Method for detecting the whole-cycle bacteria blocking performance of a sterile connector machine

Abstract

This invention provides a method for testing the antibacterial performance of a sterile pipe-connecting machine throughout the welding process. The method includes preparing a solution of *Bacillus stearothermophilus* and a solution of *Pseudomonas aeruginosa*. A first test group is established, where the outer wall of the pipe and the blade of the pipe-connecting machine are stained with *Bacillus stearothermophilus* before welding and incubated at a first temperature. A second test group is established, where the weld seam of the pipe is immersed in *Pseudomonas aeruginosa* solution for a preset time and then incubated at a second temperature. Corresponding positive and negative control groups are set up. The antibacterial performance is determined by observing the turbidity of the culture medium in each group. This invention simulates two extreme conditions—instantaneous welding contamination and long-term immersion contamination of the weld seam—using a dual-temperature stratified control system. This method simultaneously evaluates the anti-intrusion capability and long-term antibacterial performance of the weld seam, accurately distinguishing the source of contamination and improving the reliability of the test results and the accuracy of problem diagnosis.

Description

Technical Field

[0001] This invention relates to the field of bacterial detection technology, and in particular to a method for testing the antibacterial performance of a sterile pipe welding machine throughout the entire welding process. Background Technology

[0002] Microbial culture, cell therapy, gene drug development, biopharmaceutical bulk solution production, and single-use bioprocess systems have become core process systems. Aseptic control throughout the entire process is a crucial prerequisite for ensuring drug quality, preventing cell sample contamination, and guaranteeing the validity of clinical trial data. In continuous bioprocess operations, it is impossible to pre-compute integrated irradiation sterilization between different single-use pipelines, storage bags, and bioreactors. During production / experimentation, it is essential to disconnect or heat-weld two independently sterilized pipelines offline / online using an aseptic connection machine to achieve closed-loop aseptic transport of liquids, cell suspensions, viral vectors, and protein bulk solutions.

[0003] Current regulations require aseptic piping equipment to undergo bacterial challenge verification to demonstrate that the pipeline maintains its sterility throughout the welding process and post-weld. However, existing testing methods have shortcomings such as incomplete coverage of scenarios, limited simulation of contamination conditions, and inadequate control systems, making it impossible to fully reproduce the two core contamination risks in the production site: ① the risk of instantaneous intrusion of high-concentration microorganisms into the blades and outer walls of the pipeline during welding operations; ② the risk of weld penetration when the pipeline is immersed in a highly contaminated environment for a long period after welding.

[0004] Traditional methods only set up two groups of samples: a positive control and a blank control. They lack stratified controls to distinguish the source of contamination. If bacteria grow on the final sample, it is impossible to determine whether the contamination is caused by sterilization failure of the pipeline itself, instantaneous intrusion during welding, or late-stage penetration of the weld. It is also impossible to optimize the welding parameters of the pipe fitting machine, the blade cleaning process, and the pipeline weld structure in a targeted manner. In addition, traditional methods only use one type of challenge bacteria, which cannot take into account both high-temperature resistance and room-temperature penetration ability. The experimental method is not systematic and cannot simultaneously match two scenarios: instantaneous high temperature and long-term room temperature. Some heat-sensitive bacteria are prone to death or the bacterial solution has insufficient penetration ability, which may cause false negatives. Summary of the Invention In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for testing the antibacterial performance of a sterile pipe fitting machine throughout the welding process, aiming to solve the technical problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for testing the antibacterial performance of a sterile pipe-connecting machine throughout the welding process, comprising the following steps: Preparation of thermophilic Bacillus stearothermophilus and Pseudomonas aeruginosa suspensions; N first sterile tubings are provided. Culture medium is filled into the first sterile tubings and sealed. The outer wall of the first sterile tubings and the blade of the sterile connection machine are contaminated and dried by the thermophilic Bacillus stearothermophilus liquid. The first sterile tubings are disconnected and re-welded by the sterile connection machine. N-1 first sterile tubings are placed at a first temperature for culture to obtain the first experimental group. N second sterile tubings are provided. Culture medium is filled into the second sterile tubings and sealed. The second sterile tubings are disconnected and re-welded using a sterile connection machine. The weld seam of the second sterile tubings is immersed in the Pseudomonas aeruginosa solution for a preset time. Then, N-1 second sterile tubings are placed at a second temperature for incubation to obtain the second experimental group. Add the thermophilic Bacillus stearothermophilus solution to the remaining first sterile tubing and incubate the first sterile tubing at the first temperature to obtain the first positive control group; The Pseudomonas aeruginosa solution was added to the remaining second sterile tubing, and the second sterile tubing was cultured at the second temperature to obtain a second positive control group; Two third sterile tubings are provided. Culture medium is filled into the third sterile tubings and sealed. The third sterile tubings are disconnected and reconnected by a sterile connector. The first third sterile tubing is cultured according to the culture conditions in the first test group to obtain the first negative control group. The second third sterile tubing is cultured according to the culture conditions in the second test group to obtain the second negative control group. Observe the turbidity of the culture medium in the first experimental group, the second experimental group, the first positive control group, the second positive control group, the first negative control group, and the second negative control group. If the culture medium in the first experimental group and the second experimental group shows sterile growth, the culture medium in the first positive control group and the second positive control group shows bacterial growth, and the culture medium in the first negative control group and the second negative control group shows sterile growth, then the pipeline welded by the aseptic pipe-connecting machine is determined to have antibacterial properties.

[0006] According to one aspect of the above technical solution, the specific steps for preparing the thermophilic Bacillus stearothermophilus solution include: Obtain a working strain of Bacillus stearothermophilus; The thermophilic Bacillus working strain was prepared by eluting it with a 0.85%~0.95% sterile sodium chloride solution to obtain a concentration of not less than 10... 6 First working bacterial culture at CFU / mL; The concentration of the first working bacterial solution was determined by the following method: The first working bacterial solution was serially diluted 10-fold. The first working bacterial solutions of each gradient dilution were spread on the first TSA medium plates and incubated at 55℃~57℃ for 48 h~72 h. Select the first TSA culture medium plate corresponding to the required dilution for colony counting, and convert the counting results into the number of colony-forming units per milliliter of the first working bacterial solution.

[0007] According to one aspect of the above technical solution, the specific steps for preparing Pseudomonas aeruginosa suspension include: Obtain a working strain of Pseudomonas aeruginosa; The *Pseudomonas aeruginosa* working strain was prepared by eluting it with a 0.85%–0.95% sterile sodium chloride solution to a concentration of not less than 10. 6 Second working bacterial solution with CFU / mL; The concentration of the second working bacterial solution was determined by the following method: The second working bacterial solution was serially diluted 10-fold. The second working bacterial solutions of each gradient dilution were spread on the second TSA medium plates and incubated at 30℃~35℃ for 24 h~48 h. Select the second TSA culture medium plate corresponding to the required dilution for colony counting, and convert the counting results into the number of colony-forming units per milliliter of the second working bacterial solution.

[0008] According to one aspect of the above technical solution, the number N of the first sterile tubing and the second sterile tubing is 4.

[0009] According to one aspect of the above technical solution, in the step of filling and sealing the first sterile tubing with culture medium, allowing the outer wall of the first sterile tubing and the blade of the sterile tubing machine to be contaminated and dried by the thermophilic Bacillus stearothermophilus liquid, the filling volume of the culture medium is half the volume of the first sterile tubing, and the concentration of the thermophilic Bacillus stearothermophilus liquid used is greater than or equal to 10. 6 CFU / mL, the first temperature is 55℃~57℃, and the first sterile tubing is cultured for ≥7 days.

[0010] According to one aspect of the above technical solution, in the step of filling and sealing the second sterile tubing with culture medium, disconnecting and re-welding the second sterile tubing using a sterile connection machine, and immersing the weld seam of the welded second sterile tubing in the *Pseudomonas aeruginosa* solution for a preset time, the volume of culture medium filled is half the volume of the second sterile tubing, and the concentration of the *Pseudomonas aeruginosa* solution used is greater than or equal to 10. 6The concentration of CFU / mL, the preset time is 4 hours, the second temperature is 30℃~35℃, and the culture time of the second sterile tubing is greater than or equal to 7 days.

[0011] According to one aspect of the above technical solution, in the step of adding the thermophilic Bacillus stearothermophilus solution to the remaining first sterile tubing and culturing the first sterile tubing at the first temperature, 0.1 mL of the thermophilic Bacillus stearothermophilus solution is added, and the culturing time is less than or equal to 7 days.

[0012] According to one aspect of the above technical solution, in the step of adding the Pseudomonas aeruginosa solution to the remaining second sterile tubing and culturing the second sterile tubing at the second temperature, 0.1 mL of the Pseudomonas aeruginosa solution is added, and the culturing time is less than or equal to 7 days.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: By setting up a first experimental group simulating a high-bacterial-volume contamination scenario during welding operations and a second experimental group simulating a long-term immersion contamination limit scenario after welding, a single detection method was developed to simultaneously evaluate the aseptic protection capability of the aseptic pipe fitting machine during welding and the long-term bacterial penetration barrier capability of the weld. Furthermore, for the challenge bacteria species and incubation temperatures corresponding to each of the two experimental groups, independent first and second negative control groups were established, ensuring that each experimental group has its own dedicated negative baseline reference. This allows for the elimination of interference from residual sterilization of the pipeline itself or basic contamination introduced by the operation under various incubation conditions, accurately distinguishing whether the contamination originates from instantaneous intrusion during welding, later penetration of the weld, or failure of basic pipeline sterilization. Combined with the independent verification of the first and second positive control groups, the four control groups and the two experimental groups form a complete parallel logical system, significantly improving the reliability, repeatability, and accuracy of problem diagnosis of the detection results. Attached Figure Description

[0014] Figure 1 This is a flowchart of the method for detecting the antibacterial performance of the aseptic pipe fitting machine throughout the welding process in the first embodiment of the present invention; The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0015] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0016] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0018] Please see Figure 1 The image shows a method for testing the antibacterial performance of a sterile pipe-connecting machine throughout the welding process, as described in the first embodiment of the present invention. The method includes the following steps: S10, prepare thermophilic Bacillus stearothermophilus and Pseudomonas aeruginosa suspensions; S20, provide N first sterile tubing, fill the first sterile tubing with culture medium and seal it, allow the outer wall of the first sterile tubing and the blade of the sterile connection machine to be contaminated and dried by the thermophilic Bacillus stearothermophilus liquid, disconnect and re-weld the first sterile tubing by the sterile connection machine, place N-1 first sterile tubings in a first temperature for culture, and obtain the first test group; S30, provide N second sterile tubing, fill the second sterile tubing with culture medium and seal it, disconnect and reconnect the second sterile tubing using a sterile connection machine, immerse the weld of the second sterile tubing in the Pseudomonas aeruginosa solution for a preset time, and then place N-1 second sterile tubings in a second temperature for incubation to obtain the second experimental group; S40, add the thermophilic Bacillus stearothermophilus solution to the remaining first sterile tubing, and culture the first sterile tubing at the first temperature to obtain the first positive control group; S50, add the Pseudomonas aeruginosa solution to the remaining second sterile tubing, and incubate the second sterile tubing at the second temperature to obtain a second positive control group; S60, two third sterile tubings are provided, culture medium is filled into the third sterile tubings and sealed, the third sterile tubings are disconnected and re-welded using a sterile connection machine, the first third sterile tubing is cultured according to the culture conditions in the first test group to obtain the first negative control group, and the second third sterile tubing is cultured according to the culture conditions in the second test group to obtain the second negative control group; S70, observe the turbidity of the culture medium in the first experimental group, the second experimental group, the first positive control group, the second positive control group, the first negative control group, and the second negative control group. If the culture medium in the first experimental group and the second experimental group is sterile, the culture medium in the first positive control group and the second positive control group is bacterial, and the culture medium in the first negative control group and the second negative control group is sterile, then the pipeline welded by the aseptic pipe-connecting machine is determined to have antibacterial properties.

[0019] Understandably, this invention achieves a single testing method to simultaneously evaluate the aseptic protection capability of the aseptic pipe fitting machine during the welding process and the long-term bacterial penetration resistance capability of the weld by setting up a first experimental group to simulate the instantaneous high bacterial contamination scenario during welding and a second experimental group to simulate the extreme scenario of long-term immersion contamination of the weld after welding. Simultaneously, for the challenge bacteria species and incubation temperatures corresponding to each of the two experimental groups, independent first and second negative control groups are set up, ensuring that each experimental group has its own exclusive negative benchmark. This allows for the elimination of interference from basic contamination caused by residual sterilization of the pipeline itself or introduced by the operation under various incubation conditions, accurately distinguishing whether the contamination originates from instantaneous intrusion during welding, later penetration of the weld, or failure of basic sterilization of the pipeline. Combined with the independent verification of the first and second positive control groups, the four control groups and the two experimental groups form a complete parallel logical system, significantly improving the reliability, repeatability, and accuracy of problem diagnosis of the test results.

[0020] Furthermore, the specific steps for preparing the thermophilic Bacillus stearothermophilus solution include: Obtain a working strain of Bacillus stearothermophilus; The thermophilic Bacillus working strain was prepared by eluting it with a 0.85%~0.95% sterile sodium chloride solution to obtain a concentration of not less than 10... 6 The first working bacterial culture was prepared at CFU / mL; the optimal value was 0.9% sterile sodium chloride solution. The concentration of the first working bacterial solution was determined by the following method: The first working bacterial solution was serially diluted 10-fold. The first working bacterial solutions of each gradient dilution were spread on the first TSA medium plates and incubated at 55℃~57℃ for 48 h~72 h; 56℃ was the optimal value. Select the first TSA culture medium plate corresponding to the required dilution for colony counting, and convert the counting results into the number of colony-forming units per milliliter of the first working bacterial solution.

[0021] Understandably, this step clarifies the specific preparation parameters and concentration counting methods for *Bacillus stearothermophilus* bacterial suspension. The 0.85%–0.95% sodium chloride solution concentration range ensures osmotic pressure balance and maintains bacterial activity during elution, while avoiding interference from excessively high salt concentrations in subsequent experiments. The 55℃–57℃ culture temperature closely matches the optimal growth temperature of *Bacillus stearothermophilus*, ensuring the accuracy of the counting results. The standardized protocol of 10-fold serial dilution combined with TSA plate counting ensures high reproducibility of bacterial suspension concentration data across different laboratories and operators, providing a reliable data foundation for accurate control of contamination levels in subsequent challenge experiments.

[0022] Furthermore, the specific steps for preparing Pseudomonas aeruginosa suspension include: Obtain a working strain of Pseudomonas aeruginosa; The *Pseudomonas aeruginosa* working strain was prepared by eluting it with a sterile sodium chloride solution of 0.85%~0.95% (preferably 0.9%) to obtain a concentration of *Pseudomonas aeruginosa* of not less than 10. 6 Second working bacterial solution with CFU / mL; The concentration of the second working bacterial solution was determined by the following method: The second working bacterial solution was serially diluted 10-fold. The second working bacterial solutions of each gradient dilution were spread onto second TSA medium plates and incubated at 30℃~35℃ (preferably 32.5℃) for 24 h~48 h. Select the second TSA culture medium plate corresponding to the required dilution for colony counting, and convert the counting results into the number of colony-forming units per milliliter of the second working bacterial solution.

[0023] Understandably, specific parameters for bacterial suspension preparation and counting were set for the growth characteristics of *Pseudomonas aeruginosa*. The culture temperature of 30℃~35℃ matches the optimal growth temperature of *Pseudomonas aeruginosa*, ensuring the accuracy of bacterial suspension concentration counting. The culture time of 24h~48h ensures that the colonies are fully visible while avoiding counting deviations caused by over-culture. This counting method is independent of the method used to prepare *Bacillus stearothermophilus* suspension and forms a separate system. This allows the concentration control of the two challenge bacteria to be optimized according to their respective biological characteristics, solving the technical problems of inaccurate counting and incomparable challenge concentrations caused by the use of a set of parameters for different bacterial species in traditional methods.

[0024] Furthermore, the number N of the first sterile tubing and the second sterile tubing is 4.

[0025] Understandably, limiting N to 4, with 3 tubes used for experimental group culture to provide statistically significant parallel replicates and the remaining 1 tube used for the positive control experiment, ensures that each experiment has a sufficient number of replicates to meet the data reliability requirements of microbial challenge experiments, while avoiding the waste of consumables and cumbersome operation caused by excessive sample size, achieving an optimal balance between experimental reliability and cost control. At the same time, this setting allows the experimental group and the positive control group to originate from the same batch of treatment tubing, eliminating additional variables introduced by batch differences in tubing and further improving the internal consistency of experimental results.

[0026] Furthermore, in step S20, the volume of the culture medium is half the volume of the first sterile tubing, and the concentration of the thermophilic Bacillus stearothermophilus solution used is greater than or equal to 10. 6 CFU / mL, the first temperature is 55℃~57℃ (preferably 56℃), and the first sterile tubing is cultured for ≥7 days.

[0027] Understandably, the culture medium filling volume is half the tubing volume. This ensures sufficient liquid culture medium for observing microbial growth (turbidity assessment) while also reserving ample headspace to support the proliferation of aerobic or facultative anaerobic microorganisms. Overfilling prevents oxygen deficiency, which could inhibit challenge bacteria growth and cause false negatives. The concentration of *Bacillus stearothermophilus* culture is ≥10. 6 The CFU / mL concentration provides a high-concentration challenge far exceeding that of actual production environments, setting a stringent validation threshold for the aseptic connection machine's sterility assurance capabilities. The first temperature of 55℃~57℃ precisely matches the thermophilic characteristics of Bacillus stearothermophilus, and the incubation period of more than 7 days meets the pharmacopoeia's routine requirements for sterility testing, ensuring that any trace amount of live bacteria contamination can be fully proliferated to an observable level, effectively eliminating the risk of false negatives.

[0028] Furthermore, in step S30, the volume of the culture medium is half the volume of the second sterile tubing, and the concentration of the *Pseudomonas aeruginosa* solution used is greater than or equal to 10. 6 The concentration of CFU / mL is specified, the preset time is 4 hours, the second temperature is 30℃~35℃ (preferably 32.5℃), and the culture time for the second sterile tubing is greater than or equal to 7 days.

[0029] Understandably, the preset immersion time is set to 4 hours. This duration simulates the extreme exposure conditions of pipeline welds in a highly polluted environment for extended periods in production sites, while also taking into account experimental efficiency and avoiding excessively long test cycles due to excessive immersion time. The second temperature of 30℃~35℃ precisely matches the optimal growth temperature of Pseudomonas aeruginosa. Compared to traditional methods that rely solely on high-temperature culture, this temperature setting can more realistically reproduce the proliferation patterns of microorganisms in a normal-temperature environment in production workshops, avoiding false negative results caused by inactivation or slow growth of target bacteria due to temperature mismatch. The culture time of more than 7 days ensures that low-level penetrating contamination can be fully detected, significantly improving the sensitivity and real-world fit of the long-term antibacterial performance verification of welds.

[0030] Furthermore, in step S40, the culture temperature is the same as that of the first experimental group, which is 56°C, and 0.1 mL of the thermophilic Bacillus stearothermophilus solution is added, with a culture time of less than or equal to 7 days; in step S50, the culture temperature is the same as that of the second experimental group, which is 32.5°C, and 0.1 mL of the Pseudomonas aeruginosa solution is added, with a culture time of less than or equal to 7 days.

[0031] Understandably, these two steps limit the amount of bacteria added and the incubation time for the first and second positive control groups. The 0.1 mL bacterial addition, combined with the corresponding incubation temperature for the experimental group, ensures that *Bacillus stearothermophilus* and *Pseudomonas aeruginosa* proliferate rapidly and sufficiently in TSB medium, reaching the visually identifiable positive criterion (significant turbidity of the medium) within a ≤7-day incubation period. This avoids delays or failures in the positive control due to insufficient bacterial addition or temperature mismatch. The first and second positive control groups operate independently and in parallel, respectively verifying the effectiveness of the two experimental systems with different temperatures and bacterial species.

[0032] Finally, the data from the experimental group, positive group, and negative group were used to accurately determine the source of contamination. The first experimental group represented instantaneous welding contamination, the second experimental group represented weld immersion and penetration contamination, the negative control group represented the culture system corresponding to the first and second experimental groups, proving the sterility of the pipeline and equipment, and the positive control group proved that the culture medium could support the growth of the strain and ruled out false negatives due to culture medium failure.

[0033] The pipeline welded by the aseptic pipe-connecting machine is deemed to have antibacterial properties only if the culture medium in the first and second test groups is sterile, the culture medium in the first and second positive control groups is visibly growing, and the culture medium in the first and second negative control groups is sterile.

[0034] If bacteria grow in the negative control group, it indicates that the basic sterilization of the pipeline / equipment has failed; if the positive control group is sterile, it indicates that the culture medium / culture conditions have failed; if bacteria grow in the first test group, it indicates that the instantaneous sealing of the weld has failed; if bacteria grow in the second test group, it indicates that the weld seam has insufficient antibacterial ability. This invention can achieve precise fault location.

[0035] In summary, the method for testing the antibacterial performance of the aseptic pipe fitting machine throughout the welding process in the above embodiments of the present invention simulates two extreme working conditions: instantaneous contamination and long-term immersion, using a dual-test group, combined with a dual-temperature stratified control system, to achieve integrated evaluation of anti-intrusion during the welding process and antibacterial properties of the weld, as well as precise tracing of the source of contamination. In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0036] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for testing the antibacterial performance of a sterile pipe fitting machine throughout the welding process, characterized in that, Includes the following steps: Preparation of thermophilic Bacillus stearothermophilus and Pseudomonas aeruginosa suspensions; N first sterile tubings are provided. Culture medium is filled into the first sterile tubings and sealed. The outer wall of the first sterile tubings and the blade of the sterile connection machine are contaminated and dried by the thermophilic Bacillus stearothermophilus liquid. The first sterile tubings are disconnected and re-welded by the sterile connection machine. N-1 first sterile tubings are placed at a first temperature for culture to obtain the first experimental group. N second sterile tubings are provided. Culture medium is filled into the second sterile tubings and sealed. The second sterile tubings are disconnected and re-welded using a sterile connection machine. The weld seam of the second sterile tubings is immersed in the Pseudomonas aeruginosa solution for a preset time. Then, N-1 second sterile tubings are placed at a second temperature for incubation to obtain the second experimental group. Add the thermophilic Bacillus stearothermophilus solution to the remaining first sterile tubing and incubate the first sterile tubing at the first temperature to obtain the first positive control group; The Pseudomonas aeruginosa solution was added to the remaining second sterile tubing, and the second sterile tubing was cultured at the second temperature to obtain a second positive control group; Two third sterile tubings are provided. Culture medium is filled into the third sterile tubings and sealed. The third sterile tubings are disconnected and reconnected by a sterile connector. The first third sterile tubing is cultured according to the culture conditions in the first test group to obtain the first negative control group. The second third sterile tubing is cultured according to the culture conditions in the second test group to obtain the second negative control group. Observe the turbidity of the culture medium in the first experimental group, the second experimental group, the first positive control group, the second positive control group, the first negative control group, and the second negative control group. If the culture medium in the first experimental group and the second experimental group shows sterile growth, the culture medium in the first positive control group and the second positive control group shows bacterial growth, and the culture medium in the first negative control group and the second negative control group shows sterile growth, then the pipeline welded by the aseptic pipe-connecting machine is determined to have antibacterial properties.

2. The method for testing the sterile pipe welding performance throughout the welding cycle of the aseptic pipe fitting machine according to claim 1, characterized in that, The specific steps for preparing thermophilic Bacillus stearothermophilus solution include: Obtain a working strain of Bacillus stearothermophilus; The thermophilic Bacillus working strain was prepared by eluting it with a 0.85%~0.95% sterile sodium chloride solution to obtain a concentration of not less than 10... 6 First working bacterial culture at CFU / mL; The concentration of the first working bacterial solution was determined by the following method: The first working bacterial solution was serially diluted 10-fold. The first working bacterial solutions of each gradient dilution were spread on the first TSA medium plates and incubated at 55℃~57℃ for 48 h~72 h. Select the first TSA culture medium plate corresponding to the required dilution for colony counting, and convert the counting results into the number of colony-forming units per milliliter of the first working bacterial solution.

3. The method for testing the antibacterial performance of the aseptic pipe welding machine throughout the welding process according to claim 1, characterized in that, The specific steps for preparing Pseudomonas aeruginosa suspension include: Obtain a working strain of Pseudomonas aeruginosa; The *Pseudomonas aeruginosa* working strain was prepared by eluting it with a 0.85%–0.95% sterile sodium chloride solution to a concentration of not less than 10. 6 Second working bacterial solution with CFU / mL; The concentration of the second working bacterial solution was determined by the following method: The second working bacterial solution was serially diluted 10-fold. The second working bacterial solutions of each gradient dilution were spread on the second TSA medium plates and incubated at 30℃~35℃ for 24 h~48 h. Select the second TSA culture medium plate corresponding to the required dilution for colony counting, and convert the counting results into the number of colony-forming units per milliliter of the second working bacterial solution.

4. The method for testing the antibacterial performance of the aseptic pipe welding machine throughout the welding process according to claim 1, characterized in that, The number N of the first sterile tubing and the second sterile tubing is 4.

5. The method for testing the antibacterial performance of the aseptic pipe welding machine throughout the welding process according to claim 1, characterized in that, The step involves filling and sealing the first sterile tubing with culture medium, allowing the outer wall of the first sterile tubing and the blades of the sterile connector to be contaminated and dried by the thermophilic Bacillus stearothermophilus solution. In this step, the volume of culture medium filled is half the volume of the first sterile tubing, and the concentration of the thermophilic Bacillus stearothermophilus solution used is greater than or equal to 10. 6 CFU / mL, the first temperature is 55℃~57℃, and the first sterile tubing is cultured for ≥7 days.

6. The method for testing the antibacterial performance of the aseptic pipe welding machine throughout the welding process according to claim 1, characterized in that, The process involves filling the second sterile tubing with culture medium and sealing it. Then, using a sterile connection machine, the second sterile tubing is disconnected and reconnected by welding. The weld seam of the reconnected second sterile tubing is immersed in the *Pseudomonas aeruginosa* solution for a preset time. In this step, the volume of culture medium filled is half the volume of the second sterile tubing, and the concentration of the *Pseudomonas aeruginosa* solution used is greater than or equal to 10⁻⁶. 6 The concentration of CFU / mL, the preset time is 4 hours, the second temperature is 30℃~35℃, and the culture time of the second sterile tubing is greater than or equal to 7 days.

7. The method for testing the antibacterial performance of the aseptic pipe welding machine throughout the welding process according to claim 1, characterized in that, The step involves adding the thermophilic Bacillus stearothermophilus solution to the remaining first sterile tubing and culturing the first sterile tubing at the first temperature. In this step, 0.1 mL of the thermophilic Bacillus stearothermophilus solution is added, and the culturing time is less than or equal to 7 days.

8. The method for testing the antibacterial performance of the aseptic pipe welding machine throughout the welding cycle according to claim 1, characterized in that, The step involves adding the Pseudomonas aeruginosa solution to the remaining second sterile tubing and culturing the second sterile tubing at the second temperature. In this step, 0.1 mL of the Pseudomonas aeruginosa solution is added, and the culturing time is less than or equal to 7 days.

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