CN104313118A - Antibacterial efficacy detection method of diatom antibacterial material - Google Patents
Antibacterial efficacy detection method of diatom antibacterial material Download PDFInfo
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- CN104313118A CN104313118A CN201410624669.8A CN201410624669A CN104313118A CN 104313118 A CN104313118 A CN 104313118A CN 201410624669 A CN201410624669 A CN 201410624669A CN 104313118 A CN104313118 A CN 104313118A
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Abstract
The invention provides an antibacterial efficacy detection method of a diatom antibacterial material. The method comprises the steps of reagent preparation, sample determination, formula computing and the like. The detection method is wide in application range, can be used for laboratories in the industry of antibacterial materials, various bacteriological examination laboratories, laboratories in scientific research institutions, research and development departments and quality control departments in enterprises and social public detection mechanisms, is suitable for detecting the antibacterial efficacy of various antibacterial materials, and has the advantages of being simple and convenient to operate, low in cost, manpower-saving, high in repeatability, scientific and visual in effects and the like, so that the disadvantages of no detection, high cost, long repeated detection time and the like by adopting a traditional detection method are avoided, and the method can be widely applied to the detection of product quality in antibacterial material, health and environmental industries.
Description
Technical field
The present invention relates to the antibacterial efficacy detection method of diatom anti-biotic material, belong to the detection field of material composition usefulness.
Background technology
Anti-biotic material is the new function material that development in recent years is got up, and refers to the various materials with anti-microbial property through antiseptic-germicide process.At present, anti-biotic material is widely used in chemical industry, weaving, electronics and health field, just keeps the trend of sustainable growth with the amplitude of annual sales amount 30%, becomes urgently sunrise industry leaved for development.The core of anti-biotic material or its anti-microbial effect, antibacterial is again intension concept very widely, comprises sterilizing, sterilization, antibacterial and anticorrosion etc. from broadly saying.In the face of the novel antibacterial series products of constantly weeding out the old and bring forth the new, in order to ensure the development that anti-biotic material is healthy and orderly, for the selfdiscipline of anti-biotic material industry and management provide basis, sanitary inspection department needs supervision with all strength and coordinates to detect, the performance of antimicrobial form material product itself is evaluated objectively, for quality product is checked on, enable human consumer relieved safely.
Diatom anti-biotic material contains a kind of high molecular polymer, there is high-adhesiveness and filming function, the respiration channel of bacteriophage can be blocked, by organic composition, lipin dissolving is carried out to bacterium, change the permeability of bacterial cell membrane, make endobacillary metabolism generation obstacle and play the effect of sterilization, can the effective multiple harmful microorganism such as kill bacteria, fungi and virus.Therefore, the antimicrobial spectrum of diatom high molecular polymer widely, as the two-way composite antibacterial material of the novel organic-inorganic of one, and have colourless, odorless, to no skin irritation, nontoxicity, non-corrosive feature, be a kind of new selection of desirable environmental-protecting and high-efficient type two-shipper anti-biotic material.For the antibacterial efficacy of monitoring diatom anti-biotic material, although detection method referenced in reality detects is a lot, adopt different detection methods can obtain different detected results.The tested diatom that the measuring method that tradition adopts produces and standard testing pre biooxidation are obscured, and cause result inaccurate, and testing cost is expensive and waste the plenty of time.
Summary of the invention
For above-mentioned defect, the object of the present invention is to provide a kind of quantitative antibacterial detection method adopted for the diatom anti-biotic material with stronger anti-microbial effect, the method energy science reflects the antibacterial effect of anti-biotic material intuitively, detect the killing action of anti-biotic material to challenge organisms within a short period of time, the testing of inspection department can be convenient to.
For achieving the above object, the technical solution used in the present invention is as follows:
An antibacterial efficacy detection method for diatom anti-biotic material, is made up of preparation of reagents, sample determination, formulae discovery step, it is characterized in that having following steps:
(1), required reagent
(a) experimental strain: the reference cultures such as streptococcus aureus, intestinal bacteria or Candida albicans;
(b) phosphate buffered saline buffer, PBS, 0.03mol/L, pH value 7.2;
(c) vibration shaking table, 300r/min;
(d) Erlenmeyer flask
(e) nutrient agar, tryptose soya agar substratum (TSA) and husky fort nutrient agar
(2), schedule of operation
A anti-biotic material is taken 0.75g 2 parts and inserts respectively in the Erlenmeyer flask of 250mL by ();
B () adds 70mL PBS and 5mL bacteria suspension in above-mentioned Erlenmeyer flask, make the concentration of bacteria suspension in PBS be 1 × 10
4cfu/mL ~ 5 × 10
4cfu/mL;
C Erlenmeyer flask is fixed on vibration shaking table by (), be under the condition of 20 DEG C ~ 25 DEG C in operative temperature, with 300r/min jolting 2min, 1h respectively; Draw 1.0mL PBS and be suitably diluted to 10
-2, as sample liquid before the vibration of anti-biotic material test group;
D () draws vibrate front and vibrate rear sample liquid and each 1.0mL of suitable dilution diluent respectively, inoculate plate with agar tilt-pour process, each sample liquid inoculates 2 plates, carries out viable bacteria cultivation counting;
E () test is established negative control sample simultaneously and does not add sample sets; Control sample group is not to replace outside anti-biotic material containing the material of antiseptic-germicide, sample that weight is identical, and other schedule of operation is all identical with test group; Do not add sample component and do not get 5mL bacteria suspension and 70mLPBS adds in 250mL Erlenmeyer flask, mixing, respectively at 1h before vibration and after vibration, the mixed solution respectively getting 1.0mL bacteria suspension and PBS is cooked suitable dilution, carries out viable bacteria and cultivates and count;
(f) with test with batch diluent, substratum establish negative control respectively;
G () test repetition 3 times, is calculated as follows antibiotic rate
Average colony number × 100% before antibiotic rate=(after average colony number before sample vibration-sample vibration average colony number)/sample vibration
(3), evaluation method
A the contrast of () each negative all should asepsis growth;
B () does not add sample sets live bacterial count 1 × 10
4cfu/mL ~ 5 × 10
4between cfu/mL, and sample oscillation forward backward averaging colony number difference is within 10%, and test effectively;
C, in () each test, the difference bacterium > 26% of test sample antibiotic rate and control sample antibiotic rate, namely judges that this product has anti-microbial effect.
The present invention has the following advantages: (1) sensing range is wide, and the antibacterial efficacy being applicable to various diatom anti-biotic material and derived product thereof detects; (2) be suitable for mechanism many, research and development, the Quality Control department of all kinds of disinfecting inspection department, scientific research institutions, social common detection mechanism, enterprise all can evaluate diatom anti-biotic material antibacterial efficacy according to this law; (3) detection method letter, just, honest and clean, test; (4) test method favorable repeatability, reliable and stable; (5) test result is specifically objective, and the property evaluated has quantization characteristic.
Embodiment
Be clearly and completely described the technical scheme in the embodiment of the present invention below in conjunction with embodiment, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1 (adopting streptococcus aureus):
In this embodiment, be made up of preparation of reagents, sample determination, formulae discovery three partial content.
1, Cleaning Principle:
In a liquid by long-time vibration fast, increase the contact of antimicrobial component in microorganism and diatom anti-biotic material to show its anti-microbial effect, test can calculate and clearly calculate antibiotic rate, judges whether it has antibacterial ability according to antibiotic rate size.
2, required reagent and collocation method
(1) experimental strain: streptococcus aureus (ATCC 6538) is prepared into bacteria suspension.
Open strain tube in aseptic technique mode, add adequate nutrition broth culture with capillary pipet, soft pressure-vaccum for several times, makes bacterial classification melt dispersion.Get containing 5.0mL ~ 10.0mL nutrient broth medium test tube, instill a little bacteria suspension, put 37 DEG C and cultivate 18h ~ 24h.Get the bacteria suspension of 1st generation cultivation with transfering loop, streak inoculation, on nutrient agar flat board, is put 37 DEG C and is cultivated 18h ~ 24h.Or a taking-up bacterium pearl is inoculated on plate from microbank, put 37 DEG C and cultivate 18h ~ 24h, colonies typical in the above-mentioned 2nd generation culture of picking, is inoculated in nutrient agar slopes, puts 37 DEG C and cultivates 18h ~ 24h, be the 3rd culture thing.The nutrient agar getting for 3rd ~ 6 generations cultivates the fresh slant culture of 18h ~ 24h, and draw 3.0mL ~ 5.0mL diluent with 5.0mL suction pipe and add in slant tube, pressure-vaccum repeatedly, washes lower lawn.Subsequently, with 5.0mL suction pipe, washing lotion is moved in another sterile test tube, with electronic vortex mixer mixing 20s, to make bacterial suspension even.The bacteria suspension tentatively made, first with the bigness scale of bacterial concentration nephelometry its containing bacteria concentration, then with diluted to desired concn.
(2) phosphate buffered saline buffer (PBS, 0.03mol/L, pH value 7.2)
(3) vibrate shaking table (300r/min)
(4) Erlenmeyer flask
(5) nutrient agar, tryptose soya agar substratum (TSA) and husky fort nutrient agar
3, schedule of operation
(1) coloured anti-bacterial material is taken 0.75g 2 parts to insert respectively in the Erlenmeyer flask of 250mL.
(2) in above-mentioned Erlenmeyer flask, add 70mL PBS and 5mL bacteria suspension, make the concentration of bacteria suspension in PBS be 1 × 10
4cfu/mL ~ 5 × 10
4cfu/mL.
(3) being fixed on by Erlenmeyer flask on vibration shaking table, is under the condition of 20 DEG C ~ 25 DEG C in operative temperature, with 300r/min jolting 2min, 1h respectively.Draw 1.0mL PBS and be suitably diluted to 10
-2, as sample liquid before anti-biotic material test group (hereinafter referred to as test group) vibration.
(4) to draw respectively before vibration and sample liquid and each 1.0mL of suitable dilution diluent after vibration, with agar tilt-pour process inoculation plate, each sample liquid inoculates 2 plates, carries out viable bacteria and cultivates counting.Viable bacteria is cultivated counting and generally uses tilt-pour process, carries out cultivation counting, strictly undertaken by sterility requirements bacteria suspension.By test tube on demand number of packets be arranged on test-tube stand, often pipe adds 4.5mL diluent.From left to right, put on 10 by pipe for each group
-1, 10
-2, 10
-3deng.By the electronic vortex mixer mixing 20s of bacteria suspension sample, draw 0.5mL immediately and add to 10
-1in pipe.By 10
-1the electronic vortex mixer mixing 20s of Guan Yiqian method, mixing, then draw out 0.5mL and add 10
-2in pipe.So analogize, until last pipe.Select acceptable diluent degree test tube (with the every flat board of predicted growth colony number for 15cfu ~ 300cfu person is advisable), draw the suspension 1.0mL wherein mixed and be added in sterilized petri dishes.Each extent of dilution inoculates 2 plates.Generally need inoculate 2 ~ 3 different extent of dilution.By the substratum that 40 DEG C ~ 45 DEG C melt, be poured into and add in the plate of sample liquid, every plate 15mL ~ 20mL.Plate is built, at once shakes mixing gently, keep flat.After agar solidification, upset plate makes the end upwards, puts in 37 DEG C of constant incubators and cultivates.
(5) test is established negative control sample simultaneously and does not add sample sets.Control sample group is not to replace outside anti-biotic material containing the material of antiseptic-germicide, sample that weight is identical, and other schedule of operation is all identical with test group.Do not add sample component and do not get 5mL bacteria suspension and 70mLPBS adds in 250mL Erlenmeyer flask, mixing, respectively at 1h after vibrate front and vibration, the mixed solution respectively getting 1.0mL bacteria suspension and PBS is cooked suitable dilution, carries out viable bacteria cultivation and counts (concrete grammar is same as above).
(6) with test with batch diluent, substratum establish negative control respectively.
(7) test repetition 3 times, be calculated as follows antibiotic rate
Average colony number × 100% before antibiotic rate=(after average colony number before sample vibration-sample vibration average colony number)/sample vibration
4, evaluation method
(1) each negative contrast all should asepsis growth.
(2) sample sets live bacterial count is not added 1 × 10
4cfu/mL ~ 5 × 10
4between cfu/mL, and sample oscillation forward backward averaging colony number difference is within 10%, and test effectively.
(3), in each test, the difference bacterium > 26% of test sample antibiotic rate and control sample antibiotic rate, namely judges that this product has anti-microbial effect.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
Claims (1)
1. an antibacterial efficacy detection method for diatom anti-biotic material, it is characterized in that being made up of preparation of reagents, sample determination, formulae discovery step, concrete steps are as follows:
(1), required reagent
(a) experimental strain: the reference cultures such as streptococcus aureus, intestinal bacteria or Candida albicans;
(b) phosphate buffered saline buffer, PBS, 0.03mol/L, pH value 7.2;
(c) vibration shaking table, 300r/min;
(d) Erlenmeyer flask;
(e) nutrient agar, tryptose soya agar substratum (TSA) or husky fort nutrient agar;
(2), schedule of operation
A () takes 2 parts of 0.75g anti-biotic materials and inserts respectively in the Erlenmeyer flask of 250mL;
B () adds 70mL PBS and 5mL bacteria suspension in above-mentioned Erlenmeyer flask, make the concentration of bacteria suspension in PBS be 1 × 10
4cfu/mL ~ 5 × 10
4cfu/mL;
C Erlenmeyer flask is fixed on vibration shaking table by (), be under the condition of 20 DEG C ~ 25 DEG C in operative temperature, with 300r/min jolting 2min, 1h respectively; Draw 1.0mL PBS and be suitably diluted to 10
-2, as sample liquid before the vibration of anti-biotic material test group;
D () draws vibrate front and vibrate rear sample liquid and each 1.0mL of suitable dilution diluent respectively, inoculate plate with agar tilt-pour process, each sample liquid inoculates 2 plates, carries out viable bacteria cultivation counting;
E () test is established negative control sample simultaneously and does not add sample sets; Control sample group is not to replace outside anti-biotic material containing the material of antiseptic-germicide, sample that weight is identical, and other schedule of operation is all identical with test group; Do not add sample component and do not get 5mL bacteria suspension and 70mLPBS adds in 250mL Erlenmeyer flask, mixing, respectively at 1h before vibration and after vibration, the mixed solution respectively getting 1.0mL bacteria suspension and PBS is cooked suitable dilution, carries out viable bacteria and cultivates and count;
(f) with test with batch diluent, substratum establish negative control respectively;
G () test repetition 3 times, is calculated as follows antibiotic rate
Average colony number × 100% before antibiotic rate=(after average colony number before sample vibration-sample vibration average colony number)/sample vibration
(3), evaluation method
A the contrast of () each negative all should asepsis growth;
B () does not add sample sets live bacterial count 1 × 10
4cfu/mL ~ 5 × 10
4between cfu/mL, and sample oscillation forward backward averaging colony number difference is within 10%, and test effectively;
C, in () each test, the difference bacterium > 26% of test sample antibiotic rate and control sample antibiotic rate, namely judges that this product has anti-microbial effect.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925662A (en) * | 2016-06-14 | 2016-09-07 | 苏州市职业大学 | Method for detecting antibacterial property of silkworm excrement product |
| CN106823822A (en) * | 2017-01-25 | 2017-06-13 | 国家海洋局天津海水淡化与综合利用研究所 | A kind of organic Flat Membrane bacteriostasis rate assay method |
| CN109402217A (en) * | 2018-11-23 | 2019-03-01 | 中国科学院金属研究所 | A kind of rapid detection method for anti-bacteria stainless steel antibacterial effect |
| CN109609590A (en) * | 2018-12-21 | 2019-04-12 | 扬州倍加洁日化有限公司 | A kind of anti-microbial property test method of wet tissue product |
| CN110656039A (en) * | 2019-11-21 | 2020-01-07 | 浙江中跃医疗科技有限公司 | Laboratory is with antibiotic material efficiency detection device |
| CN111398260A (en) * | 2020-03-31 | 2020-07-10 | 广州立白企业集团有限公司 | Method for rapidly evaluating antibacterial efficacy and method for displaying antibacterial efficacy on surface of carrier |
-
2014
- 2014-11-07 CN CN201410624669.8A patent/CN104313118A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 赵婷 等: "纺织品抗菌性能评价方法比较", 《纺织科技进展》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925662A (en) * | 2016-06-14 | 2016-09-07 | 苏州市职业大学 | Method for detecting antibacterial property of silkworm excrement product |
| CN106823822A (en) * | 2017-01-25 | 2017-06-13 | 国家海洋局天津海水淡化与综合利用研究所 | A kind of organic Flat Membrane bacteriostasis rate assay method |
| CN109402217A (en) * | 2018-11-23 | 2019-03-01 | 中国科学院金属研究所 | A kind of rapid detection method for anti-bacteria stainless steel antibacterial effect |
| CN109402217B (en) * | 2018-11-23 | 2022-11-08 | 中国科学院金属研究所 | Rapid detection method for antibacterial effect of antibacterial stainless steel |
| CN109609590A (en) * | 2018-12-21 | 2019-04-12 | 扬州倍加洁日化有限公司 | A kind of anti-microbial property test method of wet tissue product |
| CN110656039A (en) * | 2019-11-21 | 2020-01-07 | 浙江中跃医疗科技有限公司 | Laboratory is with antibiotic material efficiency detection device |
| CN111398260A (en) * | 2020-03-31 | 2020-07-10 | 广州立白企业集团有限公司 | Method for rapidly evaluating antibacterial efficacy and method for displaying antibacterial efficacy on surface of carrier |
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Application publication date: 20150128 |