Antibacterial performance evaluation method of in-situ prepared material
By forming the samples to be tested in situ on solid-state culture medium, the accuracy of the antibacterial performance evaluation of thin and thin materials is solved, and an intuitive evaluation of the antibacterial performance of extremely thin and thin materials is achieved.
Patent Information
- Application Number
- CN202410081923.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-19
- Publication Date
- 2025-07-22
AI Technical Summary
The existing antibacterial performance evaluation methods are difficult to apply to extremely thin materials that are susceptible to curling or tearing due to external forces due to electrostatic interference, resulting in inaccurate evaluation results.
The bacterial solution is uniformly inoculated on the solid culture medium, and the samples to be tested are formed in situ at the second detection position. By observing the bacterial growth at the first and second detection positions, the samples are directly formed on the culture medium to avoid structural damage. The samples of different structures are formed by electrostatic spraying or electrospinning.
It improves the accuracy of antibacterial performance evaluation, reduces errors, and can intuitively judge the antibacterial properties of the sample. It is suitable for extremely light and thin materials.
Smart Images
Figure CN120350087A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of antibacterial performance testing, and more particularly, to a method for evaluating the antibacterial performance of in-situ prepared materials. Background Art
[0002] Currently, the main methods for evaluating antibacterial performance mainly include the spread plate method and the agar plate diffusion method.
[0003] Among them, the general operation method of the spread plate method includes: spreading about 0.1 mL of bacterial liquid in a solidified agar culture dish, and transferring a circular sample cut into a diameter of 25 mm ± 5 mm to the center of the agar medium coated with the bacterial liquid. The antibacterial drug in the sample diffuses on the agar medium, inhibiting the growth of bacteria around it. Finally, the agar culture dish is placed in an incubator and cultured for 18 - 24 h. After the bacterial liquid is spread, colonies will grow around the area where the antibacterial drug diffuses around the sample, forming a ring-shaped area without colony growth, which is called the inhibition zone. If an inhibition zone is visible around the sample, or there is no inhibition zone around the sample but there is no bacterial growth under the sample, it indicates that the sample has antibacterial performance. If no inhibition zone can be observed around the sample, and bacterial growth is observed under the sample, it indicates that the sample has no antibacterial performance. The main difference between the agar plate diffusion method and the spread plate method is that the test bacterial liquid is mixed with the non-solidified agar medium, and about 20 mL of the agar medium mixed with the bacterial liquid is poured into the solidified agar culture dish. After the agar medium containing bacteria solidifies, a circular sample cut into a diameter of 25 mm ± 5 mm is transferred to the center of the agar medium.
[0004] However, both the spread plate method and the agar plate diffusion method require processing such as transferring the sample, and are applicable to liquid antibacterial agents or textiles with stable structures, and are difficult to apply to materials prepared by instruments such as electrostatic spraying and electrospinning, which are extremely thin, easily curled by electrostatic interference, or torn by external forces. Summary of the Invention
[0005] Based on the above deficiencies, the present application provides a method for evaluating the antibacterial performance of in-situ prepared materials to partially or completely improve the problem of evaluating the antibacterial performance of in-situ prepared materials in related technologies.
[0006] The present application is implemented as follows:
[0007] An example of the present application provides a method for evaluating antibacterial performance, including:
[0008] Preparing a sample: uniformly inoculating a bacterial liquid on the surface of a solid medium. The surface of the medium inoculated with the bacterial liquid has a first detection position and a second detection position, and the sample to be detected is formed in-situ at the second detection position;
[0009] Antibacterial performance evaluation: Bacteria are cultured in the culture medium, and the growth of bacteria at the first detection position and the second detection position is observed. Compared with the first detection position, if an antibacterial zone is formed at the second detection position, it indicates that the sample to be detected has antibacterial properties; if no antibacterial zone is formed at the second detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0010] In the above implementation process, since the first detection position and the second detection position are both evenly inoculated with bacterial liquid, and the sample to be detected is set at the second detection position, if the sample to be detected has antibacterial properties, the article to be detected will inhibit the growth of bacteria at the second detection position, and an antibacterial zone will be formed. If the sample to be detected does not have antibacterial properties, the inhibitory effect of the sample to be detected on the growth of bacteria at the second detection position is poor, and no antibacterial zone will appear at the second detection position compared with the first detection position. Setting the sample to be detected and the control group at different positions on the same solid culture medium can more intuitively judge the antibacterial properties of the sample to be detected and reduce errors.
[0011] Moreover, directly in-situ forming the sample to be detected at the second detection position can avoid damaging the structure of the sample to be detected during the process of transferring the formed sample to be detected to the second detection position, and can improve the accuracy of evaluating the antibacterial performance of the sample to be detected.
[0012] In one possible implementation manner, the method for in-situ forming the sample to be detected at the second detection position includes:
[0013] Cover the first detection position with a covering, and simultaneously in-situ form the sample to be detected on the second detection position and the covering, and then remove the covering and the sample to be detected on the covering.
[0014] In the above implementation process, before forming the sample to be detected, covering the first detection position with a covering can utilize the shielding of the covering to reduce the probability of forming the sample to be detected at the first detection position and improve the accuracy of comparative detection.
[0015] In one possible implementation manner, the method for in-situ forming the sample to be detected includes: using the electrostatic spraying method to in-situ form diffusely distributed particles on the second detection position and the covering simultaneously.
[0016] In the above implementation process, by using the electrostatic spraying method to form diffusely distributed particles, the antibacterial performance of the diffusely distributed particles can be evaluated under the condition of basically not changing the particle distribution.
[0017] In one possible implementation manner, the method for in-situ forming the sample to be detected includes: using the electrospinning method to in-situ form a fiber membrane on the second detection position and the covering simultaneously.
[0018] In the above implementation process, a fibrous membrane with a network structure can be formed by electrospinning, and the antibacterial performance of the electrospun fibrous membrane can be evaluated under the condition of basically not changing the network structure of the fibrous membrane.
[0019] In a possible implementation manner, the method for removing the cover and the fibrous membrane located on the cover includes:
[0020] Using a sterile knife, cut the fibrous membrane along the edge of the cover close to the second detection position, and then remove the cover.
[0021] In the above implementation process, when in-situ forming the sample to be detected by electrospinning, use a sterile knife to cut the fibrous membrane along the edge of the cover close to the second detection position, so as to facilitate the removal of the cover and the fibrous membrane on it, reduce the probability of structural damage to the fibrous membrane at the second detection position and reduce the residual probability of the fibrous membrane at the first detection position, and improve the evaluation accuracy.
[0022] In a possible implementation manner, the spinning solution of the electrospinning method includes a non-hydrophilic spinning base material and an antibacterial agent.
[0023] In the above implementation process, in the fibrous membrane obtained by in-situ forming after the spinning solution containing a non-hydrophilic spinning base material and an antibacterial agent is prepared by electrospinning, the fiber filaments basically will not dissolve after contacting the culture medium. It is necessary to use a sterile knife to cut the fibrous membrane along the edge of the cover close to the second detection position to separate the fibrous membrane at the second detection position and the cover, so as to facilitate the removal of the cover and the fibrous membrane on it.
[0024] In a possible implementation manner, the bacterial solution is selected from at least one of Escherichia coli or Staphylococcus aureus.
[0025] In a possible implementation manner, the steps of antibacterial performance evaluation include:
[0026] Count the number of colonies at the first detection position and the second detection position; if the number of colonies at the second detection position is lower than the number of colonies at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the number of colonies at the second detection position is the same as the number of colonies at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0027] In the above implementation process, when evaluating the antibacterial performance of the sample to be detected, the number of colonies can be counted at the first detection position and the second detection position respectively, and then the number of colonies at the first detection position and the second detection position can be compared. If the number of colonies at the second detection position is lower than that at the first detection position, it indicates that the sample to be detected formed in situ at the second detection position inhibits the growth of bacteria at the second detection position, indicating that the sample to be detected has antibacterial properties. If the number of colonies at the second detection position is the same as that at the first detection position, it indicates that the sample to be detected at the second detection position basically does not inhibit the growth of bacteria at the second detection position, indicating that the sample to be detected does not have antibacterial properties.
[0028] In a possible implementation, the steps of antibacterial performance evaluation include:
[0029] Compare the colony population sizes at the first detection position and the second detection position. If the colony population at the second detection position is smaller than that at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the colony population size at the second detection position is the same as that at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0030] In the above implementation process, when evaluating the antibacterial performance of the sample to be detected, the colony population sizes at the first detection position and the second detection position can be compared. If the colony population at the second detection position is smaller than that at the first detection position, it indicates that the sample to be detected at the second detection position inhibits the growth of bacteria at the second detection position, indicating that the sample to be detected has antibacterial properties. If the colony population size at the second detection position is the same as that at the first detection position, it indicates that the sample to be detected at the second detection position basically does not inhibit the growth of bacteria at the second detection position, indicating that the sample to be detected does not have antibacterial properties.
[0031] In a possible implementation, the steps of antibacterial performance evaluation include: observing the colors at the first detection position and the second detection position. If there is an obvious color difference between the color at the second detection position and the color at the first detection position, it indicates that the sample to be detected has antibacterial properties. If the color at the second detection position is the same as the color at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0032] In the above implementation process, if the color at the second detection position is the same as the color at the first detection position, it indicates that the bacterial growth situation at the second detection position is the same as that at the first detection position, indicating that the sample to be detected at the second detection position basically does not inhibit the growth of bacteria at the second detection position, indicating that the sample to be detected does not have antibacterial properties. If there is an obvious color difference between the color at the second detection position and the color at the first detection position, it indicates that the sample to be detected has antibacterial properties. Description of the Drawings
[0033] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.
[0034] Figure 1 Flow chart of the antibacterial performance evaluation method provided for the examples of the present application;
[0035] Figure 2 Physical diagram of the agar petri dish after the end of cultivation provided for Embodiment 1 of the present application;
[0036] Figure 3 Physical diagram of the agar petri dish after the end of cultivation provided for Embodiment 2 of the present application;
[0037] Figure 4 Physical diagram of the Escherichia coli agar petri dish after the end of cultivation provided for Embodiment 3 of the present application;
[0038] Figure 5 Physical diagram of the Staphylococcus aureus agar petri dish after the end of cultivation provided for Embodiment 3 of the present application;
[0039] Figure 6 Physical diagram of the petri dish after the end of cultivation provided for Embodiment 4 of the present application;
[0040] Figure 7 Physical diagram of the petri dish after the end of cultivation provided for Embodiment 5 of the present application;
[0041] Figure 8 Physical diagram of the petri dish after the end of cultivation provided for Embodiment 6 of the present application. Detailed implementation manners
[0042] The following will describe the implementation solutions of the present application in detail in conjunction with the embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be construed as limiting the scope of the present application. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.
[0043] In some biomedical fields, antibacterial products can be prepared in situ at the wound of a patient. For example, by using a portable electrospinning technology, an electrospun antibacterial fiber membrane is prepared in situ at the wound of the patient.
[0044] Fiber membranes or other antibacterial products prepared in situ using electrospinning technology or the like are extremely thin and light in structure and are usually vulnerable to electrostatic interference and curl or be torn by external forces. In antibacterial products prepared by electrospinning or other technologies, their antibacterial properties are usually related to the structure of the products. For example, the special three-dimensional or two-dimensional network structure in electrospun fiber membranes helps the release of antibacterial agents and achieves the antibacterial effect. If the structure of the electrospun fiber membrane is damaged before contacting the wound, it will seriously affect the antibacterial properties of the antibacterial product.
[0045] During the research and development or preparation of antibacterial products, it is necessary to evaluate the antibacterial properties of antibacterial products. In current antibacterial property evaluation methods, such as the spread plate method and the agar plate diffusion method, it is usually necessary to transfer the formed product to the surface of the culture medium, and then conduct bacterial culture and observation to evaluate the antibacterial effect.
[0046] However, if the existing spread plate method and agar plate diffusion method are used for antibacterial property evaluation, transferring the extremely thin and light fiber membranes with specific structures prepared by electrospinning and other technologies to the culture medium will damage the self-structure of the fiber membranes during the transfer process, and accurate evaluation results of the antibacterial effect of the fiber membranes cannot be obtained later.
[0047] Based on this, the present application example provides an antibacterial property evaluation method to improve the antibacterial property evaluation method of extremely thin and light solid antibacterial products with specific structures prepared by electrospinning and other technologies and improve the accuracy of antibacterial property evaluation results.
[0048] The following describes in detail the antibacterial property evaluation method provided by the present application example in conjunction with the accompanying drawings.
[0049] Please refer to Figure 1 , the present application example provides an antibacterial property evaluation method including:
[0050] S1. Prepare samples
[0051] Uniformly inoculate the bacterial liquid on the surface of the solid culture medium. The surface of the culture medium inoculated with the bacterial liquid has a first detection position and a second detection position, and the sample to be detected is formed in situ at the second detection position.
[0052] Uniformly inoculate the bacterial liquid on the surface of the solid culture medium. Divide the surface of the culture medium inoculated with the bacterial liquid into a first detection position and a second detection position, so that the first detection position and the second detection position have the same initial bacterial culture conditions. Then form the sample to be detected in situ at the second detection position, so that the variable between the first detection position and the second detection position is the sample to be detected, so as to facilitate subsequent comparison of the bacterial growth conditions at the first detection position and the second detection position to determine whether the sample to be detected has an antibacterial effect.
[0053] By simultaneously demarcating a second detection position for the test and a first detection position as a control on the same culture medium, the bacterial growth conditions at the first and second detection positions can be compared more intuitively, reducing the error during the evaluation operation of the test group and the control group and improving the accuracy of the antibacterial performance evaluation.
[0054] Moreover, in-situ forming the sample to be tested at the second detection position can avoid damaging the structure of the sample to be tested during the process of transferring the sample to the culture medium, and can more precisely reflect the antibacterial effect of the sample to be tested with a specific structure.
[0055] Furthermore, this application does not limit how to evenly inoculate the bacterial liquid on the surface of the solid culture medium, and relevant personnel can make corresponding selections according to needs.
[0056] Exemplarily, 50 - 1000 μL of 10 4 -10 8 CFU bacterial liquid can be added into the already solidified agar culture dish, and the bacterial liquid can be evenly coated.
[0057] Furthermore, this application does not limit the specific type of the bacterial liquid, and relevant personnel can inoculate the corresponding bacterial liquid according to the type of antibacterial agent of the sample to be tested.
[0058] Exemplarily, the bacterial liquid can be Escherichia coli bacterial liquid or Staphylococcus aureus bacterial liquid.
[0059] Exemplarily, the type of antibacterial agent of the sample to be tested can be guanidine antibacterial agents, such as polyhexamethylene biguanide hydrochloride, polyhexamethylene guanidine, polyhexamethylene biguanide or polyaminopropyl biguanide.
[0060] Furthermore, this application does not limit the specific type of the antibacterial agent, and relevant personnel can add different types of antibacterial agents to the sample to be detected to prepare samples to be detected containing different types of antibacterial agents.
[0061] Exemplarily, the antibacterial agent can be selected from inorganic antibacterial agents or organic antibacterial agents. For example, inorganic antibacterial agents can be selected from TiO2, ZnO, CdS, SnO2 or Fe2O; organic antibacterial agents can be selected from chitosan, quaternary ammonium salts, alcohols, phenols, organometallic compounds, pyridines, imidazoles or thiophenes, etc.
[0062] Furthermore, this application does not limit the number and distribution of the first detection position and the second detection position, and relevant personnel can make corresponding selections according to needs.
[0063] In a possible embodiment, the surface of the culture medium is divided into two equal parts, one as the first detection position and the other as the second detection position.
[0064] Alternatively, in a possible embodiment, the surface of the culture medium has a plurality of first detection positions and a plurality of second detection positions.
[0065] Exemplarily, the surface of the culture medium has two first detection positions and two second detection positions. The four detection positions are evenly distributed, and there is one second detection position between two adjacent first detection positions.
[0066] Furthermore, the two second detection positions can be used to in-situ form different types of samples to be detected.
[0067] Exemplarily, a fibrous membrane can be in-situ formed at one of the detection positions by electrospinning, and particles can be in-situ formed at another detection position by electrospraying. The antibacterial properties of the same antibacterial agent under different structural conditions can be compared.
[0068] Exemplarily, a first fibrous membrane can be in-situ formed at one of the detection positions by electrospinning, and a second fibrous membrane can be in-situ formed at another detection position by electrospinning. The types of antibacterial agents in the first fibrous membrane and the second fibrous membrane can be different, and the antibacterial properties of different antibacterial agents under the same structural conditions can be compared.
[0069] Furthermore, the present application does not limit how to in-situ form the samples to be detected at the second detection positions. Relevant personnel can make corresponding selections according to needs.
[0070] Exemplarily, a fibrous membrane with a network structure can be in-situ formed at the second detection position by electrospinning.
[0071] Exemplarily, particles with a dispersed distribution structure can be in-situ formed at the second detection position by electrospraying.
[0072] Exemplarily, a fibrous membrane loaded with dispersed particles can be in-situ formed by electrospinning and electrospraying.
[0073] Furthermore, the present application does not limit how to in-situ form the samples to be detected at the second detection positions by methods such as electrospinning and / or electrospraying. In a possible embodiment, in order to avoid forming the samples to be detected in areas other than the second detection positions, such as at the first detection positions, which may affect the subsequent control for evaluating the antibacterial performance, a covering can be placed at the first detection positions, and then the samples to be detected are in-situ formed at the second detection positions and on the covering simultaneously. Then, the covering and the samples to be detected on the covering are removed.
[0074] If there are multiple second detection positions on the surface of the culture medium, and each second detection position is used to in-situ form different types of samples to be detected, the first detection position and other second detection positions can be covered simultaneously by using a covering, and then the first type of sample to be detected is formed; the covering of another second detection position is removed, and a covering is arranged above the detection position where the first sample to be detected is formed, and the covering does not contact the sample to be detected, and then the second type of sample to be detected is formed on the exposed second detection position and the covering. And so on, more different types of samples to be detected can be formed in sequence.
[0075] Further, the present application does not limit the specific type of the covering, and relevant personnel can make corresponding selections according to needs.
[0076] Exemplarily, the covering can be a mask.
[0077] The mask can be directly covered on the surface of the culture medium at the first detection position.
[0078] When it is necessary to cover the second detection position where the sample to be detected has been formed by using the mask, the mask can be supported by the edge of the culture dish. The edge of the mask close to another second detection position to be formed has a hanging curtain partially extending into the surface of the culture medium in the culture dish, and the electrospinning nozzle or the electrostatic spraying nozzle is located above the second detection position. Alternatively, the covering is a glass cover, and the glass cover is covered on the second detection position where the sample to be detected is formed.
[0079] When forming the sample to be detected by using the electrospinning method, some fibers of the electrospinning fiber membrane will span the covering and the second detection position. In order to facilitate removing a part of the fiber membrane on the covering while reducing the degree of damage to the sample structure at the second detection position, in a possible embodiment, a sterile knife can be used to cut the fiber membrane along the edge of the covering close to the second detection position, and then the covering is taken out.
[0080] Exemplarily, the electrospinning solution includes a non-hydrophilic spinning base material and an antibacterial agent. In the fiber membrane obtained by using the electrospinning method, the fibers basically do not dissolve and break on the surface of the culture medium. Therefore, the fiber membrane can be cut along the edge of the covering close to the second detection position by using a sterile knife.
[0081] The electrospinning solution can include an antibacterial agent and a hydrophilic spinning base material. After the fiber membrane is obtained by using the electrospinning method, even if the fibers dissolve and break after contacting the culture medium, it basically does not affect the release and precipitation of the antibacterial substance at the second detection position. Therefore, the antibacterial performance of the sample to be detected can still be evaluated subsequently.
[0082] Further, please continue to refer to Figure 1, the antibacterial performance evaluation method provided by the examples of this application further includes:
[0083] S2. Antibacterial performance evaluation:
[0084] Cultivate bacteria on the culture medium, and observe the bacterial growth conditions at the first detection position and the second detection position; compared with the first detection position, if an antibacterial zone is formed at the second detection position, it indicates that the sample to be detected has antibacterial properties; if no antibacterial zone is formed at the second detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0085] When cultivating bacteria on the culture medium, in the same culture environment, since the difference between the first detection position and the second detection position basically comes from the sample to be detected formed in situ at the second detection position, therefore, compared with the first detection position, if an antibacterial zone appears at the second detection position, it indicates that the sample to be detected at the second detection position has inhibited the bacterial growth at the second detection position, indicating that the sample to be detected has antibacterial properties; on the contrary, compared with the first detection position, if no obvious antibacterial zone appears at the second detection position, it indicates that the sample to be detected at the second detection position has basically not inhibited the bacterial growth at the second detection position, indicating that the sample to be detected does not have antibacterial properties.
[0086] Using the antibacterial performance evaluation method provided by the examples of this application, the antibacterial performance of the sample to be detected can be judged more intuitively and accurately.
[0087] Furthermore, this application does not limit the specific bacterial culture conditions, and relevant personnel can make corresponding selections according to the type of bacterial solution.
[0088] Exemplarily, if the bacterial solution is an Escherichia coli bacterial solution, it can be cultured in an incubator at about 37°C for 12 - 72 h.
[0089] Exemplarily, if the bacterial solution is a Staphylococcus aureus bacterial solution, it can be cultured in an incubator at about 37°C for 12 - 72 h.
[0090] Furthermore, this application does not limit the specific characteristics of the antibacterial zone, and relevant personnel can make corresponding adjustments according to needs.
[0091] In a possible implementation manner, it is possible to directly observe whether bacteria grow at the second detection position.
[0092] Exemplarily, the steps of antibacterial performance evaluation may include:
[0093] Observe the colors of the first detection position and the second detection position. If there is an obvious color difference between the color of the second detection position and the color of the first detection position, it indicates that the sample to be detected has antibacterial properties. If the color of the second detection position is the same as the color of the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0094] Some bacteria have obvious color characteristics. For example, Staphylococcus aureus is golden yellow. Therefore, when judging whether an antibacterial zone appears, the colors of the first detection position and the second detection position can be compared. If the color of the first detection position is brightly golden yellow and the color of the second detection position is the color of the sample to be detected itself or the inherent color of the culture medium, it indicates that the sample to be detected has antibacterial properties. If the color of the first detection position is brightly golden yellow and the color of the second detection position is also brightly golden yellow, it indicates that the sample to be detected basically does not inhibit the growth of Staphylococcus aureus at the second detection position, indicating that the sample to be detected does not have antibacterial properties.
[0095] Exemplarily, in one possible embodiment, the steps for evaluating antibacterial performance include:
[0096] Count the number of colonies at the first detection position and the second detection position; if the number of colonies at the second detection position is lower than that at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the number of colonies at the second detection position is the same as that at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
[0097] By comparing the number of colonies at the first detection position and the second detection position, it can be more accurately determined whether an antibacterial zone appears at the second detection position. If the number of colonies at the second detection position is lower than that at the first detection position, it means that the sample to be detected at the second detection position inhibits the growth of bacteria, indicating that the sample to be detected has antibacterial properties; if the number of colonies at the second detection position is the same as that at the first detection position, it means that the sample to be detected at the second detection position basically does not inhibit the growth of bacteria, indicating that the sample to be detected does not have antibacterial properties.
[0098] Or, in one possible embodiment, the steps for evaluating antibacterial performance include:
[0099] Compare the sizes of the colony groups at the first detection position and the second detection position. If the colony group at the second detection position is smaller than that at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the sizes of the colony groups at the second detection position and the first detection position are the same, it indicates that the sample to be detected does not have antibacterial properties.
[0100] The above-mentioned colony refers to a visible, morphologically defined cluster of bacterial daughter colonies formed by a single bacterium on a culture medium with the bacterial parent as the center. A colony group refers to a cluster composed of multiple colonies.
[0101] Further, the steps for evaluating the antibacterial performance may further include: comparing the protrusion, edge or surface morphology, or transparency of the colonies at the first detection position and the second detection position. If the colony situation at the second detection position is significantly different from that at the first detection position, it indicates that the sample to be tested has antibacterial properties; if the colony situation at the second detection position is basically similar to that at the first detection position, it indicates that the sample to be tested does not have antibacterial properties.
[0102] The following further describes the antibacterial performance evaluation method of the present application in detail with reference to embodiments.
[0103] Embodiment 1
[0104] Embodiment 1 provides an antibacterial performance evaluation method, including the following steps:
[0105] (1) Add about 100 μL of 10 5 CFU bacterial solution into the already solidified agar culture dish. After spreading the bacterial solution evenly, place the cover on the agar medium to cover half of the area on the agar medium. Among them, the bacterial solution is the bacterial solution of Escherichia coli. Under normal temperature conditions, prepare the spinning solution of polyethylene oxide (PEO). Place the electrospinning device vertically above the agar culture dish and electrospin for more than 1 minute. Due to the hydrophilicity of PEO, the fiber filaments finally deposited on the edge of the agar culture dish and the cover will automatically break, and the fiber membrane deposited on the uncovered agar medium will dissolve in situ, and there is no need to use a sterile knife to cut the fiber membrane along the edge of the cover. After electrospinning, take out the cover and the electrospun fiber membrane on it, cover the lid of the culture dish, and place it in a constant temperature incubator for cultivation for about 20 h.
[0106] (2) Steps for evaluating antibacterial performance: The actual picture of the agar culture dish after cultivation is as Figure 2 shown: Bacteria grow densely in both the left and right halves of the agar culture dish, and there is no obvious antibacterial area, indicating that the PEO electrospun fiber membrane has basically no inhibitory effect on Escherichia coli and does not have antibacterial properties.
[0107] Embodiment 2
[0108] The difference between Embodiment 2 and Embodiment 1 is that in step (1), the bacterial solution is the bacterial solution of Staphylococcus aureus; in step (2), the actual picture of the agar culture dish after cultivation is as Figure 3 shown: Staphylococcus aureus grows evenly in both the left and right halves of the agar culture dish, and there is no obvious antibacterial area, indicating that the PEO electrospun fiber membrane has basically no inhibitory effect on Staphylococcus aureus and does not have antibacterial properties.
[0109] Embodiment 3
[0110] Example 3 is different from Example 2. In step (1), a spinning solution of polyhexamethylene biguanide hydrochloride (PHMB) and PEO is prepared. Two agar culture dishes evenly coated with bacterial solutions are prepared. Each agar culture dish forms a covered first detection position and an uncovered second detection position through a covering. And the bacterial solution in one culture dish is Escherichia coli bacterial solution, and the bacterial solution in the other culture dish is Staphylococcus aureus bacterial solution. Electrospun fiber membranes are prepared on the two culture dishes respectively by the electrospinning method, and then the covering and the electrospun fiber membrane thereon are removed for bacterial culture.
[0111] In step (2), the physical diagram of the agar culture dish of Escherichia coli after the culture is as Figure 4 shown: The left half of the agar culture dish is the first detection position, and the right half is the second detection position. The left half is evenly covered with Escherichia coli, and the right half has irregular dark areas where part is not covered by Escherichia coli, forming an antibacterial area, indicating that the PHMB / PEO electrospun fiber membrane has certain antibacterial properties against Escherichia coli. The physical diagram of the agar culture dish of Staphylococcus aureus after the culture is as Figure 5 shown: The left half of the agar culture dish is the first detection position, and the right half is the second detection position. The left half is evenly covered with more Staphylococcus aureus, and most of the right half is a dark area where Staphylococcus aureus does not grow, forming a semicircle-like antibacterial area. It shows that the PHMB / PEO electrospun fiber membrane has good antibacterial properties against Staphylococcus aureus.
[0112] Example 4
[0113] Example 4 provides a method for evaluating antibacterial performance, including the following steps:
[0114] (1) Prepare four agar culture dishes evenly coated with bacterial solutions. Each agar culture dish forms a covered first detection position and an uncovered second detection position through a covering. The bacterial solutions in two of the culture dishes are Escherichia coli bacterial solutions, denoted as culture dish one and culture dish two; the bacterial solutions in the other two culture dishes are Staphylococcus aureus bacterial solutions, denoted as culture dish three and culture dish four. Prepare a polyvinylpyrrolidone (PVP) spinning solution and a spinning solution of PHMB and PVP mixture.
[0115] Form a PVP fiber membrane on culture dish one by the electrospinning method, form a PHMB / PVP fiber membrane on culture dish two by electrospinning, form a PVP fiber membrane on culture dish three by electrospinning, and form a PHMB / PVP fiber membrane on culture dish four by electrospinning. After the electrospinning is completed, remove the covering and the electrospun fiber membrane thereon. Conduct bacterial culture.
[0116] (2) Steps for evaluating antibacterial performance: The physical diagram of culture dish one after the culture is asFigure 6 As shown in No. (1), the physical diagram of Petri dish 2 after the end of cultivation is as follows Figure 6 As shown in No. (2), the physical diagram of Petri dish 3 after the end of cultivation is as follows Figure 6 As shown in No. (3), the physical diagram of Petri dish 4 after the end of cultivation is as follows Figure 6 As shown in No. (4).
[0117] (1) Escherichia coli is evenly distributed on the entire surface. An irregular dark area where Escherichia coli does not grow appears at the second detection position in the right half of (2). It shows that the PVP electrospun fiber membrane has basically no antibacterial property against Escherichia coli, and the PHMB / PVP electrospun fiber membrane has antibacterial property against Escherichia coli. Staphylococcus aureus is evenly distributed on the entire surface of (3), indicating that the PVP electrospun fiber membrane has basically no antibacterial property against Staphylococcus aureus. In (4), compared with the left half, a large semi-circular dark area where Escherichia coli does not grow appears at the second detection position in the right half. The small dark area that appears in the left half of (4) is affected by the antibacterial substance in the fiber filaments at the edge and belongs to experimental error, indicating that the PHMB / PVP electrospun fiber membrane has good antibacterial property against Staphylococcus aureus.
[0118] Example 5
[0119] The difference between Example 5 and Example 4 is that in step (1), a polyvinyl butyral (PVB) spinning solution and a spinning solution of a mixture of PHMB and PVB are prepared. Four agar Petri dishes evenly coated with bacterial liquid are prepared, and each agar Petri dish forms a covered first detection position and an uncovered second detection position through a cover. The bacterial liquid in two of the Petri dishes is Escherichia coli bacterial liquid, denoted as Petri dish 5 and Petri dish 6; the bacterial liquid in the other two Petri dishes is Staphylococcus aureus bacterial liquid, denoted as Petri dish 7 and Petri dish 8.
[0120] On Petri dish 5, a PVB fiber membrane is formed by electrospinning method. On Petri dish 6, a PHMB / PVB fiber membrane is formed by electrospinning. On Petri dish 7, a PVB fiber membrane is formed by electrospinning. On Petri dish 8, a PHMB / PVB fiber membrane is formed by electrospinning. After electrospinning, the cover and the electrospun fiber membrane thereon are removed. Bacterial cultivation is carried out. When removing the cover, use a sterile knife to cut the electrospun fiber membrane along the edge of the cover, and then remove the cover and the fiber membrane thereon.
[0121] In step (2), the physical diagram of Petri dish 5 after the end of cultivation is as follows Figure 7 As shown in No. (5), the physical diagram of Petri dish 6 after the end of cultivation is as follows Figure 7 As shown in No. (6), the physical diagram of Petri dish 7 after the end of cultivation is as follows Figure 7As shown by the serial number (7), the physical diagram of the petri dish VIII after the cultivation is as Figure 7 shown by the serial number (8).
[0122] (5) In the left half, there is no antibacterial region with a large color difference, and in the right half, there is also no antibacterial region with a large color difference, indicating that the PVB electrospun fiber membrane has basically no antibacterial property against Escherichia coli; in (5), overall, there is an obvious color difference between the left half and the right half. The reason is that the electrospun fiber membrane of PVB basically does not dissolve in the petri dish, and the electrospun fiber membrane blocks, resulting in a color difference in the right half. In (6), there is an obvious irregular dark region in the right half, and an antibacterial region appears, indicating that the PHMB / PVB electrospun fiber membrane has good antibacterial property against Escherichia coli. In (7), the morphologies of the left half and the right half are not very different, and bacteria are diffusely distributed, indicating that the PVB electrospun fiber membrane has basically no antibacterial property against Staphylococcus aureus; in (8), the left half is evenly covered with bacteria, and there are basically no bacteria in the right half. The surface of the right half is relatively smooth, and a semicircular antibacterial region appears, indicating that the PHMB / PVB electrospun fiber membrane has antibacterial property against Staphylococcus aureus.
[0123] Example 6
[0124] The difference between Example 6 and Example 4 is that: in step (1), a PEO stock solution, as well as an antibacterial agent (PHMB) and a PEO mixed stock solution, are prepared. Four agar petri dishes evenly coated with a bacterial solution are prepared, and each agar petri dish forms a covered first detection position and an uncovered second detection position through a covering. The bacterial solutions in two of the petri dishes are Escherichia coli bacterial solutions, denoted as petri dish IX and petri dish X; the bacterial solutions in the other two petri dishes are Staphylococcus aureus bacterial solutions, denoted as petri dish XI and petri dish XII.
[0125] The PEO stock solution is formed into particles on petri dish IX and petri dish XI by an electrostatic spraying method, and the antibacterial agent (PHMB) and the PEO mixed stock solution are formed into particles on petri dish X and petri dish XII by an electrostatic spraying method. After completion, the covering is removed.
[0126] In step (2), the physical diagram of the petri dish IX after the cultivation is as Figure 8 shown by the serial number (9), the physical diagram of the petri dish X after the cultivation is as Figure 8 shown by the serial number (10), the physical diagram of the petri dish XI after the cultivation is as Figure 8 shown by the serial number (11), the physical diagram of the petri dish XII after the cultivation is as Figure 8 shown by the serial number (12).
[0127] In (9), the left and right halves are evenly covered with bacteria, indicating that the PEO electrostatic spray particles have basically no antibacterial property against Escherichia coli. In (10), obvious round-shaped dark antibacterial regions appear in the right half, indicating that the PHMB / PEO electrostatic spray particles have antibacterial property against Escherichia coli. In (11), the first detection position in the left half and the second detection position in the right half are evenly covered with bacteria, indicating that the PEO electrostatic spray particles have basically no antibacterial property against Staphylococcus aureus. In (12), the left half is evenly covered with bacteria, and a similar round antibacterial region appears in the right half, indicating that the PHMB / PEO electrostatic spray particles have antibacterial property against Staphylococcus aureus.
[0128] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. An antibacterial performance evaluation method, characterized in that Including: Preparing a sample: Inoculating a bacterial solution uniformly on the surface of a solid medium. The surface of the medium inoculated with the bacterial solution has a first detection position and a second detection position. A sample to be detected is formed in situ at the second detection position. Evaluating antibacterial performance: Culturing bacteria on the medium, and observing the bacterial growth conditions at the first detection position and the second detection position. Compared with the first detection position, if an antibacterial zone is formed at the second detection position, it indicates that the sample to be detected has antibacterial properties; if no antibacterial zone is formed at the second detection position, it indicates that the sample to be detected does not have antibacterial properties.
2. The antibacterial performance evaluation method according to claim 1, characterized in that The method for forming the sample to be detected in situ at the second detection position includes: Covering the first detection position with a covering, and simultaneously forming the sample to be detected in situ on the second detection position and the covering, and then removing the covering and the sample to be detected on the covering.
3. The antibacterial performance evaluation method according to claim 2, wherein The method for forming the sample to be detected in situ includes: Using an electrostatic spraying method to simultaneously form diffusely distributed particles in situ on the second detection position and the covering.
4. The antibacterial performance evaluation method according to claim 2, characterized in that The method for forming the sample to be detected in situ includes: Using an electrospinning method to simultaneously form a fiber membrane in situ on the second detection position and the covering.
5. The antibacterial property evaluation method according to claim 4, characterized in that, The method for removing the covering and the fiber membrane on the covering includes: Using a sterile knife to cut the fiber membrane along the edge of the covering close to the second detection position, and then removing the covering.
6. The antibacterial property evaluation method according to claim 4, wherein, The spinning solution of the electrospinning method includes an antibacterial agent and a non-hydrophilic spinning base material.
7. The antibacterial property evaluation method according to any one of claims 1-6, characterized in that, The bacterial solution is selected from at least one of Escherichia coli or Staphylococcus aureus.
8. The antibacterial property evaluation method according to any one of claims 1-6, characterized in that, The steps of the antibacterial performance evaluation include: Counting the number of colonies at the first detection position and the second detection position; if the number of colonies at the second detection position is lower than that at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the number of colonies at the second detection position is the same as that at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
9. The evaluation method according to any one of claims 1 to 6, characterized in that The steps of the antibacterial performance evaluation include: Comparing the sizes of the colony groups at the first detection position and the second detection position. If the colony group at the second detection position is smaller than that at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the size of the colony group at the second detection position is the same as that at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.
10. The antibacterial property evaluation method according to claim 1, wherein The steps of the antibacterial performance evaluation include: Observing the colors at the first detection position and the second detection position. If there is an obvious color difference between the color at the second detection position and the color at the first detection position, it indicates that the sample to be detected has antibacterial properties; if the color at the second detection position is the same as the color at the first detection position, it indicates that the sample to be detected does not have antibacterial properties.