CN110272965A - ATP bioluminescence lgCB-lgIBThe method for marking bent method detection nano inorganic material anti-fungal property - Google Patents

ATP bioluminescence lgCB-lgIBThe method for marking bent method detection nano inorganic material anti-fungal property Download PDF

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CN110272965A
CN110272965A CN201910200127.0A CN201910200127A CN110272965A CN 110272965 A CN110272965 A CN 110272965A CN 201910200127 A CN201910200127 A CN 201910200127A CN 110272965 A CN110272965 A CN 110272965A
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李文杰
高月红
孙计瓒
勒慧达
郝凌云
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Abstract

The present invention relates to a kind of nano inorganic anti-biotic material anti-fungal property detection methods, and 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 and inoculation bacterium solution CBCalibration；lgCBRelative intensity of fluorescence logarithm lgIBMark Qu Jianli；Sample inoculation and shaken cultivation；Recovered liquid IBMeasurement and its viable bacteria content CBAnd TBIt 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 nano inorganic material antifungal activity with R or A characterization using ATP fluophotometer.Present invention provide that measurement shaken cultivation cross-reference sample and antimicrobial sample recovered liquid IB, with lgIBCharacterization and calculating R or A simultaneously provide evaluation of result foundation.The ATP bioluminescence lgC for the nano inorganic anti-biotic material anti-fungal property detection that the present invention researches and developsB‑lgIBBent method is marked, product quality will be supported to be promoted with advanced detection technique.

Description

ATP bioluminescence lgCB-lgIBIt marks bent method and detects nano inorganic material anti-fungal property Method
Technical field
The present invention relates to the anti-fungal property test method of nano inorganic anti-biotic material, specifically a kind of application ATP fluorescence Photometer can be carried out precisely quantitative inspection to the nano inorganic anti-biotic material antifungal activity with antibiotic rate R or antibacterial activity value A characterization The ATP bioluminescence lgC of surveyB-lgIBCalibration curve method belongs to nano inorganic material antibacterial functions detection technique field.
Background technique
In recent years, along with the increasingly reinforcing of social scientific and technological progress maked rapid progress with health of people environmental consciousness；Nanometer Inorganic antibacterial material is widely used in numerous areas such as building materials, household electrical appliances, light textile, food packet, daily use chemicals, and domestic market scale is dashed forward already Hundred billion and cumulative year after year are broken, but the lag of existing detection technique seriously restricts industry development at present.
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 nano inorganic material is antimycotic Method for testing performance standard still belongs to blank.Due to the mycelium that is formed in mycotic spore growth course can not accurate counting, can only It is qualitatively judged, it can not quantitative test；And because the fungus growth period is longer, incubation time at least 2 weeks~4 weeks, cause to test Period is longer, and time and economic cost are high.No matter Candida albicans or mould are directed to, the experimentation of existing standard method is numerous Trivial, technical difficulty is higher；Relevant operation is affected by human factors greatly, so that test error is big, lacks comparativity.In recent years in state Bacteria Detection technical field ATP fluorescence analysis development in border is increasingly mature, and testing result correlation is compared with traditional Plating 98%, accuracy is high and can realize quick detection.External anti-biotic material performance detection technical field of research for current quantification, Rapid and summary development trend has used for reference ATP fluorescence analysis principle and has formulated ISO 20743:2007-2013 " Textiles- (textiles antibacterial finishing is spun Determination of antibacterial activity of textile products The performance measurement of fabric antibacterium) " and ISO 13629-1:2012 " Textiles-Determination of antifungal Activity of textile products.Part1Luminescence (survey by textiles antibacterial finishing textile fungicidal properties It is fixed) ", 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 applicable to With water imbibition and control sample it is thin/weaving of mould increasing value > 0 or poromerics, in key technologies sides such as test conditions for validity Face is not suitable for the nanometer grade powder type inorganic antibacterial material of control sample fungi increasing value < 0；It is uncertain not provide measurement simultaneously Degree assessment.In addition, the standard method dependent antimicrobial performance characterization parameter is more single, antibacterial activity value A is only related to, and China is then Usual antibiotic rate R.
Therefore, to promote successive generations of products, industrial transformation upgrading, specification domestic market order, it would be highly desirable to research section are pushed It emulates the advanced, accuracy and reproducibility are high, easy-to-use nano inorganic anti-biotic material Performance Testing Technology.The art of this patent route The world that integrates with is designed, 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 nano inorganic anti-biotic material antifungal activity can be carried out the ATP bioluminescence lgC of detectionB-lgIBCalibration curve method, It is able to solve nano inorganic anti-biotic material or even other field anti-biotic material and the accurate quantitative test problem of 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 nano inorganic anti-biotic material 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 is living after methylene blue Bacterial content CBBlood cell plate count and inoculation bacterium solution CBCalibration；(5) viable bacteria content logarithm lgCBRelative intensity of fluorescence logarithm lgIBStandard curve is established；(6) sample inoculation and culture；(7) reclaim liquid phase is to fluorescence intensity level IBMeasurement；(8) recovered liquid viable bacteria Content CBAnd TBIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluorescence Photometer can be carried out precisely quantitative survey to the nano inorganic anti-biotic material antifungal activity with antibiotic rate R or antibacterial activity value A characterization The ATP bioluminescence lgC of examinationB-lgIBCalibration curve method, specific:
In reclaim liquid phase to fluorescence intensity level IBIn measurement:
Granularity, quality 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；It is used after methylene blue The viable bacteria content C of blood counting chamber measurement bacterium solutionB, and to inoculation bacterium solution CBIt is demarcated: 5.0 × 105CFU/mL~9.0 × 105CFU/mL.Select viable bacteria content CBIt is 3.5 × 103CFU/mL、3.5×104CFU/mL、3.5×105The bacterium solution of CFU/mL is made For standard series bacteria suspension, using ATP fluophotometer to its relative intensity of fluorescence value IBIt is measured；Draw lgCB-lgIBMark Directrix curve is derived from the linear equation Y=a of curveBX+bBAnd coefficient RB 2.Then, to equipped with each group control sample Bacterium solution is inoculated with 5mL is added dropwise in the conical flask of antimicrobial sample respectively；6 groups of 0h are contacted using ATP fluophotometer in 1min The relative intensity of fluorescence value I of sample recovered liquidBC0ij、IBT0ijIt is measured, according to fitting equation Y=aBX+bBCalculate its viable bacteria Content CBOijAnd TBOij.Meanwhile will wait for that the conical flask of culture sample is fixed on constant temperature oscillator equipped with 6 groups, in (30 ± 2) DEG C After (Candida albicans) or (28 ± 2) DEG C (mould) is with the speed oscillation culture to stipulated time of 150r/min~200r/min； Using the relative intensity of fluorescence value I of its recovered liquid of ATP fluorescent spectrophotometer measuringBCtij、IBTtij, and calculate corresponding viable bacteria content CBtijAnd TBtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to test strain standard curve lgCB-lgIBLinear equation Y=aBX+bB, with 0h contact and shaken cultivation The relative intensity of fluorescence measured value I of every control sample and antimicrobial sample recovered liquid afterwardsBC0ij、IBCtijAnd IBT0ij、IBTtijAs base Plinth data；In the case where testing condition for validity, its fungi increasing value F after shaken cultivation is calculatedij、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 nano inorganic antibacterial material Expect that the antibiotic rate R and antibacterial activity value A of sample are its 3 groups of sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and Uncertainty of measurement 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) nano inorganic anti-biotic material 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 lgCB─lgIBThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) nano inorganic anti-biotic material sample anti-fungal property level is identical, then abandons it；Take other two groups (four) remaining samples anti- Bacterium rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value it is anti-as batch (group) the nano inorganic anti-biotic material sample The evaluation result of fungi performance.
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 measure The viable bacteria amount recycled after sample culturing specific time reaches the modernization of nano inorganic material anti-fungal property detection；It can be effective Reducing human factor in experimentation influences, and breaches the qualitative analysis limitation of traditional plate culture；Realize testing result Quantification, and increase substantially the accuracy of detection data；Detection technique has certain advance.
(2) scientific: according to ATP fluorescence analysis test philosophy, to establish the viable bacteria content logarithm suitable for multi-cultur es Value lgCB- relative intensity of fluorescence logarithm lgIBStandard curve；Construct nano inorganic material anti-fungal property test ATP biology The mathematical model of fluorescence real-time quantitative analysis method.Meanwhile take into account country variant consumer perceptions habit, 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 nanometer The precision of inorganic material anti-fungal property testing result and quantification simultaneously have good reproduction and comparativity；It substantially contracts simultaneously Short test period reduces testing cost；Presently relevant the field of test technology blank can effectively be filled up.
(4) perspective: to establish with lgCB、lgIBStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously The bacteria suspension viable bacteria content measuring method and powder sample pretreatment mode for enriching fungi, specify that control sample, standard liquid are dense The technology contents such as degree, determination step, calculation formula, uncertainty；The pioneering recovered liquid viable bacteria directly measured with instrument is relatively glimmering Light intensity value IBAntibiotic rate R or antibacterial activity value A is calculated and determined for evaluation of result form, and by group and a between-group variation Coefficient CV investigates uncertainty of measurement, technically has centainly perspective.
(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, the methylene blue that this patent is established Method is simple for blood cell plate viable bacteria counting method and nano inorganic material anti-fungal property ATP fluorometric investigation after dyeing, related skill It is clear and specific that art illustrates, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for different majors Horizontal Experiment on Microbiology personnel 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 nano inorganic material anti-fungal property detection technique realization it is pervasive Change, while reference can be provided to the product scopes anti-fungal property detection technique research such as chemical product.
Further, preferred embodiment of the invention is:
The sample preparation and pretreatment carries out in the steps below:
(1) control sample: not antibacterial processed SiO 2 powder, granularity be not more than 100nm, purity 98%~ 99%；Test strain culture solution by 0.5g ± 0.05g sample powder and 44mL containing 0.1% Tween-80 pours into 100mL conical flask In, 121 DEG C of high pressure sterilization 30min after sealing.Each strain test uses 6 groups of samples；Wherein 0h contact and specific time oscillation Culture experiment respectively uses 3 groups, every group of 5 powder samples；
(2) antimicrobial sample: the nanometer grade powder type inorganic material with antibacterial action, granularity, quality, quantity and preceding place Reason etc. is identical as control sample, and every group of antimicrobial sample selects one group of control sample as object of reference and effectively identify；
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, very Bacterium cell/spore sum detection range 101CFU/mL~105CFU/mL；Amplification factor 40 ×~400 × Photobiology it is micro- Mirror；The constant incubator that (25~37) DEG C ± 1 DEG C；The constant-temperature shaking incubator of (0~50) DEG C ± 1 DEG C, (50~300) r/min； Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；(121 ± 2) DEG C, pressure steam sterilizer of (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；The pH meter of precision ± 0.1 (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；Sterile petri dish；Glass funnel；Sterile cock test tube；Diameter Oese no more than 4mm；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Absorbent cotton and gauze for biochemistry detection；Nothing Bacterium filter paper；The thermometer that (0~100) is DEG C ± 0.2 DEG C；The stopwatch of precision 0.01s；
(4) reagent: 0.1% Lv Shi alkaline methylene blue dyeing liquor；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C of storage 30d:N monomethyl ethanesulfonic acids, Tween 80, two pungent sulfonation sodium succinates choose 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 (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；
(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 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 liquid11Mol/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 ATCC6205；It produces 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 counts and inoculation bacterium solution CBCalibration, in the steps below It carries out:
(1) methylene blue: by 50 μ L dilutions being 10- with sterile working2~10-3Candida 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 to known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid be diluted, obtain CBRange is 5.0 × 105CFU/mL~9.0 × 105The inoculation bacterium solution of CFU/mL；
The viable bacteria content logarithm lgCBRelative intensity of fluorescence logarithm lgIBStandard curve is established, in the steps below It carries out:
With Cha Shi culture solution to known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid connected After continuous gradient dilution, standard series bacteria suspension 3.5 × 10 is obtained3CFU/mL、3.5×104CFU/mL、3.5×105CFU/mL is simultaneously It mixes.According to relative intensity of fluorescence value I in this patentBMeasuring method, according to viable bacteria content CBSequence from low to high, measurement is simultaneously It is relatively glimmering in 1min after eluent dilution of the 44mL containing 0.1% Tween-80 to record above-mentioned standard solution and 5mL inoculation bacterium solution The latter (is denoted as I by light intensity valueB0).Then, with the relative intensity of fluorescence logarithm lgI of standard series bacteria suspensionBAs horizontal seat Mark, with corresponding viable bacteria content logarithm lgCBFor ordinate mapping；Calibration curve is carried out to mathematical relationship between the two, is answered Fitting equation Y=a is derived from least square fitting methodBX+bBAnd linearly dependent coefficient RB 2；Work as RB 2>=0.98, confidence water When flat >=0.95, the measurement done according to this patent method is effective；
The sample inoculation and shaken cultivation carries out in the steps below:
3000r/min shaking is equipped with the conical flask 30s of each group control sample and antimicrobial sample, with sterilizing liquid-transfering gun after mixing 5mL inoculation bacterium solution is injected separately into bottle (with lgCB-lgIBCalibration curve is derived from same branch test strain stoste test tube with bacterium solution, 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used)；Shaking contacts the conical flask 10s of sample equipped with 6 groups of 0h again immediately, and will mix in bottle Recovered liquid of the even liquid as sample to be tested applies its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring in 1min.Meanwhile It will wait for that the conical flask of culture sample is fixed on constant temperature oscillator equipped with 6 groups, (30 ± 2) DEG C (Candida albicans) or (28 ± 2) DEG C (mould) carries out oscillating contact culture with the revolving speed of 150r/min~200r/min；Wherein need the nano inorganic used after dilution Material sample culture 1h~4h (Candida albicans) or 2h~8h (mould), without diluting the nano inorganic material sample directly used Product culture 4h~for 24 hours (Candida albicans) or 8h~48h (mould)；And by mixing liquid in the bottle after shaken cultivation to stipulated time Recovered liquid as sample to be tested；
The reclaim liquid phase is to fluorescence intensity level IBMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: with sterilizing liquid-transfering gun by 0.1mL Cha Shi culture solution, The ATP lysate of 0.35mL physiological saline and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；10min~20min is stood, as level-one blank sample；0.1mL level-one blank sample is moved into an other sterile test tube again In, 0.4mL physiological saline is added dropwise and mixes, as second level blank sample.Then, with sterilizing liquid-transfering gun by 0.1mL second level blank sample It successively moves in three instrument sterile test tubes, as skip test Duplicate Samples；Respectively it is added dropwise 0.1mL's into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing, and 3000r/min shakes test tube 5s, uses ATP fluorescence light immediately Degree meter measurement its relative intensity of fluorescence value IBAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).It is each flat Row sample 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 With reagent set background values (or calibrating background according to instrument operation instruction)；
(2) reclaim liquid phase is to fluorescence intensity level IBMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube by sterilizing liquid-transfering gun, 3000r/min vibration Shake test tube 30s；After being stored at room temperature 20min, the ATP that 5.0mL is added dropwise extracts reagent and mixes again；It is stored at room temperature 10min.With going out Bacterium liquid-transfering gun successively moves to the above-mentioned mixed solution of 0.1mL in three instrument sterile test tubes, tests as ATP bioluminescence Duplicate Samples.The ATP fluorescent reagent of 0.1mL is respectively added dropwise into three Duplicate Samples, 3000r/min shakes test tube 5s, glimmering with ATP immediately Its relative intensity of fluorescence value of light photometric determination IBAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Often A Duplicate Samples minute is no more than 15s, with the arithmetic average of three ATP bioluminescence test Duplicate Samples relative intensity of fluorescence values It is worth the I as sample to be tested recovered liquidBMeasured value；
The recovered liquid viable bacteria content CBAnd TBIt calculates and antibiotic rate R and antibacterial activity value A is calculated, in the steps below It carries out:
(1) recovered liquid viable bacteria content CBAnd TBIt calculates
According to test strain standard curve lgCB-lgIBLinear equation Y=aBX+bB, calculate control sample and antibacterial The viable bacteria content C of sample recovered liquid after 0h contact and shaken cultivationBAnd T.Relevant calculation is shown in formula (1)~(12):
In formula:
CB0And CBt- 3 groups of 0h contacts and the mean viable amount that control sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；
CB0iAnd CBti- every group 0h contact and the mean viable amount that control sample recycles after shaken cultivation, unit is bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3；
CB0ijAnd CBtij- every 0h contact and the viable bacteria amount that control sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；bB- standard curve lgCB-lgIBIn vertical axis intercept；
TB0And TBt- 3 groups of 0h contacts and the mean viable amount that antimicrobial sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；
TB0iAnd TBti- every group 0h contact and the mean viable amount that antimicrobial sample recycles after shaken cultivation, unit is bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3；
TB0ijAnd TBtij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation the relative intensity of fluorescence of antimicrobial sample recovered liquid measurement 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 every 0h contact control sample recovered liquid and 5mL inoculation bacterium solution contain the eluent of 0.1% Tween-80 through 44mL Relative intensity of fluorescence measured value after dilution in 1min is close, i.e.,Every control sample recovered liquid after shaken cultivation The logarithm of relative intensity of fluorescence measured valueThat is CBtij≥0.1×CB0ij.Then when 3 groups of 0h contact control sample Product reclaim liquid phase is to fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in The requirement of this patent uncertainty of measurement), 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 after shaken cultivation, fungi increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd GijThe fungi increasing value of-every control sample and antimicrobial sample after shaken cultivation, sample group i=1, 2,3, every group of sample number into spectrum j=1,2,3,4,5；
CB0ijAnd CBtij- every 0h contact and the viable bacteria amount that control sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；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 measures after shaken cultivation Value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
TB0ijAnd TBtij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every nano inorganic anti-biotic material sampleij, every group and every batch of sample it is anti- Bacterium rate RiIt is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every nano inorganic anti-biotic material 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 nano inorganic anti-biotic material sample, %；Sample group i=1,2,3；
R-every batch of nano inorganic anti-biotic material sample antibiotic rate, %；
TBtijAnd CBtijThe viable bacteria amount that-every antimicrobial sample and control sample recycle after shaken cultivation, unit are bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
WithAfter shaken cultivation, the relative intensity of fluorescence of recovered liquid is measured for-every antimicrobial sample and control sample Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every nano inorganic anti-biotic material 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 nano inorganic anti-biotic material sample；Sample group i=1,2,3；Every group of sample is compiled Number j=1,2,3,4,5；
AiThe antibacterial activity value of-every group nano inorganic anti-biotic material sample, sample group i=1,2,3；
A-every batch of nano inorganic anti-biotic material sample antibacterial activity value；
FijAnd GijThe fungi increasing value of-every control sample and antimicrobial sample after shaken cultivation, sample group i=1, 2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；
(6) bacteria suspension viable bacteria content C data revision of the convention requirement: is demarcated using blood counting chamberBWhen, with reference to GB4789.2- Relevant regulations in 2016, work as CBWhen less than 100CFU/mL, " rounding up " round numbers；Work as CBWhen not less than 100CFU/mL, the 3rd Preceding 2 bit digital is taken after bit digital " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, " four houses five Enter " afterwards using two effective digitals.Control sample and antimicrobial sample are after 0h contact and shaken cultivation, the relative fluorescence of recovered liquid Strength detection value round numbers, antibiotic rate Rij、Ri, R calculated result take three effective digitals；Fungi increasing value Fij、GijIt is living with antibacterial Property value Aij、Ai, A calculated result take two effective digitals；
(7) uncertainty of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample through 0h contact and shakes After swinging culture, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV calculated result remains into 2 significant digits), judge that ATP fluorimetric assay for biological materials method, which is applied to nano inorganic material, to be resisted very The reproducibility of bacterium performance test；In regulation group and between-group variation coefficient CV≤10%.
5 control samples, 5 antimicrobial samples and 5 control samples after shaken cultivation of every group of 0h contact, 5 Antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining value Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample, which are contacted and vibrated through 0h, to be trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after shaken cultivation that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted and are vibrated through 0h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after shaken cultivation of every group of 0h contact, 5 Antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample, which are contacted and vibrated through 0h, to be trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after shaken cultivation that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted and are vibrated through 0h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
The evaluation of result of the nano inorganic anti-biotic material 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) nano inorganic anti-biotic material samplei(Rij) or antibacterial activity value Ai(Aij) and its When the anti-fungal property of his two groups of (four) samples is compared to a levels are at least differed, one group of (part) sample is extracted again and is repeated Experiment；It is calculated through ATP bioluminescence lgCB─lgIBThe antibiotic rate or antibacterial activity value that calibration curve method measures.If front and back Two groups of (part) nano inorganic anti-biotic material sample anti-fungal property levels are identical, then abandon it；Take other two groups (four) remaining samples Product antibiotic rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) the nano inorganic anti-biotic material sample The evaluation result of product 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 nano inorganic antimicrobial powder material sample that the present embodiment is prepared with added nanometer silver-series antibacterial agent resists Fungi performance is detected as example and is illustrated.
Specific detection method carries out in the steps below:
(1) sample preparation and pretreatment
1.1 control samples: not antibacterial processed SiO 2 powder, granularity be not more than 100nm, purity 98%~ 99%；Test strain culture solution by 0.5g ± 0.05g sample powder and 44mL containing 0.1% Tween-80 pours into 100mL conical flask In, 121 DEG C of high pressure sterilization 30min after sealing.Each strain test uses 6 groups of samples；Wherein 0h contact and specific time oscillation Culture experiment respectively uses 3 groups, every group of 5 powder samples.
1.2 antimicrobial samples: the nanometer grade powder type inorganic material with antibacterial action, granularity, quality, quantity and preceding place Reason etc. is identical as control sample, and every group of antimicrobial sample selects one group of control sample as object of reference and effectively identify.
(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, very Bacterium cell/spore sum detection range 101CFU/mL~105CFU/mL；Amplification factor 40 ×~400 × Photobiology it is micro- Mirror；The constant incubator that (25~37) DEG C ± 1 DEG C；The constant-temperature shaking incubator of (0~50) DEG C ± 1 DEG C, (50~300) r/min； Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；(121 ± 2) DEG C, pressure steam sterilizer of (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；The pH meter of precision ± 0.1 (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；Sterile petri dish；Glass funnel；Sterile cock test tube；Diameter Oese no more than 4mm；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Absorbent cotton and gauze for biochemistry detection；Nothing Bacterium filter paper；The thermometer that (0~100) is DEG C ± 0.2 DEG C；The stopwatch of precision 0.01s.
2.4 reagents: 0.1% Lv Shi alkaline methylene blue dyeing liquor；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C of storage 30d:N monomethyl ethanesulfonic acids, Tween 80, two pungent sulfonation sodium succinates choose 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 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 liquid11Mol/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 ATCC6205；It produces 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 counts and inoculation bacterium solution CBCalibration
4.1 methylene blues: by 50 μ L dilutions being 10- with sterile working2~10-3Candida 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 to known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid be diluted, obtain CBRange is 5.0 × 105CFU/mL~9.0 × 105The inoculation bacterium solution of CFU/mL.
(5) viable bacteria content logarithm lgCBRelative intensity of fluorescence logarithm lgIBStandard curve is established
With Cha Shi culture solution to known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid connected After continuous gradient dilution, standard series bacteria suspension 3.5 × 10 is obtained3CFU/mL、3.5×104CFU/mL、3.5×105CFU/mL is simultaneously It mixes.According to relative intensity of fluorescence value I in this patentBMeasuring method, according to viable bacteria content CBSequence from low to high, measurement is simultaneously It is relatively glimmering in 1min after eluent dilution of the 44mL containing 0.1% Tween-80 to record above-mentioned standard solution and 5mL inoculation bacterium solution The latter (is denoted as I by light intensity valueB0).Then, with the relative intensity of fluorescence logarithm lgI of standard series bacteria suspensionBAs horizontal seat Mark, with corresponding viable bacteria content logarithm lgCBFor ordinate mapping；Calibration curve is carried out to mathematical relationship between the two, is answered Fitting equation Y=a is derived from least square fitting methodBX+bBAnd linearly dependent coefficient RB 2；Work as RB 2>=0.98, confidence water When flat >=0.95, the measurement done according to this patent method is effective.
(6) sample inoculation and shaken cultivation
3000r/min shaking is equipped with the conical flask 30s of each group control sample and antimicrobial sample, with sterilizing liquid-transfering gun after mixing 5mL inoculation bacterium solution is injected separately into bottle (with lgCB-lgIBCalibration curve is derived from same branch test strain stoste test tube with bacterium solution, 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used)；Shaking contacts the conical flask 10s of sample equipped with 6 groups of 0h again immediately, and will mix in bottle Recovered liquid of the even liquid as sample to be tested applies its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring in 1min.Meanwhile It will wait for that the conical flask of culture sample is fixed on constant temperature oscillator equipped with 6 groups, (30 ± 2) DEG C (Candida albicans) or (28 ± 2) DEG C (mould) carries out oscillating contact culture with the revolving speed of 150r/min~200r/min；Wherein need the nano inorganic used after dilution Material sample culture 1h~4h (Candida albicans) or 2h~8h (mould), without diluting the nano inorganic material sample directly used Product culture 4h~for 24 hours (Candida albicans) or 8h~48h (mould)；And by mixing liquid in the bottle after shaken cultivation to stipulated time Recovered liquid as sample to be tested.
(7) reclaim liquid phase is to fluorescence intensity level IBMeasurement
7.1 instruments and reagent set relative intensity of fluorescence background value calibration: with sterilizing liquid-transfering gun by 0.1mL Cha Shi culture solution, The ATP lysate of 0.35mL physiological saline and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；10min~20min is stood, as level-one blank sample；0.1mL level-one blank sample is moved into an other sterile test tube again In, 0.4mL physiological saline is added dropwise and mixes, as second level blank sample.Then, with sterilizing liquid-transfering gun by 0.1mL second level blank sample It successively moves in three instrument sterile test tubes, as skip test Duplicate Samples；Respectively it is added dropwise 0.1mL's into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing, and 3000r/min shakes test tube 5s, uses ATP fluorescence light immediately Degree meter measurement its relative intensity of fluorescence value IBAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).It is each flat Row sample 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 With reagent set background values (or calibrating background according to instrument operation instruction).
7.2 reclaim liquid phases are to fluorescence intensity level IBMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube by sterilizing liquid-transfering gun, 3000r/min vibration Shake test tube 30s；After being stored at room temperature 20min, the ATP that 5.0mL is added dropwise extracts reagent and mixes again；It is stored at room temperature 10min.With going out Bacterium liquid-transfering gun successively moves to the above-mentioned mixed solution of 0.1mL in three instrument sterile test tubes, tests as ATP bioluminescence Duplicate Samples.The ATP fluorescent reagent of 0.1mL is respectively added dropwise into three Duplicate Samples, 3000r/min shakes test tube 5s, glimmering with ATP immediately Its relative intensity of fluorescence value of light photometric determination IBAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Often A Duplicate Samples minute is no more than 15s, with the arithmetic average of three ATP bioluminescence test Duplicate Samples relative intensity of fluorescence values It is worth the I as sample to be tested recovered liquidBMeasured value.
(8) recovered liquid viable bacteria content CBAnd TBIt calculates and antibiotic rate R and antibacterial activity value A is calculated
8.1 recovered liquid viable bacteria content CBAnd TBIt calculates
According to test strain standard curve lgCB-lgIBLinear equation Y=aBX+bB, calculate control sample and antibacterial The viable bacteria content C of sample recovered liquid after 0h contact and shaken cultivationBAnd T.Relevant calculation is shown in formula (1)~(12):
In formula:
CB0And CBt- 3 groups of 0h contacts and the mean viable amount that control sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；
CB0iAnd CBti- every group 0h contact and the mean viable amount that control sample recycles after shaken cultivation, unit is bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3；
CB0ijAnd CBtij- every 0h contact and the viable bacteria amount that control sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；bB- standard curve lgCB-lgIBIn vertical axis intercept；
TB0And TBt- 3 groups of 0h contacts and the mean viable amount that antimicrobial sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；
TB0iAnd TBti- every group 0h contact and the mean viable amount that antimicrobial sample recycles after shaken cultivation, unit is bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3；
TB0ijAnd TBtij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation the relative intensity of fluorescence of antimicrobial sample recovered liquid measurement 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 every 0h contact control sample recovered liquid and 5mL inoculation bacterium solution contain the eluent of 0.1% Tween-80 through 44mL Relative intensity of fluorescence measured value after dilution in 1min is close, i.e. IBC0ij≈IB0, every control sample recycling after shaken cultivation Logarithm of the liquid phase to fluorescent strength determining valueThat is CBtij≥0.1×CB0ij.Then when 3 groups of 0h contact controls Sample reclaim liquid phase is to fluorescent strength determining valueGroup in and (involved calculation formula when between-group variation coefficient CV≤10% See the requirement of this patent uncertainty of measurement), 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 after shaken cultivation, fungi increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd GijThe fungi increasing value of-every control sample and antimicrobial sample after shaken cultivation, sample group i=1, 2,3, every group of sample number into spectrum j=1,2,3,4,5；
CB0ijAnd CBtij- every 0h contact and the viable bacteria amount that control sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
TB0ijAnd TBtij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after shaken cultivation, unit are bacterium colony shape At units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope.
8.4 antibiotic rate R are calculated
It tests under condition for validity, the antibiotic rate R of every nano inorganic anti-biotic material sampleij, every group and every batch of sample it is anti- Bacterium rate RiIt is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every nano inorganic anti-biotic material 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 nano inorganic anti-biotic material sample, %；Sample group i=1,2,3；
R-every batch of nano inorganic anti-biotic material sample antibiotic rate, %；
TBtijAnd CBtijThe viable bacteria amount that-every antimicrobial sample and control sample recycle after shaken cultivation, unit are bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
WithAfter shaken cultivation, the relative intensity of fluorescence of recovered liquid is measured for-every antimicrobial sample and control sample Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope.
8.5 antibacterial activity value A are calculated
It tests under condition for validity, the antibacterial activity value A of every nano inorganic anti-biotic material 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 nano inorganic anti-biotic material sample；Sample group i=1,2,3；Every group of sample is compiled Number j=1,2,3,4,5；
AiThe antibacterial activity value of-every group nano inorganic anti-biotic material sample, sample group i=1,2,3；
A-every batch of nano inorganic anti-biotic material sample antibacterial activity value；
FijAnd GijThe fungi increasing value of-every control sample and antimicrobial sample after shaken cultivation, sample group i=1, 2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope.
The requirement of the 8.6 data revisions of the convention: bacteria suspension viable bacteria content C is demarcated using blood counting chamberBWhen, with reference to GB4789.2- Relevant regulations in 2016, work as CBWhen less than 100CFU/mL, " rounding up " round numbers；Work as CBWhen not less than 100CFU/mL, the 3rd Preceding 2 bit digital is taken after bit digital " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, " four houses five Enter " afterwards using two effective digitals.Control sample and antimicrobial sample are after 0h contact and shaken cultivation, the relative fluorescence of recovered liquid Strength detection value round numbers, antibiotic rate Rij、Ri, R calculated result take three effective digitals；Fungi increasing value Fij、GijIt is living with antibacterial Property value Aij、Ai, A calculated result take two effective digitals.
8.7 uncertainties of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample through 0h contact and shakes After swinging culture, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV calculated result remains into 2 significant digits), judge that ATP fluorimetric assay for biological materials method, which is applied to nano inorganic material, to be resisted very The reproducibility of bacterium performance test；In regulation group and between-group variation coefficient CV≤10%.
5 control samples, 5 antimicrobial samples and 5 control samples after shaken cultivation of every group of 0h contact, 5 Antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample, which are contacted and vibrated through 0h, to be trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after shaken cultivation that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted and are vibrated through 0h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after shaken cultivation of every group of 0h contact, 5 Antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample, which are contacted and vibrated through 0h, to be trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after shaken cultivation that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted and are vibrated through 0h After 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) nano inorganic anti-biotic material samplei(Rij) or antibacterial activity value Ai(Aij) and its When the anti-fungal property of his two groups of (four) samples is compared to a levels are at least differed, one group of (part) sample is extracted again and is repeated Experiment；It is calculated through ATP bioluminescence lgCB─lgIBThe antibiotic rate or antibacterial activity value that calibration curve method measures.If front and back Two groups of (part) nano inorganic anti-biotic material sample anti-fungal property levels are identical, then abandon it；Take other two groups (four) remaining samples Product antibiotic rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) the nano inorganic anti-biotic material sample The evaluation result of product 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: 1300 series of secondary B2 type Biohazard Safety Equipment of Thermo Scientific, workbench Surface area 0.55m2, capacity 1130m3/ h, filter efficiency 99.99% (0.3 μm).
2.2ATP bioluminescence rapid detection system: the portable system SUREATP fluorescence of Hygiena company, the U.S. Detector, box containing matched reagent, plastics Special test tube etc.；ATP Monitoring lower-cut 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%.
2.3 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.
2.4 constant temperature and humidity incubators: Guan Sen biotechnology (Shanghai) Co., Ltd. model WS-380H, volume 380L, temperature control The constant temperature and humidity incubator of ± 0.8 DEG C of range (0~50) DEG C, wet range (30~95) %RH ± 2%RH of control.
2.5 constant-temperature shaking incubators: the permanent model WS-380H in Shanghai one, ± 0.1 DEG C of temperature control range (4~65) DEG C, oscillation frequency Rate (40~300) r/min, 1~99h59min of timing range.
2.6 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.
2.7 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.
2.8 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.
2.9 refrigerators: the cryogenic box ultra low temperature freezer of the glad experimental instruments and equipment limited model HXL-25-250AD of Dongguan City sky, ± 0.1 DEG C of its refrigerating box (2~10) DEG C, ± 0.1 DEG C of household freezer (- 30~20) DEG C.
2.10 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.
2.11 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.
2.12 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.
2.13pH meter: the accurate pH meter of Shanghai precision instrumentation Co., Ltd model MP512-03, range ability (- 2.000~19.999) pH ± 0.002pH, ± 0.4 DEG C of temperature range (- 10~110) DEG C.
2.14 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.
2.15 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 is to the anti-true of nano inorganic antimicrobial powder material sample Bacterium performance is detected, and 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 nano inorganic anti-biotic material anti-fungal property, comprising: (1) sample preparation and pretreatment；(2) it sets Alternative type and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) bacteria suspension viable bacteria after methylene blue Content CBBlood cell plate count and inoculation bacterium solution CBCalibration；(5) viable bacteria content logarithm lgCBRelative intensity of fluorescence logarithm lgIBStandard curve is established；(6) sample inoculation and shaken cultivation；(7) reclaim liquid phase is to fluorescence intensity level IBMeasurement；(8) recovered liquid Viable bacteria content CBAnd TBIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP It is precisely fixed that fluophotometer can be carried out the nano inorganic anti-biotic material antifungal activity with antibiotic rate R or antibacterial activity value A characterization Measure the ATP bioluminescence lgC of examinationB-lgIBCalibration curve method, specific:
In reclaim liquid phase to fluorescence intensity level IBIn measurement:
Specify control sample and antimicrobial sample granularity, quality 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；Blood cell is used after methylene blue The viable bacteria content C of tally measurement bacterium solutionB, and to inoculation bacterium solution CBIt is demarcated: 5.0 × 105CFU/mL~9.0 × 105CFU/ ML selects viable bacteria content CBIt is 3.5 × 103CFU/mL、3.5×104CFU/mL、3.5×105The bacterium solution of CFU/mL is as standard Serial bacteria suspension, using ATP fluophotometer to its relative intensity of fluorescence value IBIt is measured；Draw lgCB-lgIBStandard is bent Line is derived from the linear equation Y=a of curveBX+bBAnd coefficient RB 2, then, to equipped with each group control sample and anti- 5mL inoculation bacterium solution is added dropwise in the conical flask of bacterium sample respectively；Sample is contacted to 6 groups of 0h using ATP fluophotometer in 1min The relative intensity of fluorescence value I of recovered liquidBC0ij、IBT0ijIt is measured, according to fitting equation Y=aBX+bBCalculate its viable bacteria content CBOijAnd TBOij, meanwhile, it will wait for that the conical flask of culture sample is fixed on constant temperature oscillator equipped with 6 groups, in (30 ± 2) DEG C (white Candida albicans) or (28 ± 2) DEG C (mould) with the speed oscillation culture to stipulated time of 150r/min~200r/min after；Using The relative intensity of fluorescence value I of its recovered liquid of ATP fluorescent spectrophotometer measuringBCtij、IBTtij, and calculate corresponding viable bacteria content CBtijWith TBtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to test strain standard curve lgCB-lgIBLinear equation Y=aBX+bB, with every after 0h contact and shaken cultivation The relative intensity of fluorescence measured value of control sample and antimicrobial sample recovered liquidWith As basic data； In the case where testing condition for validity, its fungi increasing value F after shaken cultivation is calculatedij、GijAnd antibiotic rate RijWith antibacterial 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 nano inorganic anti-biotic material sample Antibiotic 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 are not Degree of certainty 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 is classified criterion；When The antibiotic rate R of certain group (part) nano inorganic anti-biotic material samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups (four) When the anti-fungal property of sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Calculate it Through ATP bioluminescence lgCB─lgIBThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) nanometer Inorganic antibacterial material 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) arithmetic mean of instantaneous value as batch (group) nano inorganic anti-biotic material sample antifungal activity The evaluation result of energy.
2. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Sample preparation and pretreatment, in the steps below carry out:
(1) control sample: not antibacterial processed SiO 2 powder, granularity are not more than 100nm, purity 98%~99%；It will The test strain culture solution of 0.5g ± 0.05g sample powder and 44mL containing 0.1% Tween-80 pours into 100mL conical flask, seals 121 DEG C of high pressure sterilization 30min, each strain test use 6 groups of samples afterwards；Wherein 0h contact and the test of specific time shaken cultivation Respectively with 3 groups, every group of 5 powder samples；
(2) antimicrobial sample: the nanometer grade powder type inorganic material with antibacterial action, granularity, quality, quantity and pre-treatment etc. Identical as control sample, every group of antimicrobial sample selects one group of control sample as object of reference and effectively identifies.
3. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Lectotype selection and reagent, culture medium prepare, in the steps below carry out:
(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, fungi Cell/spore sum detection range 101CFU/mL~105CFU/mL；Amplification factor 40 ×~400 × Photobiology microscope； The constant incubator that (25~37) DEG C ± 1 DEG C；The constant-temperature shaking incubator of (0~50) DEG C ± 1 DEG C, (50~300) r/min；Turn Speed >=8000r/min centrifuge and mating centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；(121± 2) DEG C, pressure steam sterilizer of (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；Sense Measure the electronic balance of 0.001g；The ultrasonic cleaner of frequency range (30~50) kHz；The pH meter of precision ± 0.1 (25 DEG C)；Electricity 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；Sterile petri dish；Glass funnel；Sterile cock test tube；Diameter is little In the oese of 4mm；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Absorbent cotton and gauze for biochemistry detection；Sterile filter Paper；Thermometer；The stopwatch of precision 0.01s；
(4) reagent: 0.1% Lv Shi alkaline methylene blue dyeing liquor；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~ 10 DEG C of storage 30d:N monomethyl ethanesulfonic acids, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and 0.05% wetting is configured to Agent aqueous 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 (Candida albicans actication of culture use): 40g glucose, 10g peptone, 20g agar are dissolved by heating In 1000mL water (agar is not added in culture solution), adjust 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 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 nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Culture presevation, activation and bacteria suspension preparation, in the steps below carry out:
(1) test strain: Candida albicans ATCC 10231；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；It produces yellow 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 nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Methylene blue after bacteria suspension viable bacteria content CBBlood cell plate counts and inoculation bacterium solution CBCalibration carries out in the steps below:
(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 is bacterium solution extension rate, then, with Cha Shi culture solution to known viable bacteria content CBIt is white Color beads bacterium suspension or mould mixing spore liquid are diluted, and obtain CBRange is 5.0 × 105CFU/mL~9.0 × 105The inoculation bacterium solution of CFU/mL.
6. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Viable bacteria content logarithm lgCBRelative intensity of fluorescence logarithm lgIBStandard curve is established, and is carried out in the steps below:
With Cha Shi culture solution to known viable bacteria content CBCandida albicans bacterium suspension or mould mixing spore liquid carry out continuous gradient After dilution, standard series bacteria suspension 3.5 × 10 is obtained3CFU/mL、3.5×104CFU/mL、3.5×105CFU/mL is simultaneously mixed, root According to relative intensity of fluorescence value I in this patentBMeasuring method, according to viable bacteria content CBSequence from low to high is measured and is recorded above-mentioned The relative intensity of fluorescence value of standard solution and 5mL inoculation bacterium solution after eluent dilution of the 44mL containing 0.1% Tween-80 in 1min (the latter is denoted as IB0), then, with the relative intensity of fluorescence logarithm lgI of standard series bacteria suspensionBAs abscissa, with corresponding Viable bacteria content logarithm lgCBFor ordinate mapping；Calibration curve is carried out to mathematical relationship between the two, using minimum two Multiply fitting process and is derived from fitting equation Y=aBX+bBAnd linearly dependent coefficient RB 2；Work as RB 2>=0.98, confidence level >=0.95 When, the measurement done according to this patent method is effective.
7. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Sample inoculation and shaken cultivation, in the steps below carry out:
3000r/min shaking is equipped with the conical flask 30s of each group control sample and antimicrobial sample, uses sterilizing liquid-transfering gun to bottle after mixing 5mL inoculation bacterium solution is inside injected separately into (with lgCB-lgIBCalibration curve is derived from same branch test strain stoste test tube with bacterium solution, and 2 DEG C ± 0.2 DEG C of preservation, 2h is interior to be used)；Conical flask 10s of the shaking equipped with 6 groups of 0h contact samples again immediately, and by mixing liquid in bottle As the recovered liquid of sample to be tested, its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring is applied in 1min, meanwhile, it will fill There are 6 groups to wait for that the conical flask of culture sample is fixed on constant temperature oscillator, (30 ± 2) DEG C (Candida albicans) or (28 ± 2) is DEG C (mould Bacterium) with the revolving speed progress oscillating contact culture of 150r/min~200r/min；Wherein need the nano inorganic material used after dilution Sample culturing 1h~4h (Candida albicans) or 2h~8h (mould), without diluting the nano inorganic material sample directly used training Support 4h~for 24 hours (Candida albicans) or 8h~48h (mould)；And using mixing liquid in the bottle after shaken cultivation to stipulated time as The recovered liquid of sample to be tested.
8. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Reclaim liquid phase to fluorescence intensity level IBMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: with sterilizing liquid-transfering gun by 0.1mL Cha Shi culture solution, The ATP lysate of 0.35mL physiological saline and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；10min~20min is stood, as level-one blank sample；0.1mL level-one blank sample is moved into an other sterile test tube again In, 0.4mL physiological saline is added dropwise and simultaneously mixes, as second level blank sample, then, with sterilizing liquid-transfering gun by 0.1mL second level blank sample It successively moves in three instrument sterile test tubes, as skip test Duplicate Samples；Respectively it is added dropwise 0.1mL's into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing, and 3000r/min shakes test tube 5s, uses ATP fluorescence light immediately Degree meter measurement its relative intensity of fluorescence value IBAnd record and (ensure that each link operating time is consistent, avoid cross contamination), Mei Geping Row sample 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 With reagent set background values (or calibrating background according to instrument operation instruction)；
(2) reclaim liquid phase is to fluorescence intensity level IBMeasurement: if blank reagent group background level meets instrument requirement, with sterilizing The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube by liquid-transfering gun, 3000r/min shaking examination Pipe 30s；After being stored at room temperature 20min, the ATP that 5.0mL is added dropwise extracts reagent and mixes again；It is stored at room temperature 10min, is moved with sterilizing Liquid rifle successively moves to the above-mentioned mixed solution of 0.1mL in three instrument sterile test tubes, parallel as the test of ATP bioluminescence The ATP fluorescent reagent of 0.1mL is respectively added dropwise into three Duplicate Samples for sample, and 3000r/min shakes test tube 5s, uses ATP fluorescence light immediately Degree meter measurement its relative intensity of fluorescence value IBAnd record and (ensure that each link operating time is consistent, avoid cross contamination), Mei Geping Row sample minute is no more than 15s, is made with the arithmetic mean of instantaneous value of three ATP bioluminescence test Duplicate Samples relative intensity of fluorescence values For the I of sample to be tested recovered liquidBMeasured value.
9. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that described Recovered liquid viable bacteria content CBAnd TBIt calculates and antibiotic rate R and antibacterial activity value A is calculated, carry out in the steps below:
(1) recovered liquid viable bacteria content CBAnd TBIt calculates
According to test strain standard curve lgCB-lgIBLinear equation Y=aBX+bB, calculate control sample and antimicrobial sample warp The viable bacteria content C of recovered liquid after 0h contact and shaken cultivationBAnd T, relevant calculation are shown in formula (1)~(12):
In formula:
CB0And CBt- 3 groups of 0h contacts and the mean viable amount that control sample recycles after shaken cultivation, unit are that bacterium colony forms list Every milliliter (CFU/mL) in position；
CB0iAnd CBti- every group 0h contact and the mean viable amount that control sample recycles after shaken cultivation, unit are formed for bacterium colony Units per ml (CFU/mL)；Sample group i=1,2,3；
CB0ijAnd CBtij- every 0h contact and the viable bacteria amount that control sample recycles after shaken cultivation, unit are that bacterium colony forms list Every milliliter (CFU/mL) in position；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；bB- standard curve lgCB-lgIBIn vertical axis intercept；
TB0And TBt- 3 groups of 0h contacts and the mean viable amount that antimicrobial sample recycles after shaken cultivation, unit are that bacterium colony forms list Every milliliter (CFU/mL) in position；
TB0iAnd TBti- every group 0h contact and the mean viable amount that antimicrobial sample recycles after shaken cultivation, unit are formed for bacterium colony Units per ml (CFU/mL)；Sample group i=1,2,3；
TB0ijAnd TBtij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after shaken cultivation, unit are that bacterium colony forms list Every milliliter (CFU/mL) in position；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation the relative intensity of fluorescence of antimicrobial sample recovered liquid measured value, RLU；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
(2) condition for validity is tested
If the eluent of every 0h contact control sample recovered liquid and 5mL inoculation bacterium solution through 44mL containing 0.1% Tween-80 dilutes The relative intensity of fluorescence measured value in 1min is close afterwards, i.e.,Every control sample reclaim liquid phase after shaken cultivation To the logarithm of fluorescent strength determining valueThat is CBtij≥0.1×CB0ij, then when 3 groups of 0h contact control sample Reclaim liquid phase is to fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this The requirement of patent uncertainty of measurement), 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 after shaken cultivation, fungi increasing value Fij、GijIt is counted respectively according to formula (13), (14) It calculates:
In formula:
FijAnd GijThe fungi increasing value of-every control sample and antimicrobial sample after shaken cultivation, sample group i=1,2,3, Every group of sample number into spectrum j=1,2,3,4,5；
CB0ijAnd CBtij- every 0h contact and the viable bacteria amount that control sample recycles after shaken cultivation, unit are that bacterium colony forms list Every milliliter (CFU/mL) in position；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
TB0ijAnd TBtij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after shaken cultivation, unit are that bacterium colony forms list Every milliliter (CFU/mL) in position；Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every nano inorganic anti-biotic material sampleij, every group and every batch of sample antibiotic rate RiIt is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every nano inorganic anti-biotic material 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 nano inorganic anti-biotic material sample, %；Sample group i=1,2,3；
R-every batch of nano inorganic anti-biotic material sample antibiotic rate, %；
TBtijAnd CBtijThe viable bacteria amount that-every antimicrobial sample and control sample recycle after shaken cultivation, unit are formed for bacterium colony Units per ml (CFU/mL)；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 after shaken cultivation, the relative intensity of fluorescence measured value of recovered liquid, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every nano inorganic anti-biotic material sampleij, every group and every batch of sample it is anti- Bacterium activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every nano inorganic anti-biotic material 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 nano inorganic anti-biotic material sample, sample group i=1,2,3；
A-every batch of nano inorganic anti-biotic material sample antibacterial activity value；
FijAnd GijThe fungi increasing value of-every control sample and antimicrobial sample after shaken cultivation, sample group i=1,2,3, Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after shaken cultivation antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3, every group of sample number into spectrum j=1,2,3,4,5；
aB- standard curve lgCB-lgIBSlope；
(6) bacteria suspension viable bacteria content C data revision of the convention requirement: is demarcated using blood counting chamberBWhen, with reference in GB4789.2-2016 Relevant regulations work as CBWhen less than 100CFU/mL, " rounding up " round numbers；Work as CBWhen not less than 100CFU/mL, the 3rd bit digital Preceding 2 bit digital is taken after " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, be adopted after " rounding up " With two effective digitals, after 0h contact and shaken cultivation, the relative intensity of fluorescence of recovered liquid is surveyed for control sample and antimicrobial sample Definite value round numbers, antibiotic rate Rij、Ri, R calculated result take three effective digitals；Fungi increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals；
(7) uncertainty of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample through 0h contact and vibrates training After supporting, reclaim liquid phase is in the group of fluorescent strength determining value 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 nano inorganic material antifungal activity The reproducibility that can be tested；In regulation group and between-group variation coefficient CV≤10%；
5 control samples, 5 antimicrobial samples and 5 control samples after shaken cultivation, 5 antibacterials that every group of 0h is contacted Sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithRespectively every group of control sample and antimicrobial sample through 0h contact and shaken cultivation after, The relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples after shaken cultivation of 3 groups of 0h contact, 15 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample, which are contacted and vibrated through 0h, to be trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after shaken cultivation, 5 antibacterials that every group of 0h is contacted Sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) (26) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2, 3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and shaken cultivation Afterwards, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples after shaken cultivation of 3 groups of 0h contact, 15 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample, which are contacted and vibrated through 0h, to be trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
10. the detection method of nano inorganic anti-biotic material anti-fungal property according to claim 1, which is characterized in that institute The evaluation of result stated 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) nano inorganic anti-biotic material samplei(Rij) or antibacterial activity value Ai(Aij) with other two When the anti-fungal property of group (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and is repeated in fact It tests；It is calculated through ATP bioluminescence lgCB─lgIBThe antibiotic rate or antibacterial activity value that calibration curve method measures, if front and back two Group (part) nano inorganic anti-biotic material sample anti-fungal property level is identical, then abandons it；Take other two groups (four) remaining samples Antibiotic rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) the nano inorganic anti-biotic material sample The evaluation result of anti-fungal property.
CN201910200127.0A 2019-03-15 2019-03-15 ATP bioluminescence lgCB-lgIBThe method for marking bent method detection nano inorganic material anti-fungal property Pending CN110272965A (en)

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