阅读说明：本技术 一种材料表面抗菌性能的检测方法 (A kind of detection method of material surface anti-microbial property ) 是由 曹永平 于兵孝 塔拉提百克·买买提居马 吴浩 焦洋 于 2019-10-14 设计创作，主要内容包括：本发明提供了一种材料表面抗菌性能的检测方法,涉及材料检测技术领域。该方法包括菌悬液的制备、材料预处理和抗菌性能检测,将菌悬液悬滴于被检测材料的表面培养,乙醇固定后染色观察。相比于传统的贴膜法检测,该检测方法省去了细菌洗脱、转种的过程,简化了操作步骤,减少了实验误差,及操作的出错率；另一方面,本发明提供的检测方法可以直接在材料表面观察细菌的生长情况来判断材料的抗菌性能,更加直观；该方法操作简单、有效、快捷,可广泛应用于各种非吸水性材料抗菌性能的检测。(The present invention provides a kind of detection methods of material surface anti-microbial property, are related to measuring for materials field.This method includes preparation, pretreatment and the anti-microbial property detection of bacteria suspension, and bacteria suspension hanging drop is cultivated in the surface for being detected material, the fixed poststaining observation of ethyl alcohol.It is detected compared to traditional film applicator coating, which eliminates the process of bacterium elution, transferred species, simplifies operating procedure, reduces experimental error, and the error rate of operation；On the other hand, the growing state that detection method provided by the invention can directly observe bacterium on the surface of the material carrys out the anti-microbial property of judgement material, more intuitively；This method is easy to operate, effective, quick, can be widely applied to the detection of various unwetted property material anti-microbial properties.)

1. a kind of detection method of material surface anti-microbial property, it is characterised in that the anti-microbial property of the detection material of quicklook, The method the following steps are included:

The preparation of S1, bacteria suspension: activation experiments bacterium, transferred species, fluid nutrient medium culture to logarithmic phase adjust bacterium with fluid nutrient medium Kind concentration, is made bacteria suspension；

S2, pretreatment: being wiped with 70% ethanol solution and be detected material surface, with aseptic water washing, under UV environment It spontaneously dries, obtains pretreated detected material；

S3, anti-microbial property detection: the bacteria suspension hanging drop that S1 is prepared is in the surface dripping quantity of the pretreated detected material of S2 For 4-6 μ l/cm², it is evenly laid out humidity be 95% sterile petri dish in, 37 DEG C constant temperature incubation 16-24 hour, room temperature nature Evaporation water, 80%-90% ethyl alcohol are fixed, and common Bacterial stain method is dyed, and the micro- sem observation of material is detected material Expect the growing state of surface bacteria, assesses anti-microbial property；

The fluid nutrient medium includes: TSB culture medium, trypsase and mannitol.

2. detection method according to claim 1, which is characterized in that fluid nutrient medium described in step S1 includes: TSB training Support base 94%-98% (v/v), trypsase 0.5%-2.5% (v/v) and mannitol 1.5%-3.5% (v/v).

3. detection method according to claim 1, which is characterized in that the condition of fluid nutrient medium culture described in step S1 Are as follows: 28-32 DEG C of temperature, 180-280r/min shaken cultivation 20-28h.

4. detection method according to claim 1, which is characterized in that the concentration of bacteria suspension described in step S1 be 1-5 × 10⁶CFU/mL。

5. detection method according to claim 1, which is characterized in that test organisms described in step S1 is Escherichia coli, gold One of staphylococcus aureus and Pseudomonas aeruginosa.

6. detection method according to claim 1, which is characterized in that detected material described in step S2 can be divided into control Material and anti-biotic material, and be unwetted property material.

7. detection method according to claim 6, which is characterized in that the non-stripping property antibacterial contained in the anti-biotic material Substance.

8. detection method according to claim 1, which is characterized in that the surface area of detected material described in step S2 is 2-8cm²。

9. detection method according to claim 1, which is characterized in that Bacterial stain method described in step S3 is simple stain One of method, Gram's stain and Ziehi-Neelsen stain.

10. the answering in unwetted property anti-biotic material and its product of detection method described in -9 any one according to claim 1 With.

Technical field

The present invention relates to measuring for materials field more particularly to a kind of detection methods of material surface anti-microbial property.

Background technique

With the continuous development of science and technology, people to disease and health deep understanding and concern, the exploitation of anti-biotic material with Using also becoming the hot spot studied at present.Anti-biotic material is a kind of new material with antibacterial functions, be can be widely applied to The fields such as medical supplies, daily necessity, packaging material for food and construction material, such as: anti-bacterial fibre, antibiotic plastic, antibacterial apply Material, antibacterial metal, anti-bacteria ceramic etc..It is needed during anti-biotic material is developed to its antibiotic property, physical property, long-acting Property, safety etc. carries out qualitative and/or evaluates dizzyly calmly.

Currently used, the detection method for anti-biotic material bacteria resistance energy includes qualitative detection and quantitative detection.It is fixed Property detection include plating medium method, plate streak and Quine's method；Quantitative detection includes infusion process, film applicator coating and succusion. Plating medium method: sample is placed on the plating medium of inoculation, and sample and tested bacterium come into full contact with after cultivating a period of time Observe fungistatic effect, the measurement suitable for the anti-biotic material containing dissolution type antibacterial agent and its product.Plate streak: in plate On culture medium after parallel scribing inoculation, after contacting sample for a period of time, commented by observation contact sample area and not in contact with sample area The bacteriostasis of valence antimicrobial product, the measurement suitable for the anti-biotic material containing dissolution type antibacterial agent and its product.Quine's method: sample Product are embedded in the culture medium of a certain amount of bacterium solution, culture after a certain period of time growing state of the observation bacterium on and around sample and Measurement sample weathers situation, suitable for evaluating the antibacterial effect of antibiotic plastic, antibiotic fabric etc..Infusion process: a certain amount of bacterium solution It equably instills in sample, sample contacts culture with bacterium after a certain period of time, calculates the bacterium number slip with " 0 " time, is suitable for The evaluation of water-absorbing material and its product.Film applicator coating: bacterium solution is added dropwise on sample, bedding film makes bacterium solution be coated uniformly on sample Product surface, contacts after a certain period of time, eluting bacterial, transferred species to solid medium culture, and count plate calculates antibacterium rate.It is suitable Evaluation for unwetted property material and its product.Succusion: being immersed in sample in certain density bacterium solution, shaken cultivation one The section time, after sample elution, transferred species to solid medium culture, count plate simultaneously calculates antibiotic rate.It is smaller suitable for area, contain There is the measurement of the anti-biotic material and its product of dissolution type antibacterial agent.Different detection methods is suitable for different detected materials, Wherein, it is most widely used that film applicator coating in practical applications.This antibiotic method needs to cultivate transferred species after bacterium on the surface of the material On bacterium to culture medium, anti-microbial property is judged according to the growing state of bacterium, step is complicated, and short time consumption is long.

With the development of anti-microbial property detection technique, Chinese patent CN108998500A discloses a kind of material anti-microbial property Rapid detection method, this method use oscillating culture, and TTC standard solution, warp is added in the bacterium solution after shaken cultivation contact Centrifugation, extraction measure OD value, according to TTC standard curve, calculate antibiotic rate.Detection suitable for stripping property material.Chinese patent CN101029866 discloses a kind of method using thiobarbituricacidα- method measurement stainless-steel antibacterial energy, and this method is Bacterium solution is covered on material, after cultivating a period of time, draws and is added with the bacterium solution after material effects by ultrasonic oscillation Thiobarbituricacidα- solution, water-bath measure solution O D value, calculate containing for end product of lipid hyperoxygen malonaldehyde by formula Amount obtains sterilizing rate.That this method avoid film applicator coating mensuration programs is complicated, time-consuming and laborious, is unsuitable for investigating a large amount of sample antibacterials The problems such as performance.Although this method avoid film applicator coating mensuration program complex operations, but increasing ultrasonic oscillation, detection solution The detecting steps such as addition, water-bath and OD value.

The detection method of existing material anti-microbial property is complicated for operation, increases experimental error, takes a long time.Therefore, urgently It is easy to operate that one kind need to be developed, quickly and effectively the detection method of material anti-microbial property.

Summary of the invention

The purpose of the present invention is to provide a kind of detection methods of material surface anti-microbial property.Overcome prior art presence Defect, the detection method is more simple and effective, reduces experimental error, and the error rate of operation.

One aspect of the present invention provides a kind of detection method of material surface anti-microbial property, and the method includes following step It is rapid:

The preparation of S1, bacteria suspension: activation experiments bacterium, transferred species, fluid nutrient medium culture to logarithmic phase, with Liquid Culture keynote Strain concentration is saved, bacteria suspension is made；

S2, pretreatment: it is wiped with 70% ethanol solution and is detected material surface, with aseptic water washing, ultraviolet wire loop It is spontaneously dried under border, obtains pretreated detected material；

S3, anti-microbial property detection: the bacteria suspension hanging drop that S1 is prepared is dripped in the surface of the pretreated detected material of S2 Dosage is 4-6 μ l/cm², it is evenly laid out humidity be 95% sterile petri dish in, 37 DEG C constant temperature incubation 16-24 hours, room temperature Natural evaporation moisture, 80%-90% ethyl alcohol are fixed, and common Bacterial stain method is dyed, and the micro- sem observation of material is tested It measures and monitor the growth of standing timber and expects the growing state of surface bacteria, assess anti-microbial property；

The fluid nutrient medium includes: TSB culture medium, trypsase and mannitol.

Preferably, fluid nutrient medium described in step S1 includes: TSB culture medium 94%-98% (v/v), trypsase 0.5%-2.5% (v/v) and mannitol 1.5%-3.5% (v/v).

It is further preferred that fluid nutrient medium described in step S1 includes: TSB culture medium 96.8% (v/v), tryptose Enzyme 1.3% (v/v) and mannitol 1.9% (v/v).

Preferably, the condition of fluid nutrient medium culture described in step S1 are as follows: 28-32 DEG C of temperature, 180-280r/min Shaken cultivation 20-28h.

Preferably, the concentration of bacteria suspension described in step S1 is 1-5 × 10⁶CFU/mL；It is further preferred that step S1 Described in bacteria suspension concentration be 3 × 10⁶CFU/mL。

Preferably, test organisms described in step S1 includes but is not limited to Escherichia coli (Escherichia coli), gold In staphylococcus aureus (Staphylococcus aureus) and Pseudomonas aeruginosa (Pesudomonaspyocyaneum) One kind can be any detectable bacterium.

Preferably, detected material described in step S2 can be divided into control material and anti-biotic material, and be unwetted Property material.

It is further preferred that the non-stripping property antibacterial material contained in above-mentioned anti-biotic material.

Preferably, surface area 2-8cm².It is further preferred that being detected material surface area is 6cm²。

Preferably, Bacterial stain method described in step S3 include but is not limited to single stain, Gram's stain and One of Ziehi-Neelsen stain；It is further preferred that Bacterial stain method described in step S3 is Gram's stain.

Specifically, detection method includes the following steps for a kind of material surface anti-microbial property:

The preparation of S1, bacteria suspension: test organisms is seeded in LB solid medium, in the incubator, 28 DEG C of activation cultures For 24 hours, strain after activating is taken, transferred species is into fluid nutrient medium, and 28-32 DEG C, 180-280r/min shaken cultivation 20-28h, until bacterium The logarithmic phase of growth takes 1mL bacterium solution, adjusts strain concentration to 1-5 × 10 with fluid nutrient medium⁶Bacteria suspension is made in CFU/mL；

S2, pretreatment: be surface area by detected material cutting being 2-8cm², wiped with 70% ethanol solution tested Material surface is surveyed, with aseptic water washing 30s, spontaneously dries 15-30min under UV environment, obtains pretreated be detected Material；

S3, anti-microbial property detection: the bacteria suspension hanging drop that S1 is prepared is dripped in the surface of the pretreated detected material of S2 Dosage is 4-6 μ l/cm², it is evenly laid out humidity be 95% sterile petri dish in, 37 DEG C constant temperature incubation 16-24 hours, room temperature Natural evaporation moisture, 80%-90% ethyl alcohol are fixed, and common Bacterial stain method is dyed, material microscope 1000-1500 The growing state for being detected material surface bacterium is observed again, assesses anti-microbial property；

The fluid nutrient medium is general fluid nutrient medium.

Another aspect of the present invention additionally provides application of the above-mentioned detection method in unwetted property anti-biotic material and product.

Compared with prior art, positive and beneficial effect of the invention is:

The present invention provides a kind of detection methods of new material surface anti-microbial property, to traditional material surface antibiotic property Detection overturned.It is detected compared to traditional film applicator coating, detection method provided by the invention eliminates traditional pad pasting The process of bacterium elution, transferred species in method, simplifies operating procedure, reduces experimental error, and the error rate of operation；Another party Face, the growing state that detection method provided by the invention can directly observe bacterium on the surface of the material carry out the antibiotic property of judgement material Can, more intuitively.This method is easy to operate, effective, quick, can be widely applied to the inspection of various unwetted property material anti-microbial properties It surveys.

Detailed description of the invention

In Fig. 1, figure (A) is the observation result figure of control material in embodiment 2, and figure (B) is anti-biotic material in embodiment 2 Observation result figure；

In Fig. 2, figure (A) is the observation result figure of pad pasting culture control material, and figure (B) is the sight of pad pasting culture anti-biotic material Examine result figure.

Specific embodiment

The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article It encloses.Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material Material, preferred method and material are enumerated by place herein.

Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention The normally understood identical meaning of those of ordinary skill.