Non-toxicity verification method of medical sterile dissolving agent

Abstract

In order to solve the problems that a non-toxicity verification method of a sterile dissolving agent in the prior art cannot fully indicate whether the sterile dissolving agent is toxic or not, and a to-be-verified dissolving agent is toxic and also can be washed away by washing liquid, the invention provides a non-toxicity verification method of a medical sterile dissolving agent. A 0.1% sterile peptone aqueous solution specified in Chinese pharmacopoeia is used as a contrast solvent; adopting one or more of seven sensitive strains as test bacteria for verification test; respectively adding the bacterial suspension containing the test bacteria into the to-be-verified dissolving agent and the contrast dissolving agent under the same condition; respectively acquiring test bacteria in the to-be-verified dissolving agent and the contrast dissolving agent by adopting a filtering method, and culturing; and judging whether the to-be-verified dissolving agent has toxicity or not according to the growth condition of the test bacteria obtained from the two dissolving agents. The method has the beneficial technical effects that the non-toxicity of the dissolving agent can be effectively verified, and the problems that the to-be-verified dissolving agent is toxic and possibly washed away by washing liquid and the like are avoided.

Description

Technical Field

[0001] The invention relates to a non-toxicity verification technology of a medical sterile dissolving agent, and in particular to a non-toxicity verification method of a medical sterile dissolving agent. Background Art

[0002] The chapter on sterility inspection of test samples in Chapter 1101 of the 2020 edition of the Chinese Pharmacopoeia, "Sterility Inspection", stipulates that if surfactants, inactivators, neutralizers, etc. are required during the sterility test, their effectiveness should be proven and they should be non-toxic to microorganisms. However, the Chinese Pharmacopoeia does not specify or explain how to verify the non-toxicity of these surfactants, inactivators, neutralizers, etc. (hereinafter collectively referred to as dissolving agents) to microorganisms. For example, the sterile solution of sodium heparin, a dissolving agent for insulin, needs to be verified for non-toxicity to ensure the effectiveness and non-toxicity of the prepared insulin solution. In actual operation, the non-toxicity of the solvent is usually verified by actual cultivation, that is, adding test bacteria to the solvent to be verified and culturing it, and judging whether the solvent to be verified is non-toxic by whether the test bacteria grows; including: (1) mixing the test sample with the solvent to be verified and dissolving it until it is clear to obtain a mixed solution; (2) filtering the mixed solution by membrane filtration; (3) washing the filter membrane twice with a washing solution, 100 ml / membrane each time; (4) adding a test bacteria with a colony count of no more than 100 cfu to 100 ml of the washing solution, and immediately filtering it with the filter membrane washed in the previous step; (5) adding thioglycollate fluid culture medium Qualitative test of FTM / tryptic soy bean liquid medium TSB; (6) Culture ≤ 5 days; Among them, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Clostridium sporogenes select medium FTM, 30-35℃; Bacillus subtilis, Candida albicans and Aspergillus niger select medium TSB, 20-25℃ (the selection of the above strains is based on Chinese Pharmacopoeia 1101 "Sterility Test" (2020 Edition) and European Pharmacopoeia 2.6.1 "Sterility Test"); (7) Observe whether the bacteria collector has test bacteria growth within 5 days; if the test bacteria grows, the dissolving agent to be verified is considered to be non-toxic; otherwise, the dissolving agent to be verified is considered to be toxic. The bacteria collector is a closed membrane filter, which can effectively reduce the risk of contamination in the sampling, dissolution, dilution, filtration, flushing, access to the culture medium and culture process during aseptic operation. However, it can be seen from the above verification process that the test bacteria were added to the filter membrane after the mixed solution of the test sample and the dissolving agent to be verified was filtered and rinsed twice. Although the filter membrane had been used to filter the mixed solution of the dissolving agent to be verified and the test sample before, after being rinsed with the rinse solution twice, even if the dissolving agent to be verified is toxic, it may be rinsed away, thereby not inhibiting the growth of the test bacteria, and thus concluding that the dissolving agent is non-toxic.

[0003] Obviously, the existing method for verifying the non-toxicity of sterile solvents cannot fully explain whether the sterile solvents are toxic, and there may be problems such as the solvent to be verified being toxic and being washed away by the flushing solution. Summary of the invention

[0004] In order to solve the problems of the prior art non-toxicity verification method of sterile dissolving agents, such as failure to fully indicate whether the sterile dissolving agents are toxic, and the possibility that the dissolving agents to be verified are toxic may also be washed away by the flushing solution, the present invention proposes a non-toxicity verification method for medical sterile dissolving agents.

[0005] The non-toxicity verification method of the medical sterile dissolving agent of the present invention adopts a 0.1% sterile peptone aqueous solution specified in the Chinese Pharmacopoeia as a comparative dissolving agent; adopts one or more of seven sensitive bacterial species as test bacteria for verification test; adds a bacterial suspension containing the test bacteria to the dissolving agent to be verified and the comparative dissolving agent under the same conditions; adopts a filtration method to obtain the test bacteria in the dissolving agent to be verified and the comparative dissolving agent respectively and culture them; judges whether the dissolving agent to be verified is toxic according to the growth of the test bacteria obtained in the two dissolving agents; wherein the seven sensitive bacterial species include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Candida albicans and Aspergillus niger.

[0006] Preferably, when multiple test bacteria are used for validation testing, each test bacteria needs to be cultured and counted separately.

[0007] Preferably, the adding of the bacterial suspension containing the test bacteria under the same conditions comprises adding a bacterial suspension containing the test bacteria with a viable count of <1000 cfu / ml to 50 ml to 80 ml of the verification dissolving agent and the comparison dissolving agent respectively and mixing them to prepare a mixed solution with a viable count of no more than 100 cfu in each 10 ml to 16 ml of solution.

[0008] Preferably, the method of judging whether the dissolving agent to be verified is toxic based on the growth conditions of the test bacteria obtained in the two dissolving agents includes quantitative testing and qualitative testing.

[0009] Preferably, the quantitative test comprises: adding a set amount of the to-be-verified dissolving agent and the comparative dissolving agent to a sterile bottle and shaking them evenly; adding a set amount of test bacteria to the sterile bottle, and obtaining the test bacteria in the to-be-verified dissolving agent and the comparative dissolving agent by filtration at 0, 10 and 20 minutes of standing; transferring the test bacteria to a plate by filter membrane transfer and culturing; and performing the above filtration sampling and culturing test in parallel three times; counting the colonies of the culture results of the to-be-verified dissolving agent and the comparative dissolving agent respectively; and calculating the recovery rate by the following formula (1): : ( = 0, 10 and 20 minutes) (1) In the formula, For the Minutes of recovery of filtration sampling culture; The solvent to be verified The average colony count of three parallel tests per minute, in cfu; For comparison, the solvent The average colony count of three parallel tests is in cfu. When it is between 50% and 200%, the solvent to be verified is considered to be non-toxic.

[0010] Preferably, the test bacteria are transferred to a plate by filter membrane transfer, including: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Clostridium sporogenes are transferred to a pre-made tryptic soy agar medium TSA plate; Candida albicans and Aspergillus niger are transferred to a Sabouraud dextrose agar medium SDA plate.

[0011] Preferably, the qualitative test comprises: adding a set amount of the to-be-verified dissolving agent and the comparison dissolving agent to a bacteria collector and shaking them evenly, adding a set amount of test bacteria to a sterile bottle, standing for 20 to 40 minutes, and then using a filtration method to obtain the test bacteria in the to-be-verified dissolving agent and the comparison dissolving agent; putting the test bacteria back into the bacteria collector and adding corresponding culture media to the bacteria collector according to the type of test bacteria added; wherein Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Clostridium sporogenes are added to the thioglycollate fluid culture medium FTM; Bacillus subtilis, Candida albicans and Aspergillus niger are added to the trypsin soybean culture medium FTM; Peptone liquid culture medium TSB; the bacteria collector is cultured under the set conditions, among which the FTM bacteria collector is cultured at 30-35℃ for ≤5 days, and the TSB bacteria collector is cultured at 20-25℃ for ≤5 days; the bacteria collectors containing the verification mixed solution and the control mixed solution samples are observed daily to see whether there is test bacteria growth; if test bacteria grow in both, the dissolving agent to be verified is deemed to be non-toxic; if test bacteria do not grow in both, it is deemed that the verification has failed; if the former has no test bacteria growth and the latter has test bacteria growth, it is deemed that the dissolving agent to be verified is toxic; if the former has test bacteria growth and the latter does not, it is deemed that the verification has failed.

[0012] The beneficial technical effect of the non-toxicity verification method of the medical sterile dissolving agent of the present invention is that it can effectively verify the non-toxicity of the dissolving agent, avoiding the problem that the dissolving agent to be verified is toxic and may be washed away by the flushing solution; and the operation is simple and the results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Attached Figure 1 A flowchart of the quantitative testing steps of the non-toxicity verification method of the medical sterile dissolving agent of the present invention; Attached Figure 2The present invention is a flowchart of the qualitative testing steps of the non-toxicity verification method of the medical sterile dissolving agent.

[0014] The non-toxicity verification method of the medical sterile dissolving agent of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments. DETAILED DESCRIPTION

[0015] The "filtration method" described in the present invention refers to a method of filtering a solution with a filter membrane to obtain the test bacteria in the solution; if necessary, after filtration, the filter membrane can be rinsed three times with 0.1% sterile peptone aqueous solution as a rinse liquid, with each rinse volume being 100 ml.

[0016] The "filter membrane transfer" mentioned in the present invention refers to transferring the test bacteria contained or possibly contained on the filter membrane to the surface of a plate in a rolling and close contact manner; the plate includes a pre-made tryptic soy agar medium TSA plate and a Sabouraud dextrose agar medium SDA plate.

[0017] The term "cultivation" as used herein refers to placing a plate containing or possibly containing test bacteria in an incubator and allowing the microorganisms to grow for a set period of time under suitable temperature and humidity conditions.

[0018] The non-toxicity verification method of the medical sterile dissolving agent of the present invention adopts 0.1% sterile peptone aqueous solution specified in the Chinese Pharmacopoeia as a comparative dissolving agent; one or more of seven sensitive bacterial species are used as test bacteria for verification test; a bacterial suspension containing test bacteria is added to the dissolving agent to be verified and the comparative dissolving agent under the same conditions; the test bacteria in the dissolving agent to be verified and the comparative dissolving agent are obtained by filtration and cultured; whether the dissolving agent to be verified is toxic is judged according to the growth of the test bacteria obtained in the two dissolving agents; wherein the seven sensitive bacterial species include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Candida albicans and Aspergillus niger. The non-toxicity verification method of the medical sterile dissolving agent of the present invention adopts 0.1% sterile peptone aqueous solution as a comparative dissolving agent and the dissolving agent to be verified to carry out test bacteria culture detection under the same conditions, and the non-toxicity of the dissolving agent to be verified is verified by comparing the test bacteria culture results of the two. Moreover, during the culture process of the test bacteria, the test bacteria are directly added to the dissolving agent to be verified and the comparison dissolving agent, thereby avoiding the possibility that the toxicity of the dissolving agent to be verified may be washed away before the test bacteria are added. The method and steps of adding, obtaining and culturing the test bacteria adopted in the non-toxicity verification method of the medical sterile dissolving agent of the present invention meet the relevant requirements of the Chinese Pharmacopoeia and the European Pharmacopoeia as much as possible, and its experimental equipment and operation methods are commonly used in the field of biotechnology, so that the entire verification operation is simple and the results are reliable. Obviously, the non-toxicity verification method of the medical sterile dissolving agent of the present invention can effectively verify the non-toxicity of the dissolving agent, avoid the occurrence of false negative results of the non-toxicity verification test of the sterile dissolving agent, and is simple to operate and the results are reliable.

[0019] In order to avoid the influence of multiple test bacteria on the test results, as one of the preferred solutions, when multiple test bacteria are used for verification testing, each test bacteria needs to be cultured and counted separately. The mixing of multiple test bacteria not only affects the growth of each test bacteria, but also affects the counting statistics.

[0020] As one of the preferred schemes, the bacterial suspension containing the test bacteria is added under the same conditions, including adding a bacterial suspension containing the test bacteria with a viable count of <1000 cfu / ml to 50ml to 80ml of the verification solvent and the comparison solvent, respectively, and then mixing to prepare a mixed solution containing no more than 100 cfu of viable bacteria in each 10ml to 16ml solution. The number of viable bacteria in the mixed solution is controlled to ensure the accuracy and reliability of the test results while taking into account the convenience of testing. If the number of viable bacteria after culture is too small, the accuracy and reliability of the test results may be affected; if the number of viable bacteria after culture is too large, the colony count is difficult and also affects the accuracy of the count.

[0021] As one of the preferred schemes, the method of judging whether the dissolving agent to be verified is toxic based on the growth of the test bacteria obtained in the two dissolving agents includes quantitative testing and qualitative testing. The quantitative detection method uses the recovery rate as the basis for judgment. In addition to determining whether the dissolving agent to be verified is toxic, it can also provide a reference for the beneficial effects of the dissolving agent to be verified on the growth of the test bacteria. The qualitative detection method, in addition to being simple to operate and having intuitive results, also uses a comparison dissolving agent as a comparison to avoid misjudgment caused by operational errors.

[0022] As one of the preferred technical solutions, the quantitative test includes, respectively, adding a set amount of the to-be-verified dissolving agent and the comparative dissolving agent to a sterile bottle and shaking them evenly; respectively adding a set amount of test bacteria to the sterile bottle, and using a filtration method to obtain the test bacteria in the to-be-verified dissolving agent and the comparative dissolving agent at 0, 10 and 20 minutes of standing; using a filter membrane transfer to transfer the test bacteria to a plate and culture them; and the above-mentioned filtration sampling and culture test is performed in parallel three times; the colony counts are performed on the culture results of the to-be-verified dissolving agent and the comparative dissolving agent; the recovery rate is calculated by the following formula (1): : ( = 0, 10 and 20 minutes) (1) In the formula, For the Minutes of recovery of filtration sampling culture; The solvent to be verified The average colony count of three parallel tests per minute, in cfu; For comparison, the solvent The average colony count of three parallel tests is in cfu. When the concentration is between 50% and 200%, the solvent to be verified is considered to be non-toxic. Filtration sampling in different time periods can be used to observe the growth rate of the test bacteria in different time periods. In addition to verifying the non-toxicity of the solvent to be verified, the growth characteristics of the test bacteria can also be observed.

[0023] As one of the preferred technical solutions, the test bacteria are transferred to the plate by using a filter membrane transfer, including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Clostridium sporogenes are transferred to a pre-made tryptic soy agar medium TSA plate; Candida albicans and Aspergillus niger are transferred to a Sabouraud glucose agar medium SDA plate. Using different plates, culture temperatures and times for different test bacteria is more conducive to the growth of the test bacteria and reduces the impact of culture conditions on the verification results.

[0024] As one of the preferred technical solutions, the qualitative test includes, respectively, adding a set amount of the to-be-verified dissolving agent and the comparison dissolving agent to a bacteria collector and shaking them evenly, respectively adding a set amount of test bacteria to a sterile bottle, and after standing for 20 to 40 minutes, using a filtration method to obtain the test bacteria in the to-be-verified dissolving agent and the comparison dissolving agent respectively; putting the test bacteria back into the bacteria collector and adding corresponding culture media to the bacteria collector according to the type of test bacteria added; wherein Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Clostridium sporogenes are added to the thioglycollate fluid culture medium FTM; Bacillus subtilis, Candida albicans and Aspergillus niger are added Tryptic soy casein broth TSB; culture the bacteria collector under the set conditions, among which the FTM bacteria collector is cultured at 30-35℃ for ≤5 days, and the TSB bacteria collector is cultured at 20-25℃ for ≤5 days; observe the bacteria collectors containing the verification mixed solution and the control mixed solution samples every day to see whether there is test bacteria growth; if test bacteria grow in both, it is regarded as the non-toxicity of the dissolving agent to be verified; if test bacteria do not grow in both, it is regarded as a verification failure; if the former has no test bacteria growth and the latter has test bacteria growth, it is regarded as toxic to the dissolving agent to be verified; if the former has test bacteria growth and the latter does not, it is regarded as a verification failure.

[0025] Example 1: Quantitative verification of the non-toxicity of the dissolving agent 1% ascorbic acid and sodium heparin solution using Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Clostridium sporogenes Take 4 portions of 30ml of the comparative dissolving agent and 50ml of the 1% ascorbic acid solution of the dissolving agent to be verified, and put them into sterile bottles 1# to 4# respectively; take 4 portions of 30ml of the comparative dissolving agent and 25ml of the heparin sodium solution of the dissolving agent to be verified, and put them into sterile bottles 5# to 8# respectively; take 4 portions of 50ml of the comparative dissolving agent and put them into sterile bottles 9# to 12# respectively; add 500μL of the bacterial suspension of Staphylococcus aureus with a colony count of <1000cfu / ml to sterile bottles 1#, 5#, and 9# respectively, and shake to mix; add 500μL of the bacterial suspension of Pseudomonas aeruginosa with a colony count of <1000cfu / ml to sterile bottles 2#, 6#, and 10# respectively, and shake to mix; Add 500 μL of bacterial suspension of Escherichia coli with colony count <1000 cfu / ml to sterile bottles 3#, 7#, and 11#, respectively, and shake to mix; add 500 μL of bacterial suspension of Clostridium sporogenes with colony count <1000 cfu / ml to sterile bottles 4#, 8#, and 12#, respectively, and shake to mix; take 1 / 5 of the solution in sterile bottles 1# to 12# at 0 minutes, 10 minutes, and 20 minutes, filter and rinse, transfer the filter membrane to the TSA plate, and place the plate in a biological incubator for 5 days at a temperature of 32.5±2.5℃; and perform three parallel tests at the same time during the above filtration sampling and culturing process; observe the colony count after the culturing. The results are shown in Table 1; Table 1: Quantitative verification results of the non-toxicity of the dissolving agent 1% ascorbic acid solution and sodium heparin solution using Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Clostridium sporogenes

[0026] Table 1 continued:

[0027] Example 2: Qualitative verification of the non-toxicity of the dissolving agent 1% ascorbic acid solution and heparin sodium solution using Bacillus subtilis, Candida albicans and Aspergillus niger Take 30ml of the comparative solvent and 50ml of the 1% ascorbic acid solution of the solvent to be verified, add 3 portions of each to 1#~3# bacteria collectors and mix evenly; take 30ml of the comparative solvent and 25ml of the heparin sodium solution of the solvent to be verified, add 3 portions of each to 4#~6# bacteria collectors and mix evenly; take 3 portions of 50ml of the comparative solvent and add 7#~9# bacteria collectors respectively; add 1ml of the bacterial suspension of Bacillus subtilis with a viable count of <100cfu / ml to 1#, 4# and 7# bacteria collectors, shake and mix evenly, and let it stand for 20 minutes; add 1ml of the bacterial suspension of Bacillus subtilis with a viable count of <100cfu / ml to 2#, 5# and 8# bacteria collectors, shake and mix evenly, and let it stand for 20 minutes; Add 1 ml of Candida albicans suspension containing viable counts <100 cfu / ml to the bacteria collector, shake and mix, and let stand for 20 minutes; add 1 ml of Aspergillus niger suspension containing viable counts <100 cfu / ml to 3#, 6# and 9# bacteria collectors, shake and mix, and let stand for 20 minutes; filter and rinse the bacterial suspensions in 1#~9# bacteria collectors respectively, and put the obtained test bacteria into the corresponding bacteria collectors; add culture medium TSB to 1#~9# bacteria collectors, and culture at 22.5±2.5℃ for 5 days; observe daily, and the observation results are shown in Table 2: Table 2: Qualitative verification results of the non-toxicity of Bacillus subtilis, Candida albicans and Aspergillus niger to the dissolving agent 1% ascorbic acid solution and heparin sodium solution

[0028] From the above embodiments, it can be seen that the beneficial technical effect of the non-toxicity verification method of the medical sterile dissolving agent of the present invention is that it can effectively verify the non-toxicity of the dissolving agent, avoiding the problem that the dissolving agent to be verified is toxic and may be washed away by the flushing solution; and the operation is simple and the results are reliable.

Claims

1. A method for verifying the non-toxicity of a medical sterile dissolving agent, characterized in that: A 0.1% sterile peptone aqueous solution specified in the Chinese Pharmacopoeia is used as a comparative dissolving agent; one or more of seven sensitive bacterial species are used as test bacteria for verification testing; a bacterial suspension containing the test bacteria is added to the dissolving agent to be verified and the comparative dissolving agent under the same conditions; the test bacteria in the dissolving agent to be verified and the comparative dissolving agent are obtained by filtration and cultured respectively; whether the dissolving agent to be verified is toxic is judged based on the growth of the test bacteria obtained in the two dissolving agents; wherein the seven sensitive bacterial species include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Candida albicans and Aspergillus niger.

2. The method for verifying the non-toxicity of the medical sterile dissolving agent according to claim 1, characterized in that: When multiple test bacteria are used for validation testing, each test bacteria needs to be cultured and counted separately.

3. The method for verifying the non-toxicity of the medical sterile dissolving agent according to claim 1, characterized in that: The adding of the bacterial suspension containing the test bacteria under the same conditions comprises adding a bacterial suspension containing the test bacteria with a viable count of <1000 cfu / ml to 50 ml to 80 ml of the verification dissolving agent and the comparison dissolving agent respectively and mixing them to prepare a mixed solution with a viable count of no more than 100 cfu in each 10 ml to 16 ml of the solution.

4. The method for verifying the non-toxicity of the medical sterile dissolving agent according to claim 1, characterized in that: The method of judging whether the dissolving agent to be verified is toxic based on the growth conditions of the test bacteria obtained in the two dissolving agents includes quantitative testing and qualitative testing.

5. The method for verifying the non-toxicity of the medical sterile dissolving agent according to claim 4, characterized in that: The quantitative test comprises: adding a set amount of the to-be-verified dissolving agent and the comparative dissolving agent to a sterile bottle and shaking them evenly; adding a set amount of test bacteria to the sterile bottle, and obtaining the test bacteria in the to-be-verified dissolving agent and the comparative dissolving agent by filtration at 0, 10 and 20 minutes of standing; transferring the test bacteria to a plate by filter membrane transfer and culturing; and performing the above filtration sampling and culturing test in parallel three times; counting the colonies of the culture results of the to-be-verified dissolving agent and the comparative dissolving agent; and calculating the recovery rate by the following formula (1): : ( = 0, 10 and 20 minutes) (1) In the formula, For the Minutes of recovery of filtration sampling culture; The average colony count of three parallel tests of the dissolving agent to be verified in the minute, in cfu; For comparison, the solvent The average colony count of three parallel tests is in cfu. When it is between 50% and 200%, the solvent to be verified is considered to be non-toxic.

6. The method for verifying the non-toxicity of the medical sterile dissolving agent according to claim 5, characterized in that: The test bacteria are transferred to the plate by filter membrane transfer, including: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Clostridium sporogenes are transferred to pre-made tryptic soy agar medium TSA plate; Candida albicans and Aspergillus niger are transferred to Sabouraud dextrose agar medium SDA plate.

7. The method for verifying the non-toxicity of a medical sterile dissolving agent according to claim 4, characterized in that: The qualitative test comprises adding a set amount of the to-be-verified dissolving agent and the comparative dissolving agent to a bacteria collector and shaking them evenly, adding a set amount of test bacteria to a sterile bottle, standing for 20 to 40 minutes, and then using a filtration method to obtain the test bacteria in the to-be-verified dissolving agent and the comparative dissolving agent respectively; Put the test bacteria back into the bacteria collector and add the corresponding culture medium to the bacteria collector according to the type of test bacteria added; among them, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Clostridium sporogenes are added to the thioglycollate fluid culture medium FTM; Bacillus subtilis, Candida albicans and Aspergillus niger are added to the tryptic soytone liquid culture medium TSB; the bacteria collector is cultured under the set conditions, among which the FTM bacteria collector is cultured at 30-35℃ for ≤5 days, and the TSB bacteria collector is cultured at 20-25℃ for ≤5 days; observe the bacteria collector containing the verification mixed solution and the comparison mixed solution samples every day to see whether there is test bacteria growth; if both have test bacteria growth, it is regarded as the non-toxicity of the dissolving agent to be verified; if neither has test bacteria growth, it is regarded as a verification failure; if the former has no test bacteria growth and the latter has test bacteria growth, it is regarded as the toxicity of the dissolving agent to be verified; if the former has test bacteria growth and the latter has test bacteria growth, it is regarded as a verification failure.

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