Medical catheter antibacterial adhesion performance testing method, inoculation liquid incubation device and application of device

Abstract

The invention belongs to the technical field of implantation and intervention medical instruments, and particularly relates to a medical catheter antibacterial adhesion performance testing method, an inoculation liquid incubation device and application of the device. The test method mainly comprises the steps of preparing inoculation liquid, incubating a catheter to be tested and testing the anti-adhesion performance, wherein the catheter to be tested is placed in a homogenized inoculation liquid incubation device containing suspended bacteria to complete inoculation and incubation of the bacteria. According to the method, the anti-bacterial adhesion performance of the surface of the medical catheter is tested by simulating the real use state of the medical catheter in the medical treatment process, and the problems that in the prior art, the content of bacteria on the upper layer and the lower layer of inoculation liquid is not uniform, the bottom of a material cannot make full contact with the inoculation liquid, and bacteria adhere to the interior of a catheter cavity and cannot be thoroughly cleaned are solved. Besides, the number of the supporting frames and the number of the supporting shafts in the incubation device can be adjusted according to the number of the to-be-tested samples, the consistency of the same batch of experimental samples can be achieved, the consistency of test conditions is guaranteed, and the test error can be greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of implantable medical devices, and in particular relates to a method for testing the anti-bacterial adhesion performance of a medical catheter, an inoculation liquid incubation device, and application of the device in testing the anti-bacterial adhesion performance of a medical catheter. Background Art

[0002] Bacterial adhesion mainly manifests itself in medicine, food safety and industry. In most cases, bacterial adhesion brings negative effects, especially in the medical field. Medical device infection is an important public health issue. Bacterial adhesion is a key step in bacterial infection. Surgical infection caused by it is one of the most common infections in hospitals. With the increasing application of medical biomaterials, the incidence of infection has also increased significantly. How to prevent and treat biomaterial-related infections is an urgent problem to be solved in clinical practice.

[0003] Bacterial adhesion is a complex process, which is affected by many factors such as the characteristics of bacteria themselves, the environment they are in, and the surface characteristics of materials. At present, there is no unified standard for testing the bacterial adhesion performance of materials. The most traditional and commonly used detection method is the colony forming unit count method (CFU method), but this method has many problems, such as the inability to evaluate irregular materials as a whole. Therefore, a series of detection methods have been developed over the years to replace CFU, among which the adenosine triphosphate method (ATP method) is the most widely used. The ATP method is to indirectly measure the number of adherent bacteria by the oxidation reaction of ATP in living cells with fluorescein. The fluorescence intensity is proportional to the number of microorganisms. Although this method is fast, sensitive, and easy to operate, the dye needs to enter the cell, and non-microbial ATP will also interfere with the detection, so the accuracy needs to be improved.

[0004] Chinese invention patent application CN109666403A discloses an anti-adhesion coating composition and an anti-adhesion coating in the field of functional coating technology, wherein the anti-adhesion coating composition includes a polysilazane base material and an anti-adhesion material, and the polysilazane base material and the anti-adhesion material are cross-linked by pre-reaction or post-reaction. The coating substrate prepared by the invention is a quartz glass slide, and the anti-adhesion property of the material is tested by colony counting method. Although the coating material has strong adhesion and good anti-adhesion property, the specific steps of the anti-adhesion test method used here are unclear, difficult to repeat, and it is aimed at sheet materials and is not suitable for medical catheters.

[0005] Chinese invention patent application CN116375963A discloses a preparation method and application of a water-based polyurethane anti-adhesion coating. The fluorinated water-based polyurethane material and its coating provided by the invention have good biocompatibility and anti-adhesion properties, and can be used in long-term interventional medical devices. However, in the application, the sheet coating material is placed in a 24-well plate and incubated with a bacterial solution, and then the adherent bacteria are obtained by washing and ultrasound. The number of adherent bacteria is counted through a plating experiment and the anti-adhesion performance of the coating is judged accordingly. This method cannot be directly applied to the test of anti-bacterial adhesion on the surface of tubular materials.

[0006] Chinese invention patent CN111662950A discloses the application of a recombinant luminescent Pseudomonas aeruginosa PAO1-lux in the detection of bacterial adhesion to medical materials. First, the luxCDA BE gene is integrated into the chromosome of the standard Pseudomonas aeruginosa strain PAO1, and the recombinant luminescent strain PAO1-lux obtained by screening with PIA selection medium is cultured with M9 medium, pH6.2, 37±1℃, and the luminescence value detection window period is 6-24h. It can be applied to the detection of bacteria adhering to medical materials such as metal aluminum, metal zinc, glass, PE gloves, and medical gauze. Compared with traditional methods, this method has the characteristics of sensitivity, simplicity, real-time observation, and observable specific adhesion location, but the preparation of recombinant luminescent strains involves molecular biology, which is not easy to achieve and has high technical requirements for experimental personnel.

[0007] There is no unified standard for testing the anti-bacterial adhesion of medical catheter materials. Currently, coated medical catheters are mainly made by directly coating the coating that inhibits bacterial adhesion on the substrate, such as silicon wafers, quartz glass slides, polyurethane sheets, etc., and then cutting the sample to an appropriate size, placing it in a 24-well plate, adding the inoculum, incubating it for a period of time, and then rinsing the sample to wash away the floating bacteria on the surface. Then, ultrasonic vibration treatment or cotton swab wiping is performed to elute the adherent bacteria, and then the anti-adhesion effect of the sample on bacteria is determined by the count of live bacteria. In addition, scanning electron microscopy (SEM) observation of the sample after simple rinsing can also determine the adhesion of the sample to bacteria. However, the former is mostly directly applied to sheet materials, while the latter uses SEM, which requires sample preparation, gold plating, etc. The operation is more complicated and has high requirements for equipment, and is not universal.

[0008] Compared with other materials, medical catheters are not easy to fix when incubated with bacterial inoculum. If the immersion treatment method is adopted, the bottom of the material cannot fully contact the inoculum, and some bacteria will adhere to the inside of the tube cavity and cannot be completely cleaned. These factors will affect the experimental results. It is particularly important to develop a test method for the anti-bacterial adhesion of the surface of medical catheters, which can reasonably fix the tubular material in the inoculum with uniform bacterial concentration during the test, and ensure that the catheter is fully in contact with the inoculum. Summary of the invention

[0009] Based on this, the purpose of the present invention is to provide a method for testing the anti-bacterial adhesion performance of medical catheters. This method uses a homogenized inoculation liquid incubation device containing suspended bacteria to inoculate and incubate bacteria on the medical catheter to be tested, so that all surfaces of the catheter material can be evenly contacted with the inoculation liquid, thereby improving the test accuracy.

[0010] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides an inoculation liquid incubation device, comprising a box 1 for containing inoculation liquid, a stirring part 7 installed at the bottom of the box 1 and a support frame 4, a support shaft 3 is installed on the top of the support frame 4; the surface of the box 1 is covered with a box cover 8. Drain holes 5 can be arranged at the bottom of the side of the box 1 to facilitate the discharge of the inoculation liquid, and the number of the drainage holes 5 can be set as needed. The support frames 4 are symmetrically distributed in pairs on both sides of the box 1, and are used to support and fix the support shafts 3 with the catheter to be tested, and the number of the support frames 4 is randomly adjusted according to the number of the catheters to be tested.

[0011] Furthermore, the stirring part 7 includes a magnetic stirring drive device, the upper surface of which is embedded in the box 1 and flush with the bottom surface of the box 1. When the catheter to be tested is incubated, the stirring rotor 6 is placed on the inner surface of the stirring drive device, and the rotor 6 is rotated to drive the flow of the bacterial suspension in the box 1 to keep the bacteria in a uniform suspension state.

[0012] Furthermore, the support shaft 3 includes a cylinder, the length of which is greater than the length of the catheter to be tested. The diameter of the cylinder is slightly smaller than the inner diameter of the catheter to be tested, and when the catheter to be tested is inserted, the elasticity of the catheter to be tested is utilized to achieve a tight combination of the two. The number of the cylinders can be randomly adjusted according to the number of catheters to be tested.

[0013] Furthermore, a sterilizer is installed on the side of the box cover 8 close to the inoculation liquid, and the sterilizer includes an ultraviolet lamp. A handle 10 can be provided on the other side of the box cover 8.

[0014] The present invention also provides an application of an inoculation liquid incubation device in the anti-bacterial adhesion performance test of a medical catheter. During the test, the support shaft 3 is inserted into the catheter to be tested and then immersed in the box 1 to complete the inoculation and incubation of bacteria; the box 1 contains an inoculation liquid that keeps the bacteria in a suspended and uniform state.

[0015] The present invention further provides a method for testing the anti-bacterial adhesion performance of a medical catheter, which mainly includes the steps of preparing an inoculation solution, incubating a catheter to be tested, and testing the anti-adhesion performance. The incubation step of the catheter to be tested mainly completes the inoculation and incubation of bacteria by placing the catheter to be tested in an inoculation solution incubation device.

[0016] Furthermore, the incubation step of the catheter to be tested mainly includes: (a) sterilizing the inoculation liquid incubation device and the catheter to be tested; (b) inserting the support shaft 3 into the catheter to be tested and placing it on the support frame 4; (c) adding the inoculation liquid into the box body 1 and completely submerging the catheter to be tested, covering the box cover 8, sealing with a sealing film, placing the incubation device in a biochemical incubator, and controlling the stirring part to continuously stir at a uniform speed for incubation at 37±1°C for 6-24 hours.

[0017] Furthermore, the inoculation solution preparation process includes: inoculating the III generation colonies into 1 / 10 sodium chloride peptone buffer, and using turbidimetry to determine the concentration of the bacterial solution to be (6±0.2)×10 8 cfu / mL, dilute the bacterial solution to 6×10 4 ~6×10 7 The material of the catheter to be tested includes latex, silicone, polyurethane, polyethylene, polyvinyl chloride, etc.

[0018] Furthermore, the inoculum preparation process includes: inoculating the III generation colony into 10 mL of 1 / 10 sodium chloride peptone buffer, and using turbidimetry to determine that the bacterial solution concentration is about 6×10 8 cfu / mL, and the bacterial solution is diluted to an appropriate concentration by 10-fold dilution method. Among them, the culture process of the third-generation bacteria is: take Escherichia coli (ATCC 8099) and Staphylococcus aureus (ATCC 6538P) frozen at -80℃ and resuscitate and culture them in a clean bench. Use an inoculation loop to pick a small amount of frozen bacteria onto a nutrient agar medium plate, and culture the first-generation culture in a 37±1℃ biochemical incubator. After 24±2h, the first-generation bacteria are inoculated onto the nutrient agar medium by streaking for the second-generation culture. Repeat the culture method until a more active third-generation colony is obtained for the experiment. All culture media and experimental consumables must be sterilized before use.

[0019] Furthermore, the anti-adhesion performance test steps mainly include: (d) using physiological saline to clean the incubated catheter to be tested 3-8 times to remove the floating bacteria on the surface; the cleaning method can be immersion or flushing or a combination of the two to ensure that the floating bacteria are completely removed; (e) placing the cleaned catheter in physiological saline for ultrasonic vibration for 10±1min or vortex vibration for 2±0.5min to remove the adherent bacteria on the surface of the catheter to be tested to obtain a bacterial suspension; (f) diluting the bacterial suspension, then taking 1mL and adding it to a disposable sterile culture dish, pouring the nutrient agar culture medium that has been sterilized and cooled to an appropriate temperature into the culture dish and shaking it to evenly disperse the bacteria; (g) after the culture medium solidifies, placing it in a biochemical incubator and culturing it at 37±1℃ for 24±2h, counting the grown bacterial colonies, and the average number of colonies in the control group is recorded as 对照 The average colony count of the sample group is recorded as 样品 , the antibacterial adhesion efficiency is recorded as GR, and the calculation formula is GR=( 对照 - 样品 ) / 对照 ×100% Furthermore, the bacteria in the inoculation solution include any one of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus and Pseudomonas aeruginosa; and the components outside the bacteria include 1 / 10 sodium chloride peptone buffer or 1 / 500 nutrient broth.

[0020] The beneficial effects of the present invention are: 1. The test method of the present invention simulates the actual use state of medical catheters in medical treatment to test the antibacterial adhesion performance of their surfaces. It can replace the method of indirectly characterizing the antibacterial adhesion performance of catheter materials by testing sheets, and can directly test the antibacterial adhesion performance of catheter materials, making the test results of the antibacterial adhesion of catheter materials more intuitive and accurate.

[0021] 2. The test method of the present invention, in the incubation step of the catheter to be tested, mainly completes the inoculation and incubation of bacteria by placing the catheter to be tested in an incubation device containing a uniform inoculation solution containing suspended bacteria, thus overcoming the problem of low bacterial content in the upper layer of the inoculation solution and high bacterial content in the lower layer in the existing test method.

[0022] 3. In the test method of the present invention, the support shaft in the incubation device is used to fix the catheters to be tested of different diameters. The catheters to be tested are tightly combined with the support shaft and then immersed in the bacterial suspension, which overcomes the problems in the existing test methods that the bottom of the material cannot fully contact the inoculation solution, and some bacteria will adhere to the inside of the tube cavity and cannot be completely cleaned.

[0023] 4. In the test method of the present invention, the number of support frames and support shafts in the incubation device can be adjusted according to the amount of test samples, which can achieve consistency between the same batch of experimental samples, ensure the consistency of test conditions, and reduce experimental errors. In conventional operation methods, samples are processed one by one, and the consistency of experimental results is poor.

[0024] 5. The test method of the present invention can measure the anti-adhesion property of the outer wall of the tube body alone. When a conventional 12-well plate is statically tested, many bacteria often adhere to the inner wall, which is not easy to be cleaned. After ultrasound, the bacteria on the inner wall will be removed, affecting the experimental results of the outer wall when counting colonies.

[0025] 6. In the test method of the present invention, the anti-adhesion performance test is performed by washing the incubated catheter for 3-8 times by immersion or flushing or a combination of the two, so as to remove the floating bacteria on the surface without destroying the adhesion of the adherent bacteria.

[0026] 7. The test method of the present invention adopts ultrasonic oscillation for 10±1 min or vortex oscillation for 2±0.5 min to remove the adhered bacteria during the anti-adhesion performance test, while ensuring the activity of the adhered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Attached Figure 1 It is a three-dimensional schematic diagram of an incubation device without a box cover; in the figure, 1 is a box body, 3 is a support shaft, 4 is a support frame, 5 is a drainage hole, 6 is a rotor, and 7 is a stirring part; Attached Figure 2 A top view of the incubation device without a cover; Attached Figure 3 This is a schematic diagram of the incubation device box cover with a handle side; in the figure, 8 is the box cover, and 9 is the sterilizer; Attached Figure 4 This is a schematic diagram of the incubation device cover including the sterilizer side; in the figure, 10 is a handle; Attached Figure 5 It is a three-dimensional schematic diagram of the incubation device without the cover after the catheter to be tested is installed on the support shaft; in the figure, 2 is the catheter to be tested; Attached Figure 6 A top view of the incubation device without the box cover after the catheter to be tested is installed on the support shaft; Attached Figure 7 The growth of adhesion bacteria in Example 1; Attached Figure 8 The growth of adhesion bacteria in Example 2; Attached Fig. 9 The growth of adhesion bacteria in Comparative Example 3; Attached Fig.10 Schematic diagram of the test device in Comparative Examples 4 and 5; Attached Fig.11 The growth of adhesion bacteria in Comparative Example 4; Attached Fig.12 This is the growth of adhesion bacteria in Comparative Example 5. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. The embodiments mentioned are all implemented on the premise of the technical solutions of the present invention, and detailed implementation processes are given, but it should be stated that the protection scope of the present invention is not limited to the following embodiments.

[0030] like Figure 1 and Figure 2 As shown, the inoculation liquid incubation device of the present invention comprises a box body 1 for holding the inoculation liquid, a stirring part 7 installed at the bottom of the box body 1, and a support frame 4, and the top of the support frame 4 is used to install a support shaft 3. A drainage hole 5 can be set at the bottom of the side of the box body 1 to facilitate the discharge of the inoculation liquid, and the number of the drainage holes 5 can be set as needed; the support frames 4 are symmetrically distributed on both sides of the box body 1, and are used to support the support shafts 3 fixed with the catheter to be tested, and the number thereof is randomly adjusted according to the number of catheters to be tested; the stirring part 7 comprises a magnetic stirring drive device, the upper surface of which is embedded in the box body 1 and flush with the bottom surface of the box body 1. When the catheter to be tested is incubated, the stirring rotor 6 is placed on the inner surface of the stirring drive device, and the rotor 6 is rotated to drive the flow of the bacterial suspension in the box body 1 so that the bacteria remain in a uniform suspended state; the support shaft 3 comprises a cylinder, the length of which is greater than the length of the catheter to be tested, and the diameter is slightly smaller than the inner diameter of the catheter to be tested. When the catheter to be tested is inserted, the elasticity of the catheter to be tested is used to achieve a close combination of the two. The number thereof can be randomly adjusted according to the number of catheters to be tested.

[0031] The surface of the box body 1 is covered with a box cover 8, such as Figure 3 and Figure 4 As shown, a sterilizer is installed on the side of the box cover 8 close to the inoculation liquid, and the sterilizer includes an ultraviolet lamp. A handle 10 can be provided on the other side of the box cover 8.

[0032] The inoculation liquid incubation device of the present invention can be widely used in the test of anti-bacterial adhesion performance of medical catheters. Figure 5 and Figure 6 As shown, during the test, after the support shaft 3 is inserted into the catheter to be tested, it is immersed in the box 1 to complete the inoculation and incubation of bacteria. Specifically, the incubation device is first sterilized, and then the catheter to be tested 2 is inserted into the support shaft 3, and the support shaft 3 is placed on the symmetrically distributed support frame 4. Then the prepared inoculation liquid is added to the box 1 and completely submerged in the catheter to be tested 2, the box cover 8 is covered and the box opening is sealed with a sealing film. Finally, the incubation device is placed in the biochemical incubator as a whole, the stirring drive device is turned on, the stirring speed is controlled, and continuous stirring is performed to keep the bacteria in the inoculation liquid in the box suspended and in a uniform concentration, so that there is no dead angle on the surface of the catheter to be tested that contacts the inoculation liquid.

[0033] Example 1 A method for testing the anti-bacterial adhesion performance of a medical catheter, wherein a commercially available anti-bacterial adhesion silicone catheter is selected as a sample for method verification, and an ordinary silicone catheter without a coating is used as a control.

[0034] Step 1: Prepare the sample, cut the sample tube into 4cm length, select a support shaft of suitable diameter, place it together with the device in the clean bench, turn on the UV lamp for UV sterilization, usually the sterilization time is 30min.

[0035] Step 2: Fix the sample. After sterilization, turn on the fan and ventilate for 20 minutes to exhaust the ozone generated in the clean bench. Insert the support shaft into the sample tube to fix it, and then put it on the support frame for use.

[0036] Step 3: Prepare the inoculum. Take out the E. coli strain (ATCC 8099) stored in a 4°C refrigerator and use the turbidimetric method to prepare an initial bacterial solution with a concentration of 6×10 8 cfu / mL of bacterial suspension, and then diluted 10-fold to 6×10 7 cfu / mL was used as the inoculum, and the solution used was 1 / 10 sodium chloride peptone buffer.

[0037] Step 4: Incubate the sample tube with the inoculation solution. Add the prepared inoculation solution into the incubator, cover the lid, seal it with a sealing film, adjust the speed and place it in a biochemical incubator, incubate at 37±1℃ for 6h.

[0038] Step 5: Clean the floating bacteria. Place the incubated sample on the clean bench, remove the sample tube, rinse it once in a beaker of sterile saline, then use a pipette to draw 5 mL of saline for rinsing, and repeat the rinsing four times.

[0039] Step 6: Collect the adherent bacteria, absorb the liquid on the cleaned sample tube with filter paper, transfer the sample to a glass test tube containing 15 mL of normal saline, and elute the adherent bacteria by ultrasonication for 10 minutes (40 kW, 100%).

[0040] Step 7: Count the number of adherent bacteria and calculate the anti-adhesion efficiency. Dilute the bacterial suspension after ultrasound by 10 times, take 1 mL of the diluted bacterial suspension and add it to the culture dish, add 15 mL of nutrient agar medium and shake to evenly disperse the bacteria. After the culture medium solidifies, place it in a biochemical incubator and culture it at 37°C for 24 hours. After the culture is completed, count the colonies. The average number of colonies in the control group is recorded as 对照 The average colony count of the sample group is recorded as 样品 , the antibacterial adhesion efficiency is recorded as GR, and the calculation formula is GR=( 对照 - 样品 ) / 对照 ×100%. The calculated anti-adhesion efficiency of the sample is 85.35%. The growth of the adherent bacteria is shown in Figure 7 shown.

[0041] Example 2 The operation steps of this embodiment are the same as those of embodiment 1, except that the method of cleaning the floating bacteria in step 5 is changed to immersing once in a beaker filled with sterile saline, replacing the saline, and then continuing to immerse, and repeating three times; the method of eluting the adherent bacteria in step 6 is changed to vortexing for 2 minutes. The anti-adhesion efficiency of the sample is calculated to be 82.26%. The growth of adherent bacteria is shown in Figure 2. Figure 8 shown.

[0042] Example 3 The operation steps of this example are the same as those of Example 1, except that the concentration of the inoculum in step 3 is adjusted to 6×10 6 cfu / mL, the incubation time of the inoculum and sample in step 4 was adjusted to 12h, and the method of eluting adherent bacteria in step 6 was changed to vortex shaking for 2min. The anti-adhesion efficiency of the sample was calculated to be 81.33%.

[0043] Example 4 The operation steps of this example are the same as those of Example 1, except that the concentration of the inoculum in step 3 is adjusted to 6×10 5 cfu / mL, the preparation solution was 1 / 500 nutrient broth, and the incubation time of the inoculation solution and the sample in step 4 was adjusted to 18h. The anti-adhesion efficiency of the sample was calculated to be 83.57%.

[0044] Example 5 The operation steps of this example are the same as those of Example 1, except that the concentration of the inoculum in step 3 is adjusted to 6×10 4 cfu / mL, and the incubation time of the inoculum and sample in step 4 was adjusted to 24h. The anti-adhesion efficiency of the sample was calculated to be 87.79%.

[0045] Example 6 The operation steps of this example are the same as those of Example 1, except that the type of bacteria in step 3 is changed from Escherichia coli strain (ATCC 8099) to Staphylococcus aureus strain (ATCC 6538P), and the concentration of the inoculum remains unchanged. The anti-adhesion efficiency of the sample is calculated to be 82.53%.

[0046] Example 7 The operation steps of this example are the same as those of Example 1, except that the type of bacteria in step 3 is changed from Escherichia coli strain (ATCC 8099) to Staphylococcus aureus strain (ATCC 6538P), and the concentration of the inoculum is adjusted to 6×10 4 cfu / mL, the incubation time in step 4 was adjusted to 18h, and the method of cleaning the floating bacteria in step 5 was changed to immerse once in a beaker filled with sterile saline, replace the saline, and then continue immersing, and repeat three times. The anti-adhesion efficiency of the sample was calculated to be 80.22%.

[0047] Comparative Example 1 The operation steps of this comparative example are the same as those of Example 1, except that the concentration of the inoculum in step 3 is adjusted to 6×10 3 cfu / mL. As a result, the number of adherent bacteria detected by ultrasound on the sample was small, and the error between parallel samples was large and not statistically significant. Therefore, it was impossible to determine whether the sample had anti-bacterial adhesion.

[0048] Comparative Example 2 The operation steps of this comparative example are the same as those of Example 1, except that the method of collecting adherent bacteria in step 6 is changed from ultrasound to wiping adherent bacteria on the sample surface with a sterile cotton swab, and then the head of the cotton swab is cut off and placed in a test tube containing physiological saline, and vortexed to disperse the bacteria evenly. However, the resistance is large when wiping adherent bacteria, and some cotton fibers will adhere to the sample tube, resulting in large errors between parallel samples in the colony counting results.

[0049] Comparative Example 3 The operation steps of this comparative example are the same as those of Example 1, except that the type of bacteria in step 3 is changed from Escherichia coli strain (ATCC 8099) to Staphylococcus aureus strain (ATCC 6538P), and the concentration of the inoculum is adjusted to 6×10 9 cfu / mL. As a result, due to the high concentration of bacterial solution, the number of adherent bacteria detected by ultrasound from the sample was too large, and the error between parallel runs was large, so it was impossible to determine whether the sample had anti-bacterial adhesion. The growth of adherent bacteria was as follows: Fig. 9 shown.

[0050] Comparative Example 4 This comparative example adopts conventional test method to test the anti-bacterial adhesion of silicone catheter. The conventional test is carried out with 12-well plate. Fig.10 The specific steps are as follows: Step 1: Prepare the sample, cut the sample tube into 1.5cm length, put it in a clean flat dish, then put it in the clean bench, turn on the UV lamp for UV sterilization, usually the sterilization time is 30min.

[0051] Step 2: Transfer the sterilized samples into a sterile 12-well plate.

[0052] Step 3: Prepare the inoculum. Take out the E. coli strain (ATCC 8099) stored in a 4°C refrigerator and use the turbidimetric method to prepare an initial bacterial solution with a concentration of about 6×10 8 cfu / mL of bacterial suspension, and then diluted 10-fold to 6×10 7 cfu / mL was used as the inoculum, and the solution used was 1 / 10 sodium chloride peptone buffer.

[0053] Step 4: Incubate the sample tube with the inoculation solution. Add an appropriate amount of inoculation solution to a 6-well plate, cover it with a lid, seal it with a sealing film, and then place it in a biochemical incubator and incubate it at 37°C for 6 hours.

[0054] Step 5: Clean the floating bacteria. Place the incubated sample on the clean bench, take out the sample tube, rinse it once in a beaker of sterile saline, then use a pipette to draw 5 ml of saline for rinsing, and repeat the rinsing four times.

[0055] Step 6: Collect the adhered bacteria, absorb the liquid on the cleaned sample tube with filter paper, transfer the sample to a glass test tube containing 10 ml of physiological saline, and elute the adhered bacteria by ultrasonication for 10 minutes (40 kW, 100%).

[0056] Step 7: Count the number of adherent bacteria and calculate the anti-adhesion efficiency. Pipette 1 mL of the bacterial suspension after ultrasound and add it to the culture dish. Add 15 mL of nutrient agar medium and shake to evenly disperse the bacteria. After the medium solidifies, place it in a biochemical incubator and culture it at 37°C for 24 hours. After the culture is completed, count the colonies and calculate the anti-adhesion efficiency of the sample to be 78.55%. The growth of adherent bacteria is as follows: Fig.11 As shown, the anti-adhesion efficiency is not much different from that in the embodiment, but it can be clearly seen that the parallelism in the control group and the sample group is poor, which may be caused by the uneven contact surface of the sample tube in the bacterial suspension, affecting the accuracy of the test results.

[0057] Comparative Example 5 The operation steps of this comparative example are the same as those of comparative example 4, except that the concentration of the inoculum in step 3 is adjusted to 6×10 6 cfu / mL, the incubation time of the inoculum and sample in step 4 was adjusted to 18h, and the anti-adhesion efficiency of the sample was calculated to be 83.27%. The growth of the adherent bacteria was as follows Fig.12 As shown, the anti-adhesion efficiency is not much different from that in the embodiment, but it can still be clearly seen that the parallelism in the control group and the sample group is poor, which affects the accuracy of the test results.

[0058] The test conditions and test results of Examples 1-7 and Comparative Examples 1-5 are summarized in Table 1: example Type of bacteria Inoculum concentration (cfu / mL) Incubation time (h) Bacteria cleaning Adherent bacteria collection Anti-adhesion efficiency (%) Example 1 Escherichia coli <![CDATA[6×10 7 ]]> 6 Dip 1 Rinse 4 Ultrasound 10min 85.35 Example 2 Escherichia coli <![CDATA[6×10 7 ]]> 6 Dip 3 Vortex for 2 minutes 82.26 Example 3 Escherichia coli <![CDATA[6×10 6 ]]> 12 Dip 1 Rinse 4 Vortex for 2 minutes 81.33 Example 4 Escherichia coli <![CDATA[6×10 5 ]]> 18 Dip 1 Rinse 4 Ultrasound 10min 83.57 Example 5 Escherichia coli <![CDATA[6×10 4 ]]> 24 Dip 1 Rinse 4 Ultrasound 10min 87.79 Example 6 Staphylococcus aureus <![CDATA[6×10 7 ]]> 6 Dip 1 Rinse 4 Ultrasound 10min 82.53 Example 7 Staphylococcus aureus <![CDATA[6×10 4 ]]> 18 Dip 3 Ultrasound 10min 80.22 Comparative Example 1 Escherichia coli <![CDATA[6×10 3 ]]> 6 Dip 1 Rinse 4 Ultrasound 10min / Comparative Example 2 Escherichia coli <![CDATA[6×10 7 ]]> 6 Dip 3 Cotton swab / Comparative Example 3 Staphylococcus aureus <![CDATA[6×10 9 ]]> 6 Dip 1 Rinse 4 Ultrasound 10min / Comparative Example 4 Escherichia coli <![CDATA[6×10 7 ]]> 6 Dip 1 Rinse 4 Ultrasound 10min 78.55 Comparative Example 5 Escherichia coli <![CDATA[6×10 6 ]]> 18 Dip 1 Rinse 4 Ultrasound 10min 83.27 Table 1 shows that in Example 1, a commercially available anti-bacterial adhesion silicone catheter was used for method verification, and a common silicone catheter without coating was used as a control to verify its anti-adhesion performance against Escherichia coli. The concentration of the inoculum of Escherichia coli was 6×10 7 cfu / mL, the incubation time was 6h, the floating bacteria cleaning method was immersion 1 time and rinse 4 times, and the adhesion bacteria were collected by ultrasound for 10min. The anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli was 85.35%. Example 2 Based on Example 1, the floating bacteria cleaning method and the adhesion bacteria collection method were adjusted to immersion 3 times and vortex 2min respectively, and the anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli was 82.26%. Example 3 Based on Example 1, the concentration of the Escherichia coli inoculum was reduced by one order of magnitude, the incubation time was 12h, and the adhesion bacteria collection method was adjusted to vortex 2min. The anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli was 81.33%. Example 4 Based on Example 1, the concentration of the Escherichia coli inoculum was reduced by two orders of magnitude, and the incubation time was extended to 18h. The anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli was 83.57%. Example 5 Based on Example 4, the concentration of the E. coli inoculum was changed to 6×10 4 cfu / mL, the incubation time was extended to 24h, and the anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli was measured to be 87.79%. The difference between Example 6 and Example 1 is that Escherichia coli is replaced with Staphylococcus aureus, and the anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli is measured to be 82.53%. In Example 7, the inoculum concentration of Staphylococcus aureus is 6×10 4 cfu / mL, the incubation time was 18h, the floating bacteria cleaning method was immersion 3 times, and the adherent bacteria were collected by ultrasound for 10min. The anti-adhesion rate of the purchased anti-bacterial adhesion silicone catheter to Escherichia coli was measured to be 80.22%. The difference between Comparative Example 1 and Example 1 is that the concentration of the inoculum in step 3 is adjusted to 6×10 3 cfu / mL. Since the inoculum concentration is too low, the experimental results are not statistically significant, so it is impossible to determine whether the sample has anti-bacterial adhesion. The difference between Comparative Example 2 and Example 1 is that the floating bacteria cleaning method and the adherent bacteria collection method are changed. Since Comparative Example 2 uses a cotton swab wiping method, the resistance is large when wiping the adherent bacteria, and some cotton fibers will adhere to the sample tube, and the colony count results have large errors between parallel samples. The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses a higher concentration of 6×10 9cfu / mL of Staphylococcus aureus was tested for anti-adhesion. Due to the high concentration of the inoculum, the anti-adhesion ability of the sample itself was exceeded, and the colony count could not be performed, and the experimental results were meaningless. In Comparative Examples 4 and 5, a 12-well plate was used for incubation, and the sample was not in sufficient contact with the inoculum. The inoculum was always in a static state. Due to the effect of gravity, the concentrations of the upper and lower inoculum were uneven, which ultimately led to poor parallelism of the experimental data.

[0059] The above results show that the anti-bacterial adhesion efficiency of catheter samples is tested by the test method provided by the present invention in combination with the incubation device of the present invention, and the test results are relatively stable and consistent with the product promotion effect.

[0060] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An inoculation liquid incubation device, characterized in that: It comprises a box body (1) for containing inoculated liquid, a stirring portion (7) and a support frame (4) installed at the bottom of the box body (1), wherein a support shaft (3) is installed at the top of the support frame (4); and a box cover (8) covers the surface of the box body (1).

2. The inoculation liquid incubation device according to claim 1, characterized in that: The stirring portion (7) comprises a magnetic stirring drive device, the upper surface of which is embedded in the box body (1) and is flush with the bottom surface of the box body (1).

3. The inoculation liquid incubation device according to claim 1, characterized in that: The support shaft (3) comprises a cylinder, the length of which is greater than the length of the catheter to be tested.

4. The inoculation liquid incubation device according to claim 1, characterized in that: A sterilizer is installed on the side of the box cover (8) close to the inoculation liquid, and the sterilizer includes an ultraviolet lamp.

5. An application of the inoculation liquid incubation device according to any one of claims 1 to 4 in the anti-bacterial adhesion performance test of medical catheters, characterized in that: During the test, the support shaft (3) is inserted into the catheter to be tested and then immersed in the box (1) to complete the inoculation and incubation of bacteria; the box (1) contains an inoculation solution that keeps the bacteria in a uniform suspended state.

6. A method for testing the anti-bacterial adhesion performance of a medical catheter, mainly comprising the steps of preparing an inoculation solution, incubating a catheter to be tested, and testing the anti-adhesion performance, characterized in that: The catheter to be tested incubation step is mainly performed by placing the catheter to be tested in the inoculation liquid incubation device according to any one of claims 1-4 to complete the inoculation and incubation of bacteria.

7. The method for testing the anti-bacterial adhesion performance of medical catheters according to claim 6, characterized in that: The incubation steps of the catheter to be tested mainly include: (a) sterilizing the inoculation liquid incubation device and the catheter to be tested; (b) inserting the support shaft (3) into the catheter to be tested and placing it on the support frame (4); (c) adding the inoculation liquid into the box body (1) and completely submerging the catheter to be tested, covering the box cover (8), sealing with a sealing film, placing the incubation device in a biochemical incubator, and controlling the stirring part to continuously stir at a uniform speed for incubation at 37±1°C for 6-24 hours.

8. The method for testing the anti-bacterial adhesion performance of medical catheters according to claim 6, characterized in that: The inoculation solution preparation process includes: inoculating the III generation colony into 1 / 10 sodium chloride peptone buffer, and using turbidimetry to determine the concentration of the bacterial solution to be (6±0.2)×10 8 cfu / mL, dilute the bacterial solution to 6×10 4 ~6×10 7 cfu / mL is obtained.

9. The method for testing the anti-bacterial adhesion performance of medical catheters according to claim 6, characterized in that: The anti-adhesion performance test steps mainly include: (d) using physiological saline to clean the incubated catheter to be tested 3-8 times; (e) placing the cleaned catheter in physiological saline and ultrasonicating for 10±1min to obtain a bacterial suspension; (f) diluting the bacterial suspension, then taking 1mL and adding it to a disposable sterile culture dish, pouring the nutrient agar culture medium cooled to 40-45°C after sterilization into the culture dish and evenly dispersing the bacteria; (g) after the culture medium solidifies, placing it in a biochemical incubator and culturing it at 37±1°C for 24±2h, counting the grown bacterial colonies, and calculating the anti-bacterial adhesion efficiency of the sample.

10. The method for testing the anti-bacterial adhesion performance of medical catheters according to claim 6, characterized in that: The bacteria in the inoculation solution include any one of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus and Pseudomonas aeruginosa; and the components outside the bacteria include 1 / 10 sodium chloride peptone buffer or 1 / 500 nutrient broth.

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