CN110272962A

CN110272962A - ATP bioluminescence lgCA-lgIAThe method that calibration curve method detects anti-bacteria ceramic bacteria resistance energy

Info

Publication number: CN110272962A
Application number: CN201910200122.8A
Authority: CN
Inventors: 李文杰; 潘北辰; 黄慧超; 张进杰; 崔宗岩
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-09-24

Abstract

The present invention relates to a kind of anti-bacteria ceramic antibacterium method for testing performance, detecting step includes: sample preparation and pretreatment；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and bacteria suspension preparation；ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AIt marks Qu Jianli and is inoculated with the viable bacteria ATP concentration C of bacterium solution_ACalibration；Sample inoculation, culture and elution recycling；Recovered liquid I_AMeasurement and its viable bacteria ATP concentration C_AAnd T_AIt calculates；Antibiotic rate R or antibacterial activity value A is calculated；Evaluation of result；Its special feature is that: accurate quantitative test can be carried out to the ceramic bacteria resistance with R or A characterization using ATP fluophotometer.Present invention provide that control sample and antimicrobial sample after elution recycling contact 0h and culture for 24 hours, measure recovered liquid I_AAnd with lgI_ACharacterization and calculating R or A；Evaluation of result foundation is provided.The ATP bioluminescence lgC for the anti-bacteria ceramic antibacterium performance detection that the present invention researches and develops_A‑lgI_ABent method is marked, product quality will be supported to be promoted and market order specification with advanced detection technique.

Description

ATP bioluminescence lgCA-lgIACalibration curve method detects anti-bacteria ceramic bacteria resistance energy Method

Technical field

The present invention relates to the antibacterium performance test methods of anti-bacteria ceramic, specifically a kind of application ATP fluophotometer pair It can be carried out the ATP bioluminescence of accurate quantitative detection with the anti-bacteria ceramic bacteria resistance that antibiotic rate R or antibacterial activity value A is characterized lgC_A-lgI_ACalibration curve method belongs to ceramic antibacterial functions detection technique field.

Background technique

Chinese pottery production history is long, and ceramic annual output accounts for about the 90% of Gross World Product, and annual export volume exceedes 4,000,000,000； Wherein domestic ceramics, architectural pottery, sanitary ceramics occupy 65%, 60% and the 40% of Gross World Product, but ceramic total yield respectively Value is but only the 10% of gross world product, and homogeneous competition caused by the technological element of a product is low grows in intensity.In recent years, using new Type antimicrobial technology transformation traditional ceramics production technology has become the main direction of domestic advantage pottery enterprise Transformation Development, and antimicrobial technology exists The application of china industry and popularize irresistible, antibacterial functions become very characteristic one " attraction " of ceramic product；But it is existing The lag of detection technique but seriously restricts industry development.

Currently, anti-biotic material performance detection technical system is mainly for bacterium and fungi, various countries' bacteria resistance energy both at home and abroad Test method principle is mostly based on the colony counting method being separately cultured to bacterium, first is that the pad pasting training that Japanese Industrial Standards propose Support and impregnate quantitative test method；Two are derived from the antibacterial around-France of U.S. textile industry；Third is that international oscillation flask method；Four It is derived from the dropping method of U.S. textile enterprise；Fifth is that Japanese enterprises are directed to the coverslip method of photocatalyst-type anti-biotic product research and development.It cuts To currently, China successively formulates JC/T 897-2002 on the basis of foreign correlation technique (revises) " antibacterial pottery for 2014 Ceramic products bacteria resistance energy " and GB/T 28116-2011 " antibacterial Bone China ".But existing antibacterium method for testing performance is in technology There are following common problems in content and practical application: first is that involved experimental strain is very few, it is difficult to meet what enterprise grew with each passing hour Research and development of new product and Quality Control demand；Second is that experimentation is cumbersome, relevant operation is influenced by laboratory technician's professional experiences, and test error is big, Lack comparativity；Third is that test period, between 48 hours~72 hours, time and economic cost are high.In recent years, international thin The development of bacterium detection technique field ATP fluorescence analysis is increasingly mature, and the correlation of ATP testing result is compared with traditional Plating 98%, accuracy is high and can realize quick detection；Developed country applies to the method in HACCP.Domestic ATP correlative study It starts late, is drawn by application demand, existing ATP fluorescence detector has become the specified health supervision of China Health department and food Safety-related special inspecting equipment.Current foreign countries' anti-biotic material performance detection technical research is to quantification, rapid and simple Change trend development, focuses on testing result accuracy and comparativity；It has used for reference ATP fluorescence analysis principle and has formulated ISO 20743: 2007-2013《Textiles-Determination of antibacterial activity of textile Products (the antibacterium performance measurement of textile-antibiotic finish textile) " and ISO 13629-1:2012 " Textiles- Determination of antifungal activity of textile products.Part1 Luminescence (measurement of textile-antibiotic finish textile fungicidal properties) ", it is specified that with ATP changes of contents characterization after sample inoculation it is anti-it is thin/ The fluorimetry of mould performance, but its be only applicable to have water imbibition and control sample it is thin/textile material of mould increasing value > 0 Or poromerics, it is not suitable for that there is unwetted property hard in terms of the key technologies such as viable bacteria way of recycling, test condition for validity Inorganic non-metallic materials such as ceramics, the glass of surface and control sample bacterium increasing value < 0 etc.；Uncertainty of measurement is not provided simultaneously Assessment.In addition, the standard method dependent antimicrobial performance characterization parameter is more single, antibacterial activity value A is only related to, and China is then used to With antibiotic rate R.

Therefore, to resist foreign technology barrier, specification domestic market order pushes industrial transformation upgrading, it would be highly desirable to research section It emulates the advanced, accuracy and reproducibility are high, easy-to-use anti-bacteria ceramic Performance Testing Technology.The art of this patent highway route design integrates with The world, detection method belong to the whole world and initiate, and can effectively fill up domestic and international correlative technology field blank.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of application ATP fluophotometers to living with antibiotic rate R or antibacterial Property value A characterization anti-bacteria ceramic bacteria resistance can be carried out the ATP bioluminescence lgC of detection_A-lgI_ACalibration curve method is able to solve Anti-bacteria ceramic or even other anti-biotic materials and product bacteria resistance can accurate quantitative test problems.

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

A kind of detection method of anti-bacteria ceramic bacteria resistance energy, comprising: (1) sample preparation and pretreatment；(2) lectotype selection And reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) ATP log concentration value lgC_ARelative fluorescence Intensity logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration；(5) it sample inoculation, culture and elutes back It receives；(6) reclaim liquid phase is to fluorescence intensity level I_AMeasurement；(7) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and anti- Bacterium activity value A is calculated；(8) evaluation of result；It is characterized in that, using ATP fluophotometer to antibiotic rate R or antibacterial activity value The anti-bacteria ceramic bacteria resistance of A characterization can be carried out the ATP bioluminescence lgC of accurate quantitative test_A-lgI_ACalibration curve method, specifically :

In reclaim liquid phase to fluorescence intensity level I_AIn measurement:

Quantity, size and the water absorption rate requirement for specifying control sample and antimicrobial sample, take at sterilizing and water suction sample Reason；After testing standard bacterial strain is passed on, activated, continuous switching 2 times Fresh bacterial cultures is taken to prepare bacteria suspension and right Its bacterium number carries out primary dcreening operation.With test strain culture solution by 1.0 × 10^-3The ATP standard stock solution of mol/L is diluted to high and low concentration mark Quasi- serial solution: 7.0 × 10^-9mol/L、7.0×10^-8mol/L、7.0×10^-7Mol/L is (if instrument upper limit of detection can not reach To 10^-7Mol/L magnitude or high standard curve it is linear poor, its ATP concentration series can be changed to 7.0 × 10^-10mol/L、 7.0×10^-9mol/L、7.0×10^-8) and 4.2 × 10 mol/L^-11mol/L、4.2×10^-10mol/L、4.2×10^-9Mol/L, and Measure its relative intensity of fluorescence value I_A；Draw the lgC of two respective concentrations_A-lgI_AStandard curve is derived from fitting equation Y =a_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and linearly dependent coefficient R_A0 ²(high concentration), R_A ²(low concentration).To bacterium Total amount is 6.0 × 10⁸After the bacteria suspension of CFU/mL carries out 10 times of gradient dilutions, its relative intensity of fluorescence value I is measured_A, according to highly concentrated Scale directrix curve equation Y=a_A0X+b_A0Calculate corresponding viable bacteria ATP concentration C_A；It is adjusted to obtain C_ARange is 5.0 × 10^- ⁸Mol/L~9.0 × 10^-8The inoculation bacterium solution of mol/L.Then, it is added dropwise respectively to each group control sample and antimicrobial sample glaze to be measured 0.3mL bacterium solution carries out elution recycling to 6 groups of 0h contact samples with 4.7mL eluent immediately；Using ATP fluorescent spectrophotometer measuring The relative intensity of fluorescence value I of recovered liquid_AC0ij、I_AT0ij, according to low concentration calibration curve equation formula Y=a_AX+b_ACalculate its viable bacteria ATP concentration C_AOijAnd T_AOij.Meanwhile contacting sample for 24 hours for 6 groups and being sealed in sterilized petri dishes, (37 ± 1) DEG C, (90 ± 2) %RH Culture is for 24 hours after ± 2h；Glaze residual bacterium is recycled using mode identical with 0h contact sample and measures the relatively glimmering of its recovered liquid Light intensity value I_ACtij、I_ATtij, calculate corresponding viable bacteria ATP concentration C_AtijAnd T_Atij；

In antibiotic rate R and antibacterial activity value A is calculated:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, contacted with 0h and cultivated for 24 hours The relative intensity of fluorescence measured value of every control sample and antimicrobial sample recovered liquid afterwardsWith Based on Data；In the case where testing condition for validity, its bacterium increasing value F after cultivating for 24 hours is calculated_ij、G_ijAnd antibiotic rate R_ijIt is living with antibacterial Property value A_ij；To the R of every group of sample_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；The antibacterial of every batch of anti-bacteria ceramic sample Rate R and antibacterial activity value A is its 3 groups of sample R_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement It is required that；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that antibacterium grading performance determines mark It is quasi-；As the antibiotic rate R of certain group (part) anti-bacteria ceramic sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups (four) samples Bacteria resistance when can be compared to a levels are at least differed, extract one group of (part) sample again and repeat to test；It is calculated through ATP Bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) anti-bacteria ceramic Sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity Value A_i(A_ij) arithmetic mean of instantaneous value as batch (group) anti-bacteria ceramic sample bacteria resistance can evaluation result.

The present invention by adopting the above technical scheme, compared with prior art, beneficial effect is:

(1) advanced: using modern precision instrument-ATP fluophotometer as test equipment, it is dense to have reached viable bacteria ATP The modernization of degree measurement and ceramic antibacterium performance detection；Can effectively reduce human factor in experimentation influences, and avoids biography The generally existing large error of system plate culture；It can ensure the quantification of testing result, while increase substantially testing number According to accuracy；Detection technique has certain advance.

(2) scientific: according to ATP fluorescence analysis test philosophy, to establish the viable bacteria ATP concentration pair suitable for multi-cultur es Numerical value lgC_A- relative intensity of fluorescence logarithm lgI_AStandard curve；It is real to construct anti-bacteria ceramic bacteria resistance energy ATP bioluminescence When quantitative analysis method mathematical model.Meanwhile country variant consumer perceptions habit is taken into account, take antibiotic rate R and antibacterial living Property value A be correlated performance evaluation index, improve the science and versatility of detection method.

(3) innovative: compared with the conventional method that existing operation is numerous, error is big, the period is long, this patent method was being tested Automation and the higher ATP fluophotometer of intelligent level are introduced in journey, are greatly simplified experimental procedure, are realized ceramics The precision of antibacterium performance test results and quantification simultaneously have good reproducibility and comparativity；Survey is greatly shortened simultaneously It tries the period, reduce testing cost；Presently relevant the field of test technology blank can effectively be filled up.

(4) perspective: to establish with lgC_A、lgI_AStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Bacteria suspension viable bacteria concentration characterization and measuring method and ceramics sample pretreatment mode are enriched, specifies control sample, standard liquid The technology contents such as concentration, determination step, calculation formula, uncertainty；The pioneering recovered liquid viable bacteria directly measured with instrument is opposite Fluorescence intensity level I_AEvaluate form as a result to calculate and determine antibiotic rate R or antibacterial activity value A, and by group and between group Coefficient of variation CV investigates uncertainty of measurement, technically has centainly perspective.

(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, this patent establish with sample Method is simple for the product inoculated and cultured ceramic bacteria resistance energy ATP fluorometric investigation that rear viable bacteria ATP concentration variation is attached most importance to for 24 hours, Description of Related Art is clear and specific, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for not With professional standards Experiment on Microbiology personnel, it may advantageously facilitate achievement transfer conversion and promote and apply.

(6) universality: because ATP is prevalent in, life entity is intracellular, and patented method can expand for experimental strain and provide tool The detection technique support for thering is wide spectrum to be worth；The introducing of pertinent instruments can greatly simplify experimental procedure, reduce testing cost；Be conducive to Expand in inspection, learn, grind, produce all circles and promote and apply, ceramic bacteria resistance energy detection technique realization generalization can be supported, while can Reference is provided to the product scopes bacteria resistance energy detection technique research such as plastics, glass.

Further, preferred embodiment of the invention is:

The sample preparation and pretreatment carries out in the steps below:

(1) control sample: in addition to being not added with any antimicrobial component, control sample classification, raw material and technique and antibacterial to be measured Ceramics sample is identical；Wherein antibacterial tests surface band glaze, another side is without glaze.Bone China and fine porcelain are thick in domestic ceramics sample Degree is not more than 6mm, water absorption rate≤0.5%；General porcelain thickness is not more than 8mm, water absorption rate≤1.0%；Stoneware and fine pottery thickness are little In 10mm, wherein stoneware water absorption rate≤5.0%；Fine pottery water absorption rate≤17.0%.Sanitary ceramics thickness of sample is 6mm~10mm, Porcelain water absorption rate≤0.5%, 8.0%≤ceramic water absorption rate < 15.5%；Architectural pottery thickness of sample is not more than 10mm, and porcelain is inhaled Water rate≤0.5%, Shi porcelain water absorption rate 0.5%~3%, Shi matter water absorption rate 6%~10%, ceramic water absorption rate >=10%.Sample ruler Very little is (50 ± 2) mm × (50 ± 2) mm, and each strain test uses 6 groups of samples；Wherein 0h contact and for 24 hours or 48h culture experiment Respectively with 3 groups, every group of 5 samples；

(2) antimicrobial sample: glaze layer adds antibacterial agent or surface applies the ceramics sample of antibiotic layer, and another side is without glaze；Sample Thickness, water absorption rate and specification, quantity etc. are identical as control sample, every group of antimicrobial sample select one group of control sample as Object of reference simultaneously effectively identifies；

(3) sample pre-treatments: each group control sample and antimicrobial sample are impregnated for 24 hours in sterile water before experiment, to its base After body and glaze layer fully absorb moisture；Again with impregnating 5min in 70% ethanol solution, and sterile water is used on superclean bench Abundant cleaning sample is to remove ethyl alcohol.Then, the excessive moisture that sample surfaces are blotted with sterilizing dry gauze puts upwardly its glaze Enter spare in sterilized petri dishes；

The lectotype selection and reagent, culture medium are prepared, and are carried out in the steps below:

(1) General Requirement: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or Deionized water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience；

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc. is spent, wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-13Mol/L~10^-7mol/L；The constant temperature of (37 ± 1) DEG C, (90 ± 2) %RH are permanent Wet incubator；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C of low temperature refrigerator；0 DEG C~8 DEG C refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency (30~50) kHz；Revolving speed (500~3000) The vortex oscillator of r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: the sterile measuring pipette of 1mL, 10mL；0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (meter Error is measured less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；The sterile conical flask of capacity 250mL, 500mL； The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Diameter connects no more than 4mm's Kind ring；L stick；Alcolhol burner；Sterilize tweezers；Sterile gauze；Medical adhesive tape；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black gel ink pen；

(4) reagent: 70% ethanol solution；(121 DEG C of high pressure sterilization 15min, 5 DEG C~10 DEG C are deposited 85% physiological saline Put 30d)；

(5) medium/liquid (commercially available medium/liquid can be used): through 121 DEG C of high pressure sterilizations after matched medium/liquid packing 15min, 2 DEG C~8 DEG C storage 30d；

Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water In, adjust pH to 7.0~7.2；

Nutrient broth (NB): 3g beef extract, 10g peptone, 5g sodium chloride are dissolved by heating in 1000mL water, pH is adjusted To 7.0~7.2；

Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g phosphoric acid hydrogen two Sodium, 2g glucose dissolve by heating in 500mL cattle heart leachate, adjust pH to 7.4 ± 0.2；

The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by 1:500/1:100 Nutrient broth (NB) mixed with 85% sterile saline solution, adjust pH to 7.0~7.2；

(6) ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction examination - 20 DEG C~-70 DEG C of agent preservations, use in 6 months；

Dilution buffer: 0.005mol/L and the disodium phosphate soln for containing 0.037% sucrose, adjusting pH to 7.2 ± 0.2；121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；

ATP standard reagent stoste: by 60.5mg trinosin (C₁₀H₁₄O₁₃P₃Na₂·3H₂O) it is dissolved in 100mL In water, being configured to concentration is 1 × 10^-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；

ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose are dissolved by heating in 250mL water In, adjust pH to 7.5 ± 0.2；It is used in 8h；

ATP lysate: 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 is international single Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, the 20mg bovine serum albumin of position/ml is dissolved in 10mL concentration is to adjust in pH to 6.0 ± 0.5,8h in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L and use (1mL cracking ATP concentration in nutrient broth can be down to 10 in 15min by liquid^-13Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, is 10% with 0.2ml concentration Benza mix after, adjust pH to 12.0 ± 0.5；

ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg cow's serum Albumen is dissolved in the ATP fluorescent reagent buffer solution of 30mL, and 15min is stored at room temperature after mixing, is used in 3h；

Culture presevation, activation and the bacteria suspension preparation, carries out in the steps below:

(1) test strain: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or provided by national corresponding culture presevation administrative center and can be traced to the source other Strain)；

(2) culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (sramana is added with capillary syring Salmonella BHI broth), pressure-vaccum makes strain melt dispersion for several times.Then, a little test strain suspension is instilled and 5mL is housed In the test tube of~10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (does not surpass Spend 1 month), inoculation times were no more than for 14 generations；

(3) actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours； Switching 1 time daily, continuous switching are no more than 15d.Test is trained using the 3rd generation~the 14th generation and in the interior Fresh bacterial transferred for 24 hours Support object；

(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 10⁸CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity)；

The ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution is living Bacterium ATP concentration C_ACalibration carries out in the steps below:

(1) standard serial solution is determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10^- ³The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10^-9mol/L、7.0×10^-8mol/L、 7.0×10^-7Mol/L is (if instrument upper limit of detection is unable to reach 10^-7Mol/L magnitude or high standard curve it is linear compared with Its ATP concentration series can be changed to 7.0 × 10 by difference^-10mol/L、7.0×10^-9mol/L、7.0×10^-8) and 4.2 × 10 mol/L^-11mol/L、4.2×10^-10mol/L、4.2×10^-9Mol/L is simultaneously mixed.Then, with sterilizing liquid-transfering gun by the ATP standard of 0.1mL Serial solution is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose is successively added dropwise and mixes It is even；The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes again, as ATP bioluminescence Test Duplicate Samples；

(2) relative intensity of fluorescence value I_AMeasurement: according to relative intensity of fluorescence value I in this patent_AMeasuring method, with test organisms After kind culture solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, to various concentration The ATP that 0.1mL is added dropwise in three Duplicate Samples of ATP standard solution respectively extracts reagent, and the ATP fluorescence examination of 0.1mL is instilled after mixing Agent；It mixes again, with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and records immediately and (ensure each link operating time Unanimously, cross contamination is avoided).Each Duplicate Samples minute is no more than 15s, with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of relative intensity of fluorescence value is its I_AMeasured value；

(3) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with ATP standard series The relative intensity of fluorescence logarithm lgI of solution_AAs abscissa, with its corresponding ATP log concentration value lgC_AFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And it applies Least square fitting method is derived from the linear equation Y=a of curve_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and phase Close coefficients R_A0 ²(high concentration), R_A ²(low concentration).Work as R_A0 ²And R_A ²>=0.98, it when confidence level >=0.95, is done according to this patent Measurement it is effective；

(4) it is inoculated with the viable bacteria ATP concentration C of bacterium solution_ACalibration: according to relative intensity of fluorescence value I in this patent_AMeasuring method is right Bacteria total amount is 6.0 × 10 after the turbidimetry primary dcreening operation of Maxwell⁸The relative fluorescence of 10 times of gradient dilution liquid of bacteria suspension of CFU/mL is strong Angle value I_AIt is measured；According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates its viable bacteria ATP concentration C_A.Through Test strain culture solution adjusts to obtain C_ARange is 5.0 × 10^-8Mol/L~9.0 × 10^-8The inoculation bacterium solution of mol/L, measures and remembers Record relative intensity of fluorescence value I of the 0.3mL inoculation bacterium solution after the dilution of 4.7mL eluent in 1min_A0；

Sample inoculation, culture and the elution recycling, carries out in the steps below:

(1) inoculated and cultured: being put into the multilayer sterile gauze being of moderate size in sterilized petri dishes bottom, by 10mL sterile physiological salt Water, which is poured on gauze, makes its saturation of just soaking；It contacts sample for 24 hours for 6 groups to be placed on sterile gauze, 6 groups of 0h contact samples are set In in the sterilized petri dishes of non-yarn releasing cloth.With sterilizing liquid-transfering gun by 0.3mL be inoculated with bacterium solution (water absorption rate be greater than 1.0% ceramics sample, The inertia such as agar thickener can be added in bacterium solution) it is added dropwise on each group control sample and antimicrobial sample glaze to be measured respectively (with song Line lgC_A-lgI_ACalibration is derived from same branch test strain stoste test tube with bacterium solution, and 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used), with L stick (attachment is inoculated with bacterium solution but does not hang drop) smears bacterium solution uniformly, its is made to cover the entire glaze of sample.Ware lid is covered, medical adhesive tape is used Sealing contacts the plate of sample equipped with 6 groups for 24 hours；(37 ± 1) DEG C, (90 ± 2) %RH culture ± 2h for 24 hours；

(2) elution recycling: (0.2% sterile surfaces activity can be added using 4.7mL test strain culture solution as eluent Agent), after 6 groups of 0h contact sample inoculation bacteriums, sufficiently eluted using solid or liquid swab method immediately；6 groups cultivate for 24 hours Sample uses type of elution identical with 0h contact sample to recycle bacterium.Solid swab method is to be soaked with saturation eluent with head Aseptic cotton carrier is uniformly embrocated 10 times in each sample surfaces back and forth anyhow respectively, and rotates with it cotton swab；Cut off experimenter's hand Cotton swab is placed in the sterile test tube equipped with remaining eluent, as recovered liquid (if recovered liquid after mixing by the swab stick of contact portion Less than 5mL, eluent is added to 5mL).Liquid swab method draws 4.7mL eluent with sterilizing liquid-transfering gun, in plate repeatedly It rinses each sample surfaces at least 4 times, sufficiently elute and moves into washing lotion in sterile test tube, as recovered liquid (if returning after mixing Liquid is received less than 5mL, adds eluent to 5mL)；

The reclaim liquid phase is to fluorescence intensity level I_AMeasurement carries out in the steps below:

(1) instrument and reagent set relative intensity of fluorescence background value calibration: use sterilizing liquid-transfering gun by 0.1mL test strain culture The ATP lysate of liquid, 0.8mL phosphate buffer solution and 0.1mL containing 0.037% sucrose is separately added into same branch sterile test tube In and mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, stand 5min~ 30min, as skip test Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (ensuring that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values)；

(2) reclaim liquid phase is to fluorescence intensity level I_AMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate for phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose that sterilize Be separately added into same branch sterile test tube, after mixing well by the above-mentioned mixed solution of 0.1mL successively move to three instruments without In bacterium test tube, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.It is added dropwise 0.1mL's respectively into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, uses its phase of ATP fluorescent spectrophotometer measuring immediately To fluorescence intensity level I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute No more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquid_AMeasurement Value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level I_A)；

The recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated, by following steps It is rapid to carry out:

(1) the viable bacteria ATP concentration C of recovered liquid_AAnd T_AIt calculates

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, calculate control sample and antibacterial After sample is contacted through 0h and is cultivated for 24 hours, the viable bacteria ATP concentration C of recovered liquid_AAnd T_A；Relevant calculation (uses instrument matched reagent box When, the viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volume_AAnd T_A) see formula (1)~(12):

In formula:

C_A0And C_AtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；

C_A0iAnd C_AtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；

C_A0ijAnd C_AtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；b_A- low concentration standard curve lgC_A-lgI_AIn vertical axis intercept；

T_A0And T_AtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；

T_A0iAnd T_AtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；

T_A0ijAnd T_AtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

(2) condition for validity is tested

If recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.7mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after cultivating for 24 hours control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is C_Atij≥0.1×C_A0ij.Then when 3 groups of 0h contact control sample reclaim liquid phase To fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlation Uncertainty of measurement requirement), the measurement carried out according to this patent method is effective.

(3) bacterium increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value F_ij、G_ijRespectively according to formula (13), (14) it calculates:

In formula:

F_ijAnd G_ijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of every anti-bacteria ceramic sample_ij, every group and every batch of sample antibiotic rate R_iAnd R It is calculated respectively according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of-every anti-bacteria ceramic sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2, 3,4,5；

R_iThe antibiotic rate of-every group anti-bacteria ceramic sample, %；Sample group i=1,2,3；

R-every batch of anti-bacteria ceramic sample antibiotic rate, %；

C_AtijAnd T_Atij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, single Position is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

WithAfter cultivating for 24 hours, the relative intensity of fluorescence of recovered liquid is measured for-every antimicrobial sample and control sample Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of every anti-bacteria ceramic sample_ij, the antibacterial of every group and every batch of sample it is living Property value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

A_ijThe antibacterial activity value of-every anti-bacteria ceramic sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2, 3,4,5；

A_iThe antibacterial activity value of-every group anti-bacteria ceramic sample, sample group i=1,2,3；

A-every batch of anti-bacteria ceramic sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(6) data revision of the convention requirement: in the concentration and inoculation bacterium solution, sample recovered liquid of calibration ATP standard serial solution Viable bacteria ATP concentration C_AWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as C_ALess than 100mol/L When, " rounding up " round numbers；Work as C_AWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals afterwards.Control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate R_ij、R_i, R calculate As a result three effective digitals are taken；Bacterium increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample and contacts through 0h and for 24 hours After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge for ATP fluorimetric assay for biological materials method to be applied to ceramic antibacterium performance test Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

The evaluation of result of the anti-bacteria ceramic sample bacteria resistance energy, carries out in the steps below:

(1) with reference to health industry common practice and the requirement of dependent antimicrobial product standard, following classification criterion is taken:

If using antibiotic rate R as correlated performance evaluation index: working as R_ij/R_i(staphylococcus aureus when/R < 90% With salmonella R_ij/R_i/ R < 80%), sample is without antibacterial actions；As 90%≤R_ij/R_i(golden yellow grape when/R < 99% 80%≤R of coccus and salmonella_ij/R_i/ R < 90%), sample has antibacterial actions；As 99%≤R_ij/R_i/ R < 99.9% When (90%≤R of staphylococcus aureus and salmonella_ij/R_i/ R < 99%), sample antibacterial actions are stronger；Work as R_ij/R_i/R (staphylococcus aureus and salmonella R when >=99.9%_ij/R_i/ R >=99%), sample antibacterial actions are extremely strong；

If using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_i(Staphylococcus aureus when/A < 1.0 Bacterium and salmonella A_ij/A_i/ A < 0.5), sample is without antibacterial actions；As 1.0≤A_ij/A_i(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonella_ij/A_i/ A < 1.0), sample has antibacterial actions；As 2.0≤A_ij/A_iIt is (golden yellow when/A < 3.0 1.0≤A of color staphylococcus and salmonella_ij/A_i/ A < 2.0), sample antibacterial actions are stronger；Work as A_ij/A_i(gold when/A >=3.0 Staphylococcus aureus and salmonella A_ij/A_i/ A >=2.0), sample antibacterial actions are extremely strong；

(2) as the antibiotic rate R of certain group (part) anti-bacteria ceramic sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the bacteria resistance of (four) sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test； It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back is anti- Bacterium ceramics sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or it is anti- Bacterium activity value A_i(A_ij) arithmetic mean of instantaneous value as batch (group) anti-bacteria ceramic sample bacteria resistance can evaluation result；

Specific embodiment

Below with reference to preferred embodiment, the present invention will be described in detail, so that advantages and features of the invention can be easier to It is readily appreciated by one skilled in the art, so as to make a clearer definition of the protection scope of the present invention.

The antiseptic sanitary sample bacteria resistance that the present embodiment is process with the added nanometer silver-series antibacterial agent of glaze layer It can be illustrated for detection.

Specific detection method carries out in the steps below:

(1) sample preparation and pretreatment

1.1 control samples: in addition to being not added with any antimicrobial component, control sample classification, raw material and technique and antibacterial to be measured Ceramics sample is identical；Wherein antibacterial tests surface band glaze, another side is without glaze.Bone China and fine porcelain are thick in domestic ceramics sample Degree is not more than 6mm, water absorption rate≤0.5%；General porcelain thickness is not more than 8mm, water absorption rate≤1.0%；Stoneware and fine pottery thickness are little In 10mm, wherein stoneware water absorption rate≤5.0%；Fine pottery water absorption rate≤17.0%.Sanitary ceramics thickness of sample is 6mm~10mm, Porcelain water absorption rate≤0.5%, 8.0%≤ceramic water absorption rate < 15.5%；Architectural pottery thickness of sample is not more than 10mm, and porcelain is inhaled Water rate≤0.5%, Shi porcelain water absorption rate 0.5%~3%, Shi matter water absorption rate 6%~10%, ceramic water absorption rate >=10%.Sample ruler Very little is (50 ± 2) mm × (50 ± 2) mm, and each strain test uses 6 groups of samples；Wherein 0h contact and for 24 hours or 48h culture experiment Respectively with 3 groups, every group of 5 samples.

1.2 antimicrobial samples: glaze layer adds antibacterial agent or surface applies the ceramics sample of antibiotic layer, and another side is without glaze；Sample Thickness, water absorption rate and specification, quantity etc. are identical as control sample, every group of antimicrobial sample select one group of control sample as Object of reference simultaneously effectively identifies.

1.3 sample pre-treatments: each group control sample and antimicrobial sample are impregnated for 24 hours in sterile water before experiment, to its base After body and glaze layer fully absorb moisture；Again with impregnating 5min in 70% ethanol solution, and sterile water is used on superclean bench Abundant cleaning sample is to remove ethyl alcohol.Then, the excessive moisture that sample surfaces are blotted with sterilizing dry gauze puts upwardly its glaze Enter spare in sterilized petri dishes.

(2) lectotype selection and reagent, culture medium are prepared

2.1 General Requirements: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or Deionized water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience.

2.2 instrument and equipments: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc. is spent, wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-13Mol/L~10^-7mol/L；The constant temperature of (37 ± 1) DEG C, (90 ± 2) %RH are permanent Wet incubator；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C of low temperature refrigerator；0 DEG C~8 DEG C refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency (30~50) kHz；Revolving speed (500~3000) The vortex oscillator of r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace.

2.3 material utensils: the sterile measuring pipette of 1mL, 10mL；0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (meter Error is measured less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；The sterile conical flask of capacity 250mL, 500mL； The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Diameter connects no more than 4mm's Kind ring；L stick；Alcolhol burner；Sterilize tweezers；Sterile gauze；Medical adhesive tape；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black gel ink pen.

2.4 reagents: 70% ethanol solution；(121 DEG C of high pressure sterilization 15min, 5 DEG C~10 DEG C are deposited 85% physiological saline Put 30d).

2.5 medium/liquids (can use commercially available medium/liquid): through 121 DEG C of high pressure sterilizations after matched medium/liquid packing 15min, 2 DEG C~8 DEG C storage 30d；

The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by 1:500/1:100 Nutrient broth (NB) mixed with 85% sterile saline solution, adjust pH to 7.0~7.2.

2.6ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction examination - 20 DEG C~-70 DEG C of agent preservations, use in 6 months；

ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg cow's serum Albumen is dissolved in the ATP fluorescent reagent buffer solution of 30mL, and 15min is stored at room temperature after mixing, is used in 3h.

(3) culture presevation, activation and bacteria suspension preparation

3.1 test strains: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or provided by national corresponding culture presevation administrative center and can be traced to the source other Strain).

3.2 culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (sramana is added with capillary syring Salmonella BHI broth), pressure-vaccum makes strain melt dispersion for several times.Then, a little test strain suspension is instilled and 5mL is housed In the test tube of~10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (does not surpass Spend 1 month), inoculation times were no more than for 14 generations.

3.3 actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours； Switching 1 time daily, continuous switching are no more than 15d.Test is trained using the 3rd generation~the 14th generation and in the interior Fresh bacterial transferred for 24 hours Support object.

The preparation of 3.4 bacteria suspensions: a small amount of Fresh bacterial is scraped from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 10⁸CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity).

(4) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration

4.1 standard serial solutions are determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10^- ³The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10^-9mol/L、7.0×10^-8mol/L、 7.0×10^-7Mol/L is (if instrument upper limit of detection is unable to reach 10^-7Mol/L magnitude or high standard curve it is linear compared with Its ATP concentration series can be changed to 7.0 × 10 by difference^-10mol/L、7.0×10^-9mol/L、7.0×10^-8) and 4.2 × 10 mol/L^-11mol/L、4.2×10^-10mol/L、4.2×10^-9Mol/L is simultaneously mixed.Then, with sterilizing liquid-transfering gun by the ATP standard of 0.1mL Serial solution is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose is successively added dropwise and mixes It is even；The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes again, as ATP bioluminescence Test Duplicate Samples.

4.2 relative intensity of fluorescence value I_AMeasurement: according to relative intensity of fluorescence value I in this patent_AMeasuring method, with test organisms After kind culture solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, to various concentration The ATP that 0.1mL is added dropwise in three Duplicate Samples of ATP standard solution respectively extracts reagent, and the ATP fluorescence examination of 0.1mL is instilled after mixing Agent；It mixes again, with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and records immediately and (ensure each link operating time Unanimously, cross contamination is avoided).Each Duplicate Samples minute is no more than 15s, with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of relative intensity of fluorescence value is its I_AMeasured value.

4.3ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with ATP standard series The relative intensity of fluorescence logarithm lgI of solution_AAs abscissa, with its corresponding ATP log concentration value lgC_AFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And it applies Least square fitting method is derived from the linear equation Y=a of curve_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and phase Close coefficients R_A0 ²(high concentration), R_A ²(low concentration).Work as R_A0 ²And R_A ²>=0.98, it when confidence level >=0.95, is done according to this patent Measurement it is effective.

The viable bacteria ATP concentration C of 4.4 inoculation bacterium solutions_ACalibration: according to relative intensity of fluorescence value I in this patent_AMeasuring method is right Bacteria total amount is 6.0 × 10 after the turbidimetry primary dcreening operation of Maxwell⁸The relative fluorescence of 10 times of gradient dilution liquid of bacteria suspension of CFU/mL is strong Angle value I_AIt is measured；According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates its viable bacteria ATP concentration C_A.Through Test strain culture solution adjusts to obtain C_ARange is 5.0 × 10^-8Mol/L~9.0 × 10^-8The inoculation bacterium solution of mol/L, measures and remembers Record relative intensity of fluorescence value I of the 0.3mL inoculation bacterium solution after the dilution of 4.7mL eluent in 1min_A0。

(5) sample inoculation, culture and elution recycling

5.1 inoculated and cultureds: being put into the multilayer sterile gauze being of moderate size in sterilized petri dishes bottom, by 10mL sterile physiological salt Water, which is poured on gauze, makes its saturation of just soaking；It contacts sample for 24 hours for 6 groups to be placed on sterile gauze, 6 groups of 0h contact samples are set In in the sterilized petri dishes of non-yarn releasing cloth.With sterilizing liquid-transfering gun by 0.3mL be inoculated with bacterium solution (water absorption rate be greater than 1.0% ceramics sample, The inertia such as agar thickener can be added in bacterium solution) it is added dropwise on each group control sample and antimicrobial sample glaze to be measured respectively (with song Line lgC_A-lgI_ACalibration is derived from same branch test strain stoste test tube with bacterium solution, and 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used), with L stick (attachment is inoculated with bacterium solution but does not hang drop) smears bacterium solution uniformly, its is made to cover the entire glaze of sample.Ware lid is covered, medical adhesive tape is used Sealing contacts the plate of sample equipped with 6 groups for 24 hours；(37 ± 1) DEG C, (90 ± 2) %RH culture ± 2h for 24 hours.

5.2 elution recycling: (0.2% sterile surfaces activity can be added using 4.7mL test strain culture solution as eluent Agent), after 6 groups of 0h contact sample inoculation bacteriums, sufficiently eluted using solid or liquid swab method immediately；6 groups cultivate for 24 hours Sample uses type of elution identical with 0h contact sample to recycle bacterium.Solid swab method is to be soaked with saturation eluent with head Aseptic cotton carrier is uniformly embrocated 10 times in each sample surfaces back and forth anyhow respectively, and rotates with it cotton swab；Cut off experimenter's hand Cotton swab is placed in the sterile test tube equipped with remaining eluent, as recovered liquid (if recovered liquid after mixing by the swab stick of contact portion Less than 5mL, eluent is added to 5mL).Liquid swab method draws 4.7mL eluent with sterilizing liquid-transfering gun, in plate repeatedly It rinses each sample surfaces at least 4 times, sufficiently elute and moves into washing lotion in sterile test tube, as recovered liquid (if returning after mixing Liquid is received less than 5mL, adds eluent to 5mL).

(6) reclaim liquid phase is to fluorescence intensity level I_AMeasurement

6.1 instruments and reagent set relative intensity of fluorescence background value calibration: use sterilizing liquid-transfering gun by 0.1mL test strain culture The ATP lysate of liquid, 0.8mL phosphate buffer solution and 0.1mL containing 0.037% sucrose is separately added into same branch sterile test tube In and mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, stand 5min~ 30min, as skip test Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (ensuring that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values).

6.2 reclaim liquid phases are to fluorescence intensity level I_AMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate for phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose that sterilize Be separately added into same branch sterile test tube, after mixing well by the above-mentioned mixed solution of 0.1mL successively move to three instruments without In bacterium test tube, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.It is added dropwise 0.1mL's respectively into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, uses its phase of ATP fluorescent spectrophotometer measuring immediately To fluorescence intensity level I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute No more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquid_AMeasurement Value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level I_A)。

(7) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated

The viable bacteria ATP concentration C of 7.1 recovered liquids_AAnd T_AIt calculates

In formula:

With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5.

7.2 test conditions for validity

7.3 bacterium increasing value F_ij、G_ijIt calculates

In formula:

a_A- low concentration standard curve lgC_A-lgI_ASlope.

7.4 antibiotic rate R are calculated

In formula:

R-every batch of anti-bacteria ceramic sample antibiotic rate, %；

a_A- low concentration standard curve lgC_A-lgI_ASlope.

7.5 antibacterial activity value A are calculated

In formula:

A-every batch of anti-bacteria ceramic sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope.

7.6 data revisions of the convention requirement: in the concentration and inoculation bacterium solution, sample recovered liquid of calibration ATP standard serial solution Viable bacteria ATP concentration C_AWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as C_ALess than 100mol/L When, " rounding up " round numbers；Work as C_AWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals afterwards.Control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate R_ij、R_i, R calculate As a result three effective digitals are taken；Bacterium increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals.

7.7 uncertainties of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample and contacts through 0h and for 24 hours After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge for ATP fluorimetric assay for biological materials method to be applied to ceramic antibacterium performance test Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

(8) evaluation of result

8.1, with reference to health industry common practice and the requirement of dependent antimicrobial product standard, take following classification criterion:

If using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_i(Staphylococcus aureus when/A < 1.0 Bacterium and salmonella A_ij/A_i/ A < 0.5), sample is without antibacterial actions；As 1.0≤A_ij/A_i(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonella_ij/A_i/ A < 1.0), sample has antibacterial actions；As 2.0≤A_ij/A_iIt is (golden yellow when/A < 3.0 1.0≤A of color staphylococcus and salmonella_ij/A_i/ A < 2.0), sample antibacterial actions are stronger；Work as A_ij/A_i(gold when/A >=3.0 Staphylococcus aureus and salmonella A_ij/A_i/ A >=2.0), sample antibacterial actions are extremely strong.

8.2 work as the antibiotic rate R of certain group (part) anti-bacteria ceramic sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the bacteria resistance of (four) sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test； It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back is anti- Bacterium ceramics sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or it is anti- Bacterium activity value A_i(A_ij) arithmetic mean of instantaneous value as batch (group) anti-bacteria ceramic sample bacteria resistance can evaluation result.

Laboratory biosafety qualification, instrument and equipment, medium/liquid, chemical reagent, reference culture used in the present embodiment:

(1) Laboratory biosafety qualification

In the two stage biological safety experiment room that institute registration number is CNAS BL0059, by having secondary advanced techniques academic title Microorganism detection professional complete related experiment.

(2) instrument and equipment

2.1 two stage biological safety cabinets: 1300 series of secondary B2 type Biohazard Safety Equipment of Thermo Scientific, workbench Surface area 0.55m², capacity 1130m³/ h, filter efficiency 99.99% (0.3 μm).

2.2 superclean benches: American blend Thermo Scientific^TMHeraguard^TM, model ECO ultra-clean work Platform, inside width × depth × height=920mm × 585mm × 645mm；Air velocity be 0.15m/s~0.25m/s and 0.36m/s~ 0.45m/s。

2.3ATP bioluminescence rapid detection system: the portable system SURE ATP fluorescence of Hygiena company, the U.S. Detector, box containing matched reagent, plastics Special test tube etc.；ATP content detection lower limit 4 × 10^-18Mol/ml, the inspection of microorganism total amount Rising limit 1.0CFU/ml, RLU range of readings 0~9999, detection time 10s, 1000 times/s of sampling rate, measurement error ± 5%.

2.4 constant temperature and humidity incubators: Guan Sen biotechnology (Shanghai) Co., Ltd. model WS-380H, volume 380L, temperature control The constant temperature and humidity incubator of ± 0.8 DEG C of range (0~50) DEG C, wet range (30~95) %RH ± 2%RH of control.

2.5 pressure steam sterilizers: the autoclave of Japanese Sanyo company model MLS-3780, volume 75L, sterilizing ± 2 DEG C of temperature (105~135) DEG C, maximum pressure 0.235MPa, timing range (1~250) min.

2.6 low temperature refrigerators: the superfreeze storage of Zhong Kemeiling low temperature Science and Technology Co., Ltd. model DW-HL398 Case, dischargeable capacity 398L, -10 DEG C~-86 DEG C of storage temperature.

2.7 refrigerating boxes: the Medical refrigerator of Zhong Kemeiling low temperature Science and Technology Co., Ltd. model YC-968L effectively holds Long-pending 968L, ± 0.1 DEG C of storage temperature (2~8) DEG C.

2.8 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.

2.9 ultrasonic cleaners: the supersonic wave cleaning machine of Shanghai Yi Jing ultrasonic instrument Co., Ltd model YQ-120C, Capacity 3.2L, supersonic frequency 40KHz/28KHz/25KHz, timing range 1min~30min/99min.

2.10 vortex oscillators: the vortex oscillator of American blend Coleparmer Votex-Genie2, the range of speeds (500~3000) r/min ± 3r/min, timing range 1s~60s.

2.11pH meter: the accurate pH meter of Shanghai precision instrumentation Co., Ltd model MP512-03, range ability (- 2.000~19.999) pH ± 0.002pH, ± 0.4 DEG C of temperature range (- 10~110) DEG C.

2.12 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.

(3) medium/liquid

The medium/liquid of Beijing overpass technical concern Co., Ltd production.

(4) chemical reagent

Trinosin standard items and prepare ATP fluorescent reagent buffer solution, ATP lysate, ATP extracting solution and Biochemical reagents needed for ATP fluorescent reagent are purchased from U.S.'s Amresco brand of Shanghai Jin Pan Biotechnology Co., Ltd agency.

(5) reference culture

Escherichia coli ATCC 8739 and staphylococcus aureus ATCC 6538 is purchased from Chinese industrial microorganism fungus kind Preservation administrative center.

The detection data and result of the present embodiment calculate:

Using ATP fluophotometer through lgC_A-lgI_ACalibration curve method can be into the bacteria resistance of antiseptic sanitary sample Row detection, coherent detection data and Calculation of Measuring Uncertainty result be shown in Table respectively 1~table 4 (Shigella and salmonella Testing result is close with escherichia coli and the testing result of staphylococcus aureus respectively).

1 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (escherichia coli)

2 relative intensity of fluorescence value Calculation of Measuring Uncertainty result (escherichia coli) of table

3 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (staphylococcus aureus)

4 relative intensity of fluorescence Calculation of Measuring Uncertainty result (staphylococcus aureus) of table

The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair Equivalent transformation made by bright description, is applied directly or indirectly in other relevant technical fields, and is included in this hair In bright scope of patent protection.

Claims

1. a kind of detection method of anti-bacteria ceramic bacteria resistance energy, comprising: (1) sample preparation and pretreatment；(2) lectotype selection and Reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) ATP log concentration value lgC_ARelative fluorescence is strong Spend logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration；(5) it sample inoculation, culture and elutes back It receives；(6) reclaim liquid phase is to fluorescence intensity level I_AMeasurement；(7) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and anti- Bacterium activity value A is calculated；(8) evaluation of result；It is characterized in that, using ATP fluophotometer to antibiotic rate R or antibacterial activity value The anti-bacteria ceramic bacteria resistance of A characterization can be carried out the ATP bioluminescence lgC of accurate quantitative test_A-lgI_ACalibration curve method, specifically :

In reclaim liquid phase to fluorescence intensity level I_AIn measurement:

Quantity, size and the water absorption rate requirement for specifying control sample and antimicrobial sample take sterilizing and water suction to handle in sample；It will After testing standard bacterial strain is passed on, activated, continuous switching 2 times Fresh bacterial cultures is taken to prepare bacteria suspension and to its bacterium number Primary dcreening operation is carried out, with test strain culture solution by 1.0 × 10^-3The ATP standard stock solution of mol/L is diluted to high and low concentration standard series Solution: 7.0 × 10^-9mol/L、7.0×10^-8mol/L、7.0×10^-7Mol/L is (if instrument upper limit of detection is unable to reach 10^- ⁷Mol/L magnitude or high standard curve it is linear poor, its ATP concentration series can be changed to 7.0 × 10^-10mol/L、7.0 ×10^-9mol/L、7.0×10^-8) and 4.2 × 10 mol/L^-11mol/L、4.2×10^-10mol/L、4.2×10^-9Mol/L, and survey Fixed its relative intensity of fluorescence value I_A；Draw the lgC of two respective concentrations_A-lgI_AStandard curve is derived from fitting equation Y= a_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and linearly dependent coefficient R_A0 ²(high concentration), R_A ²(low concentration), it is total to bacterium Amount is 6.0 × 10⁸After the bacteria suspension of CFU/mL carries out 10 times of gradient dilutions, its relative intensity of fluorescence value I is measured_A, according to high concentration Calibration curve equation formula Y=a_A0X+b_A0Calculate corresponding viable bacteria ATP concentration C_A；It is adjusted to obtain C_ARange is 5.0 × 10^-8mol/ L~9.0 × 10^-8Then 0.3mL is added dropwise to each group control sample and antimicrobial sample glaze to be measured in the inoculation bacterium solution of mol/L respectively Bacterium solution carries out elution recycling to 6 groups of 0h contact samples with 4.7mL eluent immediately；Using ATP fluorescent spectrophotometer measuring recovered liquid Relative intensity of fluorescence value I_AC0ij、I_AT0ij, according to low concentration calibration curve equation formula Y=a_AX+b_ACalculate its viable bacteria ATP concentration C_AOijAnd T_AOij, meanwhile, sample is contacted for 24 hours by 6 groups and is sealed in sterilized petri dishes, and (37 ± 1) DEG C, (90 ± 2) %RH culture are for 24 hours After ± 2h；Glaze residual bacterium is recycled using mode identical with 0h contact sample and measures the relative intensity of fluorescence value of its recovered liquid I_ACtij、I_ATtij, calculate corresponding viable bacteria ATP concentration C_AtijAnd T_Atij；

In antibiotic rate R and antibacterial activity value A is calculated:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, every after being contacted with 0h and being cultivated for 24 hours The relative intensity of fluorescence measured value of control sample and antimicrobial sample recovered liquidWith As basic data； In the case where testing condition for validity, its bacterium increasing value F after cultivating for 24 hours is calculated_ij、G_ijAnd antibiotic rate R_ijWith antibacterial activity value A_ij；To the R of every group of sample_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；The antibiotic rate R of every batch of anti-bacteria ceramic sample and Antibacterial activity value A is its 3 groups of sample R_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement require；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, antibacterium grading performance criterion is determined；When The antibiotic rate R of certain group (part) anti-bacteria ceramic sample_i(R_ij) or antibacterial activity value A_i(A_ij) anti-with other two groups (four) samples When bacterium performance is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP biology Fluorescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) anti-bacteria ceramic sample Antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i (A_ij) arithmetic mean of instantaneous value as batch (group) anti-bacteria ceramic sample bacteria resistance can evaluation result.

2. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the sample system Standby and pre-treatment carries out in the steps below:

(1) control sample: in addition to being not added with any antimicrobial component, control sample classification, raw material and technique and anti-bacteria ceramic to be measured Sample is identical；Wherein antibacterial tests surface band glaze, Bone China and fine porcelain thickness is not without glaze, in domestic ceramics sample for another side Greater than 6mm, water absorption rate≤0.5%；General porcelain thickness is not more than 8mm, water absorption rate≤1.0%；Stoneware and fine pottery thickness are not more than 10mm, wherein stoneware water absorption rate≤5.0%；Fine pottery water absorption rate≤17.0%, sanitary ceramics thickness of sample are 6mm~10mm, porcelain Matter water absorption rate≤0.5%, 8.0%≤ceramic water absorption rate < 15.5%；Architectural pottery thickness of sample is not more than 10mm, porcelain water suction Rate≤0.5%, Shi porcelain water absorption rate 0.5%~3%, Shi matter water absorption rate 6%~10%, ceramic water absorption rate >=10%, sample size For (50 ± 2) mm × (50 ± 2) mm, each strain test uses 6 groups of samples；Wherein 0h contact and for 24 hours or 48h culture experiment is each With 3 groups, every group of 5 samples；

(2) antimicrobial sample: glaze layer adds antibacterial agent or surface applies the ceramics sample of antibiotic layer, and another side is without glaze；Thickness of sample, Water absorption rate and specification, quantity etc. are identical as control sample, and every group of antimicrobial sample selects one group of control sample as reference Object simultaneously effectively identifies；

(3) sample pre-treatments: impregnating each group control sample and antimicrobial sample for 24 hours before experiment in sterile water, to its green body and After glaze layer fully absorbs moisture；Again with impregnating 5min in 70% ethanol solution, and it is abundant with sterile water on superclean bench Cleaning sample is to remove ethyl alcohol, then, the excessive moisture of sample surfaces is blotted with sterilizing dry gauze, its glaze is put into upwardly nothing It is spare in bacterium plate.

3. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the equipment choosing Type and reagent, culture medium are prepared, and are carried out in the steps below:

(1) General Requirement: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or go from Sub- water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience；

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；Fluorophotometric containing ATP The ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc., wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-13Mol/L~10^-7mol/L；The constant temperature of (37 ± 1) DEG C, (90 ± 2) %RH are permanent Wet incubator；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C of low temperature refrigerator；0 DEG C~8 DEG C refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency (30~50) kHz；Revolving speed (500~3000) The vortex oscillator of r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: the sterile measuring pipette of 1mL, 10mL；(measurement error is small by 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL In single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；The sterile conical flask of capacity 250mL, 500mL；Diameter 90mm's Sterile petri dish；Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Diameter is not more than the oese of 4mm；L stick；Alcohol Lamp；Sterilize tweezers；Sterile gauze；Medical adhesive tape；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm； Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black gel ink pen；

(4) reagent: 70% ethanol solution；85% physiological saline (121 DEG C of high pressure sterilization 15min, 5 DEG C~10 DEG C storages 30d)；

(5) medium/liquid (commercially available medium/liquid can be used): through 121 DEG C of high pressure sterilization 15min after the packing of matched medium/liquid, 2 DEG C~8 DEG C of storage 30d；

Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water, adjusted Save pH to 7.0~7.2；

Nutrient broth (NB): by 3g beef extract, 10g peptone, 5g sodium chloride dissolve by heating in 1000mL water, adjust pH to 7.0~7.2；

Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g disodium hydrogen phosphate, 2g Glucose dissolves by heating in 500mL cattle heart leachate, adjusts pH to 7.4 ± 0.2；

The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by the battalion of 1:500/1:100 It supports meat soup (NB) to mix with 85% sterile saline solution, adjusts pH to 7.0~7.2；

(6) ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction reagent- 20 DEG C~-70 DEG C preservations, use in 6 months；

Dilution buffer: 0.005mol/L and the disodium phosphate soln for containing 0.037% sucrose adjust pH to 7.2 ± 0.2； 121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；

ATP standard reagent stoste: by 60.5mg trinosin (C₁₀H₁₄O₁₃P₃Na₂·3H₂O it) is dissolved in 100mL water, Being configured to concentration is 1 × 10^-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；

ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg Magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose dissolve by heating in 250mL water, adjust Save pH to 7.5 ± 0.2；It is used in 8h；

ATP lysate: by 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 international units/ml Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, that 20mg bovine serum albumin is dissolved in 10mL is dense Degree is in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L, and adjusting use in pH to 6.0 ± 0.5,8h, (1mL lysate exists The ATP concentration in nutrient broth can be down to 10 in 15min^-13Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, the benzene for being 10% with 0.2ml concentration After pricking the mixing of oronain solution, pH to 12.0 ± 0.5 is adjusted；

ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg bovine serum albumin It is dissolved in the ATP fluorescent reagent buffer solution of 30mL, 15min is stored at room temperature after mixing, is used in 3h.

4. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the strain is protected Hiding, activation and bacteria suspension preparation, carry out in the steps below:

(1) test strain: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or other strains that is provided and can be traced to the source by national corresponding culture presevation administrative center)；

(2) culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (salmonella is added with capillary syring With BHI broth), pressure-vaccum make for several times strain melt dispersion, then, by a little test strain suspension instill equipped with 5mL~ In the test tube of 10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (is no more than 1 month), inoculation times were no more than for 14 generations；

(3) actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours；Daily Switching 1 time, continuously switching is no more than 15d, and test is using the 3rd generation~the 14th generation and in the interior Fresh bacterial cultures transferred for 24 hours；

(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain culture is added Bacteria suspension is made in liquid；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that and bacterial suspension is uniform, Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, with 0.7mm core black gel ink pen in A6 white The parallel lines that 3 length are 10cm are drawn on copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 10⁸CFU/mL) Differences in turbidity, be added dropwise culture solution until the two turbidity it is identical until.

5. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the ATP is dense Spend logarithm lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration, is pressed Following step carries out:

(1) standard serial solution is determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10^-3mol/ The ATP standard stock solution of L is diluted to high and low concentration standard serial solution: 7.0 × 10^-9mol/L、7.0×10^-8mol/L、7.0× 10^-7Mol/L is (if instrument upper limit of detection is unable to reach 10^-7Mol/L magnitude or high standard curve it is linear poor, can will Its ATP concentration series is changed to 7.0 × 10^-10mol/L、7.0×10^-9mol/L、7.0×10^-8) and 4.2 × 10 mol/L^-11mol/ L、4.2×10^-10mol/L、4.2×10^-9Mol/L is simultaneously mixed, then, with sterilizing liquid-transfering gun that the ATP standard series of 0.1mL is molten Liquid is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose and mixing is successively added dropwise；Again will The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes, parallel as the test of ATP bioluminescence Sample；

(2) relative intensity of fluorescence value I_AMeasurement: according to relative intensity of fluorescence value I in this patent_AMeasuring method is trained with test strain After nutrient solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, marked to various concentration ATP The ATP that 0.1mL is added dropwise in three Duplicate Samples of quasi- solution respectively extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；Again Secondary mixing, immediately with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and record (ensure that each link operating time is consistent, Avoid cross contamination), each Duplicate Samples minute is no more than 15s, relatively glimmering with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of light intensity value is its I_AMeasured value；

(3) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with ATP standard serial solution Relative intensity of fluorescence logarithm lgI_AAs abscissa, with its corresponding ATP log concentration value lgC_AFor ordinate mapping；It is right Mathematical relationship between the two carries out calibration curve, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application is minimum Two multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and phase relation Number R_A0 ²(high concentration), R_A ²(low concentration), works as R_A0 ²And R_A ²>=0.98, when confidence level >=0.95, the survey done according to this patent It is fixed effective；

(4) it is inoculated with the viable bacteria ATP concentration C of bacterium solution_ACalibration: according to relative intensity of fluorescence value I in this patent_AMeasuring method, to through wheat Bacteria total amount is 6.0 × 10 after family name's turbidimetry primary dcreening operation⁸The relative intensity of fluorescence value I of 10 times of gradient dilution liquid of bacteria suspension of CFU/mL_A It is measured；According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates its viable bacteria ATP concentration C_A, through test organisms Kind culture solution adjusts to obtain C_ARange is 5.0 × 10^-8Mol/L~9.0 × 10^-8The inoculation bacterium solution of mol/L, measures and records 0.3mL is inoculated with relative intensity of fluorescence value I of the bacterium solution after the dilution of 4.7mL eluent in 1min_A0。

6. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the sample connects Kind, culture and elution recycling, carry out in the steps below:

(1) inoculated and cultured: it is put into the multilayer sterile gauze being of moderate size in sterilized petri dishes bottom, 10mL sterile saline is inclined It infuses in making its saturation of just soaking on gauze；It contacts sample for 24 hours for 6 groups to be placed on sterile gauze, 6 groups of 0h contact samples are placed in not In the sterilized petri dishes of yarn releasing cloth, 0.3mL is inoculated with bacterium solution (ceramics sample of the water absorption rate greater than 1.0%, bacterium solution with sterilizing liquid-transfering gun In can add the inertia such as agar thickener) be added dropwise on each group control sample and antimicrobial sample glaze to be measured respectively (with curve lgC_A-lgI_ACalibration is derived from same branch test strain stoste test tube with bacterium solution, and 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used), with L stick (attachment is inoculated with bacterium solution but does not hang drop) smears bacterium solution uniformly, so that its is covered the entire glaze of sample, covers ware lid, use medical adhesive tape Sealing contacts the plate of sample equipped with 6 groups for 24 hours；(37 ± 1) DEG C, (90 ± 2) %RH culture ± 2h for 24 hours；

(2) elution recycling: using 4.7mL test strain culture solution as eluent (0.2% sterile surfaces activating agent can be added), 6 After group 0h contact sample inoculation bacterium, sufficiently eluted using solid or liquid swab method immediately；6 groups of samples cultivated for 24 hours Bacterium is recycled using type of elution identical with 0h contact sample, solid swab method is to be soaked with the sterile of saturation eluent with head Cotton swab is uniformly embrocated 10 times in each sample surfaces back and forth anyhow respectively, and rotates with it cotton swab；Cut off the contact of experimenter's hand Cotton swab is placed in the sterile test tube equipped with remaining eluent, as recovered liquid (if recovered liquid is insufficient after mixing by partial swab stick 5mL, addition eluent to 5mL), liquid swab method draw 4.7mL eluent, the repeated flushing in plate with sterilizing liquid-transfering gun It each sample surfaces at least 4 times, sufficiently elutes and moves into washing lotion in sterile test tube, as recovered liquid (if recovered liquid after mixing Less than 5mL, eluent is added to 5mL).

7. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the recovered liquid Relative intensity of fluorescence value I_AMeasurement carries out in the steps below:

(1) instrument and reagent set relative intensity of fluorescence background value calibration: with sterilizing liquid-transfering gun by 0.1mL test strain culture solution, The ATP lysate of phosphate buffer solution and 0.1mL of the 0.8mL containing 0.037% sucrose is separately added into same branch sterile test tube And mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, standing 5min~ 30min, as skip test Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (ensuring that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values)；

(2) reclaim liquid phase is to fluorescence intensity level I_AMeasurement: if blank reagent group background level meets instrument requirement, with sterilizing The ATP lysate of phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose is distinguished It is added in same branch sterile test tube, the above-mentioned mixed solution of 0.1mL is successively moved into three sterile examinations of instrument after mixing well Guan Zhong stands 5min~30min, and as ATP fluorometric investigation Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples is mentioned Reagent is taken, the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, with ATP fluorescent spectrophotometer measuring, it is relatively glimmering immediately Light intensity value I_AAnd record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute does not surpass 15s is crossed, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquid_AMeasured value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the relatively glimmering of three Duplicate Samples of recovered liquid Light intensity value I_A)。

8. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the recovered liquid Viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated, carry out in the steps below:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, calculate control sample and antimicrobial sample After contacting through 0h and cultivating for 24 hours, the viable bacteria ATP concentration C of recovered liquid_AAnd T_A；Relevant calculation (when using instrument matched reagent box, root The viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volume_AAnd T_A) see formula (1)~(12):

In formula:

C_A0And C_At- 3 groups of 0h contact and after cultivating for 24 hours control sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；

C_A0ijAnd C_Atij- every 0h contact and after cultivating for 24 hours control sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_A0And T_At- 3 groups of 0h contact and after cultivating for 24 hours antimicrobial sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；

T_A0ijAnd T_Atij- every 0h contact and after cultivating for 24 hours antimicrobial sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

(2) condition for validity is tested

If the recovered liquid and 0.3mL inoculation bacterium solution of every 0h contact control sample are after the dilution of 4.7mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after cultivating for 24 hours The logarithm of definite valueThat is C_Atij≥0.1×C_A0ij, then when 3 groups of 0h contact control sample reclaim liquid phases are to glimmering Luminous intensity measured valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent measurement of correlation Uncertainty requirement), the measurement carried out according to this patent method is effective；

(3) bacterium increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value F_ij、G_ijIt is counted respectively according to formula (13), (14) It calculates:

In formula:

F_ijAnd G_ijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of every anti-bacteria ceramic sample_ij, every group and every batch of sample antibiotic rate R_iDistinguish with R It is calculated according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of-every anti-bacteria ceramic sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4, 5；

R-every batch of anti-bacteria ceramic sample antibiotic rate, %；

C_AtijAnd T_Atij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, and unit is Mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every antimicrobial sample and control sample after cultivating for 24 hours, the relative intensity of fluorescence measured value of recovered liquid, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of every anti-bacteria ceramic sample_ij, every group and every batch of sample antibacterial activity value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

A_ijThe antibacterial activity value of-every anti-bacteria ceramic sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4, 5；

A-every batch of anti-bacteria ceramic sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(6) data revision of the convention requirement: in the concentration of calibration ATP standard serial solution and the viable bacteria of inoculation bacterium solution, sample recovered liquid ATP concentration C_AWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_AWhen not less than 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind use 0 replaces digit；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals, control sample and antibacterial afterwards After sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate R_ij、R_i, R calculated result Take three effective digitals；Bacterium increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method mainly passes through calculating control sample and antimicrobial sample and contacts through 0h and cultivate for 24 hours Afterwards, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV count Calculate result and remain into 2 significant digits), judge the weight that ATP fluorimetric assay for biological materials method is applied to ceramic antibacterium performance test Existing property；In regulation group and between-group variation coefficient CV≤10%；

5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after cultivating for 24 hours that every group of 0h is contacted Product, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithAfter respectively every group of control sample and antimicrobial sample are contacted through 0h and cultivated for 24 hours, respectively The relative intensity of fluorescence measured value of Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after cultivating for 24 hours of 3 groups of 0h contact, 15 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after cultivating for 24 hours that every group of 0h is contacted Product, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithAfter respectively every group of control sample and antimicrobial sample are contacted through 0h and cultivated for 24 hours, respectively The relative intensity of fluorescence measured value of Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after cultivating for 24 hours of 3 groups of 0h contact, 15 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

9. the detection method of anti-bacteria ceramic bacteria resistance energy according to claim 1, which is characterized in that the result is commented Valence carries out in the steps below:

If using antibiotic rate R as correlated performance evaluation index: working as R_ij/R_i(staphylococcus aureus and sand when/R < 90% Door Salmonella R_ij/R_i/ R < 80%), sample is without antibacterial actions；As 90%≤R_ij/R_i(staphylococcus aureus when/R < 99% With 80%≤R of salmonella_ij/R_i/ R < 90%), sample has antibacterial actions；As 99%≤R_ij/R_iWhen/R < 99.9% (90%≤R of staphylococcus aureus and salmonella_ij/R_i/ R < 99%), sample antibacterial actions are stronger；Work as R_ij/R_i/R≥ (staphylococcus aureus and salmonella R when 99.9%_ij/R_i/ R >=99%), sample antibacterial actions are extremely strong；

If using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 1.0 (staphylococcus aureus and Salmonella A_ij/A_i/ A < 0.5), sample is without antibacterial actions；As 1.0≤A_ij/A_iWhen/A < 2.0 (staphylococcus aureus and 0.5≤A of salmonella_ij/A_i/ A < 1.0), sample has antibacterial actions；As 2.0≤A_ij/A_i(golden yellow Portugal when/A < 3.0 1.0≤A of grape coccus and salmonella_ij/A_i/ A < 2.0), sample antibacterial actions are stronger；Work as A_ij/A_iIt is (golden yellow when/A >=3.0 Staphylococcus and salmonella A_ij/A_i/ A >=2.0), sample antibacterial actions are extremely strong；

(2) as the antibiotic rate R of certain group (part) anti-bacteria ceramic sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups (four) When the bacteria resistance of sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test；Calculate it Through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back anti-bacteria ceramic Sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity Value A_i(A_ij) arithmetic mean of instantaneous value as batch (group) anti-bacteria ceramic sample bacteria resistance can evaluation result.