CN110272974A

CN110272974A - ATP bioluminescence lgCB-lgIBThe method of calibration curve method evaluation chemical product killing bacteria effect

Info

Publication number: CN110272974A
Application number: CN201910200142.5A
Authority: CN
Inventors: 李文杰; 宿雅彬; 洪伟; 靳慧达; 连素梅
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-09-24

Abstract

The present invention relates to a kind of evaluation method of chemical product killing bacteria effect, detecting step includes: sample extraction and pre-treatment requirement；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and bacteria suspension preparation；OD value-viable bacteria content logarithm lgC_BMark Qu Jianli and inoculation bacterium solution C_BCalibration；lgC_BRelative intensity of fluorescence logarithm lgI_BMark Qu Jianli；Neutralizer identification and sterilization test；Recovered liquid I_BMeasurement and its viable bacteria content C_BAnd T_BIt calculates；Sterilizing rate R and sterilizing Index A calculate；Evaluation of result；Its special feature is that: accurate quantitative assessment is carried out to the chemical product killing bacteria effect with R or A characterization using ATP fluophotometer.Present invention provide that neutralizing the control sample and test specimen after recycling microbiological contamination specific time, recovered liquid I is measured_BAnd with lgI_BCharacterization and calculating R or A；Evaluation of result foundation is provided.The ATP bioluminescence lgC for the chemical product killing bacteria effect assessment that the present invention researches and develops_B‑lgI_BBent method is marked, will be promoted with the advanced detection technique support product quality of science.

Description

ATP bioluminescence lgCB-lgIBCalibration curve method evaluates chemical product killing bacteria effect Method

Technical field

The present invention relates to the killing bacteria effect evaluation method of chemical product, specifically a kind of application ATP fluophotometer pair The ATP bioluminescence of accurate quantitative test is carried out with the chemical product killing bacteria effect of sterilizing rate R or sterilizing Index A characterization lgC_A-lgI_ACalibration curve method belongs to chemical product Evaluation of Germicidal Efficacy technical field.

Background technique

In recent years, along with people's living standard improve and consumption idea change, domestic market to wash, wash shield etc. daily use chemicals Articles demand cumulative year after year；According to the publication of Zhong Shang industrial research institute " 2018-2023 is Chinese disappear fastly conduct industry development prospect and Analysis of Investment Opportunities report " display, the sustainable growth state higher than GDP speedup is integrally presented in China's chemical product consumption market at present Gesture.Cut-off 2017, domestic daily use chemicals industry size are 323,300,000,000 yuan, it is contemplated that 2018 will be up to 345,700,000,000 yuan；It is led by consumption demand Draw, has the compound average growth rate in middle and high end daily chemical products market year of antibiotic and sterilizing function 10% or more, it is contemplated that the year two thousand twenty Market scale nearly 100,000,000,000.As the segmented industry closely bound up with the people's livelihood, Chinese daily chemical product industry has entered competition and has swashed The strong transitional period；Related quality inspection technology and detection efficiency are gradually brought into schedule.

Currently, antibiotic and sterilizing effect assessment technical system is mainly for bacterium and fungi, various countries' killing bacteria effect both at home and abroad Fruit test method principle is mostly based on the colony counting method being separately cultured to bacterium, first is that the dipping that Japanese Industrial Standards propose Quantitative test method；Two are derived from the antibacterial around-France of U.S. textile industry；Third is that international oscillation flask method；Four are derived from beauty The dropping method of textile enterprise, state.Although China issued and implemented GB 15981-1995 " disinfection and the evaluation method of sterilization effect with Standard ", GB 15979-2002 " Disposable Sanitary Accessory sanitary standard " and QB/T 2738-2012 " daily chemical products antibacterial The evaluation method of fungistatic effect " etc. relevant criterions, but there are following total in technology contents and practical application for existing detection method Property problem: first is that involved experimental strain is very few, it is difficult to meet research and development of new product that enterprise grows with each passing hour and Quality Control demand；Second is that experiment Process is cumbersome, and relevant operation is influenced by laboratory technician's professional experiences, and test error is big, lacks comparativity；Third is that test period is 48 Hour~72 hours between, time and economic cost are high.In recent years, in international Bacteria Detection technical field ATP fluorescence analysis Develop increasingly mature, the correlation of ATP testing result is 98% compared with traditional Plating, and accuracy is high and can realize quick inspection It surveys；Developed country applies to the method in HACCP.Domestic ATP correlative study is started late, and is drawn by application demand, existing ATP fluorescence detector has become the specified health supervision of China Health department and the relevant special inspecting equipment of food safety.When It is quasi- to focus on testing result to quantification, rapid and summary trend development for preceding foreign countries' antibiotic and sterilizing effect detection technical research True property and comparativity；It has used for reference ATP fluorescence analysis principle and has formulated ISO 20743:2007-2013 " Textiles- Determination of antibacterial activity of textile products (spin by textile-antibiotic finish The antibacterium performance measurement of fabric) " and ISO 13629-1:2012 " Textiles-Determination of antifungal Activity of textile products.Part1 Luminescence (textile-antibiotic finish textile mildew resistance Can measurement) ", it is specified that with the fluorimetry of anti-thin/mould performance of ATP changes of contents characterization after sample inoculation, but it is 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；Chemical product killing bacteria recruitment evaluation then must be for key technologies sides such as neutralization evaluation, result calculation formula Face forms a whole set of clear and specific method, and needs to provide measurement of correlation uncertainty evaluation.In addition, the standard method is killed Bacterium Characterization result parameter is more single, only relates to antibacterial activity value A, and the usual killing rate R in China.

Therefore, to resist foreign technology barrier, specification domestic market order pushes industrial transformation upgrading, it would be highly desirable to research section It emulates the advanced, accuracy and reproducibility are high, easy-to-use daily chemical products killing bacteria measure of merit technology.The art of this patent route The world that integrates with is designed, detection method belongs to the whole world and initiates, and can 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 sterilizing rate R or sterilizing The ATP bioluminescence lgC that the chemical product killing bacteria effect of number A characterization is evaluated_B-lgI_BCalibration curve method is able to solve day Change product or even the accurate quantitative test problem of other field antimicrobial product killing bacteria effect.

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

A kind of evaluation method of chemical product killing bacteria effect, comprising: (1) sample extraction and pre-treatment requirement；(2) equipment Type selecting and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) OD value-viable bacteria content logarithm lgC_B Standard curve is established 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 sterilization test；(7) reclaim liquid phase is to fluorescence intensity level I_BMeasurement；(8) recovered liquid viable bacteria Content C_BAnd T_BIt calculates and sterilizing rate R is calculated with sterilizing Index A；(9) evaluation of result；It is characterized in that, using ATP fluorescence light The ATP biology that degree meter carries out accurate quantitative assessment to the chemical product killing bacteria effect with sterilizing rate R or sterilizing Index A characterization is glimmering Light lgC_B-lgI_BCalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_BIn measurement:

The requirement such as extraction and pre-treatment of control sample and test specimen is specified, testing standard bacterial strain is passed on, is lived After change, continuous switching 2 times Fresh bacterial cultures is taken to prepare bacteria suspension.Count plate is carried out using MTT colorimetric analysis, is built Vertical OD value-viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And related coefficient R₀ ²；And viable bacteria content C is carried out to inoculation bacterium solution_BCalibration: Then, C is selected_B It is 1.2 × 10³CFU/mL、1.2×10⁴CFU/mL、1.2×10⁵The dilution bacterium solution of CFU/mL is as standard series bacteria suspension；It surveys Fixed its relative intensity of fluorescence value I_B, draw lgC_B-lgI_BStandard curve, and it is derived from the linear equation Y=a of curve_BX+b_B And coefficient R_B ².Under conditions of neutralizer qualification result is qualified, divide into the control sample and test specimen of each group 4.8mL It 0.2mL bacterium solution and Jia Ru not mix；After effect lasts to stipulated time to be sterilized, in 4.5mL and agent solution to 0.5mL each group Microbiological contamination sample liquid carries out neutralization recycling, using the relative intensity of fluorescence value I of ATP fluorescent spectrophotometer measuring recovered liquid_BCijAnd I_BTij, and According to fitting equation Y=a_BX+b_BCalculate its viable bacteria content C_BijAnd T_Bij；

In sterilizing rate R and sterilizing Index A are calculated:

According to standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, with every control sample in specific sterilizing time The relative intensity of fluorescence measured value of product and test specimen recovered liquidWithAs basic data；Calculate every daily use chemicals sample Sterilizing rate R_ijAnd sterilizing Index A_ij, and with every group of sample R_ijAnd A_ijArithmetic mean of instantaneous value be its sterilizing rate R_iWith sterilizing Index A_i； To the R of 3 groups of samples_iOr A_iArithmetic mean of instantaneous value is taken, the sterilizing rate R or sterilizing Index A of every batch of daily use chemicals sample are obtained；Clear phase simultaneously Close the data revision of the convention and uncertainty of measurement requirement；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that chemical product killing bacteria effect is classified Criterion；As the sterilizing rate R of certain group (part) daily use chemicals sample_i(R_ij) or sterilizing Index A_i(A_ij) and other two groups of (part) samples When killing bacteria effect is compared to a levels are at least differed, one group of sample is extracted again and repeats to test；It is calculated through ATP biology Fluorescence lgC_B─lgI_BThe sterilizing rate or sterilizing index that calibration curve method measures.If the bacterium of two groups of (part) the daily use chemicals samples in front and back Killing effect level is identical, then abandons it；Take other two groups of (part) remaining sample sterilizing rates R_i(R_ij) or sterilizing Index A_i(A_ij) Evaluation result of the arithmetic mean of instantaneous value as batch (group) the daily use chemicals sample bacterium killing 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 and microplate reader as test equipment, to reach The modernization of viable bacteria content measurement and chemical product killing bacteria effect assessment technology；It can effectively reduce human factor in experimentation It influences, avoids the generally existing large error of traditional plate culture；It can ensure the quantification of testing result, while substantially Degree improves the accuracy of detection data；Detection technique has certain advance.

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

(3) innovative: compared with the conventional method that existing operation is numerous, error is big, the period is long, this patent method was being tested Automation and the higher ATP fluophotometer of intelligent level are introduced in journey, are greatly simplified experimental procedure, are realized daily use chemicals The precision of product killing bacteria effect detection result and quantification simultaneously have good reproducibility and comparativity；Substantially shorten simultaneously Test period reduces testing cost；Presently relevant the field of test technology blank can effectively be filled up.

(4) perspective: to establish with lgC_B、lgI_BStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Bacteria suspension viable bacteria content measuring method is enriched, control sample is specified, standard liquid concentration, determination step, calculation formula, does not know The technology contents such as degree；The pioneering recycling viable bacteria relative intensity of fluorescence value I directly measured with instrument_BEvaluate form as a result to count It calculates and determines sterilizing rate R or sterilizing Index A, and uncertainty of measurement is investigated by a group interior and between-group variation coefficient CV, in skill There is centainly perspective in art.

(5) operability: ATP fluophotometer and microplate reader it is cheap, it is easy to operate, be widely used, this patent is built Method is simple for the ATP fluorometric investigation of vertical viable bacteria content MTT colorimetric analysis and chemical product killing bacteria effect assessment, phase It is clear and specific that pass technology illustrates, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for difference Professional standards Experiment on Microbiology personnel may advantageously facilitate achievement transfer conversion and promote and apply.

(6) universality: because ATP is prevalent in, life entity is intracellular, and patented method can expand for experimental strain and provide tool The detection technique support for thering is wide spectrum to be worth；The introducing of pertinent instruments can greatly simplify experimental procedure, reduce testing cost；Be conducive to Expand inspection, learn, grind, produces all circles promote and apply, can support chemical product killing bacteria effect assessment technology realization generalization, together When can to the fields such as disinfectant, amenities sterilization effect assessment technique study provide reference.

Further, preferred embodiment of the invention is:

The sample extraction and pre-treatment requirement, carry out in the steps below:

(1) control sample: using test strain culture solution as control sample, dosage, quantity and standard hard water dilution behaviour Work etc. is identical as sample to be tested；

(2) test specimen: the daily chemical products such as articles for washing, skin-cleaning articles with special hygiene efficacy；Each strain makes 3 groups of sterilization test samples are derived from the different transportation and packing of same product batch number, and every group of sample is derived from same transportation and packing 3 minimum marketing packings (sample size is no less than 10g)；Single neutralizer qualification test need to be in 3 same transportation and packing Minimum marketing packing sample (sample size is no less than 10g)；3 times neutralization test sample used is taken respectively from same product batch number not Same transportation and packing.Test before with Sterile standard hard water by control sample and test specimen be diluted to respectively product description regulation it is dense 2 times of degree, the soak type that do not express and smear type fruits and vegetables, tableware washing articles activity are respectively 1:100 and 1:5；It washes The dry goods washing and nursing article activities such as clothing liquid, washing powder, fabric softener, drift stain liquid, carpet cleaner are 1:100； Direct spray-type hard-surface cleaning articles use stoste, and concentrated type sample effect concentration is 1:100.Every group of test specimen selection one Group control sample as object of reference and effectively identifies；

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: two stage biological safety cabinet or the superclean bench of lustration class >=100；Fluorophotometric containing ATP The total number of bacteria ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc., wherein ATP fluophotometer wave Long range 300nm~650nm, total number of bacteria detection range 10¹CFU/mL~10⁷CFU/mL；Wave-length coverage 400nm~760nm, The microplate reader of range of readings (0.0~4.0) Abs；The constant incubator of (37 ± 1) DEG C；The water bath with thermostatic control that (10~50) DEG C ± 1 DEG C Case；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C of low temperature refrigerator；2 DEG C~8 DEG C cold Hide case；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency (30~50) kHz；Revolving speed (300~3000) r/min Vortex oscillator；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: the sterile measuring pipette of 1mL, 10mL；0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (meter Error is measured less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；Filter sizes are not more than 0.45 μm of needle-based Filter；The sterile conical flask of capacity 150mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；96 hole flat-bottomed plates； Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Rack for test tube；Diameter is not more than the oese of 4mm；Alcolhol burner；Point The ruler of angle value 1mm； Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core Black gel ink pen；

(4) reagent: 5mg/mL, pH value 7.4 MTT (tetramethyl azo azoles salt) solution (0.45 μm of membrane filtration degerming, 4 DEG C~6 DEG C 15d is kept in dark place)；121 DEG C of high pressure sterilization 30min of following reagent, the physiology salt of 5 DEG C~10 DEG C storage 30d:85% Water；0.034g calcium chloride, 0.139g magnesium chloride are dissolved in 1000mL water, the standard hard water of hardness 342mg/L is configured to；By 5g Sodium thiosulfate is dissolved in 1000mL water (for the sample of fungicide containing chlorine type)；By 1.36g potassium dihydrogen phosphate, 2.83g phosphoric acid hydrogen two Sodium, 10g lecithin, 10g glycine, 30g tween (80) are dissolved in 1000mL water or by 1.36g potassium dihydrogen phosphates, 2.83g phosphoric acid hydrogen Disodium, 3g lecithin, 20g tween (80) are dissolved in 1000mL water (for sample containing non-oxidative bactericide)；By 20g tween (80), 1g sodium thiosulfate is dissolved in 1000mL phosphate buffer solution (for oxygen-containing type fungicide sample) etc. (or for be measured Fungicide type contained by daily use chemicals sample, the neutralizer for selecting other to be mutually applicable in)；

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

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

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

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

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

Plate count agar (PCA): 5g tryptone, 2.5g yeast extract, 1g glucose, 15g agar are dissolved by heating In 1000mL water, pH to 7.0 ± 0.2 is adjusted；

(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 nutrient broth can be down to 10 in 15min by liquid^-13Mol/L or less)；

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

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

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

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

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

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

(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 9.0 × 10⁸CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity)；

The OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculation bacterium solution C_BCalibration, in the steps below into Row:

(1) bacterium solution OD value measures: being 9.0 × 10 to bacteria total amount with test strain culture solution⁸The bacteria suspension of CFU/mL into Row 1:5,1:10,1:15,1:20 are serially diluted, and using culture solution as blank sample liquid, carry out zeroing correction to microplate reader.So Afterwards, the bacterium solution of the above-mentioned different dilutions of 100 μ L is injected separately into 96 hole flat-bottomed plates, each dilution does 3 multiple holes；To every The MTT solution of 20 μ L is added dropwise in hole, after (37 ± 1) DEG C cultivate 30min；Absorption maximum is scanned in 560nm~610nm wave-length coverage Peak determines maximum absorption wavelength；Viable bacteria OD value in each hole mixed solution is measured, the bacteria suspension viable bacteria OD value of different dilutions is it The arithmetic mean of instantaneous value of 3 multiple holes OD measured values.It is outstanding to above-mentioned bacterium meanwhile according to method as defined in national standard GB 4789.2-2016 Liquid carries out after suitably diluting, and cultivates ± 2h for 24 hours in (37 ± 1) DEG C, and carry out bacterium colony counting to plate, demarcates its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculation bacterium solution C_BCalibration: with different dilution bacterium The viable bacteria OD value of suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard curve； The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Wherein large intestine angstrom is uncommon Salmonella (or Shigella) and staphylococcus aureus (or salmonella) choose respectively dilution be 1:10,1:15,1:20 and The bacteria suspension of 1:5,1:10,1:15 draw curve as standard serial solution, work as R₀ ²>=0.98, it when confidence level >=0.95, presses The bacteria suspension viable bacteria content C measured according to this patent method_BEffectively.Then, it is adjusted through test strain culture solution, obtains C_BRange is 1.0×10⁷CFU/mL~5.0 × 10⁷The 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:

(1) determination of standard series bacteria suspension and its I_BMeasurement: use test strain culture solution by known viable bacteria content C_BBacterium solution Gradient dilution is standard series bacteria suspension: 1.2 × 10³CFU/mL、1.2×10⁴CFU/mL、1.2×10⁵CFU/mL is simultaneously mixed.So Afterwards, according to relative intensity of fluorescence value I in this patent_BMeasuring method, according to viable bacteria content C_BSequence from low to high measures and remembers Record the relative intensity of fluorescence value of above-mentioned standard series bacteria suspension；

(2) viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established: with standard series bacterium The relative intensity of fluorescence logarithm lgI of suspension_BAs abscissa, with its corresponding viable bacteria content logarithm lgC_BFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, and is derived from the linear side of curve using least square fitting method Formula Y=a_BX+b_BAnd coefficient R_B ²；Work as R_B ²>=0.98, when confidence level >=0.95, the survey done according to this patent method It is fixed effective；

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

(1) neutralizer qualification test: first mixing 1mL test specimen with 9mL with agent solution, and effect 10min is formed And reaction mixture；And test is grouped as follows: with sterilizing liquid-transfering gun by 0.2mL inoculation bacterium solution (with lgC_B-lgI_BCalibration curve is used Bacterium solution is derived from same branch test strain stoste test tube, and 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used) six test samples containing 4.8mL of packing Product (1^#、2^#), in 4.8mL and agent solution (3^#、7^#), 4.8mL neutralized reaction product solution (4^#), 4.8mL test strain culture solution (5^#) Sterile test tube in, 1000r/min shake test tube 10min, mix well each group solution.It then, will with sterilizing liquid-transfering gun 0.5mL each group mixing liquid dispenses six culture solutions of test strain containing 4.5mL (1^#、5^#、7^#), in 4.5mL and agent solution (2^#、3^#)、 4.5mL neutralized reaction product solution (4^#) sterile test tube in, and 2.5mL test strain culture solution is added with 2.5mL with agent solution Enter in same branch sterile test tube, as the 6th^#Group test sample liquid.By its mixing liquid after 1000r/min shaking each group test tube 10min As recovered liquid, according to relative intensity of fluorescence value I in this patent_BMeasuring method measures and records the opposite of above-mentioned 7 groups of recovered liquids Fluorescence intensity level；Simultaneously by the 7th^#The measured value of group recovered liquid is denoted as I_B0。

(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^#Group and the 7th^#The relative intensity of fluorescence measured value of group recovered liquid is close, i.e. I_B3≈I_B4≈I_B5≈I_B0, according to formula (1) the viable bacteria content C being calculated_B3、C_B4、C_B5Error rate Δ≤10% between group；Illustrate that this test neutralization evaluation is closed Lattice.

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 CFU/mL；

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

Above-mentioned neutralizer qualification test is repeated, if the continuous evaluation of neutralization three times is qualified, selected neutralizer Type and concentration can be used for the killing bacteria effect test of daily use chemicals sample to be measured；

(3) sterilization test: being dispensed every group of 4.8mL control sample and test specimen in sterile test tube with sterilizing liquid-transfering gun, In (20 ± 1) DEG C water bath with thermostatic control after (30 DEG C of soaps sample) heat preservation (5 ± 0.5) min, 0.2mL is added dropwise into test tube and is inoculated with bacterium Liquid mixes rapidly and starts timing according to the action time that product description marks.The hard-surface cleaning articles of time are not expressed (direct spray-type and concentrated type) acts on 5min；Soak type and smear type fruits and vegetables, tableware washing articles act on respectively 15min and 5min；The dry goods washing and nursing article such as liquid detergent, washing powder, fabric softener, carpet cleaner acts on 20min (drift stain liquid Act on 5min).After effect lasts to stipulated time to be sterilized, 0.5mL each group microbiological contamination mixing liquid is pipetted with sterilizing liquid-transfering gun, is dispensed Containing 4.5mL sterilize neutralize agent solution sterile test tube in, 1000r/min shake test tube 10min, sufficiently neutralize after as to The recovered liquid of sample；

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

(2) reclaim liquid phase is to fluorescence intensity level I_BMeasurement: if background level meets instrument requirement, then with sterilizing liquid relief The ATP lysate of disodium hydrogen phosphate buffer solution and 0.1mL of the rifle by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose is successively It is added in same branch sterile test tube, moves to 0.1mL mixed solution in three instrument sterile test tubes respectively after mixing well, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts examination Agent instills the ATP fluorescent reagent of 0.1mL after mixing；It mixes again, it is strong with its relative fluorescence of ATP fluorescent spectrophotometer measuring immediately Angle value I_BAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of sample to be tested recovered liquid_BIt surveys Definite value (or instrument matched reagent box is used, according to specification used in connection with requirement, directly upper machine measures three Duplicate Samples of recovered liquid Relative intensity of fluorescence value I_B)；

The recovered liquid viable bacteria content C_BAnd T_BCalculate and sterilizing rate R or sterilizing Index A calculate, in the steps below into Row:

(1) recovered liquid viable bacteria content C_BAnd T_BIt calculates

According to test strain standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, calculate control sample and test Sample is after dye/sterilizing specific time, the viable bacteria content C of recovered liquid_BAnd T_B.Relevant calculation (when using instrument matched reagent box, The viable bacteria content C of 1mL recovered liquid is calculated according to its practical sample volume_BAnd T_B) see formula (2)~(7):

In formula:

C_BThe mean viable amount recycled after -3 groups of control sample microbiological contamination specific times, unit are the every milli of Colony Forming Unit It rises (CFU/mL)；

C_BiThe mean viable amount recycled after-every group control sample microbiological contamination specific time, unit are the every milli of Colony Forming Unit It rises (CFU/mL)；Sample group i=1,2,3；

C_BijThe viable bacteria amount recycled after-every control sample microbiological contamination specific time, unit are every milliliter of Colony Forming Unit (CFU/mL)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；

After-every control sample microbiological contamination specific time, the relative intensity of fluorescence measured value of recovered liquid；Sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3；

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

T_BThe mean viable amount recycled after -3 groups of test specimen sterilization specific times, unit is the every milli of Colony Forming Unit It rises (CFU/mL)；

T_BiThe mean viable amount recycled after-every group test specimen sterilization specific time, unit is the every milli of Colony Forming Unit It rises (CFU/mL)；Sample group i=1,2,3；

T_BijThe viable bacteria amount recycled after-every test specimen sterilization specific time, unit is every milliliter of Colony Forming Unit (CFU/mL)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；

After-every test specimen sterilization specific time, the relative intensity of fluorescence measured value of recovered liquid；Sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3；

(2) sterilizing rate R is calculated

Under conditions of neutralizer neutralization evaluates qualified, the sterilizing rate R of every daily use chemicals sample_ij, every group and every lot sample The sterilizing rate R of product_iIt is calculated respectively according to formula (8), (9), (10) with R:

In formula:

R_ijThe sterilizing rate of-every daily use chemicals sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；

R_iThe sterilizing rate of-every group daily use chemicals sample, %；Sample group i=1,2,3；

R-every batch of daily use chemicals sample sterilizing rate, %；

a_B- standard curve lgC_B-lgI_BSlope；

(3) sterilizing Index A calculates

Under conditions of neutralizer neutralization evaluates qualified, the sterilizing Index A of every daily use chemicals sample_ij, every group and every batch of The sterilizing Index A of sample_iIt is calculated respectively according to formula (11), (12), (13) with A:

In formula:

A_ijThe sterilizing index of-every daily use chemicals sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；

A_iThe sterilizing index of-every group daily use chemicals sample, wherein sample group i=1,2,3；

A-every batch of daily use chemicals sample sterilizing index；

a_B- standard curve lgC_B-lgI_BSlope；

(4) bacteria suspension viable bacteria content C data revision of the convention requirement: is demarcated using colony counting method_BWhen, with reference to 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 Take preceding 2 bit digital after digital " rounding up ", behind with 0 instead of digit；It can also be indicated with 10 exponential form, " rounding up " Two effective digitals are used afterwards.Control sample and test specimen are after dye/sterilizing specific time, the relative intensity of fluorescence of recovered liquid Measured value round numbers, sterilizing rate R_ij、R_i, R calculated result take three effective digitals, sterilize Index A_ij、A_i, A calculated result take two Position effective digital；

(5) uncertainty of measurement: this patent method is by calculating control sample and test specimen through dye/sterilizing specific time Afterwards, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV count Calculate result and remain into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to chemical product killing bacteria measure of merit Reproducibility；In regulation group and between-group variation coefficient CV≤10%.Relevant calculation is shown in formula (14)~(16):

In formula:

μ_CiAfter-every group 3 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueArithmetic Average value；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

μ_TiAfter-every group 3 test specimens sterilization specific time, reclaim liquid phase is to fluorescent strength determining valueArithmetic Average value；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

μ_CAfter -3 groups of 9 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueArithmetic it is flat Mean value；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

μ_TAfter -3 groups of 9 test specimens sterilization specific times, reclaim liquid phase is to fluorescent strength determining valueArithmetic it is flat Mean value；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_CiAfter-every group 3 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueStandard Deviation；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_TiAfter-every group 3 test specimens sterilization specific time, reclaim liquid phase is to fluorescent strength determining valueStandard Deviation；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_CAfter -3 groups of 9 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueStandard deviation Difference；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_TAfter -3 groups of 9 test specimens sterilization specific times, reclaim liquid phase is to fluorescent strength determining valueStandard deviation Difference；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

The evaluation of result of the daily use chemicals sample bacterium killing effect carries out in the steps below:

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

If using sterilizing rate R as correlated performance evaluation index: working as R_ij/R_i(staphylococcus aureus when/R < 90% With salmonella R_ij/R_i/ R < 80%), sample is without Bactericidal Effect；As 90%≤R_ij/R_i(golden yellow Portugal when/R < 99% 80%≤R of grape coccus and salmonella_ij/R_i/ R < 90%), sample has Bactericidal Effect；As 99%≤R_ij/R_i/ R < (90%≤R of staphylococcus aureus and salmonella when 99.9%_ij/R_i/ R < 99%), sample bacterium killing effect is stronger；When R_ij/R_i(staphylococcus aureus and salmonella R when/R >=99.9%_ij/R_i/ R >=99%), sample bacterium killing effect pole By force；

If using sterilizing Index A as correlated performance evaluation index: working as A_ij/A_i(staphylococcus aureus when/A < 1.0 With salmonella A_ij/A_i/ A < 0.5), sample is without Bactericidal Effect；As 1.0≤A_ij/A_i(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonella_ij/A_i/ A < 1.0), sample has Bactericidal Effect；As 2.0≤A_ij/A_i(gold when/A < 3.0 1.0≤A of staphylococcus aureus and salmonella_ij/A_i/ A < 2.0), sample bacterium killing effect is stronger；Work as A_ij/A_i/A≥3.0 When (staphylococcus aureus and salmonella A_ij/A_i/ A >=2.0), sample bacterium killing effect is extremely strong；

(2) as the sterilizing rate R of certain group (part) daily use chemicals sample_i(R_ij) or sterilizing Index A_i(A_ij) and other two groups of (part) samples Killing bacteria effect compared to a levels are at least differed when, extract one group of sample again and repeat to test；It is raw through ATP to calculate it Object fluorescence lgC_B─lgI_BThe sterilizing rate or sterilizing index that calibration curve method measures.If two groups of front and back (part) daily use chemicals sample is thin Bacterium killing effect level is identical, then abandons it；Take other two groups of (part) remaining sample sterilizing rates R_i(R_ij) or sterilizing Index A_i(A_ij) Arithmetic mean of instantaneous value as batch (group) daily use chemicals sample bacterium killing effect evaluation result；

Specific embodiment

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

The present embodiment is commented using the potent drift stain liquid sample bacterium killing effect being process by the spirit of trichlorine card as antibacterial agent It is illustrated for valence.

Specific detection method carries out in the steps below:

(1) sample extraction and pre-treatment requirement

1.1 control samples: using test strain culture solution as control sample, dosage, quantity and standard hard water dilution behaviour Work etc. is identical as sample to be tested.

1.2 test specimens: the daily chemical products such as articles for washing, skin-cleaning articles with special hygiene efficacy；Each strain makes 3 groups of sterilization test samples are derived from the different transportation and packing of same product batch number, and every group of sample is derived from same transportation and packing 3 minimum marketing packings (sample size is no less than 10g)；Single neutralizer qualification test need to be in 3 same transportation and packing Minimum marketing packing sample (sample size is no less than 10g)；3 times neutralization test sample used is taken respectively from same product batch number not Same transportation and packing.Test before with Sterile standard hard water by control sample and test specimen be diluted to respectively product description regulation it is dense 2 times of degree, the soak type that do not express and smear type fruits and vegetables, tableware washing articles activity are respectively 1:100 and 1:5；It washes The dry goods washing and nursing article activities such as clothing liquid, washing powder, fabric softener, drift stain liquid, carpet cleaner are 1:100； Direct spray-type hard-surface cleaning articles use stoste, and concentrated type sample effect concentration is 1:100.Every group of test specimen selection one Group control sample as object of reference and effectively identifies.

(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: two stage biological safety cabinet or the superclean bench of lustration class >=100；Fluorophotometric containing ATP The total number of bacteria ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc., wherein ATP fluophotometer wave Long range 300nm~650nm, total number of bacteria detection range 10¹CFU/mL~10⁷CFU/mL；Wave-length coverage 400nm~760nm, The microplate reader of range of readings (0.0~4.0) Abs；The constant incubator of (37 ± 1) DEG C；The water bath with thermostatic control that (10~50) DEG C ± 1 DEG C Case；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C of low temperature refrigerator；2 DEG C~8 DEG C cold Hide case；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency (30~50) kHz；Revolving speed (300~3000) r/min Vortex oscillator；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace.

2.3 material utensils: the sterile measuring pipette of 1mL, 10mL；0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (meter Error is measured less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；Filter sizes are not more than 0.45 μm of needle-based Filter；The sterile conical flask of capacity 150mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；96 hole flat-bottomed plates； Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Rack for test tube；Diameter is not more than the oese of 4mm；Alcolhol burner；Point The ruler of angle value 1mm； Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core Black gel ink pen.

2.4 reagents: 5mg/mL, pH value 7.4 MTT (tetramethyl azo azoles salt) solution (0.45 μm of membrane filtration degerming, 4 DEG C~6 DEG C 15d is kept in dark place)；121 DEG C of high pressure sterilization 30min of following reagent, the physiology salt of 5 DEG C~10 DEG C storage 30d:85% Water；0.034g calcium chloride, 0.139g magnesium chloride are dissolved in 1000mL water, the standard hard water of hardness 342mg/L is configured to；By 5g Sodium thiosulfate is dissolved in 1000mL water (for the sample of fungicide containing chlorine type)；By 1.36g potassium dihydrogen phosphate, 2.83g phosphoric acid hydrogen two Sodium, 10g lecithin, 10g glycine, 30g tween (80) are dissolved in 1000mL water or by 1.36g potassium dihydrogen phosphates, 2.83g phosphoric acid hydrogen Disodium, 3g lecithin, 20g tween (80) are dissolved in 1000mL water (for sample containing non-oxidative bactericide)；By 20g tween (80), 1g sodium thiosulfate is dissolved in 1000mL phosphate buffer solution (for oxygen-containing type fungicide sample) etc. (or for be measured Fungicide type contained by daily use chemicals sample, the neutralizer for selecting other to be mutually applicable in).

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

Plate count agar (PCA): 5g tryptone, 2.5g yeast extract, 1g glucose, 15g agar are dissolved by heating In 1000mL water, pH to 7.0 ± 0.2 is adjusted.

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

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

(3) culture presevation, activation and bacteria suspension preparation

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

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

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

The preparation of 3.4 bacteria suspensions: a small amount of Fresh bacterial is scraped from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 9.0 × 10⁸CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity).

(4) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculation bacterium solution C_BCalibration

The measurement of 4.1 bacterium solution OD values: being 9.0 × 10 to bacteria total amount with test strain culture solution⁸The bacteria suspension of CFU/mL into Row 1:5,1:10,1:15,1:20 are serially diluted, and using culture solution as blank sample liquid, carry out zeroing correction to microplate reader.So Afterwards, the bacterium solution of the above-mentioned different dilutions of 100 μ L is injected separately into 96 hole flat-bottomed plates, each dilution does 3 multiple holes；To every The MTT solution of 20 μ L is added dropwise in hole, after (37 ± 1) DEG C cultivate 30min；Absorption maximum is scanned in 560nm~610nm wave-length coverage Peak determines maximum absorption wavelength；Viable bacteria OD value in each hole mixed solution is measured, the bacteria suspension viable bacteria OD value of different dilutions is it The arithmetic mean of instantaneous value of 3 multiple holes OD measured values.It is outstanding to above-mentioned bacterium meanwhile according to method as defined in national standard GB 4789.2-2016 Liquid carries out after suitably diluting, and cultivates ± 2h for 24 hours in (37 ± 1) DEG C, and carry out bacterium colony counting to plate, demarcates its viable bacteria content C_B。

4.2OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculation bacterium solution C_BCalibration: with different dilution bacterium The viable bacteria OD value of suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard curve； The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Wherein large intestine angstrom is uncommon Salmonella (or Shigella) and staphylococcus aureus (or salmonella) choose respectively dilution be 1:10,1:15,1:20 and The bacteria suspension of 1:5,1:10,1:15 draw curve as standard serial solution, work as R₀ ²>=0.98, it when confidence level >=0.95, presses The bacteria suspension viable bacteria content C measured according to this patent method_BEffectively.Then, it is adjusted through test strain culture solution, obtains C_BRange is 1.0×10⁷CFU/mL~5.0 × 10⁷The inoculation bacterium solution of CFU/mL.

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

5.1 standard series bacteria suspensions determinations and its I_BMeasurement: use test strain culture solution by known viable bacteria content C_BBacterium solution Gradient dilution is standard series bacteria suspension: 1.2 × 10³CFU/mL、1.2×10⁴CFU/mL、1.2×10⁵CFU/mL is simultaneously mixed.So Afterwards, according to relative intensity of fluorescence value I in this patent_BMeasuring method, according to viable bacteria content C_BSequence from low to high measures and remembers Record the relative intensity of fluorescence value of above-mentioned standard series bacteria suspension.

5.2 viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established: with standard series bacterium The relative intensity of fluorescence logarithm lgI of suspension_BAs abscissa, with its corresponding viable bacteria content logarithm lgC_BFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, and is derived from the linear side of curve using least square fitting method Formula Y=a_BX+b_BAnd coefficient R_B ²；Work as R_B ²>=0.98, when confidence level >=0.95, the survey done according to this patent method It is fixed effective.

(6) neutralizer identification and sterilization test

6.1 neutralizer qualification tests: first mixing 1mL test specimen with 9mL with agent solution, and effect 10min is formed And reaction mixture；And test is grouped as follows: with sterilizing liquid-transfering gun by 0.2mL inoculation bacterium solution (with lgC_B-lgI_BCalibration curve is used Bacterium solution is derived from same branch test strain stoste test tube, and 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used) six test samples containing 4.8mL of packing Product (1^#、2^#), in 4.8mL and agent solution (3^#、7^#), 4.8mL neutralized reaction product solution (4^#), 4.8mL test strain culture solution (5^#) Six sterile test tubes in, 1000r/min shake test tube 10min, mix well each group solution.It then, will with sterilizing liquid-transfering gun 0.5mL each group mixing liquid dispenses six culture solutions of test strain containing 4.5mL (1^#、5^#、7^#), in 4.5mL and agent solution (2^#、3^#)、 4.5mL neutralized reaction product solution (4^#) sterile test tube in, and 2.5mL test strain culture solution is added with 2.5mL with agent solution Enter in same branch sterile test tube, as the 6th^#Group test sample liquid.By its mixing liquid after 1000r/min shaking each group test tube 10min As recovered liquid, according to relative intensity of fluorescence value I in this patent_BMeasuring method measures and records the opposite of above-mentioned 7 groups of recovered liquids Fluorescence intensity level；Simultaneously by the 7th^#The measured value of group recovered liquid is denoted as I_B0。

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^#Group and the 7th^#The relative intensity of fluorescence measured value of group recovered liquid is close, i.e. I_B3≈I_B4≈I_B5≈I_B0, according to formula (1) the viable bacteria content C being calculated_B3、C_B4、C_B5Error rate Δ≤10% between group；Illustrate that this test neutralization evaluation is closed Lattice.

In formula:

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

Above-mentioned neutralizer qualification test is repeated, if the continuous evaluation of neutralization three times is qualified, selected neutralizer Type and concentration can be used for the killing bacteria effect test of daily use chemicals sample to be measured.

6.3 sterilization tests: being dispensed every group of 4.8mL control sample and test specimen in sterile test tube with sterilizing liquid-transfering gun, In (20 ± 1) DEG C water bath with thermostatic control after (30 DEG C of soaps sample) heat preservation (5 ± 0.5) min, 0.2mL is added dropwise into test tube and is inoculated with bacterium Liquid mixes rapidly and starts timing according to the action time that product description marks.The hard-surface cleaning articles of time are not expressed (direct spray-type and concentrated type) acts on 5min；Soak type and smear type fruits and vegetables, tableware washing articles act on respectively 15min and 5min；The dry goods washing and nursing article such as liquid detergent, washing powder, fabric softener, carpet cleaner acts on 20min (drift stain liquid Act on 5min).After effect lasts to stipulated time to be sterilized, 0.5mL each group microbiological contamination mixing liquid is pipetted with sterilizing liquid-transfering gun, is dispensed Containing 4.5mL sterilize neutralize agent solution sterile test tube in, 1000r/min shake test tube 10min, sufficiently neutralize after as to The recovered liquid of sample.

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

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

7.2 reclaim liquid phases are to fluorescence intensity level I_BMeasurement: if background level meets instrument requirement, then with sterilizing liquid relief The ATP lysate of disodium hydrogen phosphate buffer solution and 0.1mL of the rifle by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose is successively It is added in same branch sterile test tube, moves to 0.1mL mixed solution in three instrument sterile test tubes respectively after mixing well, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts examination Agent instills the ATP fluorescent reagent of 0.1mL after mixing；It mixes again, it is strong with its relative fluorescence of ATP fluorescent spectrophotometer measuring immediately Angle value I_BAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of sample to be tested recovered liquid_BIt surveys Definite value (or instrument matched reagent box is used, according to specification used in connection with requirement, directly upper machine measures three Duplicate Samples of recovered liquid Relative intensity of fluorescence value I_B)。

(8) recovered liquid viable bacteria content C_BAnd T_BReckoning and sterilizing rate R or sterilizing Index A calculate

8.1 recovered liquid viable bacteria content C_BAnd T_BIt calculates

In formula:

After-every test specimen sterilization specific time, the relative intensity of fluorescence measured value of recovered liquid；Sample group i =1,2,3；Every group of sample number into spectrum j=1,2,3.

8.2 sterilizing rate R are calculated

In formula:

R-every batch of daily use chemicals sample sterilizing rate, %；

a_B- standard curve lgC_B-lgI_BSlope.

8.3 sterilizing Index As calculate

In formula:

A-every batch of daily use chemicals sample sterilizing index；

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 colony counting method_BWhen, with reference to 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 Take preceding 2 bit digital after digital " rounding up ", behind with 0 instead of digit；It can also be indicated with 10 exponential form, " rounding up " Two effective digitals are used afterwards.Control sample and test specimen are after dye/sterilizing specific time, the relative intensity of fluorescence of recovered liquid Measured value round numbers, sterilizing rate R_ij、R_i, R calculated result take three effective digitals, sterilize Index A_ij、A_i, A calculated result take two Position effective digital.

8.5 uncertainties of measurement: this patent method is by calculating control sample and test specimen through dye/sterilizing specific time Afterwards, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV count Calculate result and remain into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to chemical product killing bacteria measure of merit Reproducibility；In regulation group and between-group variation coefficient CV≤10%.Relevant calculation is shown in formula (14)~(16):

In formula:

σ_TAfter -3 groups of 9 test specimens sterilization specific times, reclaim liquid phase is to fluorescent strength determining valueStandard deviation Difference；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3.

(9) evaluation of result

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

If using sterilizing Index A as correlated performance evaluation index: working as A_ij/A_i(staphylococcus aureus when/A < 1.0 With salmonella A_ij/A_i/ A < 0.5), sample is without Bactericidal Effect；As 1.0≤A_ij/A_i(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonella_ij/A_i/ A < 1.0), sample has Bactericidal Effect；As 2.0≤A_ij/A_i(gold when/A < 3.0 1.0≤A of staphylococcus aureus and salmonella_ij/A_i/ A < 2.0), sample bacterium killing effect is stronger；Work as A_ij/A_i/A≥3.0 When (staphylococcus aureus and salmonella A_ij/A_i/ A >=2.0), sample bacterium killing effect is extremely strong.

9.2 work as the sterilizing rate R of certain group (part) daily use chemicals sample_i(R_ij) or sterilizing Index A_i(A_ij) and other two groups of (part) samples Killing bacteria effect compared to a levels are at least differed when, extract one group of sample again and repeat to test；It is raw through ATP to calculate it Object fluorescence lgC_B─lgI_BThe sterilizing rate or sterilizing index that calibration curve method measures.If two groups of front and back (part) daily use chemicals sample is thin Bacterium killing effect level is identical, then abandons it；Take other two groups of (part) remaining sample sterilizing rates R_i(R_ij) or sterilizing Index A_i(A_ij) Arithmetic mean of instantaneous value as batch (group) daily use chemicals sample bacterium killing effect evaluation result.

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

(1) Laboratory biosafety qualification

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

(2) instrument and equipment

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

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

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

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

2.5 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 DEG C~50 DEG C), wet range (30~95) %RH ± 2%RH of control.

2.6 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.

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

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

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

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

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

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

2.13pH meter: the accurate pH meter of Shanghai precision instrumentation Co., Ltd model MP512-03, range ability (- 2.000~19.999) pH ± 0.002pH, ± 0.4 DEG C of temperature range (- 10~110) DEG C.

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

(3) medium/liquid

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

(4) chemical reagent

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

(5) reference culture

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

The detection data and result of the present embodiment calculate:

Using ATP fluophotometer through lgC_B-lgI_BCalibration curve method to antibacterial drift stain liquid sample killing bacteria effect into Row evaluation, activity 1:100, action time 5min；Select 5g/L hypo solution as neutralizer, it can be effective Neutralize residual action of the drift stain liquid sample to be measured to test strain.Related neutralizer identification, sterilization test data and bactericidal effect Evaluation and Calculation of Measuring Uncertainty result are shown in Table 1~table 3 respectively, and (testing result of Shigella and salmonella is respectively and greatly The testing result of intestines Escherichia and staphylococcus aureus is close).

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

2 sterilization test data of table and sterilizing rate R, sterilizing Index A calculated result

3 relative intensity of fluorescence value Calculation of Measuring Uncertainty result of table

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

Claims

1. a kind of evaluation method of chemical product killing bacteria effect, comprising: (1) sample extraction and pre-treatment requirement；(2) equipment is selected Type and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BMark Directrix curve is established and inoculation bacterium solution C_BCalibration；(5) viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard is bent Line is established；(6) neutralizer identification and sterilization test；(7) reclaim liquid phase is to fluorescence intensity level I_BMeasurement；(8) recovered liquid viable bacteria contains Measure C_BAnd T_BIt calculates and sterilizing rate R is calculated with sterilizing Index A；(9) evaluation of result；It is characterized in that, using ATP fluorophotometric Count the ATP bioluminescence that accurate quantitative assessment is carried out to the chemical product killing bacteria effect with sterilizing rate R or sterilizing Index A characterization lgC_B-lgI_BCalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_BIn measurement:

The requirement such as extraction and pre-treatment of control sample and test specimen is specified, after testing standard bacterial strain is passed on, is activated, It takes continuous switching 2 times Fresh bacterial cultures to prepare bacteria suspension, carries out count plate using MTT colorimetric analysis, establish OD Value-viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ²；And Viable bacteria content C is carried out to inoculation bacterium solution_BCalibration: Then, C is selected_BFor 1.2 × 10³CFU/mL、1.2×10⁴CFU/mL、1.2×10⁵The dilution bacterium solution of CFU/mL is as standard series bacteria suspension；It is opposite to measure it Fluorescence intensity level I_B, draw lgC_B-lgI_BStandard curve, and it is derived from the linear equation Y=a of curve_BX+b_BAnd phase relation Number R_B ², under conditions of neutralizer qualification result is qualified, it is separately added into each group 4.8mL control sample and test specimen 0.2mL bacterium solution simultaneously mixes；After effect lasts to stipulated time to be sterilized, in 4.5mL and agent solution to 0.5mL each group microbiological contamination sample Liquid carries out neutralization recycling, using the relative intensity of fluorescence value I of ATP fluorescent spectrophotometer measuring recovered liquid_BCijAnd I_BTij, and according to song Line equation Y=a_BX+b_BCalculate its viable bacteria content C_BijAnd T_Bij；

In sterilizing rate R and sterilizing Index A are calculated:

According to test strain standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, with every control in specific sterilizing time The relative intensity of fluorescence measured value of sample and test specimen recovered liquidWithAs basic data；Calculate every daily use chemicals sample Sterilizing rate R_ijAnd sterilizing Index A_ij, and with every group of sample R_ijAnd A_ijArithmetic mean of instantaneous value be its sterilizing rate R_iWith sterilizing index A_i；To the R of 3 groups of samples_iOr A_iArithmetic mean of instantaneous value is taken, the sterilizing rate R or sterilizing Index A of every batch of daily use chemicals sample are obtained；It is clear simultaneously The related data revision of the convention and uncertainty of measurement requirement；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that the classification of chemical product killing bacteria effect determines Standard；As the sterilizing rate R of certain group (part) daily use chemicals sample_i(R_ij) or sterilizing Index A_i(A_ij) with the bacterium of other two groups of (part) samples When killing effect is compared to a levels are at least differed, one group of sample is extracted again and repeats to test；It is calculated through ATP bioluminescence lgC_B─lgI_BThe sterilizing rate or sterilizing index that calibration curve method measures, if the killing bacteria of two groups of (part) the daily use chemicals samples in front and back Effect level is identical, then abandons it；Take other two groups of (part) remaining sample sterilizing rates R_i(R_ij) or sterilizing Index A_i(A_ij) arithmetic Evaluation result of the average value as batch (group) the daily use chemicals sample bacterium killing effect.

2. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the sample mentions It takes and pre-treatment requirement, carries out in the steps below:

(1) control sample: using test strain culture solution as control sample, dosage, quantity and standard hard water dilution operation etc. It is identical as sample to be tested；

(2) test specimen: the daily chemical products such as articles for washing, skin-cleaning articles with special hygiene efficacy；Each strain use 3 Group sterilization test sample is derived from the different transportation and packing of same product batch number, and every group of sample is derived from 3 in same transportation and packing Minimum marketing packing (sample size is no less than 10g)；Single neutralizer qualification test need to be sold with the minimum in 3 same transportation and packing Sell packaging sample (sample size is no less than 10g)；3 neutralization test sample useds are taken respectively from the different transports of same product batch number Control sample and test specimen are diluted to the 2 of product description normal concentration with Sterile standard hard water before test by packaging respectively Times, soak type and smear type fruits and vegetables, the tableware washing articles activity that do not express are respectively 1:100 and 1:5；Liquid detergent, The dry goods washing and nursing article activities such as washing powder, fabric softener, drift stain liquid, carpet cleaner are 1:100；Directly spray Type hard-surface cleaning articles are spilt using stoste, concentrated type sample effect concentration is 1:100, and every group of test specimen selects one group of control Sample as object of reference and effectively identifies.

3. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the equipment choosing Type and reagent, culture medium are prepared, and are carried out in the steps below:

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

(2) instrument and equipment: two stage biological safety cabinet or the superclean bench of lustration class >=100；Fluophotometer containing ATP is matched The total number of bacteria ATP bioluminescence rapid detection system of kit, Special test tube etc. is covered, wherein ATP fluophotometer wavelength amount Journey 300nm~650nm, total number of bacteria detection range 10¹CFU/mL~10⁷CFU/mL；Wave-length coverage 400nm~760nm, reading The microplate reader of range (0.0~4.0) Abs；The constant incubator of (37 ± 1) DEG C；The constant water bath box that (10~50) DEG C ± 1 DEG C； The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C of low temperature refrigerator；2 DEG C~8 DEG C of refrigeration Case；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic cleaner of frequency (30~50) kHz；Revolving speed (300~3000) r/min's Vortex oscillator；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: the sterile measuring pipette of 1mL, 10mL；(metering misses by 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL Difference is less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；Needle-based of the filter sizes no more than 0.45 μm filters Device；The sterile conical flask of capacity 150mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；96 hole flat-bottomed plates；Maxwell Bacterium standard opacity tube and mating test tube；Sterile test tube；Rack for test tube；Diameter is not more than the oese of 4mm；Alcolhol burner；Scale division value The ruler of 1mm； Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black Gel ink pen；

(4) reagent: 5mg/mL, pH value 7.4 MTT (tetramethyl azo azoles salt) solution (0.45 μm of membrane filtration degerming, 4 DEG C~6 DEG C 15d is kept in dark place)；121 DEG C of high pressure sterilization 30min of following reagent, the physiological saline of 5 DEG C~10 DEG C storage 30d:85%；It will 0.034g calcium chloride, 0.139g magnesium chloride are dissolved in 1000mL water, are configured to the standard hard water of hardness 342mg/L；5g is thio Sodium sulphate is dissolved in 1000mL water (for the sample of fungicide containing chlorine type)；By 1.36g potassium dihydrogen phosphate, 2.83g disodium hydrogen phosphate, 10g lecithin, 10g glycine, 30g tween (80) are dissolved in 1000mL water or by 1.36g potassium dihydrogen phosphates, 2.83g phosphoric acid hydrogen two Sodium, 3g lecithin, 20g tween (80) are dissolved in 1000mL water (for sample containing non-oxidative bactericide)；By 20g tween (80), 1g sodium thiosulfate is dissolved in 1000mL phosphate buffer solution (for oxygen-containing type fungicide sample) etc. (or for daily use chemicals sample to be measured Fungicide type contained by product, the neutralizer for selecting other to be mutually applicable in)；

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

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

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

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

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

Plate count agar (PCA): by 5g tryptone, 2.5g yeast extract, 1g glucose, 15g agar dissolve by heating in In 1000mL water, pH to 7.0 ± 0.2 is adjusted；

(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 nutrient broth can be down to 10 in 15min^-13Mol/L or less)；

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

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

4. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the strain is protected Hiding, activation and bacteria suspension preparation, carry out in the steps below:

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

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

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

(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain culture is added Bacteria suspension is made in liquid；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that and bacterial suspension is uniform, Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, with 0.7mm core black gel ink pen in A6 white The parallel lines that 3 length are 10cm are drawn on copy paper；Estimate test tube and standard opacity tube (nominal concentration 9.0 × 10⁸CFU/mL) Differences in turbidity, be added dropwise culture solution until the two turbidity it is identical until.

5. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the OD value- Viable bacteria content logarithm lgC_BStandard curve is established and inoculation bacterium solution C_BCalibration carries out in the steps below:

(1) bacterium solution OD value measures: being 9.0 × 10 to bacteria total amount with test strain culture solution⁸The bacteria suspension progress 1:5 of CFU/mL, 1:10,1:15,1:20 are serially diluted, and using culture solution as blank sample liquid, carry out zeroing correction to microplate reader, then, by 100 The bacterium solution of the above-mentioned different dilutions of μ L is injected separately into 96 hole flat-bottomed plates, and each dilution does 3 multiple holes；20 are added dropwise to every hole The MTT solution of μ L, after (37 ± 1) DEG C cultivate 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, is determined Maximum absorption wavelength；Viable bacteria OD value in each hole mixed solution is measured, the bacteria suspension viable bacteria OD value of different dilutions is its 3 multiple holes The arithmetic mean of instantaneous value of OD measured value, meanwhile, according to method as defined in national standard GB 4789.2-2016, above-mentioned bacteria suspension is fitted After dilution, ± 2h for 24 hours is cultivated in (37 ± 1) DEG C, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculation bacterium solution C_BCalibration: with different dilution bacteria suspensions Viable bacteria OD value as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard curve；Using Least square fitting method is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ², wherein escherichia coli (or Shigella) and staphylococcus aureus (or salmonella) choose respectively dilution be 1:10,1:15,1:20 and 1:5, The bacteria suspension of 1:10,1:15 draw curve as standard serial solution, work as R₀ ²>=0.98, when confidence level >=0.95, according to this The bacteria suspension viable bacteria content C that patented method measures_BEffectively, then, it is adjusted through test strain culture solution, obtains C_BRange be 1.0 × 10⁷CFU/mL~5.0 × 10⁷The inoculation bacterium solution of CFU/mL.

6. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the viable bacteria contains Measure logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established, and is carried out in the steps below:

(1) determination of standard series bacteria suspension and its I_BMeasurement: use test strain culture solution by known viable bacteria content C_BBacterium solution gradient It is diluted to standard series bacteria suspension: 1.2 × 10³CFU/mL、1.2×10⁴CFU/mL、1.2×10⁵CFU/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 is measured and is recorded State the relative intensity of fluorescence value of standard series bacteria suspension；

(2) viable bacteria content logarithm lgC_BRelative intensity of fluorescence logarithm lgI_BStandard curve is established: with standard series bacteria suspension Relative intensity of fluorescence logarithm lgI_BAs abscissa, with its corresponding viable bacteria content logarithm lgC_BFor ordinate mapping；It is right Mathematical relationship between the two carries out calibration curve, and the linear equation Y of curve is derived from using least square fitting method =a_BX+b_BAnd coefficient R_B ²；Work as R_B ²>=0.98, when confidence level >=0.95, have according to the measurement that this patent method is done Effect.

7. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the neutralizer Identification and sterilization test carry out in the steps below:

(1) neutralizer qualification test: first mixing 1mL test specimen with 9mL with agent solution, and effect 10min is formed to neutralize and be produced Object solution；And test is grouped as follows: with sterilizing liquid-transfering gun by 0.2mL inoculation bacterium solution (with lgC_B-lgI_BCalibration curve bacterium solution It is derived from same branch test strain stoste test tube, 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used) six test specimens containing 4.8mL of packing (1^#、2^#), in 4.8mL and agent solution (3^#、7^#), 4.8mL neutralized reaction product solution (4^#), 4.8mL test strain culture solution (5^#) In sterile test tube, 1000r/min shakes test tube 10min, mixes well each group solution, then, with sterilizing liquid-transfering gun by 0.5mL Each group mixing liquid dispenses six culture solutions of test strain containing 4.5mL (1^#、5^#、7^#), in 4.5mL and agent solution (2^#、3^#)、4.5mL Neutralized reaction product solution (4^#) sterile test tube in, and by 2.5mL test strain culture solution in 2.5mL and agent solution be added it is same In branch sterile test tube, as the 6th^#Group test sample liquid, 1000r/min shake each group test tube 10min after using its mixing liquid as time Liquid is received, according to relative intensity of fluorescence value I in this patent_BMeasuring method, measures and the relative fluorescence for recording above-mentioned 7 groups of recovered liquids is strong Angle value；Simultaneously by the 7th^#The measured value of group recovered liquid is denoted as I_B0；

(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^#Group and the 7th^#The relative intensity of fluorescence measured value of group recovered liquid is close, i.e. I_B3≈I_B4≈I_B5≈I_B0, according to formula (1) The viable bacteria content C being calculated_B3、C_B4、C_B5Error rate Δ≤10% between group；Illustrate that this test neutralization evaluation is qualified；

In formula:

Above-mentioned neutralizer qualification test is repeated, if the continuous evaluation of neutralization three times is qualified, the type of selected neutralizer And concentration can be used for the killing bacteria effect test of daily use chemicals sample to be measured；

(3) sterilization test: every group of 4.8mL control sample and test specimen are dispensed in sterile test tube with sterilizing liquid-transfering gun, (20 ± 1) in DEG C water bath with thermostatic control after (30 DEG C of soaps sample) heat preservation (5 ± 0.5) min, 0.2mL is added dropwise into test tube and is inoculated with bacterium solution, it is fast Speed mixes and starts timing according to the action time that product description marks, and does not express the hard-surface cleaning articles of time (directly Spray-type and concentrated type) effect 5min；Soak type and smear type fruits and vegetables, tableware washing articles act on 15min and 5min respectively； The dry goods washing and nursing article such as liquid detergent, washing powder, fabric softener, carpet cleaner acts on 20min (drift stain liquid effect 5min), after effect lasts to stipulated time to be sterilized, 0.5mL each group microbiological contamination mixing liquid is pipetted with sterilizing liquid-transfering gun, packing contains 4.5mL sterilizing neutralizes in the sterile test tube of agent solution, and 1000r/min shakes test tube 10min, as to be measured after sufficiently neutralizing The recovered liquid of sample.

8. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the recovered liquid Relative intensity of fluorescence value I_BMeasurement carries out in the steps below:

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

(2) reclaim liquid phase is to fluorescence intensity level I_BMeasurement:, then will with sterilizing liquid-transfering gun if background level meets instrument requirement The ATP lysate of 0.1mL recovered liquid, 0.8mL disodium hydrogen phosphate buffer solution and 0.1mL containing 0.037% sucrose sequentially adds In same branch sterile test tube, 0.1mL mixed solution is moved to respectively in three instrument sterile test tubes after mixing well, is stood 5min~30min, as ATP fluorometric investigation Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, mixes The ATP fluorescent reagent of 0.1mL is instilled after even；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_B And record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute is no more than 15s, with three I of the arithmetic mean of instantaneous value of a ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence value as sample to be tested recovered liquid_BMeasured value (or Using instrument matched reagent box, according to specification used in connection with requirement, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level I_B)。

9. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the recovered liquid Viable bacteria content C_BAnd T_BReckoning and sterilizing rate R or sterilizing Index A calculate, and carry out in the steps below:

(1) recovered liquid viable bacteria content C_BAnd T_BIt calculates

According to test strain standard curve lgC_B-lgI_BLinear equation Y=a_BX+b_B, calculate control sample and test specimen warp After dye/sterilizing specific time, the viable bacteria content C of recovered liquid_BAnd T_B, relevant calculation is (when using instrument matched reagent box, according to it Practical sample volume calculates the viable bacteria content C of 1mL recovered liquid_BAnd T_B) see formula (2)~(7):

In formula:

C_BThe mean viable amount recycled after -3 groups of control sample microbiological contamination specific times, unit are every milliliter of Colony Forming Unit (CFU/mL)；

C_BiThe mean viable amount recycled after-every group control sample microbiological contamination specific time, unit are every milliliter of Colony Forming Unit (CFU/mL)；Sample group i=1,2,3；

C_BijThe viable bacteria amount recycled after-every control sample microbiological contamination specific time, unit are every milliliter of Colony Forming Unit (CFU/ mL)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；

After-every control sample microbiological contamination specific time, the relative intensity of fluorescence measured value of recovered liquid；Sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3；

T_BThe mean viable amount recycled after -3 groups of test specimen sterilization specific times, unit is every milliliter of Colony Forming Unit (CFU/mL)；

T_BiThe mean viable amount recycled after-every group test specimen sterilization specific time, unit is every milliliter of Colony Forming Unit (CFU/mL)；Sample group i=1,2,3；

T_BijThe viable bacteria amount recycled after-every test specimen sterilization specific time, unit is every milliliter of Colony Forming Unit (CFU/ mL)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；

After-every test specimen sterilization specific time, the relative intensity of fluorescence measured value of recovered liquid；Sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3；

(2) sterilizing rate R is calculated

Under conditions of neutralizer neutralization evaluates qualified, the sterilizing rate R of every daily use chemicals sample_ij, every group and every batch of sample Sterilizing rate R_iIt is calculated respectively according to formula (8), (9), (10) with R:

In formula:

R-every batch of daily use chemicals sample sterilizing rate, %；

a_B- standard curve lgC_B-lgI_BSlope；

(3) sterilizing Index A calculates

Under conditions of neutralizer neutralization evaluates qualified, the sterilizing Index A of every daily use chemicals sample_ij, every group and every batch of sample Sterilizing Index A_iIt is calculated respectively according to formula (11), (12), (13) with A:

In formula:

A-every batch of daily use chemicals sample sterilizing index；

a_B- standard curve lgC_B-lgI_BSlope；

(4) bacteria suspension viable bacteria content C data revision of the convention requirement: is demarcated using colony counting method_BWhen, with reference to 2016 phase of GB4789.2- Regulation is closed, C is worked as_BWhen less than 100CFU/mL, " rounding up " round numbers；Work as C_BWhen not less than 100CFU/mL, the 3rd bit digital " four House five enters " after take preceding 2 bit digital, behind with 0 instead of digit；It can also be indicated with 10 exponential form, " rounding up " uses afterwards Two effective digitals, control sample and test specimen are after dye/sterilizing specific time, the relative intensity of fluorescence measured value of recovered liquid Round numbers, sterilizing rate R_ij、R_i, R calculated result take three effective digitals, sterilize Index A_ij、A_i, A calculated result take two effectively Number；

(5) uncertainty of measurement: this patent method, which passes through, calculates control sample and test specimen after dye/sterilizing specific time, Reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV calculate knot Fruit remains into 2 significant digits), judge the weight that ATP fluorimetric assay for biological materials method is applied to chemical product killing bacteria measure of merit Existing property；In regulation group and between-group variation coefficient CV≤10%, relevant calculation are shown in formula (14)~(16):

In formula:

μ_CAfter -3 groups of 9 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueArithmetic mean of instantaneous value； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

μ_TAfter -3 groups of 9 test specimens sterilization specific times, reclaim liquid phase is to fluorescent strength determining valueArithmetic mean of instantaneous value； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_CiAfter-every group 3 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueStandard deviation； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_TiAfter-every group 3 test specimens sterilization specific time, reclaim liquid phase is to fluorescent strength determining valueStandard deviation； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_CAfter -3 groups of 9 control sample microbiological contamination specific times, reclaim liquid phase is to fluorescent strength determining valueStandard deviation；Sample Product group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3；

σ_TAfter -3 groups of 9 test specimens sterilization specific times, reclaim liquid phase is to fluorescent strength determining valueStandard deviation；Sample Product group i=1,2,3；Every group of sample number into spectrum j=1,2,3；ATP tests Duplicate Samples number k=1,2,3.

10. the evaluation method of chemical product killing bacteria effect according to claim 1, which is characterized in that the result Evaluation carries out in the steps below:

If using sterilizing rate R as correlated performance evaluation index: working as R_ij/R_i(staphylococcus aureus and sand when/R < 90% Door Salmonella R_ij/R_i/ R < 80%), sample is without Bactericidal Effect；As 90%≤R_ij/R_i(Staphylococcus aureus when/R < 99% 80%≤R of bacterium and salmonella_ij/R_i/ R < 90%), sample has Bactericidal Effect；As 99%≤R_ij/R_i/ R < 99.9% When (90%≤R of staphylococcus aureus and salmonella_ij/R_i/ R < 99%), sample bacterium killing effect is stronger；Work as R_ij/R_i/ (staphylococcus aureus and salmonella R when R >=99.9%_ij/R_i/ R >=99%), sample bacterium killing effect is extremely strong；

If using sterilizing Index A as correlated performance evaluation index: working as A_ij/A_i(staphylococcus aureus and sand when/A < 1.0 Door Salmonella A_ij/A_i/ A < 0.5), sample is without Bactericidal Effect；As 1.0≤A_ij/A_iWhen/A < 2.0 (staphylococcus aureus and 0.5≤A of salmonella_ij/A_i/ A < 1.0), sample has Bactericidal Effect；As 2.0≤A_ij/A_iIt is (golden yellow when/A < 3.0 1.0≤A of staphylococcus and salmonella_ij/A_i/ A < 2.0), sample bacterium killing effect is stronger；Work as A_ij/A_i(gold when/A >=3.0 Staphylococcus aureus and salmonella A_ij/A_i/ A >=2.0), sample bacterium killing effect is extremely strong；

(2) as the sterilizing rate R of certain group (part) daily use chemicals sample_i(R_ij) or sterilizing Index A_i(A_ij) thin with other two groups of (part) samples When bacterium killing effect is compared to a levels are at least differed, one group of sample is extracted again and repeats to test；It is glimmering through ATP biology to calculate it Light lgC_B─lgI_BThe sterilizing rate or sterilizing index that calibration curve method measures, if the bacterium of two groups of (part) the daily use chemicals samples in front and back kills Effect of going out level is identical, then abandons it；Take other two groups of (part) remaining sample sterilizing rates R_i(R_ij) or sterilizing Index A_i(A_ij) calculation Art average value is as batch (group) daily use chemicals sample bacterium killing effect evaluation result.