CN110272951A

CN110272951A - ATP bioluminescence lgCB-lgIBThe method of calibration curve method detection antibacterial wood based panel fungicidal properties

Info

Publication number: CN110272951A
Application number: CN201910199723.1A
Authority: CN
Inventors: 李文杰; 贾月梅; 冯金艳; 曹彦强; 崔宗岩
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-09-24

Abstract

The present invention relates to a kind of antibacterial wood based panel fungicidal properties 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；OD value-viable bacteria content logarithm lgC_BMark Qu Jianli and inoculation bacterium solution C_BCalibration；lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established；Sample inoculation, culture and elution recycling；Recovered liquid I_BMeasurement and its viable bacteria content C_BAnd T_BIt calculates；Antibiotic rate R or antibacterial activity value A is calculated；Evaluation of result；Its special feature is that: accurate quantitative test is carried out to the wood based panel fungicidal properties with R or A characterization using ATP fluophotometer.Present invention provide that control sample and antimicrobial sample after elution recycling contact 0h and culture 48h, measure recovered liquid I_BAnd with lgI_BCharacterization and calculating R or A；Evaluation of result foundation is provided.The ATP bioluminescence lgC for the antibacterial wood based panel fungicidal properties detection that the present invention researches and develops_B‑lgI_BBent method is marked, will be promoted with the advanced detection technique support product quality of science.

Description

ATP bioluminescence lgCB-lgIBCalibration curve method detects antibacterial wood based panel fungicidal properties Method

Technical field

The present invention relates to the fungicidal properties test method of antibacterial wood based panel, specifically a kind of application ATP fluophotometer pair The ATP bioluminescence of accurate quantitative detection is carried out with the antibacterial wood based panel fungicidal properties that antibiotic rate R or antibacterial activity value A is characterized lgC_B-lgI_BCalibration curve method belongs to wood based panel antibacterial functions detection technique field.

Background technique

Along with the enhancing of the improvement of people's living standards and health perception, indoor environment and house fitting-up quality are by pass Note, the artificial board product that a new generation has antimildew and antibacterial performance concurrently comes into being and wide market；It can be seen that antimicrobial technology exists The application of wood based panel industry and popularize irresistible, antibacterial functions have become one very characteristic " attraction " of product；But it is existing The lag of detection technique but seriously restricts industry development.

Currently, anti-biotic material performance detection technical system is mainly for bacterium and fungi both at home and abroad, wherein mould proof detection side Method is originated from American Standard, day mark and Europe superscript substantially, and involved standard mainly includes that the external soil that is based on buries cultivation, agar plate method and moist chamber The ISO 846:1997 antibacterial culture of plastic products " assess " of cultivation, DIN 53793 " antimicrobial macromolecule material it is antimycotic Measuring method " and the domestic GB/T 1741-2007 " paint film fungus resistance measuring method " for laying particular emphasis on plate culture and suspension method, GB/T 24346-2009 " evaluation of textile fungicidal properties ", GB/T 24128-2009 " plastic mould-proof method for testing performance ", JC/T 2039-2010 " antibacterial and mouldproof Wooden decoration board ", QB/T 4199-2011 " Leather mildew-proof performance test methods ", HG/T 4301-2012 " rubber fungicidal properties test method ", LY/T 2230-2013 " evaluation of wood-based plate fungicidal properties ", GB/T 18261-2013 " test method of the mould inhibitor to timber mould and stain fungus preventing efficiency " etc..In general, existing mildew resistance There are following common problems in technology contents and practical application for energy detection method: first is that due to shape in mycotic spore growth course At mycelium can not accurate counting, can only be qualitatively judged, can not quantitative test；Second is that because the fungus growth period is longer, Incubation time at least 2 weeks~4 weeks, cause test period longer, time and economic cost are high；Third is that experimentation is cumbersome, technology Difficulty is higher；Relevant operation is affected by human factors greatly, so that test error is big, lacks comparativity.In recent years, in international bacterium The development of detection technique field ATP fluorescence analysis is increasingly mature, and testing result correlation is 98% compared with traditional Plating, quasi- Exactness is high and can realize quick detection.By demand pull, external anti-biotic material performance detection technical field of research is for current fixed Quantization, rapid and summary development trend have used for reference ATP fluorescence analysis principle and have formulated ISO 20743:2007-2013 " Textiles-Determination of antibacterial activity of textile products (textile- The antibacterium performance measurement of antibiotic finish textile) " and ISO 13629-1:2012 " Textiles-Determination of Antifungal activity of textile products.Part1 Luminescence (spin by textile-antibiotic finish The fungicidal properties of fabric measures) ", it is specified that with the fluoremetry of anti-thin/mould performance of ATP changes of contents characterization after sample inoculation Method, but its be only applicable to have water imbibition and control sample it is thin/weaving of mould increasing value > 0 or poromerics, recycled in viable bacteria It is not suitable for that there is unwetted property hard surface and control sample mould increasing value in terms of the key technologies such as mode, test condition for validity The materials such as wood based panel, the ceramics of < 0, while uncertainty of measurement assessment not being provided.In addition, the standard method dependent antimicrobial performance Characterization parameter is more single, only relates to antibacterial activity value A, and China then usual antibiotic rate R.

Therefore, to resist foreign technology barrier, specification domestic market order pushes industrial transformation upgrading, it would be highly desirable to research section It emulates the advanced, accuracy and reproducibility are high, easy-to-use antibacterial wood based panel Performance Testing Technology.The art of this patent highway route design connects Rail is international, and detection method belongs to the whole world and initiates, and can effectively fill up domestic and international correlative technology field blank.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of application ATP fluophotometers to living with antibiotic rate R or antibacterial Property value A characterization the ATP bioluminescence lgC that is detected of antibacterial wood based panel fungicidal properties_B-lgI_BCalibration curve method is able to solve Antibacterial wood based panel or even other product scope anti-biotic materials and the accurate quantitative test problem of product fungicidal properties.

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

A kind of detection method of antibacterial wood based panel fungicidal properties, comprising: (1) sample preparation and pretreatment；(2) lectotype selection And reagent, culture medium are prepared；(3) culture presevation, activation and spore suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BMark Directrix curve is established and inoculating spores liquid C_BCalibration；(5) viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard Curve is established；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level I_BMeasurement；(8) recovered liquid is living Bacterial content C_BAnd T_BIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluorescence The ATP that photometer carries out accurate quantitative test to the antibacterial wood based panel fungicidal properties with antibiotic rate R or antibacterial activity value A characterization is raw Object fluorescence lgC_B-lgI_BCalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_BIn measurement:

Quantity, size and the pre-treatment requirement for specifying control sample and antimicrobial sample, by the standard bacteria of mould test strain After strain is passed on, activated, fresh mycotic spore culture is taken to prepare spore suspension；OD value-is established using MTT colorimetric analysis Viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ².To not Spore suspension with dilution carries out viable bacteria content C_BAfter calibration, C is selected_BIt is 2.1 × 10³CFU/mL、2.1×10⁴CFU/mL、 2.1×10⁵The spore liquid of CFU/mL is as standard series bacteria suspension；Measure its relative intensity of fluorescence value I_B, draw lgC_B-lgI_B Mark is bent, and is derived from the linear equation Y=a of curve_BX+b_BAnd coefficient R_B ².Then, viable bacteria content C is chosen_BRange is 5.0×10⁵CFU/mL~9.0 × 10⁵The mixing spore liquid of CFU/mL is as inoculation bacterium solution；Distinguish to each group sample surface to be measured 0.3mL bacterium solution is added dropwise, elution recycling is carried out to 6 groups of 0h contact samples with 4.6mL eluent immediately, measures the relatively glimmering of recovered liquid Light intensity value I_BC0ij、I_BT0ij, according to lgC_B-lgI_BFitting equation Y=a_BX+b_BCalculate its viable bacteria content C_BOijAnd T_BOij.Together When, control sample that 6 groups are sealed in sterilized petri dishes and antimicrobial sample are trained under conditions of (28 ± 2) DEG C, (95 ± 2) %RH After supporting 48h ± 2h；Recycling remained on surface bacterium is eluted using mode identical with 0h contact sample and measures the relatively glimmering of recovered liquid Light intensity value I_BCtij、I_BTtij, calculate corresponding viable bacteria content C_BtijAnd T_Btij；

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

According to standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, with 0h contact and after 48h is cultivated every it is right The relative intensity of fluorescence measured value of product and antimicrobial sample recovered liquid in the same old wayWithAs basic data；? It tests under condition for validity, calculates its mould increasing value F_ij、G_ijAnd antibiotic rate R_ijWith antibacterial activity value A_ij；To every group of sample R_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；The antibiotic rate R and antibacterial activity value A of the wooden plate sample of every batch of antibacterial be Its 3 groups of sample R_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement require；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion； With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion；When certain group The antibiotic rate R of the wooden plate sample of (part) antibacterial_i(R_ij) or antibacterial activity value A_i(A_ij) mould proof with other two groups (four) samples When performance is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP bioluminescence lgC_B─lgI_BThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of the front and back wooden plate sample of (part) antibacterial is anti- Mould performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i(A_ij) Evaluation result of the arithmetic mean of instantaneous value as the wooden plate sample fungicidal properties of batch (group) antibacterial.

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

(1) advanced: using modern precision instrument-ATP fluophotometer as test equipment, precisely can quickly to survey Determine the viable bacteria amount recycled after wood based panel sample culturing specific time, reaches the modernization of wood based panel fungicidal properties detection；Can have Effect, which reduces human factor in experimentation, to be influenced, and the qualitative analysis limitation of traditional plate culture is breached；Realize detection knot The quantification of fruit, 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 lgC_B- relative intensity of fluorescence logarithm lgI_BStandard curve；It is real-time to construct antibacterial wood based panel fungicidal properties ATP bioluminescence The mathematical model of quantitative analysis method.Meanwhile country variant consumer perceptions habit is taken into account, take antibiotic rate R and antibacterial activity Value A is correlated performance evaluation index, improves the science and versatility of detection method.

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

(4) perspective: to establish with lgC_B、lgI_BStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Mycotic spore suspension viable bacteria content measuring method and wood based panel sample pre-treatments mode are enriched, it is dense to specify control sample, standard liquid 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 I_BEvaluate form as a result to calculate and determine antibiotic rate R or antibacterial activity value A, and by becoming in group and between group Different 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 OD value-that this patent is established Viable bacteria content logarithm lgC_BMethod is simple for calibration curve method and wood based panel fungicidal properties ATP fluorometric investigation, the relevant technologies It is clear and specific to illustrate, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for different majors water Flat 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 in inspection, learn, grind, produce all circles and promote and apply, wood based panel fungicidal properties detection technique realization generalization can be supported, while it can Reference is provided to the product scopes fungicidal properties detection technique research such as plastics, glass.

Further, preferred embodiment of the invention is:

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

(1) control sample: in addition to being not added with the mould proof ingredient of any Antimicrobial preservative, classification, material and the technique of control sample It is identical as the wooden plate sample of antibacterial to be measured.Sample is not more than 10mm having a size of (50 ± 1) mm × (50 ± 1) mm, thickness；Each bacterium Kind test uses 6 groups of samples；Wherein 0h contact and 48h culture experiment respectively use 3 groups, every group of 5 samples；

(2) antimicrobial sample: adding the samples such as wood (bamboo) quality plate, decorative panel and the wood-based plate of antibacterial mildew inhibitor, size, Thickness, quantity etc. are identical as control sample, and every group of antimicrobial sample selects one group of control sample as object of reference and have criterion Know；

(3) sample pre-treatments: wiping control sample and antimicrobial sample surface 2min with 75% ethanol solution before experiment, With aseptic water washing sample to remove ethyl alcohol on superclean bench, and carry out ultraviolet sterilization disinfection 5min.Then sample is to be measured Surface is put into sterilized petri dishes upwards, and sterile deionized water is injected into plate, and sample bottom is made to impregnate (non-suction for 24 hours in water Aqueous wood plate sample does not do immersion processing)；After it fully absorbs moisture, it is with sterilizing dry gauze that its surface is more to take out sample Remaining moisture blots, and surface to be measured is put into upwards spare in sterilized petri dishes；

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

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

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP Spend the ATP bioluminescence rapid detection system of meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, spore Sub- sum detection range 10¹CFU/mL~10⁵CFU/mL；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~4.0Abs Microplate reader；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) %RH ± The constant temperature and humidity incubator of 2%RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating centrifugation Pipe；The vortex oscillator of the range of speeds (500~3000) r/min；The pressuresteam sterilization of (121 ± 2) DEG C, (103 ± 5) kPa Device；- 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；Frequency range (30 ~50) ultrasonic cleaner of 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%)； Filter sizes are not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 Hole flat-bottomed plates；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the inoculation of 4mm Ring；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Alcohol Cotton balls (75%)；Aseptic filter paper；(0100) DEG C ± 0.2 DEG C of thermometer；The stopwatch of precision 0.01s；

(4) reagent: 75% ethanol solution；(0.45 μm of solution of MTT (tetramethyl azo azoles salt) of 5mg/mL, pH value 7.4 Membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C Store the physiological saline of 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are configured to 0.05% wetting agent solution；

(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；

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 and its total plate count plate count use): 300g is new Fresh potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Filter to take juice, into filtrate be added 20g glucose, 1000mL is settled to after 0.1g chloramphenicol, 20g agar；

(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 liquid^-11Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, is 10% with 0.2ml concentration Benza mix after, adjust pH to 12.0 ± 0.5 (fungal cell 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 spore suspension preparation, carries out in the steps below:

(1) test strain: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by Other strains that national Culture Collection is provided and can be traced to the source)；

(2) mould test strain culture presevation: is inoculated in simultaneously by potato-dextrose culture-medium inclined-plane with sterile working Indicate the date, 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；

(3) actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C ~30 DEG C of culture 7d~14d are until generate a large amount of spores.Mold species test tube plug must not be extracted before spore suspension by preparing, and every Only for spore liquid of preparation after test tube opening, suspension is prepared using the mycotic spore newly cultivated every time；

(4) prepared by spore suspension: the sterile water of 10mL being added into strain test tube, gently scrapes media surface with oese The spore stoste injection of wash-off is equipped with the sterile taper of jumping a queue of 15 beades and 45mL Cha Shi culture solution by fresh mycotic spore In bottle, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, sterile absorbent cotton or eight layers of yarn will be covered with The glass funnel of cloth is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；By filtrate move to sterilizing from In heart pipe, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is heavy that spore is cleaned with 50mL Cha Shi culture solution again Starch is simultaneously centrifuged, after repeated washing 3 times, with the spore sediment after the dilution centrifugation of Cha Shi culture solution.Every kind of test mould is pressed Spore suspension is prepared according to the above method, the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used；

The OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration, in the steps below It carries out:

(1) bacterium solution OD value measures: carrying out primary dcreening operation using spore total amount of the blood counting chamber to mixing spore suspension, uses Cha Shi Culture solution is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and with culture solution As blank control sample liquid, zeroing correction is carried out to microplate reader.Then, the spore liquid of the above-mentioned different dilutions of 100 μ L is distinguished 96 hole flat-bottomed plates are injected, each dilution does 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength； The viable bacteria OD value of each hole mixed solution is measured, the viable bacteria OD value of different dilution spore suspensions is its 3 multiple holes OD measured values Arithmetic mean of instantaneous value.Meanwhile the method referring to as defined in national standard GB 4789.15-2016, it is appropriate to carry out to above-mentioned mixing spore suspension After dilution, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilutions The viable bacteria OD value of spore suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard Curve；The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Work as R₀ ²≥ 0.98, when confidence level >=0.95, the spore suspension viable bacteria content C that is measured according to this patent method_BEffectively.Then, Cha Shi is used Culture solution is to known viable bacteria content C_BMould mixing spore liquid be diluted, obtain C_BRange is 5.0 × 10⁵CFU/mL~9.0 ×10⁵The inoculation bacterium solution of CFU/mL；

The viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established, in the steps below It carries out:

With Cha Shi culture solution to known viable bacteria content C_BMixing spore liquid carry out continuous gradient dilutions after, obtain standard system Column bacteria suspension: 2.1 × 10³CFU/mL、2.1×10⁴CFU/mL、2.1×10⁵CFU/mL is simultaneously mixed.According to relatively glimmering in this patent Light intensity value I_BMeasuring method, according to viable bacteria content C_BSequence from low to high measures and records above-mentioned standard solution and 0.3mL The latter (is denoted as I by relative intensity of fluorescence value of the inoculation bacterium solution after the dilution of 4.6mL eluent in 1min_B0).Then, with standard system The relative intensity of fluorescence logarithm lgI of column bacteria suspension_BAs abscissa, with corresponding viable bacteria content logarithm lgC_BFor ordinate Mapping；Calibration curve is carried out to mathematical relationship between the two, is derived from fitting equation Y=using least square fitting method a_BX+b_BAnd linearly dependent coefficient R_B ²；Work as R_B ²>=0.98, when confidence level >=0.95, have according to the measurement that this patent method is done Effect；

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

(1) 0.3mL inoculated and cultured: is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured with sterilizing liquid-transfering gun Bacterium solution is inoculated with (with lgC_B-lgI_BCalibration curve is derived from same branch with spore liquid and mixes spore liquid test tube, and 0 DEG C ± 1 DEG C saves, in 2h Using), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), its is made to cover sample whole surface.Cover ware Lid is sealed the plate equipped with 6 groups of 48h contact samples with medical adhesive tape；(28 ± 2) DEG C, (95 ± 2) %RH cultivate 48h ± 2h；

(2) elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously anti-with liquid transfer gun head Mould washing lotion in multiple pressure-vaccum plate, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL) less than 4.9mL after mixing.Each group Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould；

The reclaim liquid phase is to fluorescence intensity level I_BMeasurement 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 I_BAnd 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 I_BMeasurement: 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 I_BAnd 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 liquid_BMeasured value；

The recovered liquid viable bacteria content C_BAnd T_BIt 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 C_BAnd T_BIt calculates

According to standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, calculate that each group sample is contacted through 0h and 48h is trained The viable bacteria content C of recovered liquid after supporting_BAnd T_B.Relevant calculation is shown in formula (1)~(12):

In formula:

C_B0And C_Bt- 3 groups of 0h contacts and the mean viable amount that control sample recycles after 48h is cultivated, unit are bacterium colony shape At units per ml (CFU/mL)；

C_B0iAnd C_Bti- every group 0h contact and the mean viable amount that control sample recycles after 48h is cultivated, unit is bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3；

C_B0ijAnd C_Btij- every 0h contact and the viable bacteria amount that control sample recycles after 48h is cultivated, 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 0h contact and after 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_B- standard curve lgC_B-lgI_BSlope；b_B- standard curve lgC_B-lgI_BIn vertical axis intercept；

T_B0And T_Bt- 3 groups of 0h contacts and the mean viable amount that antimicrobial sample recycles after 48h is cultivated, unit are bacterium colony shape At units per ml (CFU/mL)；

T_B0iAnd T_Bti- every group 0h contact and the mean viable amount that antimicrobial sample recycles after 48h is cultivated, unit is bacterium colony It is formed units per ml (CFU/mL)；Sample group i=1,2,3；

T_B0ijAnd T_Btij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after 48h is cultivated, 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 0h contact and after 48h is cultivated antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

(2) condition for validity is tested

If recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.6mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after 48h is cultivated control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is C_Btij≥0.1×C_B0ij.Then when 3 groups of 0h contact control sample reclaim liquid phase To fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlation Uncertainty of measurement requirement), the measurement carried out according to this patent method is effective.

(3) mould increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample are after 48h is cultivated, mould increasing value F_ij、G_ijRespectively according to formula (13), (14) it calculates:

In formula:

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1, 2,3, every group of sample number into spectrum j=1,2,3,4,5；

a_B- standard curve lgC_B-lgI_BSlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of the wooden plate sample of every antibacterial_ij, every group and every batch of sample antibiotic rate R_i It is calculated respectively according to formula (15), (16), (17) with R:

In formula:

R_ijThe antibiotic rate of the wooden plate sample of-every antibacterial, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；

R_iThe antibiotic rate of-every group wooden plate sample of antibacterial, %；Sample group i=1,2,3；

R-every batch of antibacterial wooden plate sample antibiotic rate, %；

T_BtijAnd C_BtijThe viable bacteria amount that-every antimicrobial sample and control sample recycle after 48h is cultivated, 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 antimicrobial sample and control sample are after 48h is cultivated, the measurement of recovered liquid relative intensity of fluorescence Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_B- standard curve lgC_B-lgI_BSlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of the wooden plate sample of every antibacterial_ij, every group and every batch of sample antibacterial Activity value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

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

A_iThe antibacterial activity value of-every group wooden plate sample of antibacterial, sample group i=1,2,3；

A-every batch of antibacterial wooden plate sample antibacterial activity value；

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_B- standard curve lgC_B-lgI_BSlope；

(6) mycotic spore suspension viable bacteria content C data revision of the convention requirement: is demarcated using blood counting chamber and microplate reader_BWhen, ginseng Relevant regulations in GB 4789.2-2016 are examined, C is worked as_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BIt is not less than When 100CFU/mL, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index shape Formula indicates that " rounding up " uses two effective digitals afterwards.Control sample and antimicrobial sample return after 0h is contacted and 48h is cultivated Receive the relative intensity of fluorescence measured value round numbers of liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge to test ATP fluorimetric assay for biological materials method applied to wood based panel fungicidal properties Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is 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 48h is cultivated that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and 48h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is 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 48h is cultivated that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and 48h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

The evaluation of result of the wooden plate sample fungicidal properties of the antibacterial 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 R_ij/R_iWhen/R < 80%, sample is without mildew-proof function；When 80%≤R_ij/R_iWhen/R < 90%, sample has mildew-proof function；As 90%≤R_ij/R_iWhen/R < 99%, sample mildew-proof function is suitable In；As 99%≤R_ij/R_iWhen/R < 99.9%, sample mildew-proof function is stronger；Work as R_ij/R_iWhen/R >=99.9%, the mould proof work of sample With extremely strong；

Such as using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mould proof work With；As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function It is moderate；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function pole By force；

(2) as the antibiotic rate R of certain wooden plate sample of group (part) antibacterial_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the fungicidal properties of (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Meter It is calculated through ATP bioluminescence lgC_B─lgI_BThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back antibacterial Wooden plate sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial Activity value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as the wooden plate sample fungicidal properties of batch (group) antibacterial；

Specific embodiment

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

The present embodiment is added to the antibacterial elm multi-layer composite floor sample that nanometer silver-series antibacterial agent is process with wearing layer It is illustrated for the fungicidal properties detection of product.

Specific detection method carries out in the steps below:

(1) sample preparation and pretreatment

1.1 control samples: in addition to being not added with the mould proof ingredient of any Antimicrobial preservative, classification, material and the technique of control sample It is identical as the wooden plate sample of antibacterial to be measured.Sample is not more than 10mm having a size of (50 ± 1) mm × (50 ± 1) mm, thickness；Each bacterium Kind test uses 6 groups of samples；Wherein 0h contact and 48h culture experiment respectively use 3 groups, every group of 5 samples.

1.2 antimicrobial samples: adding the samples such as wood (bamboo) quality plate, decorative panel and the wood-based plate of antibacterial mildew inhibitor, size, Thickness, quantity etc. are identical as control sample, and every group of antimicrobial sample selects one group of control sample as object of reference and have criterion Know.

1.3 sample pre-treatments: wiping control sample and antimicrobial sample surface 2min with 75% ethanol solution before experiment, With aseptic water washing sample to remove ethyl alcohol on superclean bench, and carry out ultraviolet sterilization disinfection 5min.Then sample is to be measured Surface is put into sterilized petri dishes upwards, and sterile deionized water is injected into plate, and sample bottom is made to impregnate (non-suction for 24 hours in water Aqueous wood plate sample does not do immersion processing)；After it fully absorbs moisture, it is with sterilizing dry gauze that its surface is more to take out sample Remaining moisture blots, and surface to be measured is put into upwards spare in sterilized petri dishes.

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

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

2.2 instrument and equipments: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP Spend the ATP bioluminescence rapid detection system of meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, spore Sub- sum detection range 10¹CFU/mL~10⁵CFU/mL；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~4.0Abs Microplate reader；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) %RH ± The constant temperature and humidity incubator of 2%RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating centrifugation Pipe；The vortex oscillator of the range of speeds (500~3000) r/min；The pressuresteam sterilization of (121 ± 2) DEG C, (103 ± 5) kPa Device；- 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；Frequency range (30 ~50) ultrasonic cleaner of 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%)； Filter sizes are not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 Hole flat-bottomed plates；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the inoculation of 4mm Ring；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Alcohol Cotton balls (75%)；Aseptic filter paper；(0100) DEG C ± 0.2 DEG C of thermometer；The stopwatch of precision 0.01s.

2.4 reagents: 75% ethanol solution；(0.45 μm of solution of MTT (tetramethyl azo azoles salt) of 5mg/mL, pH value 7.4 Membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C Store the physiological saline of 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are configured to 0.05% wetting agent solution.

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；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): 300g is new Fresh potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Filter to take juice, into filtrate be added 20g glucose, 1000mL is settled to after 0.1g chloramphenicol, 20g agar.

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

ATP fluorescent reagent: 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 spore suspension preparation

3.1 test strains: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by Other strains that national Culture Collection is provided and can be traced to the source).

3.2 culture presevation: mould test strain is inoculated in simultaneously by potato-dextrose culture-medium inclined-plane with sterile working Indicate the date, 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.

3.3 actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C ~30 DEG C of culture 7d~14d are until generate a large amount of spores.Mold species test tube plug must not be extracted before spore suspension by preparing, and every Only for spore liquid of preparation after test tube opening, suspension is prepared using the mycotic spore newly cultivated every time.

The preparation of 3.4 spore suspensions: the sterile water of 10mL is added into strain test tube, gently scrapes media surface with oese The spore stoste injection of wash-off is equipped with the sterile taper of jumping a queue of 15 beades and 45mL Cha Shi culture solution by fresh mycotic spore In bottle, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, sterile absorbent cotton or eight layers of yarn will be covered with The glass funnel of cloth is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；By filtrate move to sterilizing from In heart pipe, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is heavy that spore is cleaned with 50mL Cha Shi culture solution again Starch is simultaneously centrifuged, after repeated washing 3 times, with the spore sediment after the dilution centrifugation of Cha Shi culture solution.Every kind of test mould is pressed Spore suspension is prepared according to the above method, the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used.

(4) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration

The measurement of 4.1 bacterium solution OD values: primary dcreening operation is carried out using spore total amount of the blood counting chamber to mixing spore suspension, uses Cha Shi Culture solution is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and with culture solution As blank control sample liquid, zeroing correction is carried out to microplate reader.Then, the spore liquid of the above-mentioned different dilutions of 100 μ L is distinguished 96 hole flat-bottomed plates are injected, each dilution does 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength； The viable bacteria OD value of each hole mixed solution is measured, the viable bacteria OD value of different dilution spore suspensions is its 3 multiple holes OD measured values Arithmetic mean of instantaneous value.Meanwhile the method referring to as defined in national standard GB 4789.15-2016, it is appropriate to carry out to above-mentioned mixing spore suspension After dilution, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B。

4.2OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilutions The viable bacteria OD value of spore suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard Curve；The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Work as R₀ ²≥ 0.98, when confidence level >=0.95, the spore suspension viable bacteria content C that is measured according to this patent method_BEffectively.Then, Cha Shi is used Culture solution is to known viable bacteria content C_BMould mixing spore liquid be diluted, obtain C_BRange is 5.0 × 10⁵CFU/mL~9.0 ×10⁵The inoculation bacterium solution of CFU/mL.

(5) viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established

(6) sample inoculation, culture and elution recycling

6.1 inoculated and cultureds: 0.3mL is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured with sterilizing liquid-transfering gun Bacterium solution is inoculated with (with lgC_B-lgI_BCalibration curve is derived from same branch with spore liquid and mixes spore liquid test tube, and 0 DEG C ± 1 DEG C saves, in 2h Using), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), its is made to cover sample whole surface.Cover ware Lid is sealed the plate equipped with 6 groups of 48h contact samples with medical adhesive tape；(28 ± 2) DEG C, (95 ± 2) %RH cultivate 48h ± 2h.

6.2 elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously anti-with liquid transfer gun head Mould washing lotion in multiple pressure-vaccum plate, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL) less than 4.9mL after mixing.Each group Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould.

(7) reclaim liquid phase is to fluorescence intensity level I_BMeasurement

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 I_BAnd 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 I_BMeasurement: 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 I_BAnd 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 liquid_BMeasured value.

(8) recovered liquid viable bacteria content C_BAnd T_BIt calculates and antibiotic rate R and antibacterial activity value A is calculated

8.1 recovered liquid viable bacteria content C_BAnd T_BIt calculates

In formula:

With- every 0h contact and after 48h is cultivated 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.

8.2 test conditions for validity

8.3 mould increasing value F_ij、G_ijIt calculates

In formula:

a_B- standard curve lgC_B-lgI_BSlope.

8.4 antibiotic rate R are calculated

In formula:

R-every batch of antibacterial wooden plate sample antibiotic rate, %；

a_B- standard curve lgC_B-lgI_BSlope.

8.5 antibacterial activity value A are calculated

In formula:

a_B- standard curve lgC_B-lgI_BSlope.

The requirement of the 8.6 data revisions of the convention: mycotic spore suspension viable bacteria content C is demarcated using blood counting chamber and microplate reader_BWhen, ginseng Relevant regulations in GB 4789.2-2016 are examined, C is worked as_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BIt is not less than When 100CFU/mL, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index shape Formula indicates that " rounding up " uses two effective digitals afterwards.Control sample and antimicrobial sample return after 0h is contacted and 48h is cultivated Receive the relative intensity of fluorescence measured value round numbers of liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals.

8.7 uncertainties of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge to test ATP fluorimetric assay for biological materials method applied to wood based panel fungicidal properties Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is cultivated Afterwards, the relative intensity of fluorescence measured value of each Duplicate Samples):

(9) evaluation of result

9.1 taking following classification criterion with reference to health industry common practice and the requirement of dependent antimicrobial product standard:

Such as using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mould proof work With；As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function It is moderate；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function pole By force.

9.2 work as the antibiotic rate R of certain wooden plate sample of group (part) antibacterial_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the fungicidal properties of (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Meter It is calculated through ATP bioluminescence lgC_B─lgI_BThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back antibacterial Wooden plate sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial Activity value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as the wooden plate sample fungicidal properties of batch (group) antibacterial.

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).

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

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

2.4 microplate reader: the full-automatic microplate reader of American blend Thermo Scientific, model Multiskan FC, wave Long range (340~850) nm ± 1nm, range of readings (0.0~6.0) Abs ± 0.001Abs；The corresponding measurement accuracy of 405nm is ± 1% (0.0Abs~3.0Abs) or ± 2% (3.0Abs~4.0Abs).

2.5 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.

2.6 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.

2.7 constant water bath box: the electric heating constant temperature sink of upper sea base Wei test apparatus equipment Co., Ltd model DK-S420, Volume 15L, ± 0.5 DEG C of temperature control range (5~99.9) DEG C, timing range 1min~999min.

2.8 constant temperature oscillators: the permanent model WS-380H in Shanghai one, ± 0.1 DEG C of temperature control range (4~65) DEG C, frequency of oscillation (40~300) r/min, timing range (1~99) h.

2.9 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.

2.10 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.

2.11 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.

2.12 refrigerators: the cryogenic box ultralow temperature ice of the glad experimental instruments and equipment limited model HXL-25-250AD of Dongguan City sky Case, ± 0.1 DEG C of refrigerating box (2~10) DEG C, ± 0.1 DEG C of household freezer (- 30~20) DEG C.

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

2.14 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.

2.15 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.

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

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

2.18 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

Chemical reagent

Tetramethyl azo azoles salt, trinosin standard items and preparation ATP fluorescent reagent buffer solution, ATP cracking A series of biochemical reagents needed for liquid, ATP extracting solution and ATP fluorescent reagent are purchased from Shanghai Jin Pan Biotechnology Co., Ltd agency's U.S.'s Amresco brand.

(5) reference culture

Aspergillus niger ATCC 16404, Chaetomium globosum ATCC 6205, penicillium chrysogenum ATCC 9179 are purchased from the common micro- life of China Object culture presevation administrative center.

The detection data and result of the present embodiment calculate:

Using ATP fluophotometer through lgC_B-lgI_BCalibration curve method to antibacterial elm plate sample fungicidal properties carry out Detection, coherent detection data and Calculation of Measuring Uncertainty result are shown in Table 1~table 2 respectively.

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

1 relative intensity of fluorescence Calculation of Measuring Uncertainty result of table

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

Claims

1. a kind of detection method of antibacterial wood based panel fungicidal properties, comprising: (1) sample preparation and pretreatment；(2) lectotype selection and Reagent, culture medium are prepared；(3) culture presevation, activation and spore suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BStandard Curve is established and inoculating spores liquid C_BCalibration；(5) viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard is bent Line is established；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level I_BMeasurement；(8) recovered liquid viable bacteria Content C_BAnd T_BIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluorescence light Degree meter carries out the ATP biology of accurate quantitative test to the antibacterial wood based panel fungicidal properties with antibiotic rate R or antibacterial activity value A characterization Fluorescence lgC_B-lgI_BCalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_BIn measurement:

Specify control sample and antimicrobial sample quantity, size and pre-treatment requirement, by the reference culture of mould test strain into After row passage, activation, fresh mycotic spore culture is taken to prepare spore suspension；OD value-viable bacteria is established using MTT colorimetric analysis Content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ², to different dilute The spore suspension for degree of releasing carries out viable bacteria content C_BAfter calibration, C is selected_BIt is 2.1 × 10³CFU/mL、2.1×10⁴CFU/mL、2.1× 10⁵The spore liquid of CFU/mL is as standard series bacteria suspension；Measure its relative intensity of fluorescence value I_B, draw lgC_B-lgI_BMark is bent, And it is derived from the linear equation Y=a of curve_BX+b_BAnd coefficient R_B ², then, choose viable bacteria content C_BRange be 5.0 × 10⁵CFU/mL~9.0 × 10⁵The mixing spore liquid of CFU/mL is as inoculation bacterium solution；It is added dropwise respectively to each group sample surface to be measured 0.3mL bacterium solution carries out elution recycling to 6 groups of 0h contact samples with 4.6mL eluent immediately, and the relative fluorescence for measuring recovered liquid is strong Angle value I_BC0ij、I_BT0ij, according to lgC_B-lgI_BFitting equation Y=a_BX+b_BCalculate its viable bacteria content C_BOijAnd T_BOij, meanwhile, it will 6 groups of control samples being sealed in sterilized petri dishes and antimicrobial sample cultivate 48h under conditions of (28 ± 2) DEG C, (95 ± 2) %RH After ± 2h；Recycling remained on surface bacterium is eluted using mode identical with 0h contact sample and measures the relative intensity of fluorescence of recovered liquid Value I_BCtij、I_BTtij, calculate corresponding viable bacteria content C_BtijAnd T_Btij；

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

According to standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, with 0h contact and every control sample after 48h is cultivated The relative intensity of fluorescence measured value of product and antimicrobial sample recovered liquidWithAs basic data；It is testing Under condition for validity, its mould increasing value F is calculated_ij、G_ijAnd antibiotic rate R_ijWith antibacterial activity value A_ij；To the R of every group of sample_ijWith A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；The antibiotic rate R and antibacterial activity value A of the wooden plate sample of every batch of antibacterial be its 3 Group sample R_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement require；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion；With reference to Health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion；When certain group (part) is anti- The antibiotic rate R of the wooden plate sample of bacterium_i(R_ij) or antibacterial activity value A_i(A_ij) fungicidal properties phase with other two groups (four) samples When than at least differing a levels, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP bioluminescence lgC_B─ lgI_BThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of the front and back wooden plate sample fungicidal properties of (part) antibacterial It is horizontal identical, then abandon it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i(A_ij) arithmetic Evaluation result of the average value as the wooden plate sample fungicidal properties of batch (group) antibacterial.

2. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the sample system Standby and pre-treatment carries out in the steps below:

(1) control sample: in addition to being not added with the mould proof ingredient of any Antimicrobial preservative, classification, material and the technique of control sample with to It is identical to survey the wooden plate sample of antibacterial, sample is not more than 10mm having a size of (50 ± 1) mm × (50 ± 1) mm, thickness；Each strain examination It tests and uses 6 groups of samples；Wherein 0h contact and 48h culture experiment respectively use 3 groups, every group of 5 samples；

(2) samples such as wood (bamboo) quality plate, decorative panel and the wood-based plate of antibacterial mildew inhibitor, size, thickness antimicrobial sample: are added Degree, quantity etc. are identical as control sample, and every group of antimicrobial sample selects one group of control sample as object of reference and effectively identify；

(3) sample pre-treatments: control sample and antimicrobial sample surface 2min are wiped with 75% ethanol solution before experiment, ultra-clean With aseptic water washing sample to remove ethyl alcohol on workbench, and ultraviolet sterilization disinfection 5min is carried out, then by sample surface to be measured It is put into sterilized petri dishes upwards, sterile deionized water is injected into plate, sample bottom is made to impregnate (unwetted property for 24 hours in water Wooden plate sample does not do immersion processing)；After it fully absorbs moisture, takes out sample and use sterilizing dry gauze by its excess surface water It point blots, and surface to be measured is put into upwards spare in sterilized petri dishes.

3. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the equipment choosing Type and reagent, culture medium are prepared, and are carried out in the steps below:

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

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；Fluorophotometric containing ATP The ATP bioluminescence rapid detection system of meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, spore Total detection range 10¹CFU/mL~10⁵CFU/mL；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~4.0Abs's Microplate reader；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) %RH ± 2% The constant temperature and humidity incubator of RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating centrifuge tube； The vortex oscillator of the range of speeds (500~3000) r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range (30~50) The ultrasonic cleaner of 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,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%)；Filter hole Diameter is not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 holes are flat Culture plate；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the oese of 4mm；L stick； The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Cotton ball soaked in alcohol (75%)；Aseptic filter paper；Thermometer；The stopwatch of precision 0.01s；

(4) reagent: 75% ethanol solution；MTT (tetramethyl azo azoles salt) solution (0.45 μm of filter membrane of 5mg/mL, pH value 7.4 Filtration sterilization, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min, 5 DEG C~10 DEG C storages after following reagent packing The physiological saline of 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are configured to 0.05% Wetting agent solution；

(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；

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 and its total plate count plate count use): by the fresh horse of 300g Bell potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g are added into filtrate 1000mL is settled to after chloramphenicol, 20g agar；

(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 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 antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the strain is protected Hiding, activation and spore suspension preparation, carry out in the steps below:

(1) test strain: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by country Other strains that Culture Collection is provided and can be traced to the source)；

(2) culture presevation: mould test strain is inoculated in by potato-dextrose culture-medium inclined-plane with sterile working and is indicated Date, 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；

(3) actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d must not extract mold species test tube plug, every test tube before preparing spore suspension until generate a large amount of spores Only for spore liquid of preparation after opening, suspension is prepared using the mycotic spore newly cultivated every time；

(4) prepared by spore suspension: the sterile water of 10mL being added into strain test tube, the fresh of media surface is gently scraped with oese The spore stoste injection of wash-off is equipped with the sterile conical flask of jumping a queue of 15 beades and 45mL Cha Shi culture solution by mycotic spore In, 3000r/min shakes test tube 2min, breaks up spore ball, then sterile absorbent cotton or eight layers of gauze will be covered with by mixing spore liquid Glass funnel be placed on conical flask, filtering spore suspension removes mycelia and culture medium fragment；Filtrate is moved into sterilizing centrifugation Guan Zhong, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；Spore precipitating is cleaned with 50mL Cha Shi culture solution again Object is simultaneously centrifuged, after repeated washing 3 times, with Cha Shi culture solution dilution centrifugation after spore sediment, every kind of test mould according to The above method prepares spore suspension, and the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used.

5. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the OD value- Viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration carries out in the steps below:

(1) bacterium solution OD value measures: carrying out primary dcreening operation using spore total amount of the blood counting chamber to mixing spore suspension, is cultivated with Cha Shi Liquid is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and using culture solution as Blank control sample liquid carries out zeroing correction to microplate reader, and then, the spore liquid of the above-mentioned different dilutions of 100 μ L is injected separately into 96 hole flat-bottomed plates, each dilution do 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength；It surveys The viable bacteria OD value of fixed each hole mixed solution, the viable bacteria OD value of different dilution spore suspensions are the calculation of its 3 multiple holes OD measured values Art average value, meanwhile, the method referring to as defined in national standard GB 4789.15-2016 carries out above-mentioned mixing spore suspension appropriate dilute After releasing, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilution spores The viable bacteria OD value of suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard curve； The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ², work as R₀ ²>=0.98, it sets Letter is horizontal >=0.95 when, the spore suspension viable bacteria content C that is measured according to this patent method_BEffectively, then, with Cha Shi culture solution pair Known viable bacteria content C_BMould mixing spore liquid be diluted, obtain C_BRange is 5.0 × 10⁵CFU/mL~9.0 × 10⁵The inoculation bacterium solution of CFU/mL.

6. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the viable bacteria contains Measure logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established, and is carried out in the steps below:

With Cha Shi culture solution to known viable bacteria content C_BMixing spore liquid carry out continuous gradient dilutions after, obtain standard series bacterium Suspension: 2.1 × 10³CFU/mL、2.1×10⁴CFU/mL、2.1×10⁵CFU/mL is simultaneously mixed, strong according to relative fluorescence in this patent Angle value I_BMeasuring method, according to viable bacteria content C_BSequence from low to high measures and records above-mentioned standard solution and 0.3mL inoculation The latter (is denoted as I by relative intensity of fluorescence value of the bacterium solution after the dilution of 4.6mL eluent in 1min_B0), then, with standard series bacterium The relative intensity of fluorescence logarithm lgI of suspension_BAs abscissa, with corresponding viable bacteria content logarithm lgC_BFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, is derived from fitting equation Y=a using least square fitting method_BX+b_B And linearly dependent coefficient R_B ²；Work as R_B ²>=0.98, when confidence level >=0.95, the measurement done according to this patent method is effective.

7. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the sample connects Kind, culture and elution recycling, carry out in the steps below:

(1) 0.3mL inoculation inoculated and cultured: is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured with sterilizing liquid-transfering gun Bacterium solution is (with lgC_B-lgI_BCalibration curve is derived from same branch with spore liquid and mixes spore liquid test tube, and 0 DEG C ± 1 DEG C saves, and makes in 2h With), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), so that its is covered sample whole surface, covers ware lid, The plate equipped with 6 groups of 48h contact samples is sealed with medical adhesive tape；(28 ± 2) DEG C, (95 ± 2) %RH cultivate 48h ± 2h；

(2) elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately and (is examined Family name's culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate are sufficiently eluted and are blown repeatedly with liquid transfer gun head Mould washing lotion in plate is inhaled, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL), each group less than 4.9mL after mixing Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould.

8. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the recovered liquid Relative intensity of fluorescence value I_BMeasurement 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 I_BAnd 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 I_BMeasurement: 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 I_BAnd 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 liquid_BMeasured value.

9. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the recovered liquid Viable bacteria content C_BAnd T_BIt calculates and antibiotic rate R and antibacterial activity value A is calculated, carry out in the steps below:

(1) recovered liquid viable bacteria content C_BAnd T_BIt calculates

According to standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, calculate each group sample after 0h is contacted and 48h is cultivated The viable bacteria content C of recovered liquid_BAnd T_B, relevant calculation is shown in formula (1)~(12):

In formula:

C_B0And C_Bt- 3 groups of 0h contacts and the mean viable amount that control sample recycles after 48h is cultivated, unit is Colony Forming Unit Every milliliter (CFU/mL)；

C_B0iAnd C_Bti- every group 0h contact and the mean viable amount that control sample recycles after 48h is cultivated, unit are formed for bacterium colony Units per ml (CFU/mL)；Sample group i=1,2,3；

C_B0ijAnd C_Btij- every 0h contact and the viable bacteria amount that control sample recycles after 48h is cultivated, unit is Colony Forming Unit Every milliliter (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 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_B0And T_Bt- 3 groups of 0h contacts and the mean viable amount that antimicrobial sample recycles after 48h is cultivated, unit is Colony Forming Unit Every milliliter (CFU/mL)；

T_B0iAnd T_Bti- every group 0h contact and the mean viable amount that antimicrobial sample recycles after 48h is cultivated, unit are formed for bacterium colony Units per ml (CFU/mL)；Sample group i=1,2,3；

T_B0ijAnd T_Btij- every 0h contact and the viable bacteria amount that antimicrobial sample recycles after 48h is cultivated, unit is Colony Forming Unit Every milliliter (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 48h is cultivated antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

(2) condition for validity is tested

If the recovered liquid and 0.3mL inoculation bacterium solution of every 0h contact control sample are after the dilution of 4.6mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after 48h is cultivated The logarithm of definite valueThat is C_Btij≥0.1×C_B0ij, then when 3 groups of 0h contact control sample reclaim liquid phases are to glimmering Luminous intensity measured valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent measurement of correlation Uncertainty requirement), the measurement carried out according to this patent method is effective；

(3) mould increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample are after 48h is cultivated, mould increasing value F_ij、G_ijIt is counted respectively according to formula (13), (14) It calculates:

In formula:

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；

a_B- standard curve lgC_B-lgI_BSlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of the wooden plate sample of every antibacterial_ij, every group and every batch of sample antibiotic rate R_iWith R points It is not calculated according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of the wooden plate sample of-every antibacterial, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3, 4,5；

R-every batch of antibacterial wooden plate sample antibiotic rate, %；

T_BtijAnd C_BtijThe viable bacteria amount that-every antimicrobial sample and control sample recycle after 48h is cultivated, 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 antimicrobial sample and control sample are after 48h is cultivated, the measured value of recovered liquid relative intensity of fluorescence, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_B- standard curve lgC_B-lgI_BSlope；

(5) antibacterial activity value A is calculated

In formula:

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

a_B- standard curve lgC_B-lgI_BSlope；

(6) mycotic spore suspension viable bacteria content C data revision of the convention requirement: is demarcated using blood counting chamber and microplate reader_BWhen, with reference to GB Relevant regulations in 4789.2-2016, work as C_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BNot less than 100CFU/ When mL, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals, control sample and antimicrobial sample after 0h is contacted and 48h is cultivated afterwards, the phase of recovered liquid To fluorescent strength determining value round numbers, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increasing value F_ij、G_ijWith Antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h culture Afterwards, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV count Calculate result and remain into 2 significant digits), judge the weight that ATP fluorimetric assay for biological materials method is applied to the test of wood based panel fungicidal properties Existing property；In regulation group and between-group variation coefficient CV≤10%；

5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after 48h is cultivated that every group of 0h is contacted Product, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithRespectively every group of control sample and antimicrobial sample are after 0h is contacted and 48h is cultivated, respectively The relative intensity of fluorescence measured value of Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after 48h is cultivated of 3 groups of 0h contact, 15 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after 48h is cultivated that every group of 0h is contacted Product, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithRespectively every group of control sample and antimicrobial sample are after 0h is contacted and 48h is cultivated, respectively The relative intensity of fluorescence measured value of Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after 48h is cultivated of 3 groups of 0h contact, 15 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

10. the detection method of antibacterial wood based panel fungicidal properties according to claim 1, which is characterized in that the result Evaluation carries out in the steps below:

Such as using antibiotic rate R as correlated performance evaluation index: working as R_ij/R_iWhen/R < 80%, sample is without mildew-proof function；When 80% ≤R_ij/R_iWhen/R < 90%, sample has mildew-proof function；As 90%≤R_ij/R_iWhen/R < 99%, sample mildew-proof function is moderate； As 99%≤R_ij/R_iWhen/R < 99.9%, sample mildew-proof function is stronger；Work as R_ij/R_iWhen/R >=99.9%, sample mildew-proof function pole By force；

Such as using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mildew-proof function；When 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function is moderate； As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function is extremely strong；

(2) as the antibiotic rate R of certain wooden plate sample of group (part) antibacterial_i(R_ij) or antibacterial activity value A_i(A_ij) and other two group (four Part) sample fungicidal properties compared to a levels are at least differed when, extract one group of (part) sample again and repeat to test；Calculate it Through ATP bioluminescence lgC_B─lgI_BThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back antibacterial is wooden Plate sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity Value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as the wooden plate sample fungicidal properties of batch (group) antibacterial.