strains

[0281] Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 were purchased from China Industrial Microbiological Culture Collection Center.

[0282] The test data and result calculation of this embodiment:

[0283] ATP fluorescence spectrophotometer was used to measure the B -lgI B The antibacterial performance of antibacterial sanitary ceramic samples was tested by the standard curve method. The relevant test data and the calculation results of measurement uncertainty are shown in Tables 1 to 4. The test results of Shigella and Salmonella are close to those of Escherichia coli and Staphylococcus aureus, respectively.

[0284] Table 1 Relative fluorescence intensity measurement values ​​and calculation results of antibacterial rate R and antibacterial activity value A, Escherichia coli

[0285]

[0286]

[0287] Table 2 Calculation results of measurement uncertainty of relative fluorescence intensity value (Escherichia coli)

[0288]

[0289] Table 3 Relative fluorescence intensity measurement values ​​and calculation results of antibacterial rate R and antibacterial activity value A (Staphylococcus aureus)

[0290]

[0291] Table 4 Calculation results of relative fluorescence intensity measurement uncertainty (Staphylococcus aureus)

[0292]

[0293]

[0294] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for detecting the antibacterial performance of antibacterial ceramics, comprising: ①Sample preparation and pretreatment; ② Equipment selection and reagent and culture medium preparation; ③ Bacterial strain preservation, activation and bacterial suspension preparation; ④ OD value-logarithm of viable bacterial content lgC B Standard curve establishment and inoculation of bacterial solution C B Calibration; ⑤ Logarithm of viable bacteria content lgC B -Relative fluorescence intensity log value lgI B Standard curve establishment; ⑥ Sample inoculation, culture and elution recovery; ⑦ Relative fluorescence intensity value I of the recovery solution B Determination; ⑧ Recovery liquid live bacteria content C B and T B Calculation and calculation of antibacterial rate R and antibacterial activity value A; ⑨ Result evaluation; It is characterized in that the ATP bioluminescence lgC of the antibacterial ceramics characterized by the antibacterial rate R or the antibacterial activity value A is accurately and quantitatively tested by using an ATP fluorescence photometer. B -lgI B Standard curve method, specifically: The relative fluorescence intensity value I B Measuring: The number, size and water absorption requirements of the control samples and antibacterial samples were clarified, and the samples were sterilized and water-absorbed. After the test standard strain was subcultured and activated, fresh bacterial cultures that were transferred twice were used to prepare bacterial suspensions, and the live bacteria were counted using the MTT colorimetric method. The OD value-live bacteria content logarithm lgC was established. B Standard curve, derive the linear equation of the curve Y = a0X + b0 and the correlation coefficient R0 2 ; and the inoculated bacterial solution was tested for viable bacterial content C B Calibration: 5.0×10 6 CFU / mL~9.0×10 6 CFU / mL, at the same time, select C B 4.2×10 3 CFU / mL, 4.2×10 4 CFU / mL, 4.2×10 5 CFU / mL dilution was used as the standard series bacterial suspension; its relative fluorescence intensity value I was determined B , draw lgC B -lgI B Standard curve, and derive the linear equation of the curve Y = a B X+b B And the correlation coefficient R B 2 Then, 0.3 mL of bacterial solution was added to the glaze of each group of control samples and antibacterial samples; 4.7 mL of eluent was immediately used to elute and recover the 6 groups of 0-hour contact samples, and the relative fluorescence intensity value I of the recovered solution was measured by ATP fluorescence photometer. BC0ij ,I BT0ij According to the curve equation Y = a B X+b B Calculate the viable bacterial content C B0ij and T B0ij At the same time, the six groups of 24h contact samples were sealed in sterile plates and cultured at 37℃±1℃ and 90%±2%RH for 24h±2h; the residual bacteria on the glaze surface were recovered in the same way as the 0h contact samples and the relative fluorescence intensity value I of the recovered solution was determined. BCtij ,I BTtij , calculate the corresponding viable bacterial content C Btij and T Btij ; In the calculation of antibacterial rate R and antibacterial activity value A: According to the standard curve of the test strain lgC B -lgI B The linear equation Y = a B X+b B The relative fluorescence intensity of each control sample and antibacterial sample recovery solution after 0h contact and 24h incubation was measured. and As basic data; under the effective conditions of the test, calculate the bacterial growth value F after 24h of culture ij , G ij And the antibacterial rate R ij and antimicrobial activity value A ij ; R for each group of samples ij and A ij Take the arithmetic mean to get the corresponding R i and A i ; The antibacterial rate R and antibacterial activity value A of each batch of antibacterial ceramic samples are the three groups of samples R i and A i The arithmetic mean of the relevant data and the measurement uncertainty requirements shall be clearly stated; Bacterial growth value F ij , G ij calculate: After 24 hours of culture, the bacterial growth value F of each control sample and antibacterial sample was ij , G ij Calculate according to formula (1) and (2) respectively: Where: F ij and G ij — Bacterial growth value of each control sample and antibacterial sample after 24 hours of culture, sample group i = 1, 2, 3; sample number of each group j = 1, 2, 3, 4, 5; C B0ij and C Btij —The number of viable bacteria recovered from each control sample after 0-hour contact and 24-hour incubation, in colony-forming units per milliliter, CFU / mL; and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU; T B0ij and T Btij —The number of viable bacteria recovered from each antimicrobial sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter, CFU / mL; and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU; a B —Standard curve lgC B -lgI B Slope; Calculation of antibacterial rate R: Under the effective test conditions, the antibacterial rate R of each antibacterial ceramic sample ij , the antibacterial rate R of each group and batch of samples i and R are calculated according to formulas (3), (4, and (5) respectively: Where: R ij —Antibacterial rate of each antibacterial ceramic sample, %; R i —Antibacterial rate of each group of antibacterial ceramic samples, %; R—antibacterial rate of each batch of antibacterial ceramic samples, %; T Btij and C Btij —The number of viable bacteria recovered from each antimicrobial sample and control sample after 24 h of culture, in colony forming units per milliliter (CFU / mL); and —Relative fluorescence intensity of the recovered solution of each antibacterial sample and control sample after 24h of incubation, RLU; Calculation of antibacterial activity value A: Under the effective test conditions, the antibacterial activity value A of each antibacterial ceramic sample ij , antimicrobial activity value A of each group and batch of samples i and A are calculated according to formulas (6), (7), and (8) respectively: Where: A ij —The antibacterial activity value of each antibacterial ceramic sample; A i —The antibacterial activity value of each group of antibacterial ceramic samples; A—antibacterial activity value of each batch of antibacterial ceramic samples; In the results evaluation: Refer to the general practices of the health industry and the relevant antibacterial product standards to determine the antibacterial performance classification criteria; when the antibacterial rate R of a group / piece of antibacterial ceramic samples i / R ij Or antimicrobial activity value A i / A ij When the antibacterial performance of the other two groups / four samples is at least one level lower than that of the other two groups / four samples, extract one group / sample again and repeat the experiment; calculate the ATP bioluminescence lgC B ─lgI B If the antibacterial performance level of the two groups / pieces of antibacterial ceramic samples is the same, then discard them; take the antibacterial rate R of the remaining two groups / four pieces of samples. i / R ij Or antimicrobial activity value A i / A ij The arithmetic mean value is taken as the evaluation result of the antibacterial performance of the antibacterial ceramic samples of this batch / group.

2. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The sample preparation and pretreatment are carried out according to the following steps: ① Control samples: except for the addition of no antibacterial ingredients, the control samples are exactly the same in type, raw materials and process as the antibacterial ceramic samples to be tested; the antibacterial test surface is glazed and the other side is unglazed. The thickness of bone china and fine porcelain in daily-use ceramic samples is not more than 6mm, and the water absorption rate is ≤0.5%; the thickness of ordinary porcelain is not more than 8mm, and the water absorption rate is ≤1.0%; the thickness of stoneware and fine pottery is not more than 10mm, and the water absorption rate of stoneware is ≤5.0%; the water absorption rate of fine pottery is ≤17.0%; the thickness of sanitary ceramic samples is 6mm~10mm, porcelain water absorption ≤0.5%, 8.0%≤ceramic water absorption <15.5%; building ceramic sample thickness is not more than 10mm, porcelain water absorption ≤0.5%, stoneware water absorption 0.5%~3%, stoneware water absorption 6%~10%, ceramic water absorption ≥10%, sample size is (50±2)mm×(50±2)mm, 6 groups of samples are used for each strain test; 3 groups are used for 0h contact and 24h culture tests, each with 5 samples; ② Antibacterial samples: ceramic samples with antibacterial agent added to the glaze layer or antibacterial layer applied on the surface, and no glaze on the other side; the sample thickness, water absorption rate, specifications, dimensions, quantity, etc. are the same as those of the control samples. Each group of antibacterial samples selects a group of control samples as a reference and effectively marks them; ③ Sample pretreatment: Before the experiment, soak the control samples and antibacterial samples in sterile water for 24 hours until the body and glaze layer fully absorb water; then soak them in 70% ethanol solution for 5 minutes, and wash the samples with sterile water on the clean bench to remove ethanol, then use sterilized dry gauze to absorb excess water on the surface of the samples, and place them with the glaze surface facing up in a sterile flat dish for later use.

3. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The bacterial strain preservation, activation and bacterial suspension preparation are carried out according to the following steps: ① Test bacteria: Escherichia coli ATCC 8739; Staphylococcus aureus ATCC 6538; Salmonella ATCC14028; Shigella CMCC 51105, or other bacteria provided by the corresponding national culture collection management center and traceable; ② Preservation of bacterial strains: Open the freeze-dried bacterial strain tube with aseptic operation, add an appropriate amount of nutrient broth with a capillary pipette, and use BHI culture medium for Salmonella, blow and aspirate several times to melt and disperse the bacterial strains, then drop a small amount of the test bacterial strain suspension into a test tube containing 5mL~10mL nutrient broth, and culture at 37℃±1℃ for 24h~48h; used as slant preservation bacteria, store at 5℃±1℃, the storage time shall not exceed 1 month, and the number of inoculations shall not exceed 14 generations; ③ Activation of bacterial strains: transfer the slant-preserved bacteria to nutrient agar slant medium and culture at 37℃±1℃ for 18h~24h; transfer once a day, and the continuous transfer shall not exceed 15d. The test shall use fresh bacterial cultures of the 3rd to 14th generations transferred within 24h; ④ Preparation of bacterial suspension: scrape a small amount of fresh bacteria from the strain activation culture medium with an inoculation loop, add the test strain culture solution to make a bacterial suspension; shake the test tube at 1000r / min for 1min or shake the test tube at an amplitude of 30cm for 80 times to ensure that the bacteria are evenly suspended, transfer an appropriate amount of bacterial suspension to a sterile test tube that matches the McFarland standard turbidimetric tube, and use a 0.7mm core black neutral pen to draw three parallel lines of 10cm in length on A6 white copy paper; visually observe the turbidity difference between the test tube and the standard turbidimetric tube, and add the culture solution until the turbidity of the two is the same; the nominal concentration of the standard turbidimetric tube is 6.0×10 8 CFU / mL.

4. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The OD value-logarithm of viable bacteria content lgC B Standard curve establishment and inoculation of bacterial solution C B To calibrate, follow these steps: ① Determination of bacterial solution OD value: Use the test bacterial culture solution to measure the total amount of bacteria (6.0×10 8 The bacterial suspension with a dilution ratio of 1:5, 1:10, 1:15, and 1:20 was serially diluted, and the culture solution was used as the blank control sample solution to zero the microplate reader. Then, 100 μL of the bacterial solution with different dilutions was injected into a 96-well flat-bottom culture plate, and 3 replicates were made for each dilution. 20 μL of MTT solution was added to each well, and cultured at 37°C±1°C and 90%±2%RH for 30 min. The maximum absorption peak was scanned in the wavelength range of 560nm to 610nm to determine the maximum absorption wavelength. The OD value of the live bacteria of the mixed solution in each well was determined. The OD value of the live bacteria of the bacterial suspension with different dilutions was the arithmetic mean of the OD values ​​of the 3 replicates. At the same time, according to the method specified in the national standard GB 4789.2-2016, the above bacterial suspension was appropriately diluted, cultured at 37°C±1°C for 24h±2h, and the colony count of the plate was performed to calibrate its live bacteria content C B ; ②OD value-logarithm of viable bacteria content lgC B Standard curve establishment and inoculation of bacterial solution C B Calibration: The OD value of live bacteria in bacterial suspensions of different dilutions is used as the ordinate, and the logarithm of the live bacteria content is used as the lgC B As the horizontal axis, plot OD value-lgC B Standard curve; the linear equation of the curve Y=a0X+b0 and the correlation coefficient R0 are derived by the least squares fitting method 2 , where Escherichia coli / or Shigella and Staphylococcus aureus / or Salmonella were selected with dilutions of 1:10, 1:15, 1:20 and 1:5, 1:10, 1:15 as standard series solutions to draw curves. 2 ≥0.98, confidence level ≥0.95, the measured live bacterial content of bacterial suspension C B Then, the culture fluid of the test strain was adjusted to obtain C B The range is 5.0×10 6 CFU / mL~9.0×10 6 CFU / mL of inoculum.

5. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The logarithmic value of the viable bacteria content lgC B -Relative fluorescence intensity log value lgI B To establish a standard curve, follow the steps below: ① Determination of standard series bacterial suspension and its I B Determination: Use the test bacterial culture solution to mix the known live bacterial content C B The bacterial suspension was diluted to a standard series: 4.2×10 3 CFU / mL, 4.2×10 4 CFU / mL, 4.2×10 5 CFU / mL and mix thoroughly, then, according to the relative fluorescence intensity value I B Determination method: according to the viable bacterial content C B The relative fluorescence intensity values ​​of the above standard series of bacterial suspensions and 0.3 mL of inoculated bacterial solution diluted with 4.7 mL of eluent within 1 min were measured and recorded in order from low to high; the relative fluorescence intensity value of 0.3 mL of inoculated bacterial solution diluted with 4.7 mL of eluent within 1 min was recorded as I B0 ; ②Logarithm of viable bacteria content lgC B -Relative fluorescence intensity log value lgI B Standard curve establishment: The relative fluorescence intensity logarithm of the standard series of bacterial suspensions is used as the lgI B As the horizontal axis, the corresponding logarithm of viable bacterial content lgC B Draw a graph for the ordinate; calibrate the mathematical relationship between the two curves, and use the least squares fitting method to derive the linear equation of the curve Y = a B X+b B And the correlation coefficient R B 2 ; When R B 2 When the value is ≥0.98 and the confidence level is ≥0.95, the determination is valid.

6. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The sample inoculation, cultivation and elution recovery are carried out according to the following steps: ① Inoculation culture: Place a multi-layer sterile gauze of appropriate size at the bottom of a sterile plate, and pour 10 mL of sterile physiological saline on the gauze to make it just saturated with water; place 6 groups of 24-hour contact samples on the gauze, and 6 groups of 0-hour contact samples in a plate without gauze, then use a sterile pipette to drop 0.3 mL of bacterial solution on the glaze surface of each group of control samples and antibacterial samples to be tested, respectively, and use an L-stick that is attached with the inoculated bacterial solution but not dripping to evenly spread the bacterial solution so that it covers the entire glaze surface of the sample, cover the plate lid, and seal the plate containing the 6 groups of 24-hour contact samples with medical tape; culture at 37°C±1°C and 90%±2%RH for 24h±2h; the bacterial solution and lgC B -lgI B The bacterial solution used for curve calibration is taken from the same test bacterial stock solution test tube, stored at 2℃±0.2℃, and used within 2h; ② Elution and recovery: 4.7 mL of the test strain culture fluid was used as the eluent. After the 6 groups of 0-hour contact samples were inoculated with bacteria, they were immediately fully eluted using the solid or liquid swab method. 0.2% sterile surfactant could be added to the eluent. The 6 groups of 24-hour cultured samples were eluted using the same elution method as the 0-hour contact samples. The solid swab method was to use a sterile cotton swab with a saturated eluent on the head to evenly rub each sample surface horizontally and vertically for 10 times, and then rotate the cotton swab. Cut off the part of the cotton swab that the experimenter's hand touched, place the cotton swab in a sterile test tube containing the remaining eluent, mix well and use it as the recovery liquid. If the recovery liquid is less than 5 mL, add eluent to 5 mL. The liquid swab method was to use a sterile pipette to absorb 4.7 mL of eluent, repeatedly rinse each sample surface in the plate at least 4 times, fully elute and transfer the washing liquid into a sterile test tube, mix well and use it as the recovery liquid. If the recovery liquid is less than 5 mL, add eluent to 5 mL.

7. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The relative fluorescence intensity value I of the recovery solution B The determination is carried out according to the following steps: ① Calibration of the relative fluorescence intensity background value of the instrument and reagent group: Use a sterile pipette to add 0.1 mL of the test bacterial culture solution, 0.8 mL of sodium hydrogen phosphate buffer solution containing 0.037% sucrose, and 0.1 mL of ATP lysis solution into the same sterile test tube in sequence and mix thoroughly. Then transfer 0.1 mL of the mixed solution to three sterile test tubes dedicated to the instrument, let it stand for 5 min to 30 min, and use it as a blank test parallel sample. Add 0.1 mL of ATP extraction reagent to each of the three parallel samples, mix well, and then drop 0.1 mL of ATP fluorescence reagent; mix well again, and immediately use an ATP fluorescence photometer to measure the relative fluorescence intensity value I B And record; ensure that the operation time of each link is consistent to avoid cross contamination; the measurement time of each parallel sample does not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values ​​of three blank test parallel samples is used as the background value of the instrument and reagent group, or the background value is calibrated according to the instrument instructions; ② Relative fluorescence intensity value of the recovery liquid I B Determination: If the background level meets the requirements of the instrument, use a sterile pipette to add 0.1mL of recovery solution, 0.8mL of sodium dihydrogen phosphate buffer solution containing 0.037% sucrose, and 0.1mL of ATP lysis solution into the same sterile test tube in sequence. After fully mixing, transfer 0.1mL of the mixed solution to three sterile test tubes dedicated to the instrument, let it stand for 5min to 30min, and use it as a parallel sample for ATP fluorescence test. Add 0.1mL of ATP extraction reagent to each of the three parallel samples, mix well, and then drop 0.1mL of ATP fluorescence reagent; mix again, and immediately use an ATP fluorescence photometer to measure the relative fluorescence intensity value I B And record; ensure that the operation time of each link is consistent to avoid cross contamination; The measurement time of each parallel sample shall not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values ​​of the three ATP fluorescence test parallel samples shall be taken as the I of the recovered solution of the sample to be tested. B The relative fluorescence intensity values ​​of three parallel samples of the recovery solution can be directly measured by using the instrument matching kit according to the relevant instructions. B .

8. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The viable bacteria content of the recovered liquid is C B and T B The calculation of antibacterial rate R and antibacterial activity value A is carried out according to the following steps: ①Content of live bacteria in the recovered liquid C B and T B Calculation According to the standard curve of the test strain lgC B -lgI B The linear equation Y = a B X+b B Calculate the viable bacterial content C of the recovery solution of the control sample and the antibacterial sample after 0h contact and 24h culture B and T B , the relevant calculations are shown in formulas (9) to (20). When using the instrument-matched kit, the viable bacterial content C of 1 mL of the recovered solution is estimated based on the actual injection volume. B and T B : Where: C B0 and C Bt —The average number of viable bacteria recovered from the control samples of the three groups after 0-hour contact and 24-hour incubation, in colony-forming units per milliliter, CFU / mL; C B0i and C Bti —The average number of viable bacteria recovered from the control samples after 0-hour contact and 24-hour incubation in each group, in colony-forming units per milliliter, CFU / mL; sample groups i = 1, 2, 3; C B0ij and C Btij —The number of viable bacteria recovered from each control sample after 0-hour contact and 24-hour incubation, in colony-forming units per milliliter, CFU / mL; each group of samples is numbered j=1, 2, 3, 4, 5; and —Relative fluorescence intensity of each control sample recovery solution after 0h contact and 24h incubation, RLU; b B —Standard curve lgC B -lgI B intercept on the ordinate; T B0 and T Bt —The average number of viable bacteria recovered from the antimicrobial samples of the three groups after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter, CFU / mL; T B0i and T Bti —The average number of viable bacteria recovered from the antimicrobial samples after 0-hour contact and 24-hour incubation in each group, expressed in colony-forming units per milliliter, CFU / mL; T B0ij and T Btij —The number of viable bacteria recovered from each antimicrobial sample after 0-hour contact and 24-hour incubation, expressed in colony-forming units per milliliter, CFU / mL; and —Relative fluorescence intensity of each antibacterial sample recovery solution after 0h contact and 24h incubation, RLU; ② Test validity conditions If the relative fluorescence intensity of each 0h contact control sample recovery solution and 0.3mL inoculated bacterial solution diluted with 4.7mL eluent within 1min is close, that is The logarithm of the relative fluorescence intensity of the recovered solution of each control sample after 24h of incubation C Btij ≥0.1×C B0ij , then when the relative fluorescence intensity of the recovery solution of the three groups of control samples at 0h was measured When the intra-group and inter-group variation coefficient C·V≤10%, the calculation formula involved is shown in the measurement uncertainty requirements, and the determination is valid; ③ Data revision requirements: Use plate count method to calibrate the live bacterial content of bacterial suspension C B When C B When the concentration is less than 100 CFU / mL, the integer is rounded off. B When the concentration is not less than 100 CFU / mL, the first two digits are taken after the third digit is "rounded off", and the digits are replaced by 0; it can also be expressed in the form of 10 exponentials, and two significant digits are used after "rounding off". The relative fluorescence intensity of the recovery liquid of the control sample and the antibacterial sample after 0h contact and 24h culture is taken as an integer, and the antibacterial rate R ij , R i , R calculation results are retained to three significant figures; bacterial growth value F ij , G ij and antimicrobial activity value A ij , A i 、A The calculation result shall be rounded to two significant figures; ④ Measurement uncertainty: The intra-group and inter-group coefficient of variation C·V of the relative fluorescence intensity of the recovery liquid of the control sample and the antibacterial sample after 0h contact and 24h incubation is calculated as C·V=σ÷μ×100%, and the calculation results of μ, σ and C·V are retained to two decimal places to determine the reproducibility of the ATP bioluminescence analysis method applied to the antibacterial performance test of ceramics; it is stipulated that the intra-group and inter-group coefficient of variation C·V shall not be greater than 10%; The arithmetic mean of the relative fluorescence intensity of the recovered fluid of each group of 5 control samples, 5 antibacterial samples, and 5 control samples and 5 antibacterial samples after 24h of incubation was calculated. and Calculate according to formula (21) and (22) respectively; ATP test parallel sample number k = 1, 2, 3; and Relative fluorescence intensity of each ATP test parallel sample after 0h contact and 24h incubation for each group of control samples and antibacterial samples: The arithmetic mean of the relative fluorescence intensity of the recovered fluids of 15 control samples, 15 antibacterial samples, and 15 control samples and 15 antibacterial samples after 24h of incubation in 3 groups. and Calculate according to formula (23) and (24) respectively: The standard deviation of the relative fluorescence intensity of the recovered solution of each group of 5 control samples, 5 antibacterial samples at 0h contact and 5 control samples and 5 antibacterial samples after 24h incubation and Calculate according to formula (25) and (26) respectively: Standard deviation of relative fluorescence intensity of recovered fluids of 15 control samples, 15 antibacterial samples after 0h contact and 15 control samples, 15 antibacterial samples after 24h incubation in 3 groups and Calculate according to formula (27) and (28) respectively:

9. The method for detecting the antibacterial performance of antibacterial ceramics according to claim 1, characterized in that: The result evaluation is carried out according to the following steps: ① Refer to the general practices of the health industry and the relevant antimicrobial product standards and adopt the following grading criteria: If the antibacterial rate R is used as the relevant performance evaluation index: ij / R i / R<90%, or Staphylococcus aureus and Salmonella R ij / R i / R<80%, the sample has no antibacterial effect; when 90%≤R ij / R i / R<99%, or Staphylococcus aureus and Salmonella 80%≤R ij / R i / R<90%, the sample has antibacterial effect; when 99%≤R ij / R i / R<99.9%, or Staphylococcus aureus and Salmonella 90%≤R ij / R i / R<99%, the sample has a strong antibacterial effect; when R ij / R i / R≥99.9%, or Staphylococcus aureus and Salmonella R ij / R i / R≥99%, the sample has a strong antibacterial effect; If the antibacterial activity value A is used as the relevant performance evaluation index: ij / A i / A<1.0, Staphylococcus aureus and Salmonella A ij / A i / A<0.5, the sample has no antibacterial effect; when 1.0≤A ij / A i / A<2.0, Staphylococcus aureus and Salmonella 0.5≤A ij / A i / A<1.0, the sample has antibacterial effect; when 2.0≤A ij / A i / A<3.0, Staphylococcus aureus and Salmonella 1.0≤A ij / A i / A<2.0, the sample has a strong antibacterial effect; when A ij / A i / A≥3.0, Staphylococcus aureus and Salmonella A ij / A i / A≥2.0, the sample has strong antibacterial effect; ② When the antibacterial rate R of a group / piece of antibacterial ceramic samples i / R ij Or antimicrobial activity value A i / A ij When the antibacterial performance of the samples differs by at least one level compared with the other two groups / four samples, another group / sample is extracted to repeat the experiment; Calculate the ATP bioluminescence lgC B ─lgI B The antibacterial rate or antibacterial activity value measured by the standard curve method, if the antibacterial performance level of the two groups of antibacterial ceramic samples is the same, then discard it; take the other two groups / four remaining samples of antibacterial rate R i / R ij Or antimicrobial activity value A i / A ij The arithmetic mean value is taken as the evaluation result of the antibacterial performance of the antibacterial ceramic samples of this batch / group.

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