CN110274896A

CN110274896A - ATP bioluminescence lgCB-lgIBThe method for marking bent method evaluation liquid disinfectant fungi killing effect

Info

Publication number: CN110274896A
Application number: CN201910200132.1A
Authority: CN
Inventors: 李文杰; 闫冀焕; 崔宗岩; 钱云开; 郝凌云
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-09-24
Anticipated expiration: 2039-03-15
Also published as: CN110274896B

Abstract

The present invention relates to a kind of liquid disinfectant fungi killing effect evaluation method, evaluation procedure includes: that sample extraction and minimum effective concentration determine；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and bacteria suspension preparation；Bacteria suspension viable bacteria content C after methylene blue_BBlood cell plate counts and inoculation bacterium solution C_BCalibration；lgC_BRelative intensity of fluorescence logarithm lgI_BMark Qu Jianli；Neutralizer identification and quantitative suspension test；Recovered liquid I_BMeasurement and its viable bacteria content C_BAnd T_BIt calculates；Killing rate R and sterilizing Index A calculate；Result judgement；Its special feature is that: accurate quantitative assessment is carried out to the disinfectant fungi killing effect with R or A characterization using ATP fluophotometer.Present invention provide that test sample liquid and control sample liquid after recycling effect different time, measure its I_BAnd with lgI_BCharacterization and calculating R or A；Result judgement foundation is provided.The ATP bioluminescence lgC for the disinfectant fungi killing effect evaluation that the present invention researches and develops_B‑lgI_BBent method is marked, related Evaluation of Disinfection method system can be improved.

Description

ATP bioluminescence lgCB-lgIBIt marks bent method and evaluates liquid disinfectant fungi killing effect Method

Technical field

The present invention relates to the fungi killing effect effect evaluation method of liquid disinfectant, specifically a kind of application ATP fluorescence Photometer carries out the ATP of accurate quantitative test to the liquid disinfectant fungi killing effect with killing rate R or sterilizing Index A characterization Bioluminescence lgC_B-lgI_BCalibration curve method belongs to disinfectant sterilization effect assessment technique field.

Background technique

Along with the enhancing of the improvement of people's living standards and health perception, to cut off infectious disease transmission approach, peroxidating The liquid disinfectants such as species, aldehydes, phenols, quaternary ammonium salt are increasingly wide in the fields application such as medical treatment, food, education, food and drink, house It is general, and killing effect to pathogenic microorganisms such as its bacterium, fungi and its spores and wide spectrum require it is increasingly harsh；Industry correlation work Skill innovation and research and development of products pressure are constantly soaring, and quality inspection technology and testing efficiency are brought into schedule.

Currently, antibiotic and sterilizing effect assessment technical system is mainly for bacterium and fungi, various countries' anti-mycotic efficiency both at home and abroad Test method principle rarely has mostly based on the colony counting method being separately cultured to Candida albicans and is related to mould person；Wherein fit For Candida albicans test first is that the dipping quantitative test method that Japanese Industrial Standards propose；Two are derived from U.S. textile industry It is antibacterial around-France；Third is that international oscillation flask method；Four are derived from the dropping method of U.S. textile enterprise.Although China has issued It is " disposable that cloth implements GB 15981-1995 " sterilizing the evaluation method and standard with sterilization effect ", GB 15979-2002 Amenities sanitary standard " and the relevant criterions such as QB/T 2738-2012 " evaluation method of daily chemical products antibacterial bacteriostatic effect ", But its defined test period, between 48 hours~72 hours, time and economic cost are high.In addition, mould proof effect international for many years Fruit assessment technique system be originated from substantially American Standard, day mark and Europe superscript, involved standard mainly include soil bury cultivation, agar plate method, Moist chamber culture method etc.；Due to the mycelium that is formed in mycotic spore growth course can not accurate counting, can only be qualitatively judged, It can not quantitative test；And because the fungus growth period is longer, incubation time at least 2 weeks~4 weeks, cause test period longer, the time And economic cost is high.No matter be directed to Candida albicans or mould, the experimentation of existing standard method is cumbersome, technical difficulty compared with It is high；Relevant operation is affected by human factors greatly, so that test error is big, lacks comparativity.In recent years, in international Bacteria Detection skill ATP fluorescence analysis development in art field is increasingly mature, and testing result correlation is 98% compared with traditional Plating, and accuracy is high And it can realize quick detection.By demand pull, external anti-biotic material performance detection technical field of research is for current quantification, fast Speedization and summary development trend have used for reference ATP fluorescence analysis principle and have formulated ISO 20743:2007-2013 " Textiles- Determination of antibacterial activity of textile products (spin by textile-antibiotic finish The antibacterium performance measurement of fabric) " and ISO 13629-1:2012 " Textiles-Determination of antifungal Activity of textile products.Part1Luminescence (textile-antibiotic finish textile mildew resistance Can measurement) ", it is specified that with the fluorimetry of anti-thin/mould performance of ATP changes of contents characterization after sample inoculation, but it is provided Absorption process, transfer method and transfer printing be only applicable to have water imbibition and control sample it is thin/textile material of mould increasing value > 0 or Poromerics；The assessment of liquid disinfectant fungi killing effect must be for key technologies such as neutralization evaluation, result calculation formula Aspect forms a whole set of clear and specific method, while need to provide measurement of correlation uncertainty evaluation.In addition, the standard method Anti-microbial property characterization parameter is more single, only relates to antibacterial activity value A, and China then usual killing rate R.

Therefore, to improve control and prevention of disease effect, 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 liquid disinfectant fungi killing effect measuring technology.The art of this patent road Line designs the world that integrates with, and detection method belongs to the whole world and initiates, and can 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 refer to killing rate R or sterilizing The ATP bioluminescence lgC that the liquid disinfectant fungi killing effect of number A characterization is evaluated_B-lgI_BCalibration curve method can solve Certainly liquid disinfectant or even the accurate quantitative assessment problem of other medicine and hygiene fields sterilization effects.

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

A kind of evaluation method of liquid disinfectant fungi killing effect, comprising: (1) sample extraction and minimum effective concentration are true It is fixed；(2) lectotype selection and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) bacterium after methylene blue Suspension viable bacteria content C_BBlood cell plate count and inoculation bacterium solution C_BCalibration；(5) viable bacteria content logarithm lgC_BRelative intensity of fluorescence Logarithm lgI_BStandard curve is established；(6) neutralizer identification and quantitative suspension test；(7) reclaim liquid phase is to fluorescence intensity level I_BIt surveys It is fixed；(8) recovered liquid viable bacteria content C_BAnd T_BIt calculates and sterilizing rate R is calculated with sterilizing Index A；(9) result judgement；It is characterized in that, It is precisely fixed to carry out using ATP fluophotometer to the liquid disinfectant fungi killing effect with killing rate R or sterilizing Index A characterization Measure the ATP bioluminescence lgC of evaluation_B-lgI_BCalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_BIn measurement:

Sample extraction requirement is specified, the minimum effective concentration for pressing down in disinfectant 10min and killing indicator bacteria is determined, by mycologic test After the reference culture of strain is passed on, activated, fresh Candida albicans or mycotic spore culture is taken to prepare bacteria suspension；Through beauty Using the viable bacteria content C of blood counting chamber measurement bacterium solution after indigo plant dyeing_B, and to inoculation bacterium solution C_BIt is demarcated: 5.0 × 10⁶CFU/ ML~9.0 × 10⁶CFU/mL.Select viable bacteria content C_BIt is 3.5 × 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL Bacterium solution as standard series bacteria suspension, using ATP fluophotometer to its relative intensity of fluorescence value I_BIt is measured；It draws lgC_B-lgI_BStandard curve is derived from the linear equation Y=a of curve_BX+b_BAnd coefficient R_B ².Then, disappeared with to be measured The minimum effective concentration of toxic agent carries out neutralizer qualification test, determines neutralizer type and concentration；Simultaneously by each concentration of 4.41mL Test sample liquid and control sample liquid be placed in (20 ± 2) DEG C water-bath and make it with 0.49mL inoculation bacterium solution mixes；Effect subject to sterilization Continue t₁、t₂、t₃、t₄After four sections of different times, 0.49mL microbiological contamination sample liquid is mixed with 4.41mL with agent solution respectively, is neutralized Using its mixing liquid as recovered liquid after effect progress 10min；Using the relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring recovered liquidWith And according to instruction strain standard curve side Formula Y=a_BX+b_BCalculate corresponding viable bacteria content With

In killing rate R or sterilizing Index A are calculated:

According to instruction strain standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, for t₁、t₂、t₃、t₄Four sections are not Same sterilization time, to quantify the relative intensity of fluorescence of each concentration tests sample liquid and control sample liquid recycling viable bacteria in suspension test every time Measured value is as basic data；Calculate its killing rate to fungi Or sterilizing indexAnd take the arithmetic mean of instantaneous value of its five times tests as respective concentration testing liquid disinfectant sample To the killing rate of fungi in identical sterilization time Or sterilizing index The clear related data revision of the convention and uncertainty of measurement requirement simultaneously；

In result judgement:

With reference to health industry common practice and related Disinfection Effect biological assessment standard, if certain concentration testing liquid disappears Toxic agent sample liquid quantifies in suspension test in single, through ATP bioluminescence lgC_B─lgI_BWhen the specific sterilizing that calibration curve method measures Interior fungi killing rate >=99.9% or sterilizing index >=3.0 then determines that its Disinfection Effect in this test is qualified.When this When Disinfection Effect of the concentration testing liquid disinfectant sample liquid in 5 quantitative suspension tests in identical sterilization time is qualified, side It can determine its minimum concentration and shortest time to fungi with Disinfection Effect (in practical applications with organic matter protection test Minimum concentration and shortest time are concentration and time needed for testing liquid disinfectant sample reaches practical Disinfection Effect).

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 disinfectant sample sterilizing specific time, reaches the modernization of fungi killing effect evaluation；It can be effective Reducing human factor in experimentation influences, and breaches traditional plate culture and operates limitation numerous, that the period is long；Realize evaluation The precision of the simplification and evaluation data of process；Assessment technique has certain advance.

(2) scientific: according to ATP fluorescence analysis test philosophy, to establish the viable bacteria ATP concentration pair suitable for multi-cultur es Numerical value lgC_B- relative intensity of fluorescence logarithm lgI_BStandard curve；It is glimmering to construct liquid disinfectant fungi killing effect ATP biology The mathematical model of light real-time quantitative analysis method.Meanwhile country variant consumer perceptions habit is taken into account, it takes killing rate R and goes out Bacterium Index A improves the science and versatility of evaluation method as relevant effect evaluation index.

(3) innovative: compared with existing general conventional method, this patent method introduces during the test automation with The higher ATP fluophotometer of intelligent level, greatly simplifies experimental procedure, realizes disinfectant fungi killing effect and comments The precision of valence and quantification simultaneously have good reproducibility and comparativity；Test period is greatly shortened simultaneously, reduces evaluation 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 Candida albicans and mycotic spore suspension viable bacteria concentration measuring method and disinfectant sample dilution mode are enriched, control sample is specified The technology contents such as product, standard liquid concentration, determination step, calculation formula, uncertainty；The pioneering recycling viable bacteria directly measured with instrument Relative intensity of fluorescence value I_BDetermine form as a result to calculate and determine killing rate R and sterilizing Index A, and passes through a group interior and group Between coefficient of variation CV investigate uncertainty of measurement, technically have certain perspective.

(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, the methylene blue that this patent is established Method is simple for blood cell plate viable bacteria counting method and the ATP fluorometric investigation of disinfectant fungi killing effect evaluation after dyeing, related It is clear and specific that technology illustrates, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for different special The Experiment on Microbiology personnel of industry level 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 assessment technique support for thering is wide spectrum to be worth；The introducing of pertinent instruments can greatly simplify experimental procedure, reduce testing cost；Be conducive to Expand inspection, learn, grind, produces all circles promote and apply, can support disinfectant fungi killing effect assessment technique realization generalization, together When can to the fields such as chemical product, amenities antibacterial effect assessment technique study provide reference.

Further, preferred embodiment of the invention is:

The sample extraction and minimum effective concentration determine, carry out in the steps below:

(1) control sample: using Cha Shi culture solution as control sample；

(2) liquid of 1 minimum marketing packing test specimen: is randomly selected from certain product batch number 1 complete transportation and packing Body disinfectant sample is used for neutralizer identification and quantification suspension test；Every group of test specimen selects one group of control sample as ginseng According to object and effectively identify.

(3) determination of disinfectant sample minimum effective concentration: according to having for disinfectant sample operation instructions to be measured mark Concentration range is imitated, it is diluted to high, medium and low three suitable concentrations with sterile water；It is with sterilizing liquid-transfering gun that 4.41mL difference is dense The dilution of degree dispenses three sterile test tubes, and 0.49mL inoculation bacterium solution is added dropwise respectively into each test tube；3000r/min shaking examination After pipe 30s, place it in (20 ± 2) DEG C water-bath.Start timing after solution temperature in test tube and bath temperature balance, makes to disappear Toxic agent continues 10min to the killing effect of fungi；Then, using its mixing liquid as recovered liquid, according to relative fluorescence in this patent Intensity value I_BMeasuring method measures and records the relative intensity of fluorescence value of recovered liquid.Meanwhile using 4.41mL Cha Shi culture solution as Control sample repeats above-mentioned sterilization test and recovered liquid I_BContinuous mode；And according to killing rate R calculation formula in this patent, to not Killing rate with concentration dilution liquid is calculated；If certain concentration killing rate R is 99.9%, this concentration is set as disinfection to be measured The minimum effective concentration of agent sample；

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

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

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, very Bacterium cell/spore sum detection range 10¹CFU/mL~10⁶CFU/mL；Amplification factor 40 ×~400 × Photobiology it is micro- Mirror；The constant incubator that (25~37) DEG C ± 1 DEG C；The constant water bath box that (10~50) DEG C ± 1 DEG C；(0~50) DEG C ± 1 DEG C, (50 ~300) constant temperature oscillator of r/min；Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The range of speeds (500~ 3000) the vortex oscillator of r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C Low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic wave of frequency range (30~50) kHz is clear Wash device；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；Sterile petri dish；Glass funnel；Sterile cock test tube；Diameter Oese no more than 4mm；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Absorbent cotton and gauze for biochemistry detection；Nothing Bacterium filter paper；Thermometer；The stopwatch of precision 0.01s；

(4) reagent: 0.1% Lv Shi alkaline methylene blue dyeing liquor；Calf serum；121 DEG C of high pressure sterilizations of following reagent 30min, the physiological saline of 5 DEG C~10 DEG C storage 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are appointed One kind is selected, 0.05% wetting agent solution is configured to；5g sodium thiosulfate is dissolved in 1000mL water (for chlorine type disinfectant)； 1.36g potassium dihydrogen phosphate, 2.83g disodium hydrogen phosphate, 10g lecithin, 10g glycine, 30g tween (80) are dissolved in 1000mL water Or 1.36g potassium dihydrogen phosphate, 2.83g disodium hydrogen phosphate, 3g lecithin, 20g tween (80) are dissolved in 1000mL water (for non-oxygen Change type disinfectant)；20g tween (80), 1g sodium thiosulfate are dissolved in 1000mL phosphate buffer solution (to sterilize for oxygen type Agent) etc. (or being directed to testing liquid disinfectant sample type, select the neutralizer that other phases are applicable)；

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

Sabouraud culture medium/liquid (Candida albicans actication of culture use): 40g glucose, 10g peptone, 20g agar are heated It is dissolved in 1000mL water (agar is not added in culture solution), adjusts pH to 5.6 ± 0.2 (25 DEG C)；

Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen Dipotassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose are dissolved by heating in 1000mL containing 0.05% wetting In the aqueous solution of agent, adjust pH to 6.0~6.5 (25 DEG C)；

One dextrose culture-medium of potato (mycotic spore actication of culture use): removing the peel stripping and slicing for 300g fresh potato, 20min~30min is boiled in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g chloramphenicol, 20g agar are added into filtrate After be settled to 1000mL；

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

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

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

ATP lysate: 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 is international single Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, the 20mg bovine serum albumin of position/ml is dissolved in 10mL concentration is to adjust in pH to 6.0 ± 0.5,8h in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L and use (1mL cracking ATP concentration in Sharpe culture solution can be down to 10 in 15min by liquid^-11Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, is 10% with 0.2ml concentration Benza mix after, adjust pH to 12.0 ± 0.5 (fungal cell's ATP extraction efficiency be not less than 80%)；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h；

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

(1) strain: Candida albicans ATCC 10231 is indicated；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC6205；It produces Penicillium chrysogenum ATCC 9179 (or other strains that is provided by national Culture Collection and can be traced to the source)；

(2) culture presevation: Candida albicans --- freeze-drying lactobacillus pipe is opened with sterile working, is infused with capillary syring into pipe Enter appropriate Sharpe culture solution, pressure-vaccum makes strain melt dispersion for several times；A little bacteria suspension is instilled and is trained equipped with 5mL~10mL Sharpe In the test tube of nutrient solution, 37 DEG C of culture 18h~for 24 hours.Mould --- mould test strain is inoculated in by potato-with sterile working The inoculation date is simultaneously indicated in dextrose culture-medium inclined-plane, and 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3℃ ~10 DEG C preservation 4 months, as preservation bacterium；

(3) actication of culture: Candida albicans --- with the colonies typical in oese scraping 1st generation culture, scribing line is connect Kind is in sabouraud culture medium plate；After 37 DEG C of culture 18h~for 24 hours, the colonies typical in picking 2nd generation culture is inoculated in Sharpe training Support base inclined-plane；After 37 DEG C of culture 18h~for 24 hours, 4 DEG C of sealings storages, use in 6 weeks.Mould --- preservation bacterium is scraped with oese Spore is inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d, until generating a large amount of spores.Preparation Must not extract the test tube plug of mold species before spore suspension, every test tube open after only for spore liquid of preparation, every time Suspension is prepared using the mycotic spore newly cultivated；

(4) prepared by bacteria suspension: Candida albicans test --- with oese, the fresh microbial strain culture of picking is connect from inclined-plane Kind in the sterile conical flask equipped with 50mL Sharpe culture solution, it is placed in 150r/min culture in the constant temperature oscillator of (30 ± 1) DEG C 18h~for 24 hours, 4 DEG C of sealing storages, the same day uses.Mould test --- the sterile water of 10mL is added into strain test tube, with inoculation Ring gently scrapes the fresh mycotic spore of media surface, and the spore stoste injection of wash-off is trained equipped with 15 beades and 45mL Cha Shi In the sterile conical flask of jumping a queue of nutrient solution, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, it will cover There are sterile absorbent cotton or the glass funnel of eight layers of gauze to be placed on conical flask, filtering spore suspension removes mycelia and culture medium is broken Piece；Filtrate is moved in sterile centrifugation tube, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；50mL is used again Cha Shi culture solution cleaning spore sediment is simultaneously centrifuged, and after repeated washing 3 times, is precipitated with the spore after the dilution centrifugation of Cha Shi culture solution Object.Every kind of test mould prepares spore suspension according to the method described above, and the spore liquid of each strain is mixed in equal volume；0 DEG C~7 DEG C storage uses in the same day or 4d；

Bacteria suspension viable bacteria content C after the methylene blue_BBlood cell plate counts and inoculation bacterium solution C_BCalibration, in the steps below It carries out:

(1) methylene blue: with sterile working by 50 μ L dilutions for 10^-2~10^-3Candida albicans bacterium suspension or mould The Lv Shi alkaline methylene blue dyeing liquor that mixing spore liquid and 30 μ L concentration are 0.05% moves to (bacterium solution in same branch sterile test tube respectively Dilution, which includes 4~5 albicans cells or mycotic spore with each lattice of blood counting chamber, to be advisable), 1000r/min Test tube 1min is shaken, mixes well bacteria suspension with dyeing liquor；

(2) blood cell plate counts: the bacteria suspension after ± 0.5 μ L of 5 μ L dyeing being placed in coverslip edge with aseptic straw, makes it Blood counting chamber is slowly penetrated along slide gap, bubble is not can produce between tally and slide, otherwise re-operates.With sterile Extra bacterium solution in filter paper exhaustion slot, stands 2min ± 20s, is settled down to its whole in counting chamber.When lattice tally in use 16 When, upper left, upper right, lower-left, the albicans cell in the middle lattice in bottom right 4 (i.e. 100 lattices) are taken in diagonal orientation Or mycotic spore is counted；Lattice tally in 25 is such as used, in addition to counting above-mentioned 4 diagonal orientations, also needs to count the 1 of center A middle lattice (i.e. 80 lattices)；When test strain is located on the two-wire of middle lattice, then only count online and right line or it is offline and Albicans cell or mycotic spore on left line；

(3) viable bacteria content C_BCalculate: by Photobiology microscope magnification from it is low to lofty tone to 400 × after, it is right immediately Albicans cell or mycotic spore at blood counting chamber additional space position are counted；Wherein colourless Candida albicans Bacterium cell is viable bacteria, and blue or light blue person is dead bacterium；Edge is in navy blue, inside in colourless or light blue and pink mould Bacterium spore is viable bacteria, and edge and inside are dead bacterium in navy blue person, therefore the only counting rim spore different from internal color.If Quantity without mycelia monospore in mycotic spore suspension is lower than 90%, then prepares spore liquid again.It is each to blood counting chamber small Albicans cell or mycotic spore in grid repeat microscopy and count three times, are averaged.When blood counting chamber specification is When 16 × 25, viable bacteria content (every milliliter of Colony Forming Unit, CFU/mL) C of 1mL bacterium solution_B5 × 16 × K × 10=N ÷⁴；Work as blood When ball count plate gauge lattice are 25 × 16, C_B5 × 25 × K × 10=N ÷⁴；Wherein N is that white is read in five middle lattice of blood counting chamber The total viable count of pearl bacterium cell or mycotic spore, K are bacterium solution extension rate.Then, with Cha Shi culture solution to known viable bacteria content C_BCandida albicans bacterium suspension or mould mixing spore liquid be diluted, obtain C_BRange is 5.0 × 10⁶CFU/mL~9.0 × 10⁶The 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_BCandida albicans bacterium suspension or mould mixing spore liquid connected After continuous gradient dilution, standard series bacteria suspension is obtained: 3.5 × 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL is simultaneously It mixes.Then, according to relative intensity of fluorescence value I in this patent_BMeasuring method, according to viable bacteria content C_BSequence from low to high is surveyed Determine and record the relative intensity of fluorescence value of above-mentioned standard solution.Then, with the relative intensity of fluorescence logarithm of standard series bacteria suspension Value lgI_BAs abscissa, with corresponding viable bacteria content logarithm lgC_BFor ordinate mapping；To mathematical relationship between the two into Row calibration curve is derived from fitting equation Y=a using least square fitting method_BX+b_BAnd linearly dependent coefficient R_B ²；Work as R_B ² >=0.98, when confidence level >=0.95, it is effective that measurement is done according to this patent method；

The neutralizer identification and quantitative suspension test, carry out in the steps below:

(1) neutralizer qualification test: the disinfectant minimum effective concentration that indicator bacteria 99.9% is killed in selection effect 10min suppression is made For the sample liquid concentration of neutralizer qualification test, and 1mL test sample liquid is mixed with 9mL with agent solution；During effect 10min is formed It after product, carries out test and is grouped as follows: by 0.49mL inoculation bacterium solution (with lgC_B-lgI_BCalibration curve is derived from same branch with bacterium solution Indicate strain stoste test tube, 2 DEG C ± 0.2 DEG C saves, and uses in 2h) five are dispensed containing 4.41mL test sample liquid (1^#、2^#)、 4.41mL neutralized reaction product solution (3^#), 4.41mL Cha Shi culture solution (4^#), in 4.41mL and agent solution (5^#) sterile test tube in, 1000r/min shakes test tube 10min, mixes well its solution.Then, 0.49mL each group mixing liquid is divided with sterilizing liquid-transfering gun Fill five culture solutions of Cha Shi containing 4.41mL (1^#、3^#、4^#、5^#), in 4.41mL and agent solution (2^#) sterile test tube in, while will be another 4.9mL Cha Shi culture solution in an outer sterile test tube is as the 6th^#Group test sample liquid.1000r/min shakes each group test tube After 10min, using its mixing liquid as the recovered liquid of test sample liquid, according to relative intensity of fluorescence value I in this patent_BMeasuring method, Measure and record the relative intensity of fluorescence value of above-mentioned 6 groups of recovered liquids.

(2) neutralization is evaluated: if the 1st^#、6^#The relative intensity of fluorescence measured value I of group recovered liquid_B1≥0、I_B6=0, the 2^#~5^#Relationship between the relative intensity of fluorescence measured value of group recovered liquid are as follows: I_B2> 0 and I_B2< I_B3、I_B2< I_B4、I_B2< I_B5；The 3^#、4^#、5^#The relative intensity of fluorescence measured value of group recovered liquid is close, i.e. I_B3≈I_B4≈I_B5, it is calculated according to formula (1) Viable bacteria content C_B3、C_B4、C_B5Error rate Δ≤10% between group；Illustrate that selected neutralizer type can be used for testing liquid disinfectant sample The test of product fungi killing effect, and according to repeating above-mentioned neutralizer identification examination after in equivalent and principle, adjusting neutralizer concentration It tests, determines the corresponding neutralizer concentration of disinfectant experimental concentration.

In formula:

Δ-the 3rd^#、4^#、5^#The viable bacteria content C of group recovered liquid_B3、C_B4、C_B5Error rate between group, %；

I_B3、I_B4、I_B5- the 3^#、4^#、5^#The relative intensity of fluorescence measured value of group recovered liquid, RLU；

- the 3^#、4^#、5^#The mean viable content of group recovered liquid, numerical value are

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

(3) quantitative suspension test: according to the effective concentration and action time range marked in disinfectant operation instruction to be measured, High, medium and low three suitable concentrations: C are diluted to sterile water₁、C₂、C₃(C₁It is minimum using dense specified in the description of product Degree), wherein C₂=2C₁、C₃=2C₂；Four sections of different sterilization time t are chosen simultaneously₁、t₂、t₃、t₄(t₂To be provided in the description of product Most short action time), wherein t₁=0.5t₂、t₃=1.5t₂、t₄=2t₂.Then, with sterilizing liquid-transfering gun that 4.41mL difference is dense The test sample liquid of degree dispenses in three sterile test tubes；It is placed in (20 ± 2) DEG C water-bath, to solution temperature in test tube and water-bath temperature After degree balance, 0.49mL is added dropwise respectively and is inoculated with bacterium solution.Start to clock after 3000r/min shaking test tube 30s, when sterilization functions continue To the t of setting₁、t₂、t₃、t₄After four sections of different times, the mixing liquid of 0.49mL various concentration is dispensed three with sterilizing liquid-transfering gun Containing in 4.41mL and in the sterile test tube of agent solution, 3000r/min shaking test tube 30s simultaneously starts to clock, effect lasts to be neutralized After 10min, using its mixing liquid as the recovered liquid of each concentration tests sample liquid.Then, with Cha Shi culture solution alternate test sample liquid weight Multiple above-mentioned test；

Test is repeated 5 times.

Then, calf serum is added in bacteria suspension, makes its final serum content 10% and ensures to be inoculated with bacterium solution C_BFor 5.0×10⁶CFU/mL~9.0 × 10⁶CFU/mL is repeated above-mentioned quantitative suspension test 5 times；Determine testing liquid disinfectant sample Under the conditions of existing for the organic matter, has effective concentration and the time of Disinfection Effect to fungi；

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 killing rate R is calculated with sterilizing Index A, carry out in the steps below:

(1) recovered liquid viable bacteria content C_BAnd T_BIt calculates

According to instruction strain mark song lgC_B-lgI_BLinear equation Y=a_BX+b_B, calculate the test sample of each various concentration Liquid and control sample liquid go out through four sections/after the microbiological contamination time, the viable bacteria content T of recovered liquid_BAnd C_B.Relevant calculation is shown in formula (2)~(9):

In formula:

- every time the test sample liquid of various concentration recycle after four sections of sterilization times Viable bacteria amount, unit are every milliliter of Colony Forming Unit (CFU/mL)；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；

- every time various concentration test sample liquid after four sections of sterilization times, recycling The relative intensity of fluorescence measured value of liquid, RLU；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

- every time the control sample liquid of various concentration recycle after four microbiological contamination time Viable bacteria amount, unit are every milliliter of Colony Forming Unit (CFU/mL)；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；

- every time various concentration control sample liquid after four microbiological contamination time, recycling The relative intensity of fluorescence measured value of liquid, RLU；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

(2) killing rate R is calculated

Under conditions of neutralizer qualification result is qualified, the test sample liquid of each various concentration is right in four sections of sterilization times The killing rate of fungiAnd the testing liquid disinfectant sample of respective concentration is in same sterilizing Killing rate in timeIt is calculated respectively according to formula (10)~(17):

In formula:

The test sample liquid of-various concentration every time fungi in four sections of sterilization times kills It goes out rate, %；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

The testing liquid disinfectant sample of-respective concentration is right in four sections of sterilization times The killing rate of fungi, %；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

- every time the control sample liquid of various concentration recycle after four microbiological contamination time Viable bacteria amount, unit are every milliliter of Colony Forming Unit (CFU/mL)；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

- every time the test sample liquid of various concentration recycle after four sections of sterilization times Viable bacteria amount, unit are every milliliter of Colony Forming Unit (CFU/mL)；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

a_B- standard curve lgC_B-lgI_BSlope；

(3) sterilizing Index A calculates

Under conditions of neutralizer qualification result is qualified, the test sample liquid of each various concentration is right in four sections of sterilization times The sterilizing index of fungiAnd the testing liquid disinfectant sample of respective concentration is same Sterilizing index in sterilization timeIt is calculated respectively according to formula (18)~(25):

In formula:

- every time various concentration test sample liquid in four sections of sterilization times to fungi Sterilize index；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

The testing liquid disinfectant sample of-respective concentration is right in four sections of sterilization times The sterilizing index of fungi；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

a_B- standard curve lgC_B-lgI_BSlope；

(4) bacteria suspension viable bacteria content C data revision of the convention requirement: is demarcated using blood counting chamber_BWhen, with reference to GB4789.2- Relevant regulations in 2016, work as C_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BWhen not less than 100CFU/mL, the 3rd Preceding 2 bit digital is taken after bit digital " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, " four houses five Enter " afterwards using two effective digitals.Test sample liquid and control sample liquid go out through four sections/after the microbiological contamination time, the relative fluorescence of recovered liquid Strength detection value round numbers takes three effective digitals to the sterilizing rate calculated result of fungi, and sterilizing index calculated result takes two Effective digital；

(5) uncertainty of measurement: the test sample liquid that this patent method passes through various concentration in calculating 5 times quantitative suspension tests With control sample liquid through four sections it is different go out the/microbiological contamination time, recovered liquid 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measurement Coefficient of variation CV=σ ÷ μ × 100% of value (μ, σ and CV calculated result remain into 2 significant digits)；Judge ATP Fluorimetric assay for biological materials method be applied to the reproducibility of liquid disinfectant fungi killing effect test, it is specified that coefficient of variation CV≤ 10%.Relevant calculation is shown in formula (26)~(33):

In formula:

- through t₁、t₂、t₃、t₄After four sections of different sterilization times, in 5 tests The relative intensity of fluorescence measured value of 15 ATP fluorometric investigation Duplicate Samples of test sample liquid of same concentrationArithmetic mean of instantaneous value；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1, 2,3；Duplicate Samples number k=1,2,3；

- through t₁、t₂、t₃、t₄After four sections of different sterilization times, in 5 tests 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measured values of recovered liquid of the test sample liquid of same concentrationStandard deviation；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；Duplicate Samples number k=1,2,3；

- through t₁、t₂、t₃、t₄After four different microbiological contamination times, in 5 tests The relative intensity of fluorescence measured value of 15 ATP fluorometric investigation Duplicate Samples of control sample liquid of same concentrationArithmetic mean of instantaneous value；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1, 2,3；Duplicate Samples number k=1,2,3；

- through t₁、t₂、t₃、t₄After four different microbiological contamination times, in 5 tests 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measured values of recovered liquid of the control sample liquid of same concentrationStandard deviation；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；Duplicate Samples number k=1,2,3；

The result judgement of the liquid disinfectant sample fungi killing effect carries out in the steps below:

With reference to health industry common practice and related Disinfection Effect biological assessment standard, if certain concentration testing liquid disappears Toxic agent sample liquid quantifies in suspension test in single, through ATP bioluminescence lgC_B─lgI_BWhen the specific sterilizing that calibration curve method measures Interior fungi killing rate >=99.9% or sterilizing index >=3.0 then determine that its Disinfection Effect in this test is qualified.When this is dense It, can when spending Disinfection Effect qualification of the testing liquid disinfectant sample liquid in 5 quantitative suspension tests in identical sterilization time Determine its minimum concentration and shortest time to fungi with Disinfection Effect (in practical applications most with organic matter protection test Low concentration and shortest time are concentration and time needed for testing liquid disinfectant sample reaches practical Disinfection Effect)；

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 13 α-(N, N- dimethylamino) ethylamino matrine thimerosal sample pair of 120g/L with concentration It is illustrated for the killing effect detection of fungi.

Specific evaluation method carries out in the steps below:

(1) sample extraction and minimum effective concentration determine

1.1 control samples: using Cha Shi culture solution as control sample.

1.2 test specimens: the liquid of 1 minimum marketing packing is randomly selected from certain product batch number 1 complete transportation and packing Body disinfectant sample is used for neutralizer identification and quantification suspension test；Every group of test specimen selects one group of control sample as ginseng According to object and effectively identify.

The determination of 1.3 disinfectant sample minimum effective concentrations: according to having for disinfectant sample operation instructions to be measured mark Concentration range is imitated, it is diluted to high, medium and low three suitable concentrations with sterile water；It is with sterilizing liquid-transfering gun that 4.41mL difference is dense The dilution of degree dispenses three sterile test tubes, and 0.49mL inoculation bacterium solution is added dropwise respectively into each test tube；3000r/min shaking examination After pipe 30s, place it in (20 ± 2) DEG C water-bath.Start timing after solution temperature in test tube and bath temperature balance, makes to disappear Toxic agent continues 10min to the killing effect of fungi；Then, using its mixing liquid as recovered liquid, according to relative fluorescence in this patent Intensity value I_BMeasuring method measures and records the relative intensity of fluorescence value of recovered liquid.Meanwhile using 4.41mL Cha Shi culture solution as Control sample repeats above-mentioned sterilization test and recovered liquid I_BContinuous mode；And according to killing rate R calculation formula in this patent, to not Killing rate with concentration dilution liquid is calculated；If certain concentration killing rate R is 99.9%, this concentration is set as disinfection to be measured The minimum effective concentration of agent sample.

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

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

2.2 instrument and equipments: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, very Bacterium cell/spore sum detection range 10¹CFU/mL~10⁶CFU/mL；Amplification factor 40 ×~400 × Photobiology it is micro- Mirror；The constant incubator that (25~37) DEG C ± 1 DEG C；The constant water bath box that (10~50) DEG C ± 1 DEG C；(0~50) DEG C ± 1 DEG C, (50 ~300) constant temperature oscillator of r/min；Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The range of speeds (500~ 3000) the vortex oscillator of r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C Low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic wave of frequency range (30~50) kHz is clear Wash device；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace.

2.3 material utensils: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；Sterile petri dish；Glass funnel；Sterile cock test tube；Diameter Oese no more than 4mm；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Absorbent cotton and gauze for biochemistry detection；Nothing Bacterium filter paper；Thermometer；The stopwatch of precision 0.01s.

2.4 reagents: 0.1% Lv Shi alkaline methylene blue dyeing liquor；Calf serum；121 DEG C of high pressure sterilizations of following reagent 30min, the physiological saline of 5 DEG C~10 DEG C storage 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are appointed One kind is selected, 0.05% wetting agent solution is configured to；5g sodium thiosulfate is dissolved in 1000mL water (for chlorine type disinfectant)； 1.36g potassium dihydrogen phosphate, 2.83g disodium hydrogen phosphate, 10g lecithin, 10g glycine, 30g tween (80) are dissolved in 1000mL water Or 1.36g potassium dihydrogen phosphate, 2.83g disodium hydrogen phosphate, 3g lecithin, 20g tween (80) are dissolved in 1000mL water (for non-oxygen Change type disinfectant)；20g tween (80), 1g sodium thiosulfate are dissolved in 1000mL phosphate buffer solution (to sterilize for oxygen type Agent) etc. (or being directed to testing liquid disinfectant sample type, select the neutralizer that other phases are applicable).

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 use): removing the peel stripping and slicing for 300g fresh potato, 20min~30min is boiled in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g chloramphenicol, 20g agar are added into filtrate After be settled to 1000mL.

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

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h.

(3) culture presevation, activation and bacteria suspension preparation

3.1 instruction strains: Candida albicans ATCC 10231；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC6205；It produces Penicillium chrysogenum ATCC 9179 (or other strains that is provided by national Culture Collection and can be traced to the source).

3.2 culture presevation: Candida albicans --- freeze-drying lactobacillus pipe is opened with sterile working, is infused with capillary syring into pipe Enter appropriate Sharpe culture solution, pressure-vaccum makes strain melt dispersion for several times；A little bacteria suspension is instilled and is trained equipped with 5mL~10mL Sharpe In the test tube of nutrient solution, 37 DEG C of culture 18h~for 24 hours.Mould --- mould test strain is inoculated in by potato-with sterile working The inoculation date is simultaneously indicated in dextrose culture-medium inclined-plane, and 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3℃ ~10 DEG C preservation 4 months, as preservation bacterium.

3.3 actication of culture: Candida albicans --- with the colonies typical in oese scraping 1st generation culture, scribing line is connect Kind is in sabouraud culture medium plate；After 37 DEG C of culture 18h~for 24 hours, the colonies typical in picking 2nd generation culture is inoculated in Sharpe training Support base inclined-plane；After 37 DEG C of culture 18h~for 24 hours, 4 DEG C of sealings storages, use in 6 weeks.Mould --- preservation bacterium is scraped with oese Spore is inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d, until generating a large amount of spores.Preparation Must not extract the test tube plug of mold species before spore suspension, every test tube open after only for spore liquid of preparation, every time Suspension is prepared using the mycotic spore newly cultivated.

The preparation of 3.4 bacteria suspensions: Candida albicans test --- with oese, the fresh microbial strain culture of picking is connect from inclined-plane Kind in the sterile conical flask equipped with 50mL Sharpe culture solution, it is placed in 150r/min culture in the constant temperature oscillator of (30 ± 1) DEG C 18h~for 24 hours, 4 DEG C of sealing storages, the same day uses.Mould test --- the sterile water of 10mL is added into strain test tube, with inoculation Ring gently scrapes the fresh mycotic spore of media surface, and the spore stoste injection of wash-off is trained equipped with 15 beades and 45mL Cha Shi In the sterile conical flask of jumping a queue of nutrient solution, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, it will cover There are sterile absorbent cotton or the glass funnel of eight layers of gauze to be placed on conical flask, filtering spore suspension removes mycelia and culture medium is broken Piece；Filtrate is moved in sterile centrifugation tube, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；50mL is used again Cha Shi culture solution cleaning spore sediment is simultaneously centrifuged, and after repeated washing 3 times, is precipitated with the spore after the dilution centrifugation of Cha Shi culture solution Object.Every kind of test mould prepares spore suspension according to the method described above, and the spore liquid of each strain is mixed in equal volume；0 DEG C~7 DEG C storage uses in the same day or 4d.

(4) bacteria suspension viable bacteria content C after methylene blue_BBlood cell plate counts and inoculation bacterium solution C_BCalibration

4.1 methylene blues: with sterile working by 50 μ L dilutions for 10^-2~10^-3Candida albicans bacterium suspension or mould The Lv Shi alkaline methylene blue dyeing liquor that mixing spore liquid and 30 μ L concentration are 0.05% moves to (bacterium solution in same branch sterile test tube respectively Dilution, which includes 4~5 albicans cells or mycotic spore with each lattice of blood counting chamber, to be advisable), 1000r/min Test tube 1min is shaken, mixes well bacteria suspension with dyeing liquor.

4.2 blood cell plates count: the bacteria suspension after ± 0.5 μ L of 5 μ L dyeing being placed in coverslip edge with aseptic straw, makes it Blood counting chamber is slowly penetrated along slide gap, bubble is not can produce between tally and slide, otherwise re-operates.With sterile Extra bacterium solution in filter paper exhaustion slot, stands 2min ± 20s, is settled down to its whole in counting chamber.When lattice tally in use 16 When, upper left, upper right, lower-left, the albicans cell in the middle lattice in bottom right 4 (i.e. 100 lattices) are taken in diagonal orientation Or mycotic spore is counted；Lattice tally in 25 is such as used, in addition to counting above-mentioned 4 diagonal orientations, also needs to count the 1 of center A middle lattice (i.e. 80 lattices)；When test strain is located on the two-wire of middle lattice, then only count online and right line or it is offline and Albicans cell or mycotic spore on left line.

4.3 viable bacteria content C_BCalculate: by Photobiology microscope magnification from it is low to lofty tone to 400 × after, it is right immediately Albicans cell or mycotic spore at blood counting chamber additional space position are counted；Wherein colourless Candida albicans Bacterium cell is viable bacteria, and blue or light blue person is dead bacterium；Edge is in navy blue, inside in colourless or light blue and pink mould Bacterium spore is viable bacteria, and edge and inside are dead bacterium in navy blue person, therefore the only counting rim spore different from internal color.If Quantity without mycelia monospore in mycotic spore suspension is lower than 90%, then prepares spore liquid again.It is each to blood counting chamber small Albicans cell or mycotic spore in grid repeat microscopy and count three times, are averaged.When blood counting chamber specification is When 16 × 25, viable bacteria content (every milliliter of Colony Forming Unit, CFU/mL) C of 1mL bacterium solution_B5 × 16 × K × 10=N ÷⁴；Work as blood When ball count plate gauge lattice are 25 × 16, C_B5 × 25 × K × 10=N ÷⁴；Wherein N is that white is read in five middle lattice of blood counting chamber The total viable count of pearl bacterium cell or mycotic spore, K are bacterium solution extension rate.Then, with Cha Shi culture solution to known viable bacteria content C_BCandida albicans bacterium suspension or mould mixing spore liquid be diluted, obtain C_BRange is 5.0 × 10⁶CFU/mL~9.0 × 10⁶The inoculation bacterium solution of CFU/mL.

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

With Cha Shi culture solution to known viable bacteria content C_BCandida albicans bacterium suspension or mould mixing spore liquid connected After continuous gradient dilution, standard series bacteria suspension is obtained: 3.5 × 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL is simultaneously It mixes.Then, according to relative intensity of fluorescence value I in this patent_BMeasuring method, according to viable bacteria content C_BSequence from low to high is surveyed Determine and record the relative intensity of fluorescence value of above-mentioned standard solution.Then, with the relative intensity of fluorescence logarithm of standard series bacteria suspension Value lgI_BAs abscissa, with corresponding viable bacteria content logarithm lgC_BFor ordinate mapping；To mathematical relationship between the two into Row calibration curve is derived from fitting equation Y=a using least square fitting method_BX+b_BAnd 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.

(6) neutralizer identification and quantitative suspension test

6.1 neutralizer qualification tests: the disinfectant minimum effective concentration that indicator bacteria 99.9% is killed in selection effect 10min suppression is made For the sample liquid concentration of neutralizer qualification test, and 1mL test sample liquid is mixed with 9mL with agent solution；During effect 10min is formed It after product, carries out test and is grouped as follows: by 0.49mL inoculation bacterium solution (with lgC_B-lgI_BCalibration curve is derived from same branch with bacterium solution Indicate strain stoste test tube, 2 DEG C ± 0.2 DEG C saves, and uses in 2h) five are dispensed containing 4.41mL test sample liquid (1^#、2^#)、 4.41mL neutralized reaction product solution (3^#), 4.41mL Cha Shi culture solution (4^#), in 4.41mL and agent solution (5^#) sterile test tube in, 1000r/min shakes test tube 10min, mixes well its solution.Then, 0.49mL each group mixing liquid is divided with sterilizing liquid-transfering gun Fill five culture solutions of Cha Shi containing 4.41mL (1^#、3^#、4^#、5^#), in 4.41mL and agent solution (2^#) sterile test tube in, while will be another 4.9mL Cha Shi culture solution in an outer sterile test tube is as the 6th^#Group test sample liquid.1000r/min shakes each group test tube After 10min, using its mixing liquid as the recovered liquid of test sample liquid, according to relative intensity of fluorescence value I in this patent_BMeasuring method, Measure and record the relative intensity of fluorescence value of above-mentioned 6 groups of recovered liquids.

The evaluation of 6.2 neutralizations: if the 1st^#、6^#The relative intensity of fluorescence measured value I of group recovered liquid_B1≥0、I_B6=0, the 2^#~5^#Relationship between the relative intensity of fluorescence measured value of group recovered liquid are as follows: I_B2> 0 and I_B2< I_B3、I_B2< I_B4、I_B2< I_B5；The 3^#、4^#、5^#The relative intensity of fluorescence measured value of group recovered liquid is close, i.e. I_B3≈I_B4≈I_B5, it is calculated according to formula (1) Viable bacteria content C_B3、C_B4、C_B5Error rate Δ≤10% between group；Illustrate that selected neutralizer type can be used for testing liquid disinfectant sample The test of product fungi killing effect, and according to repeating above-mentioned neutralizer identification examination after in equivalent and principle, adjusting neutralizer concentration It tests, determines the corresponding neutralizer concentration of disinfectant experimental concentration.

In formula:

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

6.3 quantitative suspension tests: according to the effective concentration and action time range marked in disinfectant operation instruction to be measured, High, medium and low three suitable concentrations: C are diluted to sterile water₁、C₂、C₃(C₁It is minimum using dense specified in the description of product Degree), wherein C₂=2C₁、C₃=2C₂；Four sections of different sterilization time t are chosen simultaneously₁、t₂、t₃、t₄(t₂To be provided in the description of product Most short action time), wherein t₁=0.5t₂、t₃=1.5t₂、t₄=2t₂.Then, with sterilizing liquid-transfering gun that 4.41mL difference is dense The test sample liquid of degree dispenses in three sterile test tubes；It is placed in (20 ± 2) DEG C water-bath, to solution temperature in test tube and water-bath temperature After degree balance, 0.49mL is added dropwise respectively and is inoculated with bacterium solution.Start to clock after 3000r/min shaking test tube 30s, when sterilization functions continue To the t of setting₁、t₂、t₃、t₄After four sections of different times, the mixing liquid of 0.49mL various concentration is dispensed three with sterilizing liquid-transfering gun Containing in 4.41mL and in the sterile test tube of agent solution, 3000r/min shaking test tube 30s simultaneously starts to clock, effect lasts to be neutralized After 10min, using its mixing liquid as the recovered liquid of each concentration tests sample liquid.Then, with Cha Shi culture solution alternate test sample liquid weight Multiple above-mentioned test.

Test is repeated 5 times.

Then, calf serum is added in bacteria suspension, makes its final serum content 10% and ensures to be inoculated with bacterium solution C_BFor 5.0×10⁶CFU/mL~9.0 × 10⁶CFU/mL is repeated above-mentioned quantitative suspension test 5 times；Determine testing liquid disinfectant sample Under the conditions of existing for the organic matter, has effective concentration and the time of Disinfection Effect to fungi.

(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 killing rate R is calculated with sterilizing Index A

8.1 recovered liquid viable bacteria content C_BAnd T_BIt calculates

In formula:

- every time various concentration control sample liquid after four microbiological contamination time, recycling The relative intensity of fluorescence measured value of liquid, RLU；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3.

8.2 killing rate R are calculated

In formula:

a_B- standard curve lgC_B-lgI_BSlope.

8.3 sterilizing Index As calculate

In formula:

a_B- standard curve lgC_B-lgI_BSlope.

The requirement of the 8.4 data revisions of the convention: bacteria suspension viable bacteria content C is demarcated using blood counting chamber_BWhen, with reference to GB4789.2- Relevant regulations in 2016, work as C_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BWhen not less than 100CFU/mL, the 3rd Preceding 2 bit digital is taken after bit digital " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, " four houses five Enter " afterwards using two effective digitals.Test sample liquid and control sample liquid go out through four sections/after the microbiological contamination time, the relative fluorescence of recovered liquid Strength detection value round numbers takes three effective digitals to the sterilizing rate calculated result of fungi, and sterilizing index calculated result takes two Effective digital.

8.5 uncertainties of measurement: the test sample liquid that this patent method passes through various concentration in calculating 5 times quantitative suspension tests With control sample liquid through four sections it is different go out the/microbiological contamination time, recovered liquid 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measurement Coefficient of variation CV=σ ÷ μ × 100% of value (μ, σ and CV calculated result remain into 2 significant digits)；Judge ATP Fluorimetric assay for biological materials method be applied to the reproducibility of liquid disinfectant fungi killing effect test, it is specified that coefficient of variation CV≤ 10%.Relevant calculation is shown in formula (26)~(33):

In formula:

- through t₁、t₂、t₃、t₄After four different microbiological contamination times, in 5 tests 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measured values of recovered liquid of the control sample liquid of same concentrationStandard deviation；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；Duplicate Samples number k=1,2,3.

(9) result judgement

With reference to health industry common practice and related Disinfection Effect biological assessment standard, if certain concentration testing liquid disappears Toxic agent sample liquid quantifies in suspension test in single, through ATP bioluminescence lgC_B─lgI_BWhen the specific sterilizing that calibration curve method measures Interior fungi killing rate >=99.9% or sterilizing index >=3.0 then determine that its Disinfection Effect in this test is qualified.When this is dense It, can when spending Disinfection Effect qualification of the testing liquid disinfectant sample liquid in 5 quantitative suspension tests in identical sterilization time Determine its minimum concentration and shortest time to fungi with Disinfection Effect (in practical applications most with organic matter protection test Low concentration and shortest time are concentration and time needed for testing liquid disinfectant sample reaches practical Disinfection Effect).

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

(1) Laboratory biosafety qualification

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

(2) instrument and equipment and test equipment

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

2.2ATP bioluminescence rapid detection system: the portable system 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%.

2.3 Photobiology microscopes: have the adjustable eyepiece of 10 times of wide visual fields and 4 times, 10 times, 20 times, 40 times and 100 times The tri- mesh research grade biomicroscope of Olympus BX43 of flat-field achromatic objective lens.

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

2.5 constant 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.

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

2.7 refrigerated centrifuges: the vertical refrigerated centrifuge of Beijing Xin Nuolihua Instrument Ltd. model DL7M-12L turns Fast 8000r/min ± 20r/min, 12300 × g of relative centrifugal force；Capacity 14400ml, timing range 1s~99h59min99s, ± 1 DEG C of temperature control range (- 20~40) DEG C, centrifuge tube specification Ф 74mm × 168mm.

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

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

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

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

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

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

2.14pH meter: the accurate pH meter of Shanghai precision instrumentation Co., Ltd model MP512-03, range ability (- 2.000~19.999) pH ± 0.002pH, ± 0.4 DEG C of temperature range (- 10~110) DEG C.

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

2.16 blood counting chambers: the blood counting chamber that refinement specification in Shanghai is 25 × 16.

(3) medium/liquid

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

(4) chemical reagent

4.1 0.1% Lv Shi alkaline methylene blue dyeing liquor: it is purchased from Shanghai innermost thoughts and feelings Biotechnology Co., Ltd.

4.2 trinosin standard items and preparation ATP fluorescent reagent buffer solution, ATP lysate, ATP extracting solution The U.S. Amresco of Shanghai Jin Pan Biotechnology Co., Ltd agency is purchased from a series of biochemical reagents needed for ATP fluorescent reagent Brand.

(5) reference culture

Candida albicans ATCC 10231 is purchased from Chinese medicine fungi preservation administrative center；Aspergillus niger ATCC 16404, ball Chactomium globosum ATCC 6205, penicillium chrysogenum ATCC 9179 are purchased from China General Microbiological culture presevation administrative center.

The evaluation data and result of the present embodiment calculate:

Using ATP fluophotometer through lgC_B-lgI_BCalibration curve method is to 13 α-(N, N- dimethylamino) ethylamino kuh-seng The fungi killing effect of alkali thimerosal sample is detected, to Candida albicans and mould killing rate up to 99.9% in 10min Minimum effective concentration be respectively 113g/L and 105g/L.Select lecithin containing 10g/L, 35g/L glycine, 100g/L tween (80) TPS solution can effectively neutralize residual action of the thimerosal sample to be measured to instruction strain as neutralizer；Correlation neutralizes Agent identification, quantitative suspension test data and Evaluation of Disinfection, Calculation of Measuring Uncertainty result are shown in Table 1~table 3 respectively.

Error rate calculated result between 1 neutralizer qualification test data of table and group

Table 2 quantifies suspension test data and killing rate R_ijt, sterilizing Index A_ijtCalculated result

3 Evaluation of Disinfection of table and relative intensity of fluorescence value Calculation of Measuring Uncertainty result

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 evaluation method of liquid disinfectant fungi killing effect, comprising: (1) sample extraction and minimum effective concentration are true It is fixed；(2) lectotype selection and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) bacterium after methylene blue Suspension viable bacteria content C_BBlood cell plate count and inoculation bacterium solution C_BCalibration；(5) viable bacteria content logarithm lgC_BRelative intensity of fluorescence Logarithm lgI_BStandard curve is established；(6) neutralizer identification and quantitative suspension test；(7) reclaim liquid phase is to fluorescence intensity level I_BIt surveys It is fixed；(8) recovered liquid viable bacteria content C_BAnd T_BIt calculates and sterilizing rate R is calculated with sterilizing Index A；(9) result judgement；It is characterized in that, It is precisely fixed to carry out using ATP fluophotometer to the liquid disinfectant fungi killing effect with killing rate R or sterilizing Index A characterization Measure the ATP bioluminescence lgC of evaluation_B-lgI_BCalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_BIn measurement:

Sample extraction requirement is specified, the minimum effective concentration for pressing down in disinfectant 10min and killing indicator bacteria is determined, by mycologic test strain Reference culture passed on, activated after, take fresh Candida albicans or mycotic spore culture to prepare bacteria suspension；It is contaminated through methylene blue Using the viable bacteria content C of blood counting chamber measurement bacterium solution after color_B, and to inoculation bacterium solution C_BIt is demarcated: 5.0 × 10⁶CFU/mL~ 9.0×10⁶CFU/mL selects viable bacteria content C_BIt is 3.5 × 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵The bacterium of CFU/mL Liquid is as standard series bacteria suspension, using ATP fluophotometer to its relative intensity of fluorescence value I_BIt is measured；Draw lgC_B- lgI_BStandard curve is derived from the linear equation Y=a of curve_BX+b_BAnd coefficient R_B ², then, with disinfectant to be measured Minimum effective concentration carries out neutralizer qualification test, determines neutralizer type and concentration；Simultaneously by the test of each concentration of 4.41mL Sample liquid and control sample liquid are placed in (20 ± 2) DEG C water-bath and mix it with 0.49mL inoculation bacterium solution；Effect lasts t subject to sterilization₁、 t₂、t₃、t₄After four sections of different times, 0.49mL microbiological contamination sample liquid is mixed with 4.41mL with agent solution respectively, neutralization carries out Using its mixing liquid as recovered liquid after 10min；Using the relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring recovered liquidWith And according to instruction strain standard curve side Formula Y=a_BX+b_BCalculate corresponding viable bacteria content With

In killing rate R or sterilizing Index A are calculated:

According to instruction strain standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, for t₁、t₂、t₃、t₄Four sections of differences are gone out The bacterium time, to quantify the relative intensity of fluorescence measurement of each concentration tests sample liquid and control sample liquid recycling viable bacteria in suspension test every time Value is as basic data；Calculate its killing rate to fungi Or sterilizing indexAnd take the arithmetic mean of instantaneous value of its five times tests as respective concentration testing liquid disinfectant sample Product are in identical sterilization time to the killing rate of fungi Or sterilizing indexThe clear related data revision of the convention and uncertainty of measurement requirement simultaneously；

In result judgement:

With reference to health industry common practice and related Disinfection Effect biological assessment standard, if certain concentration testing liquid disinfectant Sample liquid quantifies in suspension test in single, through ATP bioluminescence lgC_B─lgI_BIn the specific sterilization time that calibration curve method measures Fungi killing rate >=99.9% or sterilizing index >=3.0, then determine that its Disinfection Effect in this test is qualified, when the concentration It, can be true when Disinfection Effect of the testing liquid disinfectant sample liquid in 5 quantitative suspension tests in identical sterilization time is qualified Its fixed minimum concentration and shortest time to fungi with Disinfection Effect is (in practical applications with the minimum of organic matter protection test Concentration and shortest time are concentration and time needed for testing liquid disinfectant sample reaches practical Disinfection Effect).

2. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that the sample Product extract and minimum effective concentration determines, carry out in the steps below:

(1) control sample: using Cha Shi culture solution as control sample；

(2) test specimen: the liquid that 1 minimum marketing packing is randomly selected from certain product batch number 1 complete transportation and packing disappears Toxic agent sample is used for neutralizer identification and quantification suspension test；Every group of test specimen selects one group of control sample as object of reference And it effectively identifies；

(3) determination of disinfectant sample minimum effective concentration: according to the effective dense of disinfectant sample operation instructions to be measured mark Range is spent, it is diluted to high, medium and low three suitable concentrations with sterile water；With sterilizing liquid-transfering gun by 4.41mL various concentration Dilution dispenses three sterile test tubes, and 0.49mL inoculation bacterium solution is added dropwise respectively into each test tube；3000r/min shakes test tube It after 30s, places it in (20 ± 2) DEG C water-bath, starts timing after solution temperature in test tube and bath temperature balance, make to sterilize Agent continues 10min to the killing effect of fungi；Then, strong according to relative fluorescence in this patent using its mixing liquid as recovered liquid Angle value I_BMeasuring method measures and records the relative intensity of fluorescence value of recovered liquid, meanwhile, using 4.41mL Cha Shi culture solution as pair Product repeat above-mentioned sterilization test and recovered liquid I in the same old way_BContinuous mode；And according to killing rate R calculation formula in this patent, to difference The killing rate of concentration dilution liquid is calculated；If certain concentration killing rate R is 99.9%, this concentration is set as disinfectant to be measured The minimum effective concentration of sample.

3. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that described sets Alternative 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, fungi Cell/spore sum detection range 10¹CFU/mL~10⁶CFU/mL；Amplification factor 40 ×~400 × Photobiology microscope； The constant incubator that (25~37) DEG C ± 1 DEG C；The constant water bath box that (10~50) DEG C ± 1 DEG C；(0~50) DEG C ± 1 DEG C, (50~ 300) constant temperature oscillator of r/min；Revolving speed >=8000r/min centrifuge and mating centrifuge tube；The range of speeds (500~3000) The vortex oscillator of r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of Low-temperature Ice Case；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency range (30~50) kHz； The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL,0.1mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)；Capacity The sterile conical flask and bottle stopper of 100mL, 250mL, 500mL；Sterile petri dish；Glass funnel；Sterile cock test tube；Diameter is little In the oese of 4mm；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Absorbent cotton and gauze for biochemistry detection；Sterile filter Paper；Thermometer；The stopwatch of precision 0.01s；

(4) reagent: 0.1% Lv Shi alkaline methylene blue dyeing liquor；Calf serum；121 DEG C of following reagent high pressure sterilization 30min, 5 DEG C The physiological saline of~10 DEG C of storage 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are matched 0.05% wetting agent solution is made；5g sodium thiosulfate is dissolved in 1000mL water (for chlorine type disinfectant)；By 1.36g phosphorus Acid dihydride potassium, 2.83g disodium hydrogen phosphate, 10g lecithin, 10g glycine, 30g tween (80) are dissolved in 1000mL water or by 1.36g Potassium dihydrogen phosphate, 2.83g disodium hydrogen phosphate, 3g lecithin, 20g tween (80) are dissolved in 1000mL water and (sterilize for Non-oxidized Agent)；20g tween (80), 1g sodium thiosulfate are dissolved in (or the needle such as 1000mL phosphate buffer solution (for oxygen type disinfectant) To liquid disinfectant sample type to be measured, the neutralizer for selecting other phases applicable)；

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

Sabouraud culture medium/liquid (Candida albicans actication of culture use): 40g glucose, 10g peptone, 20g agar are dissolved by heating In 1000mL water (agar is not added in culture solution), adjust pH to 5.6 ± 0.2 (25 DEG C)；

Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen two Potassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose, which are dissolved by heating, contains 0.05% wetting agent in 1000mL Aqueous solution in, adjust pH to 6.0~6.5 (25 DEG C)；

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

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

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

ATP lysate: by 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 international units/ml Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, that 20mg bovine serum albumin is dissolved in 10mL is dense Degree is in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L, and adjusting use in pH to 6.0 ± 0.5,8h, (1mL lysate exists The ATP concentration in Sharpe culture solution can be down to 10 in 15min^-11Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, the benzene for being 10% with 0.2ml concentration After pricking the mixing of oronain solution, adjust pH to 12.0 ± 0.5 (fungal cell's ATP extraction efficiency is not less than 80%)；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL's ATP fluorescent reagent buffer solution is stored at room temperature 15min, uses in 3h after mixing.

4. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that the bacterium Kind preservation, activation and bacteria suspension preparation, carry out in the steps below:

(1) strain: Candida albicans ATCC 10231 is indicated；Aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；It produces yellow Mould ATCC 9179 (or other strains that is provided by national Culture Collection and can be traced to the source)；

(2) culture presevation: Candida albicans --- freeze-drying lactobacillus pipe is opened with sterile working, is injected with capillary syring into pipe suitable Sharpe culture solution is measured, pressure-vaccum makes strain melt dispersion for several times；A little bacteria suspension is instilled, 5mL~10mL Sharpe culture solution is housed Test tube in, 37 DEG C of culture 18h~for 24 hours, mould --- mould test strain is inoculated in by potato-grape with sterile working The inoculation date is simultaneously indicated in sugar culture-medium inclined-plane, and 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months, as preservation bacterium；

(3) actication of culture: Candida albicans --- with oese scraping 1st generation culture in colonies typical, streak inoculation in Sabouraud culture medium plate；After 37 DEG C of culture 18h~for 24 hours, the colonies typical in picking 2nd generation culture is inoculated in sabouraud culture medium Inclined-plane；After 37 DEG C of culture 18h~for 24 hours, 4 DEG C of sealings storages use, mould in 6 weeks --- with oese scraping preservation bacterium spore, It is inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d prepare spore until generating a large amount of spores Must not extract the test tube plug of mold species before suspension, every test tube open after only for spore liquid of preparation, use every time The mycotic spore newly cultivated prepares suspension；

(4) prepared by bacteria suspension: Candida albicans test --- with oese, the fresh microbial strain culture of picking is inoculated in from inclined-plane In sterile conical flask equipped with 50mL Sharpe culture solution, be placed in 150r/min culture 18h in the constant temperature oscillator of (30 ± 1) DEG C~ For 24 hours, 4 DEG C of sealing storages, the same day use, mould test --- the sterile water of 10mL is added into strain test tube, it is light with oese The spore stoste injection of wash-off is equipped with 15 beades and 45mL Cha Shi culture solution by the fresh mycotic spore for scraping media surface Sterile conical flask of jumping a queue in, 3000r/min shakes test tube 2min, breaks up spore ball, then nothing will be covered with by mixing spore liquid The glass funnel of bacterium absorbent cotton or eight layers of gauze is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；It will Filtrate moves in sterile centrifugation tube, and 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is trained again with 50mL Cha Shi Nutrient solution cleaning spore sediment is simultaneously centrifuged, and after repeated washing 3 times, dilutes the spore sediment after centrifugation with Cha Shi culture solution, often Kind test mould prepares spore suspension according to the method described above, and the spore liquid of each strain is mixed in equal volume；0 DEG C~7 DEG C are deposited It puts, is used in the same day or 4d.

5. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that the beauty Bacteria suspension viable bacteria content C after indigo plant dyeing_BBlood cell plate counts and inoculation bacterium solution C_BCalibration carries out in the steps below:

(1) methylene blue: with sterile working by 50 μ L dilutions for 10^-2~10^-3Candida albicans bacterium suspension or mould mixing The Lv Shi alkaline methylene blue dyeing liquor that spore liquid and 30 μ L concentration are 0.05% moves in same branch sterile test tube (bacterium solution dilution respectively Degree, which includes 4~5 albicans cells or mycotic spore with each lattice of blood counting chamber, to be advisable), 1000r/min shaking Test tube 1min, mixes well bacteria suspension with dyeing liquor；

(2) blood cell plate counts: the bacteria suspension after ± 0.5 μ L of 5 μ L dyeing being placed in coverslip edge with aseptic straw, makes it along glass Piece gap slowly penetrates blood counting chamber, not can produce bubble between tally and slide, otherwise re-operates, uses aseptic filter paper Extra bacterium solution in exhaustion slot, stands 2min ± 20s, is settled down to its whole in counting chamber, when using lattice tally in 16, Diagonal orientation takes upper left, upper right, lower-left, the albicans cell in the middle lattice in bottom right 4 (i.e. 100 lattices) or mould Spore is counted；Lattice tally in 25 is such as used, in addition to counting above-mentioned 4 diagonal orientations, also needs 1 middle lattice for counting center (i.e. 80 lattices)；When test strain is located on the two-wire of middle lattice, then only count on online and right line or offline and left line Albicans cell or mycotic spore；

(3) viable bacteria content C_BCalculate: by Photobiology microscope magnification from it is low to lofty tone to 400 × after, immediately to hemocytometer Albicans cell or mycotic spore at number plate additional space position are counted；Wherein colourless albicans cell For viable bacteria, blue or light blue person is dead bacterium；Edge is in navy blue, inside in colourless or light blue and pink mycotic spore For viable bacteria, edge and inside are dead bacterium in navy blue person, therefore the only counting rim spore different from internal color, if mould spore Quantity without mycelia monospore in sub- suspension is lower than 90%, then spore liquid is prepared again, in each lattice of blood counting chamber Albicans cell or mycotic spore repeat microscopy count three times, be averaged, when blood counting chamber specification be 16 × 25 When, viable bacteria content (every milliliter of Colony Forming Unit, CFU/mL) C of 1mL bacterium solution_B5 × 16 × K × 10=N ÷⁴；Work as blood count When plate gauge lattice are 25 × 16, C_B5 × 25 × K × 10=N ÷⁴；Wherein N is that Candida albicans is thin in five middle lattice of blood counting chamber The total viable count of born of the same parents or mycotic spore, K is bacterium solution extension rate, then, with Cha Shi culture solution to known viable bacteria content C_BIt is white Color beads bacterium suspension or mould mixing spore liquid are diluted, and obtain C_BRange is 5.0 × 10⁶CFU/mL~9.0 × 10⁶The inoculation bacterium solution of CFU/mL.

6. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that the work Bacterial content 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_BCandida albicans bacterium suspension or mould mixing spore liquid carry out continuous gradient After dilution, standard series bacteria suspension is obtained: 3.5 × 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL is simultaneously mixed, Then, according to relative intensity of fluorescence value I in this patent_BMeasuring method, according to viable bacteria content C_BSequence from low to high, measurement is simultaneously The relative intensity of fluorescence value for recording above-mentioned standard solution, then, with the relative intensity of fluorescence logarithm of standard series bacteria suspension lgI_BAs abscissa, with corresponding viable bacteria content logarithm lgC_BFor ordinate mapping；Mathematical relationship between the two is carried out Calibration curve is derived from fitting equation Y=a using least square fitting method_BX+b_BAnd 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 evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that in described With agent identification and quantitative suspension test, carry out in the steps below:

(1) neutralizer qualification test: during the disinfectant minimum effective concentration that indicator bacteria 99.9% is killed in selection effect 10min suppression is used as With the sample liquid concentration of agent qualification test, and 1mL test sample liquid is mixed with agent solution with 9mL；It acts on 10min and forms neutralization production It after object, carries out test and is grouped as follows: by 0.49mL inoculation bacterium solution (with lgC_B-lgI_BCalibration curve is derived from same branch with bacterium solution and indicates Strain stoste test tube, 2 DEG C ± 0.2 DEG C saves, and uses in 2h) five are dispensed containing 4.41mL test sample liquid (1^#、2^#), in 4.41mL With reaction mixture (3^#), 4.41mL Cha Shi culture solution (4^#), in 4.41mL and agent solution (5^#) sterile test tube in, 1000r/min Test tube 10min is shaken, its solution is mixed well, then, is contained the packing of 0.49mL each group mixing liquid for five with sterilizing liquid-transfering gun 4.41mL Cha Shi culture solution (1^#、3^#、4^#、5^#), in 4.41mL and agent solution (2^#) sterile test tube in, while by an other nothing 4.9mL Cha Shi culture solution in bacterium test tube is as the 6th^#Group test sample liquid, after 1000r/min shakes each group test tube 10min, by it Recovered liquid of the mixing liquid as test sample liquid, according to relative intensity of fluorescence value I in this patent_BMeasuring method is measured and is recorded State the relative intensity of fluorescence value of 6 groups of recovered liquids；

(2) neutralization is evaluated: if the 1st^#、6^#The relative intensity of fluorescence measured value I of group recovered liquid_B1≥0、I_B6=0, the 2nd^#~ 5^#Relationship between the relative intensity of fluorescence measured value of group recovered liquid are as follows: I_B2> 0 and I_B2< I_B3、I_B2< I_B4、I_B2< I_B5；3rd^#、 4^#、5^#The relative intensity of fluorescence measured value of group recovered liquid is close, i.e. I_B3≈I_B4≈I_B5, the work that is calculated according to formula (1) Bacterial content C_B3、C_B4、C_B5Error rate Δ≤10% between group；Illustrate that selected neutralizer type can be used for testing liquid disinfectant sample The test of fungi killing effect, and according to repeating above-mentioned neutralizer qualification test after in equivalent and principle, adjusting neutralizer concentration, Determine the corresponding neutralizer concentration of disinfectant experimental concentration；

In formula:

- the 3^#、4^#、5^#The mean viable content of group recovered liquid, numerical value are CFU/mL；

(3) quantitative suspension test: according to the effective concentration and action time range marked in disinfectant operation instruction to be measured, with nothing Bacterium water is diluted to high, medium and low three suitable concentrations: C₁、C₂、C₃(C₁Concentration is used to be minimum specified in the description of product), Wherein C₂=2C₁、C₃=2C₂；Four sections of different sterilization time t are chosen simultaneously₁、t₂、t₃、t₄(t₂For specified in the description of product most Short action time), wherein t₁=0.5t₂、t₃=1.5t₂、t₄=2t₂, then, with sterilizing liquid-transfering gun by 4.41mL various concentration Sample liquid is tested to dispense in three sterile test tubes；It is placed in (20 ± 2) DEG C water-bath, it is flat to solution temperature in test tube and bath temperature After weighing apparatus, 0.49mL is added dropwise respectively and is inoculated with bacterium solution, starts to clock after 3000r/min shaking test tube 30s, when sterilization functions continue to setting Fixed t₁、t₂、t₃、t₄After four sections of different times, the mixing liquid of 0.49mL various concentration is dispensed three with sterilizing liquid-transfering gun and is contained In 4.41mL and in the sterile test tube of agent solution, 3000r/min shaking test tube 30s simultaneously starts to clock, effect lasts to be neutralized After 10min, using its mixing liquid as the recovered liquid of each concentration tests sample liquid, then, with Cha Shi culture solution alternate test sample liquid weight Multiple above-mentioned test；

Test is repeated 5 times；

Then, calf serum is added in bacteria suspension, makes its final serum content 10% and ensures to be inoculated with bacterium solution C_BFor 5.0 × 10⁶CFU/mL~9.0 × 10⁶CFU/mL is repeated above-mentioned quantitative suspension test 5 times；Determine testing liquid disinfectant sample organic Under the conditions of object is existing, has effective concentration and the time of Disinfection Effect to fungi.

8. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that described returns Liquid phase is received 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 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 evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that described returns Receive liquid viable bacteria content C_BAnd T_BIt calculates and killing rate R is calculated with sterilizing Index A, carry out in the steps below:

(1) recovered liquid viable bacteria content C_BAnd T_BIt calculates

According to instruction strain mark song lgC_B-lgI_BLinear equation Y=a_BX+b_B, calculate the test sample liquid of each various concentration and right In the same old way liquid go out through four sections/after the microbiological contamination time, the viable bacteria content T of recovered liquid_BAnd C_B, relevant calculation is shown in formula (2)~(9):

In formula:

- the viable bacteria that the test sample liquid of various concentration recycles after four sections of sterilization times every time Amount, unit are every milliliter of Colony Forming Unit (CFU/mL)；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

- every time various concentration test sample liquid after four sections of sterilization times, recovered liquid Relative intensity of fluorescence measured value, RLU；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

- the viable bacteria that the control sample liquid of various concentration recycles after four microbiological contamination time every time Amount, unit are every milliliter of Colony Forming Unit (CFU/mL)；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

- every time various concentration control sample liquid after four microbiological contamination time, recovered liquid Relative intensity of fluorescence measured value, RLU；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

(2) killing rate R is calculated

Under conditions of neutralizer qualification result is qualified, the test sample liquid of each various concentration is in four sections of sterilization times to fungi Killing rateAnd the testing liquid disinfectant sample of respective concentration is in same sterilization time Interior killing rateIt is calculated respectively according to formula (10)~(17):

In formula:

- every time various concentration test sample liquid fungi in four sections of sterilization times killing Rate, %；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

The testing liquid disinfectant sample of-respective concentration is in four sections of sterilization times to fungi Killing rate, %；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

a_B- standard curve lgC_B-lgI_BSlope；

(3) sterilizing Index A calculates

Under conditions of neutralizer qualification result is qualified, the test sample liquid of each various concentration is in four sections of sterilization times to fungi Sterilizing indexAnd the testing liquid disinfectant sample of respective concentration is in same sterilizing Sterilizing index in timeIt is calculated respectively according to formula (18)~(25):

In formula:

Sterilizing of the test sample liquid in four sections of sterilization times to fungi of-various concentration every time Index；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

The testing liquid disinfectant sample of-respective concentration is in four sections of sterilization times to fungi Sterilizing index；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；

a_B- standard curve lgC_B-lgI_BSlope；

(4) bacteria suspension viable bacteria content C data revision of the convention requirement: is demarcated using blood counting chamber_BWhen, with reference in GB4789.2-2016 Relevant regulations work as C_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BWhen not less than 100CFU/mL, the 3rd bit digital Preceding 2 bit digital is taken after " rounding up ", behind with 0 replace digit；It can also be indicated with 10 exponential form, be adopted after " rounding up " With two effective digitals, test sample liquid and control sample liquid go out through four sections/after the microbiological contamination time, the relative intensity of fluorescence of recovered liquid measures It is worth round numbers, three effective digitals is taken to the sterilizing rate calculated result of fungi, sterilizing index calculated result takes two effective digitals；

(5) uncertainty of measurement: this patent method is by calculating in 5 quantitative suspension tests the test sample liquid of various concentration and right In the same old way liquid through four sections it is different go out the/microbiological contamination time, 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measured values of recovered liquid Coefficient of variation CV=σ ÷ μ × 100% (μ, σ and CV calculated result remain into 2 significant digits)；Judge ATP biology Fluorescence analysis is applied to the reproducibility of liquid disinfectant fungi killing effect test, it is specified that coefficient of variation CV≤10%, phase It closes to calculate and sees formula (26)~(33):

In formula:

- through t₁、t₂、t₃、t₄After four sections of different sterilization times, same concentration in 5 tests 15 ATP fluorometric investigation Duplicate Samples of test sample liquid relative intensity of fluorescence measured value Arithmetic mean of instantaneous value；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2,3；Duplicate Samples number k=1,2,3；

- through t₁、t₂、t₃、t₄It is same in 5 tests after four sections of different sterilization times 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measured values of recovered liquid of the test sample liquid of concentrationStandard deviation；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；Duplicate Samples number k=1,2,3；

- through t₁、t₂、t₃、t₄It is same in 5 tests after four different microbiological contamination times The relative intensity of fluorescence measured value of 15 ATP fluorometric investigation Duplicate Samples of control sample liquid of concentrationArithmetic mean of instantaneous value；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1, 2,3；Duplicate Samples number k=1,2,3；

- through t₁、t₂、t₃、t₄It is same in 5 tests after four different microbiological contamination times 15 ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence measured values of recovered liquid of the control sample liquid of concentrationStandard deviation；Test number (TN) i=1,2,3,4,5；Concentration serial number j=1,2, 3；Duplicate Samples number k=1,2,3.

10. the evaluation method of liquid disinfectant fungi killing effect according to claim 1, which is characterized in that described Result judgement carries out in the steps below:

With reference to health industry common practice and related Disinfection Effect biological assessment standard, if certain concentration testing liquid disinfectant Sample liquid quantifies in suspension test in single, through ATP bioluminescence lgC_B─lgI_BIn the specific sterilization time that calibration curve method measures Fungi killing rate >=99.9% or sterilizing index >=3.0 then determine that its Disinfection Effect in this test is qualified, when the concentration waits for When surveying Disinfection Effect qualification of the liquid disinfectant sample liquid in 5 quantitative suspension tests in identical sterilization time, it can determine Its minimum concentration and shortest time to fungi with Disinfection Effect is (in practical applications with the minimum dense of organic matter protection test Degree and shortest time are concentration and time needed for testing liquid disinfectant sample reaches practical Disinfection Effect).