CN110272961A - ATP bioluminescence lgCA-lgIAThe method of calibration curve method detection anti-bacteria stainless steel anti-fungal property - Google Patents

ATP bioluminescence lgCA-lgIAThe method of calibration curve method detection anti-bacteria stainless steel anti-fungal property Download PDF

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CN110272961A
CN110272961A CN201910199764.0A CN201910199764A CN110272961A CN 110272961 A CN110272961 A CN 110272961A CN 201910199764 A CN201910199764 A CN 201910199764A CN 110272961 A CN110272961 A CN 110272961A
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atp
culture
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solution
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李文杰
崔宗岩
曹彦忠
张进杰
杨志伟
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INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF QINHUANGDAO ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/60—Chemiluminescent detection using ATP-luciferin-luciferase system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/385—Assays involving biological materials from specific organisms or of a specific nature from fungi from Penicillium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida

Abstract

The present invention relates to a kind of anti-bacteria stainless steel anti-fungal property detection method, detecting step includes: sample preparation and pretreatment；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and bacteria suspension preparation；Bacteria suspension viable bacteria content C after methylene blueBBlood cell plate counts；ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAMark Qu Jianli and inoculation bacterium solution viable bacteria CACalibration；Sample inoculation, culture and elution recycling；Recovered liquid IAMeasurement and its viable bacteria ATP concentration CAAnd TAIt 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 stainless steel antifungal activity 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/48h, measure its IAAnd with lgIACharacterization and calculating R or A；Evaluation of result foundation is provided.The ATP bioluminescence lgC for the stainless steel antifungal activity detection that the present invention researches and developsA‑lgIABent method is marked, effectively product quality will be supported to be promoted.

Description

ATP bioluminescence lgCA-lgIACalibration curve method detects anti-bacteria stainless steel antifungal activity The method of energy
Technical field
The present invention relates to the anti-fungal property test method of anti-bacteria stainless steel, specifically a kind of application ATP fluophotometer The ATP biology that can be carried out accurate quantitative detection to the anti-bacteria stainless steel antifungal activity with antibiotic rate R or antibacterial activity value A characterization is glimmering Light lgCA-lgIACalibration curve method belongs to stainless steel antibacterial functions detection technique field.
Background technique
Stainless steel is closely bound up with human lives, is widely used in all various aspects such as industry and the people's livelihood；In recent years, by science and technology Progressive and consumption demand traction, as new structure/function integration material coating type anti-bacteria stainless steel, surface modified version Anti-bacteria stainless steel, composite bactericidal stainless steel, alloy-type anti-bacteria stainless steel come into being.It is predicted according to authoritative institution, 70% or more The stainless steel market in the fields such as Medical Devices, kitchen bathroom, public utility can be substituted by anti-bacteria stainless steel；Along with antimicrobial technology In the application of stainless steel industry and universal, relevant standardisation requires gradually to bring into schedule.
Currently, anti-biotic material performance detection technical system is mainly for bacterium and fungi, various countries' anti-mycotic efficiency both at home and abroad Test method principle rarely has mostly based on the colony counting method being separately cultured to Candida albicans and is related to mould person；Wherein fit With Candida albicans test first is that the dipping quantitative test method that Japanese Industrial Standards propose；Two are derived from U.S. textile industry It is antibacterial around-France；Third is that international oscillation flask method；Four are derived from the dropping method of U.S. textile enterprise；Fifth is that Japanese enterprises needle To the coverslip method of photocatalyst-type anti-biotic product research and development.Although China has issued and implemented the " evaluation method of disinfection and sterilization effect With standard ", " daily chemical products are anti-by GB 15979-2002 " Disposable Sanitary Accessory sanitary standard " and QB/T 2738-2012 The evaluation method of bacterium fungistatic effect " etc. relevant criterions, but its defined test period between 48 hours~72 hours, the time and Economic cost is high.In addition, international anti-mold effect assessment technique system is originated from American Standard, day mark and Europe superscript, external institute substantially for many years Relating to standard method mainly includes that soil buries cultivation, agar plate method, moist chamber culture method etc.；China is based on plate culture and suspension Method successively formulates GB/T 1741-2007 " paint film fungus resistance measuring method ", GB/T 24346-2009 " textile fungicidal properties Evaluation ", FZ/T 60030-2009 " household textiles fungicidal properties test method ", GB/T 24128-2009 " plastic mould-proof Method for testing performance ", QB/T 4199-2011 " Leather mildew-proof performance test methods ", HG/T 4301-2012 " rubber mildew resistance Can test method ", the standards such as LY/T 2230-2013 " evaluation of wood-based plate fungicidal properties "；But related stainless steel anti-fungal property inspection It surveys method standard and still belongs to blank.Due to the mycelium that is formed in mycotic spore growth course can not accurate counting, can only be determined Sex determination, can not quantitative test；And because the fungus growth period is longer, incubation time at least 2 weeks~4 weeks, cause test period compared with Long, time and economic cost are high.No matter Candida albicans or mould are directed to, the experimentation of existing standard method is cumbersome, skill Art difficulty is higher；Relevant operation is affected by human factors greatly, so that test error is big, lacks comparativity.In recent years, international thin The development of bacterium detection technique field ATP fluorescence analysis is increasingly mature, and testing result correlation is 98% compared with traditional Plating, Accuracy is high and can realize quick detection.External anti-biotic material performance detection technical field of research for current quantification, quickly Change and summary development trend have used for reference ATP fluorescence analysis principle and have formulated ISO 20743:2007-2013 " Textiles- Determination of antibacterial activity of textile products (spin by textile-antibiotic finish The antibacterium performance measurement of fabric) " and ISO 13629-1:2012 " Textiles-Determination of antifungal Activity of textile products.Part1 Luminescence (textile-antibiotic finish textile mildew resistance Can measurement) ", it is specified that with the fluorimetry of anti-thin/mould performance of ATP changes of contents characterization after sample inoculation, but it is only fitted For with water imbibition and control sample it is thin/textile material or poromerics of mould increasing value > 0, in viable bacteria way of recycling, connect It is not suitable for that there is unwetted property hard surface and control sample fungi in terms of the key technologies such as kind bacterial concentration, test condition for validity The inorganic non-metallic materials such as stainless steel, the ceramics of increasing value < 0；Uncertainty of measurement assessment is not provided simultaneously.In addition, the standard Method dependent antimicrobial performance characterization parameter is more single, only relates to antibacterial activity value A, and China then usual antibiotic rate R.
Therefore, to resist foreign technology barrier, promote successive generations of products, push 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 stainless steel Performance Testing Technology.The art of this patent highway route design connects Rail is international, and detection method belongs to the whole world and initiates, 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 stainless steel antifungal activity can be carried out the ATP bioluminescence lgC of detectionA-lgIACalibration curve method can solve Certainly anti-bacteria stainless steel or even the accurate quantitative test problem of other field anti-biotic material and product anti-fungal property.
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 stainless steel anti-fungal property, comprising: (1) sample preparation and pretreatment；(2) equipment is selected Type and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) bacteria suspension viable bacteria content after methylene blue CBBlood cell plate count；(5) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium The viable bacteria ATP concentration C of liquidACalibration；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level IAIt surveys It is fixed；(8) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；Its feature It is, the anti-bacteria stainless steel antifungal activity with antibiotic rate R or antibacterial activity value A characterization can be carried out using ATP fluophotometer The ATP bioluminescence lgC of accurate quantitative testA-lgIACalibration curve method, specific:
In reclaim liquid phase to fluorescence intensity level IAIn measurement:
Quantity, size and the pre-treatment requirement for specifying control sample and antimicrobial sample, by the standard bacteria of mycologic test strain After strain is passed on, activated, fresh Candida albicans or mycotic spore culture is taken to prepare bacteria suspension；Blood is used after methylene blue Ball count plate measures viable bacteria content C in bacterium solutionB, and C is carried out to the bacteria suspension of different dilutionsBCalibration.Then, it is tried with ATP fluorescence Agent buffer solution is 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- 8mol/L、7.0×10-7mol/L、7.0×10-6Mol/L and 2.1 × 10-9mol/L、2.1×10-8mol/L、2.1×10-7mol/ L measures its relative intensity of fluorescence value IA；Draw the lgC of two respective concentrationsA-lgIAStandard curve is derived from curvilinear equation Formula Y=aA0X+bA0(high concentration), Y=aAX+bA(low concentration) and linearly dependent coefficient RA0 2(high concentration), RA 2(low concentration).Together When, with Cha Shi culture solution to viable bacteria content CBIt is 1.0 × 108CFU/mL~5.0 × 108The test strain suspension of CFU/mL into The continuous 10 times of gradient dilutions of row, the relative intensity of fluorescence value I of measurement dilution bacterium solutionA, according to high standard fitting equation Y= aA0X+bA0Calculate its viable bacteria ATP concentration CA；It is adjusted to obtain CAIt is 5.0 × 10-7Mol/L~9.0 × 10-7The inoculation bacterium of mol/L Liquid.0.3mL inoculation bacterium solution is added dropwise respectively to each group sample surface to be measured, sample is contacted to 6 groups of 0h immediately using 4.6mL eluent Elution recycling is carried out, the relative intensity of fluorescence value I of recovered liquid is measuredAC0ij、IAT0ij, and according to low concentration calibration curve equation formula Y =aAX+bACalculate its viable bacteria ATP concentration CAOijAnd TAOij.Meanwhile under the damp condition of 95%RH ± 2%RH, by 6 groups of sealings In in sterilized petri dishes control sample and antimicrobial sample in (30 ± 2) DEG C (Candida albicans) culture ± 2h or (28 ± 2) DEG C for 24 hours After (mould) cultivates 48h ± 2h；Recycling remained on surface bacterium is eluted using mode identical with 0h contact sample and measures its recycling The relative intensity of fluorescence value I of liquidACtij、IATtij, calculate corresponding viable bacteria ATP concentration CAtijAnd TAtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, contacted with 0h and for 24 hours or 48h The relative intensity of fluorescence measured value of every control sample and antimicrobial sample recovered liquid under condition of cultureWith As basic data；Fungi increasing value F in the case where testing condition for validity, after calculating its cultureij、GijAnd antibiotic rate RijWith it is anti- Bacterium activity value Aij；To the R of every group of sampleijAnd AijArithmetic mean of instantaneous value is taken to obtain corresponding RiAnd Ai；Every batch of anti-bacteria stainless steel sample Antibiotic rate R and antibacterial activity value A be its three groups of sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and measurement Uncertainty requirement；
In evaluation of result:
With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that anti-fungal property classification determines mark It is quasi-；As the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups (four) samples When the anti-fungal property of product is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Calculate its warp ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) antibacterial is not Steel sample anti-fungal property level of becoming rusty is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or antibacterial Activity value Ai(Aij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the anti-bacteria stainless steel sample anti-fungal property.
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, precisely can quickly to survey Determine the viable bacteria amount recycled after stainless steel sample culture specific time, reaches the modernization of stainless steel anti-fungal property detection；It can Human factor in experimentation, which is effectively reduced, to be influenced, and traditional plate culture qualitative analysis limitation is breached；Realize detection knot The quantification of fruit, and greatly improve the accuracy of detection data；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 lgCA- relative intensity of fluorescence logarithm lgIAStandard curve；Construct anti-bacteria stainless steel anti-fungal property ATP bioluminescence The mathematical model of real-time quantitative analysis method.Meanwhile country variant consumer perceptions habit is taken into account, take antibiotic rate R and antibacterial Activity value A is correlated performance evaluation index, improves the science and versatility of detection method.
(3) innovative: with existing operation is numerous, the period is long, compared with the conventional method of non-quantitation, 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 stainless The precision of steel anti-fungal property testing result and quantification simultaneously have good reproducibility and comparativity；Substantially shorten simultaneously and surveys 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 lgCA、lgIAStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Candida albicans and mycotic spore suspension viable bacteria concentration measuring method are enriched, control sample, standard liquid concentration, measurement step are specified Suddenly, the technology contents such as calculation formula, uncertainty；The pioneering recovered liquid viable bacteria relative intensity of fluorescence value I directly measured with instrumentA It evaluates form as a result to calculate and determine antibiotic rate R or antibacterial activity value A, and passes through a group interior and between-group variation coefficient CV Uncertainty of measurement is investigated, technically there is centainly perspective.
(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, the methylene blue that this patent is established Blood cell plate living bacterial cells or spore concentration measuring method and the ATP fluorometric investigation method of stainless steel anti-fungal property are easy easily after dyeing Row, description of Related Art is clear and specific, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for The horizontal Experiment on Microbiology personnel of different majors 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 inspection, learn, grind, produces all circles promote and apply, can support stainless steel anti-fungal property detection technique realization generalization, simultaneously Reference can be provided to the product scopes anti-fungal property 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 not having antibacterial coating, classification, metallic matrix, technique and the appearance of control sample, Size, quantity etc. are identical with anti-bacteria stainless steel sample to be measured；Each strain test uses 6 groups of samples；Wherein 0h contact and For 24 hours or 48h culture experiment respectively uses 3 groups, every group of 5 samples；
(2) antimicrobial sample: after the processing of the electrochemical methods such as electroplated, chemical plating, antibacterial coating is formed not on surface Become rusty Steel material and product；Sample cover surface is uniform and has certain adhesive strength, no any damage for reaching substrate metal； Having a size of (50 ± 2) mm × (50 ± 2) mm, thickness is not more than 5mm.Antimicrobial sample quantity is identical as control sample, and every group to be measured Sample selects one group of control sample as object of reference and effectively identifies；
(3) sample pre-treatments: impregnating 5min in 75% ethanol solution for control sample and antimicrobial sample before experiment, and With the abundant cleaning sample of sterile water to remove ethyl alcohol on superclean bench；Sample surface to be measured is put into upwards after natural drying It is 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 degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-11Mol/L~10-6mol/L；Amplification factor 40 ×~400 × Photobiology microscope；(20~ 50) DEG C ± 1 DEG C, the constant temperature and humidity incubator of (50~95) %RH ± 2%RH；(0~50) DEG C ± 1 DEG C, (50~300) r/min Constant temperature oscillator；Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The rotation of the range of speeds (500~3000) r/min Whirlpool oscillator；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~ 10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency range (30~50) kHz；Precision ± 0.1 The pH meter of (25 DEG C)；Electric furnace；
(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；Glass funnel；Sterile rotation Fill in test tube；Diameter is not more than the oese of 4mm；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；With In the absorbent cotton and gauze of biochemistry detection；Aseptic filter paper；Thermometer；The stopwatch of precision 0.01s；
(4) reagent: 75% ethanol solution；0.1% Lv Shi alkaline methylene blue dyeing liquor；121 DEG C after following reagent packing High pressure sterilization 30min, 5 DEG C~10 DEG C storage 30d:N monomethyl ethanesulfonic acids, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, It is configured to 0.05% wetting agent solution；85% physiological saline；
(5) medium/liquid (commercially available medium/liquid can be used): 121 DEG C of high pressure sterilizations after matched medium/liquid packing 30min, 2 DEG C~8 DEG C storage 30d；
Sabouraud culture medium/liquid (the actication of culture use of Candida albicans): 40g glucose, 10g peptone, 20g agar are added Heat of solution (agar is not added in culture solution) in 1000mL water is adjusted pH to 5.6 ± 0.2 (25 DEG C)；
Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen Dipotassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose are dissolved by heating in 1000mL containing 0.05% wetting In the aqueous solution of agent, adjust pH to 6.0~6.5 (25 DEG C)；
One dextrose culture-medium of potato (mycotic spore actication of culture use): removing the peel stripping and slicing for 300g fresh potato, 20min~30min is boiled in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g chloramphenicol, 20g agar are added into filtrate After be settled to 1000mL；
(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 Sharpe culture solution can be down to 10 in 15min by liquid-11Mol/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 (fungal cell's ATP extraction efficiency be not less than 80%)；
ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h；
Culture presevation, activation and the bacteria suspension preparation, carries out in the steps below:
(1) test strain: Candida albicans ATCC 10231；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205； Penicillium chrysogenum ATCC 9179 (or other strains that is provided by national Culture Collection and can be traced to the source)；
(2) culture presevation: Candida albicans --- freeze-drying lactobacillus pipe is opened with sterile working, is infused with capillary syring into pipe Enter appropriate Sharpe culture solution, pressure-vaccum makes strain melt dispersion for several times；A little bacteria suspension is instilled and is trained equipped with 5mL~10mL Sharpe In the test tube of nutrient solution, 37 DEG C of culture 18h~for 24 hours.Mould --- mould test strain is inoculated in by potato-with sterile working The inoculation date is simultaneously indicated in dextrose culture-medium inclined-plane, and 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3℃ ~10 DEG C preservation 4 months, as preservation bacterium；
(3) actication of culture: Candida albicans --- with the colonies typical in oese scraping 1st generation culture, scribing line is connect Kind is in sabouraud culture medium plate；After 37 DEG C of culture 18h~for 24 hours, the colonies typical in picking 2nd generation culture is inoculated in Sharpe training Support base inclined-plane；After 37 DEG C of culture 18h~for 24 hours, 4 DEG C of sealings storages, use in 6 weeks.Mould --- preservation bacterium is scraped with oese Spore is inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d, until generating a large amount of spores.Preparation Must not extract the test tube plug of mold species before spore suspension, every test tube open after only for spore liquid of preparation, every time Suspension is prepared using the mycotic spore newly cultivated；
(4) prepared by bacteria suspension: Candida albicans test --- with oese, the fresh microbial strain culture of picking is connect from inclined-plane Kind in the sterile conical flask equipped with 50mL Sharpe culture solution, it is placed in 150r/min culture in the constant temperature oscillator of (30 ± 1) DEG C 18h~for 24 hours, 4 DEG C of sealing storages, the same day uses.Mould test --- the sterile water of 10mL is added into strain test tube, with inoculation Ring gently scrapes the fresh mycotic spore of media surface, and the spore stoste injection of wash-off is trained equipped with 15 beades and 45mL Cha Shi In the sterile conical flask of jumping a queue of nutrient solution, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, it will cover There are sterile absorbent cotton or the glass funnel of eight layers of gauze to be placed on conical flask, filtering spore suspension removes mycelia and culture medium is broken Piece；Filtrate is moved in sterile centrifugation tube, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；50mL is used again Cha Shi culture solution cleaning spore sediment is simultaneously centrifuged, and after repeated washing 3 times, is precipitated with the spore after the dilution centrifugation of Cha Shi culture solution Object.Every kind of test mould prepares spore suspension according to the method described above, and the spore liquid of each strain is mixed in equal volume；0 DEG C~7 DEG C storage uses in the same day or 4d；
Bacteria suspension viable bacteria content C after the methylene blueBBlood cell plate count, in the steps below carry out:
(1) methylene blue: with sterile working by 50 μ L dilutions for 10-2~10-3Candida albicans bacterium suspension or mould The Lv Shi alkaline methylene blue dyeing liquor that mixing spore liquid and 30 μ L concentration are 0.05% moves to (bacterium solution in same branch sterile test tube respectively Dilution, which includes 4~5 albicans cells or mycotic spore with each lattice of blood counting chamber, to be advisable), 1000r/min Test tube 1min is shaken, mixes well bacteria suspension with dyeing liquor；
(2) blood cell plate counts: the bacteria suspension after ± 0.5 μ L of 5 μ L dyeing being placed in coverslip edge with aseptic straw, makes it Blood counting chamber is slowly penetrated along slide gap, bubble is not can produce between tally and slide, otherwise re-operates.With sterile Extra bacterium solution in filter paper exhaustion slot, stands 2min ± 20s, is settled down to its whole in counting chamber.When lattice tally in use 16 When, upper left, upper right, lower-left, the albicans cell in the middle lattice in bottom right 4 (i.e. 100 lattices) are taken in diagonal orientation Or mycotic spore is counted；Lattice tally in 25 is such as used, in addition to counting above-mentioned 4 diagonal orientations, also needs to count the 1 of center A middle lattice (i.e. 80 lattices)；When test strain is located on the two-wire of middle lattice, then only count online and right line or it is offline and Albicans cell or mycotic spore on left line；
(3) viable bacteria content CBCalculate: by Photobiology microscope magnification from it is low to lofty tone to 400 × after, it is right immediately Albicans cell or mycotic spore at blood counting chamber additional space position are counted；Wherein colourless Candida albicans Bacterium cell is viable bacteria, and blue or light blue person is dead bacterium；Edge is in navy blue, inside in colourless or light blue and pink mould Bacterium spore is viable bacteria, and edge and inside are dead bacterium in navy blue person, therefore the only counting rim spore different from internal color.If Quantity without mycelia monospore in mycotic spore suspension is lower than 90%, then prepares spore liquid again.It is each to blood counting chamber small Albicans cell or mycotic spore in grid repeat microscopy and count three times, are averaged.When blood counting chamber specification is When 16 × 25, viable bacteria content (every milliliter of Colony Forming Unit, CFU/mL) C of 1mL bacterium solutionB5 × 16 × K × 10=N ÷4；Work as blood When ball count plate gauge lattice are 25 × 16, CB5 × 25 × K × 10=N ÷4；Wherein N is that white is read in five middle lattice of blood counting chamber The total viable count of pearl bacterium cell or mycotic spore, K are bacterium solution extension rate.Then, with Cha Shi culture solution by known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid be diluted to 1.0 × 108CFU/mL~5.0 × 108CFU/mL；
The ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution is living Bacterium ATP concentration CACalibration carries out in the steps below:
(1) determination of ATP standard serial solution and its test specimens preparation: with ATP fluorescent reagent buffer 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 × 10-8mol/L、7.0×10-7mol/ L、7.0×10-6Mol/L and 2.1 × 10-9mol/L、2.1×10-8mol/L、2.1×10-7Mol/L is simultaneously mixed well.Then, it uses Sterilizing liquid-transfering gun the above-mentioned ATP standard solution of 0.1mL is moved to respectively in three sterile test tubes, successively be added dropwise 0.05mL sterile water and 0.35mL normal saline solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution is surveyed again Sample is moved to respectively in three sterile test tubes, each that 0.4mL physiological saline is added dropwise and mixes, and is tested as second level ATP standard solution Sample；And with sterilizing liquid-transfering gun by the second level ATP standard solution test specimens of 0.1mL various concentration move to respectively three instruments without In bacterium test tube, Duplicate Samples are tested as ATP standard solution；
(2) blank background value calibration: the aqueous solution, 0.35mL with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent are raw The ATP lysate of reason salt water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；It stands Level-one blank sample is used as after 10min~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, is added dropwise 0.4mL physiological saline simultaneously mixes, as second level blank sample.Then, 0.1mL second level blank sample is moved to respectively with sterilizing liquid-transfering gun In three instrument sterile test tubes, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples is mentioned Reagent is taken, instills the ATP fluorescent reagent of 0.1mL after mixing again.3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and examination Agent group background values (or calibrating background according to instrument operation instruction)；
(3) ATP standard solution relative intensity of fluorescence value IAMeasurement: from low to high according to concentration, to various concentration ATP The ATP that 0.1mL is added dropwise in three test Duplicate Samples of standard solution respectively extracts reagent, and the ATP for instilling 0.1mL after mixing again is glimmering Light reagent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (really It is consistent to protect each link operating time, avoids cross contamination).Each Duplicate Samples minute is no more than 15s, with each concentration ATP mark The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of quasi- solution is its IAMeasured value；
(4) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with various concentration ATP The relative intensity of fluorescence logarithm lgI of standard solutionAAs abscissa, with its ATP log concentration value lgCAFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrationsA-lgIAStandard curve；And application is most Small two multiply the linear equation Y=a that fitting process is derived from curveA0X+bA0(high concentration), Y=aAX+bAIt is (low concentration) and related Coefficients RA0 2(high concentration), RA 2(low concentration).Work as RA0 2And RA 2>=0.98, it when confidence level >=0.95, is done according to this patent Measurement is effective；
(5) it is inoculated with bacterium solution viable bacteria ATP concentration CACalibration: with Cha Shi culture solution to viable bacteria content CBIt is 1.0 × 108CFU/mL ~5.0 × 108After the test strain suspension of CFU/mL carries out continuous 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA； According to high standard fitting equation Y=aA0X+bA0It calculates and demarcates corresponding viable bacteria ATP concentration CA.Through Cha Shi culture solution tune It is whole, obtain CARange is 5.0 × 10-7Mol/L~9.0 × 10-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1minA0；
Sample inoculation, culture and the elution recycling, carries out in the steps below:
(1) 0.3mL inoculated and cultured: is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured with sterilizing liquid-transfering gun Bacterium solution is inoculated with (with lgCA-lgIACalibration curve is derived from same branch test strain stoste test tube with bacterium solution, and 0 DEG C ± 1 DEG C saves, in 2h Using), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), its is made to cover sample whole surface.Cover ware Lid will be equipped with 6 groups for 24 hours with medical adhesive tape or the plate of 48h contact sample seal；Under the damp condition of (95 ± 2) %RH, (30 ± 2) DEG C culture ± 2h (Candida albicans) or (28 ± 2) DEG C culture 48h ± 2h (mould) for 24 hours；
(2) it elution recycling: after 6 groups of 0h contact sample inoculation Candida albicans or mould, is drawn immediately with sterilizing liquid-transfering gun 4.6mL eluent (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously Washing lotion is moved into sterile test tube (with mould washing lotion in liquid transfer gun head repeatedly pressure-vaccum plate, until lawn broken up after again by it Move into test tube), 3000r/min shakes test tube 2min；As sample to be tested recovered liquid (if recovered liquid is insufficient after mixing well 4.9mL, addition eluent to 4.9mL).Each group uses elution identical with 0h contact sample through the sample for 24 hours or after 48h culture Mode recycles fungi；
The reclaim liquid phase is to fluorescence intensity level IAMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: according to lgCA-lgIABlank when standard curve is established Background calibration methods, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values；
(2) reclaim liquid phase is to fluorescence intensity level IAMeasurement: if background level meets instrument requirement, with sterilizing liquid-transfering gun The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube, 3000r/min shakes test tube 30s； After being stored at room temperature 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min.It will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence.To The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquidAMeasured value；
The recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R or antibacterial activity value A is calculated, by following steps It is rapid to carry out:
(1) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate that each group sample connects through 0h Touch for 24 hours or 48h culture after recovered liquid viable bacteria ATP concentration CAAnd TA.Relevant calculation is shown in formula (1)~(12):
In formula:
CA0And CAt- 3 groups of 0h are contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, single Position is mole every liter (mol/L)；
CA0iAnd CAti- every group 0h is contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, Unit is mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtij- every 0h is contacted and control sample recycles the ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It 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 0h is contacted and the relative intensity of fluorescence of control sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAt- 3 groups of 0h are contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, single Position is mole every liter (mol/L)；
TA0iAnd TAti- every group 0h is contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, Unit is mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtij- every 0h is contacted and antimicrobial sample recycles the ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It 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 0h is contacted and the relative intensity of fluorescence of antimicrobial sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite 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 control sample recovered liquid and 0.3mL inoculation bacterium solution of every 0h contact 1min after the dilution of 4.6mL eluent Interior relative intensity of fluorescence measured value is close, i.e.,Every through the control sample reclaim liquid phase pair for 24 hours or after 48h culture The logarithm of fluorescent strength determining valueThat is CAtij≥0.1×CA0ij.Then when 3 groups of 0h contact control samples return Receive the relative intensity of fluorescence measured value of liquid(involved calculation formula is shown in this specially in group and when between-group variation coefficient CV≤10% Sharp measurement of correlation uncertainty requirement), the measurement carried out according to this patent method is effective；
(3) fungi increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample are through for 24 hours or after 48h culture, fungi increasing value Fij、GijRespectively according to formula (13), (14) calculate:
In formula:
FijAnd Gij- every control sample and antimicrobial sample are through the fungi increasing value for 24 hours or after 48h culture, sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtij- every 0h is contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, 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 0h is contacted and the relative intensity of fluorescence of control sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
TA0ijAnd TAtij- every 0h is contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture Numerical value, unit are 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 0h is contacted and the relative intensity of fluorescence of antimicrobial sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibiotic rate Ri It is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every anti-bacteria stainless steel sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
RiThe antibiotic rate of-every group anti-bacteria stainless steel sample, %；Sample group i=1,2,3；
R-every batch of anti-bacteria stainless steel sample antibiotic rate, %；
CAtijAnd TAtijThe average ATP of-every antimicrobial sample and control sample through recycling viable bacteria for 24 hours or after 48h culture is dense Degree, unit are 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 are through for 24 hours or after 48h culture, the relative intensity of fluorescence of recovered liquid Measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibacterial Activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every anti-bacteria stainless steel sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
AiThe antibacterial activity value of-every group anti-bacteria stainless steel sample, sample group i=1,2,3；
A-every batch of anti-bacteria stainless steel sample antibacterial activity value；
FijAnd Gij- every control sample and antimicrobial sample are through the fungi increasing value for 24 hours or after 48h culture, sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h is contacted and the relative intensity of fluorescence of control sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h is contacted and the relative intensity of fluorescence of antimicrobial sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(6) viable bacteria content of Candida albicans or mycotic spore suspension data revision of the convention requirement: is demarcated using blood counting chamber CBAnd concentration and inoculation bacterium solution, the viable bacteria ATP concentration C of sample recovered liquid to ATP standard solutionAWhen being demarcated, with reference to GB Data revision of the convention regulation in 4789.2-2016 in relation to clump count, works as CBLess than 100CFU/mL or CAWhen less than 100mol/L, " four House five enters " round numbers；Work as CBNot less than 100CFU/mL or CAWhen not less than 100mol/L, taken after the 3rd bit digital " rounding up " Preceding 2 bit digital, behind with 0 replace digit；It can also be indicated with 10 exponential form, " rounding up " uses two significant figures afterwards Word.After control sample and antimicrobial sample are contacted through 0h and for 24 hours/48h is cultivated, the relative intensity of fluorescence measured value of recovered liquid is rounded Number, antibiotic rate Rij、Ri, R calculated result take three effective digitals；Fungi increasing value Fij、GijWith antibacterial activity value Aij、Ai, A meter It calculates result and takes 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 Or 48h culture after, reclaim liquid phase in fluorescent strength determining value group and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV calculated result remains into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to stainless steel anti-fungal property The reproducibility of test；In regulation group and between-group variation coefficient CV≤10%.
Every group of 0h contact 5 control samples, 5 antimicrobial samples and through for 24 hours or 48h culture after 5 control samples, 5 antimicrobial samples, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value AndRespectively according to public affairs 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 contacted through 0h and for 24 hours or After 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
3 groups of 0h contact 15 control samples, 15 antimicrobial samples and through for 24 hours or 48h culture after 15 control samples Product, 15 antimicrobial samples, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value AndIt presses respectively (sample group i=1,2,3 is calculated according to formula (23), (24)；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and For 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
Every group of 0h contact 5 control samples, 5 antimicrobial samples and through for 24 hours or 48h culture after 5 control samples, 5 antimicrobial samples, the standard deviation of the relative intensity of fluorescence measured value of recovered liquid AndRespectively 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 and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and For 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
3 groups of 0h contact 15 control samples, 15 antimicrobial samples and through for 24 hours or 48h culture after 15 control samples Product, 15 antimicrobial samples, the standard deviation of the relative intensity of fluorescence measured value of recovered liquid AndRespectively (sample group i=1,2,3 is calculated according to formula (27) and (28)；Every group of sample number into spectrum j=1,2,3,4,5；ATP test is parallel Sample number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h And for 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
The evaluation of result of the anti-bacteria stainless steel sample anti-fungal property 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:
Such as using antibiotic rate R as correlated performance evaluation index: working as Rij/RiWhen/R < 80%, sample is without antifungic action； As 80%≤Rij/RiWhen/R < 90%, sample has antifungic action；As 90%≤Rij/RiWhen/R < 99%, sample is antimycotic It acts on moderate；As 99%≤Rij/RiWhen/R < 99.9%, sample antifungic action is stronger；Work as Rij/RiWhen/R >=99.9%, sample Product antifungic action is extremely strong；
Such as using antibacterial activity value A as correlated performance evaluation index: working as Aij/AiWhen/A < 0.5, sample is without antimycotic work With；As 0.5≤Aij/AiWhen/A < 1.0, sample has antifungic action；As 1.0≤Aij/AiWhen/A < 2.0, sample is antimycotic It acts on moderate；As 2.0≤Aij/AiWhen/A < 3.0, sample antifungic action is stronger；Work as Aij/AiWhen/A >=3.0, sample is antimycotic It acts on extremely strong；
(2) as the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups When the anti-fungal property of (four) sample 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 bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back is anti- Bacterium stainless steel sample anti-fungal property level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or Antibacterial activity value Ai(Aij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the anti-bacteria stainless steel sample anti-fungal property；
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 present embodiment is illustrated by taking the detection of the anti-fungal property of alloy-type anti-bacteria stainless steel sink sample as an example.
Specific detection method carries out in the steps below:
(1) sample preparation and pretreatment
1.1 control samples: in addition to not having antibacterial coating, classification, metallic matrix, technique and the appearance of control sample, Size, quantity etc. are identical with anti-bacteria stainless steel sample to be measured；Each strain test uses 6 groups of samples；Wherein 0h contact and For 24 hours or 48h culture experiment respectively uses 3 groups, every group of 5 samples.
1.2 antimicrobial samples: after the processing of the electrochemical methods such as electroplated, chemical plating, antibacterial coating is formed not on surface Become rusty Steel material and product；Sample cover surface is uniform and has certain adhesive strength, no any damage for reaching substrate metal； Having a size of (50 ± 2) mm × (50 ± 2) mm, thickness is not more than 5mm.Antimicrobial sample quantity is identical as control sample, and every group to be measured Sample selects one group of control sample as object of reference and effectively identifies.
1.3 sample pre-treatments: impregnating 5min in 75% ethanol solution for control sample and antimicrobial sample before experiment, and With the abundant cleaning sample of sterile water to remove ethyl alcohol on superclean bench；Sample surface to be measured is put into upwards after natural drying It is 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 degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-11Mol/L~10-6mol/L；Amplification factor 40 ×~400 × Photobiology microscope；(20~ 50) DEG C ± 1 DEG C, the constant temperature and humidity incubator of (50~95) %RH ± 2%RH；(0~50) DEG C ± 1 DEG C, (50~300) r/min Constant temperature oscillator；Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The rotation of the range of speeds (500~3000) r/min Whirlpool oscillator；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~ 10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency range (30~50) kHz；Precision ± 0.1 The pH meter of (25 DEG C)；Electric furnace.
2.3 material utensils: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；Glass funnel；Sterile rotation Fill in test tube；Diameter is not more than the oese of 4mm；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；With In the absorbent cotton and gauze of biochemistry detection；Aseptic filter paper；Thermometer；The stopwatch of precision 0.01s.
2.4 reagents: 75% ethanol solution；0.1% Lv Shi alkaline methylene blue dyeing liquor；121 DEG C after following reagent packing High pressure sterilization 30min, 5 DEG C~10 DEG C storage 30d:N monomethyl ethanesulfonic acids, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, It is configured to 0.05% wetting agent solution；85% physiological saline.
2.5 medium/liquids (can use commercially available medium/liquid): 121 DEG C of high pressure sterilizations after matched medium/liquid packing 30min, 2 DEG C~8 DEG C storage 30d；
Sabouraud culture medium/liquid (Candida albicans actication of culture use): 40g glucose, 10g peptone, 20g agar are heated It is dissolved in 1000mL water (agar is not added in culture solution), adjusts pH to 5.6 ± 0.2 (25 DEG C)；
Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen Dipotassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose are dissolved by heating in 1000mL containing 0.05% wetting In the aqueous solution of agent, adjust pH to 6.0~6.5 (25 DEG C)；
One dextrose culture-medium of potato (mycotic spore actication of culture use): removing the peel stripping and slicing for 300g fresh potato, 20min~30min is boiled in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g chloramphenicol, 20g agar are added into filtrate After be settled to 1000mL.
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；
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 Sharpe culture solution can be down to 10 in 15min by liquid-11Mol/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 (fungal cell's ATP extraction efficiency be not less than 80%)；
ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h.
(3) culture presevation, activation and bacteria suspension preparation
3.1 test strains: Candida albicans ATCC 10231；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205； Penicillium chrysogenum ATCC 9179 (or other strains that is provided by national Culture Collection and can be traced to the source).
3.2 culture presevation: Candida albicans --- freeze-drying lactobacillus pipe is opened with sterile working, is infused with capillary syring into pipe Enter appropriate Sharpe culture solution, pressure-vaccum makes strain melt dispersion for several times；A little bacteria suspension is instilled and is trained equipped with 5mL~10mL Sharpe In the test tube of nutrient solution, 37 DEG C of culture 18h~for 24 hours.Mould --- mould test strain is inoculated in by potato-with sterile working The inoculation date is simultaneously indicated in dextrose culture-medium inclined-plane, and 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3℃ ~10 DEG C preservation 4 months, as preservation bacterium.
3.3 actication of culture: Candida albicans --- with the colonies typical in oese scraping 1st generation culture, scribing line is connect Kind is in sabouraud culture medium plate；After 37 DEG C of culture 18h~for 24 hours, the colonies typical in picking 2nd generation culture is inoculated in Sharpe training Support base inclined-plane；After 37 DEG C of culture 18h~for 24 hours, 4 DEG C of sealings storages, use in 6 weeks.Mould --- preservation bacterium is scraped with oese Spore is inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d, until generating a large amount of spores.Preparation Must not extract the test tube plug of mold species before spore suspension, every test tube open after only for spore liquid of preparation, every time Suspension is prepared using the mycotic spore newly cultivated.
The preparation of 3.4 bacteria suspensions: Candida albicans test --- with oese, the fresh microbial strain culture of picking is connect from inclined-plane Kind in the sterile conical flask equipped with 50mL Sharpe culture solution, it is placed in 150r/min culture in the constant temperature oscillator of (30 ± 1) DEG C 18h~for 24 hours, 4 DEG C of sealing storages, the same day uses.Mould test --- the sterile water of 10mL is added into strain test tube, with inoculation Ring gently scrapes the fresh mycotic spore of media surface, and the spore stoste injection of wash-off is trained equipped with 15 beades and 45mL Cha Shi In the sterile conical flask of jumping a queue of nutrient solution, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, it will cover There are sterile absorbent cotton or the glass funnel of eight layers of gauze to be placed on conical flask, filtering spore suspension removes mycelia and culture medium is broken Piece；Filtrate is moved in sterile centrifugation tube, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；50mL is used again Cha Shi culture solution cleaning spore sediment is simultaneously centrifuged, and after repeated washing 3 times, is precipitated with the spore after the dilution centrifugation of Cha Shi culture solution Object.Every kind of test mould prepares spore suspension according to the method described above, and the spore liquid of each strain is mixed in equal volume；0 DEG C~7 DEG C storage uses in the same day or 4d.
(4) bacteria suspension viable bacteria content C after methylene blueBBlood cell plate count, in the steps below carry out:
4.1 methylene blues: with sterile working by 50 μ L dilutions for 10-2~10-3Candida albicans bacterium suspension or mould The Lv Shi alkaline methylene blue dyeing liquor that mixing spore liquid and 30 μ L concentration are 0.05% moves to (bacterium solution in same branch sterile test tube respectively Dilution, which includes 4~5 albicans cells or mycotic spore with each lattice of blood counting chamber, to be advisable), 1000r/min Test tube 1min is shaken, mixes well bacteria suspension with dyeing liquor.
4.2 blood cell plates count: the bacteria suspension after ± 0.5 μ L of 5 μ L dyeing being placed in coverslip edge with aseptic straw, makes it Blood counting chamber is slowly penetrated along slide gap, bubble is not can produce between tally and slide, otherwise re-operates.With sterile Extra bacterium solution in filter paper exhaustion slot, stands 2min ± 20s, is settled down to its whole in counting chamber.When lattice tally in use 16 When, upper left, upper right, lower-left, the albicans cell in the middle lattice in bottom right 4 (i.e. 100 lattices) are taken in diagonal orientation Or mycotic spore is counted；Lattice tally in 25 is such as used, in addition to counting above-mentioned 4 diagonal orientations, also needs to count the 1 of center A middle lattice (i.e. 80 lattices)；When test strain is located on the two-wire of middle lattice, then only count online and right line or it is offline and Albicans cell or mycotic spore on left line.
4.3 viable bacteria content CBCalculate: by Photobiology microscope magnification from it is low to lofty tone to 400 × after, it is right immediately Albicans cell or mycotic spore at blood counting chamber additional space position are counted；Wherein colourless Candida albicans Bacterium cell is viable bacteria, and blue or light blue person is dead bacterium；Edge is in navy blue, inside in colourless or light blue and pink mould Bacterium spore is viable bacteria, and edge and inside are dead bacterium in navy blue person, therefore the only counting rim spore different from internal color.If Quantity without mycelia monospore in mycotic spore suspension is lower than 90%, then prepares spore liquid again.It is each to blood counting chamber small Albicans cell or mycotic spore in grid repeat microscopy and count three times, are averaged.When blood counting chamber specification is When 16 × 25, viable bacteria content (every milliliter of Colony Forming Unit, CFU/mL) C of 1mL bacterium solutionB5 × 16 × K × 10=N ÷4；Work as blood When ball count plate gauge lattice are 25 × 16, CB5 × 25 × K × 10=N ÷4；Wherein N is that white is read in five middle lattice of blood counting chamber The total viable count of pearl bacterium cell or mycotic spore, K are bacterium solution extension rate.Then, with Cha Shi culture solution by known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid be diluted to 1.0 × 108CFU/mL~5.0 × 108CFU/mL。
(5) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration
The determination of 5.1ATP standard serial solution and its test specimens preparation: with ATP fluorescent reagent buffer 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 × 10-8mol/L、7.0×10-7mol/ L、7.0×10-6Mol/L and 2.1 × 10-9mol/L、2.1×10-8mol/L、2.1×10-7Mol/L is simultaneously mixed well.Then, it uses Sterilizing liquid-transfering gun the above-mentioned ATP standard solution of 0.1mL is moved to respectively in three sterile test tubes, successively be added dropwise 0.05mL sterile water and 0.35mL normal saline solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution is surveyed again Sample is moved to respectively in three sterile test tubes, each that 0.4mL physiological saline is added dropwise and mixes, and is tested as second level ATP standard solution Sample；And with sterilizing liquid-transfering gun by the second level ATP standard solution test specimens of 0.1mL various concentration move to respectively three instruments without In bacterium test tube, Duplicate Samples are tested as ATP standard solution.
5.2 blank background value calibrations: the aqueous solution, 0.35mL with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent are raw The ATP lysate of reason salt water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；It stands Level-one blank sample is used as after 10min~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, is added dropwise 0.4mL physiological saline simultaneously mixes, as second level blank sample.Then, 0.1mL second level blank sample is moved to respectively with sterilizing liquid-transfering gun In three instrument sterile test tubes, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples is mentioned Reagent is taken, instills the ATP fluorescent reagent of 0.1mL after mixing again.3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and examination Agent group background values (or calibrating background according to instrument operation instruction).
5.3ATP standard solution relative intensity of fluorescence value IAMeasurement: from low to high according to concentration, to various concentration ATP The ATP that 0.1mL is added dropwise in three test Duplicate Samples of standard solution respectively extracts reagent, and the ATP for instilling 0.1mL after mixing again is glimmering Light reagent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (really It is consistent to protect each link operating time, avoids cross contamination).Each Duplicate Samples minute is no more than 15s, with each concentration ATP mark The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of quasi- solution is its IAMeasured value.
5.4ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with various concentration ATP The relative intensity of fluorescence logarithm lgI of standard solutionAAs abscissa, with its ATP log concentration value lgCAFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrationsA-lgIAStandard curve；And application is most Small two multiply the linear equation Y=a that fitting process is derived from curveA0X+bA0(high concentration), Y=aAX+bAIt is (low concentration) and related Coefficients RA0 2(high concentration), RA 2(low concentration).Work as RA0 2And RA 2>=0.98, it when confidence level >=0.95, is done according to this patent Measurement is effective.
5.5 inoculation bacterium solution viable bacteria ATP concentration CsACalibration: with Cha Shi culture solution to viable bacteria content CBIt is 1.0 × 108CFU/mL ~5.0 × 108After the test strain suspension of CFU/mL carries out continuous 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA； According to high standard fitting equation Y=aA0X+bA0It calculates and demarcates corresponding viable bacteria ATP concentration CA.Through Cha Shi culture solution tune It is whole, obtain CARange is 5.0 × 10-7Mol/L~9.0 × 10-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1minA0。
(6) sample inoculation, culture and elution recycling
6.1 inoculated and cultureds: 0.3mL is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured with sterilizing liquid-transfering gun Bacterium solution is inoculated with (with lgCA-lgIACalibration curve is derived from same branch test strain stoste test tube with bacterium solution, and 0 DEG C ± 1 DEG C saves, in 2h Using), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), its is made to cover sample whole surface.Cover ware Lid will be equipped with 6 groups for 24 hours with medical adhesive tape or the plate of 48h contact sample seal；Under the damp condition of (95 ± 2) %RH, (30 ± 2) DEG C culture ± 2h (Candida albicans) or (28 ± 2) DEG C culture 48h ± 2h (mould) for 24 hours.
6.2 elution recycling: it after 6 groups of 0h contact sample inoculation Candida albicans or mould, is drawn immediately with sterilizing liquid-transfering gun 4.6mL eluent (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously Washing lotion is moved into sterile test tube (with mould washing lotion in liquid transfer gun head repeatedly pressure-vaccum plate, until lawn broken up after again by it Move into test tube), 3000r/min shakes test tube 2min；As sample to be tested recovered liquid (if recovered liquid is insufficient after mixing well 4.9mL, addition eluent to 4.9mL).Each group uses elution identical with 0h contact sample through the sample for 24 hours or after 48h culture Mode recycles fungi.
(7) reclaim liquid phase is to fluorescence intensity level IAMeasurement
7.1 instruments and reagent set relative intensity of fluorescence background value calibration: according to lgCA-lgIABlank when standard curve is established Background calibration methods, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values.
7.2 reclaim liquid phases are to fluorescence intensity level IAMeasurement: if background level meets instrument requirement, with sterilizing liquid-transfering gun The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube, 3000r/min shakes test tube 30s； After being stored at room temperature 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min.It will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence.To The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquidAMeasured value.
(8) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R or antibacterial activity value A is calculated
8.1 recovered liquid viable bacteria ATP concentration CsAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate that each group sample connects through 0h Touch for 24 hours or 48h culture after recovered liquid viable bacteria ATP concentration CAAnd TA.Relevant calculation is shown in formula (1)~(12):
In formula:
CA0And CAt- 3 groups of 0h are contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, single Position is mole every liter (mol/L)；
CA0iAnd CAti- every group 0h is contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, Unit is mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtij- every 0h is contacted and control sample recycles the ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It 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 0h is contacted and the relative intensity of fluorescence of control sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAt- 3 groups of 0h are contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, single Position is mole every liter (mol/L)；
TA0iAnd TAti- every group 0h is contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, Unit is mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtij- every 0h is contacted and antimicrobial sample recycles the ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It 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 0h is contacted and the relative intensity of fluorescence of antimicrobial sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5.
8.2 test conditions for validity
If the control sample recovered liquid and 0.3mL inoculation bacterium solution of every 0h contact 1min after the dilution of 4.6mL eluent Interior relative intensity of fluorescence measured value is close, i.e.,Every through the control sample reclaim liquid phase pair for 24 hours or after 48h culture The logarithm of fluorescent strength determining valueThat is CAtij≥0.1×CA0ij.Then when 3 groups of 0h contact control sample The relative intensity of fluorescence measured value of recovered liquid(involved calculation formula is shown in this in group and when between-group variation coefficient CV≤10% The requirement of patent measurement of correlation uncertainty), the measurement carried out according to this patent method is effective.
8.3 fungi increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample are through for 24 hours or after 48h culture, fungi increasing value Fij、GijRespectively according to formula (13), (14) calculate:
In formula:
FijAnd Gij- every control sample and antimicrobial sample are through the fungi increasing value for 24 hours or after 48h culture, sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtij- every 0h is contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, 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 0h is contacted and the relative intensity of fluorescence of control sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
TA0ijAnd TAtij- every 0h is contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture Numerical value, unit are 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 0h is contacted and the relative intensity of fluorescence of antimicrobial sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope.
8.4 antibiotic rate R are calculated
It tests under condition for validity, the antibiotic rate R of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibiotic rate Ri It is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every anti-bacteria stainless steel sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
RiThe antibiotic rate of-every group anti-bacteria stainless steel sample, %；Sample group i=1,2,3；
R-every batch of anti-bacteria stainless steel sample antibiotic rate, %；
CAtijAnd TAtijThe average ATP of-every antimicrobial sample and control sample through recycling viable bacteria for 24 hours or after 48h culture is dense Degree, unit are 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 are through for 24 hours or after 48h culture, the relative intensity of fluorescence of recovered liquid Measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope.
8.5 antibacterial activity value A are calculated
It tests under condition for validity, the antibacterial activity value A of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibacterial Activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every anti-bacteria stainless steel sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
AiThe antibacterial activity value of-every group anti-bacteria stainless steel sample, sample group i=1,2,3；
A-every batch of anti-bacteria stainless steel sample antibacterial activity value；
FijAnd Gij- every control sample and antimicrobial sample are through the fungi increasing value for 24 hours or after 48h culture, sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h is contacted and the relative intensity of fluorescence of control sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h is contacted and the relative intensity of fluorescence of antimicrobial sample recovered liquid is surveyed through for 24 hours or after 48h culture Definite value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope.
The requirement of the 8.6 data revisions of the convention: the viable bacteria content of Candida albicans or mycotic spore suspension is demarcated using blood counting chamber CBAnd concentration and inoculation bacterium solution, the viable bacteria ATP concentration C of sample recovered liquid to ATP standard solutionAWhen being demarcated, with reference to GB Data revision of the convention regulation in 4789.2-2016 in relation to clump count, works as CBLess than 100CFU/mL or CAWhen less than 100mol/L, " four House five enters " round numbers；Work as CBNot less than 100CFU/mL or CAWhen not less than 100mol/L, taken after the 3rd bit digital " rounding up " Preceding 2 bit digital, behind with 0 replace digit；It can also be indicated with 10 exponential form, " rounding up " uses two significant figures afterwards Word.After control sample and antimicrobial sample are contacted through 0h and for 24 hours/48h is cultivated, the relative intensity of fluorescence measured value of recovered liquid is rounded Number, antibiotic rate Rij、Ri, R calculated result take three effective digitals；Fungi increasing value Fij、GijWith antibacterial activity value Aij、Ai, A meter It calculates result and takes two effective digitals.
8.7 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 Or 48h culture after, reclaim liquid phase in fluorescent strength determining value group and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV calculated result remains into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to stainless steel anti-fungal property The reproducibility of test；In regulation group and between-group variation coefficient CV≤10%.
Every group of 0h contact 5 control samples, 5 antimicrobial samples and through for 24 hours or 48h culture after 5 control samples, 5 antimicrobial samples, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value AndRespectively according to public affairs 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 through 0h contact and for 24 hours Or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
3 groups of 0h contact 15 control samples, 15 antimicrobial samples and through for 24 hours or 48h culture after 15 control samples Product, 15 antimicrobial samples, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value AndIt presses respectively (sample group i=1,2,3 is calculated according to formula (23), (24)；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and For 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
Every group of 0h contact 5 control samples, 5 antimicrobial samples and through for 24 hours or 48h culture after 5 control samples, 5 antimicrobial samples, the standard deviation of the relative intensity of fluorescence measured value of recovered liquid AndRespectively 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 and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and For 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
3 groups of 0h contact 15 control samples, 15 antimicrobial samples and through for 24 hours or 48h culture after 15 control samples Product, 15 antimicrobial samples, the standard deviation of the relative intensity of fluorescence measured value of recovered liquid AndRespectively (sample group i=1,2,3 is calculated according to formula (27) and (28)；Every group of sample number into spectrum j=1,2,3,4,5；ATP test is parallel Sample number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h And for 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
(9) evaluation of result
9.1, with reference to health industry common practice and the requirement of dependent antimicrobial product standard, take following classification criterion:
Such as using antibiotic rate R as correlated performance evaluation index: working as Rij/RiWhen/R < 80%, sample is without antifungic action； As 80%≤Rij/RiWhen/R < 90%, sample has antifungic action；As 90%≤Rij/RiWhen/R < 99%, sample is antimycotic It acts on moderate；As 99%≤Rij/RiWhen/R < 99.9%, sample antifungic action is stronger；Work as Rij/RiWhen/R >=99.9%, sample Product antifungic action is extremely strong；
Such as using antibacterial activity value A as correlated performance evaluation index: working as Aij/AiWhen/A < 0.5, sample is without antimycotic work With；As 0.5≤Aij/AiWhen/A < 1.0, sample has antifungic action；As 1.0≤Aij/AiWhen/A < 2.0, sample is antimycotic It acts on moderate；As 2.0≤Aij/AiWhen/A < 3.0, sample antifungic action is stronger；Work as Aij/AiWhen/A >=3.0, sample is antimycotic It acts on extremely strong.
9.2 work as the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups When the anti-fungal property of (four) sample 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 bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back is anti- Bacterium stainless steel sample anti-fungal property level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or Antibacterial activity value Ai(Aij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the anti-bacteria stainless steel sample anti-fungal property.
Detection qu alification, instrument and equipment used in the present embodiment and test equipment, medium/liquid, chemical reagent, reference culture:
(1) bio-safety 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 and test equipment
2.1 two stage biological safety cabinets: Thermo Scientific1300 series of secondary B2 type Biohazard Safety Equipment, workbench Surface area 0.55m2, capacity 1130m3/ h, filter efficiency 99.99% (0.3 μm).
2.2 superclean benches: American blend Thermo ScientificTMHeraguardTM, 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。
2.3 ATP bioluminescence rapid detection systems: the portable system SURE ATP of Hygiena company, the U.S. is glimmering Optical detector, box containing matched reagent, plastics Special test tube etc.；ATP content detection lower limit 4 × 10-18Mol/ml, microorganism total amount Detection limit 1.0CFU/ml, RLU range of readings 0~9999, detection time 10s, 1000 times/s of sampling rate, measurement error ± 5%.
2.4 Photobiology microscopes: have the adjustable eyepiece of 10 times of wide visual fields and 4 times, 10 times, 20 times, 40 times and 100 times The tri- mesh research grade biomicroscope of Olympus BX43 of flat-field achromatic objective lens.
2.5 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.
2.6 constant temperature oscillators: the permanent model WS-380H in Shanghai one, ± 0.1 DEG C of temperature control range (4~65) DEG C, frequency of oscillation (40~300) r/min, timing range (1~99) h.
2.7 refrigerated centrifuges: the vertical refrigerated centrifuge of Beijing Xin Nuolihua Instrument Ltd. model DL7M-12L turns Fast 8000r/min ± 20r/min, 12300 × g of relative centrifugal force；Capacity 14400ml, timing range 1s~99h59min99s, ± 1 DEG C of temperature control range (- 20~40) DEG C, centrifuge tube specification Ф 74mm × 168mm.
2.8 pressure steam sterilizers: Japanese Sanyo company model MLS-3780 autoclave, volume 75L, sterilizing temperature ± 2 DEG C, maximum pressure 0.235MPa of degree (105~135) DEG C, timer (1~250) min.
2.9 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.
2.10 refrigerators: the cryogenic box ultralow temperature ice of the glad experimental instruments and equipment limited model HXL-25-250AD of Dongguan City sky Case, ± 0.1 DEG C of refrigerating box (2~10) DEG C, ± 0.1 DEG C of household freezer (- 30~20) DEG C.
2.11 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.
2.12 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.
2.13 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.
2.14 pH meters: 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.
2.15 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.
2.16 blood counting chambers: the blood counting chamber that refinement specification in Shanghai is 25 × 16.
(3) medium/liquid
The medium/liquid of Beijing overpass technical concern Co., Ltd production.
(4) chemical reagent
4.1 0.1% Lv Shi alkaline methylene blue dyeing liquor: it is purchased from Shanghai innermost thoughts and feelings Biotechnology Co., Ltd.
4.2 trinosin standard items and preparation ATP fluorescent reagent buffer solution, ATP lysate, ATP extracting solution The U.S. Amresco of Shanghai Jin Pan Biotechnology Co., Ltd agency is purchased from a series of biochemical reagents needed for ATP fluorescent reagent Brand.
(5) reference culture
Candida albicans ATCC 10231 is purchased from Chinese medicine fungi preservation administrative center；Aspergillus niger ATCC 16404, ball Chactomium globosum ATCC 6205, penicillium chrysogenum ATCC 9179 are purchased from China General Microbiological culture presevation administrative center.
The detection data and result of the present embodiment calculate:
Using ATP fluophotometer through lgCB-lgIBCalibration curve method can be carried out the antifungal activity of antibacterial stainless steel sample Detection, coherent detection data and Calculation of Measuring Uncertainty result are shown in Table 1~table 4 respectively.
1 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (Candida albicans)
2 relative intensity of fluorescence value Calculation of Measuring Uncertainty result (Candida albicans) of table
3 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (mould)
4 relative intensity of fluorescence Calculation of Measuring Uncertainty result (mould) 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 (10)

1. a kind of detection method of anti-bacteria stainless steel anti-fungal property, comprising: (1) sample preparation and pretreatment；(2) lectotype selection And reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) bacteria suspension viable bacteria content C after methylene blueB Blood cell plate count；(5) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution Viable bacteria ATP concentration CACalibration；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level IAMeasurement； (8) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；Its feature exists In, using ATP fluophotometer to antibiotic rate R or antibacterial activity value A characterization anti-bacteria stainless steel antifungal activity can be carried out essence The ATP bioluminescence lgC of quasi- quantitative testA-lgIACalibration curve method, specific:
In reclaim liquid phase to fluorescence intensity level IAIn measurement:
Specify control sample and antimicrobial sample quantity, size and pre-treatment requirement, by the reference culture of mycologic test strain into After row passage, activation, fresh Candida albicans or mycotic spore culture is taken to prepare bacteria suspension；Hemocytometer is used after methylene blue Viable bacteria content C in number plate measurement bacterium solutionB, and C is carried out to the bacteria suspension of different dilutionsBCalibration, it is then, slow with ATP fluorescent reagent Solution is rushed by 1.0 × 10-3The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10-8mol/L、 7.0×10-7mol/L、7.0×10-6Mol/L and 2.1 × 10-9mol/L、2.1×10-8mol/L、2.1×10-7Mol/L, measurement Its relative intensity of fluorescence value IA；Draw the lgC of two respective concentrationsA-lgIAStandard curve is derived from fitting equation Y= aA0X+bA0(high concentration), Y=aAX+bA(low concentration) and linearly dependent coefficient RA0 2(high concentration), RA 2(low concentration), meanwhile, it uses Cha Shi culture solution is to viable bacteria content CBIt is 1.0 × 108CFU/mL~5.0 × 108The test strain suspension of CFU/mL carries out continuous 10 times of gradient dilutions, the relative intensity of fluorescence value I of measurement dilution bacterium solutionA, according to high standard fitting equation Y=aA0X+bA0 Calculate its viable bacteria ATP concentration CA；It is adjusted to obtain CAIt is 5.0 × 10-7Mol/L~9.0 × 10-7The inoculation bacterium solution of mol/L, to each 0.3mL inoculation bacterium solution is added dropwise in group sample surface to be measured respectively, is washed immediately to 6 groups of 0h contact samples using 4.6mL eluent De- recycling, measures the relative intensity of fluorescence value I of recovered liquidAC0ij、IAT0ij, and according to low concentration calibration curve equation formula Y=aAX+ bACalculate its viable bacteria ATP concentration CAOijAnd TAOij, meanwhile, under the damp condition of 95%RH ± 2%RH, 6 groups are sealed in sterile Control sample and antimicrobial sample in plate cultivate ± 2h or (28 ± 2) DEG C (mould) for 24 hours at (30 ± 2) DEG C (Candida albicans) After cultivating 48h ± 2h；Recycling remained on surface bacterium is eluted using mode identical with 0h contact sample and measures the phase of its recovered liquid To fluorescence intensity level IACtij、IATtij, calculate corresponding viable bacteria ATP concentration CAtijAnd TAtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, with 0h contact and for 24 hours or 48h culture Under the conditions of every control sample and antimicrobial sample recovered liquid relative intensity of fluorescence measured valueWithAs Basic data；Fungi increasing value F in the case where testing condition for validity, after calculating its cultureij、GijAnd antibiotic rate RijIt is living with antibacterial Property value Aij；To the R of every group of sampleijAnd AijArithmetic mean of instantaneous value is taken to obtain corresponding RiAnd Ai；Every batch of anti-bacteria stainless steel sample resists Bacterium rate R and antibacterial activity value A is its three groups of sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and measurement be not true Fixed degree requires；
In evaluation of result:
With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that anti-fungal property is classified criterion；When The antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) with other two groups (four) samples When anti-fungal property is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is raw through ATP to calculate it Object fluorescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) anti-bacteria stainless steel Sample anti-fungal property level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or antibacterial activity Value Ai(Aij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the anti-bacteria stainless steel sample anti-fungal property.
2. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the sample Preparation and pre-treatment carry out in the steps below:
(1) control sample: in addition to not having antibacterial coating, classification, metallic matrix, technique and the appearance of control sample, size, Quantity etc. is identical with anti-bacteria stainless steel sample to be measured；Each strain test uses 6 groups of samples；Wherein 0h contact and for 24 hours or 48h culture experiment respectively uses 3 groups, every group of 5 samples；
(2) antimicrobial sample: after the processing of the electrochemical methods such as electroplated, chemical plating, the stainless steel of antibacterial coating is formed on surface Material and product；Sample cover surface is uniform and has certain adhesive strength, no any damage for reaching substrate metal；Size For (50 ± 2) mm × (50 ± 2) mm, thickness is not more than 5mm, and antimicrobial sample quantity is identical as control sample, every group of sample to be tested It selects one group of control sample as object of reference and effectively identifies；
(3) sample pre-treatments: control sample and antimicrobial sample are impregnated into 5min in 75% ethanol solution before experiment, and super With the abundant cleaning sample of sterile water to remove ethyl alcohol on net workbench；Sample surface to be measured is put into upwards after natural drying sterile It is spare in plate.
3. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the equipment Type selecting 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, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-11Mol/L~10-6mol/L；Amplification factor 40 ×~400 × Photobiology microscope；(20~50) DEG C ± 1 DEG C, the constant temperature and humidity incubator of (50~95) %RH ± 2%RH；The perseverance of (0~50) DEG C ± 1 DEG C, (50~300) r/min Warm oscillator；Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The vortex of the range of speeds (500~3000) r/min shakes Swing device；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C Refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency range (30~50) kHz；Precision ± 0.1 (25 DEG C) pH meter；Electric furnace；
(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL,0.1mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)；Capacity The sterile conical flask and bottle stopper of 100mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock examination Pipe；Diameter is not more than the oese of 4mm；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；For giving birth to Change the absorbent cotton and gauze of detection；Aseptic filter paper；Thermometer；The stopwatch of precision 0.01s；
(4) reagent: 75% ethanol solution；0.1% Lv Shi alkaline methylene blue dyeing liquor；121 DEG C of high pressures after following reagent packing Sterilize 30min, and 5 DEG C~10 DEG C storage 30d:N monomethyl ethanesulfonic acids, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and prepares At 0.05% wetting agent solution；85% physiological saline；
(5) medium/liquid (commercially available medium/liquid can be used): 121 DEG C of high pressure sterilization 30min after the packing of matched medium/liquid, 2 DEG C ~8 DEG C of storage 30d；
Sabouraud culture medium/liquid (the actication of culture use of Candida albicans): 40g glucose, 10g peptone, the heating of 20g agar is molten Solution (agar is not added in culture solution) in 1000mL water is adjusted pH to 5.6 ± 0.2 (25 DEG C)；
Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen two Potassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose, which are dissolved by heating, contains 0.05% wetting agent in 1000mL Aqueous solution in, adjust pH to 6.0~6.5 (25 DEG C)；
One dextrose culture-medium of potato (mycotic spore actication of culture use): removing the peel stripping and slicing for 300g fresh potato, 20min~30min is boiled in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g chloramphenicol, 20g agar are added into filtrate After be settled to 1000mL；
(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 Sharpe culture solution can be down to 10 in 15min-11Mol/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, adjust pH to 12.0 ± 0.5 (fungal cell's ATP extraction efficiency is not less than 80%)；
ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL's ATP fluorescent reagent buffer solution is stored at room temperature 15min, uses in 3h after mixing.
4. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the strain Preservation, activation and bacteria suspension preparation, carry out in the steps below:
(1) test strain: Candida albicans ATCC 10231；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC6205；It produces yellow green Mould ATCC 9179 (or other strains that is provided by national Culture Collection and can be traced to the source)；
(2) culture presevation: Candida albicans --- freeze-drying lactobacillus pipe is opened with sterile working, is injected with capillary syring into pipe suitable Sharpe culture solution is measured, pressure-vaccum makes strain melt dispersion for several times；A little bacteria suspension is instilled, 5mL~10mL Sharpe culture solution is housed Test tube in, 37 DEG C of culture 18h~for 24 hours, mould --- mould test strain is inoculated in by potato-grape with sterile working The inoculation date is simultaneously indicated in sugar culture-medium inclined-plane, and 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months, as preservation bacterium；
(3) actication of culture: Candida albicans --- with oese scraping 1st generation culture in colonies typical, streak inoculation in Sabouraud culture medium plate；After 37 DEG C of culture 18h~for 24 hours, the colonies typical in picking 2nd generation culture is inoculated in sabouraud culture medium Inclined-plane；After 37 DEG C of culture 18h~for 24 hours, 4 DEG C of sealings storages use, mould in 6 weeks --- with oese scraping preservation bacterium spore, It is inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d prepare spore until generating a large amount of spores Must not extract the test tube plug of mold species before suspension, every test tube open after only for spore liquid of preparation, use every time The mycotic spore newly cultivated prepares suspension；
(4) prepared by bacteria suspension: Candida albicans test --- with oese, the fresh microbial strain culture of picking is inoculated in from inclined-plane In sterile conical flask equipped with 50mL Sharpe culture solution, be placed in 150r/min culture 18h in the constant temperature oscillator of (30 ± 1) DEG C~ For 24 hours, 4 DEG C of sealing storages, the same day use, mould test --- the sterile water of 10mL is added into strain test tube, it is light with oese The spore stoste injection of wash-off is equipped with 15 beades and 45mL Cha Shi culture solution by the fresh mycotic spore for scraping media surface Sterile conical flask of jumping a queue in, 3000r/min shakes test tube 2min, breaks up spore ball, then nothing will be covered with by mixing spore liquid The glass funnel of bacterium absorbent cotton or eight layers of gauze is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；It will Filtrate moves in sterile centrifugation tube, and 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is trained again with 50mL Cha Shi Nutrient solution cleaning spore sediment is simultaneously centrifuged, and after repeated washing 3 times, dilutes the spore sediment after centrifugation with Cha Shi culture solution, often Kind test mould prepares spore suspension according to the method described above, and the spore liquid of each strain is mixed in equal volume；0 DEG C~7 DEG C are deposited It puts, is used in the same day or 4d.
5. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the methylene blue Bacteria suspension viable bacteria content C after dyeingBBlood cell plate count, in the steps below carry out:
(1) methylene blue: with sterile working by 50 μ L dilutions for 10-2~10-3Candida albicans bacterium suspension or mould mixing The Lv Shi alkaline methylene blue dyeing liquor that spore liquid and 30 μ L concentration are 0.05% moves in same branch sterile test tube (bacterium solution dilution respectively Degree, which includes 4~5 albicans cells or mycotic spore with each lattice of blood counting chamber, to be advisable), 1000r/min shaking Test tube 1min, mixes well bacteria suspension with dyeing liquor；
(2) blood cell plate counts: the bacteria suspension after ± 0.5 μ L of 5 μ L dyeing being placed in coverslip edge with aseptic straw, makes it along glass Piece gap slowly penetrates blood counting chamber, not can produce bubble between tally and slide, otherwise re-operates, uses aseptic filter paper Extra bacterium solution in exhaustion slot, stands 2min ± 20s, is settled down to its whole in counting chamber, when using lattice tally in 16, Diagonal orientation takes upper left, upper right, lower-left, the albicans cell in the middle lattice in bottom right 4 (i.e. 100 lattices) or mould Spore is counted；Lattice tally in 25 is such as used, in addition to counting above-mentioned 4 diagonal orientations, also needs 1 middle lattice for counting center (i.e. 80 lattices)；When test strain is located on the two-wire of middle lattice, then only count on online and right line or offline and left line Albicans cell or mycotic spore；
(3) viable bacteria content CBCalculate: by Photobiology microscope magnification from it is low to lofty tone to 400 × after, immediately to hemocytometer Albicans cell or mycotic spore at number plate additional space position are counted；Wherein colourless albicans cell For viable bacteria, blue or light blue person is dead bacterium；Edge is in navy blue, inside in colourless or light blue and pink mycotic spore For viable bacteria, edge and inside are dead bacterium in navy blue person, therefore the only counting rim spore different from internal color, if mould spore Quantity without mycelia monospore in sub- suspension is lower than 90%, then spore liquid is prepared again, in each lattice of blood counting chamber Albicans cell or mycotic spore repeat microscopy count three times, be averaged, when blood counting chamber specification be 16 × 25 When, viable bacteria content (every milliliter of Colony Forming Unit, CFU/mL) C of 1mL bacterium solutionB5 × 16 × K × 10=N ÷4；Work as blood count When plate gauge lattice are 25 × 16, CB5 × 25 × K × 10=N ÷4；Wherein N is that Candida albicans is thin in five middle lattice of blood counting chamber The total viable count of born of the same parents or mycotic spore, K are bacterium solution extension rate, then, with Cha Shi culture solution by known viable bacteria content CBIt is white Color beads bacterium suspension or mould mixing spore liquid are diluted to 1.0 × 108CFU/mL~5.0 × 108CFU/mL。
6. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the ATP Log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration, It carries out in the steps below:
(1) determination of ATP standard serial solution and its test specimens preparation: use ATP fluorescent reagent buffer 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 × 10-8mol/L、7.0×10-7mol/L、 7.0×10-6Mol/L and 2.1 × 10-9mol/L、2.1×10-8mol/L、2.1×10-7Mol/L is simultaneously mixed well, then, with going out Bacterium liquid-transfering gun moves to the above-mentioned ATP standard solution of 0.1mL in three sterile test tubes respectively, successively be added dropwise 0.05mL sterile water and 0.35mL normal saline solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution is surveyed again Sample is moved to respectively in three sterile test tubes, each that 0.4mL physiological saline is added dropwise and mixes, and is tested as second level ATP standard solution Sample；And with sterilizing liquid-transfering gun by the second level ATP standard solution test specimens of 0.1mL various concentration move to respectively three instruments without In bacterium test tube, Duplicate Samples are tested as ATP standard solution；
(2) blank background value calibration: aqueous solution, 0.35mL physiology salt with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent The ATP lysate of water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；Stand 10min Level-one blank sample is used as after~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, it is raw that 0.4mL is added dropwise Reason salt water simultaneously mixes, and as second level blank sample, then, 0.1mL second level blank sample is moved to three Zhi Yi respectively with sterilizing liquid-transfering gun In device special sterile test tube, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples extracts reagent, Instill the ATP fluorescent reagent of 0.1mL after mixing again, 3000r/min shakes test tube 5s, immediately with ATP fluorescent spectrophotometer measuring its Relative intensity of fluorescence value IAAnd record and (ensure that each link operating time is consistent, avoid cross contamination), when each Duplicate Samples measure Between be no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and reagent set sheet Floors (or calibrating background according to instrument operation instruction)；
(3) ATP standard solution relative intensity of fluorescence value IAMeasurement: from low to high according to concentration, to various concentration ATP standard The ATP that 0.1mL is added dropwise in three test Duplicate Samples of solution respectively extracts reagent, instills the ATP fluorescence examination of 0.1mL after mixing again Agent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records and (ensures each The link operating time is consistent, avoids cross contamination), each Duplicate Samples minute is no more than 15s, molten with each concentration ATP standard The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of liquid is its IAMeasured value；
(4) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with various concentration ATP standard The relative intensity of fluorescence logarithm lgI of solutionAAs abscissa, with its ATP log concentration value lgCAFor ordinate mapping；To two Mathematical relationship between person carries out calibration curve, draws the lgC of two respective concentrationsA-lgIAStandard curve；And application minimum two Multiply the linear equation Y=a that fitting process is derived from curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and related coefficient RA0 2(high concentration), RA 2(low concentration), works as RA0 2And RA 2>=0.98, when confidence level >=0.95, the measurement done according to this patent Effectively；
(5) it is inoculated with bacterium solution viable bacteria ATP concentration CACalibration: with Cha Shi culture solution to viable bacteria content CBIt is 1.0 × 108CFU/mL~5.0 ×108After the test strain suspension of CFU/mL carries out continuous 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA；According to High standard fitting equation Y=aA0X+bA0It calculates and demarcates corresponding viable bacteria ATP concentration CA, it is adjusted through Cha Shi culture solution, Obtain CARange is 5.0 × 10-7Mol/L~9.0 × 10-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution warp Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1minA0。
7. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the sample Inoculation, culture and elution recycling, carry out in the steps below:
(1) 0.3mL inoculation inoculated and cultured: is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured with sterilizing liquid-transfering gun Bacterium solution is (with lgCA-lgIACalibration curve is derived from same branch test strain stoste test tube with bacterium solution, and 0 DEG C ± 1 DEG C saves, and makes in 2h With), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), so that its is covered sample whole surface, covers ware lid, 6 groups will be equipped with for 24 hours with medical adhesive tape or the plate of 48h contact sample seals；Under the damp condition of (95 ± 2) %RH, (30 ± 2) DEG C culture ± 2h (Candida albicans) or (28 ± 2) DEG C culture 48h ± 2h (mould) for 24 hours；
(2) elution recycling: after 6 groups of 0h contact sample inoculation Candida albicans or mould, 4.6mL is drawn with sterilizing liquid-transfering gun immediately Eluent (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution and by washing lotion It moves into sterile test tube and (with mould washing lotion in liquid transfer gun head repeatedly pressure-vaccum plate, moves it into examination again after lawn is broken up Pipe), 3000r/min shakes test tube 2min；After mixing well as sample to be tested recovered liquid (if recovered liquid less than 4.9mL, Eluent is added to 4.9mL), each group uses type of elution identical with 0h contact sample to return through the sample for 24 hours or after 48h culture Receive fungi.
8. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the recycling Liquid phase is to fluorescence intensity level IAMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: according to lgCA-lgIABlank background when standard curve is established Calibration method, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values；
(2) reclaim liquid phase is to fluorescence intensity level IAMeasurement:, will with sterilizing liquid-transfering gun if background level meets instrument requirement The ATP lysate of 4.9mL recovered liquid and 0.1mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；Room After temperature stands 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min, it will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence, to The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value IAAnd record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquidAMeasured value.
9. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the recycling Liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R or antibacterial activity value A is calculated, carry out in the steps below:
(1) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate each group sample through 0h contact and For 24 hours or 48h culture after recovered liquid viable bacteria ATP concentration CAAnd TA, relevant calculation is shown in formula (1)~(12):
In formula:
CA0And CAt- 3 groups of 0h are contacted and control sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, and unit is to rub You are every liter (mol/L)；
CA0iAnd CAti- every group 0h is contacted and control sample recycles the average ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It is mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtij- every 0h is contacted and control sample recycles the ATP concentration of viable bacteria through for 24 hours or after 48h culture, and unit is 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 through the relative intensity of fluorescence measured value of control sample recovered liquid for 24 hours or after 48h culture, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAt- 3 groups of 0h are contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria through for 24 hours or after 48h culture, and unit is to rub You are every liter (mol/L)；
TA0iAnd TAti- every group 0h is contacted and antimicrobial sample recycles the average ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It is mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtij- every 0h is contacted and antimicrobial sample recycles the ATP concentration of viable bacteria through for 24 hours or after 48h culture, and unit is 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 through the relative intensity of fluorescence measured value of antimicrobial sample recovered liquid for 24 hours or after 48h culture, 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 control sample recovered liquid and 0.3mL inoculation bacterium solution of every 0h contact are after the dilution of 4.6mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every through the control sample recovered liquid relative fluorescence for 24 hours or after 48h culture The logarithm of strength detection valueThat is CAtij≥0.1×CA0ij, then when 3 groups of 0h contact control sample recovered liquid Relative intensity of fluorescence measured value(involved calculation formula is shown in this patent phase in group and when between-group variation coefficient CV≤10% Close uncertainty of measurement requirement), the measurement carried out according to this patent method is effective；
(3) fungi increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample are through for 24 hours or after 48h culture, fungi increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd Gij- every control sample and antimicrobial sample are through the fungi increasing value for 24 hours or after 48h culture, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtij- every 0h is contacted and control sample recycles the average ATP concentration of viable bacteria, unit through for 24 hours or after 48h culture It 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 0h contact and through the relative intensity of fluorescence measured value of control sample recovered liquid for 24 hours or after 48h culture, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
TA0ijAnd TAtij- every 0h is contacted and antimicrobial sample recycles the average ATP concentration values of viable bacteria through for 24 hours or after 48h culture, 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 0h contact and through the relative intensity of fluorescence measured value of antimicrobial sample recovered liquid for 24 hours or after 48h culture, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibiotic rate RiWith R points It is not calculated according to formula (15), (16), (17):
In formula:
RijThe antibiotic rate of-every anti-bacteria stainless steel sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3, 4,5；
RiThe antibiotic rate of-every group anti-bacteria stainless steel sample, %；Sample group i=1,2,3；
R-every batch of anti-bacteria stainless steel sample antibiotic rate, %；
CAtijAnd TAtijThe average ATP concentration of-every antimicrobial sample and control sample through recycling viable bacteria for 24 hours or after 48h culture, it is 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；
With- every antimicrobial sample and control sample are through for 24 hours or after 48h culture, the relative intensity of fluorescence of recovered liquid is measured Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibacterial activity Value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every anti-bacteria stainless steel sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3, 4,5；
AiThe antibacterial activity value of-every group anti-bacteria stainless steel sample, sample group i=1,2,3；
A-every batch of anti-bacteria stainless steel sample antibacterial activity value；
FijAnd Gij- every control sample and antimicrobial sample are through the fungi increasing value for 24 hours or after 48h culture, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and through the relative intensity of fluorescence measured value of control sample recovered liquid for 24 hours or after 48h culture, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and through the relative intensity of fluorescence measured value of antimicrobial sample recovered liquid for 24 hours or after 48h culture, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(6) the viable bacteria content C of Candida albicans or mycotic spore suspension data revision of the convention requirement: is demarcated using blood counting chamberBAnd Concentration and inoculation bacterium solution, the viable bacteria ATP concentration C of sample recovered liquid to ATP standard solutionAWhen being demarcated, with reference to GB Data revision of the convention regulation in 4789.2-2016 in relation to clump count, works as CBLess than 100CFU/mL or CAWhen less than 100mol/L, " four House five enters " round numbers；Work as CBNot less than 100CFU/mL or CAWhen not less than 100mol/L, taken after the 3rd bit digital " rounding up " Preceding 2 bit digital, behind with 0 replace digit；It can also be indicated with 10 exponential form, " rounding up " uses two significant figures afterwards After word, control sample and antimicrobial sample are contacted through 0h and for 24 hours/48h is cultivated, the relative intensity of fluorescence measured value of recovered liquid is rounded Number, antibiotic rate Rij、Ri, R calculated result take three effective digitals；Fungi increasing value Fij、GijWith antibacterial activity value Aij、Ai, A meter It calculates result and takes two effective digitals；
(7) uncertainty of measurement: this patent method mainly pass through calculate control sample and antimicrobial sample contacted through 0h and for 24 hours or 48h culture after, reclaim liquid phase in fluorescent strength determining value group and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV calculated result remains into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to stainless steel anti-fungal property The reproducibility of test；In regulation group and between-group variation coefficient CV≤10%；
Every group of 0h contact 5 control samples, 5 antimicrobial samples and through for 24 hours or 48h culture after 5 control samples, 5 Antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value AndRespectively 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 contacted through 0h and for 24 hours or After 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
3 groups of 0h contact 15 control samples, 15 antimicrobial samples and through for 24 hours or 48h culture after 15 control samples, 15 Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value AndRespectively 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 contacted through 0h and for 24 hours or After 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
Every group of 0h contact 5 control samples, 5 antimicrobial samples and through for 24 hours or 48h culture after 5 control samples, 5 Antimicrobial sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquid AndRespectively according to public affairs 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 contacted through 0h and for 24 hours or After 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
3 groups of 0h contact 15 control samples, 15 antimicrobial samples and through for 24 hours or 48h culture after 15 control samples, 15 Part antimicrobial sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquid AndRespectively 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 and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and For 24 hours or after 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
10. the detection method of anti-bacteria stainless steel anti-fungal property according to claim 1, which is characterized in that the knot Fruit evaluation, 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:
Such as using antibiotic rate R as correlated performance evaluation index: working as Rij/RiWhen/R < 80%, sample is without antifungic action；When 80%≤Rij/RiWhen/R < 90%, sample has antifungic action；As 90%≤Rij/RiWhen/R < 99%, the antimycotic work of sample With moderate；As 99%≤Rij/RiWhen/R < 99.9%, sample antifungic action is stronger；Work as Rij/RiWhen/R >=99.9%, sample Antifungic action is extremely strong；
Such as using antibacterial activity value A as correlated performance evaluation index: working as Aij/AiWhen/A < 0.5, sample is without antifungic action； As 0.5≤Aij/AiWhen/A < 1.0, sample has antifungic action；As 1.0≤Aij/AiWhen/A < 2.0, sample antifungic action It is moderate；As 2.0≤Aij/AiWhen/A < 3.0, sample antifungic action is stronger；Work as Aij/AiWhen/A >=3.0, sample antifungic action It is extremely strong；
(2) as the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two group (four Part) sample anti-fungal property compared to a levels are at least differed when, extract one group of (part) sample again and repeat to test；It calculates It is through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back antibacterial is not Steel sample anti-fungal property level of becoming rusty is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or antibacterial Activity value Ai(Aij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the anti-bacteria stainless steel sample anti-fungal property.
CN201910199764.0A 2019-03-15 2019-03-15 ATP bioluminescence lgCA-lgIAThe method of calibration curve method detection anti-bacteria stainless steel anti-fungal property Withdrawn CN110272961A (en)

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